A Paal–Knorr agent for chemoproteomic profiling of targets of isoketals in cells

Natural systems produce various γ-dicarbonyl-bearing compounds that can covalently modify lysine in protein targets via the classic Paal–Knorr reaction. Among them is a unique class of lipid-derived electrophiles – isoketals that exhibit high chemical reactivity and critical biological functions. However, their target selectivity and profiles in complex proteomes remain unknown. Here we report a Paal–Knorr agent, 4-oxonon-8-ynal (herein termed ONAyne), for surveying the reactivity and selectivity of the γ-dicarbonyl warhead in biological systems. Using an unbiased open-search strategy, we demonstrated the lysine specificity of ONAyne on a proteome-wide scale and characterized six probe-derived modifications, including the initial pyrrole adduct and its oxidative products (i.e., lactam and hydroxylactam adducts), an enlactam adduct from dehydration of hydroxylactam, and two chemotypes formed in the presence of endogenous formaldehyde (i.e., fulvene and aldehyde adducts). Furthermore, combined with quantitative chemoproteomics in a competitive format, ONAyne permitted global, in situ, and site-specific profiling of targeted lysine residues of two specific isomers of isoketals, levuglandin (LG) D2 and E2. The functional analyses reveal that LG-derived adduction drives inhibition of malate dehydrogenase MDH2 and exhibits a crosstalk with two epigenetic marks on histone H2B in macrophages. Our approach should be broadly useful for target profiling of bioactive γ-dicarbonyls in diverse biological contexts.

Natural systems produce various γ-dicarbonyl-bearing compounds that can covalently modify lysine in protein targets via the classic Paal–Knorr reaction.

Synthetic chemistry methods have been increasingly underscored by their potential to be repurposed as biocompatible methods for both chemical biology and drug discovery. The most-known examples of such a repurposing approach include the Staudinger ligation¹ and the Huisgen-based click chemistry.² Moreover, bioconjugation of cysteine and lysine can be built upon facile chemical processes,³ while chemoselective labelling of other polar residues (e.g., histidine,⁴ methionine,⁵ tyrosine,⁶ aspartic and glutamic acids^7,8) requires more elaborate chemistry, thereby offering a powerful means to study the structure and function of proteins, even at a proteome-wide scale.The classical Paal–Knorr reaction has been reported for a single-step pyrrole synthesis in 1884.^9,10 The reaction involves the condensation of γ-dicarbonyl with a primary amine under mild conditions (e.g., room temperature, mild acid) to give pyrrole through the intermediary hemiaminals followed by rapid dehydration of highly unstable pyrrolidine adducts (Fig. S1^†).Interestingly, we and others have recently demonstrated that the Paal–Knorr reaction can also readily take place in native biological systems.^11–13 More importantly, the Paal–Knorr precursor γ-dicarbonyl resides on many endogenous metabolites and bioactive natural products.¹⁴ Among them of particular interest are isoketals¹⁵ (IsoKs, also known as γ-ketoaldehydes) which are a unique class of lipid derived electrophiles (LDEs) formed from lipid peroxidation (Fig. S2^†)¹⁶ that has emerged as an important mechanism for cells to regulate redox signalling and inflammatory responses,¹⁷ and drive ferroptosis,¹⁸ and this field has exponentially grown over the past few years. It has been well documented that the γ-dicarbonyl group of IsoKs can rapidly and predominantly react with lysine via the Paal–Knorr reaction to form a pyrrole adduct in vitro (Fig. 1).¹⁵ Further, the pyrrole formed by IsoKs can be easily oxidized to yield lactam and hydroxylactam products in the presence of molecular oxygen (Fig. 1). These rapid reactions are essentially irreversible. Hence, IsoKs react with protein approximately two orders of magnitude faster than the most-studied LDE 4-hydoxynonenal (4-HNE) that contains α,β-unsaturated carbonyl to generally adduct protein cysteines by Michael addition (Fig. S3^†).¹⁵ Due to this unique adduction chemistry and rapid reactivity, IsoKs exhibit intriguing biological activities, including inhibition of the nucleosome complex formation,¹⁹ high-density lipoprotein function,²⁰ mitochondrial respiration and calcium homeostasis,²¹ as well as activation of hepatic stellate cells.²² Furthermore, increases in IsoK-protein adducts have been identified in many major diseases,²³ such as atherosclerosis, Alzheimer''s disease, hypertension and so on.Open in a separate windowFig. 1The Paal–Knorr precursor γ-dicarbonyl reacts with the lysine residue on proteins to form diverse chemotypes via two pathways. The red arrow shows the oxidation pathway, while the blue one shows the formaldehyde pathway.Despite the chemical uniqueness, biological significance, and pathophysiological relevance of IsoKs, their residue selectivity and target profiles in complex proteomes remain unknown, hampering the studies of their mechanisms of action (MoAs). Pioneered by the Cravatt group, the competitive ABPP (activity-based protein profiling) has been the method of choice to analyse the molecular interactions between electrophiles (e.g., LDEs,²⁴ oncometabolites,²⁵ natural products,^26,27 covalent ligands and drugs^28–30) and nucleophilic amino acids across complex proteomes. In this regard, many residue-specific chemistry methods and probes have been developed for such studies. For example, several lysine-specific probes based on the activated ester warheads (e.g., sulfotetrafluorophenyl, STP;³¹N-hydroxysuccinimide, NHS³²) have recently been developed to analyse electrophile–lysine interactions at a proteome-wide scale in human tumour cells, which provides rich resources of ligandable sites for covalent probes and potential therapeutics. Although these approaches can also be presumably leveraged to globally and site-specifically profile lysine-specific targets IsoKs, the reaction kinetics and target preference of activated ester-based probes likely differ from those of γ-dicarbonyls, possibly resulting in misinterpretation of ABPP competition results. Ideally, a lysine profiling probe used for a competitive ABPP analysis of IsoKs should therefore possess the same, or at least a similar, warhead moiety. Furthermore, due to the lack of reactive carbonyl groups on IsoK-derived protein adducts, several recently developed carbonyl-directed ligation probes for studying LDE-adductions are also not suitable for target profiling of IsoKs.^33,34Towards this end, we sought to design a “clickable” γ-dicarbonyl probe for profiling lysine residues and, in combination with the competitive ABPP strategy, for analysing IsoK adductions in native proteomes. Considering that the diversity of various regio- and stereo- IsoK isomers¹⁵ (a total of 64, Fig. S2^†) in chemical reactivity and bioactivities is likely attributed to the substitution of γ-dicarbonyls at positions 2 and 3, the “clickable” alkyne handle needs to be rationally implemented onto the 4-methyl group in order to minimize the biases when competing with IsoKs in target engagement. Interestingly, we reasoned that 4-oxonon-8-ynal, a previously reported Paal–Knorr agent used as an intermediate for synthesizing fatty acid probes³⁵ or oxa-tricyclic compounds,³⁶ could be repurposed for the γ-dicarbonyl-directed ABPP application. With this chemical in hand (herein termed ONAyne, Fig. 2A), we first used western blotting to detect its utility in labelling proteins, allowing visualization of a dose-dependent labelling of the proteome in situ (Fig. S4^†). Next, we set up to incorporate this probe into a well-established chemoproteomic workflow for site-specific lysine profiling in situ (Fig. 2A). Specifically, intact cells were labelled with ONAyne in situ (200 μM, 2 h, 37 °C, a condition showing little cytotoxicity, Fig. S5^†), and the probe-labelled proteome was harvested and processed into tryptic peptides. The resulting probe-labelled peptides were conjugated with both light and heavy azido-UV-cleavable-biotin reagents (1 : 1) via Cu^I-catalyzed azide–alkyne cycloaddition reaction (CuAAC, also known as click chemistry). The biotinylated peptides were enriched with streptavidin beads and photoreleased for LC-MS/MS-based proteomics. The ONAyne-labelled peptides covalently conjugated with light and heavy tags would yield an isotopic signature. We considered only those modified peptide assignments whose MS1 data reflected a light/heavy ratio close to 1.0, thereby increasing the accuracy of these peptide identifications. Using this criterium, we applied a targeted database search to profile three expected probe-derived modifications (PDMs), including 13 pyrrole peptide adducts (Δ273.15), 77 lactam peptide adducts (Δ289.14), and 557 hydroxylactam peptide adducts (Δ305.14), comprising 585 lysine residues on 299 proteins (Fig. S6 and S7^†). Among them, the hydroxylactam adducts were present predominately, since the pyrrole formed by this probe, the same as IsoKs, can be easily oxidized when being exposed to O₂. This finding was in accordance with a previous report where the pyrrole adducts formed by the reaction between IsoK and free lysine could not be detected, but rather their oxidized forms.³⁷ Regardless, all three types of adducts were found in one lysine site of EF1A1 (K387, Fig. S8^†), further confirming the intrinsic relationship among those adductions in situ.Open in a separate windowFig. 2Adduct profile and proteome-wide selectivity of the γ-dicarbonyl probe ONAyne. (A) Chemical structure of ONAyne and schematic workflow for identifying ONAyne-adducted sites across the proteome. (B) Bar chart showing the distribution of six types of ONAyne-derived modifications formed in situ and in vitro (note: before probe labelling, small molecules in cell lysates were filtered out through desalting columns).State-of-the-art blind search can offer an opportunity to explore unexpected chemotypes (i.e., modifications) derived from a chemical probe and to unbiasedly assess its proteome-wide residue selectivity.^38,39 We therefore sought to use one of such tools termed pChem³⁸ to re-analyse the MS data (see Methods, ESI^†). Surprisingly, the pChem search identified three new and abundant PDMs (Fig. 1 and Table S1^†), which dramatically expand the ONAyne-profiled lysinome (2305 sites versus 585 sites). Overall, these newly identified PDMs accounted for 74.6% of all identifications (Fig. 2B and Table S2^†). Among them, the PDM of Δ287.13 (Fig. 1 and S7^†) might be an enlactam product via dehydration of the probe-derived hydroxylactam adduct. The other two might be explained by the plausible mechanism as follows (Fig. 1). The endogenous formaldehyde (FA, produced in substantial quantities in biological systems) reacts with the probe-derived pyrrole adduct via nucleophilic addition to form a carbinol intermediate, followed by rapid dehydration to a fulvene (Δ285.15, Fig. S7^†) and immediate oxidation to an aldehyde (Δ301.14, Fig. S7^†). In line with this mechanism, the amount of FA-derived PDMs was largely eliminated when the in vitro ONAyne labelling was performed in the FA-less cell lysates (Fig. 2B and Table S3^†). Undoubtedly, the detailed mechanisms underlying the formation of these unexpected PDMs require further investigation, and so does the reaction kinetics. Regardless, all main PDMs from ONAyne predominantly target the lysine residue with an average localization probability of 0.77, demonstrating their proteome-wide selectivity (Fig. S9^†).Next, we adapted an ABPP approach to globally and site-specifically quantify the reactivity of lysine towards the γ-dicarbonyl warhead through a dose-dependent labelling strategy (Fig. 3A) that has been proved to be successful for other lysine-specific probes (e.g., STP alkyne).³¹ Specifically, MDA-MB-231 cell lysates were treated with low versus high concentrations of ONAyne (1 mM versus 0.1 mM) for 1 h. Probe-labelled proteomes were digested into tryptic peptides that were then conjugated to isotopically labelled biotin tags via CuAAC for enrichment, identification and quantification. In principle, hyperreactive lysine would saturate labelling at the low probe concentration, whereas less reactive ones would show concentration-dependent increases in labelling. For fair comparison, the STP alkyne-based lysine profiling data were generated by using the same chemoproteomic workflow. Although 77.5% (3207) ONAyne-adducted lysine sites can also be profiled by STP alkyne-based analysis, the former indeed has its distinct target-profile with 930 lysine sites newly identified (Fig. S10 and Table S4^†). Interestingly, sequence motif analysis with pLogo⁴⁰ revealed a significant difference in consensus motifs between ONAyne- and STP alkyne-targeting lysines (Fig. S11^†).Open in a separate windowFig. 3ONAyne-based quantitative reactivity profiling of proteomic lysines. (A) Schematic workflow for quantitative profiling of ONAyne–lysine reactions using the dose-dependent ABPP strategy (B) Box plots showing the distribution of R_10:1 values quantified in ONAyne- and STP alkyne-based ABPP analyses, respectively. Red lines showing the median values. ***p ≤ 0.001 two-tailed Student''s t-test. (C) Representative extracted ion chromatograms (XICs) showing changes in the EF1A1 peptide bearing K273 that is adducted as indicated, with the profiles for light and heavy-labelled peptides in blue and red, respectively.Moreover, we quantified the ratio (R_{1 mM:0.1 mM}) for a total of 2439 ONAyne-tagged lysines (on 922 proteins) and 17904 STP alkyne-tagged lysines (on 4447 proteins) across three biological replicates (Fig. S12 and Table S5^†). Strikingly, only 26.7% (651) of quantified sites exhibited nearly dose-dependent increases (R_{1 mM:0.1 mM} > 5.0) in reactivity with ONAyne, an indicative of dose saturation (Fig. 3B and C). In contrast, such dose-dependent labelling events accounted for >69.1% of all quantified lysine sites in the STP alkyne-based ABPP analysis.³¹ This finding is in accordance with the extremely fast kinetics of reaction between lysine and γ-dicarbonyls (prone to saturation). Nonetheless, by applying 10-fold lower probe concentrations, overall 1628 (80.2%) detected lysines could be labelled in a fully concentration-dependent manner with the median R_10:1 value of 8.1 (Fig. 3B, C, S12 and Table S5^†). Next, we asked whether the dose-depending quantitation data (100 μM versus 10 μM) can be harnessed to predict functionality. By retrieving the functional information for all quantified lysines from the UniProt Knowledgebase, we found that those hyper-reactive lysines could not be significantly over-represented with annotation (Fig. S12^†). Nonetheless, among all quantified lysines, 509 (25.1%) possess functional annotations, while merely 2.5% of the human lysinome can be annotated. Moreover, 381 (74.8%) ONAyne-labelled sites are known targets of various enzymatic post-translational modifications (PTMs), such as acetylation, succinylation, methylation and so on (Fig. S13^†). In contrast, all known PTM sites accounted for only 59.6% of the annotated human lysinome. These findings therefore highlight the intrinsic reactivity of ONAyne towards the ‘hot spots’ of endogenous lysine PTMs.The aforementioned results validate ONAyne as a fit-for-purpose lysine-specific chemoproteomic probe for competitive isoTOP-ABPP application of γ-dicarbonyl target profiling. Inspired by this, we next applied ONAyne-based chemoproteomics in an in situ competitive format (Fig. 4A) to globally profile lysine sites targeted by a mixture of levuglandin (LG) D₂ and E₂, two specific isomers of IsoKs that can be synthesized conveniently from prostaglandin H₂ (ref. 41) (Fig. S2^†). Specifically, mouse macrophage RAW264.7 cells (a well-established model cell line to study LDE-induced inflammatory effects) were treated with 2 μM LGs or vehicle (DMSO) for 2 h, followed by ONAyne labelling for an additional 2 h. The probe-labelled proteomes were processed as mentioned above. For each lysine detected in this analysis, we calculated a control/treatment ratio (R_C/T). Adduction of a lysine site by LGs would reduce its accessibility to the ONAyne probe, and thus a higher R_C/T indicates increased adduction. In total, we quantified 2000 lysine sites on 834 proteins across five biological replicates. Among them, 102 (5.1%) sites exhibited decreases of reactivity towards LGs treatment (P < 0.05, Table S6^†), thereby being considered as potential targets of LGs. Notably, we found that different lysines on the same proteins showed varying sensitivity towards LGs (e.g., LGs targeted K3 of thioredoxin but not K8, K85 and K94, Table S6^†), an indicative of changes in reactivity, though we could not formally exclude the effects of changes in protein expression on the quantified competition ratios. Regardless, to the best of our knowledge, the proteome-wide identification of potential protein targets by IsoKs/LGs has not been possible until this work.Open in a separate windowFig. 4ONAyne-based in situ competitive ABPP uncovers functional targets of LGs in macrophages. (A) Schematic workflow for profiling LGs–lysine interactions using ONAyne-based in situ competitive ABPP. (B) Volcano plot showing the log₂ values of the ratio between the control (heavy) and LGs-treated (light) channels and the −log₁₀(P) of the statistical significance in a two-sample t-test for all quantified lysines. Potential targets of LGs are shown in blue (R_C/T>1.2, P < 0.05), with the validated ones in red. (C) Bar chart showing the inhibitory effect of 2 μM LGs on the cellular enzymatic activity of MDH2. Data represent means ± standard deviation (n = 3). Statistical significance was calculated with two-tailed Student''s t-tests. (D) Pretreatment of LGs dose-dependently blocked ONAyne-labelling of MDH2 in RAW264.7 cells, as measured by western blotting-based ABPP. (E and F) LGs dose-dependently decreased the H2BK5 acetylation level in RAW 264.7 cells, as measured either by western blotting (E) or by immunofluorescence imaging (F). n = 3. For G, nuclei were visualized using DAPI (blue).We initially evaluated MDH2 (malate dehydrogenase, mitochondrial, also known as MDHM), an important metabolic enzyme that possesses four previously uncharacterized liganded lysine sites (K157, K239, K301 and K329, Fig. 4B) that are far from the active site (Fig. S14^†). We found that LGs dramatically reduced the catalytic activity of MDH2 in RAW264.7 cells (Fig. 4C), suggesting a potentially allosteric effect. We next turned our attention to the targeted sites residing on histone proteins, which happen to be modified by functionally important acetylation, including H2BK5ac (Fig. 4B) that can regulate both stemness and epithelial–mesenchymal transition of trophoblast stem cells.⁴² We therefore hypothesized that rapid adduction by LGs competes with the enzymatic formation of this epigenetic mark. Immunoblotting-based competitive ABPP confirmed that LGs dose-dependently blocked probe labelling of H2B (Fig. 4D). Further, both western blots and immunofluorescence assays revealed that LG treatment decreased the level of acetylation of H2BK5 (average R_C/T = 1.3, P = 0.007) in a concentration-dependent manner (Fig. 4E and F). Likewise, a similar competitive crosstalk was observed between acetylation and LG-adduction on H2BK20 (average R_C/T = 1.2, P = 0.01) that is required for chromatin assembly⁴³ and/or gene regulation⁴⁴ (Fig. 4B and S15^†). Notably, these findings, together with several previous reports by us and others about histone lysine ketoamide adduction by another important LDE, 4-oxo-2-noenal,^11,45,46 highlight again the potentially important link between lipid peroxidation and epigenetic regulation. In addition to the targets validated as above, many other leads also merit functional studies considering diverse biological or physiologic effects of LGs in macrophages.     

One-electron bonds in copper–aluminum and copper–gallium complexes

Odd-electron bonds have unique electronic structures and are often encountered as transiently stable, homonuclear species. In this study, a pair of copper complexes supported by Group 13 metalloligands, M[N((o-C₆H₄)NCH₂PⁱPr₂)₃] (M = Al or Ga), featuring two-center/one-electron (2c/1e) σ-bonds were synthesized by one-electron reduction of the corresponding Cu(i) ⇢ M(III) counterparts. The copper bimetallic complexes were investigated by X-ray diffraction, cyclic voltammetry, electron paramagnetic spectroscopy, and density functional theory calculations. The combined experimental and theoretical data corroborate that the unpaired spin is delocalized across Cu, M, and ancillary atoms, and the singly occupied molecular orbital (SOMO) corresponds to a σ-(Cu–M) bond involving the Cu 4p_z and M ns/np_z atomic orbitals. Collectively, the data suggest the covalent nature of these interactions, which represent the first examples of odd-electron σ-bonds for the heavier Group 13 elements Al and Ga.

Hanging on by a thread. Formally zerovalent copper complexes with an Al(iii) or Ga(iii) support were investigated. The combined experimental and theoretical data corroborate the presence of an odd-electron σ-bond between Cu and the Group 13 center.

Odd-electron σ-bonds, where the electrons are delocalized between two atoms, can occur as two-center/one-electron (2c/1e) or two-center/three-electron (2c/3e) interactions. Proposed by Pauling in 1931,¹ odd-electron σ-bonds have garnered attention because of their fundamental importance to chemical bonding and their relationship to radical species generated during oxidative stress in biological systems.^2–14 Examples of compounds exhibiting odd-electron bonding are typically homonuclear (like H₂⁺, He₂⁺, and alkali metal dimers) and transiently stable, limiting them to spectroscopic characterization.^1,11,15–18The first solid-state structure of a formally one-electron σ-bond was a tetraphosphabenzene species (Fig. 1a) which was formed by the coupling of two diphosphirenyl radicals.¹⁹ Following this discovery, the formation of discrete 2c/1e σ-bonds, where the odd-electron is delocalized between two homonuclear main group centers, was reported for B·B and then extended to P·P.^8,17,20 Of note, the first solid-state structure of a B·B compound was reported in only 2014 (Fig. 1b).²¹ Examples of 2c/1e σ-bonds between the heavier Group 13 congeners are even more lacking because of the greater propensity for their unpaired spins to couple, forming larger more stable clusters.⁸ To our knowledge, there are only three structurally characterized examples of odd-electron bonds for the heavy Group 13 atoms,²² and these examples are all homonuclear π-radicals (Fig. 1c).^23–26Open in a separate windowFig. 1Select examples of structurally characterized molecules (a–d) featuring odd-electron bonds.Heteronuclear odd-electron σ-bonds are also rare. The Cu(TPB) complex, where TPB is a trisphosphinoborane, is the single structural example of a 2c/1e bond between heteroatoms (Fig. 1d).²⁷ The authors described the bonding as Cu·B, where the unpaired electron is heavily polarized toward B. A theoretical study predicted that such a bond would also exist between Cu and Al, but no heavier analogues of Cu(TPB) have been synthesized to date.²⁸ Furthermore, the heavier Group 13 elements by virtue of their lower electronegativity compared to B should facilitate greater covalent interactions with the Cu center.Hence, we sought to target formally zerovalent Cu complexes supported by Al(III) or Ga(III) as an extension of the previously reported isoelectronic nickelate species and Cu(TPB).²⁹ Herein, we describe the synthesis, structure, spectroscopic characterization, and DFT calculations of cationic [CuML]⁺ complexes (L = [N((o-C₆H₄)NCH₂PⁱPr₂)₃]³⁻; M = Al and Ga) as well as their one-electron reduced metalloradical counterparts that feature discrete 2c/1e bonds.     

Photoinduced inverse Sonogashira coupling reaction

Alkynes are widely used in chemistry, medicine and materials science. Here we demonstrate a transition-metal and photocatalyst-free inverse Sonogashira coupling reaction between iodoalkynes and (hetero)arenes or alkenes under visible-light irradiation. Mechanistic and computational studies suggest that iodoalkynes can be directly activated by visible light irradiation, and an excited state iodoalkyne acted as an “alkynyl radical synthetic equivalent”, reacting with a series of C(sp²)–H bonds for coupling products. This work should open new windows in radical chemistry and alkynylation method.

A transition-metal and photocatalyst-free, photoinduced inverse Sonogashira coupling reaction was developed. Under visible-light irradiation, the excited state iodoalkyne acted as an “alkynyl radical synthetic equivalent”.

Alkynes are among the most important class of compounds in organic chemistry. Because of their structural rigidity, special electronic properties and numerous methods available for the functionalization of the triple bond, alkynes are important tools and structural elements both in medicinal chemistry and materials sciences.¹ Therefore, the development of a new methodology to introduce carbon–carbon triple bonds is of great importance in organic chemistry. The Sonogashira coupling reaction is typically used for the formation of C(sp)–C(sp²) bonds starting from hetero(aryl) halides and terminal alkynes.² Recently, “inverse Sonogashira coupling” involving the direct alkynylation of unreactive C(sp²)–H bonds with readily available alkynyl halides has received growing interest in the development of a complementary strategy (Fig. 1a). Various main-group and transition metals have been developed to promote this transformation.³ In addition, a photomediated Sonogashira reaction without a photocatalyst was also developed by several groups (Fig. 1b).⁴Open in a separate windowFig. 1Models of alkynylation. (a) Conventional inverse Sonogashira reaction. (b) Photomediated Sonogashira reaction. (c) SOMOphilic alkynylation. (d) Photoinduced inverse Sonogashira reaction.In recent years, SOMOphilic alkylnylation (SOMO = singly occupied molecular orbital) has become an excellent method of introducing alkynyl groups (Fig. 1c).⁵ Based on photoredox and transition metal catalysis, numerous in situ generated radicals undergo α-addition and β-elimination to alkynyl reagents, like the broadly applicable ethynylbenziodoxolone (EBX) reagent. Various radical alkynylations were thus discovered by Li,⁶ Chen,⁷ Waser,⁸ and many other groups.⁹ However, extending the scope of radical precursors, more atom–economic reactions, and a deeper understanding of the mechanism in these transformations are still highly desirable.After the discovering of trityl radicals by Gomberg in 1900, the “rational” era of radical chemistry has since begun.¹⁰ Now, the development of radical reactions, especially those involving C(sp³) and C(sp²) radicals, enables rapid access to drug discovery, agrochemistry, materials science, and other disciplines.¹¹ However, the C(sp) radical remains a baffling species. Due to their very high energy, short life time, and limited and harsh preparation methods, alkynyl radicals remain an elusive species, which just exists in some extreme environments, like outer-space and the petrochemical industry.¹² Even though alkynyl radicals have been proposed as intermediates for some alkynylation methods, they were regarded as mysterious species and ignored by organic chemists for a long time.¹³ Recently, two approaches have been developed to aid the alkynyl radical generation step. In 2015, Hashmi and collaborators reported a [Au₂(μ-dppm)₂]²⁺ catalyzed free radical–radical C(sp)–C(sp³) bond coupling reaction between iodoalkynes and aliphatic amines.¹⁴ Under irradiation of sunlight, the dimeric gold complex was proposed to reduce the iodine acetylide to an alkynyl radical. In 2017, Li developed a transition-metal-free alkynylation reaction between iodoalkyne and 2-indolinone.¹⁵ Iodoalkynes could release alkynyl radicals under high temperature conditions. In 2019, we reported an Au(i) and Ir(iii) catalyzed alkynylative cyclization of o-alkylnylphenols with iodoalkynes, wherein the photosensitized energy transfer promoted the oxidative addition of a gold(i) complex with iodoalkynes.¹⁶ Based on our continuous interest in haloalkyne and photo-chemistry, we proposed that an iodoalkyne could be a potential “alkynyl radical precursor” under light irradiation. In this work, we uncovered a novel mode of transition-metal and photocatalyst-free, direct photoexcitation of iodoalkynes for the inverse Sonogashira coupling reaction with arenes, heteroarenes, and alkenes via an “alkynyl-radical type” transfer (Fig. 1d).     

Computational design of an amidase by combining the best electrostatic features of two promiscuous hydrolases

While there has been emerging interest in designing new enzymes to solve practical challenges, computer-based options to redesign catalytically active proteins are rather limited. Here, a rational QM/MM molecular dynamics strategy based on combining the best electrostatic properties of enzymes with activity in a common reaction is presented. The computational protocol has been applied to the re-design of the protein scaffold of an existing promiscuous esterase from Bacillus subtilis Bs2 to enhance its secondary amidase activity. After the alignment of Bs2 with a non-homologous amidase Candida antarctica lipase B (CALB) within rotation quaternions, a relevant spatial aspartate residue of the latter was transferred to the former as a means to favor the electrostatics of transition state formation, where a clear separation of charges takes place. Deep computational insights, however, revealed a significant conformational change caused by the amino acid replacement, provoking a shift in the pK_a of the inserted aspartate and counteracting the anticipated catalytic effect. This prediction was experimentally confirmed with a 1.3-fold increase in activity. The good agreement between theoretical and experimental results, as well as the linear correlation between the electrostatic properties and the activation energy barriers, suggest that the presented computational-based investigation can transform in an enzyme engineering approach.

A computational strategy, based on combining the best electrostatic properties of enzymes with activity on a common reaction, is presented and applied to the re-design of the protein scaffold of an promiscuous esterase to enhance its secondary amidase activity.

