The design, synthesis and biological evaluation of novel thiamin diphosphate analog inhibitors against the pyruvate dehydrogenase multienzyme complex E1 from Escherichia coli.

Pyruvate dehydrogenase multienzyme complex E1 (PDHc E1) is a potential target enzyme when looking for inhibitors to combat microbial disease. In this study, we designed and synthesized a series of novel thiamin diphosphate (ThDP) analogs with triazole ring and oxime ether moieties as potential inhibitors of PDHc E1. Their inhibitory activities against PDHc E1 were examined both in vitro and in vivo. Most of the tested compounds exhibited moderate inhibitory activities against PDHc E1 (IC50 = 6.1-75.5 µM). The potent inhibitors 4g, 4h and 4j, had strong inhibitory activities with IC50 values of 6.7, 6.9 and 6.1 µM against PDHc E1 in vitro and with inhibition rates of 35%, 50% and 33% at 100 µg mL(-1) against Gibberella zeae in vivo, respectively. The binding mode of 4j to PDHc E1 was analyzed by a molecular docking method. Furthermore, the possible interactions of the important residues of PDHc E1 with compound 4j were examined by site-directed mutagenesis, enzymatic assays and spectral fluorescence studies. The theoretical and experimental results are in good agreement and suggest that compound 4j could be used as a lead compound for further optimization, and may have potential as a new microbicide.

Thiaminylated adenine nucleotides. Chemical synthesis, structural characterization and natural occurrence.

Thiamine and its three phosphorylated derivatives (mono-, di- and triphosphate) occur naturally in most cells. Recently, we reported the presence of a fourth thiamine derivative, adenosine thiamine triphosphate, produced in Escherichia coli in response to carbon starvation. Here, we show that the chemical synthesis of adenosine thiamine triphosphate leads to another new compound, adenosine thiamine diphosphate, as a side product. The structure of both compounds was confirmed by MS analysis and 1H-, 13C- and 31P-NMR, and some of their chemical properties were determined. Our results show an upfield shifting of the C-2 proton of the thiazolium ring in adenosine thiamine derivatives compared with conventional thiamine phosphate derivatives. This modification of the electronic environment of the C-2 proton might be explained by a through-space interaction with the adenosine moiety, suggesting U-shaped folding of adenosine thiamine derivatives. Such a structure in which the C-2 proton is embedded in a closed conformation can be located using molecular modeling as an energy minimum. In E. coli, adenosine thiamine triphosphate may account for 15% of the total thiamine under energy stress. It is less abundant in eukaryotic organisms, but is consistently found in mammalian tissues and some cell lines. Using HPLC, we show for the first time that adenosine thiamine diphosphate may also occur in small amounts in E. coli and in vertebrate liver. The discovery of two natural thiamine adenine compounds further highlights the complexity and diversity of thiamine biochemistry, which is not restricted to the cofactor role of thiamine diphosphate.

Synthesis and biological evaluation of pyrophosphate mimics of thiamine pyrophosphate based on a triazole scaffold.

Novel triazole-based pyrophosphate analogues of thiamine pyrophosphate (TPP) have been synthesised and tested for inhibition of pyruvate decarboxylase (PDC) from Zymomonas mobilis. The thiazolium ring of thiamine was replaced by a triazole in an efficient two-step procedure. Pyrophosphorylation then gave extremely potent triazole inhibitors with K(I) values down to 20 pM, compared to a K(D) value of 0.35 microM for TPP. This triazole scaffold was used for further investigation and six analogues containing mimics of the pyrophosphate group were synthesised and tested for inhibition of PDC. Several effective analogues were found with K(I) values down to around 1 nM.

A DFT study of solvation effects on the tautomeric equilibrium and catalytic ylide generation of thiamin models.

Thiamin diphosphate (ThDP) is the biologically active form of vitamin B1 and an essential cofactor for a number of enzymes. The effect of solvent polarity on the tautomeric equilibria of ThDP using three model systems of the 4'-aminopyrimidine ring is studied by density functional theory calculations (B3LYP/6-311+G(d,p)//B3LYP/6-31G(d)) in the gas phase and selected solvents (cyclohexane, ether, dichloroethane, and water). Solvation effects are investigated using three different schemes: implicit solvation by a continuum model, explicit solvation by inclusion of three water molecules mimicking the first solvation shell of the enzymatic environment, and by a mixed implicit/explicit solvation model. The 4'-aminopyrimidine tautomer is more stable than the 1',4'-iminopyrimidine tautomer in all solvation schemes employed; however, the trend for the stabilities of the 1',4'-iminopyrimidine tautomer in the solvents depends on the specific ThDP-model. Formation of the catalytic important ylide for ThDP-dependent enzymes by deprotonation of ThDP(C2) is also investigated by localization of transition states for two possible pathways. Only the less stable tautomer, 1',4'-iminopyrimidine ThDP, is able to form the catalytic active ylide. Generation of the ylide through a direct intramolecular proton transfer from ThDP(C2) to the ThDP(N4') nitrogen lone pair is favored by 6 kcal/mol in the gas phase, as compared to a water-mediated ylide generation. However, inclusion of a dielectric medium reduces this difference dramatically. Furthermore, inclusion of two water molecules to model the apoenzymatic environment lowers the activation energies of both direct and water-mediated ylide generation.

