Tautomeric Shift Research Articles

Synthesis and reactivity of alkali metal aluminates bearing bis(organoamido)phosphane ligand.

In this study, we report a group of alkali metal aluminates bearing bis(organoamido)phosphane ligand. The starting complex {[PhP(NtBu)2]AlMe2}Li·OEt2 (1) was prepared by stepwise deprotonation of the parent PhP(NHtBu)2 by nBuLi and AlMe3. Further derivatization of aluminate 1 was performed by the virtual substitution of lithium -{[PhP(NtBu)2]AlMe2}K (2), methyl substituents - {[PhP(NtBu)2]AlH2}Li·THF (3), modification of steric bulk and induction effects on the phosphorus atom - {[tBuP(N-2,6-iPr2C6H3)2]AlMe2}Li·(OEt2)2 (4), and phosphorus atom oxidation state {[Ph(Y)P(NtBu)2]AlMe2}Li (Y = O (5), S (6), Se (7), Te (8)). The structure causing non-covalent interactions in 1-4 were evaluated with the help of theoretical calculations and topological analysis ranging from π-electron system-metal to agostic interactions of various types. The further reactions of 1 with various nucleophiles were found to be a versatile tool for the preparation of iminophosphonamides via the formation of P-E bond (E = Si, Ge, Sn, Pb, P, and C) and followed by P(III) → P(V) tautomeric shift.

Yttrium and Lithium Complexes with Diamidophosphane Ligand Bearing 2,1,3-Benzothiazolyl Substituent: Polydentate Complexation and Reversible NH–PH Tautomery

Deprotonation of a bis(amino)phosphane H2L = PhP(HNBtd)2 bearing a heterocyclic Btd = 2,1,3-benzothiadiazol-4-yl substituents at nitrogen atoms by silylamides LiNTms2 and Y(NTms2)3 (Tms = trimethylsilylamide) results in lithium and yttrium complexes with the deprotonated HL– and L2– forms as κ2-N and κ4-N chelating ligands. A binuclear complex [LiHL]2 was crystallized from Et2O, and was shown to reversibly dissociate in thf (tetrahydrofuran) with the NH(soln)–PH(crystal) tautomeric shift; the compound [Li2L] was spectroscopically characterized. Yttrium readily forms stable bis-ligand complexes [YL2]– and [YL(HL)]. In the latter, the H atom in HL resides on phosphorus; the coordination sphere remains accessible to another ligands, and it was crystallized as [{YL(HL)}2(µ-dioxane)] species (YN8O coordination). In the former complex, the coordination sphere was saturated (YN8) by closer bound ligands; it was crystallized as a salt with [Li(thf)4]+. The monoligand complex could not be cleanly obtained in a 1:1 reaction of H2L and Y(NTms2)3, and was only crystallographically characterized as a dimer [YL(NTms)2]2. Partial oxidation of the central P atom with the formation of phosphine-oxide ligands PhP(O)(NBtd)2– was observed. They co-crystallize in the same position as non-oxidized ligands in [YL2]– and [YL(NTms2)]2 species and participate in bonding between two units in the latter. TD-DFT calculations reveal that main transitions in the visible region of electronic spectra correspond to the charge transfer bands mostly associated with the orbitals located on Btd fragments.

Free-Energy Landscape of a pH-Modulated G·C Base Pair Transition from Watson-Crick to Hoogsteen State in Duplex DNA.

In double-helical DNAs, the most stable Watson-Crick (WC) base pair (bp) can be in thermal equilibrium with much less abundant Hoogsteen (HG) bp by the spontaneous rotation of the glycosidic angle in purine bases. Previous experimental studies showed that in the case of a G·C bp, the population of the transient HG is enhanced as a protonated form (HG+) through the protonation of the cytosine base under weakly acidic conditions. Hence, pH is a key factor that can modulate this transition event from the WC to HG+ bp. In this study, to computationally probe the overall free-energy landscapes of this pH-modulated G·C HG breathing, a comprehensive classical molecular dynamics (MD) simulation protocol is proposed using an enhanced sampling MD in conjunction with the standard thermodynamic integration method. From this MD protocol proposed, the free-energy surfaces of the G·C bp transition from the WC to HG bp were constructed successfully at any pH range, producing pH-dependent free-energy quantities in close agreement with previously reported experimental results. The simulation protocol is expected to provide valuable atomistic insight into the DNA bp transition events coupled with protonation or tautomeric shift in a target bp.

