Rigid nonproteinogenic cyclic amino acids as ligands for glutamate receptors: trans-tris(homoglutamic) acids

The second-generation asymmetric synthesis of the trans-tris(homoglutamic) acids reported herein proceeds via Strecker reaction of chiral ketimines, obtained from condensation of racemic 2-ethoxycarbonylmethylcyclopentanone and commercially available (S)- and (R)-1-phenylethylamine, respectively. In the key stereodifferentiating step, the cyanide addition leads to mixtures of diastereomeric alpha-amino nitrile-esters, the composition of which is independent of the reaction temperature and the type of the solvent, respectively. Hydrolysis of the alpha-amino nitrile-esters with concentrated H(2)SO(4) yielded diastereomeric mixtures of secondary alpha-amino amido-esters, which after separation were hydrogenolyzed and hydrolyzed each to the enantiomeric trans-1-amino-2-carboxymethylcyclopentanecarboxylic acids. Their configuration was completely established by NMR methods, CD spectra, and X-ray analysis of the trans-1S,2R-configured secondary alpha-amino amido-ester. In receptor binding assays and functional tests, trans-1S,2R-1-amino-2-carboxymethylcyclopentanecarboxylic acid hydrochloride was found to behave as a selective mGluR(2)-antagonist without relevant binding properties at iGluRs.     

Preparation of (25R)- and (25S)-26-functionalized steroids as tools for biosynthetic studies of cholic acids

A new synthesis of both epimeric forms of 26-cholestanoic acids and 26-alcohols containing a 3beta-hydroxy-Delta(5)- or a Delta(4)-3-keto-functionality in ring A is described starting from stigmasterol or (20S)-3beta-acetoxy-pregn-5-en-20-carboxylic acid. The obtained compounds are useful as standards for studies of cholic acids. Construction of the side chain was achieved by linkage of steroidal 23-iodides to sulfones prepared from (2R)- and (2S)-3-hydroxy-2-methylpropanoates. Oxidation of intermediate 26-alcohols into the corresponding carboxylic acids ensuring preservation of stereochemistry at C-25 and functional groups in the cyclic part was achieved with sodium chlorite catalyzed by TEMPO and bleach.     

Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.     

Chiral synthesis of (2S,3S)-2-(2-morpholin-2-yl-2-phenylmethoxy)phenol

Resolution of (2RS,3RS)-2-[alpha-(2-methoxymethoxyphenoxy)phenylmethyl]morpholine, 11, with (+) mandelic acid led to the formation of (+)-(2S,3S)-2-[alpha-(2-methoxymethoxyphenoxy)phenyl methyl] morpholine (11a). Compound 11 was synthesized in seven steps from (2RS,3RS)-cinnamyl alcohol-2,3-epoxide (4), with an overall yield of 17%. Cleavage of the methoxymethyl group of the Fmoc derivative 12 with catalytic amounts of p-toluenesulfonic acid in methanol afforded (+)-(2S,3S)-2-(2-morpholin-2-yl-2-phenylmethoxy)phenol 2. The synthetic utility as well as the configuration of compound 2 has been demonstrated by converting (S,S)-2-(2-morpholin-2-yl-2-phenylmethoxy)phenol 2 to (2S,3S)-2-[alpha-(2-ethoxyphenoxy)phenylmethyl]morpholine (1) and (2S,3S)-2-(2-methoxyphenoxy) benzyl)morpholine (16), two potential norepinephrine reuptake inhibitors under clinical evaluation.     

A straightforward stereoselective synthesis of meso-, (S,S)- and (R,R)-2,6-diaminopimelic acids from cis-1,4-diacetoxycyclohept-2-ene

A straightforward synthesis of meso-2,6-diaminopimelic acid (DAP) meso-1 was developed from 1,4-diacetoxycyclohept-2-ene (2) via an oxidative ring cleavage. Subsequently, an enantio-divergent synthesis of (S,S)- and (R,R)-1 was performed using a homochiral monoacetate 7 available from 2 by enzymatic desymmetrization.     

