Preparation of (R) and (S) alpha methyldopa from a chiral hydantoin containing the alpha phenylethyl group

Chiral hydantoin (S)-1 was prepared in good yield from phenyl isocyanate and N-[(S)-alpha-phenylethyl]glycinate, (S)-3. Enolate (S)-1-Li was methylated in high yield and good diastereoselectivity. In contrast, a second alkylation reaction of methylated enolate (S)-4-Li proceeded with essentially no diastereoselectivity. Nevertheless, dialkylated hydantoins, (S,S)-7 and (S,R)-7, could be readily separated by flash chromatography and subsequent hydrolysis of either derivative afforded the desired (S)-L-alpha-methyldopa or (R)-D-alpha-methyldopa in good yield.     

Mechanism of enantioselective protonation of an enolate in the presence of a chiral phenolic additive

The synthesis of enantiomerically enriched (+)-(R)-2-methyl-1-tetralone 1 (up to 29% e.e.) was achieved by enantioselective protonation of the achiral enolate such as 2, using the oxazoline (S)-3 as the source of chirality. Copyright 1999 Wiley-Liss, Inc.     

Synthetic applications of enantioselective protonation and case study for (S)-alpha-damascone

Among the fragrance compounds synthesized by enantioselective protonation, (S)-alpha-damascone, (R)-muscone, and (S,S)-Vulcanolide are the most prominent ones. (S)-alpha-damascone has been prepared by four different procedures: from the magnesium enolate, from the lithium enolate, from the enol, and from the corresponding thiol ester. We now present a new, industrially viable protocol for the addition of allyl magnesium chloride to the 'cyclogeranoketene' by a Barbier reaction, followed by protonation of the ensuing magnesium enolate by an aggregate formed from (-)-N-isopropylephedrine, lithium isopropylate, and acetic acid, furnishing (S)-alpha-damascone in 91% yield and with 71% ee.     

Inhibitors designed for the active site of dihydroorotase

Four new compounds have been synthesized as potential inhibitors of dihydroorotase from Escherichia coli. NMR spectroscopy was used to show that 4,6-dioxo-piperidine-2(S)-carboxylic acid (3), exists in solution as a mixture of the hydrate (7), enol (8), and enolate (9) tautomeric forms. This compound was found to be a competitive inhibitor versus dihydroorotate and thio-dihydroorotate at pH values of 7-9. The K(i) of 76 microM was lowest at pH7.0 where the ketone and hydrate forms of the inhibitor 3 predominate in solution. Compound 3 was reduced to the two diastereomeric 4-hydroxy derivatives (4 and 5) and then dehydrated to yield the alkene derivative, 1,2,3,6-tetrahydro-6-oxopyridine-2(S)-carboxylic acid (6). Compounds 4-6 were competitive inhibitors versus thio-dihydroorotate at pH 8.0 with K(i) values of 3.0, 1.6, and 2.3 mM. Dihydroorotase was unable to dehydrate the 4-hydroxy derivative 4 or 5 to the alkene 6 or catalyze the reverse reaction.     

EPR and Mössbauer studies of benzoyl-CoA reductase

Benzoyl-CoA reductase catalyzes the two-electron transfer from a reduced ferredoxin to the aromatic ring of benzoyl-CoA; this reaction is coupled to stoichiometrical ATP hydrolysis. A very low reduction potential (less than -1 V) is required for the first electron transfer to the aromatic ring. In this work the nature of the redox centers of purified benzoyl-CoA reductase from Thauera aromatica was studied by EPR and M?ssbauer spectroscopy. The results obtained indicated the presence of three [4Fe-4S] clusters. Redox titration studies revealed that the reduction potentials of all three clusters were below -500 mV. The previously reported S = 7/2 state of the enzyme during benzoyl-CoA-independent ATPase activity (Boll, M., Albracht, S. J. P., and Fuchs, G. (1997) Eur. J. Biochem. 244, 840-851) was confirmed by M?ssbauer spectroscopy. Inactivation by oxygen was associated with the irreversible conversion of part of the [4Fe-4S] clusters to [3Fe-4S] clusters. Acetylene stimulated the benzoyl-CoA-independent ATPase activity and induced novel EPR signals with g(av) >2. The presence of simple cubane clusters in benzoyl-CoA reductase as the sole redox-active metal centers demonstrates novel aspects of [4Fe-4S] clusters since they adopt the role of elemental sodium or lithium which are used as electron donors in the analogous chemical Birch reduction of aromatic rings.     

