Enantiomeric Substrates Research Articles

An enantiomerically pure 1,5,7-trimethyl-3-azabicyclo[3.3.1]nonan- 2-one as 1H NMR shift reagent for the ee determination of chiral lactams, quinolones, and oxazolidinones.

The chiral lactam 1 (or its enantiomer ent-1) was shown to be an effective (1)H NMR shift reagent for the ee determination of chiral lactams, quinolones, and oxazolidinones. It was successfully employed in many cases in which a detection of enantiomers by chromatographic methods failed. The method was extended to a broader range of simple substrates bearing a lactam moiety to evaluate its scope. The NH signals of the substrate enantiomers showed the strongest separation and were used for (1)H NMR integration. In most cases, compound 1 (1.5 equiv; 0.06 M solution) induced a baseline separation of the NH signals and it can consequently be regarded as a generally applicable shift reagent for chiral products with a lactam moiety.

Catalysis, Stereochemistry, and Inhibition of Ureidoglycolate Lyase

Ureidoglycolate lyase (UGL, EC 4.3.2.3) catalyzes the breakdown of ureidoglycolate to glyoxylate and urea, which is the final step in the catabolic pathway leading from purines to urea. Although the sequence of enzymatic steps was worked out nearly 40 years ago, the stereochemistry of the uric acid degradation pathway and the catalytic properties of UGL have remained very poorly described. We now report the first direct investigation of the absolute stereochemistry of UGL catalysis. Using chiral chromatographic analyses with substrate enantiomers, we demonstrate that UGL catalysis is stereospecific for substrates with the (S)-hydroxyglycine configuration. The first potent competitive inhibitors for UGL are reported here. These inhibitors are compounds which contain a 2,4-dioxocarboxylate moiety, designed to mimic transient species produced during lyase catalysis. The most potent inhibitor, 2,4-dioxo-4-phenylbutanoic acid, exhibits a KI value of 2.2 nM and is therefore among the most potent competitive inhibitors ever reported for a lyase enzyme. New synthetic alternate substrates for UGL, which are acyl-alpha-hydroxyglycine compounds, are described. Based on these alternate substrates, we introduce the first assay method for monitoring UGL activity directly. Finally, we report the first putative primary nucleotide and derived peptide sequence for UGL. This sequence exhibits a high level of similarity to the fumarylacetoacetate hydrolase family of proteins. Close mechanistic similarities can be visualized between the chemistries of ureidoglycolate lyase and fumarylacetoacetate hydrolase catalysis.

Lipase-catalyzed Irreversible Transesterification of Secondary Alcohols Using Isopropenyl Acetate

Asymmetric acetylation of a set of secondary alcohols with the innocuous acyl donor isopropenyl acetate catalyzed by a lipase from Pseudomonas cepacia immobilized on ceramic particles (PSL-C) in toluene as organic medium afforded the chiral alcohols and the corresponding acetates in high enantiomeric excess (up to 99%). An effective baseline separation of the enantiomers of both substrate and product was performed in one analysis without derivatization using gas chromatography on a new chiral stationary phase (CSP) Chirasil-β-Dex containing an undecamethylene spacer (C11-Chirasil-Dex).

Stereoselective catalytic oxidations of biomimetic copper complexes with a chiral trinucleating ligand derived from 1,1-binaphthalene

Abstract The new octadentate ligand R-(−)-N,N′-dimethyl-N,N′-bis{3-[bis(1-methyl-2-benzimidazolyl)amino]propyl}1,1′-binaphthalenyl-2,2′-diamine (L) was employed for the synthesis of dinuclear and trinuclear copper(II) complexes. Two terminal binding sites with tridentate aminobis(benzimidazole) linkages (A sites) and one central binding site with the bidentate diamino-binaphthalenyl residue (B site) are used by the ligand to bind divalent metal centres in the trinuclear complex [Cu3L][ClO4]6. Spectroscopic measurements suggest that in the dinuclear complex [Cu2L][ClO4]4 the copper ions are five-coordinated, with ligation by the aminobis(benzimidazole) residues, one of the tertiary amine donors of the diamino-binaphthalenyl moiety, and one water molecule. The complexes bind azide in the μ-1,3 fashion at low concentration and in the terminal mode at high concentration. The copper(II) complexes derived from L are catalytically active in the oxidation of 3,5-di-tert-butylcatechol (DTBC) by dioxygen. The oxidations are biphasic, with a fast initial stoichiometric phase corresponding to reduction of a pair of copper(II) centres and oxidation of DTBC to quinone, followed by the catalytic reaction, that follows substrate saturation behaviour. The complexes act as stereoselective catalysts in the biomimetic oxidations of the optically active catechol derivatives l - and d -Dopa and their methyl esters. In all the cases, the preferred enantiomeric substrate has the L configuration. This preference is dictated by the chirality of the binaphthalenyl residue.