Enantioselective synthesis of (2R, 3S)- and (2S, 3R)-4,4,4-trifluoro-N-Fmoc-O-tert-butyl-threonine and their racemization-free incorporation into oligopeptides via solid-phase synthesis

An efficient method for the enantioselective synthesis of (2R, 3S)- and (2S, 3R)-4,4,4-trifluoro-N-Fmoc-O-tert-butyl-threonine on multigram scales was developed. Absolute configurations of the two stereoisomers were ascertained by X-ray crystallography. Racemization-free coupling conditions for the incorporation of tfT into oligopeptides were then explored. For solution-phase synthesis, tfT racemization was not an issue under conventional coupling conditions. For solid-phase synthesis, the following conditions were identified to achieve racemization-free synthesis: if tfT (3.0 equiv) was not the first amino acid to be linked to the resin (1.0 equiv), the condition is 2.7 equiv DIC/3.0 equiv HOBt as the coupling reagent at 0 degrees C for 20 h; if tfT (3.0 equiv) was the first amino acid to be linked to the resin (1.0 equiv), then 1.0 equiv of CuCl(2) needs to be added to the coupling reagent.     

Inhibition of African swine fever virus DNA synthesis by (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine

The acyclic nucleotide analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] is a potent and selective inhibitor of African swine fever virus (ASFV) replication. Using the DNA-DNA hybridization technique with plasmid pRPEL-2 as probe, we have shown that (S)-HPMPA exerts a specific, dose-dependent, inhibitory effect on viral DNA synthesis. Also, (S)-HPMPA inhibits the production of late viral proteins, especially IP-73, in ASFV-infected MS and Vero cells. When evaluated under the same experimental conditions, phosphonoacetic acid (PAA) also caused an inhibition of viral DNA and late viral protein synthesis but only so at a concentration which was 10- to 20-fold higher than that required for (S)-HPMPA.     

Partial resolution of racemic trans-4-[5-(4-alkoxyphenyl)-2,5-dimethylpyrrolidine-1-oxyl-2-yl]benzoic acids by the diastereomer method with (R)- or (S)-1-phenylethylamine

The partial resolution is described of a series of racemic trans-4-[5-(4-alkoxyphenyl)-2,5-dimethylpyrrolidine-1-oxyl-2-yl]benzoic acids (1), which are the key intermediates for the synthesis of chiral organic radical liquid crystalline compounds and are crystallized to give racemic compounds. Racemic acid 1 [(+/-)-1] with a long alkyl chain (C7 to C13) could be resolved by the conventional diastereomeric salt formation using (R)- or (S)-1-phenylethylamine (2) as the resolving agent, whereas resolution of (+/-)-1 with a short alkyl chain (C4 to C6) was unsuccessful. Use of six equiv of (R)- or (S)-2 for the initial diastereomeric salt formation of (+/-)-1 with a C7-C13 alkyl chain, followed by recrystallization of the resulting salts once or twice, gave 2S,5S- or 2R,5R-enriched 1, respectively, in an ee range of 75-92% and with an overall recovery of 11-27%, based on the original quantity of (+/-)-1.     

Asymmetric synthesis and teratogenic activity of (R)- and (S)-2-ethylhexanoic acid, a metabolite of the plasticizer di-(2-ethylhexyl)phthalate