Stereoselective Syntheses of (-)-Podorhizol Lignan and its Derivatives: erythro and threo Preferential Aldol Condensation of Potassium Enolate from γ-Butyrolactone with Alkoxybenzaldehyde

(-)-Podorhizol (1) was stereoselectively synthesized by erythro preferential aldol condensation of 3,4,5-trimethoxy- benzaldehyde with potassium enolate from (+)-(R)-3- (3,4-methylenedioxybenzyl)-4-butanolide (2) (erythro:threo=85:15). Erythro selectivity was observed in the aldol condensation of many alkoxybenzaldehydes with potassium enolate from (+)-γ-butyrolactone 2. However, benzaldehydes having methoxy groups at both the 2 and 6 positions gave threo selectivity in the aldol condensation with potassium enolate from (+)-γ-butyrolactone 2.     

Stereoselective synthesis of 2,3,7-trimethylcyclooctanone and related compounds and determination of their relative and absolute configurations by the MalphaNP acid method

The copper/chiral phosphoramidite (L(1))-catalyzed conjugate addition of dimethylzinc to cycloocta-2,7-dienone 4, followed by the methylation of the intermediate enolate, yielded a single isomer of 7,8-dimethylcyclooct-2-enone (+)-5. Compound (+)-5 was subjected to the second conjugate addition with ent-L(1) giving only one stereoisomer of 2,3,7-trimethylcyclooctanone (+)-6, which was converted to 2,3,7-trimethylcyclooctanol 7. To determine the relative and absolute configurations of these compounds, the (1)H NMR anisotropy method using (S)-(+)-2-methoxy-2-(1-naphthyl)propionic acid {(S)-(+)-MalphaNP acid} 1 was applied. Racemic alcohol (+/-)-7 was esterified with (S)-(+)-MalphaNP acid 1 yielding diastereomeric esters, which were efficiently separated by HPLC on silica gel affording the first-eluted MalphaNP ester (-)-10a and the second-eluted one (-)-10b. The relative and absolute configurations of ester (-)-10a were determined to be (S;1R,2S,3R,7S) by analyzing the (1)H and (13)C NMR spectra of (-)-10a and (-)-10b, especially their HSQC-TOCSY and NOESY spectra, and by applying the MalphaNP anisotropy method. The alcohol 7 formed from (+)-6 was similarly esterified with (S)-(+)-MalphaNP acid 1 yielding an MalphaNP ester, which was identical with (-)-10a, and the relative and absolute configurations of 2,3,7-trimethylcyclooctanone (+)-6 were determined to be (2S,3R,7S).     

Energetics of the proposed rate-determining step of the glyoxalase I reaction.

The proposed rate-limiting step of the reaction catalyzed by glyoxalase I is the proton abstraction from the C1 carbon atom of the substrate by a glutamate residue, resulting in a high-energy enolate intermediate. This proton transfer reaction was modelled using molecular dynamics and free energy perturbation simulations, with the empirical valence bond method describing the potential energy surface of the system. The calculated rate constant for the reaction is approximately 300-1500 s(-1) with Zn2+, Mg2+ or Ca2+ bound to the active site, which agrees well with observed kinetics of the enzyme. Furthermore, the results imply that the origin of the catalytic rate enhancement is mainly associated with enolate stabilization by the metal ion.     

