Flavin Analogues Research Articles

Tuning the Flavin Core via Donor Appendage for Selective Subcellular Bioimaging and PDT Application.

Herein, we report a novel flavin analogue as singular chemical component for lysosome bioimaging, and inherited photosensitizer capability of the flavin core was demonstrated as a promising candidate for photodynamic therapy (PDT) application. Fine-tuning the flavin core with the incorporation of methoxy naphthyl appendage provides an appropriate chemical design, thereby offering photostability, selectivity, and lysosomal colocalization, along with the aggregation-induced emissive nature, making it suitable for lysosomal bioimaging applications. Additionally, photosensitization capability of the flavin core with photostable nature of the synthesized analogue has shown remarkable capacity for generating reactive oxygen species (ROS) within cells, making it a promising candidate for photodynamic therapy (PDT) application.

Subtle chemical modification around the flavin core to create diverse morphological assembly and sensing application

Herein, an intricate interplay of the non-covalent interactions induced via subtle chemical modification around the flavin core was envisaged to generate a diverse array of morphological features. Three different flavin analogues with the covalently linked carboxylic group, a glycine or proline entity were chosen along with a simple flavin entity and were investigated for their photophysical and morphological behavior. A systematic structural variation around flavin core has resulted in the formation of irregular shaped, fibrous, tapered fibers, sphere shaped and punctured disk shaped self-assembled structures. Interestingly, the carboxylic-flavin analogue was found to differentiate between monovalent alkali versus divalent alkaline earth metal ions. Overall, the present study highlights the importance of subtle structural engineering in regulating the diverse modes for self-assembled structures, which might be playing a role in designing chemical structures for practical applications.

Bioinspired flavin analogues as organic electrode materials for supercapacitor applications

With the increasing interest in incorporating redox-active organic molecules as potential materials in energy storage systems, we envisaged a chemical design around naturally occurring redox-active flavin core structure.

Pendent supported rare coordination for flavin: Structural, photophysical and morphological characterization of flavin-silver(I) complexes

The present manuscript discusses our effort in stabilizing the rare bidentate coordinate mode for the flavin entity, via pendent supported secondary coordination; thereby resulting in a polymeric structure. Subtle chemical modification around the flavin core structure incorporating allyl and picolyl functionality in proximity to the secondary coordinate site was envisaged and two novel flavin analogues allylflavin (AF) and picolylflavin (PiF) was synthesized and solid-state X-ray diffraction studies was performed to elucidate the structural features of their silver complexes. Interestingly, two crystallographically unique silver ions was found to be characteristic feature, with different geometry around silver ion is observed depending on its involvement in coordination at primary or secondary site of the flavin unit. Furthermore, complexation behavior has been probed via photophysical techniques and morphological studies were undertaken to ascertain the facile transfer of the coordination polymeric motif onto the surface.

Inducing atypical higher order architecture for silver-flavin complex via nitrile pendent: Structural and antibacterial study

Herein, we report rare examples of higher order structural architecture for metal-flavin complexes and investigation pertaining to their antibacterial properties. Notably, while mononuclear discrete units are preferred and frequently reported for flavin-metal complexes; incorporation of a nitrile pendent at N10 position of the modified flavin analogue (nitrile flavin-NF) has resulted in rare higher order architecture for silver-flavin complexes. To the best of our knowledge, a tridentate coordination mode for flavin as observed in complex 2 has never been reported. Significant structural variation and hence the functional behaviour was observed with strongly/weakly coordination nature of the counter-anion. The antimicrobial behaviour was investigated against gram-positive and gram-negative bacteria displaying broad-spectrum antibacterial activity; with complex 1 displaying prominent antibacterial activity among the two silver-flavin complexes studied.

Substrate Electronics Dominate the Rate of Reductive Dehalogenation Promoted by the Flavin-Dependent Iodotyrosine Deiodinase.