The stereoselectivity of the teratogenic activity of 2-ethylhexanoic acid (EHXA), a metabolite of the widely-used plasticizer di-(2-ethylhexyl)phthalate, was investigated. The enantiomers of EHXA were prepared via asymmetric synthesis with the aid of the chiral auxiliaries (R)- and (S)-1-amino-2-(methoxymethyl)pyrrolidine (RAMP, SAMP). The aqueous solutions of the sodium salts of (R)- and (S)-EHXA and the racemic EHXA [+/- )-EHXA) were injected each morning and evening of day 7 and 8 of gestation in the NMRI mouse (500 mg/kg, i.p.), a period highly sensitive in regard to the production of neural tube defects (exencephaly) by branched-chain carboxylic acids. (S)-EHXA did not yield any teratogenic or embryotoxic response in this model, while (R)-EHXA was highly teratogenic (59% of living fetuses exhibited exencephaly) and embryotoxic (as indicated by embryolethality and fetal weight retardation); the exencephaly rate induced by (+/- )-EHXA was between those of the two enantiomeres (32%). It is therefore likely that stereoselective interactions of the enantiomers of EHXA with chiral molecules in the embryo are decisive in regard to the teratogenic response. This first example of the stereoselectivity of the teratological activity of an environmental pollutant suggests that the safety of man-made chemicals can be improved by the use of pure enantiomers instead of racemates.     

Enantioconvergent synthesis of (-)-(S)- and (+)-(R)-2-acetyl-3,6-dihydroxycyclohex-2-enone starting from rac-6-hydroxy-3-methoxycyclohex-2-enone

The synthesis of enantiomerically pure (-)-(S)- and (+)-(R)-2-acyl-3,6-dihydroxycyclohex-2-enone starting from diastereomerically pure N-tosyl-(S)-proline esters 3-methoxy-6-hydroxycyclohex-2-enone 1 is presented. An enantioconvergent synthesis of either (-)-(S)- and (+)-(R)-2-acyl-3,6-dihydroxycyclohex-2-enone starting with the racemic alpha-ketol 1 through a conversion of ( approximately 1:1) mixture of diastereomeric esters into one diastereomer by a repeated crystallization, followed by dimethylaminopyridine-catalyzed equilibration as key steps is described.     

Conformational studies on host-guest peptides containing chiral alpha-methyl-alpha-amino acids. Comparison of the helix-inducing potential of alpha-aminoisobutyric acid, (S)-2-ethylalanine and (S)-2-methylserine

The conformational behaviour of host-guest peptides of the type Ac-Ala-Xxx-Ala-Ala-Xxx-Ala-Ala-Xxx-Ala-Ala-NH-PEGM (Xxx = alpha-aminoisobutyric acid (Aib), (S)-2-ethylalanine ((S)-Iva), (S)-2-methylserine ((S)-alpha-MeSer)) has been studied by CD spectroscopy in CF3CH2OH, CH3OH, and water and by i.r. spectroscopy in CHCl3 and in the solid state. In this way the relative helix-inducing potential of the two chiral alpha-methyl-alpha-amino acids (S)-Iva and (S)-alpha-MeSer could be established in comparison to the strong helix-former Aib. The results show that (S)-Iva exerts a comparable helix-inducing effect as Aib, making this amino acid a valuable complementary tool for the stabilization or induction of helices. No significant helix-promoting effect was observed for (S)-alpha-MeSer in polar solvents; however, the i.r.-spectroscopic data in CHCl3 and in the solid state point to a helical conformation under these conditions. Possible reasons for the different behaviour of (S)-Iva and (S)-alpha-MeSer are briefly discussed.     

Diaryldimethylpiperazine ligands with mu- and delta-opioid receptor affinity: Synthesis of (+)-4-[(alphaR)-alpha-(4-allyl-(2S,5S)-dimethylpiperazin-1-yl)-(3-hydroxyphenyl)methyl]-N-ethyl-N-phenylbenzamide and (-)-4-[(alphaR)-alpha-(2S,5S)-dimethylpiperazin-1-yl)-(3-hydroxyphenyl)methyl]-N-ethyl-N-phenylbenzamide

