A novel chromogenic and fluorogenic scaffold for detection of oxidative radicals

Radicals have profound biorelevance and their robust detections are warranted. We have devised a novel “covalent-assembly” type scaffold for sensitive detection of oxidative radicals.     

Protein nanopatterns by oxime bond formation

Patterning proteins on the nanoscale is important for applications in biology and medicine. As feature sizes are reduced, it is critical that immobilization strategies provide site-specific attachment of the biomolecules. In this study, oxime chemistry was exploited to conjugate proteins onto nanometer-sized features. Poly(Boc-aminooxy tetra(ethylene glycol) methacrylate) was synthesized by free radical polymerization. The polymer was patterned onto silicon wafers using an electron beam writer. Trifluoroacetic acid removal of the Boc groups provided the desired aminooxy functionality. In this manner, patterns of concentric squares and contiguous bowtie shapes were fabricated with 150-170-nm wide features. Ubiquitin modified at the N-terminus with an α-ketoamide group and N(ε)-levulinyl lysine-modified bovine serum albumin were subsequently conjugated to the polymer nanopatterns. Protein immobilization was confirmed by fluorescence microscopy. Control studies on protected surfaces and using proteins presaturated with O-methoxyamine indicated that attachment occurred via oxime bond formation.     

An autocatalytic chromogenic and fluorogenic photochemical reaction controlled by nucleic acids

The autocatalytic photochemical reaction, which is potentially controlled by any selected nucleic acid, is highly sequence specific and not inhibited by its products, was developed. This reaction generates colored and fluorescent products, which can be monitored by the naked eye.     

The Beckmann fragmentation of quadricyclanone oxime

The attempted O-tosylation of tetracyclo[3.2.0.0^2,7.0^4,6]heptan-3-one (quadricyclanone) oxime with p-toluenesulfonyl chloride in dichloromethane in the presence of triethylamine/DMAP or pyridine resulted in the Beckmann fragmentation to give a mixture of 4-exo-/4-endo-tosyloxy- and 4-exo-/4-endo-chlorobicyclo[3.1.0]hex-2-ene-6-endo-carbonitriles in 90% overall yield. Solvolysis of all four products in 2,2,2-trifluoroethanol afforded the corresponding 4-exo-trifluoroethoxy derivative as the sole product. Quadricyclanone itself undergoes the fragmentation reaction with hydroxylamine-O-sulfonic acid, selectively affording the 4-exo-hydroxy-6-endo-nitrile in 90% isolated yield.     

Hydrazine-selective chromogenic and fluorogenic probe based on levulinated coumarin

Chemosignaling of hydrazine by selective deprotection of levulinated coumarin was investigated. In the presence of hydrazine, levulinated coumarin was selectively deprotected, resulting in chromogenic and fluorescent turn-on type signaling. The selective naked-eye detectable signaling of hydrazine was possible in the presence of representative metal ions and common anions in an aqueous environment.     

A fluorescein-based fluorogenic and chromogenic chemodosimeter for the sensitive detection of sulfide anion in aqueous solution

A highly sensitive chromo- and fluorogenic chemodosimeter for sulfide anion was developed based on its nucleophilicity. 2,4-Dinitrobenzenesulfonyl-fluorescein (I) is a weakly fluorescent compound. Upon mixing with sulfide anion in aqueous acetone solution, the 2,4-dinitrobenzenesulfonyl group of I was efficiently removed and highly fluorescent fluorescein was released, hence leading to the dramatic increases in both fluorescence and absorbance of the reaction solution. The fluorescence increment is linear with sulfide anion concentration in the range 50-1000 nmol L⁻¹ with a detection limit of 4.3 nmol L⁻¹ (3σ). The proposed chemodosimeter showed excellent selectivity toward sulfide anion and was successfully applied to the determination of sulfide anion in synthetic wastewater samples.     