The application of enzymes for desired chemical transformations has been demonstrated by the report of novel and functional designed structures.^1–5 Recent advances in molecular biology and screening technologies have enabled the creation of enzymes via directed evolution. By mimicking the process of natural evolution, iterative cycles of (semi-)random mutations facilitate the improvement of proteins in the laboratory through screening and selection, and hence the identification of active variants.^6–12 Minimal structural information is needed for this strategy and distal sites critical for enzyme catalysis can also be identified. Nevertheless, directed evolution is limited by the fact that, even with the most efficient high-throughput system, only a fraction of all the possible mutants of a given enzyme can be sampled within a set timeframe.¹³ Furthermore, the development of an efficient screening system for a tailored reaction remains challenging. Recently machine-learning (ML) methods have been proposed to expedite evolution and expand the number of properties that can be optimized.^14,15 However, in order to create enzymes with novel reactivities by means of ML methods, protein engineers will have to use proteins with sequences not assigned to the designated reaction or with properties other than those of specific interest, which currently is a technical challenge. Sequence–function data from engineering experiments must be collected to catalogue the natural diversity of proteins in order to convert ML into a useful tool.¹⁵An alternative approach is a rational design, a technique that modifies selected residues at specific positions of an already existing protein scaffold through the analysis of existing mechanistic and structural data.¹⁶ To reveal the structures of the protein in the full catalytic process under physiological conditions, including metastable transition state (TS) structures, computer simulations are essential. Among all the computer-assisted design strategies, two philosophies can be identified: the redesign of the active site of an existing substrate-promiscuous enzyme and the de novo design that constructs an enzyme “from scratch”. The use of promiscuous enzymes is found to be a very promising starting point for the design of new and highly efficient biocatalysts.^17,18 However, the knowledge about the particular molecular mechanisms that allow enzymes to catalyze more than one chemical reaction is still under debate.^19–22Because both the enzyme redesign and the de novo design approach require knowledge of the TS of the reaction to be catalyzed, quantum mechanical (QM) calculations offer crucial complementary information that accelerates the development of novel designed reactions. Moreover, multiscale methods are the only tool that can offer a detailed atomistic picture of the reactions in the active site of the enzyme, which can be dramatically different from that in the gas phase or solution. In multiscale methods, electrons of the reacting fragments are explicitly described by QM methods and the large and complex interacting environment (the fully solvated protein) is described by molecular mechanics (MM) force fields. The mechanism of a reaction in the active site of an enzyme can be determined within these hybrid QM/MM methods through the extensive exploration of the Free Energy Surface (FES). This allows the determination of the rate-limiting step in a multi-step process and, within the framework of Transition State Theory (TST),²³ the prediction of rate constants directly comparable with experiments. Previous studies combining computer simulations with experimental kinetic measurements have demonstrated the good agreement that can be achieved,^24,25 which obviously depends on the quality of both simulations and experiments. In this regard, the error in the determination of activation free energies associated with the use of computational methods such as the umbrella sampling method,^26,27 employed in the present study, is usually accepted to be within 1 kcal mol⁻¹.²⁸Optimizing the secondary activity of promiscuous enzymes is a non-trivial challenge as can be illustrated by analysing the Bacillus subtilis esterase Bs2. While Bs2 is recognized as a serine hydrolase whose primary reaction is the hydrolysis of esters, it can also catalyze the hydrolysis of the amide bond of N-(4-nitrophenyl)-butyramide as a secondary reaction (Fig. 1).^22,29 Previously directed evolution experiments by Arnold and co-workers resulted in a 7-mutation variant with a 100-fold enhancement of the esterase activity (using para-nitrobenzyl butyrate as the substrate).³⁰ Bornscheuer and co-workers used a combination of directed evolution and rational design based on docking and classical energy minimization to get a 3-fold increase of the amidase activity of Bs2 after two single mutations.²² In a larger context, despite the successes of different computer-assisted designs of new enzymes, it has been argued that the high activities of the best artificial enzymes have been largely due to directed evolution and the contribution of computation was comparatively modest.³¹Open in a separate windowFig. 1Schematic representation of the reaction mechanism of the hydrolysis of N-(4-nitrophenyl)-butyramide catalyzed by Bs2. (a) Acylation step: the nucleophilic addition of Ser189 to the carbonyl followed by the breaking of the C–N bond is triggered by His399-assisted proton shuffling, and the leaving group, in this case, is 4-nitroaniline. (b) Hydrolysis step: the nucleophilic addition of a water molecule followed by the resolution of the acyl–enzyme complex is triggered by His399-assisted protein shuffling, yielding butyric acid as a product.We envisaged that creating mutations to optimize the preorganization of the protein environment will result in a variant that exhibits improved activity for the desired reaction.³² Based on our recent QM/MM studies of different enzymatic reactions, we have quantified and shown how the reactivity of different proteins can be rationalized from their electrostatic properties,^{24,25,33–36} as the pioneering studies reported by Warshel and co-workers.^37–39 The computed changes of the electrostatic potential or the electric field exerted by the studied proteins on the key atoms of the substrates reflect that there is a small reorganization of these entities when evolving from the reactant state (RS) to the TS at the lowest energy cost.^24,33–36 The electrostatic effects within the active site of the enzyme, therefore, appear to be critical for the electronic reorganization of the reactants during chemical transformations. These studies support the idea that the electrostatic properties of enzymes are the origin of their catalytic features;⁴⁰ consequently, we view that a detailed understanding of the molecular mechanism, including the evolution of electrostatic potential generated in the active site of the enzyme, could be useful in future computer-assisted protein design methods.To engineer enzymes with optimal electrostatic preorganization, comparative analysis between unrelated natural enzymes that catalyze the same chemical reactions can be a reliable strategy. In previous studies, we have shown that Candida antarctica lipase B (CALB) also displays amidase activity similar to that of Bs2, though being non-homologous with each other.^24,25 QM/MM studies of the amidase reaction catalyzed by wild-type Bs2 and CALB enzymes were previously conducted.^24,25 We expect that the favorable features of each enzyme could be isolated and combined to create a redesigned enzyme with improved catalytic activity for the secondary amidase reaction. In the present paper, based on our knowledge derived from previous comparative studies, and by applying the concept of electrostatic pre-organization,^{24,33–36,40–43} a variant with improved activity for the designated amidase reaction was generated. After overlapping the structures of both proteins in one of the located TSs, through the use of a rotation quaternion around selected atoms of the substrate, a catalytically improved Bs2 variant was delineated. In particular, residues of Bs2 with an unfavorable electrostatic effect on catalysis were substituted by those placed in an equivalent spatial position in CALB with a favorable effect, as explained in detail below. The QM/MM FES of the full catalytic reaction in the proposed variant, combined with the experimental characterization, will be used to propose a general computer-based strategy that can be potentially used to design new enzymes.     

Single-atom cobalt-hydroxyl modification of polymeric carbon nitride for highly enhanced photocatalytic water oxidation: ball milling increased single atom loading

Expediting the oxygen evolution reaction (OER) is the key to achieving efficient photocatalytic overall water splitting. Herein, single-atom Co–OH modified polymeric carbon nitride (Co-PCN) was synthesized with single-atom loading increased by ∼37 times with the assistance of ball milling that formed ultrathin nanosheets. The single-atom Co-N₄OH structure was confirmed experimentally and theoretically and was verified to enhance optical absorption and charge separation and work as the active site for the OER. Co-PCN exhibits the highest OER rate of 37.3 μmol h⁻¹ under visible light irradiation, ∼28-fold higher than that of common PCN/CoO_x, with the highest apparent quantum yields reaching 4.69, 2.06, and 0.46% at 400, 420, and 500 nm, respectively, and is among the best OER photocatalysts reported so far. This work provides an effective way to synthesize efficient OER photocatalysts.

Single-atom Co^II-OH modified polymeric carbon nitride synthesized with increased single-atom loading under the assistance of ball milling exhibits high photocatalytic water oxidation activity with Co-N₄OH as the highly active site.

Massive fuel energy consumption induced environmental and ecological problems, especially the greenhouse effect, and the resultant extreme climates and rise in sea level are threatening human life.¹ As a potential substitution for fuel energy, hydrogen energy conversion from solar energy via photocatalytic water splitting attracts great attention from scientists.^2–5 However, the photocatalytic hydrogen evolution efficiency from overall water splitting is still restricted by the sluggish oxygen evolution reaction (OER) that involves energy absorption, four-electron transfer, breakage of O–H bonds, and formation of O–O bonds,^6,7 and thus efficient OER photocatalysts become the key to achieving efficient overall water splitting. Though numerous hydrogen evolution photocatalysts have been reported, research on OER photocatalysts is mainly around a few semiconductors including BiVO₄, WO₃, Ag₃PO₄, α-Fe₂O₃, etc.8–11 and their activity is not high enough yet for practical applications. Therefore, exploring high-efficiency OER photocatalysts is still necessary.Polymeric carbon nitride (PCN) was first reported in 2009 (ref. 12) as a photocatalyst with a layered melon-type orthorhombic structure,¹³ and thereafter quickly became a “star” photocatalyst thanks to its advantages of being visible-light responsive and metal-free, non-toxic, and low cost, and its relatively high chemical stability.¹⁴ Because of several self-deficiencies including fast photogenerated charge recombination and a narrow optical absorption spectrum, PCN exhibits relatively low photocatalytic activity.¹⁵ Then, a series of strategies were put forward successively to enhance the photoactivity of PCN, such as enhancement of crystallinity,¹⁶ morphological control,¹⁷ structural modification¹⁸ (including extensively researched single atom modification in recent years^19,20), exfoliation,²¹ construction of hetero-(homo-)junctions,²² and loading of noble metals.²³ Though photocatalytic water splitting on PCN was extensively researched in the past, the research was mainly around the hydrogen evolution half-reaction used for exploring properties and the catalytic mechanism of photocatalysts, and little research was focused on the industrially useable overall water splitting process owing to the sluggish OER.¹⁵ Therefore, enhancing the photocatalytic OER activity of PCN becomes the key to practical applications.To increase OER rates of PCN, several kinds of methods were proposed, such as rational design of compound cocatalysts (e.g., CoO_x, IrO₂, CoP, CoPi, RhO_x, RuO_x, PtO_x, MnO_x, Co(OH)₂, Ni(OH)₂, and CoAl₂O₄ (ref. 24–30)), modification of carbon dots and carbon rings,^31,32 fabrication of special architectures of PCN (e.g., PCN quantum dot stacked nanowires³³), and single-atom (e.g., B, Co, and Mn^34–36) modification. For instance, Zhao and coauthors prepared B and N-vacancy comodified PCN that exhibits the highest OER rate of ∼28 μmol h⁻¹ (ref. 36) and recently their group further used these B doped PCN ultrathin nanosheets to fabricate a Z-scheme heterojunction for overall water splitting with a solar-to-hydrogen efficiency reaching ∼1.2%.³⁷ Comparatively, PCN loaded with compound cocatalysts can only enhance OER activity to a limited degree and there are finite methods for carbon modification and special architecture fabrication. Single-atom modification shows a bright prospect, on account of metal atoms capable of being inserted into the framework of PCN and effectively increasing the OER activity. However, reported single metal atom modification routes are all based on direct ion adsorption on PCN or calcination of mixtures of metal salts and PCN feedstocks.^34,35,38 New routes need be explored to increase effective loading of single atoms in PCN. Besides, the metal-OH structure is considered efficient for the OER,^30,39,40 and a single metal atom-OH structure has never been reported for modification of PCN, though Mn–OH was thought to play a key role in the OER process.³⁴Ball milling is an extensively used versatile and scalable way for preparation of heterogeneous catalysts and even single-atom catalysts,^41,42 but was rarely used in synthesis of PCN-based single-atom photocatalysts. In this work, we synthesized single-atom Co–OH modified PCN (Co-PCN) with the single-atom content in PCN highly increased with the assistance of ball milling. The simple synthetic route is shown in Fig. 1a. PCN was ball-milled to obtain BM-PCN that then adsorbed Co²⁺ till saturation to form BM-PCN/Co which was calcined to obtain BM-PCN/Co-c (Co-PCN). For comparison, PCN was directly used to adsorb Co²⁺ till saturation to form PCN/Co which was calcined to obtain PCN/Co-c. PCN mainly comprises large blocks with the size of several micrometers (Fig. S1^†), while BM-PCN contains massive irregular particles with the size reduced to several hundreds of nanometers (Fig. S2^†), indicative of high efficacy of ball milling. BM-PCN/Co-c exhibits a similar morphology as BM-PCN (Fig. 1b and S3^†) and PCN/Co-c exhibits a similar morphology to PCN (Fig. S4^†), but the surface area and mesopore volume of BM-PCN and BM-PCN/Co-c are not higher than those of PCN and PCN/Co-c (Fig. S5^†), manifesting that ball-milling and subsequent calcination did not form massive mesopores, which accords well with the particle size variation from several micrometers (before ball milling) to several hundreds of nanometers (after ball milling). However, the Co content in BM-PCN/Co-c, BM-PCN/Co, PCN/Co-c, and PCN/Co was measured to be 0.75, 0.50, 0.02, and ∼0.02 wt%, respectively, by inductively coupled plasma mass spectrometry (ICP-MS). The ∼37 times higher Co content in BM-PCN/Co-c than in PCN/Co-c suggests the ball-milling enhanced adsorption of Co²⁺ on surfaces of BM-PCN, which should arise mainly from the ball-milling induced increase of surface energy and adsorption sites.⁴³Open in a separate windowFig. 1(a) Schematic illustration for synthesis of single-atom Co^II-OH modified PCN (BM-PCN/Co-c); and (b) SEM, (inset in b) TEM, (c) AFM, (d) EDS elemental mapping, and (e) HAADF-STEM images of BM-PCN/Co-c.The TEM image shows the existence of small and ultrathin nanosheets in BM-PCN/Co-c (inset in Fig. 1b) which can also be observed in the atomic force microscopy (AFM) image with a thickness of ∼7–10 nm and lateral size of <70 nm (Fig. 1c), and formation of these ultrathin nanosheets results from the ball milling of PCN.⁴⁴ It should be noted that most formed ultrathin nanosheets with high surface energy may stack into compact particles upon ball milling, leading to no increase of the total surface area. Energy dispersive X-ray spectroscopy (EDS) elemental mapping images of BM-PCN/Co-c indicate homogeneous distribution of C, N, O, and Co elements in the sample (Fig. 1d). The high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image of BM-PCN/Co-c shows massive white spots (marked by circles) with a mean size of <1 Å dispersed in the sample (Fig. 1e and S6^†), which should correspond to single-atom Co.To further verify the single-atom Co structure in BM-PCN/Co-c, Co K-edge X-ray absorption near-edge structure spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS) analysis were performed. As shown in Fig. 2a, the absorption-edge position of BM-PCN/Co-c is quite close to that of CoO and their peak positions are similar and far from those of other reference samples, indicating that the valence of Co in BM-PCN/Co-c is about +2. The bonding structure around Co was determined by Fourier transformed (FT) k³-weighted EXAFS analysis. As shown in Fig. 2b, a distinct single Co-ligand peak at ∼1.6 Å for BM-PCN/Co-c is observed, which prominently differs from the Co–Co coordination peak at ∼2.2 Å for Co foil and the Co^II–O coordination peak at ∼1.7 Å for CoO. The wavelet transform (WT) contour plot of BM-PCN/Co-c shows only one intensity maximum (Fig. S7^†), and the Cl 2p core-level XPS spectrum of BM-PCN/Co-c reveals no residue of Cl (Fig. S8^†). These further indicate the single-atom dispersion of Co species. Apparently, the Co-ligand peak is almost consistent with the Co^II–N peak for Co porphyrin, suggesting that the single-atom Co in BM-PCN/Co-c mainly coordinates with N. Least-square EXAFS curve fitting was performed to confirm quantitative structural parameters of Co^II in BM-PCN/Co-c, as shown in Fig. 2c, S9, and S10 and Table S1.^† Simple Co–N single-shell fitting of BM-PCN/Co-c (Fig. S10^†) gave a coordination number of 5.6 ± 0.4 (Table S1^†), that is, Co^II coordinates with five atoms. Considering that the PCN monolayer provides four appropriate N coordination sites at most,⁴⁵ Co^II likely coordinates with four N atoms and one OH atom. Thus, we further performed Co–N₄/Co–O double-shell fitting (Fig. 2c) and the obtained R-factor (0.0011) remarkably reduces relative to that from Co–N single-shell fitting (0.0035), indicative of rationality of the proposed Co^II–N₄OH structure. Confirmed Co–N and Co–O bond lengths are 2.04 and 2.15 Å, respectively (Table S1^†).Open in a separate windowFig. 2(a) Co K-edge XANES and (b) EXAFS spectra of Co foil, Co porphyrin (Copr), CoO, Co₃O₄, Co₂O₃, and BM-PCN/Co-c; EXAFS (c) R space-fitting and (inset in c) K space-fitting curves of BM-PCN/Co-c; (d) optimized structure of PCN and Co-doped PCN with different doping configurations and calculated formation energies (e) of Co doped PCN; and (e) Co 2p and (f) O 1s core-level XPS spectra of samples.To further confirm rationality of the Co–N₄OH coordination structure, density functional theory (DFT) calculations were conducted. As shown in Fig. 2d, three possible Co^II coordination structures in the PCN monolayer were explored. The Co–N₄OH structure without removal of H from PCN exhibits a much lower formation energy (∼0.15 eV) than Co–N₄ and Co–N₃ structures with removal of two H atoms from PCN (∼2.51 and 3.55 eV), demonstrating a high probability of existence of the Co–N₄OH structure in BM-PCN/Co-c. This structure can also be evidenced by X-ray photoelectron spectroscopy (XPS). As shown in Fig. 2e, the Co 2p core-level XPS spectrum of BM-PCN/Co-c shows two distinct peaks at binding energies of 796.8 and 781.4 eV beside satellite peaks, corresponding to Co 2p_1/2 and 2p_3/2 of Co^II ions.⁴⁶ The spectrum of BM-PCN/Co also shows two Co 2p peaks but at a binding energy ∼1.1 eV higher, suggesting variation of the Co coordination structure from BM-PCN/Co to BM-PCN/Co-c. PCN/Co-c exhibits no peaks because of its low Co content. Fig. 2f shows O 1s core-level spectra of PCN, BM-PCN, BM-PCN/Co-c, and PCN/Co-c. All the samples exhibit one peak at a binding energy of ∼532.0 eV, ascribed to surface hydroxyl species,⁴⁷ but an additional peak could be obtained for BM-PCN or BM-PCN/Co-c after deconvolution. The peak at a binding energy of ∼531.3 eV for BM-PCN should be assigned to adsorbed H₂O at new active adsorption sites generated by ball milling. This peak can also be observed in the spectrum of BM-PCN/Co, but with a ∼0.1 eV shift to a higher binding energy (Fig. S11^†) owing to the influence of adsorbed Co^II ions. The peak at ∼531.2 eV for BM-PCN/Co-c should be assigned to Co–OH,⁴⁸ given that there is only one O 1s peak for BM-PCN-c (synthesized by direct calcination of BM-PCN) (Fig. S11^†). The calculated Co/O(–Co) molar ratio, based on the XPS data, is ∼1.07 (Table S2^†), close to 1, consistent with the Co–N₄OH coordination structure.In C 1s and N 1s core-level XPS spectra, BM-PCN, BM-PCN/Co-c, PCN/Co-c, and BM-PCN/Co exhibit similar peaks to PCN (Fig. S12a–d^†), indicative of their similar framework structure which can also be evidenced by their similar N/C molar ratios, 1.53 (Table S3^†), but the N–H peak of BM-PCN shifts ∼0.2 eV to a lower binding energy relative to that of PCN, likely arising from the ball-milling induced destruction of intralayer hydrogen bonds (Fig. S13^†). The Co content in BM-PCN/Co, BM-PCN/Co-c, and PCN/Co-c is too low to cause detectable variation of C 1s and N 1s peaks. Similar FT-IR absorption bands of the samples (Fig. S14a and b^†) also indicate their basic frame structure, but in enlarged spectra (Fig. S14c^†), ν(C–N) and ν(C N) absorption bands of BM-PCN shift 16 cm⁻¹ to a higher wavenumber and 19 cm⁻¹ to a lower wavenumber, respectively, relative to those of PCN at 1242 and 1640 cm⁻¹,⁴⁹ likely resulting from the ball-milling induced hydrogen bond destruction, and the shift of these two absorption bands turns smaller for BM-PCN/Co-c, suggesting calcination-induced reforming of the destroyed hydrogen bonds, which is consistent with the XPS results (Fig. S12c^†). Besides, BM-PCN exhibits a wider and relatively stronger ν(N–H)/ν(O–H) absorption band than PCN (Fig. S14a^†), probably owing to the hydrogen bond destruction and new adsorbed H₂O, while this absorption band for BM-PCN/Co-c becomes much weaker, suggesting hydrogen bond reforming and loss of new adsorbed H₂O (Fig. 2f). Zeta potentials of the samples dispersed in water reflect variation of surface adsorbed hydroxyl species. As shown in Fig. S15a,^† all the samples exhibit negative zeta potentials because of dissociation of surface hydroxyl species. The zeta potentials, following the order PCN (−24 mV) > BM-PCN (−41 mV) < BM-PCN/Co-c (−30 mV) ≈ PCN/Co-c (−28 mV), suggest the ball milling-induced increase of surface hydroxyls in BM-PCN and calcination-induced decrease in BM-PCN/Co-c, consistent with the FT-IR results.Solid-state ¹³C magic-angle-spinning nuclear magnetic resonance (NMR) spectra of PCN, BM-PCN, BM-PCN/Co-c, and PCN/Co-c show two similar peaks at chemical shifts of ∼164 and 156 ppm (Fig. S15b^†), ascribed to C−NH_x and N C–N, respectively,⁵⁰ indicating their similar molecular framework, but in enlarged spectra, BM-PCN exhibits ∼0.3° movement of the N C–N peak to a lower chemical shift compared with PCN, because of the ball-milling induced hydrogen bond destruction, and the C−NH_x peak of BM-PCN/Co-c moves ∼0.2° to a lower chemical shift, likely owing to formation of the C–N–Co structure whose peak lies close to the C−NH_x peak.⁵¹ The XRD patterns of the samples are shown in Fig. S15c.^† PCN and PCN/Co-c exhibit typical diffraction peaks of melon-type carbon nitride with a layered orthorhombic structure and peaks at 13.1° and 27.6° correspond to (210) and (002) facets, respectively,^13,52 but BM-PCN reveals remarkably decreased peak intensity and ∼0.2° shift of the (002) peak to a lower 2θ (indicative of the increased interlayer distance) relative to PCN, demonstrating the ball-milling induced hydrogen bond destruction and substantial decrease of crystallinity. The remarkable decrease of crystallinity and almost no change of the surface area of BM-PCN, compared with those of PCN, further suggest that ball milling may form massive thin nanosheets (Fig. 1c) most of which stack into compact particles (Fig. 1b) owing to their high surface energy. In comparison with BM-PCN, BM-PCN/Co-c exhibits a narrower (002) peak, suggesting enhanced crystallinity owing to the calcination-induced hydrogen bond reforming, consistent with the FT-IR results. On the whole, it is likely the ball-milling induced destruction of hydrogen bonds that contributes largely to the increase of surface energy and new active adsorption centers and thus Co²⁺ adsorption on BM-PCN.Optical absorption capability of samples was investigated by UV-vis diffuse reflectance spectroscopy (DRS). As shown in Fig. 3a, BM-PCN/Co-c, BM-PCN, and PCN/Co-c exhibit considerably higher, lower, and similar optical absorption than/to PCN, respectively. For BM-PCN/Co-c, the optical absorption enhancement at a wavelength of <400 nm may benefit from the electron-rich Co that enhances π–π* transitions in heptazine rings,⁵³ and the Urbach tail absorption should arise from the Co–OH doping.^54,55 Bandgaps (E_g) of PCN, BM-PCN, BM-PCN/Co-c, and PCN/Co-c were roughly confirmed as 2.70, 2.81, 2.56, and 2.73 eV, respectively, via the formula E_g/eV = 1240/(λ_ed/nm)⁵⁶ where λ_ed is the absorption edge determined by solid lines in the spectra. The wider E_g of BM-PCN probably results from the quantum size effect of massive ultrathin crystal nanosheets (Fig. 1c) formed by ball milling, and the narrower E_g of BM-PCN/Co-c arises from the Co–OH doping that was then verified by DFT calculations. As shown in Fig. S16,^† the calculated E_g of BM-PCN/Co-c, ∼1.90 eV, is much smaller than that of PCN (2.57 eV), in accordance with the experimental results. For PCN, the conduction band (CB) is contributed by C 2p and N 2p orbitals and the valence band (VB) mainly by N 2p orbitals, while for BM-PCN/Co-c, the CB is contributed by Co 3d, C 2p, and N 2p orbitals and the VB mainly by Co 3d and N 2p orbitals (Fig. S16c and d^†), effectively manifesting that the narrowing of E_g of BM-PCN/Co-c results from the Co–OH doping. In addition, there are prominent doping levels (E_d) in the bandgap of BM-PCN/Co-c, mainly contributed by Co 3d and O 2p orbitals (Fig. S16d^†), effectively proving the Co–OH doping effect in BM-PCN/Co-c. Similar calculation results have been reported for Pt–OH modified carbon nitride.⁵⁷ Given that the experimental Co content (0.75 wt%) is much lower than the theoretical (6.71 wt%), practical doping levels in the bandgap may approach more to the VB. CB edges of the samples (E_CB) could be roughly determined by using Mott-Schottky plots (Fig. S17^†) and their Fermi levels (E_f) were subsequently confirmed based on VB-XPS spectra (Fig. S18^†). Energy band levels of the samples are shown in Fig. 3b, and it seems that ball milling causes a slight downshift of the VB edge (E_VB) of BM-PCN, favorable for photocatalytic water splitting, but the Co–OH doping causes a slight downshift of E_CB and upshift of E_VB of BM-PCN/Co-c. It is noteworthy that the E_d close to the VB edge (E_VB) can capture photogenerated holes⁵⁸ and thus the single-atom Co–OH works as the active site for the OER (Fig. 3b).Open in a separate windowFig. 3(a) UV-vis diffuse reflectance spectra of PCN, BM-PCN, BM-PCN/Co-c, and PCN/Co-c; (b) energy band levels of the samples and schematic illustration for water oxidation on BM-PCN/Co-c; (c) photoluminescence spectra, (d) time-resolved fluorescence spectra, and (e) anodic photocurrent (J_a) response of the samples; and (f) EPR spectra of the samples in the dark and under visible light irradiation. Data in (d) are the results of fitting decay curves to a tri-exponential model. Dark J_a in (e) was set as zero for distinct comparison.Spectroscopy and photoelectrochemical tests were conducted to evaluate photogenerated charge separation and transfer performance. As shown in Fig. 3c, photoluminescence (PL) spectra of all the samples show one emission peak, basically corresponding to their bandgap emission. BM-PCN exhibits weaker PL intensity than PCN, revealing a decreased photogenerated charge recombination efficiency, which originates from faster charge transfer from the inside to the surface of ultrathin nanosheets (Fig. S19^†) and trapped by surface states.⁵⁹ BM-PCN/Co-c exhibits the lowest PL intensity and the PL intensity of PCN/Co-c is lower than that of PCN, which arises from the E_d capturing photogenerated holes to reduce their direct recombination with electrons beside the ultrathin nanosheet effect in BM-PCN/Co-c. Fig. 3d shows time-resolved fluorescence spectra of the samples. Decay curves were well fitted to a tri-exponential model (S3) and the obtained results are shown in Fig. 3d. Three lifetimes (τ₁–τ₃) and their mean lifetime (τ_m, 89.2 ns) of BM-PCN are all much longer than those of PCN (τ_m = 17.9 ns), further suggesting the faster charge transfer from the inside to the surface of ultrathin nanosheets in BM-PCN, decreasing the direct charge recombination efficiency, but with subsequent surface radiative recombination.⁶⁰ Interestingly, the τ₁–τ₃ and τ_m (10.8 ns) of BM-PCN/Co-c are much shorter than those of PCN, which should result from faster transfer of holes to E_d that effectively decreases the charge recombination efficiency, with subsequent nonradiative energy transformation.⁶¹ The Co–OH doping effect also makes PCN/Co-c exhibit shorter τ₁–τ₃ and τ_m (16.5 ns) than PCN. Fig. 3e shows the photocurrent response of the samples. Their anodic photocurrent density follows the order PCN < PCN/Co-c < BM-PCN < BM-PCN/Co-c, indicating gradually increased photogenerated charge separation efficiencies,⁶² basically consistent with the PL results. The relatively high photocurrent response of BM-PCN benefits from the applied bias that effectively inhibits surface recombination of photogenerated charge carriers.To assess charge mobility of the samples, their electrochemical impedance spectroscopy (EIS) spectra were tested with high-frequency data simply fitted to an equivalent circuit (Fig. S20^†). The obtained charge transfer resistance (R_ct) follows the order PCN (26 Ω) > BM-PCN (18 Ω) ≈ PCN/Co-c (19 Ω) > BM-PCN/Co-c (13 Ω). Apparently, BM-PCN/Co-c exhibits smaller R_ct than BM-PCN and PCN/Co-c, and PCN/Co-c exhibits smaller R_ct than PCN, indicating the highest charge transfer performance of BM-PCN/Co-c⁶³ which originates from the single-atom Co modification⁶⁴ that may increase the electron density to facilitate charge transport. The smaller R_ct of BM-PCN than that of PCN indicates the additional favorable effect of ultrathin nanosheets.⁶⁵Fig. 3f shows electron paramagnetic resonance (EPR) spectra of the samples. All reveal one single Lorentzian line centered at a g of 2.0039, attributed to unpaired electrons in heptazine rings.⁶⁶ In the dark, the EPR signal intensity follows the order PCN < BM-PCN < PCN/Co-c < BM-PCN/Co-c, and the stronger signal of BM-PCN than that of PCN results from formation of ultrathin nanosheets that enhances delocalization of unpaired electrons, while the stronger signal of BM-PCN/Co-c and PCN/Co-c mainly benefits from the Co doping that increases the delocalized electron density.⁶⁷ Under visible light irradiation, the samples exhibit remarkable signal enhancement, following the sequence PCN < BM-PCN < PCN/Co-c < BM-PCN/Co-c, similar to that of the signal intensity in the dark, suggesting that the increase in the delocalized electron density facilitates charge photoexcitation. The high delocalized electron density favors charge transport, consistent with the EIS results, and the high photoexcited charge density benefits enhancement of photocatalytic activity.Photocatalytic OER activity of various samples was well evaluated using Ag⁺ as the sacrificial agent (Fig. S21^†). The Co content in BM-PCN/Co-c was optimized according to the photocatalytic OER rates and BM-PCN-c exhibits no detectable OER activity (Fig. S22^†), indicating indispensability of the Co–OH structure for the OER. The influence of the calcination temperature (T_c °C) of BM-PCN/Co on OER rates of BM-PCN/Co-c (T_c = 460) and BM-PCN/Co-cT_c was investigated and BM-PCN/Co-c exhibits the highest photoactivity (Fig. 4a), manifesting that the optimal calcination temperature is 460 °C. Under both simulated solar light and visible light irradiation (λ ≥ 420 nm), BM-PCN/Co-c exhibits substantially higher OER activity than PCN/Co-c (Fig. 4b), further suggesting the significance of the single-atom Co loading amount, and remarkably higher activity than common PCN/CoO_x (with 0.75 wt% Co, obtained via photodeposition) and BM-PCN-c/Co(OH)₂ (with 0.75 wt% Co), demonstrating the high efficacy of the single-atom distribution of Co–OH in BM-PCN/Co-c. Besides, urea was used as the feedstock to synthesize carbon nitride (marked as PCN-urea) with a larger surface area (76 m² g⁻¹ (ref. 68)) than PCN, and PCN-urea was further used to synthesize PCN-urea/Co-c similar to the synthesis of BM-PCN/Co-c. The OER activity of BM-PCN/Co-c is prominently higher than that of PCN-urea/Co-c (with the optimized Co content and Co single atom distribution, Fig. S23^†), suggesting the significant role of ball milling in fabricating the single-atom Co–N₄OH structure. To quantitively compare photoactivity of the samples, their mean OER rates under visible light illumination for 2 h are shown in Fig. 4c. The OER rate of BM-PCN/Co-c can reach ∼37.3 μmol h⁻¹, about 13.8, 28.7, 2.6, and 2.0 times those of PCN/Co-c, PCN/CoO_x, BM-PCN-c/Co(OH)₂, and PCN-urea/Co-c, respectively. Comparatively, less N₂ was generated for BM-PCN/Co-c (Fig. S24^†), further demonstrating the significance of single-atom Co–OH modification.Open in a separate windowFig. 4(a) The influence of the calcination temperature (T_c °C) of BM-PCN/Co on photocatalytic OER activity of BM-PCN/Co-c (T_c = 460) and BM-PCN/Co-cT_c, under Xe-lamp illumination, with AgNo₃ as the sacrificial agent; (b) photocatalytic oxygen evolution on various samples under Xe-lamp illumination with or without using a 420-nm filter; (c) corresponding OER rates of the samples in 2 h; (d) photocatalytic OER rates of BM-PCN/Co-c under irradiation with various monochromatic light sources for 12 h; (e) apparent quantum yields (AQYs) of BM-PCN/Co-c at different wavelengths and reaction times and the highest AQY at every wavelength, along with the UV-DRS spectrum; and (f) proposed mechanism for photocatalytic water oxidation on the single-atom Co^II-OH structure.Photocatalytic oxygen evolution on BM-PCN/Co-c was also tested under monochromatic light irradiation (Fig. S25^†). Apparently, BM-PCN/Co-c can exhibit OER activity even at a wavelength of 500 nm. The mean OER rate in 12 h decreases from 1.85 to 0.54 μmol h⁻¹ with increasing wavelengths from 400 to 500 nm (Fig. 4d), independent of light intensity of the Xe lamp and is mainly dependent on optical absorption capability of BM-PCN/Co-c at various wavelengths (Fig. 3a). Fig. 4e shows apparent quantum yields (AQYs) of BM-PCN/Co-c at different reaction times and wavelengths. Basically, there are maxima of AQYs with increasing reaction time at every wavelength, suggesting the adverse effect of excessive photodeposited Ag on surfaces of samples. These maxima are shown in Fig. 4e and accord well with the UV-vis DRS spectrum with increasing wavelengths. The maxima of AQYs at 400, 420, 450, and 500 nm can reach 4.69, 2.06, 1.07, and 0.46%, respectively. Compared with the reported photocatalytic OER results for PCN (Table S4^†), BM-PCN/Co-c exhibits the top-class performance.To investigate chemical stability of BM-PCN/Co-c, the cyclic OER experiment was conducted. After five consecutive runs, OER rates of BM-PCN/Co-c decrease less (Fig. S26a^†), with the morphology similar to the original (Fig. S26b^†). Co single atoms in the sample could still be distinctly observed by HAADF-STEM (Fig. S26c and d^†). In addition, N 1s core-level XPS spectra of BM-PCN/Co-c are almost similar before and after the cyclic experiment (Fig. S26e^†). These indicate the high stability of the basic framework structure of the sample. However, Co 2p core-level spectra show remarkable differences before and after the experiment, not only the Co^II peak shift, probably owing to ion (e.g., IO₄⁻) adsorption, but also formation of a large amount of Co^III (Fig. S26f^†). Coexistence of Co^II/Co^III may suggest the photocatalytic OER mechanism.The proposed OER mechanism based on the Co–OH structure is shown in Fig. 4f, according to the reported results in Mn doped PCN.³⁴ Four holes are needed to complete four oxidation steps and obtain one O₂ molecule. The first step starting with one hole may involve formation of the Co^III O bond. The Co–N₄OH structure should facilitate the water oxidation more compared with that of Co–N₄ without OH coordination, by leaving out the initial adsorption process of H₂O molecules.³⁴ On the whole, the high photocatalytic OER activity of Co-PCN benefits from the Co–N₄OH structure that not only effectively enhances optical absorption, and charge separation and transport, but also works as the highly active site for the OER.     