Inhibition of pyruvate decarboxylase from Z. mobilis by novel analogues of thiamine pyrophosphate: investigating pyrophosphate mimics.

Replacement of the thiazolium ring of thiamine pyrophosphate with a triazole gives extremely potent inhibitors of pyruvate decarboxylase from Z. mobilis, with K(I) values down to 20 pM; this system was used to explore pyrophosphate mimics and several effective analogues were discovered.

Thiamine models and perspectives on the mechanism of action of thiamine-dependent enzymes.

Thiamine dependent enzymes catalyze ligase and lyase reactions near a carbonyl moiety. Chemical models for these reactions serve as useful tools to substantiate a detailed mechanism of action. This tutorial review covers all such studies performed thus far, emphasizing the role of each part around the active site and the conformation of the cofactor during catalysis.

The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography.

Enzymes that use the cofactor thiamin diphosphate (ThDP, 1), the biologically active form of vitamin B(1), are involved in numerous metabolic pathways in all organisms. Although a theory of the cofactor's underlying reaction mechanism has been established over the last five decades, the three-dimensional structures of most major reaction intermediates of ThDP enzymes have remained elusive. Here, we report the X-ray structures of key intermediates in the oxidative decarboxylation of pyruvate, a central reaction in carbon metabolism catalyzed by the ThDP- and flavin-dependent enzyme pyruvate oxidase (POX)3 from Lactobacillus plantarum. The structures of 2-lactyl-ThDP (LThDP, 2) and its stable phosphonate analog, of 2-hydroxyethyl-ThDP (HEThDP, 3) enamine and of 2-acetyl-ThDP (AcThDP, 4; all shown bound to the enzyme's active site) provide profound insights into the chemical mechanisms and the stereochemical course of thiamin catalysis. These snapshots also suggest a mechanism for a phosphate-linked acyl transfer coupled to electron transfer in a radical reaction of pyruvate oxidase.

Inhibition of thiamin diphosphate dependent enzymes by 3-deazathiamin diphosphate.

3-Deazathiamin diphosphate (deazaTPP) and a second thiamin diphosphate (TPP) analogue having a benzene ring in place of the thiazolium ring have been synthesised. These compounds are both extremely potent inhibitors of pyruvate decarboxylase from Zymomonas mobilis; binding is competitive with TPP and is essentially irreversible even though no covalent linkage is formed. DeazaTPP binds approximately seven-fold faster than TPP and at least 25,000-fold more tightly (K(i) less than 14 pM). DeazaTPP is also a potent inhibitor of the E1 subunit of alpha-ketoglutarate dehydrogenase from E. coli and binds more than 70-fold faster than TPP. In this case slow reversal of the inhibition could be observed and a K(i) value of about 5 nM was calculated (ca. 500-fold tighter binding than TPP).

The reaction of dimethyltin(IV) dichloride with thiamine diphosphate (H2TDP): synthesis and structure of [SnMe2(HTDP)(H2O)]Cl.H2O, and possibility of a hitherto unsuspected role of the metal cofactor in the mechanism of vitamin-B1-dependent enzymes.

The complex [SnMe(2)(HTDP)(H(2)O)]Cl.H(2)O, synthesized by reaction between dimethyltin(IV) dichloride and thiamine diphosphate hydrochloride (H(3)TDPCl) in water, was characterized by X-ray diffractometry and IR and Raman spectroscopy in the solid state, and by electrospray mass spectrometry (ESMS) and NMR spectroscopy ((1)H, (13)C, (31)P, (119)Sn and inverse-detection (1)H,(15)N HMBC) in aqueous solution. In the solid state the HTDP(-) anion chelates the metal via one oxygen atom of each phosphate group [Sn-O = 2.062(3), 2.292(3) A], and another oxygen atom belonging to the terminal phosphate links the SnMe(2)(2+) cations into chains. The tin atom has distorted octahedral coordination involving the trans methyl groups, the above-mentioned diphosphate oxygen atoms, and the oxygen atom of the coordinated water molecule. The thiamine moiety has F conformation. NMR studies suggest that the interaction between the organometallic cation and the HTDP(-) ligand persists in D(2)O solution, which is in keeping with the ESMS spectrum showing a peak corresponding to [SnMe(2)(HTDP)]. Both in the solid state and in solution, the acidic HTDP(-) proton in the complex is located on the N(1') atom of the pyrimidine ring. The enzymatic behavior of native pyruvate decarboxylase (EC 4.1.1.1, PDC), obtained from baker's yeast, was compared in a coupled assay with that shown by the "SnMe(2)-holoenzyme" created by incubation of apoPDC with [SnMe(2)(HTDP)(H(2)O)]Cl.H(2)O. The SnMe(2)-holoenzyme exhibited about 34% of the activity of the native enzyme (with a Michaelis-Menten constant of 2.7 microM, as against 6.4 microM for native PDC), so confirming the very low specificity of PDC regarding the identity of its metal ion cofactor. In view of the observed protonation of N(1'), it is suggested that the role of divalent cations in the mechanism of thiamine-diphosphate-dependent enzymes may be not only to anchor the cofactor in its binding site but also to shift the acidic proton of HTDP(-) from the diphosphate group to N(1'); protonation of N(1') is widely believed to be important for enzyme function, but the origin of the proton has never been clarified.

