Identification of the enol tautomer of imidazolone propionate as the urocanase reaction product

A fluorescent product was transiently formed during catalysis by urocanase from Pseudomonas putida. The fluorophore showed an emission maximum at 430 nm when excited at 330 nm, essentially identical to that exhibited by the enol tautomer of imidazolone propionate. The keto isomer was not fluorescent under these conditions. In aqueous acid solutions where imidazolone propionate is relatively stable, an equilibrium mixture of tautomeric forms contained approximately 1% of the enol isomer. In ethanolic solutions, the equilibrium concentration of enol tautomer increased to approximately 25%. The differing content of imidazolone propionate tautomers as a function of solvent conditions permitted a comparison of the keto and enol forms as substrates for the reverse reaction. This revealed an almost complete preference for the enol tautomer. These results are taken as direct proof that enol imidazolone propionate is the true urocanase reaction product.     

Isomerization and Racemization of Menthols

Menthols were converted to Δ³-menthone enol acetate (VII) via menthones having only one asymmetric carbon atom. It was shown that the optical rotation of menthone enol acetate was proportional to the optical purity of starting menthols. Optical purity of original menthol could, therefore, be determined by optical rotation of menthone enol acetate derived from.     

Synthesis and kinetic analysis of some phosphonate analogs of cyclophostin as inhibitors of human acetylcholinesterase

Two new monocyclic analogs of the natural AChE inhibitor cyclophostin and two exocyclic enol phosphates were synthesized. The potencies and mechanisms of inhibition of the bicyclic and monocyclic enol phosphonates and the exocyclic enol phosphates toward human AChE are examined. One diastereoisomer of the bicyclic phosphonate exhibits an IC₅₀ of 3 μM. Potency is only preserved when the cyclic enol phosphonate is intact and conjugated to an ester. Kinetic analysis indicates both a binding and a slow inactivation step for all active compounds. Mass spectrometric analysis indicates that the active site Ser is indeed phosphorylated by the bicyclic phosphonate.     

Vibrational circular dichroism of 3‐(trifluoroacetyl)‐camphor and its interaction with chiral amines

Vibrational circular dichroism (VCD) spectroscopy and density functional theory (DFT) calculations are used to investigate the keto–enol equilibrium of 3‐(trifluoroacetyl)‐camphor (TFC) and to study the interaction of TFC with chiral amines in deuterated Chloroform. It is shown that the VCD spectra of the enol‐ and keto forms of TFC can clearly be distinguished and that the enol form is favored. By deprotonation of the TFC enol with chiral amines, no indication of a mutual diasteriomeric influence on the VCD spectra induced by transfer of stereochemical information between the chiral ionic species is found, neither experimentally nor theoretically. Chirality 2010. © 2010 Wiley‐Liss, Inc.     

A comparative study of enol aldehyde formation from betamethasone, dexamethasone, beclomethasone and related compounds under acidic and alkaline conditions

Enol aldehydes are one type of key degradation and metabolic intermediates from a group of corticosteroids containing the 1,3-dihydroxyacetone side chain on their D-rings, such as betamethasone, dexamethasone, beclomethasone, and related compounds. The formation of enol aldehydes from these corticosteroids is via acid-catalyzed β-elimination of water from the side chain, a process known as Mattox rearrangement. It was recently reported by our group that enol aldehydes could also be formed directly from the corresponding 17,21-diesters of these corticosteroids but only under alkaline condition, which was proposed to follow a variation pathway of the original Mattox rearrangement. In this paper, we report the results of a comparative study of enol aldehyde formation from these structurally similar corticosteroids (under the original acidic Mattox condition) and their 17,21-diesters (under the alkaline Mattox variation condition), respectively. In general, enol aldehydes were found to be formed under both conditions; however, the ratios of the E- and Z-isomers of the enol aldehyde were different in each case. The only exception was beclomethasone 17,21-diester under the alkaline condition, where a competing elimination of HCl from the 9,11-positions became predominant. These results can be explained by their structural differences with regard to the Mattox mechanism and its variation pathway. Lastly, solvent effect under acidic condition was studied between an aprotic and a protic solvent and the result suggests that enol aldehyde formation is greatly favored in an aprotic environment.     

Computational studies on pterins and speculations on the mechanism of action of dihydrofolate reductase

The significance of the enol form of the pterin ring in enzymatic reduction of dihydrofolate by DHFR is discussed on the basis of the results of ab initio calculations carried out on the keto/enol tautomers of 6-methyl-7, 8-dihydropterin as the model compound for the natural substrate, dihydrofolate.     