Chelation-assisted C–C bond activation of biphenylene by gold(i) halides

A chelation-assisted oxidative addition of gold(i) into the C–C bond of biphenylene is reported here. The presence of a coordinating group (pyridine, phosphine) in the biphenylene unit enabled the use of readily available gold(i) halide precursors providing a new, straightforward entry towards cyclometalated (N^C^C)- and (P^C)-gold(iii) complexes. Our study, combining spectroscopic and crystallographic data with DFT calculations, showcases the importance of neighboring, weakly coordinating groups towards the successful activation of strained C–C bonds by gold.

Pyridine and phosphine directing groups promote the C–C activation of biphenylene by readily available gold(i) halides rendering a new entry to (N^C^C)- and (P^C)-gold(iii) species.

Activation of C–C bonds by transition metals is challenging given their inertness and ubiquitous presence alongside competing C–H bonds.¹ Both the intrinsic steric hindrance as well as the highly directional character of the p orbitals involved in the σ_C–C bond impose a high kinetic barrier for this type of processes.^2,3 Biphenylene, a stable antiaromatic system featuring two benzene rings connected via a four-membered cycle, has found widespread application in the study of C–C bond activation. Since the seminal report from Eisch et al. on the oxidative addition of a nickel(0) complex into the C–C bond of biphenylene,⁴ several other late transition metals have been successfully applied in this context.⁵ Interestingly, despite the general reluctance of gold(i) to undergo oxidative addition,⁶ its oxidative insertion into the C–C bond of biphenylene was demonstrated in two consecutive reports by the groups of Toste^7a and Bourissou,^7b respectively. The high energy barrier associated with the oxidation of gold could be overcome by the utilization of gold(i) precursors bearing ligands that exhibit either a strongly electron-donating character (e.g. IPr = [1,3-bis(2,6-diisopropylphenyl)imidazole-2-ylidene])^7a or small bite angles (e.g. DPCb = diphosphino-carborane).^7b,8 In line with these two approaches, more sophisticated bidentate (N^C)- and (P^N)-ligated gold(i) complexes have also been shown to aid the activation of biphenylene at ambient temperature (Scheme 1a).^7c,dOpen in a separate windowScheme 1(a) Previous reports on oxidative addition of ligated gold(i) precursors onto biphenylene. (b) This work: pyridine- and phosphine-directed C–C bond activation of biphenylene by commercially available gold(i) halides.In this context, we hypothesized that the oxidative insertion of gold(i) into the C–C bond of biphenylene could be facilitated by the presence of a neighboring chelating group.⁹ This approach would not only circumvent the need for gold(i) precursors featuring strong σ-donor or highly tailored bidentate ligands but also offer a de novo entry towards interesting, less explored ligand templates. However, recent work by Breher and co-workers showcased the difficulty of achieving such a transformation.¹⁰Herein, we report the oxidative insertion of readily available gold(i) halide precursors into the C–C bond of biphenylene. The appendage of both pyridine and phosphine donors in close proximity to the σ_C–C bond bridging the two aromatic rings provides additional stabilization to the metal center and results in a de novo entry to cyclometalated (N^C^C)- and (P^C)gold(iii) complexes (Scheme 1b).Our study commenced with the preparation of 5-chloro-1-pyridino-biphenylene system 2via Pd-catalyzed Suzuki cross coupling reaction between 2-bromo-3-methylpyridine and 2-(5-chlorobiphenylen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 1 (Scheme 2).¹¹ To our delight, the reaction of 2 with gold(i) iodide in toluene at 130 °C furnished complex κ³-(N^C^C)Au(iii)–I 3 in 60% yield.^12,13 Complex 3 was isolated as yellow plate-type crystals from the reaction mixture and its molecular structure was unambiguously assigned by NMR spectroscopy, high-resolution mass spectrometry (HR-MS) and crystallographic analysis. Complex 3 exhibits the expected square-planar geometry around the metal center, with a Au–I bond length of 2.6558(3) Å.¹⁴ The choice of a neutral weakly bound gold(i)-iodide precursor is key for a successful reaction outcome: similar reactions in the presence of [(NHC)AuCl + AgSbF₆] failed to deliver the desired biscyclometalation adducts, as reported by Breher et al. in ref. 10. The oxidative insertion of gold(i) iodide into the four-membered ring of pyridino-substituted biphenylene provides a novel and synthetically efficient entry to κ³-(N^C^C)gold(iii) halides. These species have recently found widespread application as precursors for the characterization of highly labile, catalytically relevant gold(iii) intermediates,^15a–d as well as for the preparation of highly efficient emitters in OLEDs.^15e–g Previous synthetic routes towards these attractive biscyclometalated gold(iii) systems involved microwave-assisted double C–H functionalization reactions that typically proceed with low to moderate yields.^15aOpen in a separate windowScheme 2Synthesis of complex 3via oxidative addition of Au(i) into the C–C bond of pyridine-substituted biphenylene. X-ray structures of complex 3 with atoms drawn using 50% probability ellipsoids. Hydrogen atoms have been omitted for clarity. Additional selected bond distances [Å]: N–Au = 2.126(2), C1–Au = 1.973(2), C2–Au = 2.025(2), Au–I = 2.6558(3) and bond angles [deg]: N–Au–I = 99.25(6), N–Au–C1 = 79.82(9), C1–Au–C2 = 81.2(1), C2–Au–I = 99.73(8). For experimental details, see ESI.^†Encouraged by the successful results obtained with the pyridine-substituted biphenylene and considering the prominent use of phosphines in gold chemistry,^6,16 we wondered whether the same reactivity would be observed for a P-containing system. To this end, both adamantyl- and tert-butyl-substituted phosphines were appended in C1 position of the biphenylene motif. Starting from 5-chlorobiphenylene-1-carbaldehyde 4, phosphine-substituted biphenylenes 5a and 5b could be accessed in 3 steps (aldehyde reduction to the corresponding alcohol, Appel reaction and nucleophilic displacement of the corresponding benzylic halide) in 64 and 57% overall yields, respectively.¹³ The reactions of 5a and 5b with commercially available gold(i) halides (Me₂SAuCl and AuI) furnished the corresponding mononuclear complexes 7a–b and 8a–b, respectively (Scheme 3).¹³ All these complexes were fully characterized and the structures of 7a, 7b and 8a were unambiguously characterized by X-ray diffraction analysis.¹³ Interestingly, the nature of the halide has a clear effect on the chemical shift of the phosphine ligand so that a Δδ of ca. 5 ppm can be observed in the ³¹P NMR spectra of 7a–b (Au–Cl) compared to 8a–b (Au–I), the latter being the more deshielded. The Au–X bond length is also impacted, with a longer Au–I distance (2.5608(1) Å for 8a) compared to that measured in the Au–Cl analogue (2.2941(7) Å for 7a) (Δd = 0.27 Å).¹³Open in a separate windowScheme 3Synthesis and reactivity of complexes 7a–b, 8a–b, 9 and 10. X-ray structure of complexes 11b, 12 and 14 with atoms drawn using 50% probability ellipsoids. Hydrogen atoms have been omitted for clarity. For experimental details and X-ray structures see ESI.^†Despite numerous attempts to promote the C–C activation in these complexes,^10,13 all reactions resulted in the formation of highly stable cationic species 11a–b and 12, which could be easily isolated from the reaction media. In the case of cationic mononuclear-gold(i) complexes 11, a ligand scrambling reaction in which the chloride ligand is replaced by a phosphine in the absence of a scavenger, a process previously described for gold(i) species, can be used to justify the reaction outcome.¹⁷ The formation of dinuclear gold complex 12 can be ascribed to the combination of a strong aurophilic interaction between the two gold centers (Au–Au = 2.8874(4) Å) and the stabilizing η²-coordination of the metal center to the aromatic ring of biphenylene. Similar η²-coordinated gold(i) complexes have been reported but, to the best of our knowledge, only as mononuclear species.¹⁸Taking into consideration the observed geometry of complexes 7a–b in the solid state,¹³ the facile formation of stable cationic species 11 and 12 and the lack of reactivity of the gold(i) iodides 8a–b, we hypothesized that the free rotation around the C–P bond was probably restricted, placing the gold(i) center away from the biphenylene system and thus preventing the desired oxidative insertion reaction. To overcome this problem, we set out to elongate the arm bearing the phosphine unit with an additional methylene group, introduced via a Wittig reaction from compound 4 to yield ligand 6, prepared in 4 steps in 27% overall yield. Coordination with Me₂SAuCl and AuI resulted in gold(i) complexes 9 and 10, respectively (Scheme 3). The structure of 9 was unambiguously assigned by X-ray diffraction analysis and a similar environment around the metal center to that determined for complex 7a was observed for this complex.¹³With complexes 9 and 10 in hand, we explored their reactivity towards C–C activation of the four-membered ring of biphenylene.¹⁹ After chloride abstraction and upon heating at 100 °C for 5 hours, ring opening of the biphenylene system was observed for complex 9. Interestingly, formation of mono-cyclometalated adduct 13 was exclusively observed (the structure of 13 was confirmed by ¹H, ¹³C, ³¹P, ¹⁹F, ¹¹B and 2D NMR spectroscopy and HR-MS).¹³ The solvent appears to play a major role in this process, as performing the reaction in non-chlorinated solvents resulted in stable cationic complexes similar to 11.^13,20,21 The presence of adventitious water is likely responsible for the formation of the monocyclometalated (P^C)gold(iii) complex 13 as when the reaction was carried out in C₂H₄Cl₂ previously treated with D₂O, the corresponding deuterated adduct 13-d could be detected in the reaction media. These results showcase the difficulties associated with the biscyclometalation for P-based complexes as well as the labile nature of the expected biscyclometalated adducts. Interestingly though, these processes can be seen as a de novo entry towards relatively underexplored (P^C)gold(iii) species.²²The C–C activation was further confirmed by X-ray diffraction analysis of the phosphonium salt 14, which arise from the reductive elimination at the gold(iii) center in 13 upon exchange of the BF₄⁻ counter-anion with the weakly coordinating sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaBAr^F).^13,23 The phosphorus atom is four-coordinate, with weak bonding observed to the distant counter-anion and a distorted tetrahedral geometry (C1–P–C2 = 95.05(17), C2–P–C3 = 112.1(1), C3–P–C4 = 116.6(1), C4–P–C1 = 107.4(2) deg). These results represent the third example in which the C(sp²)–P bond reductive elimination at gold(iii) has been reported.²⁴Further, it is important to note that, in contrast to the reactivity observed for the pyridine-substituted biphenylene, neither P-coordinated gold(i) iodo complexes 8a, 8b nor 10 reacted to give cyclometalated products despite prolonged heating, which highlights the need for highly reactive cationized gold(i) species to undergo oxidative addition when phosphine ligands are flanking the C–C bond.¹³To get a deeper understanding on the observed differences in reactivity for the N- vs. P-based directing groups, ground- and transition-state structures for the oxidative insertion of gold(i) halides in C1-substituted biphenylenes were computed by DFT calculations. The reactions of Py-substituted 2 with AuI to give 3 (I) and those of P-substituted 7a (II) and 9 (III) featuring the cationization of the gold(i) species were chosen as models for comparative purposes with the experimental conditions (Fig. 1 and S1–S10 in the ESI^†).^25–27 The computed activation energies for the three processes are in good agreement with the experimental data. The pyridine-substituted biphenylene I exhibits the lowest activation barrier for the oxidative insertion process (ΔG^‡ = 34.4 kcal mol⁻¹). The reaction on the phosphine-substituted derivatives II and III proved to be, after cationization of the corresponding gold(i) halide complexes (II-BF₄, III-BF₄) higher in energy (ΔG^‡ = 39.6 and 46.3 kcal mol⁻¹ respectively), although the obtained values do not rule out the feasibility of the C–C activation process. The transition state between I and I′ exhibits several interesting geometrical features: (a) the biphenylene is significantly bent, (b) the cleavage of the C–C bond is well advanced (d_C–C = 1.898 Å in TSIvs. d_C–C = 1.504 Å in I), and (c) the two C and the I atoms form a Y-shape around gold with minimal coordination from the pyridine (d_N–Au = 2.742 Å in TSIvs. d_N–Au = 2.093 Å in I and 2.157 Å in I′, respectively). The transition-state structures found for the P-based ligands (TSII and TSIII) also show an elongation of the C–C bond and display a bent biphenylene. However, much shorter P–Au distances (d_P–Au = 2.330 Å for TSII and 2.314 Å for TSIII) can be observed compared to the pyridine-based system, as expected due to the steric and electronic differences between these two coordinating groups. Analogously, longer C–Au distances were also found for the P-based systems (d_C1–Au = 2.152 Å for TSIvs. 2.235 Å and 2.204 Å for TSII and TSIII; d_C2–Au = 2.143 Å for TSIvs. 2.219 Å and 2.162 Å for TSII and TSIII), with a larger deviation of square planarity for Au in TSIII compared to TSII.^28,29 These results suggest that, provided the appropriate distance to the C–C bond is in place, the strong coordination of phosphorous to the gold(i) center does not prevent the C–C activation of biphenylene but other reactions (i.e. formation of diphosphine gold(i) cationic species, protodemetalation) can outcompete the expected biscyclometalation process. In contrast, a weaker donor such as pyridine offers a suitable balance bringing the gold in close proximity to the C–C bond and enables both the oxidative cleavage as well as the formation of the double metalation product.Open in a separate windowFig. 1Energy profile (ΔG and ΔG^‡ in kcal mol⁻¹), optimized structures, transition states computed at the IEFPCM (toluene/1,2-dichloroethane)-B3PW91/DEF2QZVPP(Au,I)/6-31++G(d,p)(other atoms) level of theory for the C–C activation of biphenylene with gold(i) iodide from I and gold(i) cationic from II and III. Computed structures of the transition states (TSI, TSII and TSIII) and table summarizing relevant distances.     

Strong non-Arrhenius behavior at low temperatures in the OH + HCl → H2O + Cl reaction due to resonance induced quantum tunneling

The OH + HCl → H₂O + Cl reaction releases Cl atoms, which can catalyze the ozone destruction reaction in the stratosphere. The measured rate coefficients for the reaction deviate substantially from the Arrhenius limit at low temperatures and become essentially independent of temperature when T < 250 K, apparently due to quantum tunneling; however, the nature of the quantum tunneling is unknown. Here, we report a time-dependent wave packet study of the reactions on two newly constructed potential energy surfaces. It is found that the OH + HCl reaction possesses many Feshbach resonances trapped in a bending/torsion excited vibrational adiabatic potential well in the entrance channel due to hydrogen bond interaction. These resonance states greatly induce quantum tunneling of a hydrogen atom through the reaction barrier, causing the reaction rates to deviate substantially from Arrhenius behavior at low temperature, as observed experimentally.

The OH + HCl reaction possesses many Feshbach resonances trapped in the hydrogen bond well in the entrance channel, which substantially enhance the reaction rates at low temperatures.