Single chemosensor for multiple analytes: chromogenic and fluorogenic detection for fluoride anions and copper ions

A o-hydroxybenzoyl-(N-butyl-4,6-naphthamimide) hydrazone (1) has been synthesized. Compound 1 displayed high selectivity for F^? and Cu²⁺ with UV/vis absorption and fluorescence spectra, respectively. In the presence of F^?, while the absorption peak of 1 showed the red shift, its fluorescent intensity decreased due to deprotonation interaction. In the presence of Cu²⁺, both the absorption and fluorescence peaks decreased because of the coordination interaction.     

Continuous kinetic assay of arylsulfatases with new chromogenic and fluorogenic substrates

Arylsulfatases are determined, evenin weakly acidic solution, by a direct and continuous kinetic method using new coumarin-derived sulfates as substrates. After enzymatic hydrolysis, the substrate dissociates to form intensely colored and strongly fluorescent phenolates, with absorption maxima ranging from 383 to 497 nm, and fluorescence emission maxima between 470 and 577 nm. Rates of enzymatic hydrolysis, optimum pH values and detection limits of arylsulfatase from Aerobacter aerogenes and Patella vulgata are determined.     

Highly enantioselective one-pot synthesis of spirocyclopentaneoxindoles containing the oxime group by organocatalyzed Michael addition/ISOC/fragmentation sequence

A highly diastereo- and enantioselective organocatalytic protocol for the synthesis of biologically important spirocyclopentaneoxindoles containing the oxime functional group from easily accessible 3-allyl-substituted oxindoles and nitroolefins has been developed by a one-pot Michael addition/ISOC/fragmentation sequence.     

Bis-pyrene carbocations for chromogenic and fluorogenic dual-detection of fluoride anion in situ

We have designed and synthesized a new carbocation precursor (carbinol) based on bis-pyrene derivative. The carbinol can be transformed to stable carbocations with quenched fluorescence and long wavelength absorption. The stable carbocations can be further used as fluoride anion detection in acidic environment, which show both chromogenic and fluorogenic responses in the presence of fluoride anion. UV–vis and fluorescence spectroscopy were utilized to monitor the changes during detection process. The responsive mechanism was studied by quantum chemical calculations and the results indicate that the carbocation mediated formation of C–F covalent bond leads to this dual responsive phenomenon. The bis-pyrene carbocations and corresponding F adduct was confirmed MALDI-TOF mass spectrum. The successful elucidation of this new mechanism opens a new avenue for carbocation-based sensor or biological labeling in the future.     

Iterative oxime bond chemistry leads to protease inhibitors

[structure: see text] In the present paper, we have looked at iterative coupling as a strategy to form new druglike molecules. We have developed an iterative coupling chemistry based on oxime bond formation between hydroxyaromatic aldehyde building blocks to form linear oxime oligomers. The strategy is validated by the discovery of micromolar protease inhibitors.     

Barrierless electron transfer bond fragmentation reactions

The ultrafast N-O bond fragmentation in a series of N-methoxypyridyl radicals, formed by one-electron reduction of the corresponding N-methoxypyridiniums, has been investigated as potentially barrierless electron-transfer-initiated chemical reactions. A model for the reaction involving the electronic and geometric factors that control the shape of the potential energy surface for the reaction is described. On the basis of this model, molecular structural features appropriate for ultrafast reactivity are proposed. Femtosecond kinetic measurements on these reactions are consistent with a kinetic definition of an essentially barrierless reaction, i.e., that the lifetime of the radical is a few vibrational periods of the fragmenting bond, for the p-methoxy-N-methoxypyridyl radical.     

