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).     

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.     

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.     

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.     

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.     

Correction: Influence of the primary and secondary coordination spheres on nitric oxide adsorption and reactivity in cobalt(ii)–triazolate frameworks

Correction for ‘Influence of the primary and secondary coordination spheres on nitric oxide adsorption and reactivity in cobalt(ii)–triazolate frameworks’ by Julia Oktawiec et al., Chem. Sci., 2021, DOI: 10.1039/d1sc03994f.

The authors regret that incorrect details were given for ref. 35, 37 and 59 in the original article. The correct versions of ref. 35, 37 and 59 are given below as ref. 1, 2 and 3, respectively.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.     

Assembly of multicyclic isoquinoline scaffolds from pyridines: formal total synthesis of fredericamycin A

The construction of an isoquinoline skeleton typically starts with benzene derivatives as substrates with the assistance of acids or transition metals. Disclosed here is a concise approach to prepare isoquinoline analogues by starting with pyridines to react with β-ethoxy α,β-unsaturated carbonyl compounds under basic conditions. Multiple substitution patterns and a relatively large number of functional groups (including those sensitive to acidic conditions) can be tolerated in our method. In particular, our protocol allows for efficient access to tricyclic isoquinolines found in hundreds of natural products with interesting bioactivities. The efficiency and operational simplicity of introducing structural complexity into the isoquinoline frameworks can likely enable the collective synthesis of a large set of natural products. Here we show that fredericamycin A could be obtained via a short route by using our isoquinoline synthesis as a key step.

A concise approach for rapid assembly of multicyclic isoquinoline scaffolds from pyridines and β-ethoxy α,β-unsaturated carbonyl compounds was developed, which enabled the formal total synthesis of fredericamycin A.

Isoquinolines and their derivatives are common structural motifs in numerous natural products. Among them, the analogues of isoquinolines fused with rings from the benzene side such as 8-hydroxyisoquinolin-1[2H]-one (Fig. 1a) have been found in hundreds of natural products with interesting bioactivities.¹ For example, fredericamycin A and the related family members, isolated from Streptomyces griseus, show both antimicrobial and anti-tumor activities.² Ericamycin is a natural product isolated in the culture of Streptomyces varius n. sp. with anti-staphylococcal activities.³ Due to the widespread presence of isoquinolines in both natural and synthetic molecules, numerous approaches have been developed to assemble this class of scaffolds.⁴ The dominated strategies reported to date focus on forming the new pyridine ring of isoquinolines (Fig. 1b, left part). Classic methods include Bischler–Napieralski isoquinoline synthesis,^4a,b Pictet–Gams isoquinoline synthesis,^4a and Pomeranz–Fritsch reaction.^4a These reactions, proven to be useful since as early as 1893,⁵ have their own merits and limitations. For instance, high reaction temperature (e.g. reflux in toluene) and strong acids are typically required and thus functional group tolerance can become challenging. On the other side, the introduction of structural complexities and substitution patterns is constrained as the substrates have to be pre-settled to favor the formation of pyridine moieties. Here we report a new approach to prepare isoquinoline scaffolds by constructing a new benzene ring (Fig. 1b, right part).⁶ Our method starts with pyridine derivatives as the substrates to react with readily available β-ethoxy α,β-unsaturated carbonyl compounds. The reaction cascade involves five main plausible mechanistic processes (Michael addition, Dieckmann condensation, elimination, aromatization and in situ methylation) to furnish isoquinoline-based products with medium to good yields. The tricyclic isoquinoline-containing products might serve as formal common starting points for rapid total synthesis of a large number of natural products, such as those exemplified in Fig. 1a. In the present study, we demonstrate that starting from the tricyclic isoquinoline adduct 6a prepared using our method, fredericamycin A can be synthesized in 8 steps (Fig. 1c). Our strategy for isoquinoline assembly offers complementary and in certain cases better solutions not readily provided by the classic methods. We expect our method to find impressive applications in concise modular synthesis of complex natural products and molecular libraries, especially those bearing isoquinoline units fused with additional cyclic structures.Open in a separate windowFig. 1Isoquinoline analogues and their synthesis.Our design and initial studies are illustrated in Scheme 1.⁷ We first used pyridine 1a to react with α-substituted cycloenones (2a–2d), in the hope of obtaining isoquinoline 3a as the target product (Scheme 1a). The use of 2a and 2b was inspired by studies from Tamura, in which α-Br in 1,4-naphthoquinone was used as a leaving group to form an aromatic ring.⁸ Unfortunately, no product was formed and most of the starting materials were recovered. When SPh (2c) or SOPh (2d) was incorporated at the α site of the cycloenone, side products 4a and 4b were isolated respectively in moderate yields. The Michael products 4a and 4b could not be further transformed into our desired cyclic product 3a under various conditions. We then studied the use of β-substituted cycloenones (2e–2g) to react with 1a (Scheme 1b). No reactions were observed when 2e or 2f was used. To our delight, when the halogen of 2e/2f was replaced with a methoxy unit (OCH₃, substrate 2g), an encouraging amount of annulation product 3a was detected (10% yield). A side product 5a was also obtained (5% yield) in this initial study and it couldn''t be further transformed into the annulation product 3a under various alkaline conditions. It is noteworthy that, while β-alkoxy cycloenones (specifically, only β-alkoxy cyclohexenones) have been used in Staunton–Weinreb annulation⁹ to prepare fused aromatic compounds, no examples for those containing a heterocyclic aromatic ring were reported.¹⁰ Even for the construction of an aromatic ring without any heteroatom, low yields (mostly ranging from 0 to 30%) often occurred for this type of annulation starting with β-alkoxy cycloenones,⁹ which severely hampered its usage in Staunton–Weinreb annulation for the total synthesis of natural products. Our initial results showcased the possibility of direct assembly of isoquinoline scaffolds from β-methoxy cyclopentenone for the first time, though also in a low yield of 10%.Open in a separate windowScheme 1Proposed routes and initial studies for isoquinoline synthesis.With the initial results in hand, we performed additional condition optimization (11 The β-methoxy cyclopentenone 2g could also react to give 6a in a lower yield of 65% (entry 3). Other bases [such as triethylenediamine (DABCO), diazabicyclo[5.4.0]undec-7-ene (DBU), 4-dimethylaminopyridine (DMAP), lithium bis(trimethylsilyl)amide (LiHMDS) and potassium bis(trimethylsilyl)amide (KHMDS)] gave poorer results with yields ranging from 0 to 42% (entry 4). When THF was changed to other solvents, lower yields (<41%) were obtained (entry 5). Revising the ratio of 1a to 2h from 1 : 1.5 to 1.5 : 1 delivered 6a in 39% to 54% yields (entries 6–8). Lower reaction temperature (e.g. −78 °C) could not improve the outcome of this cascade transformation, but gave 23% yield of 6a together with 16% yield of recovered starting material 2h (entry 9). Long exposure to low temperature in step 1 could also lead to a considerable amount of the undesired elimination product 5a (ca. 29% yield), which was decomposed under the following methylation conditions (step 2). No product was observed in the absence of the methoxy group in 1a as it could stabilize the transition state via the formation of a metallate complex (entry 10).Screening of conditions^a

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 (

Catalytic enantioselective synthesis of benzocyclobutenols and cyclobutanols via a sequential reduction/C–H functionalization

We report here a sequential enantioselective reduction/C–H functionalization to install contiguous stereogenic carbon centers of benzocyclobutenols and cyclobutanols. This strategy features a practical enantioselective reduction of a ketone and a diastereospecific iridium-catalyzed C–H silylation. Further transformations have been explored, including controllable regioselective ring-opening reactions. In addition, this strategy has been utilized for the synthesis of three natural products, phyllostoxin (proposed structure), grandisol and fragranol.