Directed evolution of N-acetylneuraminic acid aldolase to catalyze enantiomeric aldol reactions

Expanding the scope of substrate specificity and stereoselectivity is of current interest in enzyme catalysis. Using error-prone PCR for in vitro directed evolution, the Neu5Ac aldolase from Escherichia coli has been altered to improve its catalytic activity toward enantiomeric substrates including N-acetyl- l-mannosamine and l-arabinose to produce l-sialic acid and l-KDO, the mirror-image sugars of the corresponding naturally occurring d-sugars. The first generation variant containing two mutations (Tyr98His and Phe115Leu) outside the (α,β) 8-barrel active site exhibits an inversion of enantioselectivity toward KDO and the second generation variant contains an additional amino acid change Val251Ile outside the α,β-barrel active site that improves the enantiomeric formation of l-sialic acid and l-KDO. The X-ray structure of the triple mutant epNanA.2.5 at 2.3 Å resolution showed no significant difference between the wild-type and the mutant enzymes. We probed the potential structural ‘hot spot’ of enantioselectivity with saturation mutagenesis at Val251, the mutated residue most proximal to the Schiff base forming Lys165. The selected variant had an increase in k cat via replacement with another hydrophobic residue, leucine. Further sampling of a larger sequence space with error-prone PCR selected a third generation variant with significant improvement in l-KDO catalysis and a complete reversal of enantioselectivity.

Lipase-catalyzed access to enantiomerically pure ( R)- and ( S)- trans-4-phenyl-3-butene-2-ol

The enzymatic kinetic resolution of ( RS)- trans-4-phenyl-3-butene-2-ol was investigated by screening a range of lipases both for enantioselective transesterification and for enantioselective hydrolysis of its acetate. The lipase from Pseudomonas cepacia immobilized on diatomaceous earth (PSL-D)-catalyzed asymmetric transesterification was performed on gram scale using isopropenyl acetate as an innocuous acyl donor in organic media affording the ( S)-alcohol in high enantiomeric excess (>99% ee) and enantiomeric ratio E >150. The lipase ( Candida antarctica B, CAL-B)-catalyzed asymmetric hydrolysis of the racemic acetate was performed on gram scale in phosphate buffer affording the ( R)-alcohol in high enantiomeric excess (>99% ee) and enantiomeric ratio E >150. The investigation demonstrates that the transesterification of the racemic alcohol in organic solvent was faster than the hydrolysis of the corresponding acetate in phosphate buffer. A GC method was developed to achieve an effective analytical separation of the enantiomers of both substrate and product in one analysis using the chiral stationary phase heptakis(2,3-di- O-methyl-6- O-tert-butyldimethylsilyl)-β-cyclodextrin.

How does active site water affect enzymatic stereorecognition?

The E value for ketone reduction catalyzed by a secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus is temperature dependent, due to a relatively large activation entropy difference, ΔΔS‡, which favors the formation (or reaction) of the (R)-enantiomer of 2-butanol and 2-pentanol. In contrast, the activation enthalpy, ΔΔH‡, favors the formation of the (S)-enantiomer of 2-butanol and 2-pentanol. C295A mutant SADH shows very large reductions in ΔΔS‡ compared to wild-type SADH, in addition to the expected reductions in ΔΔH‡. The decrease in ΔΔH‡ can be readily explained on the basis of steric interaction and van der Waals contacts of the enantiomeric substrates in the active site. The comparison of ΔΔS‡ of the wild-type and C295A mutant SADH reduces the possible entropy contributions to those associated with the Cys-295 sulfhydryl group. Examination of the crystal structure shows an ordered water molecule in the small pocket, located at a distance of 4.1Å from the sulfur atom of Cys-295. Thus, the stereospecificity of SADH may be at least partly determined by the selective expulsion of this bound water from the small alkyl binding pocket upon binding of large substituents in the small alkyl pocket. The data obtained with wild-type, S39T and C295A SADH with NADP, SNADP and APADP show a reasonable enthalpy–entropy compensation relationship, suggesting a contribution of solvation. Our results suggest that selective release of bound solvent from the active site upon binding of enantiomeric substrates may contribute to the differential activation entropy, ΔΔS‡.