Highly selective chromogenic and fluorogenic probe based on benzothiazole-thiosemicarbazone Schiff base for detection of copper (II) in real samples

Copper is the third most abundant essential transition metal ion in the human body. It's responsible for important activities in many living things, but excessive intake of Cu²⁺ can lead to a range of diseases. A colorimetric and turn-off fluorescent probe (E)-2-(5-(benzothiazol-2-yl)-2-(diethylamino)-4-hydroxybenzylidene)-N-phenylhydrazine-1-carbothioamide ( ZTR ) was designed and synthesized by thiosemicarbazone Schiff base as a specific complexes site strategy to achieve highly specific Cu²⁺ detection. The fluorescence of the probe ZTR solution fell dramatically when Cu²⁺ was added, and its appearance changed from dazzling blue to nearly colorless. The simple structure and readily available fluorescent probe provide a novel approach for the quantitative detection of Cu²⁺ in the linear range from 0 to 0.12 μM, with a detection limit down to 16 nM, and with high selectivity for Cu²⁺ over 15 other metal ions. Job’s plot analysis showed that probe ZTR and Cu²⁺ formed a 1:1 coordination complex. In addition, because of its low detection limits and fast response time, the created fluorescent molecule was effectively used to study the target ions on test paper strips and in water samples.))     

A photo-induced fluorogenic reaction for the detection of catecholamine-O-sulfates

Catecholamine-O-sulfates react with 1,2-diaminoethane to give fluorescent products if exposed to u.v. irradiation (312 nm) in the presence of oxygen. The fluoresence spectra of the reaction mixtures are similar to those obtained form the fress catecholamines and diaminoethane, indicating that the reaction proceeds by photo-cleavage of the O-sulfate bond, followed by reaction of the free catecholamine withdiaminoethane and oxygen. The factors that affect the reaction are described. After a 40-min irradiation of the catecholamine-O-sulfates in 0.71 M diaminoethane solution at 7°C, the intensities compared to those generated from free catecholamines were 46% for dopamine-3-O-sulfate, 84% for dopamine-4-O-sulfate, 42% for norepinephrine-3-O-suflate, 50% for nonrepinephrine-4-O-sulfate, 64% for epinephrine-3-O-sulfate and 136% for epinephrine-4-O-sulfate.     

Selective detection and identification of phosphopeptides by dansyl MS/MS/MS fragmentation

Protein phosphorylation regulates many cellular processes and pathways, such as cell cycle progression, signal transduction cascades and gene expression. Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel approach to label selectively phospho-Ser/-Thr residues by exploiting the features of a novel linear ion trap mass spectrometer. Using dansyl labelling and MS3 fragmentation, we developed a method useful for the large-scale proteomic profiling of phosphorylation sites. The new residues in the sequence were stable and easily identifiable under general conditions for tandem mass spectrometric sequencing.     

Water induced chromogenic and fluorogenic signal modulation in a bi-fluorophore appended acyclic amino-receptor system

An acyclic amino-receptor based bi-fluorophoric signaling system 3 exhibits water-induced simultaneous dual channel chromogenic and fluorogenic signal modulation. Its micromolar solution in various organic solvents exhibits an enhancement in absorption in the presence of water in trace amounts through the water-induced delactonization of rhodamine dye, rendering a visual perception as a function of colour change. The presence of water molecules also facilitates a fluorescence resonance energy transfer (FRET) from the excited nitro-benz-oxa-diazole fluorophore to rhodamine dye of 3 and leads to an enhancement of emission up to a second order of magnitude. The failure of the reference compounds 2 and 4 to respond under similar conditions reveals the role of structural variation of the receptor on the perturbation of photophysical processes.     

Radical generation enabled by photoinduced N–O bond fragmentation

Recent advances in synthetic chemistry have seen a resurgence in the development of methods for visible light-mediated radical generation. Herein, we report the development of a photoactive ester based on a quinoline N-oxide core structure, that provides a strong oxidant in its excited state. The heteroaromatic N-oxide provides access to primary, secondary, and tertiary radical intermediates, and its application toward the development of a photochemical Minisci alkylation is reported.

Recent advances in synthetic chemistry have seen a resurgence in the development of methods for visible light-mediated radical generation.