We report here a sequential enantioselective reduction/C–H functionalization to install contiguous stereogenic carbon centers of benzocyclobutenols and cyclobutanols.

Molecules with inherent ring strain have gained considerable interest in the synthetic community.¹ Among them, four-membered ring molecules have been recognized as powerful building blocks in organic synthesis.² Driven by ring strain releasing, the reactions of carbon–carbon bond cleavage have been extensively studied in recent years.³ Meanwhile, cyclobutane motifs represent important structural units in natural product and bioactive molecules as well (Scheme 1).⁴ Therefore, a general and robust method to constitute four-membered ring derivatives is of great value, especially in an enantiomerically pure form.⁵Open in a separate windowScheme 1Representative cyclobutane-containing bioactive molecules.[2 + 2]-Cycloaddition⁶ and the skeleton rearrangement reaction⁷ are two primary methods to prepare chiral cyclobutane derivatives. Recently, the precision modification of four-membered ring skeletons to access enantioenriched cyclobutane derivatives has attracted emerging attention. Several strategies have been developed, including allylic alkylation,⁸ α-functionalization,⁹ conjugate addition¹⁰ and C–H functionalization¹¹ of prochiral or racemic cyclobutane derivatives (Scheme 2a).¹² However, the enantioselective synthesis of chiral benzocyclobutene derivatives is still underdeveloped.¹³ Although two efficient palladium-catalyzed C–H activation strategies have been developed by Baudoin¹⁴ and Martin¹⁵ groups via similar intermediate five-membered palladacycles, no enantioenriched benzocyclobutene derivative has been prepared by employing the above two methods. In 2017, Kawabata reported an elegant example of asymmetric intermolecular α-arylation of enantioenriched amino acid derivatives to afford benzocyclobutenones with tetrasubstituted carbon via memory of chirality (Scheme 2b).¹⁶ In 2018, Zhang reported an iridium-catalyzed asymmetric hydrogenation of α-alkylidene benzocyclobutenones in good enantioselectivities (3 examples, 83–88% ee).^12c To the best of our knowledge, there is no report on enantioselective synthesis of benzocyclobutene derivatives with all-carbon quaternary centers.Open in a separate windowScheme 2Asymmetric synthesis of cyclobutanes and their derivatives. (a) Enantioselective functionalization of four-membered ring substrates. (b) Synthesis of chiral benzocyclobutenone via memory of chirality. (c) This work: sequential enantioselective reduction/C–H functionalization.In line with our continued interest in precision modification of four-membered ring skeletons,^9d,10c,12a we initiated our studies on the synthesis of chiral benzocyclobutenes via enantioselective functionalization of highly strained benzocyclobutenones. It is well known that benzocyclobutene derivatives are labile to undergo a ring-opening reaction to release their inherent ring strains.¹⁷ Therefore, it is a challenging task to modify the benzocyclobutenone and preserve the four-membered ring skeleton at the same time. We envisioned that a carbonyl group directed C–H functionalization¹⁸ of the gem-dimethyl group could furnish enantioenriched α-quaternary benzocyclobutenones (Scheme 2c). This could be viewed as an alternative approach to achieve the alkylation of benzocyclobutenone, which was otherwise directly inaccessible using enolate chemistry through the unstable anti-aromatic intermediate.¹⁹ In addition, a highly regioselective C–H activation would be required to functionalize the methyl group instead of the aryl ring. Here we report our work on sequential enantioselective reduction and intramolecular C–H silylation to provide enantioenriched benzocyclobutenols and cyclobutanols with all-carbon quaternary centers. The excellent diastereoselectivity and regioselectivity of silylation were attributed to rigid structural organization of the 4/5 fused ring. Furthermore, this strategy has been utilized to accomplish the total synthesis of natural products phyllostoxin (proposed structure), grandisol and fragranol.We commenced our studies with enantioselective reduction of readily prepared dimethylbenzocyclobutenone 1a (Scheme 3).^15,20 Surprisingly, enantioselective reduction of the carbonyl group of cyclobutanone derivatives received little attention. The first reduction of parent benzocyclobutenone was studied in 1996 by Kündig using chlorodiisopinocamphenylborane²¹ or chiral oxazaborolidines (CBS reduction),²² and only moderate enantioselectivity (44–68% ee) was obtained.²³ Although copper-catalyzed asymmetric hydrosilylation of benzocyclobutenone 1a using CuCl/(R)-BINAP gave the benzocyclobutenol ent-2a in 88% ee, optimization of ligands gave no further improvement (Scheme 3a, see Tables S1–S4^† for details).²⁴ Gladly, excellent enantioselective reduction could be achieved in 94% yield and 97% ee under Noyori''s asymmetric transfer hydrogenation conditions (Scheme 3b, conditions A, RuCl[(S,S)-Tsdpen](p-cymene)).²⁵ The product 2a showed remarkable stability and no ring-opening byproduct 2a′ was observed. The reduction of parent benzocyclobutenone was examined under conditions A, and benzocyclobutenol was obtained in 90% yield and 81% ee. Apparently, the steric influence imposed by the α-dimethyl group enhanced the enantioselectivity of the reduction. Similarly, the CBS reduction ((S)-B–Me) of benzocyclobutenone 1a gave better results compared with parent benzocyclobutenone, affording the product 2a in 86% yield and 92% ee (Scheme 3c).Open in a separate windowScheme 3Enantioselective reduction of benzocyclobutenone 1a. (a) Copper hydride reduction. (b) Ru-catalyzed asymmetric transfer hydrogenation. (c) CBS reduction.We then examined the substrate scope of the reduction reaction (26 was chosen to improve the yield and enantioselectivity. Besides, benzocyclobutenol 2g with nitro substitution could be obtained in 96% yield and 93% ee. Treatment of pyrrolidinyl substituted benzocycobutenone 1h with catalyst (S,S)-Ts-DENEB afforded desired product 2h in 49% yield and 89% ee, together with ring-opening product 2h′ (18%).Enantioselective reduction of benzocyclobutenones^a

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.     

Mapping paratopes of nanobodies using native mass spectrometry and ultraviolet photodissociation

Following immense growth and maturity of the field in the past decade, native mass spectrometry has garnered widespread adoption for the structural characterization of macromolecular complexes. Routine analysis of biotherapeutics by this technique has become commonplace to assist in the development and quality control of immunoglobulin antibodies. Concurrently, 193 nm ultraviolet photodissociation (UVPD) has been developed as a structurally sensitive ion activation technique capable of interrogating protein conformational changes. Here, UVPD was applied to probe the paratopes of nanobodies, a class of single-domain antibodies with an expansive set of applications spanning affinity reagents, molecular imaging, and biotherapeutics. Comparing UVPD sequence fragments for the free nanobodies versus nanobody·antigen complexes empowered assignment of nanobody paratopes and intermolecular salt-bridges, elevating the capabilities of UVPD as a new strategy for characterization of nanobodies.

Ultraviolet photodissociation mass spectrometry is used to probe the paratopes of nanobodies, a class of single-domain antibodies, and to determine intersubunit salt-bridges and explore the nanobody·antigen interfaces.