Iodotyrosine deiodinase (IYD) is unusual in its reliance on flavin to promote reductive dehalogenation of halotyrosines under aerobic conditions. Applications of this activity can be envisioned for bioremediation, but expansion of its specificity requires an understanding of the mechanistic steps that limit the rate of turnover. Key processes capable of controlling steady-state turnover have now been evaluated and described in this study. While proton transfer is necessary for converting the electron-rich substrate into an electrophilic intermediate suitable for reduction, kinetic solvent deuterium isotope effects suggest that this process does not contribute to the overall efficiency of catalysis under neutral conditions. Similarly, reconstituting IYD with flavin analogues demonstrates that a change in reduction potential by as much as 132 mV affects kcat by less than 3-fold. Furthermore, kcat/Km does not correlate with reduction potential and indicates that electron transfer is also not rate determining. Catalytic efficiency is most sensitive to the electronic nature of its substrates. Electron-donating substituents on the ortho position of iodotyrosine stimulate catalysis and conversely electron-withdrawing substituents suppress catalysis. Effects on kcat and kcat/Km range from 22- to 100-fold and fit a linear free-energy correlation with a ρ ranging from -2.1 to -2.8 for human and bacterial IYD. These values are consistent with a rate-determining process of stabilizing the electrophilic and nonaromatic intermediate poised for reduction. Future engineering can now focus on efforts to stabilize this electrophilic intermediate over a broad series of phenolic substrates that are targeted for removal from our environment.

Enhancing the photosensitizing activity of natural flavins: Tuning the heavy-atom effect in the isoalloxazine series

Structure-photosensitizing activity relationships for a series of flavin analogues were investigated with the final goal of identifying the most potent photosensitizer in these series. The main structural modifications involved the introduction of various halogen atoms in C7- and/or C8-positions on the isoalloxazine ring. These compounds were synthesized by reacting judiciously-functionalized anilines with alloxan. The SAR trends showed that the photosensitizing activity increased with the size of the halogen atoms, confirming the importance of the heavy-atom effect on the photosensitizer’s activity. The halogens in C8 were more active than the di-substituted halogens, which in turn were more active than the C7-substituted equivalents. However, even if the photosensitizing activity is slightly less important for the 7- compared to the 8-substituted derivatives, the 7-haloisoalloxazines are promising photosensitizers, as they present a better cellular toxicity profile than the 8-substituted analoges. The photosensitizing activity perfectly correlated with the determined fluorescence for the same compounds. Except for the dihalogeno derivatives, all the compounds were not toxic up to a 50 μM range.

Flavin based supramolecular gel displaying multi-stimuli triggered sol-gel transition.

Herein, we report the design and synthesis of an amphiphilic flavin analogue as a robust low molecular weight gelator involving minimal structural modification. Four flavin analogues were evaluated for their gelation capabilities and the flavin analogue with antipodal positioning of the carboxyl and octyl functionalities was found to be the most efficient gelator with the minimum gelation concentration being as low as 0.03 M. A wide range of solvents were used for gelation studies suggesting its widespread applicability. Morphological, photophysical and rheological characterization studies were performed to fully characterize the nature of the gel. Interestingly, reversible multiple stimuli responsive sol-gel transition was observed with changing pH and redox activity, while metal screening showed specific transition in the presence of ferric ions. The gel was able to differentiate between ferric and ferrous species with well-defined sol-gel transition. The current results potentially offer a redox-active flavin-based material as a low molecular weight gelator for the development of next-generation materials.

Stabilizing hydroperoxyflavin intermediate formation via a peptide appendage: a neutral flavoenzyme model.

A bioinspired mimic for the stabilization of hydroperoxyflavin intermediate formation was designed and investigated for monooxygenase like catalytic properties. A suitable peptide appendage was covalently linked to the C7-position of the neutral isoalloxazine core to synthesize Fl-G, Fl-F, Fl-P, and Fl-βA analogues. While the presence and identity of the peptide appendage were found to be crucial for catalytic efficiency, corroborative observations were made from theoretical studies as well, supporting the precise conformational and accessibility requirements for the stabilization of the key hydroperoxyflavin intermediate. A simple yet elegant flavopeptide model (Fl-G) was found to achieve almost quantitative catalytic efficiency compared to the control flavin analogue without a peptide appendage.

Cooperative chemoenzymatic synthesis of N-heterocycles via synergizing bio- with organocatalysis.