A comprehensive study of the active site residues of DT-diaphorase: rational design of benzimidazolediones as DT-diaphorase substrates.

A series of quinone substrates were modeled into the active site of human DT-diaphorase and minimized. Correlation of these models with the substrate specificity k(cat)/K(m) provided insights into the structural requirements of quinone substrates. The W105, F106, and H194 residues can influence the position of the quinone substrate in the active site resulting in formation of one of the two possible Michael anions resulting from hydride transfer from FADH(2). Electron withdrawing groups on the substrate can stabilize these anions resulting in excellent substrate specificity. Inspection of models indicated that the W-105 and F-106 residues form parallel walls that will accommodate large polycyclic substrates. Thus excellent polycyclic substrates of DT-diaphorase were designed. However, the placement of tetrahedral centers on these polycyclic substrates interfered with the W-105 and the F-106 residues resulting in their exclusion from the active site. The histidine (H194) residue permits recognition of substrate enantiomers as a result of hydrogen bonding interactions. As a result of this study, it will be possible to design poor to excellent substrates of DT-diaphorase and take advantage of varying levels of this enzyme in histologically different cancers.

Interfacial binding and activity of lipases at the lipid–water interface: effects of Gum Arabic and surface pressure

We investigated the effects of Gum Arabic (GA) on the interfacial binding and activity of human pancreatic lipase (HPL) on trioctanoylglycerol (TC8) emulsion. Using the pH-stat technique, in the presence of sodium taurodeoxycholate (NaTDC) at concentration of 0.5 mM or higher, GA had no effect on the HPL activity measured in the presence of colipase. However, in the absence of bile salts or at low bile salt concentration, GA inhibited the HPL activity when TC8 emulsion was used as substrate. At 3% (w/v, final concentration), GA strongly desorbed pure HPL from the TC8 interface and the classical anchoring effect of colipase was clearly observed. An excellent correlation was therefore observed between the interfacial binding and the lipase activity of HPL as measured on a TC8 emulsion. In conclusion, GA was found to be surface active and to have similar effects to those of bile salts on the interfacial binding and activity of HPL. Using the monomolecular film technique, coupled to an ELISA, the penetration of Humicola lanuginosa lipase (HLL) into monomolecular films of non-hydrolysable enantiomeric substrate analogues (1,2-didecanoyl-3-deoxyamino-3- O-methyl glycerol (R3) or 2,3-didecanoyl-3-deoxyamino-1- O-methyl glycerol (S3)) spread at the air–water interface was monitored at constant surface pressure. By combining the surface area variations and the surface excess of HLL, the cross-sectional area of HLL molecule was estimated and a model for its penetration/orientation into the monomolecular films of R3 or S3 was proposed. No significant difference was found in term of HLL molecular area when using these films, indicating that the chirality of the lipid has no significant effect on HLL penetration. Nevertheless, This penetration was found to be deeper at low surface pressures.

Enantioselectivity in Candida antarctica lipase B: a molecular dynamics study.

A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme-substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics-based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3-methyl-2-butanol with various aliphatic carboxylic acids and also 2-butanol, as well as 3,3-dimethyl-2-butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity.

Lactones 7: 1. Enantioselective lactonization of racemic ethyl (5,5-dimethyl-2,3-epoxycyclohex-1-yl)acetate

Seven fungi strains were checked as biocatalysts of lactonization of ethyl (5,5-dimethyl-2,3-epoxycyclohex-1-yl)acetate. Two of them transformed the racemic substrate with high efficiency and enantioselectivity. Rhodotorula rubra transformed preferentially the (−) enantiomer of substrate, whereas Fusarium semitectum, the (+) one.

The enantioselectivity of haloalkane dehalogenases

Two haloalkane dehalogenases were tested for their ability to perform kinetic resolutions of a series of racemic substrates and to convert meso substrates enantioselectively. For the kinetic resolutions E-values of up to 9 were measured and, in the conversions of the meso substrates, products were obtained with an enantiomeric excess of up to 47%. A kinetic analysis revealed that despite modest overall chiral recognition (expressed as E-values), there are large differences between the K m values (>100 fold) of two enantiomeric substrates but that these differences are compensated by correspondingly large differences in k cat.