Antibodies must possess high specificity toward target antigens to enable recognition and activation of an immune response. Because of this specificity, antibodies or antibody fragments are being increasingly explored for deployment in diagnostic assays, vaccine design and other therapeutic applications.¹ As such, unravelling the collection of noncovalent interactions and structural features that endow antibodies with specificity is a primary goal in biomedical research and critical for development of new biotherapeutics.² Methods to decipher antibody–antigen interactions have advanced significantly in recent years, and well-established methods including X-ray crystallography, mutagenesis techniques, cryo-EM, and hydrogen–deuterium exchange mass spectrometry remain the most versatile methods.^2–5 Another new strategy, native mass spectrometry (MS) has emerged as a powerful tool for structural biology, including analysis of macromolecular complexes and antibodies.^6–10 In this technique, rapid but gentle ionization and transfer of protein complexes into the gas-phase via electrospray ionization (ESI) of aqueous solutions of near-physiological ionic strength preserves noncovalent interactions, allowing elucidation of ligand binding and stoichiometry of complexes.^6–9 Coupled to advanced methodologies including ion mobility (IMS) or tandem mass spectrometry (MS/MS), native MS has proven an innovative strategy for interrogating protein structure, revealing topology, distinguishing conformations, identifying ligand binding sites, and determining folding thermodynamics.^6–9,11,12 As one example of a premier application in biotherapeutic development, native MS has been utilized to rapidly measure stoichiometry, heterogeneity, and stability of antibody·antigen complexes.^10,13–17Detailed structural analyses by native MS often rely on controlled dissociation or disassembly of protein complexes via ion activation in MS/MS workflows.^7,8,18–21 Collision-based dissociation methods comprise the most ubiquitous ion activation techniques and are capable of dismantling protein assemblies into subunits, detaching ligands, and facilitating determination of stoichiometries.^7,8,18 However, the slow-heating process of collision induced dissociation (CID) causes protein unfolding and less effective fragmentation of the peptide backbone, limiting characterization of native protein complexes.^7,8,18 Significantly more structural information can be acquired by employing alternative ion activation techniques.^7,8,18 For example, surface induced dissociation (SID) is a collision-based method that promotes disassembly of protein complexes into subunits through a single high energy collision that minimizes protein unfolding and enables robust characterization of native protein assemblies.^20,21 SID has proven especially versatile for the analysis of quaternary structure, as protein complexes disassemble to produce subcomplexes and subunits that reflect the native architecture.^20,21 Most impressive, the outcome of SID correlates with the magnitude of protein interfacial area (cleavage of the weakest protein:protein interfaces) and has been integrated into computational modelling workflows to enhance the accuracy of protein assembly predictions, emphasizing the value of native MS/MS for structural interrogation.^22–24 Alternatively, electron-based dissociation methods result in cleavage of the protein backbone to produce sequence fragments that are enhanced at surface exposed or flexible protein regions, informing topology (through the preservation of noncovalent interactions that prevent separation of reaction products) and degree of protein disorder.^7,8,19,25Also a method sensitive to protein structure, ultraviolet photodissociation (UVPD) is a photon-based ion activation method that has demonstrated exceptional use for structural biology. In particular, propensities of polypeptide backbone cleavages induced by 193 nm UVPD correlate with backbone flexibility and arrangement of non-covalent interactions, an outcome related to the likelihood of separation (and detection) of nascent product ions after a backbone cleavage event. Product ions enmeshed by stabilizing non-covalent interactions are less likely to separate (i.e. UVPnoD), causing an apparent suppression of backbone fragmentation. This correlation empowers characterization of conformational changes induced by point mutations, ligand binding, and protein complexation by UVPD.^26–33 Additionally, the high sequence coverage and rapid timescale of photodissociation enable detailed analyses of protein gas-phase structure, even informing proton sequestration with single residue resolution.³⁴ As native MS and UVPD increasingly gain broader utility for new protein applications, development and establishment of strategies to routinely study protein structure become imperative to cement these methodologies as cornerstones in the fields of structural biology and biotechnology that encompass development of new therapeutics, imaging agents, diagnostics and drug delivery agents.One fascinating new class of biotherapeutics are nanobodies, single domain antibodies derived from the variable domain of functional heavy chain antibodies found in camelids and certain shark species.^35,36 In contrast to conventional ∼150 kDa heterotetrameric immunoglobulin (IgG) antibodies, nanobodies feature a single peptide chain and overall reduced size of ∼15 kDa. Nanobodies offer high stability, solubility, affinity, and specificity, features that have propelled these single domain antibodies as a valuable alternative biotechnology.^37–42 Concurrently, these same features facilitate native MS analysis, for which decreased size, increased solubility and antigen affinity are favorable for rapid and routine analysis of nanobody complexes, circumventing analytical challenges and tedious sample preparations, such as proteolysis and deglycosylation steps,^14,17,43 often required for native MS of typical IgG·antigen complexes. One recent native MS study mapped the location of the epitope of influenza A hemagglutinin (HA1) bound to an antibody based on a decrease in backbone cleavages of HA1 when bound in the Ab·2HA1 complex relative to the free HA1 antigen during UVPD-MS analysis.³¹ This innovative approach motivated our interest in exploring an intriguing inverse strategy to map paratopes of nanobodies. In the present work, native MS and 193 nm UVPD are showcased as a valuable combination for determination of intersubunit salt-bridges and nanobody·antigen interfaces, ultimately localizing nanobody paratopes.Three nanobodies^44–46 with distinct proteinaceous antigens of green fluorescent protein (GFP), ribonuclease A (RNAseA), and porcine pancreatic amylase (PPA), referred to here as Gnb,⁴⁴ Rnb,⁴⁵ and Anb,⁴⁶ respectively, were targeted to evaluate native MS and UVPD for characterization of nanobody·antigen complexes. Each of these nanobodies interact with the antigen via differing contributions of the complementarity-determining regions (CDR) 1–3 and framework residues to the protein interface, which vary in surface area from 554 Ų to 683 Ų to 1062 Ų for Rnb, Gnb, and Anb, respectively. These three pairs of nanobodies and respective antigens were first ionized individually using native conditions (Fig. S1^†), and as nanobody·antigen complexes (Fig. 1). In each case, native MS produced the expected 1 : 1 complex in the full mass spectrum (MS1), in accordance with known crystal structures, in a range of charge states. A single charge state of the antigen-bound (bound state) nanobody was isolated and subjected to 193 nm UVPD (Fig. 1B, D and F), resulting in disassembly to release the free nanobody and antigen as well as sequence fragments from backbone cleavage of each protein, the latter of which will be the focus of UVPD analysis. UVPD mass spectra of each free nanobody are shown in Fig. S2.^† The corresponding backbone cleavage maps for the free nanobodies and corresponding nanobody·antigen complexes are shown in Fig. S3.^†Open in a separate windowFig. 1Native MS and UVPD of nanobody·antigen complexes. (A) MS1 of Gnb·GFP complex and (B) UVPD of the 13+ charge state. (C) MS1 of Rnb·RNAseA complex and (D) UVPD of the 11+ charge state. (E) MS1 of Anb·PPA complex and (F) UVPD of the 15+ charge state. Insets display expanded views of m/z regions populated by sequence fragments of low relative abundance. For UVPD, 1 laser pulse at 3 mJ was applied.Backbone cleavages induced by UVPD have been shown to be favored at protein regions with higher flexibility, typically ones less stabilized by networks of non-covalent interactions which might prevent separation and release of fragment ions.^26–31,47 Accordingly, comparing abundances of fragment ions produced by free and bound nanobodies should reveal regions in which backbone fragmentation is suppressed or enhanced, thus uncovering those residues involved in stabilizing interactions with the antigen and effectively localizing the paratope. Ten types of fragment ions (a, a + 1, b, x, x + 1, y, y − 1, y − 2, and z, where +1, −1, and +2 indicate the gain or loss of hydrogen atoms) commonly generated by UVPD were monitored across the three nanobodies in both the free and bound states. Among the collection of ions detected, a- and x-type ions are the most prevalent and dominant for the nanobodies and complexes, thus providing the greatest sequence coverage. Those fragment ions displaying statistically significant differences in abundances (p < 0.05, n = 5) upon complexation of the nanobody to the antigen are shown in Fig. S4–S6^† and mapped onto the backbone position cleaved to generate the fragment ions. The high diversity of fragment types characteristic of UVPD originates from competing pathways: direct dissociation from excited electronic states yields a/a + 1/x/x + 1 ions, and internal conversion to the ground state following intramolecular vibrational energy redistribution (IVR) produces b/y fragments.^32,33 IVR processes may preferentially sever weak non-covalent interactions, whereas direct dissociation from excited states occurs on a faster time-scale minimizing disruption of non-covalent interactions.^32,33 Consequently, the latter dissociation pathways and respective products, a/a + 1/x/x + 1 ions, are best suited to evaluate antigen-induced changes in nanobody topology. Variations in abundances of these four fragment types tended to be greater at the interface and CDRs, and inspection of the color-coded maps in Fig. S4–S6^† reveals that in most cases the abundances of these a/x-type ions decreased for the nanobody·antigen complexes relative to the free nanobodies, signifying suppression of backbone fragmentation.To underscore the impact of antigen binding on UVPD, significant changes in backbone fragmentation (ΔUVPD) based on differences in summed abundances of a/a + 1/x/x + 1 fragments for each free nanobody versus nanobody·antigen complex are shown in Fig. 2, along with color maps highlighting residues at the CDRs and protein interfaces. Generally, apparent suppression of backbone fragmentation (e.g., less efficient separation of nascent fragment ions owing to stabilizing non-covalent interactions) is the greatest at or adjacent to the interface residues, an outcome which is especially notable and consistent for patches of residues near CDR3 in Gnb (Fig. 2A), near CDR1 and CDR3 in Rnb (Fig. 2B), and near CDR2 and CDR3 in Anb (Fig. 2C). These patterns in suppression of backbone fragmentation upon antigen binding correlate with structural features of the respective crystal structures: main antigen contacts in Gnb are predominantly present on CDR3;⁴⁴ only CDR1 and CDR3 participate in antigen binding for Rnb;⁴⁵ CDR2 and CDR3 primarily mediate antigen binding for Anb.