Observations on the reactivity of thiyl radicals derived from 3,6-epidithiodiketopiperazine-2,5-diones and related congeners

A range of thiyl radicals derived from the reduced form of epidithiodiketopiperazines (ETPs) act as polarity reversal catalysts for the hydrosilylation of an enol lactone but not for H-atom abstraction from a model ribose ester.     

Proline-valine pseudo peptide enol lactones. Effective and selective inhibitors of chymotrypsin and human leukocyte elastase

Pro-Val pseudo dipeptides incorporating protio and halo enol lactones were tested for inhibitory activity against the serine proteases human leukocyte elastase (HLE), porcine pancreatic elastase, alpha-chymotrypsin, trypsin, thrombin, and urokinase. The protio enol lactones 1a-c were found to be HLE substrates but were poor alternate substrate inhibitors. The bromo enol lactone trans isomer 2a was found to be a very effective inhibitor of HLE and chymotrypsin, as shown by the binding constants (KI), acylation rates (ka), inactivation rates, and partition ratios determined for each enzyme. This inhibitor shows better specificity toward its target enzyme HLE than monosubstituted halo enol lactones; we attribute this to a pseudo dipeptide acyl enzyme whose structure is similar to that adopted by good peptide substrates of HLE. Inactivation of chymotrypsin by the bromo enol lactone 2a is permanent, but inactivation of HLE is partially recoverable upon treatment with the nucleophile hydrazine, indicating that lactone 2a produces two species of inactivated HLE. The more stable of these species could be the result of alkylation of His-57 by the electrophilic bromomethyl ketone revealed in the acyl enzyme, and the less stable, hydrazine-reactivatable species could be the result of alkylation of Asp-102 or the hydrolysis of the bromomethyl ketone group in the initially formed acyl enzyme to form a new, more stable acyl enzyme.     

Mechanism of Chloride Elimination from 3-Chloro- and 2,4-Dichloro-cis,cis-Muconate: New Insight Obtained from Analysis of Muconate Cycloisomerase Variant CatB-K169A

Chloromuconate cycloisomerases of bacteria utilizing chloroaromatic compounds are known to convert 3-chloro-cis,cis-muconate to cis-dienelactone (cis-4-carboxymethylenebut-2-en-4-olide), while usual muconate cycloisomerases transform the same substrate to the bacteriotoxic protoanemonin. Formation of protoanemonin requires that the cycloisomerization of 3-chloro-cis,cis-muconate to 4-chloromuconolactone is completed by protonation of the exocyclic carbon of the presumed enol/enolate intermediate before chloride elimination and decarboxylation take place to yield the final product. The formation of cis-dienelactone, in contrast, could occur either by dehydrohalogenation of 4-chloromuconolactone or, more directly, by chloride elimination from the enol/enolate intermediate. To reach a better understanding of the mechanisms of chloride elimination, the proton-donating Lys169 of Pseudomonas putida muconate cycloisomerase was changed to alanine. As expected, substrates requiring protonation, such as cis,cis-muconate as well as 2- and 3-methyl-, 3-fluoro-, and 2-chloro-cis,cis-muconate, were not converted at a significant rate by the K169A variant. However, the variant was still active with 3-chloro- and 2,4-dichloro-cis,cis-muconate. Interestingly, cis-dienelactone and 2-chloro-cis-dienelactone were formed as products, whereas the wild-type enzyme forms protoanemonin and the not previously isolated 2-chloroprotoanemonin, respectively. Thus, the chloromuconate cycloisomerases may avoid (chloro-)protoanemonin formation by increasing the rate of chloride abstraction from the enol/enolate intermediate compared to that of proton addition to it.     

Enzymatic synthesis and inhibitory characteristics of tartronate semialdehyde phosphate