In the classical picture, a chemical reaction with an energetic barrier can only occur at collision energies higher than the barrier, which leads to the well-known Arrhenius formula for chemical reaction rates. However, chemical reactions can happen at energies below the reaction barrier through quantum tunneling,^1–3 resulting in the deviation of the reaction rates from the Arrhenius behavior at low temperatures. The effect of quantum tunneling on the reaction rates increases with decreasing reaction temperature, and hence becomes especially important in low-temperature environments such as the interstellar medium and atmospheric processes.^4,5 Reaction resonances are quasi-trapped quantum states in the transition state region with some lifetime, and can substantially promote quantum tunneling through the reaction barrier. Over the past decades, great efforts have been devoted to detecting resonances in chemical reactions and to studying their structures and dynamics.^6–14 Theoretical studies on the O(³P) + HCl → OH + H reaction using the accurate ³A′′ potential energy surface revealed that the tunneling induced by the resonances trapped in the van der Waals well in the reactant channel can substantially enhance the thermal rate constants at low temperatures.^15–17 A combined experimental and theoretical investigation discovered that resonance-induced quantum tunneling dramatically enhances the reactivity of the F + p-H₂ → HF + H reaction,¹⁴ and is fully responsible for the unusually high chemical reactivity of the reaction in the low temperature interstellar medium. Recently, a quantum dynamics calculation showed that the presence of two resonance peaks strongly influence the rotational quenching of HF (j = 1, 2) with H, leading to an up to two-fold increase in the thermal rate coefficients at the low temperatures characteristic of the interstellar medium.¹⁸ Thus, understanding quantum tunneling, and in particular, resonance-induced quantum tunneling, in chemical reactions is of general interest and fundamental importance to low-temperature chemistry.The OH + HCl → H₂O + Cl reaction is of great importance in atmospheric chemistry because it releases Cl atoms from one of the principal chlorine-containing species in the stratosphere, HCl. The Cl atoms generated from the reaction can catalyze the ozone destruction reaction in the stratosphere, which was responsible for the formation of the ozone hole over Antarctica.¹⁹ Since the chlorine-catalyzed ozone destruction is proportional to the steady-state Cl atom concentration, which is directly controlled by the rate of the reaction, extensive studies have been carried out to measure the rate coefficient for the reaction with high accuracy over a wide range of temperatures.^20–29 The measured rate coefficients exhibit a small activation energy of a few hundred K, deviate substantially from the Arrhenius limit at low temperatures and become essentially independent of temperature when T < 250 K.^25,28,29 In addition, a large H/D kinetic isotope effect has also been found.^21,22,26,28 All these observations suggest the presence of an important quantum tunneling effect in the reaction.The dynamics of this reaction and its reverse have also attracted great attention in the past decades. In particular, the endothermic Cl + H₂O → HCl + OH reaction with a late barrier has been extensively investigated as a benchmark system for mode specificity and bond selectivity chemistry.^30–35 Recently, the construction of two high-quality potential energy surfaces (PESs) in its ground electronic state using the PIP-NN method have substantially advanced the theoretical study of the dynamics and kinetics of the system. The first PES was based on a large number of ab initio data points calculated at the multi-reference configuration interaction (MRCI) level of theory by Li, Dawes, and Guo (LDG),³³ and the second one was fitted to ab initio energy points obtained using an explicitly correlated unrestricted coupled-cluster method with single, double, and perturbative triple excitations (UCCSD(T)-F12b) and the augmented correlation-consistent polarized valence triple-zeta (aug-cc-pVTZ, or AVTZ) basis set by Zuo, Zhao, Guo, and Xie (ZZGX) with a fitting error of 6.9 meV.³⁶ The static barrier height is 2.86 and 2.23 kcal mol⁻¹, respectively, for these two PESs. On both PESs, there exists a well of about 3.5 kcal mol⁻¹ in the OH + HCl entrance channel due to the hydrogen bond (HB) interaction between OH and HCl. Extensive quantum dynamics studies on the PESs have revealed many interesting features of the reaction in both directions. In particular, time-dependent wave packet calculations for the title reaction with OH in the ground and vibrational excited states found one or two broad peaks in the total reaction probabilities, which are presumed to be the signature of the resonances supported by the reactant complex well.^36,37 However, the impact of these peaks on the reaction rates has not been investigated. Ring-polymer molecular dynamics (RPMD) calculations^38,39 were also carried out on both PESs to compute the thermal rate coefficients for the reaction.^40–42 It was found that the RPMD rates on the LDG PES underestimate the experimental data, while the RPMD rates on the latest ZZGX PES agree with the experimental results much better, and do not decrease further when the temperature falls below 300 K, apparently due to quantum tunneling. Unfortunately, RPMD calculations cannot provide any clue regarding the nature of the quantum tunneling.Therefore, despite the significant progress that has been made in theoretical studies of the system, some key issues still remain to be addressed: Are the broad peaks in the total reaction probabilities obtained from the time-dependent wave packet calculations indeed the signature of the resonances in the reaction? If not, do there exist resonances in the reaction? How do resonances affect the reaction rates at low temperature? Here, we report a quantum dynamics study of the reaction on two new and more accurate PESs. Good agreement is achieved between the rate coefficients calculated on these two PESs and the experimental data. Our calculations reveal that the HB well in the entrance channel of OH + HCl supports many low energy resonance states. These resonance states substantially enhance the quantum tunneling effect and have an important impact on the reaction rates at low temperatures.In order to improve the fitting accuracy of the ZZGX PES, we constructed two new PESs using the fundamental invariant neural network (FI-NN) method, fitted to ∼70 000 ab initio energy points calculated at the UCCSD(T)-F12a and UCCSD(T)-F12b levels of theory, respectively, both with the AVTZ basis set. The fitting RMSE is 3.07 and 3.12 meV, respectively, for the F12a and F12b PES, which is about half that for the ZZGX PES.³⁶ The spin–orbit coupling of the channels of both the reagent OH and the product Cl have been included using FI-NN fitting to about 38 000 points calculated at the MRCI/aug-cc-pVTZ level of theory with a fitting error of 0.19 meV. The CASSCF wave-function with an active space of (5e, 3o) was used as a reference for MRCI. Details of the new PESs are provided in the ESI^†. The static barrier height for PESa is 0.088 eV (0.095 eV with SO correction included), and that for PESb is 0.097 eV (0.104 eV). As can be seen from Tables S1 and S2,^† the geometries and energies of all the stationary points for the F12b PES without SO correction are in good agreement with the ZZGX PES. Table S2^† also shows the corresponding complete basis set (CBS) energies for these stationary points based on AVTZ, AVQZ and AV5Z calculations. Because the F12b energies are slightly closer to the CBS results, we will present the dynamical results obtained on the F12b PES in the main text and provide those for the F12a PES in the SI.On the new PESs, we carried out potential-averaged five-dimensional (PA5D) time-dependent wave packet^43,44 calculations to obtain the total reaction probabilities for the reaction by freezing the non-reacting OH bond in its ground vibrational state. Tests revealed that the PA5D treatment is capable of providing reaction probabilities for the ground rovibrational initial state that are essentially identical to those obtained using the full six-dimensional approach, as shown in Fig. S2.^†Fig. 1(A) shows the total reaction probabilities for the HCl + OH reaction as a function of collision energy calculated on the F12b PES with both reagents in the ground rovibrational state at propagation times of 60 000, 120 000, 360 000, and 2 400 000 a.u. At high collision energies, the reaction probabilities converge quickly with respect to the propagation time, and one can barely see any difference among the four reaction probability curves, which exhibit smooth increases with collision energy. However, in the low collision energy region, large differences appear for different propagation times. At t = 60 000 a.u., the reaction probability presents a smooth curve with some small and broad oscillations, as was observed in the wave packet calculations of Guo and coworkers.³⁶Open in a separate windowFig. 1(A) Total reaction probabilities for the ground initial state of the OH + HCl → Cl + H₂O reaction on the F12b PES at wave packet propagation times of T = 60 000, 120 000, 360 000, and 2 400 000 a.u. (B) Same as (A), except showing the collision energy between 0.0 and 0.04 eV. The crosses mark the points for which the wavefunctions are shown in Fig. 2. (C) Total reaction probabilities for some partial waves J = 0, 30, 60, and 90 as a function of the collision energy. (D) Same as (C) except showing the collision energy between 0.0 and 0.04 eV.When the propagation time is increased to t = 120 000, the reaction probabilities at collision energies below 0.05 eV increase substantially. With further increasing the propagation time to 360 000 a.u., many oscillatory structures emerge at collision energies below the barrier height of 0.104 eV, in particular in the very low collision energy region as shown in Fig. 1(B). These sharp structures become fully converged after around 2 400 000 a. u. of wave packet propagation (∼58 ps). The reaction probability even at a collision energy close to zero reaches 3–4%. The convergence of the total reaction probabilities on the F12a PES is very similar to that on the F12b PES (Fig. S3^†), except that the final converged reaction probabilities for these two PESs exhibit a small shift, apparently due to slightly different barrier heights. Therefore, it is clear that reaction resonances exist in the title reaction in the very low collision energy region, and that these resonance states substantially induce quantum tunneling and enhance the reactivity. The lifetimes for these resonance states are quite long, with many being longer than 6.5 ps and having corresponding widths smaller than 0.1 meV. Fig. 1(C) presents converged (t = 2 400 000 a.u. ≈ 58 ps) total reaction probabilities for the total angular momentum J = 0, 30, 60, and 90. With increasing J, the reaction probability curve shifts to higher energy. In the low collision energy region (<0.05 eV), the total reaction probabilities for J = 30 exhibit rich oscillatory structures as in the J = 0 case (Fig. 1(D)), which are expected to have a great influence on the rate constant at low temperature. With further increase of the total angular momentum, the influence of these resonances on the total reaction fades due to the centrifugal barrier, which prevents the low-energy wave function from entering the well. They only leave a small trace in the total reaction probabilities at low energies for J = 60, and do not have any effect for J = 90.To understand the nature of these resonances, we calculated scattering wave functions at two collision energies (1.08 and 4.26 meV) with the peak reaction probabilities indicated by x in Fig. 1(B). Fig. 2(A) shows the two dimensional (2D) contour at the collision energy of 1.08 meV in the Jacobi coordinates HCl bond length (r_H–Cl) and center of mass distance between OH and HCl (R_HCl–OH), with the other coordinates integrated. As can be seen, the wave function is localized in the HB well region in the entrance channel with a few nodes in the R coordinate and no node in the r_H–Cl coordinate. Inspection of the scattering wave function for the bending and torsion coordinates reveals nodes exist in these coordinates (Fig. S4 and S5^†). The 2D contour in the coordinates of R_HCl–OH and r_H–Cl at the collision energy of 4.26 meV shown in Fig. 2(B) looks similar to that shown in Fig. 2(A), except with more nodes in the R direction. Therefore, the observed resonance states in the reaction are Feshbach resonances trapped in a bending/torsion excited vibrationally adiabatic potential (VAP) well in the reactant complex region due to the HB interaction.Open in a separate windowFig. 2Reactive scattering wave functions for the OH + HCl → Cl + H₂O reaction on the F12b PES in the two Jacobi coordinates R(HCl–OH) and r(H–Cl) with other coordinates integrated at the collision energies of 1.08 (A) and 4.26 meV (B). The contour lines are the corresponding 2D PESs along the two reactive bonds R(HCl–OH) and r(H–Cl) with other coordinates optimized. The geometries for the saddle point and the HB minimum are displayed in (A). The coordinate units in the figures are a₀. Fig. 3(A) shows the accurate rate constants for the initial ground rovibrational state, k_g, based on the probabilities for J = 0, 30, 60, 90 using the uniform J-shifting approach with a temperature-dependent shifting constant.^45–47 A test shows that the J-shifting scheme based on these four individual J values only introduces a few percent error to the rate constants in the temperature region considered here (Fig. S6^†). As can be seen from the figure, with decreasing temperature, k_g first decreases rapidly from 1000 K to 700 K, then decreases slowly. It reaches a minimum at T ≈ 260 K, and increases slowly with further decrease of the temperature. At temperatures lower than 300 K, the rate constants for the ground rovibrational initial state are larger than the measured thermal rate coefficients, with k_g being larger than k_exp by ∼70% at T = 200 K.Open in a separate windowFig. 3(A) Accurate rate constants, k_g, for the initial ground rovibrational state of the HCl + OH → H₂O + Cl reaction calculated on the F12b PES, in comparison with k^JS_g (obtained using the J-shifting approximation) and k^NR_g (based on the background reaction probabilities for J = 0 with the resonance contribution removed shown below); (B) the background reaction probabilities up to E = 0.1 eV by connecting some valleys of the reaction probabilities marked by x.Also shown in Fig. 3(A) are the rate constants for the ground rovibrational initial state, k^JS_g, obtained from the J = 0 reaction probabilities using the J-shifting approximation (see ESI^† for details). As can be seen, the J-shifting approximation works very well at high temperatures around 1000 K, but begins to overestimate the rates with decreasing temperature. At T = 500 K, k^JS_g is about 10% higher than the true rate. At temperatures below 300 K, the J-shifting approximation underestimates the reaction rate, with k^JS_g being smaller than k_g by about 16% at T = 200 K. Overall, the J-shifting approximation works fairly well for the ground rovibrational initial state, although there are numerous resonance peaks in the reaction probabilities in the low collision energy region.Now we consider the issue of the effect of the resonance structures found in the reaction on the rate constant. For a reaction system with isolated resonances, it is rather straightforward to remove the resonance contributions from the reaction probabilities by fitting the resonance peaks to some Lorentzian functions and to obtain smooth background scattering probabilities, as demonstrated in the F + HD reaction and recently in the inelastic scattering of H + HF (ref. 6 and 18). However, the OH + HCl reaction possesses numerous highly overlapped resonances in the low energy region, as shown in Fig. 1(A), and it is impractical to fit these resonance peaks as accurately as the isolated resonances. Instead, we obtained an approximate background reaction probability curve, which is shown in Fig. 3(B), by smoothly connecting some resonance valleys as shown in the figure. The rate constants k^NR_g calculated using the background curve shown in Fig. 3(B) with the J-shifting approximation were compared with the original k_g in Fig. 3(A). Given the fact that the J-shifting approximation works fairly well for k^JS_g, as shown in Fig. 3(A), it is very reasonable to expect that will work even better for the k^NR_g values obtained from the background reaction probabilities with the resonance contribution removed. As can be seen, k^NR_g exhibits rate behavior typical for systems with a low barrier with some quantum tunneling effects. At T = 1000 K, k^NR_g is essentially identical to k_g, but decreases much faster than k_g as the temperature drops. At T = 200 K, k_g is larger than k^NR_g by a factor of about 5.6 (9.8 × 10⁻¹³vs. 1.74 × 10⁻¹³ cm³ s⁻¹), indicating that the reaction probabilities in the low collision energy region due to resonances substantially enhance the rate constants for the reaction at low temperatures. It is worthwhile to note that for systems with overlapped resonances like that shown in Fig. 3(B), the reaction probabilities at the valleys must be considerably higher than the true background reaction probabilities; therefore, the background curve shown in Fig. 3(B) is the upper limit of the background reaction probabilities and the rate based on the curve shown in Fig. 3(A) is also the upper limit of the rate for the ground rovibrational initial state without the resonance contributions. Therefore, the true enhancement due to the resonances must be larger than that shown in Fig. 3(A).For reliable comparison with the measured thermal rate coefficients, we must take into account the contributions from all the thermally populated initial states of the reagents. Due to the very large number of thermally populated rotational states for this reaction even at 200 K, we opted to calculate the cumulative reaction probabilities, NE(E), (the sum of the reaction probabilities for all the initial states with a fixed total energy) for the reaction from which the thermal rate constants can be reliably evaluated.^48–52 The transition state wave packet calculations were carried out to obtain NE(E) using the details given in the ESI^†. Due to huge computational efforts required to obtain the cumulative reaction probabilities for J > 0, we only calculated NE(E) for J = 0 and employed the J–K-shifting approximation^45,49,53 to obtain the thermal rate constant. NE(J = 0, E) as a function of total energy measured with respect to the ground rovibrational energy of OH and HCl is presented in Fig. S7.^†In Fig. 4, we present thermal rate constants for the reaction calculated on both the F12a and F12b PES, together with the rate constants for the ground rovibrational state (k_g) and the previous experimental measurements.^{21,22,24–29} As can be seen, the thermal rate coefficients are smaller than k_g over the entire temperature region, indicating that reagent rotation excitations diminish the reaction rates. Overall, the thermal rate coefficients calculated on both PESs agree with the experimental results rather well, with the F12a PES slightly overestimating and the other PES slightly underestimating compared to the experimental measurements. As observed in the experiments, the thermal rate coefficients decrease quickly with decreasing temperature in the high-temperature region, but decrease much more slowly at low temperatures, in particular at T < 300 K, substantially deviating from Arrhenius behavior.Open in a separate windowFig. 4Thermal rate constants of the HCl + OH → H₂O + Cl reaction calculated on both the F12a and F12b PES, compared with rate constants for the ground rovibrational state, the RPMD rates on ZZGX PES and the previous experimental measurements.^{21,22,24–29} Experimental data are taken from Husain et al. (in circles),^21,22 Molina et al. (in diamonds),²⁴ Ravishankara et al. (in upward-pointing triangles),²⁵ Smith et al. (in squares),²⁶ Sharkey et al. (in triangle left),²⁷ Battin-Leclerc et al. (in downward-pointing triangles),²⁸ and Bryukov et al. (in leftward-pointing triangles).²⁹Also shown in Fig. 4 are the RPMD rates calculated (see ESI^† for details) on the F12a, F12b and ZZGX PESs.⁴² As shown, the RPMD rates on the F12b PES agree with those on the ZZGX PES extremely well, although the barrier heights for the F12b PES are higher by 9 meV. The present RPMD rates agree with the J–K-shifting quantum rates rather well, except at 200 K, at which the RPMD rates are overestimated by about 40%. As a result, the RPMD rate is higher than the experimental value at T = 200 K, in particular for the F12a PES. Previous studies have shown that RPMD tends to underestimate reaction rates in the strong quantum tunneling region, even for systems with resonances such as the O(³P) + HCl → OH + H reaction;⁵⁴ however, it overestimates the rates for the F + H₂ reaction with pronounced post-barrier Feshbach resonances⁵⁵ and some insertion reactions.⁵⁶ The discrepancy between the RPMD and J–K-shifting quantum rate at T = 200 K could be caused by a possible underestimation of the rate by the J–K-shifting approximation, as in the J-shifting approximation of the ground rovibrational initial state shown in Fig. 3(A). On the other hand, the rapid increase in the rate with decreasing temperature (from 250 K to 200 K) could also be a feature of bimolecular reaction rates in the high-pressure limit for reactions with a pre-reactive minimum.⁵⁷ RPMD rate theory in its original form employs a free energy calculation,^38,39 which allows thermalization in the pre-reactive minimum, sampling tunneling pathways with energies not accessible in the low-pressure limit. Therefore, RPMD models a rate process that resembles rates in the high-pressure limit, which could lead to an overestimation of the rate at low temperature. A recent study also reported a similar issue with RPMD.⁵⁸ The authors attributed it to spurious resonances in RPMD favoring energy transfer in the pre-reactive minimum, which is similar to our argument. Certainly, more quantum dynamics calculations to provide rigorous quantum rates at low temperatures to assess the accuracy of the J–K-shifting approximation and the RPMD method on this reaction system with strong HB interaction in the entrance channel would be highly desirable.Therefore, the OH + HCl reaction possesses many long-lifetime Feshbach resonances trapped in a bending excited VAP well in the entrance channel due to HB interaction. These resonance states substantially induce quantum tunneling of the hydrogen atom through the reaction barrier and enhance the reactivity in the low-collision region. Consequently, the reaction rate for the reaction becomes essentially independent of temperature in the low-temperature region and deviates substantially from Arrhenius behavior, as observed experimentally. The resonance states in the reaction are very different in location from those in the F + H₂/H₂O reactions,^{8,11,12,14,59–63} which are trapped in the VAP well in the product channel. Furthermore, they are trapped in the bending/torsion excited VAP well with HCl in the ground vibrational state, unlike those in the F + H₂/H₂O reactions with HF in vibrationally excited states.^8,63,64 In nature, these resonance states in the reaction arise from HB interaction as in the F + H₂O (v = 0) reaction,^11,62,63 but are different from those in the F + H₂/HD/HOD (v = 1) reactions^{8,10,12,14,64–66} due to chemical bond softening. Because the resonance states are trapped in the bending/torsion excited VAP well, their lifetimes are much longer than those observed in the F + H₂/H₂O reactions. As a result, they should have an important impact on the differential cross sections. More efforts, and in particular more joint efforts involving theory and experiment, should be devoted to studying these Feshbach resonances in this reaction of great atmospheric importance in detail.     

Monitoring single Au38 nanocluster reactions via electrochemiluminescence

Herein, we report for the first time single Au₃₈ nanocluster reaction events of highly efficient electrochemiluminescence (ECL) with tri-n-propylamine radicals as a reductive co-reactant at the surface of an ultramicroelectrode (UME). The statistical analyses of individual reactions confirm stochastic single ones influenced by the applied potential.

Herein, we report for the first time single Au₃₈ nanocluster reaction events of highly efficient electrochemiluminescence (ECL) with tri-n-propylamine radicals as a reductive co-reactant at the surface of a Pt ultramicroelectrode (UME).

Single entity measurements have been introduced by Bard and Wightman based on the collisions/reactions of individual nanoparticles and molecules at an ultramicroelectrode (UME).^1–9 Since then, the field of single entity electrochemistry has gradually attracted several research groups and has become a frontier field of nanoelectrochemistry and electroanalytical chemistry.^8,10–14 For instance, it has been shown that the chemistry of the electrode surface plays an important role in the collision/reaction events and the kinetics of reaction processes.^15–21 Dasari et al. reported that hydrazine oxidation and proton reduction can be detected using single Pt nanoparticles on the surface of a mercury or bismuth modified Pt UME, and the material of the electrode was found to affect the shape of current–time transients.^22,23 Fast scan cyclic voltammetry provides better chemical information about transient electrode–nanoparticle interactions, which is otherwise difficult to obtain with constant-potential techniques.²⁴ There are only a few reports on photoelectrochemical systems including semiconductor nanoparticles designed to detect single nanoparticles in the course of photocatalysis processes.^25–28 More importantly, owing to the nature of stochastic processes of single entity reactions, statistical analyses have shown substantial influences on the understanding of the underlying processes.Electrochemiluminescence or electrogenerated chemiluminescence (ECL),²⁹ as a background-free technique,^30–32 was also utilized to detect individual chemical reactions and single Pt nanoparticle collisions based on the reaction between the Ru(bpy)₃²⁺ complex and tri-n-propylamine (TPrA) radicals on the surface of an ITO electrode.^2,33,34 It was found that the size of the nanoparticles, the origin of the interaction between particles and the electrode surface, the concentration of species generation, and the lifetime of individual electrogenerated nanocluster species (i.e., Au₃₈²⁺, Au₃₈³⁺, and Au₃₈⁴⁺) in conjunction with the reactivity of those oxidized species with co-reactant radical intermediates (i.e., TPrA radical) play crucial roles in the frequency of the ECL reaction events leading to individual ECL responses. More strikingly, a higher ECL reaction frequency is directly proportional to the amount of collected ECL light.²¹ Chen and co-workers also employed ECL to study stationary single gold-platinum nanoparticle reactivity on the surface of an ITO electrode.³⁵ Lin and co-workers monitored the hydrogen evolution reaction in the course of “ON” and “OFF” ECL signals.³⁶ Recently, we performed a systematic and mechanistic ECL study of a series of gold nanoclusters, with the general formula of Au_n(SC₂H₄Ph)_m^z (n = 25, 38, 144, m = 18, 24, 60 and z = −1, 0, +1), where near-infrared (NIR) ECL emission was observed.³⁷ There are several enhancement factors, such as catalytic loops^38,39 that improve the signal to noise ratio. The Wightman group was able to report single ECL reactions based on the capability of ECL.⁷ Furthermore, thus far, we have explored ECL mechanisms and reported the ECL efficiency of five different gold nanoclusters i.e., Au₂₅(SR)₁₈¹⁻, Au₂₅(SR)₁₈⁰, Au₂₅(SR)₁₈¹⁺, Au₃₈(SR)₂₄⁰ and Au₁₄₄(SR)₆₀⁰, among which the Au₃₈(SR)₂₄⁰/TPrA system revealed outstanding ECL efficiency, ca. 3.5 times higher than that of Ru(bpy)₃²⁺/TPrA as a gold standard. Therefore, we decided to focus on the Au₃₈ (SR)₂₄⁰/TPrA system. It was discovered that the ECL emission of these nanomaterials can be tuned through varying the applied potential and local concentration of the desired co-reactant.Herein, for the first time we report on ECL via a single Au₃₈(SC₂H₄Ph)₂₄ nanocluster (hereafter denoted as Au₃₈ NC) reaction (eq. (1)) in the vicinity of an UME in the presence of TPrA radicals as a reductive co-reactant.1where x is the oxidation number that can be either 0, 1, 2, 3 or 4. Single ECL spikes (Fig. 1A) along with ECL spectroscopy were used for elucidating individual reaction events. Indeed, each single ECL spike demonstrates a single Au₃₈^(x−1)* reaction product. Au₃₈ NCs were synthesized according to procedures reported by us and others, and fully characterized using UV-Visible-NIR, photoluminescence, ¹HNMR spectroscopy and MALDI mass spectrometry to confirm the Au₃₈ nanocluster synthesis (details are provided in ESI, Sections 1–3, Fig. S1–S4^†).^38,40,41Fig. 2 (left) shows a differential pulse voltammogram (DPV) in an anodic scan of a 2 mm Pt disc electrode immersed in 0.1 mM Au₃₈ acetonitrile/benzene solution containing 0.1 M TBAPF₆ as the supporting electrolyte. There are five discrete electrochemical peaks at which Au₃₈⁰ was oxidized to Au₃₈⁺ (E°′ = 0.39 V), Au₃₈²⁺ (E°′ = 0.60 V), and Au₃₈^3+/4+ (E°′ = 0.99 V) and reduced to Au₃₈⁻ (E°′ = −0.76 V) and Au₃₈²⁻ (E°′ = −1.01 V).^38,40,41Open in a separate windowFig. 1(A) An example of the reaction event transient of 10 μM Au₃₈ in benzene/acetonitrile (1 : 1) containing 0.1 M TBAPF₆ in the presence of 20 mM TPrA at 0.9 V vs. SCE, acquired at 15 ms time intervals using a 10 μm Pt UME. The white dashed-line indicates the threshold to identify single ECL spikes. (B) Illustration of a single nanocluster ECL spike. (C) ECL instrumentation with an inset showing ECL spike generation in the vicinity of the Pt UME.Open in a separate windowFig. 2Anodic DPV for Au₃₈ (left), reaction energy diagram of Au₃₈²⁺ and TPrA· (middle) along with the ECL–voltage curve (right) in an anodic potential scan at a 2 mm Pt disk electrode immersed in a solution of 10 μM Au₃₈ with 20 mM TPrA.The rich electrochemistry of Au₃₈ NCs is well-matched with that of co-reactants such as TPrA to generate near infrared-ECL (NIR-ECL), and the ECL emission efficiency of the Au₃₈/TPrA system is 3.5 times larger than that of the Ru(bpy)₃²⁺/TPrA co-reactant ECL system.²⁷Thus, it is of utmost interest to investigate the ECL generation of the above co-reactant system in single reactions, which improves the ECL signal detection sensitivity. To perform the ECL experiment a solution of 10 μM Au₃₈ NC with 20 mM TPrA was prepared. We first confirmed the ECL light generation of such solution along with its blank solution containing only TPrA using a typical 2 mm diameter Pt disk electrode (Fig. 2, S5 and S6^†).A 10 μm Pt UME electrode, which is electrochemically inert (Fig. S7^†), was utilized to investigate the ECL of single NC reactions under potentiostatic conditions, at which a specific positive bias potential was applied to oxidize both Au₃₈ and TPrA. Fig. 1A shows a typical ECL–time transient current curve (ECL intensity versus time) at 0.90 V vs. SCE, which was acquired using a photomultiplier tube (PMT, R928) for a duration of 1800 s at data acquisition time intervals of 15 ms (Fig. 1C and ESI, Section 3^†). Fig. 1B represents an exemplary event of a single ECL spike with a sharp increase followed by a decay in the ECL intensity. It is observed from the many spikes in Fig. 1B that this process can reoccur with a high probability in the vicinity of the UME, probably due to an electrocatalytic reaction loop (Fig. 1C). Indeed, ECL intensity was enhanced in this way as an already relaxed species, i.e., Au₃₈^z+1*, participates in an oxidation step to regenerate Au₃₈^z+1 to react with the TPrA radical (TPrA˙).Once photons resulting from the excited state relaxation in the vicinity of the UME are captured by the PMT, individual reaction events can be observed (Fig. 1A with the instrumentation schematic shown in Fig. 1C). As shown in Fig. 3A, there are many ECL spikes during 1800 s of measurement, each of which represents an individual ECL generation reaction in the vicinity of the UME surface. It is worth noting that there are several spikes with various intensities. This is most likely due to the Brownian motion which is random movement due to the diffusion of individual nanocluster species such as Au₃₈⁰, Au₃₈¹⁺, Au₃₈²⁺, etc., electrogenerated at the local applied potentials. Long and co-workers⁴² proposed that silver nanoparticle collision on the surface of a gold electrode follows Brownian motion, leading to several types of surface-nanoparticle response peak shapes. In fact, the observed ECL spikes, shown in Fig. 1C, with a rise and an exponential decay suggested that Au₃₈ nanocluster species diffuse directly through the electrode double-layer, move towards the tunneling region of the electrode surface, collide⁴² and become oxidized, react with TPrA radicals thereafter to produce excited states, and emit ECL. It is worth emphasizing that this path could be partially different for each individual nanocluster owing to the angle and direction relative to the electrode surface. The single Au₃₈ NC reaction behaviour at various bias potentials was investigated following the electrochemical energy diagram shown in Fig. 2, middle. For example, at a bias potential of 0.70 V (the green spot on the DPV in Fig. 2), Au₃₈⁰ undergoes two successive oxidation reactions to Au₃₈²⁺ and TPrA oxidation and deprotonation start to generate TPrA·. In fact, at a very close oxidation potential to Au₃₈²⁺, TPrA is also oxidized to its corresponding cation radical (ca. 0.80 V vs. SCE) Fig. S6,^† followed by deprotonation to form the TPrA radical.³⁸ The TPrA· with a very high reduction power (E°′ = −1.7 eV)⁴³ injects one electron to the LUMO orbital of the nanocluster and forms excited state Au₃₈⁺*, as illustrated in the reaction energy diagram in Fig. 2, middle.³⁸ Then, Au₃₈⁺* emits ECL light while relaxing to the ground state. For another instance, at 1.10 V vs. SCE (the red spot on the DPV in Fig. 2), Au₃₈⁰ is oxidized to Au₃₈^3/4+ feasibly. At this potential, the TPrA radical is generated massively in the vicinity of the electrode. The efficient electron transfer between the TPrA radical and Au₃₈^3/4+ generates both Au₃₈²⁺* and Au₃₈³⁺* that emit light at the same wavelength of 930 nm.³⁸ The results of such interactions produced a transient composed of many ECL events (Fig. 3A), which is an indication of bias potential enforcement on the nanocluster light emission.Open in a separate windowFig. 3Single-nanocluster ECL photoelectron spectroscopy of Au₃₈. ECL–time transients (A), statistics of the number of photons (B), histogram of the single reaction time between sequential spikes (C) and accumulated ECL spectrum (D) for a 10 μm Pt UME at 1.1 V immersed in a 10 μM Au₃₈ nanocluster solution in benzene/acetonitrile (1 : 1) containing 0.1 M TBAPF₆ in the presence of 20 mM TPrA. (E)–(H) The counterpart plots to (A)–(D) for the UME biased at 0.7 V. # represents the number.We further tried to collect the current–time traces of such events; however, owing to the high background current originating from the high concentration of TPrA relative to that of the nanocluster, no noticeable spikes in the current were observed.In order to study the photochemistry and understand deeply the single nanocluster reactions, ECL–time transients were collected at different applied potentials (i.e., 0.7, 0.8, 0.9 and 1.1 V vs. SCE) as labelled in green, brown, purple, and red on the DPV in Fig. 2, respectively. The transients were further analysed using our home-written MATLAB algorithm adapted from that for nanopore electrochemistry.⁴⁴ The population of individual events was identified by applying an appropriate threshold to discriminate ECL spikes from the noise as demonstrated in Fig. 1A. In fact, the applied algorithm also assisted us to learn about the raising time and intensity of each spike, as well as photons of individual spikes. For instance, Fig. 3A shows another typical transit for 1800 s at an UME potential bias of 1.1 V for the ECL events. Indeed, the integrated area of each peak, the charge of the photoelectrons at the PMT, is directly proportional to the number of photons emitted from individual reactions (see ESI, Section 5^†). Basically, the PMT amplifies the collected single photon emitted in the course of light-to-photoelectron conversion (see ESI, Section 6 and Fig. S8^†) and translates a single photon into photoelectrons. The extracted charge of each ECL reaction, Q_ECL, was then converted to the corresponding number of photons by dividing by the gain factor, g, which is 1.55 × 10⁶ (Fig. S8^†), following eqn (2):2The histograms of the number of photons show a Gaussian distribution (Fig. 3B) with a reaction frequency of 53.5 ± 2.9 at E = 1.1 V, whereas at a lower potential of 0.7 V the reaction frequency drops to 18.5 ± 1.7 (Fig. 3F). This indicates that there is a three-fold lower reaction occurrence at the lower potential. The integration of the Gaussian fitting at 1.1 V and 0.7 V also reveals a three-fold drop from 3.3 × 10⁵ to 1.2 × 10⁵ photons over 1800 s.To further explore the effect of electrode potential bias on the single Au₃₈ NCs ECL reaction, potentials lower than 1.1 and higher than 0.7 V, ca. 0.8 and 0.9 V (brown and purple labels in Fig. 2), were applied. In fact, the resulting ECL–time transients show a lower population of single spikes (Fig. S12A and ESI,^†). The integrated Gaussian curve values support the ECL–time transient observations with ∼4.1 × 10⁴ and ∼6.5 × 10⁴ photons, respectively. In fact, it is unlikely that the PMT would get more than two events in the duration, owing to the following reasons: (i) it has been shown that only 5.5% of incoming photons can be effectively converted to photoelectron signals by our R928 PMT during our absolute efficiency calibration, ESI Section 6 and Fig. S8–S19^†;⁴⁵ (ii) spherical ECL emission is proven to be detected for a substantial small part upon examination of our detection system for the absolute ECL quantum efficiency;⁴⁵ (iii) Au₃₈ nanocluster ECL emissions occur at 930 nm, which is almost at the wavelength detection limit of our PMT response curve.^38,45In addition, we evaluated the stochastic (a series of random events at various probability distributions) nature of the observed events and extracted the reaction time interval (τ) at various potentials. The resulting graph shows an exponential decay (Fig. 3C) as expressed in eqn (3):3where frequency (λ) gives the mean rate of the event and A represents the fitting amplitude. One can expect to obtain the distribution of the number of emitted photons and spatial brightness function. In fact, the exponential decay is a clear indication of random single reaction events as Whiteman and co-workers described for a 9,10-diphenylanthracene (DPA) ECL system in the annihilation pathway.^7,46 At a potential of 1.1 V, λ and A are found to be 4.98 ± 0.02 ms⁻¹ and 80.4 ± 3.2, whereas at 0.7 V, λ and A turned out to be 32.9 ± 1.6 ms⁻¹ and 9.5 ± 0.1 (Fig. 3C and G). Indeed, the lower potential of 0.70 V vs. SCE is high enough to generate the TPrA radical along with Au₃₈²⁺, thereby leading to excited Au₃₈⁺*, Fig. 3E. One can conclude that at the applied potentials of 0.7 V and 1.1 V, Au₃₈⁰ is oxidized to Au₃₈²⁺ and Au₃₈⁴⁺, resulting in the generation of Au₃₈⁺* and Au₃₈³⁺* under static conditions. Thus, there are higher populations of ECL spikes with no discrepancy in the number of collected photon distributions. However, at two intermediate potentials, i.e., 0.8 and 0.9 V, a dynamic behaviour which is due to the mixed oxidation of Au₃₈ species, in the vicinity of the UME, is observed. In fact, at these two applied potentials, the local concentration of the corresponding gold nanoclusters (i.e., Au₃₈³⁺ and Au₃₈⁴⁺) is not sufficient to produce significant ECL spikes. We also attempted to collect the ECL spectrum using a charge-coupled device (CCD) camera, which is relatively more sensitive in the NIR region (e.g., λ > 900 nm, Fig. S16^†). Fig. 3D and H display an accumulated spectrum at 1.1 and 0.7 V vs. SCE, which is collected for 30 minutes. The fitted accumulated ECL spectrum indicates an ECL peak emission at 930 nm and supports higher reactivity at 1.1 V than that at 0.7 V.³⁸ To confirm that the observed ECL spikes and accumulated spectra are generated based on the oxidation of Au₃₈ nanoclusters in the presence of TPrA radicals, ECL–time transients were recorded upon holding an applied potential at which no faradaic process occurs. Fig. S11^† represents ECL–time curves and accumulated ECL spectra at 0.0 V and 0.4 V. One can notice that no appreciable ECL signal can be observed.In addition, we investigate the Pearson cross-correlation (ρ) between the intensities of ECL spikes with τ as shown in Fig. S14^† in which there is a positive correlation at 0.7 and 1.0 V and a negative correlation at 0.8 and 0.9 V. In fact, ρ evaluates whether there is a stationary random process between the two defined parameters (see ESI, Section 6^†). Interestingly, the frequency of the reaction at different applied potentials revealed decay from 0.7 to 0.8 V, followed by an upward trend to 0.9 and 1.1 V vs. SCE (Fig. S15^†). This could be additional support for the transition stage at 0.8 and 0.9 V, where the applied potential as the major driving force to generate oxidized forms (e.g., Au₃₈³⁺ and Au₃₈⁴⁺) governs the flux of the nanocluster species that reach the vicinity of the electrode. Furthermore, the effectiveness of electron transfer reaction kinetics between the radical species, i.e., Au₃₈^z+1 and TPrA radical, competes with the flow of the incoming nanoclusters. It is worth mentioning that each of the ECL single event experiments was repeated three times, and very similar results were obtained. Moreover, lower (5 μM) and higher (20 μM) concentrations of Au₃₈ in the presence of 20 mM were tested. In fact, the former shows a smaller number of single reactions; however the later revealed a larger number of multiple reactions (Fig. S13^†).In summary, in this communication we demonstrated that Au₃₈ NC ECL at the single reaction level can be monitored using a simple photoelectrochemical setup following a straightforward protocol. Indeed, we have rich basic knowledge about the ECL mechanisms of various gold nanoclusters with different charge states (Au₂₅(SR)₁₈¹⁺, Au₂₅(SR)₁₈⁰, Au₂₅(SR)₁₈¹⁻) and various sizes (Au₂₅(SR)₁₈⁰, Au₃₈(SR)₂₄⁰, Au₁₄₄(SR)₆₀⁰) in fine detail. Thus, the ECL emission mechanisms of gold clusters, including the contribution of each charge state and influence of various concentrations of co-reactants, are well known. For instance, in our previous studies^{38,39,47–49} we clearly identified three charge states of an Au₂₅(SR)₁₈¹⁻/TPrA system and we discovered that at a high concentration of TPrA the reduction in the bulk solution of gold nanoclusters influences the ECL emission wavelength. We also have learnt that the Au₃₈/TPrA system is a co-reactant independent of co-reactant concentration. Furthermore, an extensively higher concentration of TPrA provides a dominant reaction over any unknown decomposition reaction at higher oxidation states of Au₃₈. It was discovered that the population of ECL reactions is directly governed by the applied bias potential on a Pt UME. This work is a strong indication of the high sensitivity of the ECL technique in detecting single ECL reactions in a simple solution, which complements those reported by the Bard group using rubrene, for instance, embedded in an organic emulsion in the presence of TPrA or oxalate as a co-reactant.^50,51 These systems needed a substantial ECL enhancement in the presence of an ionic liquid as the supporting electrolyte and emulsifier. The current approach can be further extended to investigate other molecules and nanomaterials'' electrocatalytic processes at the single reaction level.     