Why Is DNA Double Stranded? The Discovery of DNA Excision Repair Mechanisms.

The persistence of hereditary traits over many generations testifies to the stability of the genetic material. Although the Watson-Crick structure for DNA provided a simple and elegant mechanism for replication, some elementary calculations implied that mistakes due to tautomeric shifts would introduce too many errors to permit this stability. It seemed evident that some additional mechanism(s) to correct such errors must be required. This essay traces the early development of our understanding of such mechanisms. Their key feature is the cutting out of a section of the strand of DNA in which the errors or damage resided, and its replacement by a localized synthesis using the undamaged strand as a template. To the surprise of some of the founders of molecular biology, this understanding derives in large part from studies in radiation biology, a field then considered by many to be irrelevant to studies of gene structure and function. Furthermore, genetic studies suggesting mechanisms of mismatch correction were ignored for almost a decade by biochemists unacquainted or uneasy with the power of such analysis. The collective body of results shows that the double-stranded structure of DNA is critical not only for replication but also as a scaffold for the correction of errors and the removal of damage to DNA. As additional discoveries were made, it became clear that the mechanisms for the repair of damage were involved not only in maintaining the stability of the genetic material but also in a variety of biological phenomena for increasing diversity, from genetic recombination to the immune response.

Free energy landscapes of prototropic tautomerism in pyridoxal 5'-phosphate schiff bases at the active site of an enzyme in aqueous medium.

We have performed hybrid quantum-classical metadynamics simulations and quantum chemical calculations to investigate the free energy landscapes of intramolecular proton transfer and associated tautomeric equilibrium in pyridoxal 5 '-phosphate (PLP) Schiff Bases, namely the internal and external aldimines, at the active site of serine hydroxymethyltransferase (SHMT) enzyme in aqueous medium. It is important to determine the relative stability of the two tautomers (ketoenamine and enolimine) of the PLP aldimines to study the catalytic activity of the concerned enzyme. Both the internal PLP aldimine (PLP-LYS) and the external PLP aldimine (PLP-SER) of SHMT are found to have a higher stability for the ketoenamine tautomer over the enolimine form. The higher stability of the ketoenamine tautomer can be attributed to the more number of favorable interactions of the ketoenamine form with its surroundings at the active site of the enzyme. The ketoenamine is found to be stabilized by about 2.5 kcal/mol in the PLP-LYS internal aldimine, while this stabilization is about 6.7 kcal/mol for the PLP-SER external aldimine at the active site of the enzyme compared to the corresponding enolimine forms. The interactions faced by the PLP aldimines at the active site pocket determine the relative dominance of the tautomers and could possibly alter the tautomeric shift in different PLP dependent enzymes. © 2018 Wiley Periodicals, Inc.

Molecular and Spectroscopic Characterization of Aspergillus flavipes and Pseudomonas putida L-Methionine γ-Lyase in Vitro.

Pseudomonas putida L-methionine γ-lyase (PpMGL) has been recognized as an efficient anticancer agent, however, its antigenicity and stability remain as critical challenges for its clinical use. From our studies, Aspergillus flavipes L-methionine γ-lyase (AfMGL) displayed more affordable biochemical properties than PpMGL. Thus, the objective of this work was to comparatively assess the functional properties of AfMGL and PpMGL via stability of their internal aldimine linkage, tautomerism of pyridoxal 5'-phosphate (PLP) and structural stability responsive to physicochemical factors. The internal Schiff base of AfMGL and PpMGL have the same stability to hydroxylamine and human serum albumin. Acidic pHs resulted in strong cleavage of the internal Schiff base, inducing the unfolding of MGLs, compared to neutral-alkaline pHs. At λ 280nm excitation, both AfMGL and PpMGL have identical fluorescence emission spectra at λ 335nm for the intrinsic tryptophan and λ 560nm for the internal Schiff base. The maximum PLP tautomeric shift of ketoenamine to enolimine was detected at acidic pH causing complete enzyme unfolding, subunits dissociation and tautomeric shift of intrinsic PLP, rather than neutral-alkaline ones. The T m of AfMGL and PpMGL in presence of thermal stabilizer/ destabilizer was assayed by DSF. The T m of AfMGL and PpMGL was 73.1°C and 74.4°C, respectively, suggesting the higher proximity to the tertiary structure of both enzymes. The T m of AfMGL and PpMGL was slightly increased by trehalose and EDTA in contrast to guanidine HCl and urea. The active site and PLP-binding domains are identically conserved in both AfMGL and PpMGL.