Lipase-catalyzed irreversible transesterification of 1-(2-furyl)ethanol using isopropenyl acetate

Asymmetric acetylation of racemic 1-(2-furyl)ethanol with the innocuous acyl donor isopropenyl acetate catalyzed by lipases in organic media afforded the chiral alcohol and acetate in high enantiomeric excess (up to 99%). The effect of molecular sieves as well as organic solvents on the kinetic resolution were studied. An effective separation of the enantiomers of both substrate and product was performed using gas chromatography on the chiral stationary phase heptakis-(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin.

Design and realization of a tailor-made enzyme to modify the molecular recognition of 2-arylpropionic esters by Candida rugosa lipase

Within a research project aimed at probing the substrate specificity and the enantioselectivity of Candida rugosa lipase (CRL), computer modeling studies of the interactions between CRL and methyl (±)-2-(3-benzoylphenyl)propionate (Ketoprofen methyl ester) have been carried out in order to identify which amino acids are essential to the enzyme/substrate interaction. Different binding models of the substrate enantiomers to the active site of CRL were investigated by applying a computational protocol based on molecular docking, conformational analysis, and energy minimization procedures. The structural models of the computer generated complexes between CRL and the substrates enabled us to propose that Phe344 and Phe345, in addition to the residues constituting the catalytic triad and the oxyanion hole, are the amino acids mainly involved in the enzyme–ligand interactions. To test the importance of these residues for the enzymatic activity, site-directed mutagenesis of the selected amino acids has been performed, and the mutated enzymes have been evaluated for their conversion and selectivity capabilities toward different substrates. The experimental results obtained in these biotransformation reactions indicate that Phe344 and especially Phe345 influence CRL activity, supporting the findings of our theoretical simulations.

Enantioselective oxidation of amphetamine by copper-containing quinoprotein amine oxidases from Escherichia coli and Klebsiella oxytoca

The enantioselective properties of copper-containing quinoprotein amine oxidase (EC 1.4.3.6) from Escherichia coli K12 and Klebsiella oxytoca in the kinetic resolution of ( R, S)-1-phenyl-2-aminopropane, amphetamine, have been determined. Determination of the enantiomeric ratio, E=( k cat/ K M) R /( k cat/ K M) S , the ratio of specificity constants for the enantiomeric substrates, can be accomplished in several ways. For practical reasons, we calculated E using non-linear regression analysis of initial rate data obtained at a fixed overall concentration of amphetamine mixtures of chiral composition ranging from 0 to 50% ( R)-(−)-amphetamine [Jongejan et al., Recl. Trav. Chim. Pays-Bas 110 (1990) 247]. It is found that both enzymes catalyze the enantioselective oxidation of amphetamine with E-values of sufficient magnitude ( E≈15) which may open the possibility for future application of amine oxidase-catalyzed kinetic resolutions of racemic amphetamine. The preference for the ( R)-enantiomer of amphetamine is in agreement with the pro- S specificity that has been observed for the conversion of 2-phenylethylamine. Rationalization of this observation, based on the structure of the E. coli amine oxidase, is discussed.

Improving lipase enantioselectivity in organic solvents by forming substrate salts with chiral agents.

We recently demonstrated (J Am Chem Soc 121:3334-3340, 1999) that enzymatic enantioselectivity in organic solvents can be markedly enhanced by temporarily enlarging the substrate via salt formation. In the present study, this approach was expanded by finding that, in addition to its size, the stereochemistry of the counterion can greatly affect the enantioselectivity enhancement. For example, the enantioselectivity [E = (k(cat)/K(M))(S)/(k(cat)/K(M))(R)] of crystalline Pseudomonas cepacia lipase in the propanolysis of phenylalanine methyl ester (PheOMe) in anhydrous acetonitrile was found to be 5.8 +/- 0.6; the E value doubled when PheOMe's salt with S mandelic acid was used as a substrate instead of the free ester, and rose sevenfold with R mandelic acid as a Bronsted-Lowry acid. Similar effects were observed with other bulky, but not petite, counterions. The greatest enantioselectivity enhancement was afforded by 10-camphorsulfonic acid: the E value increased to 18 +/- 2 for a salt with its R enantiomer and jumped to 53 +/- 4 for the S. These effects, also observed in other organic solvents, were explained by means of structure-based molecular modeling of the lipase-bound transition states of the substrate enantiomers and their diastereomeric salts.