Novel oxorhenium and oxotechnetium MO(NS)(S)2 complexes in the development of 5-HT1A receptor imaging agents

The [NS][S](2) mixed-ligand system was applied to synthesize oxorhenium and oxotechnetium complexes of the general formula MO(o-CH(3)OC(6)H(4)N(CH(2)CH(2))(2)NCH(2)CH(2)S)(p-CH(3)C(6)H(4)S)(2) (M=Re in 1, M=(99)Tc in 2, and M=(99m)Tc in 3). The bidentate [NS] ligand includes the 1-(2-methoxyphenyl)piperazine moiety which is a fragment of the true 5-HT(1A) antagonist WAY 100635. The oxorhenium complex 1 was prepared by a ligand exchange reaction using ReOCl(3)(PPh(3))(2) as precursor while [Bu(4)N][(99)TcOCl(4)] and (99)Tc-gluconate were used as precursors in the synthesis of the oxotechnetium-99 complex 2. Both complexes were characterized by elemental analysis and spectroscopic methods. Crystallographic analysis of 1 showed that the rhenium coordination geometry is trigonal bipyramidal. The basal plane of the trigonal bipyramid is defined by the oxo group and two sulphur atoms, one belonging to the [NS] ligand and the other to an aromatic thiol, while the apical positions are occupied by the nitrogen of the [NS] ligand and the sulphur of the second aromatic thiol. The oxotechnetium-99 complex 2 has almost identical unit cell parameters to those of the oxorhenium complex 1 indicating, in combination with the other analytical data, that the complexes are isostructural. The binding affinity of the oxorhenium complex 1 for the 5-HT(1A) receptor subtype was determined in rat brain hippocampal preparations (IC(50)=106 nM). The oxotechnetium-99m complex 3 was prepared by a ligand exchange reaction using (99m)Tc-glucoheptonate as the precursor. Its structure was established by comparative HPLC studies using the oxotechnetium-99 complex 2 as a reference. Complex 3 was administered by intravenous injection in rats. At 2 min post injection, 0.153% of the injected dose per gram of tissue was measured in rat brain.     

Intramolecular aldol cyclization of C-4-ulopyranosyl-2'-oxoalkanes controlled by steric effects. Asymmetric synthesis of substituted 8-oxabicyclo[3.2.1]octanones and -octenones and cyclopentenones

Whereas C-2- and 4-ulopyranosyl compounds (C-2- and C-4-ulosides) can be converted to cyclopentenones under base conditions through beta-elimination and ring contraction, base-initiated beta-elimination of C-glycosyl 2'-aldehydes and 2'-ketones results in the formation of acyclic alpha,beta-unsaturated aldehydes or ketones. By combining both molecular features we synthesized 1-C-(4-ulopyranosyl)-2-oxoalkanes 6, 13, and 20 and investigated their reactions when they were treated with base. Both alpha- and beta-anomers of C-(4-ulopyranosyl)acetaldehydes 6 and 13 underwent a fast intramolecular aldol reaction between the C-5 enolate and 2'-aldehyde to form optically pure 8-oxabicyclo[3.2.1]octanones, which further transformed to 8-oxabicyclo[3.2.1]octenones 14 and 15 by beta-elimination. However, this aldol reaction did not occur when 1-C-(4-ulopyranosyl)propan-2-one 20 was treated with base because of steric hindrance exerted by the additional methyl group. Instead, an alternate C-3 enolization led to beta-elimination and further electro-ring opening to form an acyclic enol, which was then converted through a disrotatory intramolecular aldol cyclization to a cis-substituted cyclopentenone 21.     

Chiral alcohol production by NADH-dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH.