Double hydrogen bond interaction in 7-azaindole complexes with protic solvents

The double hydrogen bond interaction between 7-azaindole (7AI) and protic solvents including methanol (MeOH), formamide (FM), formic acid (FA), pyridone (PY) and 7AI has been investigated as a proper model of mutations generated by tautomeric shifts in hydrogen-bonded bases of DNA. The results confirm electrostatic nature for all considered hydrogen bonds except for NH hydrogen bond in 7AI-FA. The largest interaction and polarization energies are obtained for 7AI-FA by means of energy decomposition analyses (EDA). The EDA results show an inverse competitive correlation between polarization and electrostatic components of attraction energy to determine the nature of hydrogen bond. The red-shifted hydrogen bonds are identified for all complexes as a result of hyperconjugation, except for 7AI-MeOH, which its blue-shift effect is attributed to the rehybridization dominating of CH bond. Investigation of aromaticity indices for 7AI complexes represents an increase in aromaticity of pentagonal ring and a decrease in hexagonal ring. Finally, the double hydrogen bond between 7AI and FA is identified as dominant interaction to achieve the tautomerization of 7AI in all applied approaches.

Germylenes and stannylenes stabilized within N2PE rings (E = Ge or Sn): combined experimental and theoretical study.

The deprotonation of aminophosphanes PhP(NHR)2 (R = t-Bu or Dip; Dip = 2,6-i-Pr2C6H3) and t-BuP(NHDip)2 using n-BuLi gave, depending on the stoichiometry, both the dilithium compounds {[PhP(Nt-Bu)2]Li2}2 (), [PhP(Nt-Bu)(NDip)]Li2·(Et2O) (), [t-BuP(NDip)2]Li2·(Et2O)2 () and [t-BuP(NDip)2]Li2·(tmeda)2 (), and the monolithium compounds [PhP(NHt-Bu)(NR)]Li·(tmeda) (R = t-Bu , Dip ) and [t-BuP(NHDip)(NDip)]Li·(tmeda) (). Treatment of , and with GeCl2·dioxane or SnCl2 in a 1 : 1 stoichiometric ratio gave the corresponding tetrylenes [PhP(Nt-Bu)2]E (E = Ge , Sn ), [PhP(Nt-Bu)(NDip)]Ge () and [t-BuP(NDip)2]E (E = Ge , Sn ). The heteroleptic germylene [Ph(H)P(Nt-Bu)2]GeCl () was obtained by the reaction of the monolithium compound [PhP(NHt-Bu)(Nt-Bu)]Li·(tmeda) () with GeCl2·dioxane in a 1 : 1 stoichiometric ratio, as a result of a spontaneous NH → PH tautomeric shift in the ligand backbone. In contrast, an analogous reaction with SnCl2 produced only stannylene along with the PhP(NHt-Bu)2 starting material, suggesting scrambling of the ligands rather than a NH → PH tautomeric shift. Finally, heating in solution led to P-C bond cleavage and formation of the bis(imino)phosphide [DipNPNDip]Li·(tmeda) (). The reaction of with GeCl2·dioxane, SnCl2 or PbCl2 in a 2 : 1 stoichiometric ratio yielded the unprecedented tetrylenes [DipNPNDip]2E (E = Ge , Sn and Pb ), in which the tetrylene center is incorporated within two N2PE rings. Treatment of the monolithium compound with n-BuLi and K (or KC8) gave [t-BuNPNt-Bu]Li·(tmeda) () and{[t-BuNPNt-Bu]K(tmeda)}2 (), respectively. In contrast to the reaction with , similar reactions of with GeCl2·dioxane and SnCl2 resulted in the known compounds cis-[P(μ-Nt-Bu)2P(t-BuN)2]E (E = Ge, Sn); evidently the t-Bu groups do not provide sufficient steric shielding to protect the bis(imino)phosphide backbone as in the case of . The bonding situation in a set of selected compounds (, ) has been subjected to a theoretical survey with particular emphasis on the nature of the bonding between the ligand and the central metal and the bonding within the NPN core of the ligand, showing significant differences among the studied complexes.