PH dependence of penicillin amidase enantioselectivity for charged substrates

The pH dependence of E (enantiomeric ratio or enantioselectivity, a quantitative measure for enzyme stereospecificity) was studied for penicillin amidase catalysed hydrolysis of charged enantiomeric substrates. Theoretical analysis shows that a pH dependence can only be observed around the p K values of groups in the active site whose ionisation control the enzyme activity. For charged substrates that may perturb these p K values, a pH dependence of E is also expected. This was experimentally verified around these p K values. The S′ 1-stereospecificity of penicillin amidase was studied for the hydrolysis of the enantiomeric phenylacetyl- S/ R-Phe and for the racemic phenylacetyl- S, R-PhG. The S 1-stereospecificity was investigated for the hydrolysis of the enantiomeric S/ R-PhG-NH 2. The observed pH modulation of E (more than 3-fold for the studied substrates in the pH range 4.5–9) was found to be a result of compensatory effects for binding and catalysis. The ratios k cat, S / k cat, R and K m, S / K m, R for the hydrolysis of the enantiomeric phenylacetyl-Phe were found to decrease from 1000 to 10 and from 0.1 to 0.01, respectively in the pH range 5–8. The dependence was stronger for the S′ 1- than for the S 1-subsite. This is probably due to the stronger influence of the substrate carboxyl group in the S′ 1-subsite than that of the substrate amino group in the S 1-subsite on the p K of the N-terminal Ser B1 that is essential for the activity. The observed pH dependence of E was used to discuss the importance of ground–state interactions for discrimination between enantiomers and for enzyme catalysis in general. The experimental results conform to the split site model according to which a better binding must not be fundamentally inhibitory.

Potential of enzymatic kinetic resolution using solid substrates suspension: improved yield, productivity, substrate concentration, and recovery

In the literature the enzymatic kinetic resolution of a suspension of a solid substrate has largely been treated as a conventional kinetic resolution of a fully dissolved substrate. In this paper it is shown that this type of kinetic resolution is different in several important aspects. Quantitative models are developed for two types of such suspension processes. These models are used to compare the merits of these processes with the conventional kinetic resolution process where fully dissolved substrate is used. In the suspension processes the liquid phase concentration of substrate enantiomer that should be converted can be kept close to the maximum value, i.e., the solubility, when process conditions are properly chosen, whereas in a conventional process this concentration gradually decreases. Calculations show that this leads to a productivity that is about 6-fold higher in the suspension processes. Also, for enzymes with a low enantioselectivity, a severalfold increase in yield of remaining enantiopure substrate is predicted compared to the conventional kinetic resolution of dissolved enantiomers. Other potential advantages of using suspension reactions are that the initial substrate concentration may be higher (up to 25% (w/w)) and that the desired remaining substrate may be recovered by simply filtering off the solid crystals. Experimental evidence that these merits can be exploited is only partly given, using the few available examples from the literature.

Oxidation of Ultrafast Radical Clock Substrate Probes by the Soluble Methane Monooxygenase from Methylococcus capsulatus(Bath)

Radical clock substrate probes were used to assess the viability of a discrete substrate radical species in the mechanism of hydrocarbon oxidation by the soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). New substituted cyclopropane probes were used with very fast ring-opening rate constants and other desirable attributes, such as the ability to discriminate between radical and cationic intermediates. Oxidation of these substrates by a reconstituted sMMO system resulted in no rearranged products, allowing an upper limit of 150 fs to be placed on the lifetime of a putative radical species. This limit strongly suggests that there is no such substrate radical intermediate. The two enantiomers of trans-1-methyl-2-phenyl-cyclopropane were prepared, and the regioselectivity of their oxidation to the corresponding cyclopropylmethanol and cyclopropylphenol products was determined. The results are consistent with selective orientation of the two enantiomeric substrates in the hydrophobic cavity at the active site of sMMO, specific models for which were examined by molecular modeling.

Markedly Enhancing Enzymatic Enantioselectivity in Organic Solvents by Forming Substrate Salts

A new approach is proposed to enhance enzymatic enantioselectivity in organic solvents. It is based on the presumption that the less reactive substrate enantiomer experiences greater steric hindran...