⁴⁶ Although nanobodies characteristically feature a disulfide bond spanning C23 to a paired cysteine directly N-terminal to the CDR3, cleavage of the disulfide bond via UVPD was sufficient to unlock the nanobody and allow release of sequence ions from other concomitant backbone cleavages within this region, including CDR1 and CDR2. Moreover, any statistically significant enhancement of backbone cleavages induced by antigen binding was sparse and remote from interaction sites. These infrequent increases in UVPD fragmentation for nanobody·antigen complexes relative to the free nanobody possibly indicate disruption of non-covalent interactions and increased flexibility in those limited regions of the nanobody. However, this sporadic enhancement of backbone cleavages upon antigen binding occurs in stark contrast to more prevalent suppression of backbone cleavages spanning larger swaths of neighboring residues in the nanobody·antigen complexes. Overall, the observed suppression of fragmentation at the protein·protein interface serves as a strong basis for the development of native MS-UVPD strategies to discern nanobody paratopes.Open in a separate windowFig. 2Suppression and enhancement of backbone cleavage sites based on abundances of UVPD fragment ions induced for each nanobody by antigen binding for (A) Gnb, (B) Rnb, and (C) Anb. ΔUVPD heat plots display significant differences (p < 0.05, n = 5) for the abundances of a- and x-type fragment ions between the free and bound nanobody. Blue and red indicate suppression and enhancement of fragment abundances, respectively, for the nanobody upon complexation. Positions that display no significant change are shown in grey, while white indicates small significant changes. Color maps highlighting interface residues and CDRs are also included for each nanobody. ΔUVPD values correspond to the fragment abundance per residue for the bound state minus the fragment abundance per residue for the free state. All product ions had a signal-to-noise ratio > 3.Contrary to classical IgGs where paratopes are primarily localized to CDRs,⁴⁸ nanobody paratopes feature greater diversity in terms of residue identity and position, and also include the involvement of framework residues as well as the potential absence of interactions from certain CDRs, obfuscating assignment of paratopes.⁴⁸ By leveraging the trends in the reduction of fragmentation observed in Fig. 2 for the three nanobody·antigen complexes relative to the free nanobodies, a strategy for the approximation of surface patches contributing to the paratope was developed. Because the most structurally significant changes of fragmentation related to antigen binding are demarcated by apparent suppression of backbone cleavages for stretches of neighboring residues, the UVPD data was analyzed by averaging abundances of fragment ions originating from backbone cleavages across every five residues (non-overlapping box-car average) prior to comparing fragment abundances for the free and bound states using Welch''s t-test (n = 5). A stringent significance cutoff of p < 0.001 was subsequently applied to limit false assignments of protein sections involved at the interface. Protein sections (5 residues long) displaying significant suppression of backbone cleavages (i.e. reduction in abundances of fragment ions originating from backbone cleavages in each 5 residue segment) according to this method are plotted onto the crystal structures in Fig. 3. Impressively, only protein sections adjacent to the protein·protein interfaces displayed significant suppression for Gnb and Anb. Similarly, Rnb mostly featured suppression adjacent to the interface, while only two sections remote from the interface (spanning residues 15–24 and 45–49) were also suppressed. Furthermore, a significant enhancement of fragmentation was only observed for Rnb near the N-terminus and C-terminus (Fig. S7^†), remote from the paratope. This Rnb complex featured the smallest interfacial area, which may lead to instability, compaction, or distortion during ion transmission, resulting in the unexpected suppression and enhancement at these regions. Regardless, UVPD suppression was predominant at the interfaces of each of the three nanobody complexes, presenting a new strategy for the approximation of nanobody residue patches that contribute to the paratope.Open in a separate windowFig. 3The a and x fragment ion abundances originating from backbone cleavages were averaged across every 5 residues for the free and bound forms of each nanobody. Sections displaying significant UVPD suppression upon complexation (p < 0.001, n = 5) were mapped onto the crystal structure as green spheres for (A) Gnb, (B) Rnb, and (C) Anb. Residue positions displaying significant suppression of backbone cleavages, interface residues, and CDR regions are shown for each nanobody as color maps. For comparative purposes, the CDR regions of each nanobody are demarcated on the crystal structures in Fig. S8.^†Tracking charge states of specifically a- and x-type ions produced by 193 nm UVPD also informs proton sequestration along the protein sequence,³⁴ a feature that is capitalized on here to identify the formation of inter-subunit salt-bridges. In this strategy, charge states for each detected a- or x-type ion are weighted based on intensity and plotted for each backbone cleavage position of the nanobody. For example, if both a₄²⁺ and a₄³⁺ have equal intensities, the weighted average charge state at backbone cleavage position 4 (between residues 4 and 5) would be 2.5. Because electrostatic interactions, such as salt-bridges, are highly stable in the gas-phase,^49,50 applying this method to monitor changes in proton sequestration across free and bound states promises to reveal the locations of inter-subunit salt bridges introduced by complexation, if charge partitioning during subunit ejection is due to heterolytic scission of salt-bridges as previously proposed.^26,51,52 Indeed, this is demonstrated in the analysis of the Rnb·RNAseA complex, for which one inter-chain salt bridge has been identified by X-ray crystallography between nanobody R107 and antigen E111. Charge site analysis of the a-ion series for free Rnb (6+ charge state) (Fig. 4A) displays discrete step-changes in charge states at residues R39, between residues 43–47 (suggesting protonation at R45), R68, Q111, and H128, indicating localized protons at these sites. Although coverage of the x-ion series is sparser, it nonetheless corroborates proton localization at Q111 as well as near the N-terminus in the span of residues Q2–L5. Protonation of side-chains is not unexpected for basic amino acids like R and K, but protonation of backbone heteroatoms is also possible for non-basic residues like Q at either the amide oxygen or the amide nitrogen of the peptide bond.^53,54 The step analysis reported here localizes all 6 protons of the 6+ charge state of free Rnb.Open in a separate windowFig. 4Weighted average charge of a-type and x-type fragment ions attributed to Rnb produced by UVPD of (A) free Rnb (6+ charge state) and (B) Rnb·RNAseA (11+ charge state), delineated based on the backbone cleavage site along the sequence of the nanobody.Charge site analysis of Rnb in the Rnb·RNAseA complex was not as comprehensive, but nonetheless many charge sites were elucidated (Fig. 4B). Specifically, R29, R46, and R68 remained protonated, while a shift occurred from protonation at Q111 in free RNAseA to protonation of the span of residues 108–106 in the Rnb·RNAseA complex, indicating proton sequestration at R107, a residue engaged in a salt-bridge with the antigen in solution. Charge migration observed upon antigen binding based on this charge-site analysis thus evinces formation of salt-bridges between binding partners. Similarly for Gnb·GFP, charge site analysis enabled localization of multiple charges in both the free and bound states (Fig. S9^†) including protonation at R36 on Gnb, which is involved in electrostatic interactions with E142 on the GFP antigen. Additionally, a charge was located at R58 for bound Gnb that is absent for free Gnb. Although slightly higher than canonical distance cutoff of 4 Å for salt bridges, Gnb R58 is within 4.5–5.5 Å of the E172 and D173 side-chains of GFP according to the crystal structure. Salt-bridge formation spanning this distance may be possible in the gas-phase, according to charge site analysis derived from the UVPD data. The companion residues, E45 and E104, of the nanobody are also engaged in putative interchain salt-bridges when bound to GFP based on the X-ray structure, however, localization of deprotonation sites of acidic residues is not possible in the positive ion mode.For Anb, coverage of a- and x-type fragments from the Anb·PPA complex was less comprehensive and precluded detailed charge site analyses (Fig. S10^†). Regardless, data for Gnb and Rnb demonstrate that basic residues engaged in inter-subunit salt-bridges maintain a proton upon disassembly of the nanobody·antigen complexes, highlighting an exceptional use of MS/MS to localize gas-phase salt-bridges between protein subunits. However, it is also noted that these changes in protonation sites may be caused by reasons other than those postulated here. Specifically, it is possible that proton migration is caused by vibrational redistribution of deposited energy from photoabsorption, leading to proton mobilization,^53,54 and is not due to heterolytic scission of salt bridges. These results are nonetheless intriguing and may guide future interpretations of charge partitioning during UVPD of protein complexes.Following incredible advances in native MS, routine and rapid analysis of biotherapeutics for quality control and drug development has become commonplace.^13–17 At the same time, UVPD has been propelled over the past decade as a premier ion activation method capable of uncovering structural features of proteins that are not revealed by other MS/MS methods.⁴⁷ As shown in this study, we extended the combination of native MS and UVPD to characterize nanobody·antigen complexes, particularly aiming to showcase UVPD for mapping the binding interface. The pattern of fragment ions generated by UVPD for nanobody complexes resulted in the discernment of inter-chain salt-bridges. Additionally, tracking trends in suppression of fragmentation enabled the localization of nanobody paratopes using only micromolar quantities of nanobodies and circumventing some of the limitations of traditional structural biology methods such as NMR and X-ray crystallography. We anticipate that further improvements in data analysis/informatics methods will further extend the level of structural detail gleaned from the very dense UVPD mass spectra generated for large macromolecular assemblies akin to nanobody·antigen complexes.     