The immediate product of the pyruvate kinase catalyzed phosphorylation of beta-hydroxypyruvate is the enol of tartronate semialdehyde phosphate (TSP). The reaction has the same pH profile as that for the phosphorylation of pyruvate with pK's of 8.2 and 9.7 observed in H2O. This enol tautomerizes in solution to the aldehyde, which in turn becomes hydrated. 31P NMR spectra indicate that the enol resonates approximately 1 ppm upfield from the hydrated aldehyde. By following the tautomerization spectrophotometrically at 240 nm, we have found it to be independent of pH (0.2 min-1 below pH 6 in water), except that it is 2-fold slower above the pK of the phosphate group (6.3 in H2O and 6.7 in D2O). It is 3.6-fold slower in D2O. When this TSP is reduced with NaBH4, approximately 50% of the product is D-2-phosphoglyceric acid (substrate for enolase). Thus, while the immediate product of the phosphorylation rection is the enol of TSP, the eventual product is D,L-TSP. Both the enol and the aldehyde forms of TSP were found to be potent inhibitors of yeast enolase with apparent Ki values of 100 nM and 5 microM, respectively. However, since the aldehyde form is 95-99% hydrated [Stubbe, J., & Abeles, R. (1980) Biochemistry 19, 5505], the true Ki for the aldehyde species is 50-250 nM. The enol of TSP shows slow binding behavior, as expected for an intermediate analogue, with a t1/2 for this process of approximately 15 s (k = 0.046 s-1) and an initial Ki of approximately 200 nM.     

Synthetic libraries of tyrosine-derived bacterial metabolites

The preparation of a collection of 131 small molecules, reminiscent of families of long chain N-acyl tyrosines, enamides and enol esters that have been isolated from heterologous expression of environmental DNA (eDNA) in Escherichia coli, is reported. The synthetic libraries of N-acyl tyrosines and their 3-keto counterparts were prepared via solid-phase routes, whereas the enamides and enol esters were synthesized in solution-phase.     

Production of 16beta-(acetoxy)acetoxy derivatives by reaction of 17-keto steroid enol acetates with lead (IV) acetate

Treatment of enol acetates of 3beta-acetoxyandrost-5-en-17-one and its 5alpha-reduced analog, 5alpha-androstan-17-one, and estrone acetate, 1-4, with Pb(OCOCH(3))(4) in acetic acid and acetic anhydride gave the previously unreported products, 16beta-(acetoxy)acetoxy-17-ketones 8-10 and 12, in 9-15% yields along with the known major products, 16beta-acetoxy-17-ketones 5-7 and 11. Similar treatment of the 16beta-acetoxy-17-ketones with the lead reagent did not yield the corresponding (acetoxy)acetates. Reaction of the enol acetate 3 with Pb(OCOCD(3))(4) in CD(3)COOD yielded principally the labeled (acetoxy)acetate 10-d(3), which had a CD(3)COOCH(2)COO moiety at C-16beta. In contrast, when the deuterated enol acetate 3-d(3), which was obtained by treatment of the 17-ketone 14 with (CD(3)CO)(2)O in the presence of LDA and which had a CD(3)COO moiety at C-17, was reacted with Pb(OCOCH(3))(4), the resulting product was the labeled compound 10-d(2). This product had a CH(3)COOCD(2)COO function at C-16beta. Based on these results, along with further isotope-labeling experiments, it seems likely that the (acetoxy)acetate is produced through a lead (IV) acetate-catalyzed migration of the 17-acetyl function of the enol acetate to the C-16beta-position followed by attack of an acetoxy anion of the lead reagent.     

Synthesis of l-muscone by asymmetric methylation via enol esters

An effective synthetic route of l-muscone (1) by asymmetric methylation, followed by enolate-trapping to generate enol esters as intermediates, was described. Interestingly, the enol esters can be used as substrates for enzymatic optical resolution to improve optical purity. Additionally, several excellent new chiral ligands were discovered for asymmetric methylation of (E)-cyclopentadec-2-enone to produce l-muscone with high optical purity.     

Enols of aldehydes in the peroxidase/oxidase-promoted generation of excited triplet species

General (acid and base) or specific (fluoride ion) catalysis generates the enol of isobutanal and propanal from the corresponding trimethylsilyl enol ethers. The enols are directly rapidly oxidized by peroxidase (acting as an oxidase) to triplet acetone or triplet acetaldehyde, respectively, and formic acid. Due to the faster rate of reaction and the absence of quenching by excess aldehyde, the excited carbonyl emits more strongly than when the aldehyde itself is the substrate. With both enols the emission is pure phosphorescence. Both triplet acetone and triplet acetaldehyde are generated within the enzyme, as shown by the different quenching by D- and L-tryptophan, and are somewhat protected from oxygen quenching, as attested by the very fact that phosphorescence is observed. The use of enol precursors as substrates opens wide possibilities for photochemical investigations in the absence of light over a much broader range of experimental conditions.     