Inspired by Nature’s ingenuity, considerable progress has been made in recent years to develop chemoenzymatic processes by the integration of environmentally friendly feature of biocatalysis with versatile reactivity of chemocatalysis. However, the current types of chemoenzymatic processes are relatively few and mostly rely on metal catalysts. Here, we report a previously unexplored cooperative chemoenzymatic system for the synthesis of N-heterocycles. Starting from alcohols and amines, benzimidazole, pyrazine, quinazoline, indole, and quinoline can be obtained in excellent yields in water with O2 as the terminal oxidant. Synthetic bridged flavin analog is served as a bifunctional organocatalyst for the regeneration of cofactor nicotinamide adenine dinucleotide in the bioprocess and oxidative cyclodehydrogenation in the chemoprocess. Compared to the classical acceptorless dehydrogenative coupling strategy, being metal and base free, requiring only water as solvent, and not needing atmosphere protection were observed for the present method, exhibiting a favorable green and sustainable alternative.

Binding of Dual-Function Hybridized Metal-Organic Capsules to Enzymes for Cascade Catalysis.

The combination of chemo- and biocatalysis for multistep syntheses provides attractive advantages in terms of evolvability, promiscuity, and sustainability striving for desirable catalytic performance. Through the encapsulation of flavin analogues by both NADH and heme mimics codecorated heteroleptic metal–organic capsules, herein, we report a progressive host–guest strategy to imitate cytochrome P450s catalysis for cascade oxidative coupling catalysis. Besides the construction of stable dual-function metal–organic capsules and the modification of cofactor-decorated capsules at the domain of enzymes, this supramolecular strategy involves multistage directional electron flow, affording reactive ferric peroxide species for inducing oxygenation. Under light irradiation, the metal–organic capsule selectively converts stilbene to oxidative coupling products (including 2-oxo-1,2-diphenylethyl formate, 2-alkoxy-1,2-diphenylethanone) in tandem with enzymatic reactions respectively, at the domain of natural enzymes. The ingenious combination of capsules and enzymes with the in situ-regenerated capsule-loaded NADH cofactor promises non-native coupling reactions by forming regional cooperation and division. This abiotic–biotic conjugated host–guest strategy is conducive to the de novo creation of multifunctional components approaching active enzymatic sites for reinforced matter and energy transporting, demonstrating a key role of multicomponent supramolecular catalysts for one-pot integrated catalytic conversions.

Broadening the Scope of the Flavin-Tag Method by Improving Flavin Incorporation and Incorporating Flavin Analogs.

Methods for facile site‐selective modifications of proteins are in high demand. We have recently shown that a flavin transferase can be used for site‐specific covalent attachment of a chromo‐ and fluorogenic flavin (FMN) to any targeted protein. Although this Flavin‐tag method resulted in efficient labeling of proteins in vitro, labelling in E. coli cells resulted in partial flavin incorporation. It was also restricted in the type of installed label with only one type of flavin, FMN, being incorporated. Here, we report on an extension of the Flavin‐tag method that addresses previous limitations. We demonstrate that co‐expression of FAD synthetase improves the flavin incorporation efficiency, allowing complete flavin‐labeling of a target protein in E. coli cells. Furthermore, we have found that various flavin derivatives and even a nicotinamide can be covalently attached to a target protein, rendering this method even more versatile and valuable.

Comparing ultrafast excited state quenching of flavin 1,N6-ethenoadenine dinucleotide and flavin adenine dinucleotide by optical spectroscopy and DFT calculations.