Correction: Mass spectrometric detection of fleeting neutral intermediates generated in electrochemical reactions

Correction for ‘Mass spectrometric detection of fleeting neutral intermediates generated in electrochemical reactions’ by Jilin Liu et al., Chem. Sci., 2021, 12, 9494–9499, DOI: 10.1039/D1SC01385H.

The authors regret that the details for ref. 15 and 17 were inadvertently swapped in the original article. The correct versions of ref. 15 and 17 are given below as ref. 1 and 2, respectively.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.     

HydroFlipper membrane tension probes: imaging membrane hydration and mechanical compression simultaneously in living cells

HydroFlippers are introduced as the first fluorescent membrane tension probes that report simultaneously on membrane compression and hydration. The probe design is centered around a sensing cycle that couples the mechanical planarization of twisted push–pull fluorophores with the dynamic covalent hydration of their exocyclic acceptor. In FLIM images of living cells, tension-induced deplanarization is reported as a decrease in fluorescence lifetime of the dehydrated mechanophore. Membrane hydration is reported as the ratio of the photon counts associated to the hydrated and dehydrated mechanophores in reconvoluted lifetime frequency histograms. Trends for tension-induced decompression and hydration of cellular membranes of interest (MOIs) covering plasma membrane, lysosomes, mitochondria, ER, and Golgi are found not to be the same. Tension-induced changes in mechanical compression are rather independent of the nature of the MOI, while the responsiveness to changes in hydration are highly dependent on the intrinsic order of the MOI. These results confirm the mechanical planarization of push–pull probes in the ground state as most robust mechanism to routinely image membrane tension in living cells, while the availability of simultaneous information on membrane hydration will open new perspectives in mechanobiology.

HydroFlippers respond to membrane compression and hydration in the same fluorescence lifetime imaging microscopy histogram: the responses do not correlate.