An efficient enzymatic synthesis of thiamin pyrophosphate.

Thiamin pyrophosphate was synthesized in 71% yield, on a multi-milligram scale, using overexpressed thiazole kinase, pyrimidine kinase, thiamin phosphate synthase, and thiamin phosphate kinase. This provides a facile route to isotopically labeled thiamin pyrophosphate from its readily available pyrimidine and thiazole precursors.

[Enzymatic synthesis of dihydroxyethylthiamine pyrophosphate, its optical characteristics, and quantitative determination]. / Enzimaticheskii sintez digidroksiétiltiaminpirofosfata, ego opticheskie kharakteristiki i kolichestvennoe opredelenie.

Modification of the method of Holzer et al. for the synthesis and purification of dihydroxyethylthiamine pyrophosphate, an intermediate of the transketolase reaction, is described. Absorption and circular dichroism spectra as well as the molar coefficient of circular dichroism absorption at 273 nm are presented. A convenient and rapid procedure for quantitative determination of dihydroxyethylthiamine pyrophosphate in solution where it is present as the only component or as a mixture with thiamine pyrophosphate (or other optically inactive compounds), is proposed.

Synthesis and properties of 2-acetylthiamin pyrophosphate: an enzymatic reaction intermediate.

The synthesis of 2-acetylthiamin pyrophosphate (acetyl-TPP) is described. The synthesis of this compound is accomplished at 23 degrees C by the oxidation of 2-(1-hydroxyethyl)thiamin pyrophosphate using aqueous chromic acid as the oxidizing agent under conditions where Cr(III) coordination to the pyrophosphoryl moiety and hydrolysis of both the pyrophosphate and acetyl moieties were prevented. Although the chemical properties exhibited by acetyl-TPP are similar to those of the 2-acetyl-3,4-dimethylthiazolium ion examined by Lienhard [Lienhard, G.E. (1966) J. Am. Chem. Soc. 88, 5642-5649], significant differences exist because of the pyrimidine ring in acetyl-TPP. Characterization of acetyl-TPP by ultraviolet spectroscopy, 1H NMR, 13C NMR, and 31P NMR provided evidence that the compound in aqueous solution exists as an equilibrium mixture of keto, hydrate, and intramolecular carbinolamine forms. The equilibria for the hydration and carbinolamine formation reactions at pD 1.3 as determined by 1H NMR are strongly dependent on the temperature, showing an increase in the hydrate and carbinolamine forms at the expense of the keto form with decreasing temperature. The concentration of keto form also decreases with increasing pH. Acetyl-TPP is stable in aqueous acid but rapidly deacetylates at higher pH to form acetate and thiamin pyrophosphate. Trapping of the acetyl moiety in aqueous solution occurs efficiently with 1.0 M hydroxylamine at pH 5.5-6.5 to form acetohydroxamic acid and to a much smaller extent with 1.0 M 2-mercaptoethanol at pH 4.0 and 5.0 to form thio ester. Transfer of the acetyl group to 0.5 M dihydrolipoic acid at pH 5.0 and 1.0 M phosphate dianion at pH 7.0 is not observed to any significant extent in water. The kinetic and thermodynamic reactivity of acetyl-TPP with water and other nucleophiles is compatible with a hypothetical role for acyl-TPPs as enzymatic acyl-transfer intermediates.

Interaction of thiamin diphosphate and thiamin thiazolone diphosphate with wheat germ pyruvate decarboxylase.

The interactions of the apoenzyme of wheat germ pyruvate decarboxylase with thiamin diphosphate and with thiamin thiazolone diphosphate have been investigated. The results test hypotheses concerning the structure of the transition state for decarboxylation of the enzyme-bound adduct of pyruvate and thiamin diphosphate. Thiamin thiazolone diphosphate, a possible transition state analogue, binds to the apoenzyme by a two-step process. The first is slow and reversible (k = 200 M-1 s-1; K = 5 X 10(-7) M). The second step is irreversible (k = 1 X 10(-6) s-1). The rate constant for activation by thiamin diphosphate is 160 M-1 s-1. Thiamin diphosphate is released very slowly from the holoenzyme (k = 2 X 10(-5) s-1). Thiamin thiazolone diphosphate competitively inhibits activation of the apoenzyme by thiamin diphosphate, Ki = 2 X 10(-6) M. Km for thiamin diphosphate is only 3 times larger. Thiamin thiazolone is solvated preferentially to thiamin in 2-butanol, a medium whose polarity should resemble that of the binding site. It is concluded that the observed high affinity of thiamin thiazolone diphosphate for the apoenzyme is the result of a combination of effects which do not require the assumption that it is an analogue of the transition state for the decarboxylation of enzyme-bound 2-(2-lactyl) thiamin diphosphate.