Photoinduced radical generation has become a focal point of contemporary chemical research. Historically, the photochemical formation of radicals has been achieved via the direct irradiation of organic chromophores with high energy UV light, with notable examples including Norrish – type 1 reactions and the photochemical decomposition of azo compounds, peroxides, N-(acyloxy)-pyridones, and xanthates.¹ While a powerful tool, the propensity of organic functional groups to absorb UV light leads to undesired excitation events that ultimately result in uncontrolled, deleterious reactivity.² The use of visible light irradiation to drive reactivity offers a solution to the problems presented by UV light photochemistry, as typical organic functionalities do not absorb in the visible region of the electromagnetic spectrum. In this regard, photoredox catalysis has emerged as a powerful tool for the generation of radical intermediates, as the visible light absorbing catalysts can access high energy excited states that engage organic substrates in redox events.³ While incredibly versatile, the manipulation of substrate oxidation states can limit the scope of reactivity, as functionalities that are predisposed to oxidation (or reduction) may undergo undesired side reactions. To circumvent this limitation, redox auxiliaries are employed to alter the redox properties of the substrate and facilitate reactivity under mild reducing (or oxidizing) conditions.⁴Radical formation via photoinduced dissociation of an auxiliary, or complex, represents a complementary strategy that, in principle, should be tolerant of redox sensitive functionalities. However, despite the ubiquity of this strategy in UV light mediated reaction manifolds, the development of reagents that undergo efficient photodissociation upon visible light irradiation is limited. In 2017, Melchiorre and coworkers reported the dissociation of 4-alkyl-1,4-dihydropyridines (alkyl-DHPs) upon irradiation with 405 nm light (Fig. 1A).⁵ Study of the photoactive alkyl-DHPs revealed that they are reductants in their excited state (D⁺˙/D* = −2.0 V vs. SCE) and, as such, both the dissociative and redox properties of the excited state could be exploited for ipso-substitution of aryl nitriles and nickel catalyzed C(sp²)–C(sp³) cross-coupling reactions.Open in a separate windowFig. 1Overview of photochemical radical generation in synthetic chemistry.In 2020, Ohmiya and colleagues reported that boracene, when reacted with organolithium or Grignard reagents, forms a photoactive alkyl borate salt that liberates an equivalent of an alkyl radical upon irradiation with 440 nm light (Fig. 1B).⁶ Characterization of the boracene-based alkyl borate revealed a strongly reducing excited state (B⁺˙/B* = −2.2 V vs. SCE), and this auxiliary was demonstrated to be effective for nickel catalyzed C(sp²)–C(sp³) cross-coupling reactions with tertiary alkyl radicals.Pioneering work by Barton and coworkers in the 1980''s demonstrated that the thermal or photochemical decomposition of N-(acyloxy)-2-thiopyridones (commonly referred to as Barton esters) could efficiently generate carbon centered radicals, ultimately delivering the corresponding alkane or thioether products.⁷ However, state-of-the-art methods employing pyridine N-oxide or its derivatives as radical precursors have demonstrated limited intermolecular reactivity, as the generated radical intermediates are competitively trapped by the pyridine-based auxiliary.