The detection and study of membrane mechanics in living cells is a topic of current concern.^1–14 To enable this research, appropriate chemistry tools, that is small-molecule fluorescent probes that allow imaging of membrane tension, are needed.¹⁵ With the direct imaging of physical forces being intrinsically impossible, design strategies toward such probes have to focus on the suprastructural changes caused by changes in membrane tension.¹⁵ These suprastructural changes are divers, often interconnected, and vary with the composition of the membrane.^15–25 Beyond the fundamental lipid compression and decompression, they include changes in membrane curvature, from rippling, buckling and budding to tubules extending from the membrane and excess lipid being ejected. Of similar importance are changes in membrane organization, particularly tension-induced phase separation and mixing, i.e. assembly and disassembly of microdomains. Consequences of these suprastructural changes include microdomain strengthening and softening and changes in membrane hydration and viscosity.^16–25The currently most developed fluorescent flipper probes have been introduced^26,27 to image membrane tension by responding to a combination of mechanical compression and microdomain assembly in equilibrium in the ground state.¹⁵ Extensive studies, including computational simulations,²⁸ have shown that flipper probes align non-invasively along the lipid tails of one leaflet and report changes in membrane order and tension as changes in fluorescent lifetimes and shifts of excitation maxima.¹⁵ Among other candidates, solvatochromic probes respond off-equilibrium in the excited state to changes in membrane hydration and have very recently been considered for the imaging of membrane tension in living cells.^29–36 So far not considered to image tension, ESIPT probes also report off equilibrium in the excited state on membrane hydration, but for different reasons.^37,38 Mechanosensitive molecular rotors respond off equilibrium in the excited state to changes in microviscosity.^{17,30,32,39–53} The same principle holds for the planarization of bent, papillon or flapping fluorophores.^54–57 The response of all possible probes to tension can further include less desired changes in positioning and partitioning between different domains, not to speak of more catastrophic probe aggregation, precipitation, disturbance of the surrounding membrane structure, and so on. Although the imaging of membrane tension is conceivable in principle with most of above approaches, the complex combination of parameters that has to be in place can thus far only be identified empirically, followed by much optimization.¹⁵The force-induced suprastructural changes are accompanied by the alteration in several unrelated physical properties of membranes. It is, for instance, well documented that membrane hydration increases with membrane disorder, from solid-ordered (S_o) to liquid-disordered (L_d) phases.^29,58 Increasing cholesterol content decreases membrane hydration in solid- and liquid-ordered membranes.⁵⁹ However, studies in model membranes also indicate that membrane hydration and membrane fluidity do not necessarily correlate.⁵⁹ The dissection of the individual parameters contributing to the response of fluorescent membrane tension probes would be important for probe design and understanding of their responses, but it remains a daunting challenge. In this study, we introduce fluorescent flipper probes that simultaneously report on mechanical membrane compression and membrane hydration at equilibrium in the ground state. Changes of both in response to changes in membrane tension and membrane composition are determined in various organelles in living cells.The dual hydration and membrane tension probes are referred to as HydroFlippers to highlight the newly added responsiveness to membrane hydration. The mechanosensing of lipid compression in bilayer membranes by flipper probes has been explored extensively.¹⁵ Fluorescent flippers²⁷ like 1 are designed as bioinspired⁶⁰ planarizable push–pull probes²⁶ (Fig. 1). They are constructed from two dithienothiophene fluorophores that are twisted out of co-planarity by repulsion of methyls and σ holes on sulfurs^61,62 next to the twistable bond. The push–pull system is constructed first from formal sulfide and sulfone redox bridges in the two twisted dithienothiophenes. These endocyclic donors and acceptors are supported by exocyclic ones, here a trifluoroketone acceptor and a triazole donor.⁶³ To assure stability, these endo- and exocyclic donors are turned off in the twisted ground state because of chalcogen bonding and repulsion, respectively.⁶²Open in a separate windowFig. 1The dual sensing cycle of HydroFlippers 1–5, made to target the indicated MOIs in living cells and responding to membrane compression by planarization and to membrane hydration by dynamic covalent ketone hydration. With indication of excitation maxima (ref. 63) and fluorescence lifetimes (this study).Mechanical planarization of the flipper probe establishes conjugation along the push–pull systems, electrons flow from endocyclic donors to acceptors, which turns on the exocyclic donors and acceptors to finalize the push–pull system.⁶² This elaborate, chalcogen-bonding cascade switch has been described elsewhere in detail, including high-level computational simulations.⁶² The planar high-energy conformer 1dp excels with red shifted excitation and increased quantum yield and lifetime compared to the twisted conformer 1dt because the less twisted Franck-Condon state favors emission through planar intramolecular charge transfer (PICT) over non-radiative decay through twisted ICT, or conical intersections.¹⁵Flipper probe 1 was considered for dual responsiveness to membrane tension and hydration because of the trifluoroketone acceptor.⁶³ Dynamic covalent hydration of 1dt yields hydrate 1ht.^64–76 Blue-shifted excitation and short lifetime of 1ht are not expected to improve much upon planarization because the hydrate is a poor acceptor and thus, the push–pull system in 1hp is weak. The dynamic covalent chemistry of the trifluoroketone acceptor has been characterized in detail in solution and in lipid bilayer membranes.⁶³To explore dual responsiveness to membrane tension in any membrane of interest (MOI) in living cells, HydroFlippers 2–5 were synthesized. While HydroFlipper 1 targets the plasma membrane (PM), HydroFlippers 2–4 were equipped with empirical targeting motifs.⁷⁷ HydroFlipper 5 terminates with a chloroalkane to react with the self-labeling HaloTag protein, which can be expressed in essentially any MOI.⁷⁸ Their substantial multistep synthesis was realized by adapting reported procedures (Schemes S1–S4^†).The MOIs labeling selectivity of HydroFlippers was determined in HeLa Kyoto (HK) cells by confocal laser scanning microscopy. Co-localization experiments of flippers 1–4 with the corresponding trackers gave Pearson correlation coefficients (PCCs) >0.80 for the targeting of mitochondria, lysosomes and the endoplasmic reticulum (ER, Fig. S4–S6^†). HydroFlipper 5 was first tested with stable HGM cells, which express both HaloTag and GFP on mitochondria (referred to as 5_M).^78,79 The well-established chloroalkane penetration assay demonstrated the efficient labeling of HaloTag protein by 5 as previously reported HaloFlippers (Fig. S3^†).⁷⁸ By transient transfection, HydroFlippers 5 were also directed to lysosomes (5_L), Golgi apparatus (GA, 5_G)⁸⁰ and peroxisomes (5_P) with HaloTag and GFP expressed on their surface.⁷⁸ PCCs >0.80 for co-localization of flipper and GFP emission confirmed that MOI labeling with genetically engineered cells was as efficient as with empirical trackers (Fig. S7–S11^†).Dual imaging of membrane compression and hydration was envisioned by analysis of fluorescence lifetime imaging microscopy (FLIM) images using a triexponential model (Fig. 2).⁸¹ FLIM images of ER HydroFlipper 4 in iso-osmotic HK cells were selected to illustrate the concept (Fig. 3a). Contrary to classical flipper probes, the fluorescence decay curve of the total FLIM image (Fig. 2a, grey) showed a poor fit to a biexponential model (Fig. 2a, cyan, b). Consistent with their expected dual sensing mode, a triexponential fit was excellent (Fig. 2a, dark blue, c). Lifetimes τ_1i = 4.3 ns (†) were obtained besides background. This three-component model was then applied to every pixel of FLIM images (Fig. 3c). The resulting reconvoluted FLIM histogram revealed three clearly separated populations for τ₁ (red), τ₂ (green), and background (τ₃, blue, Fig. 2d). Maxima of these three clear peaks were at the lifetimes estimated by triexponential fit of the global decay curve, thus demonstrating the validity of the methodology at necessarily small photon counts. Irreproducible fitting would give randomly scattered data without separated peaks.Open in a separate windowFig. 2(a) Fluorescence decay curve (grey, corresponding to the total image, not to a single pixel) with biexponential (cyan) and triexponential fit (dark blue). (b, c) Residual plots for bi- (b) and triexponential fit (c). (d) Histogram with the intensities associated with the τ₁ (red), τ₂ (green), and τ₃ (blue, background) components obtained by triexponential fit of the fluorescence decay curve of each pixel of the FLIM image, fit to Gaussian function (black solid curves).Open in a separate windowFig. 3FLIM images of HK cells labelled with ER flipper 4 before (a, c) and after (b, d) hyper-osmotic shock, showing average lifetimes τ_av (a, b) and τ₁ (c, d) from triexponential reconvolution; scale bars = 10 μm. (e) Distribution of the photon counts associated with the τ₁ component of 4 in HK cells after triexponential reconvolution of FLIM images before (c, τ_1i) and after (d, τ_1h) hyper-osmotic shock, showing decreasing lifetimes for τ₁ (4d). (f) The dehydration factor dh_i defined as total integrated photon counts for τ₁ (Στ₁) divided by Στ₂ (i.e., dh_i = area Στ_1i/area Στ_2i) for 4 in strongly hydrated ER (dh_i < 2, turquoise) and 1 in weakly hydrated plasma membrane (dh_i > 6, purple) of HK Kyoto cells under iso-osmotic conditions.Dual response of HydroFlippers to changes in membrane tension^a

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)

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.     