Watson–Crick-like pairs in CCUG repeats: evidence for tautomeric shifts or protonation

RNA transcripts that include expanded CCUG repeats are associated with myotonic dystrophy type 2. Crystal structures of two CCUG-containing oligomers show that the RNA strands associate into slipped duplexes that contain noncanonical C–U pairs that have apparently undergone tautomeric transition or protonation resulting in an unusual Watson–Crick-like pairing. The overhanging ends of the duplexes interact forming U–U pairs, which also show tautomerism. Duplexes consisting of CCUG repeats are thermodynamically less stable than the trinucleotide repeats involved in the TRED genetic disorders, but introducing LNA residues increases their stability and raises the melting temperature of the studied oligomers by ∼10°C, allowing detailed crystallographic studies. Quantum mechanical calculations were performed to test the possibility of the tautomeric transitions or protonation within the noncanonical pairs. The results indicate that tautomeric or ionic shifts of nucleobases can manifest themselves in biological systems, supplementing the canonical “rules of engagement.”

Ambit-Tautomer: An Open Source Tool for Tautomer Generation.

We present a new open source tool for automatic generation of all tautomeric forms of a given organic compound. Ambit-Tautomer is a part of the open source software package Ambit2. It implements three tautomer generation algorithms: combinatorial method, improved combinatorial method and incremental depth-first search algorithm. All algorithms utilize a set of fully customizable rules for tautomeric transformations. The predefined knowledge base covers 1-3, 1-5 and 1-7 proton tautomeric shifts. Some typical supported tautomerism rules are keto-enol, imin-amin, nitroso-oxime, azo-hydrazone, thioketo-thioenol, thionitroso-thiooxime, amidine-imidine, diazoamino-diazoamino, thioamide-iminothiol and nitrosamine-diazohydroxide. Ambit-Tautomer uses a simple energy based system for tautomer ranking implemented by a set of empirically derived rules. A fine-grained output control is achieved by a set of post-generation filters. We performed an exhaustive comparison of the Ambit-Tautomer Incremental algorithm against several other software packages which offer tautomer generation: ChemAxon Marvin, Molecular Networks MN.TAUTOMER, ACDLabs, CACTVS and the CDK implementation of the algorithm, based on the mobile H atoms listed in the InChI. According to the presented test results, Ambit-Tautomer's performance is either comparable to or better than the competing algorithms. Ambit-Tautomer module is available for download as a Java library, a command line application, a demo web page or OpenTox API compatible Web service.

Study of tautomeric transformations of 1,4,5,8-tetraaminoanthraquinone by electronic spectroscopy

Based on the example of 1,4,5,8-tetraamino-9,10-anthraquinone, a method is described for establishing the tautomeric compositions and sequences of tautomeric transformations that occur in known samples of quinones. An analysis of electronic absorption spectra shows that this substance is a dynamic equilibrium mixture of 4,5,8,9-tetraamino-1,10-anthraquinone and the imines tautomeric to it. The tautomeric compositions of different samples of matter differ and can contain 1,4,8-triamino-5-hydroxy-9,10-anthraquinone-10-imine, 4,8,9-triamino-10-hydroxy-1,5-anthraquinone-5-imine, 5,8-diamino-4,9-dihy-droxy-1,10-antraquinonediimine, and 8,10-diamino-5,9-dihydroxy-1,4-antraquinonediimine. Tautomeric equilibrium shifts can accompany not only chemical reactions but physical processes as well, so any investigation of the properties of substances without establishing their tautomeric compositions would be incorrect.