^8,9 To overcome these limitations, we sought to design a pyridine N-oxide based auxiliary that does not undergo undesired alkylation reactions. Inspired by Barton''s work and informed by our previous studies,^8,9 we envisioned that the fast fragmentation of N–O bonds in pyridine N-oxide derivatives could be leveraged in the design of a photocleavable activator that would deliver carbon centered radicals from readily available carboxylic acids as precursors. Importantly, the development of a photoactive ester derived from simple pyridine N-oxide core structure delivers a species that is a strong oxidant in its excited state, complementing the reductive excited states of the previously developed photoactive auxiliaries. Moreover, by using a core structure that is derived from an abundant heteroaromatic building block, the designed photoactive esters will contain highly tunable core structures, allowing for control over the photoexcitation and fragmentation events. Herein, we report the development of a photoactive ester derived from a quinoline N-oxide core structure and its application to achieve an efficient intermolecular Minisci alkylation (Fig. 1C).At the outset of our study, we established several requirements to be satisfied by potential photoactive esters (Fig. 2B): (1) the pyridine N-oxide core structure needed to be preserved, (2) the heteroaromatic core would need to have blocking substituents at the sites of alkylation to slow deleterious functionalization of the ester (i.e. 2-, 4-, and 6-substituted pyridine N-oxide derivatives), (3) the heteroaromatic N-oxide would need to maintain sufficient nucleophilicity to form the activated N-acyloxy pyridinium, (4) the heteroaromatic backbone would need to deliver a sterically accessible N-oxide functionality. Additionally, it was recognized that pyridine N-oxide derivatives bearing alkyl substituents with benzylic C–H bonds were not suitable photoactive esters, as a deleterious Boekelheide reaction occurred upon acylation of the N-oxide functionality. Ultimately, 2,4,6-triphenyl pyridine N-oxide (TPPNO), methyl 2-phenylquinoline-4-carboxylate N-oxide (PQCNO), and methyl acridine-9-carboxylate N-oxide (ACNO) were identified as potential photoactive ester precursors that met all aforementioned requirements.Open in a separate windowFig. 2(A) Comparison of pyridinium esters. (B) Overview of photoactive ester design principles. (C) Photophysical characterization of Ac-TPPNO, Ac-PQCNO, and Ac-ACNO.Initial investigations focused on the photophysical characterization of the three heteroaromatic N-oxides (Fig. 2C). Uv-vis spectroscopy in acetonitrile revealed TPPNO to have two maximum absorbances at 319 nm and 365 nm, with the latter absorbance tailing off beyond 400 nm. PQCNO and ACNO also displayed a strong absorbance feature at 365 nm, however, for ACNO two additional lower energy absorbance features at 418 nm and 444 nm were observed. Addition of acetyl chloride to TPPNO and PQCNO resulted in an increase of intensity for the absorbance features, with no apparent shift in the absorbance maxima. Acylation of the ACNO auxiliary with acetyl chloride caused the maximum at 274 nm to diverge into two distinct sharp absorbance features with maxima at 264 nm and 275 nm. Additionally, the absorbance feature at 364 nm both increased in intensity and became more structured upon acylation, revealing an apparent shoulder at 352 nm. Measurement of the fluorescence spectra for the heteroaromatic N-oxides (irradiated at 361 nm) revealed that Ac-TPPNO, Ac-PQCNO, and Ac-ACNO have emission maxima at 435 nm, 468 nm and 519 nm, respectively (Fig. 3A). In the absence of an acyl equivalent, no emission was observed for the heteroaromatic N-oxides. This finding is consistent with previous reports that pyridine N-oxides and quinoline N-oxides possess non-emissive excited states under basic conditions due to a propensity to undergo a fast rearrangement on the singlet surface.¹⁰Open in a separate windowFig. 