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.     

Construction of vicinal 4°/3°-carbons via reductive Cope rearrangement

Herein reported is a strategy for constructing vicinal 4°/3° carbons via reductive Cope rearrangement. Substrates have been designed which exhibit Cope rearrangement kinetic barriers of ∼23 kcal mol⁻¹ with isoenergetic favorability (ΔG ∼ 0). These fluxional/shape-shifting molecules can be driven forward by chemoselective reduction to useful polyfunctionalized building blocks.

Herein reported is a strategy for constructing vicinal 4°/3° carbons via reductive Cope rearrangement.

Constructing sterically congested vicinal quaternary–tertiary carbons (4°/3° carbons) via Cope rearrangement is currently quite limited with only a handful of papers on the subject published over the past 40 years. This stands in stark contrast to the plethora of other methods for establishing sterically congested vicinal carbons.^1–5 Central to the challenge are kinetic and thermodynamic issues associated with the transformation. In the simplest sense, Cope rearrangements proceed in the direction that results in highest alkene substitution (Fig. 1).^6,7 To forge 4°/3° motifs by Cope rearrangement, additional driving forces must be introduced to reverse the [3,3] directionality and compensate for the energetic penalty associated with the steric and torsional strain of the targeted vicinal 4°/3° motif. With limited reports in all cases, oxy-Cope substrates (Scheme 1, eqn (1)),^8–14 divinylcyclopropanes (Scheme 1, eqn (2)),^15–20 and vinylidenecyclopropane-based 1,5-dienes²¹ (Scheme 1, eqn (3)) have demonstrated favourability for constructing vicinal 4°/3° carbons. Malachowski et al. put forth a series of studies on the construction of quaternary centers via Cope rearrangement driven forward by a conjugation event (Scheme 1, eqn (4)).^22–25 In their work, a single example related to the construction of vicinal 4°/3° centers was disclosed, though kinetic (180 °C) and thermodynamic (equilibrium mixtures) challenges are also observed.²³ And of particular relevance to this work, Wigfield et al. demonstrated that 3,3-dicyano-1,5-dienes with the potential to generate vicinal 4°/3° carbons instead react via an ionic mechanism yielding the less congested products (Scheme 1, eqn (5)).²⁶Open in a separate windowFig. 1Cope equilibrium of 1,1,6-trisubstituted 1,5-dienes.Open in a separate windowScheme 1(A) Cope rearrangements for constructing vicinal 4°/3°-centers (B) this report.Our group has been examining strategies to decrease kinetic barriers and increase the thermodynamic favourability of 3,3-dicyano-1,5-diene-based Cope substrates.^27–31 Beyond the simplest, unsubstituted variants, this class of 1,5-diene is not particularly reactive in both a kinetic and thermodynamic sense (e.g.Scheme 1, eqn (5)).^26,32 Reactivity issues aside, these substrates are attractive building blocks for two main reasons: (1) they have straightforward accessibility from alkylidenemalononitriles and allylic electrophiles by deconjugative allylic alkylation.³³ (2) The 1,5-diene termini are substantially different (malononitrile vs. simple alkene) thus allowing for orthogonal functional group interconversion facilitating target and analogue synthesis.³⁴ Herein we report that a combination of 1,5-diene structural engineering^28,31 and reductive conditions (the reductive Cope rearrangement^29,30) can result in the synthesis of building blocks containing vicinal gem-dimethyl 4°/3° carbons along with orthogonal malononitrile and styrene functional groups for interconversion (Scheme 1B). On this line, malononitrile can be directly converted to amides³⁴ yielding functionally dense β-gem-dimethylamides, important pharmaceutical scaffolds.³⁵This project began during the Covid-19 pandemic lockdown (ca. March–May 2020). As such, we were not permitted to use our laboratory out of an abundance of caution. We took this opportunity to first computationally investigate a Cope rearrangement that could result in vicinal 4°/3° carbons (Scheme 2). Then, when permitted to safely return to the lab, we would experimentally validate our findings (vide infra). From our previous work, it is known that by adding either a 4-aromatic group²⁸ or a 4-methyl group³¹ to a 3,3-dicyano-1,5-diene, low barrier (rt – 80 °C) diastereoselective Cope rearrangements can occur. Notably, the 4-substituent was found to destabilize the starting material (weaken the C3–C4 bond, conformationally bias the substrate for [3,3]), and stabilize the product side of the equilibrium via resonance (phenyl group) or hyperconjugation (methyl group). In this study, we modelled substrates 1, 3, and 5 that have variable 4-substitution and would result in vicinal gem-dimethyl- and phenyl-containing 4°/3° carbons upon Cope rearrangement to 2, 4, or 6, respectively. We chose to target this motif due to likely synthetic accessibility from simple starting materials but also because of the important and profound impact that gem-dimethyl groups impart on pharmaceuticals.³⁵ Substrate 1 lacking 4-substitution had an extremely unfavourable kinetic and thermodynamic profile (ΔG^‡ = 31.6; ΔG = +5.3 kcal mol⁻¹). When a 4-methyl group was added, the kinetic barrier (ΔG^‡) dropped appreciably to 28.2 kcal mol; however, the thermodynamics were still quite endergonic (ΔG = +4.4 kcal mol⁻¹). Most excitingly, it was uncovered that the 4-phenyl group dramatically impacted the kinetics and thermodynamics: the [3,3] has a barrier of 22.9 kcal mol⁻¹ (ΔG^‡) and is ∼isoenergetic (ΔG = +0.17 kcal mol⁻¹). Thus, the reaction appears to be fluxional/shape-shifting at room temperature.^36–40 For this substrate, we also modelled the dissociative pathway (Scheme 2D). It was found that bond breakage to two allylic radical intermediates is a higher energy process than the concerted transition state (Scheme 2Cvs.Scheme 2D). Specifically, the dissociative pathway was found to be kinetically less favourable (ΔG^‡ ∼ 27.6 kcal mol; ΔG = 26.2 kcal mol⁻¹) than the concerted process (ΔG^‡ = 22.9 kcal mol⁻¹). While the dissociative pathway is less favourable than the concerted transformation, we surmised that the two-step process becomes accessible at elevated temperature (vide infra). Finally, the ionic pathway was calculated to be significantly higher for this substrate (see the ESI^†).Open in a separate windowScheme 2Computational analysis of 3,3-dicyano-1,5-diene that in theory could result in vicinal 4°/3° carbons. (A) 4-Unsubstituted 3,3-dicyano-1,5-diene. (B) 4-Methyl 3,3-dicyano-1,5-diene. (C) 4-Phenyl 3,3-dicyano-1,5-diene. (D) The dissociative mechanism for substrate 5 is higher than the closed transition state. (E) visualization of the kinetic- and thermodynamic differences of transformations (A–D).The class of substrate uncovered from our computational investigation could be accessed from γ,γ-dimethyl-alkylidenemalononitrile (7a) and 1,3-diarylallyl electrophiles (such as 8a) by Pd-catalyzed deconjugative allylic alkylation (Scheme 3A).