Direct electrochemical hydrodefluorination of trifluoromethylketones enabled by non-protic conditions

CF₂H groups are unique due to the combination of their lipophilic and hydrogen bonding properties. The strength of H-bonding is determined by the group to which it is appended. Several functional groups have been explored in this context including O, S, SO and SO₂ to tune the intermolecular interaction. Difluoromethyl ketones are under-studied in this context, without a broadly accessible method for their preparation. Herein, we describe the development of an electrochemical hydrodefluorination of readily accessible trifluoromethylketones. The single-step reaction at deeply reductive potentials is uniquely amenable to challenging electron-rich substrates and reductively sensitive functionality. Key to this success is the use of non-protic conditions enabled by an ammonium salt that serves as a reductively stable, masked proton source. Analysis of their H-bonding has revealed difluoromethyl ketones to be potentially highly useful dual H-bond donor/acceptor moieties.

The electrochemical hydrodefluorination of trifluoromethylketones under non-protic conditions make this single-step reaction at deeply reductive potentials uniquely amenable to challenging electron-rich substrates and reductively sensitive functionalities.

The difluoromethyl group (CF₂H) has attracted significant recent attention in medicinal chemistry,^1,2 which complements the well-documented importance and growing use of fluorine in small molecule pharmaceuticals.^3–6 The CF₂H group is an H-bond donor^7,8 that is also lipophilic,^9,10 a unique combination that positions it as an increasingly valuable tool within drug-discovery.¹¹ CF₂H has been used as a bioisostere of OH and SH in serine and cystine moieties, respectively, as well as NH₂ groups, where greater lipophilicity and rigidity provide advantages to pharmacokinetics and potency.^12–14The hydrogen-bond acidity of CF₂H groups is exceptionally dependent on the atom or group to which it is appended (Fig. 1A).^1,2 The H-bond acidity of alkyl-CF₂H groups is half that of O–CF₂H and even a quarter of SO₂–CF₂H groups.¹ This mode of control allows the H-bonding strength and, therefore its function, to be finely tuned. While much research has focused on the synthesis, behaviour and use of XCF₂H groups, where X = O, S, SO, SO₂, Ar, it is surprising that the corresponding carbonyl containing moiety (X = CO) has remained relatively elusive in these contexts. Not only would difluoromethyl ketones (DFMK) be expected to provide a relatively strong H-bond, but the carbonyl unit provides a complementary, yet proximal mode of intermolecular interaction (Fig. 1B). Indeed, the dual action of neighbouring H-bond donor and acceptor functionalities provides the fundamental basis for many biological systems, including in the secondary structure assembly mechanisms for proteins and DNA/RNA nucleobase pairing, as well as in enzyme/substrate complexes. Indeed, the DFMK functionality has demonstrated important utility in biological applications, including anti-malarial and -coronaviral properties.¹⁵ Finally, the carbonyl provides a useful synthetic handle for further derivatization.Open in a separate windowFig. 1H-Bonding in DFMKs and their synthesis via hydrodefluorination.While some progress has been made on the synthesis of DFMKs,¹⁶ there still remains a need for a general and more broadly accessible route to their preparation. Current strategies for DFMK preparation require multi-step processes, expensive reagents, installation of activating groups, or are inherently low yielding.^15a,16–25 The hydrodefluorination of trifluoromethyl ketones (1) potentially represents the most accessible strategy, as the starting materials are most readily prepared through a high-yielding trifluoroacetylation of C–H or C–X bonds.^26–29 In 2001, Prakash demonstrated the viability of this approach using 2 equivalents of magnesium metal as stoichiometric reductant to drive the defluorination, with a second hydrolysis step (HCl (3–5 M) or fluoride, overnight stirring) to reveal the product.³⁰ The scope in this 2-step process (6 substrates) reflects the limitations of using a reductant, such as Mg, that has a fixed reduction potential, as well as incompatibilities arising from Mg/halide exchange with aryl halides. Similar limitations with the use of electron-rich substrates were revealed in related contributions from Uneyama.³¹In order to access more electron-rich and reductively challenging substrates, such as those containing medicinally relevant heterocycles, we postulated that electrochemical reduction could be employed (Fig. 1C). Electrosynthesis is becoming an increasingly valuable enabling technology and has seen a recent resurgence due to the precise control, unique selectivity, and the potential scalability and sustainability benefits that it offers.^32–36 This strategy would avoid the undesirable use of stoichiometric metals and the ‘deep-reduction’ potentials required are readily accessed by simply selecting the applied potential. Pioneering early work from Uneyama on the cathodic formation of silylenol ether intermediate 2, suggested this approach could be viable.^37,38 The fundamental challenge in designing a practical, single-step process under highly reducing potentials (<−2.0 V vs. Fc/Fc⁺), is to avoid the reduction of the proton source, which would otherwise compete to generate H₂ gas and leave the starting material untouched. Uneyama does not demonstrate hydrodefluorination, presumably due to this problem. Additional challenges posed by ‘deep-reduction’ include a lack of tolerance for reduction-sensitive functionality (alkene, C–X bonds etc.), low mass balance due to substrate decomposition and the undesirable use of sacrificial metal anodes.³⁹ Solving these problems should provide generally applicable, safe and scalable conditions for the hydrodefluorination of readily accessible trifluoromethyl ketones (1).Given the electron-rich nature of indoles, their ubiquity in bioactive compounds, and their ease of functionalisation, we chose indole 1a as the model substrate for optimisation. The highly reductive potentials required will render it a challenging substrate, which should lead to more general conditions suitable for other important substrate classes. Indeed, when we applied the Mg conditions of Prakash to this substrate, no silyl enol ether intermediate (2a) was observed, nor product 3a, and the starting material remained completely untouched (

Hydroxy-directed fluorination of remote unactivated C(sp3)–H bonds: a new age of diastereoselective radical fluorination

We report a photochemically induced, hydroxy-directed fluorination that addresses the prevailing challenge of high diastereoselectivity in this burgeoning field. Numerous simple and complex motifs showcase a spectrum of regio- and stereochemical outcomes based on the configuration of the hydroxy group. Notable examples include a long-sought switch in the selectivity of the refractory sclareolide core, an override of benzylic fluorination, and a rare case of 3,3′-difluorination. Furthermore, calculations illuminate a low barrier transition state for fluorination, supporting our notion that alcohols are engaged in coordinated reagent direction. A hydrogen bonding interaction between the innate hydroxy directing group and fluorine is also highlighted for several substrates with ¹⁹F–¹H HOESY experiments, calculations, and more.

We report a photochemical, hydroxy-directed fluorination that addresses the prevailing challenge of high diastereoselectivity. Numerous motifs showcase a range of regio- and stereochemical outcomes based on the configuration of the hydroxy group.

The hydroxy (OH) group is treasured and versatile in chemistry and biology.¹ Its ubiquity in nature and broad spectrum of chemical properties make it an attractive source as a potential directing group.² The exploitation of the mild Lewis basicity exhibited by alcohols has afforded several elegant pathways for selective functionalization (e.g., Sharpless epoxidation,³ homogeneous hydrogenation,⁴ cross-coupling reactions,⁵ among others⁶). Recently, we reported a photochemically promoted carbonyl-directed aliphatic fluorination, and most notably, established the key role that C–H⋯O hydrogen bonds play in the success of the reaction.⁷ Our detailed mechanistic investigations prompt us to postulate that other Lewis basic functional groups (such as –OH) can direct fluorination in highly complementary ways.⁸ In this communication, we report a hydroxy-directed aliphatic fluorination method that exhibits unique directing properties and greatly expands the domain of radical fluorination into the less established realm governing high diastereoselectivity.⁹Our first inclination that functional groups other than carbonyls may influence fluorination regiochemical outcomes was obtained while screening substrates for our published ketone-directed radical-based method (Scheme 1).^8a In this example, we surmised that oxidation of the tertiary hydroxy group on substrate 1 cannot occur and would demonstrate functional group tolerance (directing to C11, compound 2). Surprisingly, the two major regioisomers (products 3 and 4) are derivatized by Selectfluor (SF) on C12 and C16 – indicative of the freely rotating hydroxyl directing fluorination. Without an obvious explanation of how these groups could be involved in dictating regiochemistry, we continued the mechanistic study of carbonyl-directed fluorination (Scheme 2A). We established that the regioselective coordinated hydrogen atom abstraction occurs by hydrogen bonding between a strategically placed carbonyl and Selectfluor radical dication (SRD).⁷ However, we noted that the subsequent radical fluorination is not diastereoselective due to the locally planar nature of carbonyl groups. Thus, we posed the question: are there other directing groups that can provide both regio- and diastereoselectivity? Such a group would optimally be attached to a sp³ hybridized carbon; thus the “three dimensional” hydroxy carbon logically comes to mind as an attractive choice, and Scheme 1 illustrates the first positive hint.Open in a separate windowScheme 1Observed products for the fluorination of compound 1.Open in a separate windowScheme 2(A) Proposed mechanism, (B) β-caryophyllene alcohol hypochlorite derivative synthetic probe, (C) isodesmic relation of transition states showing the general importance of the hydroxy group to reactivity (ωB97xd/6-31+G*), and (D) ¹H NMR experiment with Selectfluor and various additives at different concentrations.We began our detailed study with a simple substrate that contains a tertiary hydroxyl group. Alcohol 5 was synthesized stereoselectively by the reaction of 3-methylcyclohexanone, FeCl₃, and 4-chlorophenylmagnesium bromide;¹⁰ the 4-chlorophenyl substituent allows for an uncomplicated product identification and isolation (aromatic chromophore). We sought to determine optimal reaction conditions by examination of numerous photosensitizers, bases, solvents, and light sources (7 Although we utilize cool blue LEDs (sharp cutoff ca. 400 nm), CFLs (small amount of UVB (280–315 nm) and UVA (315–400 nm)) are useable as well.¹¹ A mild base additive was also found to neutralize adventitious HF and improve yields in the substrates indicated ( EntrySensitizer ¹⁹F yield1None0% 2 Benzil 83% 3Benzil, no base63%4Benzil, K₂CO₃68%5Benzil, CFL light source75%65-Dibenzosuberenone15%74,4′-Difluorobenzil63%89,10-Phenantherenequinone71%9Perylene8%10Methyl benzoylformate42%Open in a separate window^aUnless stated otherwise: substrate (0.25 mmol, 1.0 equiv.), Selectfluor (0.50 mmol, 2.0 equiv.), NaHCO₃ (0.25 mmol, 1.0 equiv.), and sensitizer (0.025 mmol, 10 mol%) were dissolved in MeCN (4.0 mL) and irradiated with cool white LEDs for 14 h.Substrate scope^a

A therapeutic keypad lock decoded in drug resistant cancer cells

A molecular keypad lock that displays photodynamic activity when exposed to glutathione (GSH), esterase and light in the given order, is fabricated and its efficacy in drug resistant MCF7 cancer cells is investigated. The first two inputs are common drug resistant tumor markers. GSH reacts with the agent and shifts the absorption wavelength. Esterase separates the quencher from the structure, further activating the agent. After these sequential exposures, the molecular keypad lock is exposed to light and produces cytotoxic singlet oxygen. Among many possible combinations, only one ‘key’ can activate the agent, and initiate a photodynamic response. Paclitaxel resistant MCF7 cells are selectively killed. This work presents the first ever biological application of small molecular keypad locks.

Information processing therapeutics with an implemented keypad lock logic gate selects input order for activation in drug resistant cancer cells.

The complex nature of diseases such as cancer necessitates smarter drugs that can discriminate each disease state or regulate drug efficacy spatially and/or temporally. With this intention, activatable drugs, drugs with on demand release properties are developed with promising selectivity.^1–4 Information processing therapeutics which are based on molecular logic gate operations are another approach to solve this problem.^5–7 Molecular logic gates are small compounds using Boolean logic operations to process inputs (i.e. the analyte concentration), and give an output as a result (fluorescence, and therapeutic activity etc.).⁸ Selective drug activation, release, multiple-analyte sensing and theranostic applications of these devices have been explored by us and others.^5,9–19Among the operations that can be carried out using small molecules, keypad locks provide an alternative application in information security.²⁰ This logic operation can give a specific output when the inputs are given in the correct form and correct sequence. For the device, each input is considered as an AND logic operation where the history of the process is also considered. A pioneering example was reported by Margulies and Shanzer in 2007 where energy transfer is modulated by chelation of Fe³⁺ in a pH dependent manner.²¹ Later, various other devices were introduced with advanced properties such as more than 2 input responsiveness and error detection capability.^22–24 All-photonic logic gates to address chemical waste production is extensively studied by Gust, Andréasson and Pischel.^25,26 Beside small molecule keypad locks, enzymes, antibodies, and DNA hybrids are used to achieve the same goal.^27–30 Although their potential use in molecular cryptology is highlighted, so far, there is no solid biological application of small molecule keypad locks.In the research presented here, a molecular keypad lock is developed which displays a photodynamic therapeutic output when a molecule is exposed to analytes in the correct order and type (PS3, Fig. 1). Two inputs of the system are chosen to be the common markers of drug resistant tumours: glutathione (GSH) and esterase enzyme (E). Cancer cells develop resistance to traditional chemotherapy in time by changing the protein expression or metabolite content of the cell. This adaptation of cancer cells is an obstacle for their treatment and needs to be addressed. Glutathione is a tripeptide used in reductive biochemical synthesis and it is known to be present in elevated levels in rapidly dividing cells such as cancer cells.³¹ A high GSH level is reported to contribute to drug resistance, since GSH adducts of the drugs are exported out of the cell much more rapidly.^32,33 Likewise, esterase enzyme activity is known to be associated with drug detoxification as this enzyme contributes to the chemical conversion of the drug.^34,35 Glutathione and esterase enzyme are chosen to be the first two inputs of the molecular keypad lock, the first two digits of the password. In the research, light is used as the final input. Although trivial, light is essential for photodynamic activity and spatiotemporal control of irradiation, further improving selectivity of the therapy.Open in a separate windowFig. 1Chemical structures of model photosensitizers (PS1 and PS2) and a molecular keypad lock (PS3). Ester bonds (red) are prone to hydrolysis by the esterase enzyme. Distyryl sites of the photosensitizers (blue) can react with thiol nucleophile provided that it is bound to an electron deficient group (i.e. pyri-dinium).Keypad lock PS3 is a photodynamic therapy (PDT) agent. PDT is a non-invasive method used for the treatment of surface cancers and certain other diseases ranging from atherosclerosis to macular degeneration.^36–39 In this therapy, a photosensitizer is excited with light, and produces cytotoxic singlet oxygen (¹O₂) thereby triggering apoptosis or necrosis of the cell, initiating an immune response and blocking microvasculature.⁴⁰ In the research, a boradiazaindecene (BODIPY) photosensitizer is used to benefit from versatile chemistry and spectroscopic properties.^41–45Near-IR absorbing PS3 shown in Fig. 1 is the molecular keypad lock and it is synthesized in 13 steps (Scheme S1^†). PS3 and model compound PS2 have heavy atoms on the structure to favour intersystem crossing required for transition to the triplet state and hence ¹O₂ generation occurs.⁴³ Ester bonds on the structure of PS3 are prone to cleavage by esterase enzyme. Distryryl bonds on the PS3 (blue) tend to reduce or form an adduct with thiol nucleophiles when it is activated by the pyridinium electron withdrawing group.⁴⁶ This property lies at the heart of sequential operation of esterase and GSH. When GSH reacts with electron poor double bonds, the extended conjugated structure is broken and PS3-a is generated (Fig. 2). This structure has absorption below 550 nm, like brominated core BODIPY molecules (compound 8, Scheme S1^†), and therefore can be excited with a green light. A quencher (green) is attached to ensure that photodynamic activity is OFF until esterase cleaves the ester bond. This is because of the energy transfer from the photosensitizer to this module, until esterase separates the photosensitizer. Since PS3 lacks absorption around the 500–550 nm region, it is inactive until GSH reacts with the compound. However, the GSH reacted photosensitizer does show absorption in this region; so, in order to avoid full activation just by GSH, a quencher module is attached. Spectral overlap between the BODIPY core (see the structure of compound 8 in the ESI,^† similar to that of PS3-a in terms of conjugation) and quencher (Q) can be seen from UV-Vis absorption and fluorescence spectra (Fig. 3 and S1^†). By this way, the photosensitizer is chemically modulated by GSH to ensure excitation, and then esterase enzyme inhibits energy transfer by removing the quencher. Lastly a green light is used to excite the photosensitizer leading to generation of photodynamic action. Since light is necessary for the final excitation of the molecule, it should always be the last input. If the order of esterase and GSH changes, as shown in Fig. 2, activation is not expected to take place since cleavage of the ester bonds generates 4-hydroxybenzyl derivative on PS3, which spontaneously faces 1,4-elimination to generate pyridine (Fig. S2^†).⁴⁷ Pyridine on its own is not sufficiently electron withdrawing to favour nucleophilic attack of double bonds by GSH and to activate it as demonstrated below. Therefore, the photosensitizer preserves extended conjugation and essentially lacks absorption at the wavelength of excitation.Open in a separate windowFig. 2Sequential operation of GSH and esterase. GSH can only react with BODIPY distyryl units when the structure has electron withdrawing pyridinium, either reducing it or forming an adduct. Esterase enzyme cleaves ester bonds, liberating the photosensitizer from the quencher module (green). Initial esterase activity converts the pyridinium unit to pyridine, thereby decreasing the reactivity of double bonds with GSH.Open in a separate windowFig. 3Normalized UV-Vis absorption and fluorescence spectra of PS1–3 in 2% water in THF (a and b). Samples are excited at 600 nm. Spectral changes of PS3 (10 μM) alone (black) or PS3 upon exposure to 0.5 mM GSH (c) and 10U esterase (d) for 90 min and 60 min at 37 °C, in 2% water in THF, respectively. A new peak at 544 nm appears upon incubation with GSH which is attributed to reduced PS3 and/orthe GSH-adduct. Esterase treatment increases the relative intensity of the shoulder peak around 600 nm.In order to understand the response of the PS3 to GSH, a molecule is incubated with 0.5 mM of GSH at 37 °C for 90 min. A new peak at 544 nm appears in UV-Vis absorption spectra consistent with the hypothesis (Fig. 3c, S1 and S9^†). The formation of the GSH adduct (PS3-a) is demonstrated by Liquid Chromatography Mass spectrometry analysis (Fig. S3^†). When control module PS1 is exposed to the same conditions, this new peak is not detected indicating that the pyridine bearing structure is neither activated enough for the nucleophilic substitution by GSH nor did it display PDT activity (Fig. S4 and S5^†). On the other hand, GSH treated pyridinium bearing PS2 immediately displayed a colour change indicative of broken conjugation (Fig. S6^†). When PS3 is incubated with esterase for 1 h, a small hypsochromic shift in the absorption peak is detected as a shoulder to the parent peak which is attributed to the conversion of pyridinium to pyridine (PS3-c, Fig. 3d). The control PS3 sample which is incubated under the same conditions but lacks esterase does not show an enhancement of this peak (Fig. 3d, black). High Resolution Mass Spectrometry analysis of the esterase treated PS2 samples confirm the hydrolysis of the ester and subsequent formation of the pyridine compound (Fig. S7^†). Esterase treated samples display an increase in the emission intensity when excited at 620 nm (Fig. S8^†). This is attributed to the initial quenching of the quencher module by the pyridinium photosensitizer. Analysis of the absorption and emission spectra suggest that the quencher module of PS3 can induce energy transfer to the pyridinium photosensitizer (Fig. 3). Once separated by esterase, fluorescence of the quencher module increases. In the case of GSH treated sample, a small enhancement in emission upon excitation at 500 nm is observed (Fig. S9^†). Note that the GSH adduct (or PS3 with reduced double bonds) has higher absorption at this wavelength, which would be the reason for the increase in emission intensity. In the spectral analysis organic solvents with a low water content are used to monitor the formation of water-insoluble, neutral, pyridine-bearing intermediate species.In the project, the molecular keypad lock is aimed to unlock in the presence of drug resistant tumour markers and get activated. Activation cannot take place when the input order differs. To demonstrate this, photodynamic action in the presence of all three inputs in a different order is investigated. ¹O₂ production can be followed by using trap molecule, 1,3-diphenylisobenzofuran (DPBF).⁴⁸ This molecule reacts with ¹O₂ and loses its absorption at 418 nm. The effect of different input combinations on the PDT action are given in Fig. 4. In the first 15 min, all samples are kept in the dark. Under such conditions no ¹O₂ generation is detected, which indicates lack of dark activity. DPBF is exposed to light from a LED source (peak 505 nm) under the same experimental conditions and no decrease in the absorption is detected. This control experiment eliminates the photodegradation of DPBF in the absence of a photosensitizer. Upon irradiation before the activation of the photosensitizer by GSH and esterase, no ¹O₂ generation is observed as expected. The results show that ¹O₂ generation, and the subsequent decrease in DPBF absorption, are significantly more in the input order of glutathione, esterase enzyme and light, consistent with the proposed mode of activation.Open in a separate windowFig. 4 ¹O₂ generation ability of PS3 (0.1 μM) when three inputs are given in a different order. All samples contain 50 μM of ¹O₂ trap molecule DPBF. In the first 15 minutes samples are kept in the dark. GSH is added in 0.5 mM concentration and incubated for 90 min at 37 °C. Samples are incubated with 10U esterase for 1 h at 37 °C. An LED light is irradiated from a 30 cm distance for 45 min.To analyse the effect of PDT action in the cell, a drug resistant cell line is generated. MCF7 cells are exposed to an increased dose of traditional cancer therapeutic agent paclitaxel as described in the literature.⁴⁹ When the spindle-shaped morphology is obtained following maximum drug dose application, cells are reported to have drug resistance. At this stage, PS3 is applied to both normal and drug resistant cells. When cell viabilities at various concentrations are analysed, it has been found that the light toxicity of PS3 is significantly enhanced in drug resistant cells (Fig. 5). The IC50 values of irradiated samples are calculated to be 124.8 μM for MCF7 cells. This value is reduced to 52.5 μM in paclitaxel resistant MCF7 (Pac-MCF7) indicating improved cytotoxicity in these cells. Efficient induction of apoptosis is also proved by Annexin V and PI staining (Fig. 6). Under dark conditions, cells do not have significant loss of viability. Upon irradiation, resistant cells are more prone to apoptosis by the photosensitizer. Relative singlet oxygen generation abilities and results of cell culture experiments altogether confirm selective activation in drug resistant cells.Open in a separate windowFig. 5Change in the cell viability of normal and paclitaxel resistant MCF7 cells (Pac-MCF7) in the presence of PS3 at various concentrations. For each group, cell viability is analysed both after incubation in the dark or after irradiation with a 505 nm LED light source from a distance of 10 cm. Average values of three independent experiments are used.Open in a separate windowFig. 6Apoptosis induction by PS3 (25 μM) in normal and paclitaxel resistant MCF7 cancer cells under dark conditions and upon irradiation with a 505 nm LED light from 10 cm distance. Scale bars: 50 μm.     