3(A) Absorbance and emission spectra for Ac-TPPNO, Ac-PQCNO, and Ac-ACNO. Emission recorded upon irradiation at 361 nm. (B) Fluorescence spectra of Ac-PQCNO monitored over 25 successive scans (excitation 335 nm). (C) Decomposition of Ac-PQCNO upon irradiation by a 427 nm Kessil lamp, monitored by Uv-vis. (D) Proposed mechanism for photochemical decomposition of Ac-PQCNO.Investigation of the photoinduced N–O bond cleavage of Ac-PQCNO revealed that upon successive fluorescence scans (excitation at 335 nm), Ac-PQCNO is observed to decompose steadily with the concomitant appearance of the fluorescence signal corresponding to the generation of deoxygenated quinoline (Fig. 3B). Interrogation of the decomposition by Uv-vis spectroscopy revealed that after 60 s of irradiation of Ac-PQCNO with a 427 nm Kessil lamp, a decrease in the absorbance feature at 365 nm was observed. Upon extending irradiation time out to 30 min, significant degradation of the Ac-PQCNO was observed.Together, the photophysical characterization shows the photoactive esters possess conserved absorption features at 365 nm. Consistent with previous studies on quinoline N-oxide photochemistry, the absorption feature at 365 nm is thought to be π,π* in character and responsible for the observed deoxygenation reactivity.¹¹ The deoxygenation of the aromatic N-oxides is hypothesized to arise from crossing over of the π,π* excited state to a dissociative π,σ* state. Computational evidence in support of the hypothesized mechanism can be found in Fukuda and Ehara''s study on the deoxygenation of structurally related N-hydroxypyridine-2(1H)-thione.¹² Experimental support for this proposed mechanism can be found in Hata and Tanaka''s study of the gas phase photolysis of pyridine N-oxide¹³ as well as Hata''s subsequent translation of this work to the solution phase photodeoxygenation of heteroaromatic N-oxides in the presence of a strong Lewis acid, BF₃·Et₂O.¹⁴Interrogation of the thermal reactivity of Ac-PQCNO revealed there was no deoxygenation of the heteroaromatic N-oxide after heating to 90 °C for 3 hours. This demonstrates that the remarkable reactivity of Ac-PQCNO is due to dissociation of the N–O bond from a low energy photoexcited state.Electrochemical analysis of the heteroaromatic N-oxide photoactive esters using cyclic voltammetry in acetonitrile revealed the Ac-TPPNO, Ac-PQCNO, and Ac-ACNO to have low energy reduction waves, with measured E_1/2 of −0.79 V vs. SCE, − 0.45 V vs. SCE, and −0.47 V vs. SCE. Applying information gathered from absorbance and emission spectroscopy, as well as the electrochemical data, the standard potential for oxidation of Ac-TPPNO, Ac-PQCNO and Ac-ACNO in the excited state was estimated to be +2.30 V [E⁰(T⁺*/T˙)], +2.57 V [E⁰(P⁺*/P˙)], and +2.32 V [E⁰(A⁺*/A˙)] vs. SCE according to the Rehm–Weller approximation (Fig. 2C).¹⁵Due to the mild photolytic conditions for decarboxylative radical generation from N-oxides and characteristics as excited state oxidants, we sought to assess the reactivity of the photocleavable esters towards the development of an intermolecular Minisci alkylation (Fig. 4). Initial studies focused on the addition of the tert-butyl radical to 4-chloroquinoline in acetonitrile. Assessment of reactivity revealed TPPNO and PQCNO to perform identically under unoptimized conditions delivering 27% of the desired 2-tert-butyl-4-chloroquinoline product, while ACNO produced less than 5% of the desired product. Due to the low cost of the parent quinoline and its high yielding N-oxidation,¹⁶ we elected to focus our investigations on the application of PQCNO as a photoactive ester. Degassing the reaction resulted in a substantial increase in yield, providing 90% isolated yield of the desired product. Further investigation of solvents and additives did not result in increased reactivity.Open in a separate windowFig. 4Scope of intermolecular Minisci alkylation. Isolated yields unless otherwise noted. Standard conditions: substrate (0.2 mmol, 1 equiv.), PQCNO (2 equiv.), acyl chloride (2.2 equiv.), CaCl₂ (1 equiv.), MeCN (0.5 mL), N₂ atmosphere, 427 nm Kessil lamp, 30 min. ^aReaction run for 1 hour. ^bReaction run for 2 hours. ^cReaction run for 1 hour with a 1 : 1 mixture of MeCN to DCM as solvent. NMR yield with methyl tert-butyl ether as internal standard, in brackets.Assessment of the scope for the intermolecular Minisci alkylation revealed the tert-butyl radical addition was the most efficient of the simple