Enzymatic generation of triplet acetone by deglycosylated horseradish peroxidase

A study of the effects of deglycosylation of horseradish peroxidase on protein conformation, as well as on its catalytic activity of oxidation of isobutyraldehyde or its enol form to triplet acetone and formic acid, was performed. The loss of carbohydrates leads to structural modifications of this enzyme. This is confirmed by a change in the circular dichroism spectrum, an increase in tryptophan's environment polarity, and a loss of the chiral specificity toward D- and L-tryptophan. Deglycosylation does not destroy either the peptide backbone or the amino acid residues and does not affect the heme group content of the protein. The rates of oxygen uptake and light emission observed when horseradish peroxidase oxidizes isobutyraldehyde or the trimethylsilyl enol ether form of the latter are reduced when the enzyme is 70% deglycosylated. Concomitantly, the acting deglycosylated enzyme becomes inactivated during the course of the reaction. It appears that the carbohydrate moiety plays an important role in the protection of the peroxidase from damaging effects induced by triplet acetone and in the stabilization of the three-dimensional structure of this enzyme.     

Transient state kinetics of the reactions of isobutyraldehyde with compounds I and II of horseradish peroxidase

Elementary reactions have been studied quantitatively in the complex overall process catalyzed by horseradish peroxidase whereby isobutyraldehyde and molecular oxygen react to form triplet state acetone and formic acid. The rate constant for the reaction of the enol form of isobutyraldehyde with compound I of peroxidase is (8 +/- 1) X 10(6) M-1 s-1 and with compound II (1.3 +/- 0.3) X 10(6) M-1 s-1. Neither the enolate anion nor the keto form is reactive. The reactivity of enols with peroxidase parallels that of unionized phenols and a common mechanism is proposed. The overall catalyzed reaction of isobutyraldehyde and oxygen consists of an initial burst followed by a steady state phase. The burst is caused by the following sequence: 1) an initial high yield of compound I is formed from reaction of native enzyme with the autoxidation product of isobutyraldehyde, a peracid and 2) compound I rapidly depletes the equilibrium pool of enol which is present. After this burst a steady state phase is observed in which the rate-limiting step is the conversion of the keto to the enol form of the aldehyde catalyzed by phosphate buffer. The rate constant for the keto form reacting with phosphate is (8.7 +/- 0.6) X 10(-5) M-1 s-1. All constants were measured in dilute aqueous ethanol at 35 degrees C, pH 7.4, and ionic strength 0.67 M. Both the initial burst of light and the steady state emission from triplet acetone can be observed with the naked eye. Since the magnitude of the burst is a measure of the equilibrium amount of enol, the keto-enol equilibrium constant is readily calculated and hence also the rate constant for conversion of enol to keto. The keto-enol equilibrium constant is unaffected by phosphate which therefore acts as a true catalyst.     

Ketonization of enols in aqueous solution: is carbon protonation always rate-determining?

The pH-rate profiles for the ketonization of the (E)- and (Z)-photoenols of o-methylacetophenone (MA) in aqueous solution were determined by nanosecond laser flash photolysis. Carbon protonation of the enol anions of MA by solvent water is exceptionally fast, k(0)'(K)≈ 2.0 × 10(7) s(-1), too fast to permit establishment of the acid-base equilibrium on the enol oxygen prior to ketonization. Analysis of the pH-rate profile of the (E)-enol using the common assumption of rate-determining carbon protonation would lead to an erroneous value for the acidity constant of that enol, pK(a,c)(E) = 11.3, which is too high by about two pK units.     

Novel galbonolide derivatives as IPC synthase inhibitors: design, synthesis and in vitro antifungal activities

A series of novel galbonolide derivatives having a modified methyl enol ether moiety were prepared in total synthetic procedures and evaluated for their in vitro antifungal activities. The antifungal activity was labile to modification of the enol ether functionality and almost all of the modified compounds lacked the activity except for the analogue with an introduction of a methylthio group at the C-6 position, which retained a modest antifungal potency against Cryptococcus neoformans.     

In vivo and in vitro preparation of divinyl-132,173-cyclopheophorbide-a enol

Divinyl-13²,17³-cyclopheophorbide-a enol was in vivo produced as a metabolite of divinyl-chlorophyll-a by protists and in vitro prepared by the intramolecular cyclization of methyl divinyl-pyropheophorbide-a, one of the divinyl-chlorophyll-a derivatives. The ¹H NMR spectra in CDCl₃ showed that the obtained product took exclusively its enol form in the solution. The intramolecular cyclization of chlorin π-system at the C13² and C17³ positions affected the optical properties of such chlorophyll derivatives including the non-fluorescent emission of the enol.