Directed evolution of a cyclodipeptide synthase with new activities via label-free mass spectrometric screening

Directed evolution is a powerful approach to engineer enzymes via iterative creation and screening of variant libraries. However, assay development for high-throughput mutant screening remains challenging, particularly for new catalytic activities. Mass spectrometry (MS) analysis is label-free and well suited for untargeted discovery of new enzyme products but is traditionally limited by slow speed. Here we report an automated workflow for directed evolution of new enzymatic activities via high-throughput library creation and label-free MS screening. For a proof of concept, we chose to engineer a cyclodipeptide synthase (CDPS) that synthesizes diketopiperazine (DKP) compounds with therapeutic potential. In recombinant Escherichia coli, site-saturation mutagenesis (SSM) and error-prone PCR (epPCR) libraries expressing CDPS mutants were automatically created and cultivated on an integrated work cell. Culture supernatants were then robotically processed for matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS analysis at a rate of 5 s per sample. The resulting mass spectral data were processed via custom computational algorithms, which performed a multivariant analysis of 108 theoretical mass-to-charge (m/z) values of 190 possible DKP molecules within a mass window of 115–373 Da. An F186L CDPS mutant was isolated to produce cyclo(l-Phe–l-Val), which is undetectable in the product profile of the wild-type enzyme. This robotic, label-free MS screening approach may be generally applicable to engineering other enzymes with new activities in high throughput.

A robotic workflow for directed evolution of new enzymatic activities via high-throughput library creation and label-free MS screening.

Mimicking random mutagenesis and natural selection in the laboratory,^1–3 directed enzyme evolution is valuable in improving a variety of properties of biocatalysts (i.e., catalytic activity, stability, substrate specificity, and enantioselectivity).^4–6 While it is straightforward to generate thousands of enzyme variants using established methods such as error-prone PCR (epPCR), site-saturation mutagenesis (SSM), and DNA shuffling, phenotypic screening remains a major bottleneck due to the substrate and product versatility of biocatalysts.^7,8 Optical assays are widely applied in high-throughput screening (HTS) during directed evolution, often requiring a surrogate substrate or a coupling assay with a colorimetric or fluorogenic readout.^9,10 However, it is very challenging to apply such approaches to discover new catalytic activities. On the other hand, mass spectrometry (MS) is label-free and suitable for untargeted molecular profiling.¹¹ The high sensitivity and chemical resolution of MS further highlight its potential in discovering new products, because new catalytic activities are often weak when first emerging from enzyme promiscuity.¹²We and others have recently developed a range of high-throughput MS screening methods for directed protein evolution.^13–16 However, the label-free advantage of MS has not been fully demonstrated in engineering new enzymatic activities, possibly due to the difficulties in untargeted MS screening. Particularly, it requires careful standardization and optimization of sample preparation, MS acquisition, and data processing, which are necessary to minimize experimental noise and spot the weak signals of a new product. On the other hand, biofoundries provide an emerging infrastructure to assist the design–build–test–learn (DBTL) cycles in biological engineering via robotic standardization and parallelization.^17–19 Using an integrated biofoundry, here we report a workflow for unlabeled MS screening of recombinant libraries to rapidly isolate enzyme mutants that catalyze the formation of new products. This new workflow extends our previous matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS-based screening approach from agar colonies¹⁶ to liquid cultures in industry-standard microplates for better uniformity. Unlike colony biomass, liquid culture media often contain high concentrations of nonvolatile components, which interfere with MALDI matrix crystallization and cause severe ion suppression during MS analysis. Therefore, sample preparation steps, such as liquid–liquid extraction and solid-phase extraction, need to be incorporated and automated.Cyclodipeptide synthases (CDPSs) are a family of enzymes that catalyze the formation of a diketopiperazine (DKP) from two aminoacyl-tRNA substrates (aa-tRNAs), which is an important pharmacophore for modern drug development^20–22 (Fig. 1A and S1^†). To produce new DKP derivatives for therapeutic and industrial applications, the catalytic mechanism of CDPSs has been studied^23,24 to guide engineering. AlbC (239 aa) is the first identified CDPS protein, taking phenylalanyl-tRNA^Phe (Phe-tRNA^Phe) and leucyl-tRNA^Leu (Leu-tRNA^Leu) as substrates to synthesize cyclo(l-Phe–l-Leu) (cFL) in its native host Streptomyces noursei.^25,26 Recombinant E. coli with AlbC overexpression is able to produce more cyclodipeptide derivatives in addition to cFL.²⁷ To date, eight CDPSs have been structurally characterized,^26,28–32 and mutagenesis studies reveal that the residues within the P1 and P2 catalytic pockets are the key determinants of substrate specificity. However, the detailed mechanism of substrate recognition and catalysis is still elusive, and CDPSs are recognized as recalcitrant targets for rational engineering.^33,34 To our knowledge, large-scale screening of CDPS variants has not been reported, possibly due to the lack of applicable HTS assays.Open in a separate windowFig. 1(A) Scheme of diketopiperazine (DKP) biosynthesis catalyzed by cyclodipeptide synthase (CDPS). (B) Workflow of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS)-based high-throughput screening method for directed evolution of AlbC protein in E. coli.Here we develop and apply untargeted MS screening on a biofoundry for directed evolution of AlbC mutants that form new CDP products. The workflow consists of strain library creation, colony picking, microtiter cultivation, organic solvent extraction, transfer of the sample to a MALDI target and acquisition of mass spectra, followed by data processing and visualization (Fig. 1B). For library creation, AlbC variants were generated by either SSM or epPCR approaches and expressed from a pET28a plasmid under control of a T7 promoter. Upon genetic transformation of E. coli Rosetta(DE3), individual clones were transferred by a colony picker into microtiter plates. Subsequent cultivation, inducible production by addition of isopropyl β-d-1-thiogalactopyranoside (IPTG), ethyl acetate extraction, and MS sample preparation were performed using an integrated robotic workcell in the Shenzhen Synthetic Biology Infrastructure. This workcell integrates common instruments for synthetic biology, including a liquid-handling station, shaking incubators, centrifuge, plate reader, and so on. Organic extracts of the liquid cultures were spotted onto a MALDI target and overlaid with 4-CHCA matrix solution. MS targets were then manually transferred to a stand-alone MALDI mass spectrometer in this study, although a robotic configuration has been previously reported to automate this step.³⁵We first applied the workflow to analyze the wild-type (WT) AlbC-expressing strain, Rosetta(DE3)/pET28-AlbC(WT). After cell cultivation, inducible production, and sample preparation, MS analysis was performed using an Autoflex MALDI-ToF mass spectrometer in the reflector positive ion mode (Fig. 1). In the resulting MALDI mass spectra, we observed [M + H]⁺ ion peaks with m/z values corresponding to cFL (m/z 261.17), cFY (m/z 311.15), cFM (m/z 279.12), cYL (m/z 277.16), cFF(cYM) (m/z 295.15), cLL (m/z 227.16) and cMM (m/z 263.12), all of which were absent from the control strain Rosetta(DE3)/pET28 (Fig. S2^†). Production of these CDP molecules was further confirmed by examining tandem MS results using LC-MS in the multiple reaction monitoring (MRM) mode (Table S3^†), where ion fragmentation patterns were consistent with the literature.^26,27 Moreover, the observed production profile was also consistent with previous studies.^26,27 These results validated our workflow to detect CDP products from microplate cultures of AlbC-expressing E. coli using MALDI-ToF MS.To engineer AlbC mutants that synthesize new products, we first focused on the key residues in the substrate-binding pockets. AlbC employs a “ping-pong” catalytic mechanism: the initial step transfers the aminoacyl moiety of the first aa-tRNA onto a conserved serine, leading to the formation of an aminoacyl enzyme intermediate; then, the aminoacyl enzyme reacts with the aminoacyl moiety of the second aa-tRNA to form a dipeptidyl enzyme.³² Previous biochemical experiments demonstrated that two binding pockets P1 and P2 of AlbC accommodate the aminoacyl moieties of the two aa-tRNA during the biosynthesis²⁴ (Fig. 2). Only a limited number of mutations were examined on P1 and P2 residues,^25,26 possibly due to the lack of HTS assays for large-scale analysis. Instead, we applied our workflow to create and screen the SSM libraries of select residues, including 10 residues in the binding pockets (L33, V65, L119, L185, L200, M152, M159, I204, T206 and P207) and 4 residues (R99, R101, R102 and D205) outside the pockets which have interactions of the tRNA moiety reported in the literature (Fig. 2A).Open in a separate windowFig. 2(A) Structural analysis of the AlbC (PDB ID, 3OQV). Enlarged view of the catalytically active pocket. The possible catalytic residues are shown in green (pocket-1) and orange (pocket-2) and basic residues on helix α4 are labelled in cyan. (B) Heatmap of the relative activity change in the cFL production levels of mutation of 14 amino acid residues in AlbC. WT residues are labeled with a black dashed rectangle. Dark blue boxes indicate that a specific mutant was not covered in randomly picked clones (activity assigned as −1).For SSM libraries, we adopted the “22c-trick” strategy to design degenerative primers, and 94 library clones were randomly picked for each residue library to reach a >98.6% probability of full library coverage of the 20 canonical amino acids.³⁶ In addition to library variants, the WT and control strains were also included in the same 96-well plate. Overall, the abovementioned workflow (Fig. 1B) takes approximately 5 s for sample preparation and MS analysis for each mutant culture. Tentative ion peaks of cyclodipeptides were assigned based on theoretical m/z values (ESI Section 1^†) using the MetaboAnalyst webserver.³⁷ For each ion peak, one-way analysis of variance (ANOVA) was used to evaluate statistical differences between the mean peak areas of a library variant and those of the control group. AlbC gene mutations were revealed by Sanger sequencing for all 14 SSM library members.To visualize the MALDI-ToF MS screening results, a heatmap was generated based on the cFL production of library members relative to that of WT (Fig. 2B), and we observed a general consistency between our results and literature data (Fig. 3A), respectively, which was similar to a previous study.²⁶ Also, mutagenesis of basic residues (R99, R101 and R102) outside the pockets led to the decline of cFL production in the mutants, although R101 was more tolerant to mutations than R99 and R102. Furthermore, the substitution of D205 in the β6-α8 loop to alanine enhanced AlbC catalytic activity as observed previously.²⁵ On the other hand, some previously unnoticed phenomena were observed. For example, in the D205 SSM library, relative ion intensities of cFL (m/z 261.17) in many mutants are higher than that of WT (Fig. S3^†). The top 3 mutants, D205M, D205K and D205R, were subsequently analyzed by LC-MS/MS. The results not only confirmed augmented synthesis of cFL, but also revealed increased production of other leucine-containing products including cYL, cLL and cML (Fig. 3B). Also interestingly, the discovery that most mutations of the T206 residue in the P2 pocket abolished the biosynthesis of the main product cFL was unprecedented (Fig. S3 and ESI Section 2^†). When examining MALDI mass spectra in detail, we noticed that although the T206F mutation greatly reduced cFL production, cFF and cFY products were largely not affected. Replacing the small threonine side chain with a bulky aromatic phenylalanine side chain could affect the activity of WT, because the phenyl ring structure increased the steric hindrance of T206F, thus impairing binding with its substrate and causing loss of its enzyme activity for cFL (Fig. 5A). These results suggested that T206 is also a key residue for the selectivity of the second Leu-tRNA substrate binding in the pocket. Unfortunately, we did not observe any new CDP molecules produced from the 14 SSM library mutants.Impact of AlbC mutations on the main product cFL

0D–1D hybrid nanoarchitectonics: tailored design of FeCo@N–C yolk–shell nanoreactors with dual sites for excellent Fenton-like catalysis

Heterogeneous Fenton-like processes are very promising methods of treating organic pollutants through the generation of reactive oxygen containing radicals. Herein, we report novel 0D–1D hybrid nanoarchitectonics (necklace-like structures) consisting of FeCo@N–C yolk–shell nanoreactors as advanced catalysts for Fenton-like reactions. Each FeCo@N–C unit possesses a yolk–shell structure like a nanoreactor, which can accelerate the diffusion of reactive oxygen species and guard the active sites of FeCo. Furthermore, all the nanoreactors are threaded along carbon fibers, providing a highway for electron transport. FeCo@N–C nano-necklaces thereby exhibit excellent performance for pollutant removal via activation of peroxymonosulfate, achieving 100% bisphenol A (k = 0.8308 min⁻¹) degradation in 10 min with good cycling stability. The experiments and density-functional theory calculations reveal that FeCo dual sites are beneficial for activation of O–O, which is crucial for enhancing Fenton-like processes.

Novel 0D–1D hybrid nanoarchitectonics consisting of FeCo@N–C yolk–shell nanoreactors are developed for Fenton-like reaction. With the multilevel advantages of this design, FeCo@N–C nano-necklaces exhibit excellent performance for BPA removal.

Advanced oxidation processes (AOPs) are one of the most promising strategies to eliminate organic contaminants, sustainably generating reactive oxygen species (ROS) to ideally destroy all non-biodegradable, recalcitrant, toxic, or membrane-permeable organic impurities.^1–4 Among these AOPs, sulfate radical (SO₄˙⁻)-based Fenton-like processes have gained increasing attention as a water treatment strategy because of the strong oxidation potential of SO₄˙⁻ (3.1 V vs. normal hydrogen electrode) at wider pH ranges. SO₄˙⁻ is mainly produced by physical or chemical methods for activation of persulfate salts, such as peroxymonosulfate (PMS) and persulfate.^5–9 Over the past two decades, heterogeneous catalysis has emerged as the most effective approach to water treatment, with much effort dedicated to developing better catalysts, including transition metal-based and carbonaceous materials.^10,11 Unfortunately, most metal-based catalysts suffer from leaching of toxic metal ions, which can thwart their practical application,^12,13 and although carbonaceous catalysts produce no secondary pollution, their cycle performance is always depressed.¹⁴ There is therefore an urgent need to find robust catalysts with adequate activity and stability for Fenton-like processes.To achieve superior performance, an ideal Fenton-like catalyst should contain oxidants with favorably reactive centers for cleavage of peroxyl bonds (O–O), have structure optimized for target pollutant attraction, and have chainmail to protect the vulnerable active sites for long periods.^15–17 Recent studies have demonstrated Co–N–C active sites prefer to activate the O–O of PMS.¹⁸ Furthermore, introducing Fe-doping into the Co–N–C system not only suppresses Co²⁺ leaching, but also modulates the pyrrolic-N content, which is the adsorption site for capture of bisphenol A (BPA).¹⁹ We previously discovered that Co@C yolk–shell nanoreactors could enhance the catalytic activity because of the confinement effect in the nano-spaces between the core and shell, while the carbon shell acted like a chainmail protecting the Co active sites, keeping them highly reactive after five cycles.^20,21Combining different kinds of materials to generate novel hybrid material interfaces can enable the creation of new kinds of chemical and physical functionalities that do not currently exist. However, one cannot simply mix these materials in an uncontrolled manner, because the ensemble of interfaces created by random mixing tends to favour thermodynamically stable interfaces that are functionally less active. Therefore, to prepare new materials with high functionality, it is necessary to carefully control the hybridization of components in interfacial regions with nanometric or atomic precision. By further hybridization of different components e.g., zero to one dimension (0D–1D) hybrid structures, we can prepare the structure to increase not only the specific surface area but also the interfacial region between different materials.In this work, we report novel 0D–1D hybrid nanoarchitectonics (necklace-like structures) consisting of FeCo@N–C yolk–shell nanoreactors as a PMS activator for Fenton-like processes. This catalyst has multilevel advantages: (i) each FeCo@N–C unit is a well-formed yolk–shell nanoreactor, which can guarantee sufficient contact of reactants and active sites, as well as defend them for good durability; (ii) all single nanoreactors are threaded along the carbon fibers, providing a highway for electron transport; and (iii) all the carbon fibers constructed into a thin film with macroscopic structure, which overcomes the complex recyclability of powder catalysts. Benefiting from favorable composition and unique structure, the FeCo@N–C catalyst delivers excellent performance for BPA removal via activation of PMS accompanied with good stability.The synthesis processes of necklace-like nanoarchitecture containing FeCo@N–C yolk–shell nanoreactors are illustrated in Fig. 1a. First, uniform Fe–Co Prussian blue analogue (Fe–Co PBA) nanocubes with an average size of 800–900 nm (Fig. 1b) are encapsulated in polyacrylonitrile (PAN) nanofibers by electrospinning. The obtained necklace-like FeCo PBA–PAN fibers (Fig. 1c) are then pyrolyzed at 800 °C in N₂ atmosphere to produce FeCo@N–C nano-necklaces. The scanning electron microscopy (SEM) image (Fig. 1d) of the FeCo@N–C shows this necklace-like morphology with its large aspect ratio, with the FeCo@N–C particles strung along the PAN-derived carbon fibers. A broken particle (Fig. 1e) shows that the FeCo@N–C has a yolk–shell architecture, which is also identified by transmission electron microscopy (TEM). Fig. 1f and g show the well-defined space between the inner yolk and outer shell, which is attributed to the volume shrinkage of the original Fe–Co PBAs. During pyrolysis, Fe–Co PBA is reduced to FeCo (inner yolk) and PAN is carbonized (outer carbon shell), resulting in the unique necklace-like nanoarchitecture.^22–24 The high-resolution TEM in Fig. 1h shows a lattice fringe of 0.20 nm, which matches well with the (110) plane of FeCo alloy.²⁵ The scanning transmission electron microscopy (STEM) image (Fig. 1i) and corresponding elemental map (Fig. 1j) indicate that FeCo nanocrystals are well distributed in the inner core with some small FeCo nanocrystals located on external carbon shells. Furthermore, the control samples of Fe@N–C and Co@N–C nano-necklaces, prepared by only replacing the Fe–Co PBA nanocubes with Fe–Fe PB and Co–Co PBA (Fig. S1^†), also demonstrate the versatility of this synthetic strategy. The formation of hierarchical porous structure, beneficial to the PMS transportation on the surface of catalysts, could be determined by N₂ adsorption–desorption isotherms and corresponding pore volume analysis (Fig. S2 and Table S1^†).Open in a separate windowFig. 1(a) Preparation of FeCo@N–C necklace-like nanoarchitecture. SEM images of (b) Fe–Co PBA cubic particles and (c) the electrospun FeCo PBA–PAN fibers. (d and e) SEM, (f and g) TEM, and (h) high-resolution TEM images of FeCo@N–C nano-necklaces. (i) STEM and (j) the corresponding elemental mappings of C, N, Fe, and Co.The X-ray diffraction patterns of the as-prepared products are depicted in Fig. S3,^† with one prominent diffraction peak centered at 44.8° corresponding to the (110) lattice plane of FeCo alloy. All the products also have a characteristic signal at 26°, implying that graphite carbon is formed during pyrolysis. Raman spectroscopy further analyzed the crystal structures and defects of the FeCo@N–C nano-necklaces (Fig. S4^†), where peaks found at 1349 cm⁻¹ and 1585 cm⁻¹ index the disordered (D band) and graphitic carbon (G band), respectively.²⁶ X-ray photoelectron spectroscopy investigated the composition and valence band spectra of FeCo@N–C nano-necklaces. The survey spectrum (Fig. S5a^†) reveals the presence of Fe (1.4%), Co (1.2%), C (86.4%), N (4.5%), and O (6.5%) in the composite. The high-resolution N 1s spectrum (Fig. S5b^†) exhibits broad peaks at 398.1, 401.1, and 407.4 eV, corresponding to the pyridinic-N, graphitic-N, and σ* excitation of C–N, respectively.²⁷ The high-resolution Fe 2p spectrum (Fig. S5c^†) shows a broad peak at 707.4 eV, attributed to Fe⁰. Similarly, the 777.5 eV peak observed in the Co 2p spectrum (Fig. S5d^†) corresponds to Co⁰, implying that FeCo dual sites have formed.²⁸ The oxidation state of these sites was investigated by ⁵⁷Fe Mössbauer spectroscopy, which found a sextet in the Mössbauer spectrum of the FeCo@N–C nano-necklaces attributed to FeCo dual sites (Fig. 2a and Table S2^†).²⁹ The coordination environment of the FeCo dual sites was also verified by X-ray absorption fine structure (XAFS) spectroscopy. Fig. 2b shows that the X-ray absorption near-edge structure (XANES) spectra of the Fe K-edge, which demonstrates a similar near-edge structure to that of Fe foil, illustrating that the main valence state of Fe in FeCo@N–C nano-necklaces is Fe⁰. Furthermore, the extended-XAFS (EXAFS) spectra (Fig. 2c) displays a peak at 1.7 Å, which is ascribed to the Fe–N bond, and a remarkable peak at approximately 2.25 Å corresponding to the metal–metal band.^10,30 The Co K-edge and EXAFS spectra (Fig. S6^†) also confirm the presence of Co–N and the metal–metal band. These results provide a potential structure of the FeCo dual sites in the FeCo@N–C nano-necklaces, as illustrated in Fig. 2d.Open in a separate windowFig. 2(a) ⁵⁷Fe Mössbauer spectra of FeCo@N–C nano-necklaces at 298 K. (b) Fe K-edge XANES spectra of FeCo@N–C nano-necklaces and Fe foil. (c) Corresponding Fourier transformed k³-weighted of the EXAFS spectra for Fe K-edge. (d) Possible structure of the FeCo dual sites.This dual-metal center and necklace-like structure may be beneficial to enhance catalytic performance. Fig. 3a shows the Fenton-like performance for BPA degradation compared to Fe@N–C nano-necklaces, Co@N–C nano-necklaces, and FeCo@N–C particles (Fe–Co PBA directly carbonized without electrospinning). Here, the FeCo@N–C nano-necklaces display a higher catalytic performance, with BPA completely removed in 7 min. To clearly compare their catalytic behavior, the kinetics of BPA degradation was fitted by the first-order reaction. As shown in Fig. 3b, FeCo@N–C nano-necklaces exhibit the highest apparent rate constant (k = 0.83 min⁻¹), which is approximately 6.4, 2.6, and 1.2 times that of FeCo@N–C particles, Fe@N–C nano-necklaces, and Co@N–C nano-necklaces, respectively. The significantly enhanced performance of FeCo@N–C nano-necklaces suggests that the FeCo dual sites and necklace-like nanoarchitecture are crucial. Furthermore, the concentration of BPA and PMS in the solution is higher than that in yolk–shell nanoreactor, resulting a concentration gradient which helps to accelerate the diffusion rates of reactants (Fig. 3c).^31,32 For these nano-necklaces, the carbon shell acts like a chainmail protecting the FeCo active sites from attack by molecules and ions, and all the nanoreactors are threaded along the carbon fibers, providing a highway for electron transport, which is important for SO₄˙⁻ generation (SO₄˙⁻ production as eqn, HSO₅⁻ + e⁻ → SO₄˙⁻ + OH⁻). Electrochemical impedance spectroscopy further confirms the good conductivity of the FeCo@N–C nano-necklaces (Fig. 3d). In addition, the concentration of metal-ion leaching and cycling performance (Fig. 3e and f) reveal the high reusability of FeCo@N–C nano-necklaces, with 95% BPA removal in 20 min after five cycles, which is also proved by the SEM and TEM characterization (Fig. S7^†). The effect of other reaction parameters on the BPA degradation, such as pH, reaction temperature, PMS or catalysts dosage, and common anions, were investigated in detail (Fig. S8–S11^†). All the results demonstrate that FeCo@N–C nano-necklaces deliver a better performance for PMS catalysis. In addition, the turnover frequency (TOF) value of FeCo@N–C nano-necklaces is 5.5 min⁻¹ for BPA degradation, which is higher than many previously reported catalysts (detailed catalytic performance comparison as shown in Table S3^†).Open in a separate windowFig. 3(a) BPA degradation efficiency in different reaction systems and (b) the corresponding reaction rate constants. (c) Schematic illustration of PMS activation in FeCo@N–C nano-necklaces. (d) Nyquist plots of the catalysts. (e) The metal leaching in different reaction systems. (f) Cycling performance of FeCo@N–C nano-necklaces for BPA removal. Reaction conditions: [catalyst] = 0.15 g L⁻¹, [BPA] = 20 mg L⁻¹, [PMS] = 0.5 g L⁻¹, T = 298 K, and initial pH = 7.0.To examine the enhanced catalytic activity, radical quenching experiments were conducted. As shown in Fig. 4a, when NaN₃ is added to the reaction solution as a scavenger for ¹O₂, there is no significant reduction of BPA decomposition, implying that non-radicals are not the dominant reactive species. By comparison, when tert-butanol (TBA) (radical scavenger for ˙OH) is added, there is a slight (2.8%) decrease in BPA removal. However, if methanol (radical scavenger for SO₄˙⁻ and ˙OH) is added, the efficiency of BPA degradation declines by up to 59.2%, indicating that the major radicals generated from the PMS activation are SO₄˙⁻;³³ the presence of these radicals is also verified by electron paramagnetic resonance (EPR) (Fig. 4b). Furthermore, the significant inhibition ratio can be observed when KI (quencher for the surface) is added, demonstrating that BPA degradation is mainly attributed to reactions with SO₄˙⁻, which is produced by a surface catalytic process.³⁴Open in a separate windowFig. 4(a) Effects of the radical scavengers on BPA degradation. (b) EPR spectra of SO₄˙⁻ and ˙OH. (c) The energy profiles of PMS on FeCo@N–C nano-necklaces surface. (d) Optimized configurations of PMS adsorbed on FeCo@N–C nano-necklaces.Density-functional theory was applied to calculate the surface energy of PMS activation at FeCo dual sites (Fig. 4c, d and S12^†). The dissociation barrier of PMS into SO₄˙⁻ and OH⁻ is −2.25 eV, which is much lower than that on an Fe or Co single site, suggesting that cleavage of O–O bonds of PMS occurs more easily on FeCo dual sites. This is because FeCo dual sites provide two anchoring sites for the dissociated O atoms, leading to more efficient activation of O–O. The FeCo@N–C nano-necklaces can reduce the energy barrier of O–O bond breaking, which results in high activity for PMS activation and thus high productivity of SO₄˙⁻.     

Correction: The oxygen-resistant [FeFe]-hydrogenase CbA5H harbors an unknown radical signal

Correction for ‘The oxygen-resistant [FeFe]-hydrogenase CbA5H harbors an unknown radical signal’ by Melanie Heghmanns et al., Chem. Sci., 2022, 13, 7289–7294, https://doi.org/10.1039/D2SC00385F.

The authors realized that incorrect references were cited following the sentence “In conjunction with the signal''s significant width, the frequency dependence clearly indicates spin–spin interaction between the F-clusters.” The correct references are shown below as ref. 1 and 2.Additionally ref. 36 and 37 were reversed in the reference list. The correct ref. 36 is shown below as ref. 3 and the correct ref. 37 is shown below as ref. 4.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.     