alkyl fragments (90%, 4a), followed by isopropyl addition (50%, 4b), ethyl addition (48%, 4c), and finally methyl addition (43%, 4d) (Fig. 4). Simple carbocyclic radical fragments such as cyclohexyl (4e), cyclobutyl (4f), and cyclopropyl (4g) provided the corresponding alkylation products in 88%, 57%, and 34% yield, respectively. Interestingly, Minisci alkylation products from more complex carbocyclic radical fragments were also accessible under-developed reaction conditions as 1-phenylcyclopropyl (4h), adamantyl (4i), 4-(methoxycarbonyl)-[2.2.2]-bicycloctyl (4j), and 3-(methoxycarbonyl)-[1.1.1]-bicyclopentyl (4k) fragments gave the corresponding radical addition products in 37–80% yield. Application of tetrahydropyran-4-carbonyl chloride (4l), N-Boc azetidine 3-carbonyl chloride (4m), N-Boc piperidine 4-carbonyl chloride (4n), and N-Boc piperidine 3-carbonyl chloride (4o) provided the desired coupling products in 41–61% yield, respectively.Assessment of the heterocyclic coupling partners revealed that substituted pyridine and quinoline derivatives performed well in the Minisci alkylation, however, over-alkylation of the heteroarene was often observed (4p–4t). Notably, 4-cyanopyridine (4p) and lepidine (4s) performed well under the developed intermolecular reaction conditions; these substrates were either low yielding or inaccessible under our previously reported fragment coupling conditions.⁸ Modestly complex heteroaromatic scaffolds such as quinoxaline (4u), and 3-chloro-6-phenylpyridazine (4v) also performed well under the tert-butylation reaciton conditions. Biologically active scaffolds such as the 4-chloroquinazoline core of erlotinib (4w) and the imidazopyrazine core structure of gandotinib (4x) each provided a single regioisomer of the tert-butyl addition product in high yield. Finally, nicotine (4y) was observed to undergo tert-butyl addition in 37% yield with retention of the configuration at the benzylic stereocenter.During the exploration of the Minisci alkylation reaction, it was realized that the deoxygenated quinoline could be recovered in high yields (87% recovery). Resubjecting the recovered quinoline to oxidation conditions, PQCNO could be re-generated in 71% yield. Regenerated PQCNO was observed to show no decrease in reactivity when recycled through three consecutive reactions (see ESI^†).To probe the mechanism of the photoinduced Minisci alkylation reaction, we monitored the reaction by employing an in situ LED NMR device equipped with a 430 nm LED source. Investigation of the N–O bond fragmentation revealed that the rate of deoxygenation was not dependent on the rate of decarboxylation for the acyloxy group. Benzoyloxy and pivaloyloxy substituted N-oxides provided similar rates of deoxygenation (k_obs = −5.8 × 10⁻⁵ M s⁻¹ (BzO–) vs. k_obs = −6.3 × 10⁻⁵ M s⁻¹ (PivO–); see ESI^†), a result that is interesting given the difference in rates of decarboxylation form the respective carboxylic acids.^17,18 Alteration of the aryl substituent in the 2-position of the PQCNO auxiliary was observed to impact the rate of deoxygenation, as ortho substituted 2,6-dimethylphenyl substituent provided an increased rate of deoxygenation (k_obs = −7.9 × 10⁻⁵ M s⁻¹), whereas electron rich para-methoxyphenyl substituent led to a substantial decrease in the observed rate for deoxygenation (k_obs = −2.2 × 10⁻⁵ M s⁻¹).Monitoring the deoxygenation of Piv-PQCNO under reaction conditions revealed an increase in the observed rate of deoxygenation in the presence of 4-chloroquinoline (Fig. 5A). We hypothesize that the change in rate for deoxygenation is indicative of a propagative reaction mechanism in which electron transfer from the radical addition product (III) to acylated PQCNO (I) leads to formation of the final C–H alkylated product as well as generating a second equivalent of R˙ through a reductive decarboxylation of acylated PQCNO (I). Determination of the quantum yield (Φ) for the decomposition of Piv-PQCNO supported the