Flavins are photoenzymatic cofactors often exploiting the absorption of light to energize photoinduced redox chemistry in a variety of contexts. Both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are used for this function. The study of these photoenzymes has been facilitated using flavin analogs. Most of these analogs involve modification of the flavin ring, and there is recent evidence that adenine (Ade)-modified FAD can affect enzyme turnover, but so far this has only been shown for enzymes where the adenine and flavin rings are close to each other in a stacked conformation. FAD is also stacked in aqueous solution, and its photodynamics are quite different from unstacked FAD or FMN. Oxidized photoexcited FAD decays rapidly, presumably through PET with Ade as donor and Fl* as acceptor. Definitive identification of the spectral signatures of Ade∙+ and Fl∙- radicals is elusive. Here we use the FAD analog Flavin 1,N6-Ethenoadenine Dinucleotide (εFAD) to study how different photochemical outcomes depend on the identity of the Ade moiety in stacked FAD and its analog εFAD. We have used UV-Vis transient absorption spectroscopy complemented by TD-DFT calculations to investigate the excited state evolution of the flavins. In FAD*, no radicals were observed, suggesting that FAD* does not undergo PET. εFAD* kinetics showed a broad absorption band that suggests a charge transfer state exists upon photoexcitation with evidence for radical pair formation. Surprisingly, significant triplet flavin was produced from εFAD* We hypothesize that the dipolar (ε)Ade moieties differentially modulate the singlet-triplet energy gap, resulting in different intersystem crossing rates. The additional electron density on the etheno group of εFAD supplies better orbital overlap with the flavin S1 state, accelerating charge transfer in that molecule.

(Invited) Supramolecular Chemistry-Based One-Pot Separation of Solubilizer-Free Pure Semiconducting Single-Walled Carbon Nanotubes: Molecular Strategy and Mechanism

We summarize a supramolecular chemistry-based one-pot, and highly efficient separation of a solubilizer/adsorbent-free pure semiconducting SWCNT (sc-SWCNT) using flavin/isoalloxazine analogues with different substituents. On the basis of both experimental and computational simulations (DFT study), we have revealed the molecular requirements of the solubilizers as well as provided a possible mechanism for such a highly efficient selective sc-SWCNT separation[1]. We introduced thio-substituents at 7- and 8-positions of a flavin analogue, which allows for coordination of metal ions. Based on both experimental and theoretical methods, we have demonstrated that the formed Cu2+-based supramolecular assembled complex shows very high selective sorting of semiconducting single-walled carbon nanotubes with (8,6)- and (9,4)-chiralities[2]. The present sorting methods are very simple (one-pot) and gives a promising sc-SWCNT separation methodology.

Flavin-mediated reductive iron mobilization from frog M and Mycobacterial ferritins: impact of their size, charge and reactivities with NADH/O2.

In vitro, reductive mobilization of ferritin iron using suitable electron transfer mediators has emerged as a possible mechanism to mimic the iron release process, in vivo. Nature uses flavins as electron relay molecules for important biological oxidation and oxygenation reactions. Therefore, the current work utilizes three flavin analogues: riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which differ in size and charge but have similar redox potentials, to relay electron from nicotinamide adenine dinucleotide (NADH) to ferritin mineral core. Of these, the smallest/neutral analogue, RF, released more iron (~ threefold) in comparison to the larger and negatively charged FMN and FAD. Although iron mobilization got marred during the initial stages under aerobic conditions, but increased with a greater slope at the later stages of the reaction kinetics, which gets inhibited by superoxide dismutase, consistent with the generation of O2∙- in situ. The initial step, i.e., interaction of flavins with NADH played critical role in the iron release process. Overall, the flavin-mediated reductive iron mobilization from ferritins occurred via two competitive pathways, involving the reduced form of flavins either alone (anaerobic condition) or in combination with O2∙- intermediate (aerobic condition). Moreover, faster iron release was observed for ferritins from Mycobacterium tuberculosis than from bullfrog, indicating the importance of protein nanocage and the advantages they provide to the respective organisms. Therefore, these structure-reactivity studies of flavins with NADH/O2 holds significance in ferritin iron release, bioenergetics, O2-based cellular toxicity and may be potentially exploited in the treatment of methemoglobinemia. Smaller sized/neutral flavin analogue, riboflavin (RF) exhibits faster reactivity towards both NADH and O2 generating more amount of O2∙- and releases higher amount of iron from different ferritins, compared to its larger sized/negatively charged derivatives such as FMN and FAD.