Exciton interactions in helical crystals of a hydrogen-bonded eumelanin monomer

Eumelanin, a naturally occurring group of heterogeneous polymers/aggregates providing photoprotection to living organisms, consist of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) building blocks. Despite their prevalence in the animal world, the structure and therefore the mechanism behind the photoprotective broadband absorption and non-radiative decay of eumelanin remain largely unknown. As a small step towards solving the incessant mystery, DHI is crystallized in a non-protic solvent environment to obtain DHI crystals having a helical packing motif. The present approach reflects the solitary directional effect of hydrogen bonds between the DHI chromophores for generating the crystalline assembly and filters out any involvement of the surrounding solvent environment. The DHI single crystals having an atypical chiral packing motif (P2₁2₁2₁ Sohncke space group) incorporate enantiomeric zig-zag helical stacks arranged in a herringbone fashion with respect to each other. Each of the zig-zag helical stacks originates from a bifurcated hydrogen bonding interaction between the hydroxyl substituents in adjacent DHI chromophores which act as the backbone structure for the helical assembly. Fragment-based excited state analysis performed on the DHI crystalline assembly demonstrates exciton delocalization along the DHI units that connect each enantiomeric helical stack while, within each stack, the excitons remain localized. Fascinatingly, over the time evolution for generation of single-crystals of the DHI-monomer, mesoscopic double-helical crystals are formed, possibly attributed to the presence of covalently connected DHI trimers in chloroform solution. The oligomeric DHI (in line with the chemical disorder model) along with the characteristic crystalline packing observed for DHI provides insights into the broadband absorption feature exhibited by the chromophore.

Single crystals of DHI monomer, a eumelanin precursor, adopt an atypical chiral packing arrangement incorporating enantiomeric zig-zag helical stacks while its covalently connected DHI trimer forms double-helical crystals in the mesoscopic scale.

Eumelanin, which represents a broad class of natural pigments found in the animal kingdom, acts as a biological shield for protecting the skin cells against harsh UV radiation.¹ Eumelanin, a black coloured pigment, obtained from the oxidative polymerization of 5,6-dihydroxyindoles (DHIs) and 5,6-dihydroxyindole carboxylic acid (DHICA) is one of the extensively explored archetypes of the melanin family (Fig. 1a).² Eumelanin has synergistic merits of possessing broadband UV absorption and proficient dissipation of the excessive electronic energy via non-radiative deactivation of the excited states, thereby resulting in the photoprotective nature of the pigment.³ Apart from the photoprotective behaviour, melanin possesses exceptional antioxidant activity via its free radical scavenging traits.^4–7 However, much less has been understood about the fundamental photophysics and structural features of eumelanin due to the enormous heterogeneity in the molecular framework^8,9 coupled with poor solubility in common solvents.¹⁰ Recent years have witnessed a growing interest towards unravelling the excited state processes occurring in the eumelanin pigment upon interaction with light.^11,12 A better correlation between the structure–property relationship and photoexcited state processes in eumelanin can guide the development of inspired functional materials for potential application in biomedical and dermo-cosmetic fields.^1,13–15Open in a separate windowFig. 1(a) Chemical diagrams of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole carboxylic acid (DHICA). (b) Various unconventional hydrogen bonding interactions identified in DHI crystals.In the natural world, the chromophoric architecture of eumelanin can symbolize an organized and efficient organic system for photoprotection that nature developed through evolution. The outcome of the research done so far indicates the presence of continuous π-stacks of oligomers in eumelanin which induce different levels of aggregation to construct the eumelanin framework.¹⁶ Furthermore, eumelanin has been reported to exhibit weak fluorescence which indicates the presence of competing non-radiative channels that provide efficient de-excitation pathways for repopulating the ground states.^17–20 The broadband absorption of eumelanin has theoretically and experimentally been evaluated, in part, to be a result of π-stacking interactions between the oligomers of DHI/DHICA in multiple oxidation states.^21–24 While dealing with biomacromolecules, non-covalent interactions such as hydrogen bonding and π–π stacking take the centre stage in controlling the supramolecular architecture, especially in the 3-dimensional structures of proteins and DNA. Modulating the balance between each of these noncovalent interactions over another will produce significant changes not only in the structure but also in the functional properties.Hydrogen bonding is simultaneously both ubiquitous and diverse and therefore its significance in biochemical systems comes as no surprise particularly due to the surrounding aqueous environment. Apart from the classical hydrogen bonding interactions, an array of hydrogen bond-like weak interactions which include a delocalized π-system acting as the acceptor group to the X–H hydrogen donor (X = O, N, C) is identified to provide additional contributions for stabilizing the biomolecular structure and controlling intrinsic functions (Fig. 1b).^25,26 Investigations aimed at identification (using X-ray crystallography) followed by energetic quantification of the major stabilizing interactions such as those with the aromatic π-rings in biological complexes are of paramount importance for developments in diverse areas including drug design. There has been extensive research conducted on eumelanin building blocks showcasing their ability to form hydrogen bonds through the –OH and –NH functional groups.^27,28 Most reports almost exclusively focus on the hydrogen bonding with the solvent environment surrounding the eumelanin monomer units.²⁹ Findings from these theoretical studies have demonstrated the role of several deactivation pathways in the presence of a protic solvent, namely –OH and –NH bond elongation and 5-/6-membered ring puckerings.¹⁸Chemical and spectral evidence from the eumelanin polymeric structures identified so far points to five main levels of chemical disorder leading to the supramolecular structure, which includes (i) disorder from the simultaneous presence of different building blocks; (ii) molecular size disorder; (iii) disorder from the position of coupling; (iv) electronic/redox disorder of the constituent units and (v) supramolecular disorder.³⁰ Given the complex structure of melanin, a bottom-up approach using the building blocks or basic constituent molecules of eumelanin is a pertinent strategy for the mechanistic study of the photoproperties of eumelanin. This can be followed by understanding the more intricate structures of melanin formed from the constituents with less complex approaches. Due to the abundant presence of water in the natural media, the corresponding solute–solvent interactions can have profound significance in driving the fast polymerization and the consequent heterogeneity of natural melanin. The tedious task of extracting melanin from natural sources and the lack of solubility of the polymeric melanin material in organic/aqueous solvents have called for basic model systems to understand the complex eumelanin architecture. In this regard, we have adopted a facile approach to decode the perpetual puzzle by single crystal X-ray crystallographic and spectroscopic analyses of DHI crystalline aggregates derived from a non-aqueous environment (chloroform). Due to the highly autooxidative³¹ nature of eumelanin precursors even in the slight presence of protic solvents, the simple model implemented here precludes the contribution of solute–solvent hydrogen bonding interaction towards the formation and resultant structure of DHI crystalline aggregates. In chloroform, each DHI molecule experiences weak interactions solely from the neighbouring DHI chromophores thereby leading to helical aggregation.Our efforts towards recognizing and monitoring the photogenerated excitons and charge-transfer dynamics in crystalline and contorted polyaromatic assemblies^32–36 prompted us to explore the structure–optical property relationship in the eumelanin precursor molecule DHI. Unlike the commonly understood π-stacking in eumelanin derivatives, the single crystals of DHI arrange in a helical zig-zag fashion with a completely edge-to-face aggregate structure driven by both conventional and unconventional hydrogen bonds (Fig. 1b). The structural heterogeneity imposed by the different hydrogen bonds has led to varied levels of exciton delocalization between the neighbouring chromophores in the crystalline DHI aggregates. Along with the diffracting single crystals of monomeric DHI, covalently connected trimeric units of DHI are also identified in chloroform solution, which form double-helical crystals in the mesoscopic scale. Such double-helical architectures are omnipresent in nature as exemplified by the DNA structure.DHI was synthesized by following a previously reported procedure having l-dopa as the starting material (Scheme S1, Appendix C1–C4 and C7, ESI^†).³⁷ Slow evaporation from dry chloroform solution of DHI produced colourless diamond shaped single crystals of DHI (Fig. S1a^†). Interestingly, the DHI molecule with no chiral centre atypically crystallized in the Sohncke space group, P2₁2₁2₁ (Table S1^†). Single-crystal X-ray structure analysis revealed the presence of conventional and unconventional hydrogen bonds directing the crystalline self-assembly of DHI chromophores about a zig-zag helical backbone. Fig. 2 presents the four different types of non-covalent dimers (D1–4) distinguished within the DHI crystal. The zig-zag helical stacks proceed along the crystallographic a-axis (Fig. S2^†) and are fabricated by bifurcated O–H⋯O hydrogen bonds (d_O1⋯H = 2.25 Å, d_O2⋯H = 2.34 Å, <O1–H–O2 = 67.89°) between the two hydroxyl substituents in the DHI chromophore (D3 in Fig. 2). Such bifurcated hydrogen bonded assemblies are prevalent in the secondary and tertiary structures of proteins.³⁸ Interestingly, in the DHI crystal, enantiomeric helical stacks (Fig. S2^†) that are arranged with respect to different screw axes are observed, wherein each stack aligns in a herringbone fashion to the other zig-zag helix (as represented by the dimers D1, D2 and D4). The stacks are interconnected majorly through the unconventional hydrogen bonds such as C–H⋯π (d_C–H⋯π = 2.66–2.99 Å), O–H⋯π (d_O–H⋯π = 2.59 Å), C–H⋯N (d_C–H⋯N = 2.72 Å), C–H⋯O (d_C–H⋯O = 2.66 Å) and the classical N–H⋯O (d_N–H⋯O = 2.72 Å) hydrogen bonds. The absence of π–π stacking interaction is validated by the Hirshfeld surface analysis wherein the formation of the DHI crystalline assembly is majorly stabilized by the C⋯H (40.5%), H⋯H (29.7%), N⋯H (4.3%) and O⋯H (25.4%) noncovalent interactions (Fig. S3 and Table S2^†).Open in a separate windowFig. 2Different orientations of DHI (D1–4) and the directing hydrogen bonds observed in the single crystal.Detailed examination of the interchromophoric interactions supporting the zig-zag helical stacks in DHI crystals using Bader''s quantum theory of atoms in molecules (QTAIM) analysis revealed the presence of supramolecular synthons in the crystal system (Fig. S4^†). This is evidenced by the (3,+1) ring critical points in each of the representative dimers. For a molecular self-assembly to occur efficiently, recognition between the intermolecular functionalities is important, which often culminates in the formation of smaller repetitive units or supramolecular synthons.³⁹ The recognition information which is then carried by these units forms the kernel of self-aggregation or crystallization processes. In the case of DHI crystals, all the dimer assemblies D1–D4 display synthon formation with the D1 and D3 synthons showing greater energetic stabilities. The dimer unit representing the helical backbone, D3, forms two supramolecular synthons orchestrated by the bifurcated O–H⋯O hydrogen bonds and a weak C–H⋯O interaction (Fig. S4^†). The five- and six-membered rings so formed fabricate the helical zig-zag backbone of the DHI crystal. Similarly, the dimer D1 also forms two synthons through a classical N–H⋯O interaction along with the weak C–H⋯π and O–H⋯π interactions. D2 and D4 dimers are stabilized by one synthon each, materialized by C–H⋯π and C–H⋯N interactions in D2 and N–H⋯O and C–H⋯O interactions in D4.The synthon formation and the concomitant electron delocalization involving the π-rings in dimers D1 and D2 have resulted in the aromatic stabilization of the π-rings upon comparison with the monomer DHI. The nucleus independent chemical shift (NICS(1)) values evaluated for D1–4 and monomer DHI in the ground state indicate the aromatic stabilization of the π-surface leading to the favourable alternate stacking of the enantiomeric zig-zag helices facilitated by the unconventional hydrogen bonds. The negative NICS(1) values for the six- and five-membered (6C, 5C) rings of molecule A (Fig. S5 and Table S3^†) increased to −27.22 ppm (6C) and −29.13 ppm (5C) when compared to the monomer DHI (6C: −26.14 ppm, 5C: −28.03 ppm). Similarly, in D2, the π-surface of molecule B that is involved in the weak interaction undergoes aromatic stabilization (6C: −29.43 ppm, 5C: −31.48 ppm). Truncated symmetry adapted perturbation theory (SAPT(0)) analysis⁴⁰ performed on the DHI dimers shows higher stabilization for D1 (E^SAPT_int = −9.70 kcal mol⁻¹) and D3 (E^SAPT_int = −6.57 kcal mol⁻¹) dimers, which could be attributed to the two supporting supramolecular synthons in both the dimers (Table S4^†). The total stabilization of D1 and D3 orientations is facilitated by the higher contributions of electrostatic energy (E⁽¹⁾_elc = −6.17 to −6.14 kcal mol⁻¹) and induction energy (E⁽¹⁾_elc = −1.60 to −1.38 kcal mol⁻¹) towards the total SAPT energy. The dominant role of the stronger classical hydrogen bonds in the fabrication of D1 and D3 synthons when compared to the other DHI orientations (having equal contribution from the weak unconventional interactions) explains the observed energy distribution in the SAPT(0) analysis.The crystalline architecture of the DHI precursor molecule identified herein could provide a sound model for understanding the inherent nature of the excited energy states leading to the characteristic photo-function of the eumelanin pigment. Several experimental and theoretical investigations on eumelanin aggregates revealed the occurrence of excitation energy transfer within the aggregates.^41,42 The extent of energy delocalization within the four dimer orientations in the DHI crystal structure is determined using the fragment-based excited state analysis developed by Plasser, executed in the TheoDORE package.^43–45 The expanse of excitation delocalization amongst the fragment units is described by the value of participation ratio (PR). The contribution towards the exciton delocalization from the fragments is defined by the mean position or the POS value, wherein the indicated number shows the involvement of one or more units. The charge-transfer or Frenkel character of the excited states is defined by the CT number which assumes values closer to one for pure CT states and closer to zero for pure Frenkel states. In dimers D1 and D2, the first singlet excited state of highest oscillator strength (S2) shows Frenkel exciton character with effective delocalization of the excitons between the individual constituting units during the excitation process (Tables S5 and S6^†). The low CT values of the S2 states in D1 (PR = 1.79, POS = 1.33, CT = 0.08) and D2 (PR = 1.98, POS = 1.45, CT = 0.02) along with a PR close to a value of two indicated the delocalization of the Frenkel excitons on the two monomers. Hence, there exists a possible excitation energy delocalization along the adjacent enantiomeric stacks in the DHI crystal. However, for D3 with the bifurcated hydrogen bonding interaction between the monomeric units, both S1 and S2 states have significant oscillator strength. The Frenkel excitons in S1 and S2 states remain localized on only one fragment of D3 (Table S7^†), while in D4, there exists a partial delocalization of the Frenkel excitons in the S1 state (Table S8^†). Thus, it is understood that within each enantiomeric stack the initial Frenkel excitons remain localized on one fragment. The natural transition orbitals (NTOs) of dimers D1–4 give an idea about the nature of the dominant orbital transitions for the allowed electronic excitations. The allowed electronic excitation in all the dimers could only be well represented by taking two distinct orbital transitions with significant coefficients into consideration. The absence of a single dominant orbital transition in the dimers proposed the need for fragment-based hole–electron analysis for the better understanding of the electronic energy delocalization. The hole–electron isosurface analysis provides a pictorial representation of the corresponding delocalized and localized nature of the initial Frenkel excitons in the four DHI orientations (Fig. 3). The delocalization of the hole and electron density on both the fragments puts forward the possibility of effective energy transfer in dimers D1, D2 and D4.⁴⁶ In dimer D3, a very weak delocalization of the electron–hole densities was observed suggesting a localized exciton formation.Open in a separate windowFig. 3Hole–electron isosurface plots of the DHI orientations in the crystal. (a) D1, (b) D2, (c) D3 and (d) D4.Several factors including slight exposure to air,⁴⁷ humidity and/or light have been observed to cause the autooxidative polymerization of DHI, wherein lowered temperatures decrease the kinetics of this solid-state polymerization. Although, a major fraction of the solid oligomer mixture remains as the DHI monomer (≥80%), oligomeric units up to DHI-hexamers have been identified with varying solubilities in different solvents. MALDI-MS spectra (Appendix C5, ESI^†) sequentially collected for the oligomer mixture in CHCl₃ and DMSO indicated the major presence of DHI trimers (in CHCl₃; m/z = 442.138) and DHI-hexamers (in DMSO; m/z = 882.382) along with the smaller counterparts. Interestingly, over the time course for nucleation and the subsequent growth mechanism of the single crystals of monomeric DHI in chloroform, oligomerization of DHI is found to occur concurrently to form the covalently connected DHI trimer (DHI-T). Along with the diffracting single crystals of DHI, small right-handed double-helical crystals (non-diffracting, Fig. S1b–f and S6^†) are observed for the first time, which could be attributed to the self-assembled morphology of DHI-T (Fig. 4a). Observed only in chloroform, we speculate that the prolonged exposure of the chlorinated solvent plays a significant role in the chemical transformation of the DHI monomer to the covalent trimer, possibly through a radical initiated reaction.⁴⁸ The ¹H-NMR spectrum obtained for the bulk crystalline sample dissolved in CDCl₃ along with the observed MALDI-MS data of the DHI trimer evidenced the formation of DHI-T, although in very low yields when compared to the monomer (Appendix C5 and C6^†).Open in a separate windowFig. 4(a) Optical microscopy images of the right-handed double-helical crystals of the DHI trimer. (b) Normalized absorbance and excitation spectra showing DHI-T formation in chloroform solution and the Kubelka–Munk transformed diffuse reflectance spectrum of the DHI bulk crystal. (c) Optimized structure of DHI-T at the CAM-B3LYP/6-311g+(d,p) level of theory.With the understanding that DHI readily aggregates/crystallizes in chloroform, the directionality of the hydrogen bonding interactions (to the π-ring) within the definite spatial arrangements of D1 and D2 orientations hints towards the mode/position of coupling for the associated generation of DHI-T (Appendix C9^†). The molecular structure of the DHI trimer that best fits the observed characterization data (Fig. S7a and Appendix C8, ESI^†) is in line with the chemical disorder model having semiquinone and catechol units connected covalently as an extension of the D1 and D2 noncovalent interactions. Geometry optimization of the predicted structure of the DHI-T performed using the CAM-B3LYP/6-311g+(d,p) level of theory in Gaussian 16 suite shows a twisted conformation having the possibility of forming intramolecular hydrogen bonds from the –OH and –NH functionalities (Fig. 4 and S7b^†). Separation of DHI-T from DHI is a real challenge since exposure of DHI to the adsorbent in column chromatography accelerates the oxidative polymerization of DHI, resulting in a black, insoluble material difficult to characterize. Also, the presence of higher oligomer units of DHI (hexamers etc.) was not identified in the multiple data sets collected for the DHI sample dissolved in CHCl₃.Solvent-dependent steady-state UV-vis absorption and fluorescence emission measurements of DHI were performed and the line shapes of the absorption spectra of monomeric DHI in different solvents match consistently (Fig. S8^†). Two major absorption bands at λ_a1 ∼ 270 nm and λ_a2 ∼ 300 nm form the characteristic absorption spectrum of the DHI monomer. The fluorescence emission of monomeric DHI exhibits a single broad spectral feature peaking at λ_em ∼ 330 nm in a majority of the solvents. The relative fluorescence quantum yields are found to be exceptionally low in chloroform, dichloromethane, THF and water (Table S9^†) indicating the presence of non-radiative decay channels for dissipating the excitation energy. In chloroform, the emergence of a red-shifted tail in the absorption spectrum of the DHI-monomer is observed over time possibly signifying the onset of DHI-T formation. The fluorescence emission in CHCl₃ also shows a new band peaking at λ_em ∼ 460 nm along with the emission band at λ_em ∼ 335 nm (Fig. S9^†). A broad red-shifted band arising at 370 nm in the excitation spectrum of DHI solution collected in chloroform at λ_em ∼ 460 nm (Fig. 4b) evidenced the presence of the DHI trimer. A similar decrease in fluorescence quantum yield in chloroform and the concomitant emergence of new bands in the fluorescence emission profiles have been noted previously in tryptophan and other indole species synthesized for eumelanin investigation.^49,50 In such cases, the photoionization of excited indole leads to the ejection of a solvated electron which attacks the chloroform molecule, releasing a chloride ion, and further undergoes reactions to yield photoproducts.Spectroscopic investigation of the crystalline DHI (containing both monomer single crystals and covalent trimer crystals) sample showed broad, red-shifted absorption bands spanning from 210 to 560 nm (Fig. 4b and S10^†). The solid-state absorption spectrum shows two prominent bands at λ_a1 ∼ 280 nm and λ_a2 ∼ 305 nm which could be attributed to the red-shifted absorption bands of the crystalline DHI monomer (compared to the monomer absorption bands in the solution state). The observed red-shift in the absorption band of the DHI crystal arises from the nonplanar packing motif and the ensuing intermolecular interactions in the solid state. The presence of a broad shoulder band centred at λ_a3 ∼ 375 nm in the absorption spectrum could be assigned to the double-helical crystal of the covalent DHI trimer. The crystalline state fluorescence emission spectrum of DHI spans from 390 to 490 nm (Fig. S10^†). The excitation energies and the allowed vertical transitions of the monomer and DHI-T have been computed at the CAM-B3LYP/6-311g+(d,p) level of theory. Unlike the precursor DHI monomer which undergoes higher energy electronic transitions (at 270 nm and 300 nm), the favorable transition in the covalent trimer is red-shifted with the S₀ → S₁ electronic excitation occurring at ∼434 nm. Hence, the spectroscopic and theoretical investigation of bulk crystalline DHI indicates that the broad absorption profile of the eumelanin precursor could be ascribed to the combined effects of the non-planar chromophore stacking and the presence of covalent DHI trimers that exist as double-helical aggregates. The photoprotective nature of eumelanin arises from the signature broadband absorption of eumelanin which spans throughout the UV and visible region tailing around 800 nm as explained by the chemical disorder model. In line with this understanding, the spectroscopic data of the DHI crystal also exhibit broadband absorption which expands up to 600 nm unlike the DHI monomer. Moreover, the low relative fluorescence quantum yields of DHI suggest the presence of non-radiative decay pathways within the DHI units. The oligomeric trimer which in itself shows structural heterogeneity aggregates as double helical structures and shows a red-shifted absorption band which is comparable to the computed TDDFT vertical excitation energies. Thus, our report on the characterization of DHI and the oligomeric trimer could possibly be beneficial in advancing melanin structure characterization and elucidating the photoprotective function of eumelanin.The solid-state CD spectrum of crystalline DHI (in KBr, Fig. S11a^†) showed the signatures for the presence of helical packing^51–55 (possibly from the zig-zag helical motif along with the double helical arrangement). However, the basis of the CD couplet of significant intensity spanning from ∼250 to 600 nm (including ranges outside of the absorption maxima) could not be exclusively assigned to chiral absorption from the chromophoric packing.⁵⁶ In the case of macromolecular systems having long-range organization, differential scattering of incident left and right circularly polarized light can provide significant contributions to the observed circular dichroism.^57–59 The occurrence of broad CD bands outside the absorption bands of the macromolecule can signify the possible role of differential scattering in the circular dichroism.⁶⁰ Although, for the DHI sample, the characteristic CD spectrum has been found to be concurrent for the different data sets collected using freshly prepared crystalline samples on different days (Fig. S11a^†), the ratios of the intensities of the positive and negative bands have been observed to vary. Such a heterogeneity in the ellipticity values of the positive and negative bands could be attributed to the possible presence of different enantiomeric assemblies that exhibit varying abilities to undergo chiral absorption and differential scattering.⁵⁷ The possibility of having linear dichroism (LD) artifacts in the CD data was evaluated for the DHI sample (Fig. S11b^†).⁶¹ The LD artifact fell within the error bar of the order of 10⁻³–10⁻⁴ mdeg and hence, the contribution of LD to the strong CD signal of DHI could be ignored.^62,63 Also, the idea of having a chiral nucleation centre, probably from any conformationally chiral DHI oligomer units, leading to the double-helical aggregation and the consequent mesoscopic chirality could not be ignored while assessing the origin of the observed CD spectrum. The existing uncertainties in solving the source of the double-helical aggregation of the DHI chromophores and identifying the intermolecular forces acting behind the same remain a challenge that requires detailed examination in future studies.     

Simplifying and expanding the scope of boron imidazolate framework (BIF) synthesis using mechanochemistry

Mechanochemistry enables rapid access to boron imidazolate frameworks (BIFs), including ultralight materials based on Li and Cu(i) nodes, as well as new, previously unexplored systems based on Ag(i) nodes. Compared to solution methods, mechanochemistry is faster, provides materials with improved porosity, and replaces harsh reactants (e.g. n-butylithium) with simpler and safer oxides, carbonates or hydroxides. Periodic density-functional theory (DFT) calculations on polymorphic pairs of BIFs based on Li⁺, Cu⁺ and Ag⁺ nodes reveals that heavy-atom nodes increase the stability of the open SOD-framework relative to the non-porous dia-polymorph.

Mechanochemistry enables rapid access to boron imidazolate frameworks (BIFs), including ultralight materials based on Li and Cu(i) nodes, as well as new, previously unexplored systems based on Ag(i) nodes.