³³ As such, model 1,5-diene 5a was prepared to verify the computational results. It was found that upon synthesis of 5a, an inseparable 21 : 79 mixture of 1,5-diene 5a and the 1,5-diene 6a was observed. The predicted ratio of 5a to 6a was 57 : 43 (Scheme 2C). These two results are within the error of the calculations (predicted; slightly endergonic, observed; slightly exergonic). To determine whether the transformation was progressing through the predicted concerted pathway (Scheme 2C) over the dissociative pathway (Scheme 2D), substrate 5b was prepared by an analogous deconjugative allylic alkylation reaction. Similarly, two Cope equilibrium isomers 5b and 6b are observed at room temperature in a 12 : 88 ratio. Upon heating at 100 °C for 3 h, the 1,5-dienes “scramble” (e.g. iso-6b is observed; 0.2 : 1.0 : 1.5 ratio of 5b : 6b : iso-6b) indicating that the dissociative pathway is only accessible at elevated temperature. This is all in good agreement with the calculated kinetics and thermodynamics of this system (Scheme 2).Open in a separate windowScheme 3(A) Observation of fluxional [3,3] and confirmation of calculated predictions. (B) Optimization of a reductive Cope rearrangement protocol for constructing vicinal 4°/3° centers. (C) The Pd-catalyzed deconjugative allylic alkylation must be regioselective.With respect to the synthetic methodology, we aimed to increase the overall efficiency and applicability of the sequence (Scheme 3B). Specifically, we wanted to avoid [3,3] equilibrium mixtures and sensitive/unstable substates and intermediates. It was found that the direct coupling of 7a with diphenylallyl alcohol 9a could take place in the presence of DMAP, Ac₂O, and Pd(PPh₃)₄. When the coupling was complete, methanol and NaBH₄ were added to drive the Cope equilibrium forward, yielding the reduced Cope rearrangement product 10a in 76% isolated yield. In terms of practicality and efficiency, this method utilizes diphenylallyl alcohols, which are more stable and synthetically accessible than their respective acetates, and the [3,3] equilibrium mixture can be directly converted dynamically to a single reduced product.With an efficient protocol in hand for constructing malononitrile–styrene-tethered building blocks featuring central vicinal 4°/3° carbons, we next examined the scope of the transformation (Scheme 4). We chose diarylallyl alcohols with the propensity to react regioselectively via an electronic bias (Scheme 3C).^41,42 The combination of p-nitrophenyl and phenyl (10b) or p-methoxyphenyl (10c) yielded regioselective outcomes with the electron-deficient arene at the allylic position. This is consistent with the expected regiochemical outcome where the nucleophile reacts preferentially at the α-position and the electrophile reacts at the allylic position bearing the donor-arene (Scheme 3C).^41,42 Then, reductive Cope rearrangement occurs to position the electron-deficient arene adjacent to the gem-dimethyl quaternary center. This is an exciting outcome as many pharmaceutically relevant (hetero)arenes are electron deficient. Thus, fluorinated arenes were installed at the allylic position of products 10d–10k. While the phenyl group resulted in poor regioselectivity (1 : 1–3 : 1), the p-methoxyphenyl group enhanced the regiomeric ratios in all cases (3 : 1–15 : 1). The degree of selectivity is correlated with the number and position of fluorine atoms. N-Heterocycles could be incorporated with excellent regioselectivity, generally speaking (10l–10q). For example, 3-chloro-4-pyridyl (10l/10m) groups were installed at the allylic position with >20 : 1 rr. 4-Chloro-3-pyridyl was poorly regioselective (10n), but the combination of 4-trifluomethyl-3-pyridyl/p-methoxyphenyl (10o) gave good regioselectivity of 11 : 1. 2-Pyridyl/p-methoxyphenyl (10q) was also a regioselective combination. We also examined a few other heterocycles including quinoline (10s) and thiazole (10t and 10u) with excellent and modest regioselectivity observed, respectively. As a general trend, when the arenes on the allylic electrophile become less polarized, poor regioselectivity is observed in the Pd-catalyzed allylic alkylation. For example, the combination of p-chlorophenyl and p-methoxyphenyl (10v) or phenyl (10w) yields regioisomeric mixtures of products. This can be circumvented by utilizing symmetric electrophiles (to 10x).Open in a separate windowScheme 4Scope of the 4°/3°-center-generating reductive Cope rearrangement.The phenyl or the p-methoxyphenyl group is necessary to achieve the 4°/3° carbon-generating Cope rearrangement: it functions as an “activator” by lowering the kinetic barrier and increasing thermodynamic favourability. These activating groups can be removed through alkene C C cleavage reactions (e.g. metathesis (Scheme 5) and ozonolysis (Scheme 6B)). In this regard, highly substituted cycloheptenes 11 were prepared by allylation and metathesis (Scheme 4).^28,43 The yields were modest to excellent over this two-step sequence. In many cases, where 10 exists as a mixture of regioisomers, the major allylation/RCM products 11 could be chromatographically separated from their minor constituents. As shown in Scheme 6A, the malononitrile can be transformed via oxidative amidation³⁴ to products 12 containing a dense array of pharmaceutically relevant functionalities (amides, gem-dimethyl, fluoroaromatics, and heteroaromatics). Following this transformation, ozonolysis terminated with a NaBH₄ quench installs an alcohol moiety on small molecule 13a.Open in a separate windowScheme 5Removal of the “activating group” by ring-closing metathesis.Open in a separate windowScheme 6(A) oxidative amidation of malononitrile. (B) Removal of “activating group” by ozonolysis.These first computational and experimental studies utilizing 3,3-dicyano-1,5-dienes as substrates for constructing vicinal 4°/3° centers sets the stage for much further examination and application. For example, while we focused our efforts on gem-dimethyl-based quaternary carbons, it is likely that other functionality can be installed at this position. For example, while unoptimized, it appears the protocol is reasonably effective at incorporating a piperidine moiety in addition to heteroarenes from the allylic electrophile (7b + 9f → 14a; Scheme 7A). Similar functional group interconversion chemistry as described in Schemes 5 and ​and66 can thus yield functionally dense building blocks 15 and 16 in good yields.Open in a separate windowScheme 7(A) The construction of 4/3° centres on piperidines. (B) Promoting endergonic [3,3] rearrangements is possible, assuming the [3,3] kinetic barrier is sufficiently low.While the 4,6-diaryl-3,3-dicyano-1,5-dienes offered the most attractive energetic profile (low kinetic barrier, isoenergetic [3,3] equillibrium; Scheme 2C), the 4-methyl analogue is also intriguing to consider as a viable substrate class for reductive Cope rearrangement (Scheme 2B). The challenge here is that the kinetics and thermodynamics are quite unfavourable (not observable by NMR), but potentially not prohibitively so. It is extremely exciting to find that Cope equilibria that are significantly endergonic in the desired, forward direction (e.g.3a to 4a) can be promoted by a related reductive protocol (Scheme 7B). While unoptimized, we were able to isolate product 17 in xx% yield by heating at 90 °C in the presence of Hantzsch ester in DMF.