Phenylacetaldehyde reductase (PAR) produced by styrene-assimilating Corynebacterium strain ST-10 was used to synthesize chiral alcohols. This enzyme with a broad substrate range reduced various prochiral aromatic ketones and beta-ketoesters to yield optically active secondary alcohols with an enantiomeric purity of more than 98% enantiomeric excess (e.e.). The Escherichia coli recombinant cells which expressed the par gene could efficiently produce important pharmaceutical intermediates; (R)-2-chloro-1-(3-chlorophenyl)ethanol (28 mg.mL-1) from m-chlorophenacyl chloride, ethyl (R)-4-chloro-3-hydroxy butanoate) (28 mg.mL-1) from ethyl 4-chloro-3-oxobutanoate and (S)-N-tert-butoxycarbonyl(Boc)-3-pyrrolidinol from N-Boc-3-pyrrolidinone (51 mg.mL-1), with more than 86% yields. The high yields were due to the fact that PAR could concomitantly reproduce NADH in the presence of 3-7% (v/v) 2-propanol in the reaction mixture. This biocatalytic process provided one of the best asymmetric reductions ever reported.     

The sterochemistry at carbon 3 of pyruvate lyase condensation products. 2-Keto-3-deoxygluconate 6-phosphate and 2-keto-3-deoxygalactonate-6-phosphate aldolase of Pseudomonas saccharophila.

In Pseudomonas saccharophila 2-keto-3-deoxygalactonate-6-P aldolase (EC 4.1.2.21) is induced by growth on galatose while 2-keto-3-deoxygluconate-6-P aldolase (EC 4.1.2.14) is constitutive. These enzymes catalyze identical reactions except for the configuration fixed at C-4 during the condensation reaction. It was found with each enzyme that in a condensation between [3-3H3]pyruvate and D-glyceraldehyde-3-P, the respective condensation products were formed 8 to 10 times faster than tritium was released to water. Since pyruvate deprotonation is obligatory for condensation, the above result requires a hydrogen isotope effect in enolpyruvate formation, which must be then at least partially rate limiting for C--C synthesis. Further, condensation between D-glyceraldehyde-3-P and (3R)-[3-3H, 2H,H]pyruvate or (3S)-[3-3H, 2H,H]pyruvate, as catalyzed by each enzyme, enriched for (3R)- and (3S)-3-3H, 2H-labeled condensation product, respectively. Thus, each enzyme catalyzes C--C and C--H synthesis with retention of configuration at C-3. This shows that the active sites of both enzymes are asymmetric since solutes can only approach a single face of the bound pyruvyl enolate. In addition, the respective aldehyde specific portions of the two active sites must have opposite chiralities, with respect to each other, for correctly orienting the carbonyl faces of the incoming D-glyceraldehyde-3-P, to generate the correct configuration at C-4 of the respective condensation products.     

Hydrolysis of phosphoenolpyruvate catalyzed by phosphoenolpyruvate carboxylase from Zea mays.

In addition to the normal carboxylation reaction, phosphoenolpyruvate carboxylase from Zea mays catalyzes a HCO3(-)-dependent hydrolysis of phosphoenolpyruvate to pyruvate and Pi. Two independent methods were used to establish this reaction. First, the formation of pyruvate was coupled to lactate dehydrogenase in assay solutions containing high concentrations of L-glutamate and aspartate aminotransferase. Under these conditions, oxalacetic acid produced in the carboxylation reaction was efficiently transaminated, and decarboxylation to form spurious pyruvate was negligible. Second, sequential reduction of oxalacetate and pyruvate was achieved by initially running the reaction in the presence of malate dehydrogenase with NADH in excess over phosphoenolpyruvate. After the reaction was complete, lactate dehydrogenase was added, thus giving a measure of pyruvate concentration. At pH 8.0 in the presence of Mg2+, the rate of phosphoenolpyruvate hydrolysis was 3-7% of the total reaction rate. The hydrolysis reaction catalyzed by phosphoenolpyruvate carboxylase was strongly metal dependent, with rates decreasing in the order Ni2+ greater than Co2+ greater than Mn2+ greater than Mg2+ greater than Ca2+. These results suggest that the active site metal ion binds to the enolate oxygen, thus stabilizing the proposed enolate intermediate. The more stable the enolate, the less reactive it is toward carboxylation and the greater the opportunity for hydrolysis.     