Mechanochemistry^1–7 has emerged as a versatile methodology for the synthesis and discovery of advanced materials, including nanoparticle systems^8–10 and metal–organic frameworks (MOFs),^11–15 giving rise to materials that are challenging to obtain using conventional solution-based techniques.^16–18 Mechanochemical techniques such as ball milling, twin screw extrusion¹⁹ and acoustic mixing^20,21 have simplified and advanced the synthesis of a wide range of MOFs, permitting the use of simple starting materials such as metal oxides, hydroxides or carbonates,^22,23 at room temperature and without bulk solvents, yielding products of comparable stability and, after activation, higher surface areas than solution-generated counterparts.^24–29 The efficiency of mechanochemistry in MOF synthesis was recently highlighted by accessing zeolitic imidazolate frameworks (ZIFs)^30,31 that were theoretically predicted, but not accessible under conventional solution-based conditions.¹⁷The advantages of mechanochemistry in MOF chemistry led us to address the possibility of synthesizing boron imidazolate frameworks (BIFs),^32–34 an intriguing but poorly developed class of microporous materials analogous to ZIFs, comprising equimolar combinations of tetrahedrally coordinated boron(iii) and monovalent Li⁺ or Cu⁺ cations as nodes (Fig. 1A–C). Although BIFs offer an attractive opportunity to access microporous MOFs with lower molecular weights, particularly in the case of “ultralight” systems based on Li⁺ and B(iii) centers, this family of materials has remained largely unexplored – potentially due to the need for harsh synthetic conditions, including the use of n-butyllithium in a solvothermal environment.^32–34Open in a separate windowFig. 1Structures of previously reported BIFs with: (A) zni-, (B) dia-, or (C) SOD-topology (M = Li, Cu); (D) tetrakis(imidazolyl)boric acids used herein for mechanochemical BIF synthesis; and (E) schematic representation of the herein developed mechanosynthesis of dia- and SOD BIF polymorphs based on Li, Cu or Ag metal nodes.We now show how switching to the mechanochemical environment enables lithium- and copper(i)-based BIFs to be prepared rapidly (i.e., within 60–90 minutes), without elevated temperatures or bulk solvents, and from readily accessible solid reactants, such as hydroxides and oxides (Fig. 1D and E). While the mechanochemically-prepared BIFs exhibit significantly higher surface areas than the solvothermally-prepared counterparts, mechanochemistry allows for expanding this class of materials towards previously not reported Ag⁺ nodes. The introduction of BIFs isostructural with those based on Li⁺ or Cu⁺ but comprising of Ag⁺ ions, enables a periodic density-functional theory (DFT) evaluation of their stability. This reveals that switching to heavier elements as tetrahedral nodes improves the stability of sodalite topology (SOD) open BIFs with respect to close-packed diamondoid (dia) topology polymorphs.As a first attempt at mechanochemically synthesis of BIFs, we targeted the synthesis of previously reported zni-topology LiB(Im)₄ and CuB(Im)₄ frameworks (Li-BIF-1 and Cu-BIF-1, respectively, Fig. 1A) using a salt exchange reaction between LiCl or CuCl with commercially available sodium tetrakis(imidazolyl)borate (Na[B(Im)₄]) (Fig. 2A). Milling of LiCl and Na[B(Im)₄] in a 1 : 1 stoichiometric ratio for up to 60 minutes led to the appearance of Bragg reflections consistent with the target Li-BIF-1 (CSD MOXJEP) and the anticipated NaCl byproduct. The reaction was, however, incomplete, as seen by X-ray reflections of Na[B(Im)₄] starting material. In order to improve reactant conversion, we explored liquid-assisted grinding (LAG), i.e. milling in the presence of a small amount of a liquid phase (measured by the liquid-to-solid ratio η³⁵ in the range of ca. 0–2 μL mg⁻¹). Using LAG conditions with acetonitrile (MeCN, 120 μL, η = 0.5 μL mg⁻¹) led to the complete disappearance of reactant X-ray reflections, concomitant with the formation of Li-BIF-1 alongside NaCl within 60 minutes.Open in a separate windowFig. 2(A) Reaction scheme for the mechanochemical synthesis of Li-BIF-1 by a salt metathesis strategy. Selected PXRD patterns for: (B) Na[B(Im)₄] (C) LiCl, (D) simulated Li-BIF-1 (CSD MOXJPEP) and (E) synthesized BIF-1-Li by LAG for 60 minutes with MeCN (η = 0.5 μL mg⁻¹), (F) CuCl, (G) simulated Cu-BIF-1 (CSD MOXJIT), and (H) synthesized BIF-1-Cu by LAG for 60 minutes with MeOH (η = 0.50 μL mg⁻¹). Asterisks denote NaCl, a byproduct of the metathesis reaction. (Fig. 2B–E, also see ESI^†). The copper-based zni-CuB(Im)₄ (Cu-BIF-1) was readily obtained from CuCl within 60 minutes using similar LAG conditions. We also explored LAG with methanol (MeOH), revealing that the exchange reaction to form NaCl took place with both LiCl and CuCl starting materials. With LiCl, however, the PXRD pattern of the product could not be matched to known phases involving Li⁺ and B(Im)₄⁻ (see ESI^†). With CuCl as a reactant, LAG with MeOH (η = 0.5 μL mg⁻¹) cleanly produced Cu-BIF-1 alongside NaCl (see ESI^†).Next, we explored an alternative synthesis approach, analogous to that previously used to form ZIFs and other MOFs: an acid–base reaction between a metal oxide or hydroxide and the acid form of the linker: tetrakis(imidazolato)boric acid, HB(Im)₄ (Fig. 3A).^36–40 Neat milling LiOH with one equivalent of HB(Im)₄ in a stainless steel milling assembly led to the partial formation of Li-BIF-1, as evidenced by PXRD analysis (see ESI^†). Complete conversion of reactants into Li-BIF-1 was achieved in 60 minutes by LAG with MeCN (η = 0.25 μL mg⁻¹), as indicated by PXRD analysis (Fig. 3B–E), Fourier transform infrared attenuated total reflectance spectroscopy (FTIR-ATR), thermogravimetric analysis (TGA) in air, and analysis of metal content by inductively-coupled plasma mass spectrometry (ICP-MS) (see ESI^†).Open in a separate windowFig. 3(A) Reaction scheme for the mechanochemical synthesis of Li-BIF-1 using the acid–base strategy. Selected PXRD patterns for: (B) H[B(Im)₄] (C) LiOH, (D) simulated Li-BIF-1 (CSD MOXJPEP), (E) synthesized BIF-1-Li by LAG for 60 minutes with MeCN (η = 0.25 μL mg⁻¹), (F) Cu₂O, (G) simulated Cu-BIF-1 (CSD MOXJIT), and (H) synthesized Cu-BIF-1 by ILAG for 60 minutes with MeOH (η = 0.50 μL mg⁻¹) and NH₄NO₃ additive (5% by weight).Neat milling of HB(Im)₄ with Cu₂O under similar conditions gave a largely non-crystalline material, as evidenced by PXRD (see ESI^†). Switching to the ion- and liquid-assisted grinding (ILAG) methodology, in which the reactivity of a metal oxide is enhanced by a small amount of a weakly acidic ammonium salt, and which was introduced to prepare zinc and cadmium ZIFs from respective oxides,^37–40 enabled the synthesis of Cu-BIF-1 from Cu₂O. Specifically, PXRD analysis revealed complete disappearance of the oxide in samples obtained by ILAG with either MeOH or MeCN (η = 0.5 μL mg⁻¹) in the presence of NH₄NO₃ additive (5% by weight, see ESI^†). Notably, achieving complete disappearance of Cu₂O reactant signals also required switching from stainless steel to a zirconia-based milling assembly, presumably due to more efficient energy delivery.⁴¹ After washing with MeOH, the material was characterized by FTIR-ATR, TGA in air, and analysis of metal content by ICP-MS (see ESI^†).Whereas both the metathesis and acid–base approaches can be used to mechanochemically generate Li- and Cu-BIF-1, the latter approach has a clear advantage of circumventing the formation of the NaCl byproduct. Consequently, in order to further the development of mechanochemical routes to other BIFs, we focused on the acid–base strategy. As next targets, we turned to MOFs based on tetrakis(2-methylimidazole)boric acid H[B(Meim)₄],³⁶ previously reported³² to adopt either a non-porous diamondoid (dia) topology (BIF-2) or a microporous sodalite (SOD) topology (BIF-3) with either Li⁺ or Cu⁺ as nodes (Fig. 4). Attempts to selectively synthesize either Li-BIF-2 or Li-BIF-3 by neat milling or LAG (using MeOH or MeCN as liquid additives) with LiOH and a stoichiometric amount of HB(Meim)₄ were not successful. Exploration of different milling times and η-values produced only mixtures of residual reactants with Li-BIF-2, Li-BIF-3, and/or not yet identified phases (see ESI^†). Consequently, we explored milling in the presence of 2-aminobutanol (amb), which is a ubiquitous component of solvent systems used in the solvothermal syntheses of BIFs.^32,33 Gratifyingly, using a mixture of amb and MeCN in a 1 : 3 ratio by volume as the milling liquid led to an effective strategy for the selective synthesis of both the dia-topology Li-BIF-2 (CSD code MOXKUG), and the SOD-topology Li-BIF-3 (CSD code MUCLOM).^‡ The selective formation of phase-pure samples of Li-BIF-2 and Li-BIF-3 was confirmed by PXRD analysis, which revealed an excellent match to diffractograms simulated based on the previously reported structures (Fig. 4B–G). Systematic exploration of reaction conditions, including time (between 15 and 60 minutes) and η value (between 0.25 and 1 μL mg⁻¹) revealed that the open framework Li-BIF-3 is readily obtained at η either 0.75 or 1 μL mg⁻¹ after milling for 45 minutes or longer (Fig. 4B–G, also see ESI^†).^§ Lower η-values of 0.25 and 0.5 μL mg⁻¹ preferred the formation of the dia-topology Li-BIF-2, which was obtained as a phase-pure material upon 60 minutes milling at η = 0.5 μL mg⁻¹, following the initial appearance of a yet unidentified intermediate. The preferred formation of Li-BIF-2 at lower η-values is consistent with our previous observations that lower amounts of liquid promote mechanochemical formation of denser MOF polymorphs.³⁷Open in a separate windowFig. 4(A) Reaction scheme for the mechanochemical synthesis of Li-BIF-3. Comparison of selected PXRD patterns for the synthesis of Li-BIF-2 and Li-BIF-3: (B) H[B(Meim)₄] reactant; (C) LiOH reactant; (D) simulated for Li-BIF-3 (CSD MUCLOM); (E) simulated for Li-BIF-2 (CSD MOXKUG); (F) Li-BIF-3 mechanochemically synthesized by LAG for 60 minutes with a 1 : 3 by volume mixture of amb and MeCN (η = 1 μL mg⁻¹); and (G) Li-BIF-2 mechanochemically synthesized by LAG for 60 minutes with a 1 : 3 by volume mixture of amb and MeCN (η = 0.5 μL mg⁻¹). Comparison of selected PXRD patterns for the synthesis of Cu-BIF-2 and Li-BIF-3: (H) Cu₂O; (I) Cu-BIF-3 (CSD MOXJOZ); (J) Cu-BIF-2 (CSD MUCLIG); (K) Cu-BIF-3 mechanochemically synthesised by ILAG for 60 minutes using NH₄NO₃ ionic additive (5% by weight) and MeOH (η = 1 μL mg⁻¹); and (L) mechanochemically synthesised Cu-BIF-2 by ILAG for 90 minutes using NH₄NO₃ ionic additive (5% by weight) and MeOH (η = 0.5 μL mg⁻¹).Samples of both Li-BIF-2 and Li-BIF-3 after washing with MeCN were further characterized by FTIR-ATR, TGA in air, and analysis of metal content by ICP-MS (see ESI^†). Nitrogen sorption measurement on the mechanochemically obtained Li-BIF-3, after washing with MeCN and evacuation at 85 °C, revealed a highly microporous material with a Brunauer–Emmett–Teller (BET) surface area of 1010 m² g⁻¹ (Fig. 5A), which is close to the value expected from the crystal structure of the material (1200 m² g⁻¹, 32 For direct comparison with previous work,³² we also calculated the Langmuir surface area, revealing an almost 40% increase (1060 m² g⁻¹) compared to samples made solvothermally (762.5 m² g⁻¹) (Fig. 5A, inset).Experimental Brunauer–Emmett–Teller (BET) and Langmuir surface area (in m² g⁻¹) of mechanochemically synthesized SOD-topology BIFs, compared to previously measured and theoretically calculated values, along with average particle sizes (in nm) established by SEM and calculated energies (in eV) for all Li-, Cu-, and Ag-BIF polymorphs. The difference between calculated energies for SOD- and dia-polymorphs in each system is given as ΔE (in kJ mol⁻¹)

Stereoselective tandem iridium-catalyzed alkene isomerization-cope rearrangement of ω-diene epoxides: efficient access to acyclic 1,6-dicarbonyl compounds

The Cope rearrangement of 2,3-divinyloxiranes, a rare example of epoxide C–C bond cleavage, results in 4,5-dihydrooxepines which are amenable to hydrolysis, furnishing 1,6-dicarbonyl compounds containing two contiguous stereocenters at the 3- and 4-positions. We employ an Ir-based alkene isomerization catalyst to form the reactive 2,3-divinyloxirane in situ with complete regio- and stereocontrol, which translates into excellent control over the stereochemistry of the resulting oxepines and ultimately to an attractive strategy towards 1,6-dicarbonyl compounds.

Iridium catalyzed alkene isomerization-cope rearrangement of ω-diene epoxide furnishes 3,4-dihydrooxepines. These oxepines are hydrolyzed to diastereomerically pure 1,6-dicarbonyl compound containing two contiguous stereocenters within acyclic system.

1,6-Dicarbonyl compounds are widespread as targets and intermediates in organic synthesis.¹ Due to the “dissonant” polarizing effect induced by the two carbonyl groups,² these motifs are challenging to retrosynthetically disconnect into classical synthons. Unsurprisingly, many approaches toward 1,6-dicarbonyls rely on dimerization of α,β-unsaturated carbonyl compounds (Scheme 1a)³ or oxidative cleavage of substituted cyclohexene derivatives⁴ which significantly limits the range of possible products. Alternative strategies, such as the ring-opening of donor–acceptor cyclopropanes with enolate nucleophiles, efficiently form the 1,6-dicarbonyl skeleton, albeit with limited substrate scope (Scheme 1b).⁵ The Cope rearrangement of 1,5-dienes, featuring oxygen functionality in the 3- and 4-positions,⁶ represents a promising strategy towards 1,6-dicarbonyl compounds but suffers from lack of stereocontrol over the diene substrates, resulting in diastereomeric mixtures of products (Scheme 1c).Open in a separate windowScheme 1Selected approaches towards the formation of 1,6-dicarbonyl compounds and our proposed approach.A conceptually related approach towards the preparation of 1,6-dicarbonyl compounds is through the hydrolysis of 3,4-dihydrooxepines (Scheme 1d), which are in turn generated through the Cope rearrangement of 2,3-divinyloxiranes.⁷ Such a sigmatropic rearrangement is also noteworthy as a rare example where an epoxide C–C bond is selectively cleaved over the usually more reactive C–O bond. This intriguing rearrangement has been studied but its use in synthesis is scarce, presumably due to difficulties in the stereoselective synthesis and handling of the key divinyl epoxides.In line with our interest in the strategic application of alkene isomerization to generate reactive synthetic intermediates in stereodefined form,⁸ we posited to form the reactive 2,3-divinyloxiranes in situ, through alkene isomerization^9,10 of the simpler allyl epoxides, which are accessible in enantiomerically enriched form.¹¹ Such a strategy might greatly facilitate access to these intermediates and therefore uncover a synthetically attractive route toward 1,6-dicarbonyl compounds featuring two contiguous stereocenters.With this idea in mind, we first explored the isomerization and subsequent Cope rearrangement of allyl-vinyl epoxides 1 (Scheme 2). To induce isomerization, we employed a cationic iridium-based catalytic system,¹² which is known to reliably isomerize alkenes with high degrees of regio- and stereocontrol.¹³Open in a separate windowScheme 2Substrate scope for the tandem iridium-catalyzed alkene isomerization-Cope rearrangement of allyl-vinyl epoxides.In line with our expectations, our model substrate 1a (R² = R³ = H, R⁴ = Me, R⁵ = CO₂Et) was smoothly isomerized at 65 °C in the presence of 1.5 mol% of Ir dimer to obtain the corresponding divinyl epoxide with a complete E-selectivity. With suitable conditions for alkene isomerization in hand, we exposed substrate 1a to the Ir-based catalytic system at 120 °C and were equally pleased to observe the 4,5-dihydrooxepine product 2a, resulting from the tandem isomerization-Cope rearrangement as a single diastereoisomer in 81% yield. We proceeded to test the generality of our protocol with respect to different alkene and epoxide substitution patterns. Pleasingly, product 2b was generated with complete stereoselectivity, showcasing the compatibility of the reaction conditions with potentially labile tertiary stereocenters α to the ester group. We then wondered whether the anti-diastereomer could be accessed starting from the corresponding cis allyl-vinyl epoxide. Indeed, in line with the known stereospecific behavior of the Cope rearrangement, we obtained the complementary diastereomer 2c. Turning our attention to more highly substituted epoxides, we were pleased to observe the formation of dihydrooxepines 2d and 2e, which correspond to 1,6-keto-aldehyde and diketone products, respectively. Substrate 1f (R² = R⁴ = R⁵ = H, R³ = Ph), which features an unactivated vinyl group, also underwent the rearrangement, demonstrating that an activated alkenyl group is not required for a successful outcome. Similarly, product 2g featuring two alkyl groups is also generated, with high diastereoselectivity albeit in moderate yield. Products featuring ethyl and methyl ester 2h, 2i could also be obtained in good yields and diastereoselectivity. We next tested substrate 1j (R² = Me, R³ = Ph, R⁴ = CH₂CH₂Ph, R⁵ = H), as a geometric-mixture of the double bond (E : Z = 1.1 : 1) and in accordance with the stereospecificity of the process, the oxepine 2j was obtained as a mixture of two diastereomers with the same ratio. Disappointingly, substrate 1k did not undergo isomerization, presumably due to the Lewis basic nature of the ketone, likely poisoning the Ir-catalyst.During our study, we noticed that allyl-vinyl epoxides bearing electron donating groups on the vinyl moiety tend to decompose during purification by column chromatography on silica gel. This obstacle further motivated us to explore diallyl epoxides 3 as substrates, where the reactive divinyl epoxide would be generated by isomerization of both allyl fragments. Notably, these diallyl epoxides are much more stable compared to their vinyl counterparts and can be readily prepared in two steps from simple alkynes.¹⁴ To our delight, diallyl epoxide 3a (R = CH₂OMe) smoothly underwent the double isomerization-Cope rearrangement cascade at 140 °C, furnishing oxepine 2l with impressive yield and diastereoselectivity (Scheme 3). The use of alkene isomerization to form the reactive divinyl epoxide in situ avoids the isolation of the unstable divinyl epoxide, while controlling the stereochemistry of both double bonds, particularly not trivial to achieve using classical olefination reactions. Products 2m and 2n feature ester and silyl groups, highlighting the functional group tolerance of the catalytic system.Open in a separate windowScheme 3Substrate scope for tandem iridium-catalyzed double alkene isomerization-Cope rearrangement of diallyl epoxides.Our next objective was to hydrolyze the diastereomerically pure oxepines obtained through the rearrangement in a stereoretentive fashion, revealing the acyclic 1,6-dicarbonyl motif. Pleasingly, diversely substituted oxepines 2 underwent smooth hydrolysis either using 5 mol% of Pd(MeCN)₂Cl₂¹⁵ at 50 °C or an acidic aqueous solution to form 1,6-dicarbonyls 4 in diastereomerically pure form (Scheme 4).¹⁶ Dicarbonyl products featuring labile tertiary centers 4a and 4b are formed under these conditions with excellent diastereoselectivities and yields. Without surprise, oxepine 2f (R² = R⁴ = R⁵ = H, R³ = Ph) furnished the keto-substituted product 4c in good yield. The relative stereochemistry of 4b was unambiguously confirmed by single crystal X-ray diffraction analysis of the corresponding carboxylic acid 7 (Scheme 4b).¹⁷ The reaction is scalable to ½ gram of substrate and could be performed in a single-pot operation without isolation of the intermediate oxepine (Scheme 4b). By using this approach, 1h provides 4b in 61% yield as a single diastereomer, underlining the synthetic potential and efficiency of this method.Open in a separate windowScheme 4Hydrolysis of oxepines and one-pot sequence.     

Electrooxidative o-carborane chalcogenations without directing groups: cage activation by copper catalysis at room temperature

Copper-catalyzed electrochemical direct chalcogenations of o-carboranes was established at room temperature. Thereby, a series of cage C-sulfenylated and C-selenylated o-carboranes anchored with valuable functional groups was accessed with high levels of position- and chemo-selectivity control. The cupraelectrocatalysis provided efficient means to activate otherwise inert cage C–H bonds for the late-stage diversification of o-carboranes.

Copper-catalyzed electrochemical cage C–H chalcogenation of o-carboranes has been realized to enable the synthesis of various cage C-sulfenylated and C-selenylated o-carboranes.

Carboranes are polyhedral molecular boron–carbon clusters, which display unique properties, such as a boron enriched content, icosahedron geometry and three-dimensional electronic delocalization.¹ These features render carboranes as valuable building blocks for applications to optoelectronics,² as nanomaterials, in supramolecular design,³ organometallic coordination chemistry,⁴ and boron neutron capture therapy (BNCT) agents.⁵ As a consequence, considerable progress has been witnessed in transition metal-catalyzed regioselective cage B–H functionalization of o-carboranes⁶ and different functional motifs have been incorporated into the cage boron vertices.^7–10 However, progress in this research arena continues to be considerably limited by the shortage of robust and efficient methods to access carborane-functionalized molecules. While C–S bonds are important structural motifs in various biologically active molecules and functional materials,¹¹ strategies for the assembly of chalcogen-substituted carboranes continue to be scarce. A major challenge is hence represented by the strong coordination abilities of thiols to most transition metals, which often lead to catalyst deactivation.¹² While copper-catalyzed B(4,5)–H disulfenylation of o-carboranes was achieved,^7e elevated reaction temperature was required, and 8-aminoquinoline was necessary as bidentate directing group. The bidentate directing group¹³ needs to be installed and removed, which jeopardizes the overall efficacy. Likewise, an organometallic strategy was recently devised for cysteine borylation with a stoichiometric platinum(ii)-based carboranes.¹⁴ Meanwhile, oxidative cage B/C–H functionalizations largely call for noble transition metal catalysts¹⁵ and stoichiometric amounts of chemical oxidants, such as expensive silver(i) salts.¹⁶In recent years, electricity has been identified as an increasingly viable, sustainable redox equivalent for environmentally-benign molecular synthesis.^17,18 While significant advances have been realized by the merger of electrocatalysis with organometallic bond activation,¹⁹ electrochemical carborane functionalizations continue unfortunately to be underdevelopment. In sharp contrast, we have now devised a strategy for unprecedented copper-catalyzed electrochemical cage C–H chalcogenations of o-carboranes in a dehydrogenative manner, assembling a variety of C-sulfenylated and C-selenylated o-carboranes (Fig. 1a). It is noteworthy that our electrochemical cage C–S/Se modification approach is devoid of chemical oxidants, and does not need any directing groups, operative at room temperature.Open in a separate windowFig. 1Electrochemical diversification of o-carboranes and optimization of reaction conditions. ^aReaction conditions: procedure A: 1a (0.10 mmol), 2a (0.3 mmol), CuOAc (15 mol%), 2-PhPy (15 mol%), LiOtBu (0.2 mmol), TBAI (2.0 equiv.), solvent (3 mL), platinum cathode (10 mm × 15 mm × 0.25 mm), graphite felt (GF) anode (10 mm × 15 mm × 6 mm), 2 mA, under air, r.t., 16 h. ^bYield was determined by ¹H NMR with CH₂Br₂ as the internal standard. ^cIsolated yields in parenthesis. ^dKI (1.0 equiv.) as additive. ^eProcedure B: 2 (0.3 mmol), LiOtBu (0.2 mmol), TBAI (2.0 equiv.), solvent (3.0 mL), 2 mA, r.t., 3 h, then adding 1a (0.10 mmol), 2-PhPy (15 mol%), CuOAc (15 mol%), 2 mA, rt, 16 h. ^f2b (0.3 mmol), LiOtBu (0.2 mmol), KI (1.0 equiv.), TBAI (2.0 equiv.), solvent (3.0 mL), 2 mA, r.t., 3 h, then adding 1a (0.10 mmol), 2-PhPy (15 mol%), CuOAc (15 mol%), r.t., 16 h. TBAI = tetrabutylammonium iodide, TBAPF₆ = tetrabutylammonium hexafluorophosphate. DCE = 1,2-dichloroethane, THF = tetrahydrofuran.We commenced our studies by probing various reaction conditions for the envisioned copper-catalyzed cage C–H thiolation of o-carborane in an operationally simple undivided cell setup equipped with a GF (graphite felt) anode and a Pt cathode (Fig. 1b and Table S1^†). After extensive experimentation, we observed that the thiolation of substrate 1 proceeded efficiently with catalytic amounts of CuOAc and 2-phenylpyridine, albeit in the presence of 2 equivalents LiOtBu as the base, and 2 equivalents n-Bu₄NI as the electrolyte at room temperature under a constant current of 2 mA (entry 1). The yield was reduced when other copper sources or additives were used (entries 2–5). Surprisingly, n-Bu₄NPF₆ as the electrolyte failed to facilitate the carborane modification, indicating that n-Bu₄NI operates not only as electrolyte, but also as a redox mediator (entry 6). Altering the stoichiometry of the electrolyte or using KI did not improve the performance (entries 7–8). Product formation was not observed, when the reaction was conducted with DCE as the solvent, while CH₃CN resulted in a drop of the catalytic performance (entries 9–10). Control experiments confirmed the essential role of the electricity and the catalyst (entries 11–12), while a sequential procedure was found to be beneficial (entries 13–15).With the optimized reaction conditions in hand, we explored the versatility of the cage C–H thiolation of o-carborane 1a with different thiols 2 (Scheme 1). Electron-rich as well as electron-deficient substituents on the arenes were found to be amenable to the electrocatalyzed C–H activation, providing the corresponding thiolation products 3aa–3ao in good to excellent yields. Thereby, a variety of synthetically useful functional groups, such as fluoro (3ae, 3am), chloro (3af, 3ak, 3an) and bromo (3ag, 3al), were fully tolerated, which should prove instrumental for further late-stage manipulations. Various disubstituted aromatic and heterocyclic thiols afforded the corresponding cage C–S modified products 3ap–3as. Notably, aliphatic thiols efficiently underwent the electrochemical transformation to provide the corresponding cage alkylthiolated products 3at–3au. Notably, the halogen-containing thiols (2e–2f, 2k–2n and 2q) reacted selectively with o-carboranes to deliver the desired products without halide coupling byproducts being observed. The connectivity of the products 3aa, 3am and 3ao was unambiguously verified by X-ray single crystal diffraction analysis.²²Open in a separate windowScheme 1Electrochemical C–H thiolation of o-carborane 1a. (a) Procedure B. (b) KI (1 equiv.). (c) Cul as the catalyst.Encouraged by the efficiency of the cupraelectro-oxidative cage C–H thiolation, we became intrigued to explore the chalcogenantion of differently-decorated o-carboranes 1 (Scheme 2). Electronically diverse carboranes 1 served as competent coupling partners, giving the corresponding thiolation products 4bo–4do with high levels of efficacy in position-selective manner. The strategy was not restricted to phenyl-substituted o-carboranes. Indeed, substrates bearing benzyl and even alkyl groups also performed well to deliver the desired products 4eo–4ga. It is noteworthy that the C–H activation approach was also compatible with selenols to give the o-carboranes 4av–4fv. The molecular structures of the carborane 4br and 4av were unambiguously verified by single-crystal X-ray diffraction.²²Open in a separate windowScheme 2Electrochemical cage C–H chalcogenation of o-carboranes. (a) Procedure B. (b) KI (1 equiv.).Scaffold functionalization of the thus obtained carborane 3ag provided the alkynylated derivative 5a and amine 5b (Scheme 3), giving access to carborane-based host materials of relevant to phosphorescent organic light-emitting diodes.²⁰Open in a separate windowScheme 3Late-stage diversification.Next, we became attracted to delineating the mode of the cupraelectro-catalyzed cage C–H chalcogenation. To this end, control experiments were performed (Scheme 4a). First, electrocatalysis in the presence of TEMPO or Ph₂C CH₂ gave the desired product 3aa. EPR studies of thiol 2a, LiOtBu and THF under the electrochemical conditions showed a small radical signal, which might be attributed to a thiol radical.²¹ Second, the cupraelectrocatalysis occurred efficiently in the dark. Third, detailed cyclovoltammetric analysis of the thiol and iodide mediator (Scheme 4b and ESI^†)²¹ revealed an irreversible oxidation of the thiol anion at E_p = −0.62 V vs. Ag/Ag⁺ and two oxidation events for the iodide, including an irreversible oxidation at E_p = 0.12 V vs. Ag/Ag⁺ and a reversible oxidation at E_p = 0.44 V vs. Ag/Ag⁺, which is in good agreement with the literature reported iodide oxidation potentials,^18c,d and is suggestive of the preferential oxidation of the iodide as a redox mediator. In this context, the use of n-Bu₄NI as a redox mediator to achieve copper-catalyzed electrochemical arene C–H aminations had been documented.^18d Furthermore, we calculated the redox potential of complex C by means of DFT calculations at the PW6B95-D4/def2-TZVP + SMD(MeCN)//TPSS-D3BJ/def2-SVP level of theory.²¹ These studies revealed a calculated oxidation half-wave potential for complex C is E^o,calc_1/2 = −0.08 V vs. SCE. Hence, iodide is a competent redox mediator to achieve the transformation from complex C to complex D. Analysis of non-covalent interactions²¹ in complex C (Fig. 2) show the presence of a weak stabilization interaction between the chalcogen''s anisole group and the 2-phenylpyridine. In contrast, in complex D these interactions were found more relevant between the o-carborane phenyl group and the chalcogen aromatic motif.Open in a separate windowFig. 2Non-covalent interaction plots for the complexes C and D. Strong attractive interactions are shown in blue, weak attractive interactions are given in green, while red corresponds to repulsive interactions. Ar = 4-MeOC₆H₄.Open in a separate windowScheme 4Control experiments and cyclic voltammograms.On the basis of the aforementioned findings,¹⁸ a plausible reaction mechanism is proposed in Scheme 5, which commences with an anodic single electron-transfer (SET) oxidation of the thiol anion E to form the sulfur-centered radical F. Subsequently, the copper(i) species A reacts with the sulfur radical F to deliver copper(ii) complex B, which next reacts with o-carborane 1 in the presence of LiOtBu to generate a copper(ii)-o-carborane complex C. Thereafter, the complex C is oxidized by the anodically generated redox mediator I₂ to furnish the copper(iii) species D,^18d which subsequently undergoes reductive elimination, affording the final product and regenerating the catalytically active complex A. Alternatively, the direct oxidation of copper(ii) complex C by electricity to generate copper(iii) species D can not be excluded at this stage.^18a,bOpen in a separate windowScheme 5Proposed reaction mechanism.In conclusion, a sustainable electrocatalytic C–H chalcogenation of o-carboranes with thiols and selenols was realized at room temperature by earth abundant copper catalysis. The C–H activation was characterized by mild reaction conditions and high functional group tolerance, leading to the facile assembly of various o-carboranes. Thereby, a transformative platform for the design of cage C–S and C–Se o-carboranes was established that avoids chemical oxidants by environmentally-sound electricity in the absence of directing groups. A plausible mechanism of paired electrolysis was established by detailed mechanistic studies.