proposed propagative chain mechanism, in the presence of 4-chloroquinoling Φ = 0.983 whereas in the absence of a substrate Φ = 0.218 for Piv-PQCNO decomposition. The increase in observed quantum yield is indicative of chain mechanism promoting Piv-PQCNO decomposition in the presence of a substrate. Further support was found when subjecting 2-phenylpropionyl chloride to the reaction conditions in the absence of a heteroaromatic substrate, 1-chloro-1-phenylethane was observed (see ESI^†), demonstrating the ability of acyl PQCNO to oxidize stabilized radical intermediates.Open in a separate windowFig. 5(A) Reaction profile in the presence (blue) and absence (yellow) of substrate, monitored using 430 nm LED NMR apparatus. (B) Proposed mechanism for photomediated Minisci reaction.¹⁹On the basis of these findings, we propose the following mechanism (Fig. 5B). Initiation of the Minisci alkylation occurs by the photoinduced decomposition of acyl-PQCNO (I) to generate an equivalent of a reactive radical intermediate (R˙). Addition of R˙ to an equivalent of the protonated heteroaromatic substrate provides intermediate (II). Deprotonation of (II) generates radical intermediate (III) that is in turn oxidized by a second equivalent of acyl-PQCNO (I), providing the desired C–H alkylated product while generating a second equivalent of R˙ through the decomposition of reduced acyl-PQCNO (IV), thereby propagating the reaction.Finally, we sought to explore alternate radical transformations that can be promoted by PQCNO-based esters (Scheme 1). Lactonization of 2-phenylbenzoyl chloride was carried out, providing moderate yield for the 3,4-benzocoumarin product.²⁰ By employing trifluoroacetic anhydride (TFAA) as an acyl equivalent in the presence of tert-butyl anisole, radical trifluoromethylation of the electron-rich arene was achieved in moderate yields.⁸ Further assessments of reactivity are currently ongoing within our laboratory.Open in a separate windowScheme 1Alternate radical transformations.In conclusion, we have developed a photoactive ester based upon the quinoline N-oxide core which delivers a strong oxidant in its excited state. The designed photocleavable ester enabled the development of a photochemical Minisci alkylation, providing a reaction platform that leveraged both the photochemical dissociation and the oxidizing characteristics of the photoactive esters. The photochemical reactivity of the PQCNO ester was also demonstrated to effect radical lactonization and trifluoromethylation reactions.     

Enzyme fingerprints of activity,and stereo- and enantioselectivity from fluorogenic and chromogenic substrate arrays

A series of stereochemically and structurally diverse fluorogenic and chromogenic substrates for hydrolytic enzymes has been synthesized and used to characterize enzyme activity profiles of esterases, lipases, proteases, peptidases, phosphatases, and epoxide hydrolases. The substrates used are particularly resilient to nonspecific reactions due to their mechanism of activation. The activities recorded with the individual substrates are therefore remarkably reproducible, and enable us to use the overall pattern of activity as a specific fingerprint for the enzyme sample. Fingerprints of activity, and enantio- and stereoselectivity are displayed as arrays of color-scale squares that are easily analyzed visually. Such fingerprints might be useful for quality control, enzyme discovery, and possibly for addressing the issue of functional convergence in enzymes.     

Nerve agent simulant detection by using chromogenic triaryl methane cation probes

Two triaryl methane cations have been used as probes for colorimetric detection of nerve agent simulants. Buffered mixed aqueous solutions of 1 and 2 showed bathochromic shifts in the presence of DCNP (diethylcyanophosphonate) and DCP (diethylchlorophosphate). The colour modulation can be observed to the naked eye. Appropriate mechanisms for the recognition event are proposed.