Asymmetric aldol reaction of enol trichloroacetate catalyzed by (S, S)-(EBTHI)TiCl(OMe)

(S,S)-Ethylenebis(tetrahydroindenyl)titanium chloride methoxide, (S, S)-(EBTHI)TiCl(OMe) (3) was synthesized from the corresponding titanium dichloride. The asymmetric aldol reaction of enol trichloroacetate of cyclohexanone 1 with aromatic aldehydes was studied in the presence of a catalytic amount of the chiral titanium complex 3, with the result that the optically active syn aldol adduct 2 was preferentially obtained with up to 91% ee.     

Evolution of enzymatic activity in the enolase superfamily: functional studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis

o-Succinylbenzoate synthase (OSBS) from Amycolatopsis, a member of the enolase superfamily, catalyzes the Mn2+-dependent exergonic dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate (SHCHC) to 4-(2'-carboxylphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB) in the menaquinone biosynthetic pathway. This enzyme first was identified as an N-acylamino acid racemase (NAAAR), with the optimal substrates being the enantiomers of N-acetyl methionine. This laboratory subsequently discovered that this protein is a much better catalyst of the OSBS reaction, with the value of k(cat)/K(M), for dehydration, 2.5 x 10(5) M(-1) s(-1), greatly exceeding that for 1,1-proton transfer using the enantiomers of N-acetylmethionine as substrate, 3.1 x 10(2) M(-1) s(-1) [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-8]. The efficiency of the promiscuous NAAAR reaction is enhanced with alternate substrates whose structures mimic that of the SHCHC substrate for the OSBS reaction, for example, the value of k(cat)/K(M) for the enantiomers of N-succinyl phenylglycine, 2.0 x 10(5) M(-1) s(-1), is comparable to that for the OSBS reaction. The mechanisms of the NAAAR and OSBS reactions have been explored using mutants of Lys 163 and Lys 263 (K163A/R/S and K263A/R/S), the putative acid/base catalysts identified by sequence alignments with other OSBSs, including the structurally characterized OSBS from Escherichia coli. Although none of the mutants display detectable OSBS or NAAAR activities, K163R and K163S catalyze stereospecific exchange of the alpha-hydrogen of N-succinyl-(S)-phenylglycine with solvent hydrogen, and K263R and K263 catalyze the stereospecific exchange the alpha-hydrogen of N-succinyl-(R)-phenylglycine, consistent with formation of a Mn2+-stabilized enolate anion intermediate. The rates of the exchange reactions catalyzed by the wild-type enzyme exceed those for racemization. That this enzyme can catalyze two different reactions, each involving a stabilized enediolate anion intermediate, supports the hypothesis that evolution of function in the enolase superfamily proceeds by pathways involving functional promiscuity.     

Synthesis of 2-thiohydantoins and their S-glucosylated derivatives as potential antiviral and antitumor agents

A series of 3-alkyl-5-((Z))-arylidene-2-thiohydantoins 4a-1 were synthesized from the direct condensation of the aromatic aldehydes with 3-alkyl-2-thiohydantoins 3a-c, which in turn were prepared from the reaction of glycine (1) and alkyl isothiocyanates 2a-c. The alkylation of 4a-1 with methylthioethyl chloride gave 5-((Z))-arylidene-3-alkyl-S-(2-methylthioethyl)-2-thiohydantoins 5a-e. S-Glucosylation took place on the reaction of 4a-1 with 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide under anhydrous alkaline conditions. These structures have been confirmed from a model study of the coupling of 4a with methylthioethyl chloride and alpha-D-glucose pentaacetate, respectively under Lewis acid conditions.     