112 The importance of being modified: tautomeric forms of pyrimidines provide expanded use of the genetic code

Tautomeric shifts of five-modified uridines have been established in, Escherichia coli tRNAVal cmo5UAC, as well as in the human cytoplasmic tRNALys3 mcm5s2UUU. These shifts enable the recognition of multiple codons with the wobble base pair in Watson–Crick geometry, suggesting an energetic preference for perhaps an enhanced efficiency with this conformation. Thus, modifications at the fifth-position of wobble position uridines appear to be a common mechanism for expanding tRNA’s codon recognition. One deviation from the universal code which is particularly interesting is the reading of AUG and the universal isoleucine codon AUA as methionine in both the A- and P-sites of the mitochondrial ribosome of most metazoans. We have discovered a mechanism by which five-formylcytidine (f5C) at the wobble position of human mitochondrial tRNAMet (hmtRNAMet f5CAU) expands the decoding ability of tRNA, enabling it to read AUA as methionine. We have also found that another modified cytidine can restrict wobble-base formation. This underscores the ability of cytidines, as well as uridine, modifications within the anticodon loop to modulate the decoding ability of the tRNA, providing an insight into decoding mechanisms and evolution of the genetic code.

Solvent Polarity-Induced Conformational Unlocking of Asparagine

Classical and Car-Parrinello molecular dynamics simulations are performed to study the solvent effect on the conformational distribution of asparagine. Conformational populations obtained from the simulations in gas phase and in nonpolar chloroform solvent are in agreement with the most probable single conformation of asparagine in the gas phase measured in recent laser ablation with molecular beam Fourier transform microwave spectroscopy experiments. We rationalize that intramolecular hydrogen bonding and dipole-dipole interactions between carbonyl groups dictate such a conformational locking to a single asparagine conformer. The solvent polarity induced interlocking or intermolecular hydrogen bonding with water solvent molecules destabilizes the (NH···O═C) bonding between side chain and terminal groups of asparagine, while not essentially affecting the (NH···O═C) intramolecular hydrogen bondings within the side chain nor within the terminal groups. Such a conformational unlocking or cage effect is observed in asparagine within aqueous solution. We observed a spontaneous conversion of neutral to zwitterionic isomer of asparagine in aqueous solution, which is in agreement with interpretation of Raman spectroscopy results. Using Møller-Plesset second order perturbation theory, we show that a tautomeric shift from neutral to zwitterionic occurs on asparagine in between DMSO and water solvents. The ramification of these findings for the conformational character of asparagine is briefly discussed.

Unexpected Metal-Induced Isomerisms and Phosphoryl Migrations in Pt(II) and Pd(II) Complexes of the Functional Phosphine 2-(Bis(diphenylphosphino)methyl)-oxazoline

The reaction of the functional diphosphine 1 [1 = 2-(bis(diphenylphosphino)methyl-oxazoline] with [PtCl(2)(NCPh)(2)] or [PdCl(2)(NCPh)(2)], in the presence of excess NEt(3), affords [Pt{(Ph(2)P)(2)C···C(···NCH(2)CH(2)O)}(2)] ([Pt(1(-H)-P,P)(2)], 3a) and [Pd{(Ph(2)P)(2)C···C(···NCH(2)CH(2)O)}(2)] ([Pd(1(-H)-P,P)(2)], 3b), respectively, in which 1(-H) is (oxazoline-2-yl)bis(diphenylphosphino)methanide. The reaction of 3b with 2 equiv of [AuCl(tht)] (tht = tetrahydrothiophene) afforded [Pd(1(-H)-P,N)(2)(AuCl)(2)] (4), as a result of the opening of the four-membered metal chelate since ligand 1(-H), which was P,P-chelating in 3b, behaves as a P,N-chelate toward the Pd(II) center in 4 and coordinates to Au(I) through the other P donor. In the absence of a base, the reaction of ligand 1 with [PtCl(2)(NCPh)(2)] in MeCN or CH(2)Cl(2) afforded the isomers [Pt{(Ph(2)P)(2)C═C(OCH(2)CH(2)NH)}(2)]Cl(2) ([Pt(1'-P,P)(2)]Cl(2) (5), 1' = 2-(bis(diphenylphosphino)methylene)-oxazolidine) and [Pt{(Ph(2)P)(2)C═C(OCH(2)CH(2)NH)}{Ph(2)PCH═C(OCH(2)CH(2)N(PPh(2))}]Cl(2) ([Pt(1'-P,P)(2'-P,P)]Cl(2) (6), 2' = (E)-3-(diphenylphosphino)-2-((diphenylphosphino)methylene)oxazolidine]. The P,P-chelating ligands in 5 result from a tautomeric shift of the C-H proton of 1 to the nitrogen atom, whereas the formation of one of the P,P-chelates in 6 involves a carbon to nitrogen phosphoryl migration. The reaction of 5 and 6 with a base occurred by deprotonation at the nitrogen to afford 3a and [Pt{(Ph(2)P)(2)C···C(···NCH(2)CH(2)O)}{Ph(2)PCH═COCH(2)CH(2)N(PPh(2))}]Cl ([Pt(1(-H)-P,P)(2'-P,P)]Cl (7)], respectively. In CH(2)Cl(2), an isomer of 3a, [Pt{Ph(2)P)(2)C···C(···NCH(2)CH(2)O)}{Ph(2)PC(PPh(2))═COCH(2)CH(2)N}] ([Pt(1(-H)-P,P)(1(-H)-P,N)] (8)), was obtained as a side product which contains ligand 1(-H) in two different coordination modes. Complexes 3b·4CH(2)Cl(2), 4·CHCl(3), 6·2.5CH(2)Cl(2), and 8·CH(2)Cl(2) have been structurally characterized by X-ray diffraction.

Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase

Threonine synthase (TS), which is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the elimination of the γ-phosphate group from O-phospho-L-homoserine (OPHS) and the subsequent addition of water at Cβ to form L-threonine. The catalytic course of TS is the most complex among the PLP enzymes, and it is an intriguing problem how the elementary steps are controlled in TS to carry out selective reactions. When L-vinylglycine was added to Thermus thermophilus HB8 TS in the presence of phosphate, L-threonine was formed with k(cat) and reaction specificity comparable with those when OPHS was used as the substrate. However, in the absence of phosphate or when sulfate was used in place of phosphate, only the side reaction product, α-ketobutyrate, was formed. Global analysis of the spectral changes in the reaction of TS with L-threonine showed that compared with the more acidic sulfate ion, the phosphate ion decreased the energy levels of the transition states of the addition of water at the Cβ of the PLP-α-aminocrotonate aldimine (AC) and the transaldimination to form L-threonine. The x-ray crystallographic analysis of TS complexed with an analog for AC gave a distinct electron density assigned to the phosphate ion derived from the solvent near the Cβ of the analog. These results indicated that the phosphate ion released from OPHS by γ-elimination acts as the base catalyst for the addition of water at Cβ of AC, thereby providing the basis of the reaction specificity. The phosphate ion is also considered to accelerate the protonation/deprotonation at Cγ.

ChemInform Abstract: Solid-State Reactions Studied by 13C Rotor-Synchronized Magic Angle Spinning Two-Dimensional Exchange NMR. Part 1. Self-Diffusion and the Tautomeric Hydrogen Shift in Tropolone.

ChemInform Abstract: Solid-State Reactions Studied by 13C Rotor-Synchronized Magic Angle Spinning Two-Dimensional Exchange NMR. Part 1. Self-Diffusion and the Tautomeric Hydrogen Shift in Tropolone.

A UV/VIS spectra investigation of pH‐sensitive dyes using time‐dependent density functional theory

AbstractThe UV absorption spectra of neutral and charged hydroxy‐, amino‐, benzene‐, naphtene, and quinoline derivated dyes are investigated with the PCM‐TDDFT/6‐311+g(2d,p)//PCM‐PBE0/6‐311g(d,p) theoretical scheme using three popular hybrid functionals (O3LYP, PBE0, and BHandHLYP) and taking into account solvation effects (water, methanol, or ethanol). More precisely, we report herein the theoretical λmax corresponding to the first dipole‐allowed π → π*. The raw results show that BHandHLYP is the less accurate (MAE of 36 nm) functional, though, after the statistical treatment, this hybrid becomes the most reliable and accurate functional for the evaluation of the absorption wavelengths. Indeed, the agreement between theory and experiment is satisfactory: The error on the excitation energies is close to 13 nm. In addition, our methodology has been applied to evaluate the UV spectra of nitrocatechol derivatives in aqueous solution, and the keto‐enol tautomeric shift on the UV/VIS absoprtion spectrum of a β‐diketone derivative. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2010

Heme Regulation of Human Cystathionine β-Synthase Activity: Insights from Fluorescence and Raman Spectroscopy