A host\u2013guest approach to combining enzymatic and artificial catalysis for catalyzing biomimetic monooxygenation

Direct transfer of protons and electrons between two tandem reactions is still a great challenge, because overall reaction kinetics is seriously affected by diffusion rate of the proton and electron carriers. We herein report a host–guest supramolecular strategy based on the incorporation of NADH mimics onto the surface of a metal-organic capsule to encapsulate flavin analogues for catalytic biomimetic monooxygenations in conjunction with enzymes. Coupling an artificial catalysis and a natural enzymatic catalysis in the pocket of an enzyme, this host–guest catalyst–enzyme system allows direct proton and electron transport between two catalytic processes via NADH mimics for the monooxygenation of both cyclobutanones and thioethers. This host–guest approach, which involves the direct coupling of abiotic and biotic catalysts via a NADH-containing host, is quite promising compared to normal catalyst–enzyme systems, as it offers the key advantages of supramolecular catalysis in integrated chemical and biological synthetic sequences.

Determination of acid dissociation constants of flavin analogues by capillary zone electrophoresis.

Acid dissociation constants (pKa ) of nine kinds of flavin analogues as molecular catalyst candidates were determined by CZE. Although some of the analogues are instable and degradable under the light exposure or in alkaline aqueous solutions, the effective electrophoretic mobility of the flavin analogue of interest has been measured with the residual substance. The pKa values of the flavin analogues were analyzed through the changes in the effective electrophoretic mobility with varying pH of the separation buffer. One or two steps pKa values were determined by the analysis. One of the degraded species from the flavin analogues, lumichrome, was also detected in the CZE analysis, and its pKa values were also determined. While coexisting impurities generated over the storage conditions were found in some analogues, the pKa values of the target analogues were successfully determined with the help of the CZE separations. A pressure-assisted CZE was utilized for the determination or the estimation of the pKa values of such analogues as possessing carboxylic acid moiety.

A flavin-Cu2+ supramolecular complex for highly selective sorting of semiconducting single-walled carbon nanotubes with specific chiralities

We introduce thio-substituents at the 7- and 8-positions of a flavin analogue, which allows for the coordination of metal ions, and demonstrate, based on both experimental and theoretical methods, that the formed Cu2+-based supramolecular assembled complex shows very high selective sorting of semiconducting single-walled carbon nanotubes with (8,6)- and (9,4)-chiralities.

Methyl groups matter: Photo-CIDNP characterizations of the semiquinone radicals of FMN and demethylated FMN analogs.

In this contribution, the relative hyperfine couplings are determined for the 1H nuclei of the flavin mononucleotide (FMN) radical in an aqueous environment. In addition, three structural analogs with different methylation patterns are characterized and the influence of the substituents at the isoalloxazine moiety on the electronic structure of the radicals is explored. By exploiting nuclear hyperpolarization generated via the photo-CIDNP (chemically induced dynamic nuclear polarization) effect, it is possible to study the short-lived radical species generated by in situ light excitation. Experimental data are extracted by least-squares fitting and supported by quantum chemical calculations and published values from electron paramagnetic resonance and electron-nuclear double resonance. Furthermore, mechanistic details of the photoreaction of the investigated flavin analogs with l-tryptophan are derived from the photo-CIDNP spectra recorded at different pH values. Thereby, the neutral and anionic radicals of FMN and three structural analogs are, for the first time, characterized in terms of their electronic structure in an aqueous environment.

Unraveling flavoenzyme reaction mechanisms using flavin analogues and linear free energy relationships.

Flavoenzymes mediate a large number of different chemical transformations that employ the flavin coenzyme for many different purposes. Flavins are commonly utilized in a variety of both 1- and 2-electron transfers and reactions involving oxygen activation. In addition, flavins have also been shown to function as acid/base catalysts or nucleophilic catalysts, to electrostatically stabilize transition states, and to serve simply as structural components in some enzymes. In all of these functions, the electronic properties of the flavin play a critical role. Studies carried out over a number of years have shown that these electronic properties (and subsequently, the catalytic properties of the flavoenzyme) can be manipulated by altering the substituents on the isoalloxazine ring system of the flavin. Here, we detail methods for the chemoenzymatic preparation and purification of flavin analogues, the reconstitution of apo-flavoenzymes with these analogues, and the use of linear free energy relationships (LFERs) to correlate the perturbations induced by the altered substituents. Using examples from the literature, we highlight how the use of flavin analogues and LFERs can provide mechanistic insight into the transition state structures that define flavoenyzme chemical mechanisms.