Tartrate dehydrogenase reductive decarboxylation: stereochemical generation of diastereotopically deuterated hydroxymethylenes

Tartrate dehydrogenase catalyzes the reductive decarboxylation of meso-tartrate to glycerate. Concomitant with the ketonization of the intermediate enolate the C3 hydroxymethylene of glycerate necessarily acquires a proton from solvent. In D2O, the proton is shown to be added stereospecifically to form (2R,3R)-[3-2H]glycerate. The 1H-NMR assignments of the diastereotopic C3 protons of glycerate were confirmed by the enzymatic conversion of [1R-2H]fructose-6-phosphate to (2R,3R)-[3-2H]glycerate. The decarboxylation-protonation occurs with retention of configuration, implying that the general acid is positioned on the same face of the intermediate as the departing carboxylate. The stereochemically pure (2R,3R)-[3-2H]glycerate is readily synthesized and serves as a chiral hydroxymethylene synthon as demonstrated by the synthesis of (2S,3R)-[3-2H]serine.     

4-Amino-2-alkyl-butyramides as small molecule CCR2 antagonists with favorable pharmacokinetic properties

A systematic examination of the central aromatic portion of the lead (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-(4-fluorophenyl)-4-(1'H-spiro[indene-1,4'-piperidin]-1'-yl)butanamide (9) led to the discovery of a novel class of CCR2 receptor antagonists, which carry small alicyclic groups such as cyclopropyl, cylobutyl, or cyclopropylmethyl attached at C2 of the carbon backbone. The most potent compound discovered, namely (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-cyclopropyl-4-[(1R,3'R)-3'-methyl-1'H-spiro[indene-1,4'-piperidin]-1'-yl]butanamide (29), showed very high binding affinity (IC50 = 4 nM, human monocyte) and excellent selectivity toward other related chemokine receptors. The excellent pharmacokinetic profile of this new lead compound allows for extensive in vivo evaluation.     

Total synthesis and biological evaluation of (-)-pectinatone employing a methyl-branched wax ester as key building block

Unnatural (-)-pectinatone ((-)-3) was prepared in five steps starting from the highly methyl-branched wax ester 4, employing bromination of the ester enolate and subsequent base-induced elimination to the enoate 6 as the key step. Both (-)-3 and the amides 8b and 8c, which were isolated as by-products in the reaction sequence, displayed antimicrobial activity and cytotoxicity.     

Synthesis of glycosides of 3-deoxy-4-thiopentopyranosid-2-uloses and their reduction products: 3-deoxy-4-thiopentopyranosides

Michael addition of common thiols to the enone system of (2S)-2-benzyloxy-2H-pyran-3(6H)-one (1) afforded the corresponding 3-deoxy-4-thiopentopyranosid-2-ulose derivatives (2-4). The reaction was highly diastereoselective, and the addition was governed by the quasiaxially disposed 2-benzyloxy substituent of the starting pyranone. As expected from the enantiomeric excess of 1 (ee > 86%) the corresponding thiouloses 2-4 exhibited the same optical purity. However, the enantiomerically pure thioulose 5 was obtained by reaction of 1 with the chiral thiol, N-(tert-butoxycarbonyl)-L-cysteine methyl ester. The thio derivative 7 was also synthesized by reaction of 6 (enantiomer of 1) with the same chiral thiol. Alternatively, 4-thiopent-2-uloses 9-12 were prepared in high optical purity by 1,4-addition of thiols to (2S)-[(S)-2'-octyloxy]dihydropyranone 8. Similarly, reaction of 13 (enantiomer of 8) with benzenemethanethiol afforded 14 (enantiomer of 10). This way, the stereocontrol exerted by the anomeric center on the starting dihydropyranone led to 4-thiopentuloses of the D and L series. Sodium borohydride reduction of the carbonyl function of uloses 10 and 12 gave the corresponding 3-deoxy-4-thiopentopyranosid-2-uloses (16-19). The diastereomers having the beta-D-threo configuration (16, 18) slightly predominated over the beta-D-erythro (17, 19) analogues. However, the reduction of the enantiomeric pyranones 10 and 14 with K-Selectride was highly diastereofacial selective in favor of the beta-D- and beta-L-threo isomers 16 and 20, respectively.