Cystathionine beta-synthase (CBS) plays a central role in homocysteine metabolism, and malfunction of the enzyme leads to homocystinuria, a devastating metabolic disease. CBS contains a pyridoxal 5'-phosphate (PLP) cofactor which catalyzes the synthesis of cystathionine from homocysteine and serine. Mammalian forms of the enzyme also contain a heme group, which is not involved in catalysis. It may, however, play a regulatory role, since the enzyme is inhibited when CO or NO are bound to the heme. We have investigated the mechanism of this inhibition using fluorescence and resonance Raman spectroscopies. CO binding is found to induce a tautomeric shift of the PLP from the ketoenamine to the enolimine form. The ketoenamine is key to PLP reactivity because its imine C horizontal lineN bond is protonated, facilitating attack by the nucleophilic substrate, serine. The same tautomer shift is also induced by heat inactivation of Fe(II)CBS, or by an Arg266Met replacement in Fe(II)CBS, which likewise inactivates the enzyme; in both cases the endogenous Cys52 ligand to the heme is replaced by another, unidentified ligand. CO binding also displaces Cys52 from the heme. We propose that the tautomer shift results from loss of a stabilizing H-bond from Asn149 to the PLP ring O3' atom, which is negatively charged in the ketoenamine tautomer. This loss would be induced by displacement of the PLP as a result of breaking the salt bridge between Cys52 and Arg266, which resides on a short helix that is also anchored to the PLP via H-bonds to its phosphate group. The salt bridge would be broken when Cys52 is displaced from the heme. Cys52 protonation is inferred to be the rate-limiting step in breaking the salt bridge, since the rate of the tautomer shift, following CO binding, increases with decreasing pH. In addition, elevation of the concentration of phosphate buffer was found to diminish the rate and extent of the tautomer shift, suggesting a ketoenamine-stabilizing phosphate binding site, possibly at the protonated imine bond of the PLP. Implications of these findings for CBS regulation are discussed.

Mechanically Induced Phase Change in Barbituric Acid

By grinding a commercial sample of barbituric acid in its trioxo form (polymorph II, 99%) for 24 h, a new compound has been isolated. The new phase has been identified as the trihydroxyl isomer. The characterization of the new isomer has been carried out by means of X-ray powder diffraction, solid-state NMR (1H MAS, 13C and 15N CPMAS, 2D PASS, and FSLG-HETCOR), IR and Raman spectroscopies. The conversion results from the complete tautomeric shift of a methylene and of two N−H hydrogen atoms. 1H MAS spectra allow the characterization of the hydrogen bond interactions on the basis of their strength in the starting compound and in the new isomer. In solution, the trihydroxyl isomer immediately converts to the trioxo form as demonstrated by 1H NMR experiments in protic, aprotic, and amphiprotic solvents (D2O, MeOH-d4, DMSO-d6, acetone-d6).

Towards a Tunable Tautomeric Switch in Azobenzene Biomimetics: Implications for the Binding Affinity of 2‐(4′‐Hydroxyphenylazo)benzoic Acid to Streptavidin

The tautomeric equilibria of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) and 2-(3',5'-dimethyl-4'-hydroxyphenylazo)benzoic acid (3',5'-dimethyl-HABA) have been studied by a combination of spectroscopic and computational methods. For neutral HABA in solvents of different polarity (toluene, chloroform, DMSO, DMF, butanol, and ethanol) the azo tautomer (AT) is largely predominant. For monoanionic HABA, the hydrazone tautomer (HT) is the only detected species in apolar solvents such as toluene and chloroform, while the AT is the only detected species in water and a mixture of both tautomers is detected in ethanol. Comparison of the results obtained for HABA and its 3',5'-dimethylated derivative shows that dimethylation of the hydroxybenzene ring shifts the tautomeric preferences towards the hydrazone species. These findings have been used to examine the differences in binding affinity to streptavidin, as the lower affinity of HABA can be explained in terms of the larger energetic cost associated with the tautomeric shift to the bioactive hydrazone species. Overall, these results suggest that a balanced choice of chemical substituents, embedding environment, and pH can be valuable for exploitation of the azo-hydrazone tautomerism of HABA biomimetics in biotechnological applications.