We have explored the synthesis of compounds that have good affinity for both mu- and delta-opioid receptors from the (alphaR,2S,5S) class of diaryldimethylpiperazines. These non-selective compounds were related to opioids that have been found to interact selectively with mu- or delta-opioid receptors as agonists or antagonists. In our initial survey, we found two compounds, (+)-4-[(alphaR)-alpha-(4-allyl-(2S,5S)-dimethylpiperazin-1-yl)-(3-hydroxyphenyl)methyl]-N-ethyl-N-phenylbenzamide (14) and its N-H relative, (-)-4-[(alphaR)-alpha-(2S,5S)-dimethylpiperazin-1-yl)-(3-hydroxyphenyl)methyl]-N-ethyl-N-phenylbenzamide (15), that interacted with delta-receptors with good affinity, and, as we hoped, with much higher affinity at mu-receptors than SNC80. The relative configuration of the benzylic position in (+)-4-[(alphaR)-alpha-(4-allyl-(2S,5S)-dimethyl-1-piperazinyl)-(3-methoxyphenyl)methyl]-benzyl alcohol (10) was determined by X-ray crystallographic analysis of a crystal that was an unresolved twin. The absolute stereochemistry of that benzylic stereogenic center was unequivocally derived by the X-ray crystallographic analysis from the two other centers of asymmetry in the molecule that were known. Those were established from the synthesis via a dipeptide cyclo-L-Ala-L-Ala in which the absolute stereochemistry was established.     

Spacer length effect of a chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

A new chiral stationary phase (CSP) containing 11 methylene-unit spacer was prepared by bonding (+)-(18-crown-6)-2,3,11,12-carboxylic acid to aminoundecylsilica gel. The new CSP was superior to the one containing three methylene-unit spacer in the resolution of alpha-amino acids, beta-amino acids, amines, and amino alcohols in terms of both the separation (alpha) and the resolution factors (R(S)). In the resolution of alpha-amino acids on the new CSP containing a long spacer, the retention factors (k(1)) were quite small compared to those on the CSP containing a short spacer. However, in the resolution of relatively more lipophilic beta-amino acids, amines, and amino alcohols, the retention factors (k(1)) were generally greater on the CSP containing a long spacer than on the CSP containing a short spacer. All of these resolution behaviors have been rationalized by the effective competition of the ammonium ions (R-NH(3)(+)) generated by the residual undecylamino groups of the new CSP under acidic condition with the ammonium ions (R-NH(3)(+)) of analytes for the complexation inside the cavity of the crown ether ring of the CSP and the effective lipophilic interaction between the CSP and the relatively more lipophilic analytes.     

Primary deuterium isotope effects for the 3-methylaspartase-catalyzed deamination of (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, and (2S,3S)-3-ethylaspartic acid

3-Methylaspartate ammonia-lyase catalyzes the deamination of (2S)-aspartic acid 137 times more slowly than the deamination of (2S,3S)-3-methylaspartic acid but catalyzes the amination of fumaric acid 1.8 times faster than the amination of mesaconic acid [Botting, N.P., Akhtar, M., Cohen, M. A., & Gani, D. (1988) Biochemistry (preceding paper in this issue)]. In order to understand the mechanistic basis for these observations, the deamination reaction was examined kinetically with (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, (2S,3S)-3-ethylaspartic acid, and the corresponding C-3-deuteriated isotopomers. Comparison of the double-reciprocal plots of the initial reaction velocities for each of the three pairs of substrates revealed that the magnitude of the primary isotope effect on both Vmax and V/K varied with the substituent at C-3 of the substrate. 3-Methylaspartic acid showed the largest isotope effect (1.7 on Vmax and V/K), 3-ethylaspartic acid showed a smaller isotope effect (1.2 on Vmax and V/K), and aspartic acid showed no primary isotope effect at all. These results, which are inconsistent with earlier reports that there is no primary isotope effect for 3-methylaspartic acid [Bright, H. J. (1964) J. Biol. Chem. 239, 2307], suggest that for both 3-methylaspartic acid and 3-ethylaspartic acid elimination occurs via a predominantly concerted mechanism whereas for aspartic acid an E1cb mechanism prevails.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and biological effects of a beta2- or beta3-amino acid insertion in a peptide.

Molecular mechanics calculations on conformers of Ac-HGly-NHMe, Ac-beta2-HAla-NHMe and Ac-beta3-HAla-NHMe indicate that low-energy conformations of the beta-amino acids backbone, corresponding to gauche rotamers around the Calpha-Cbeta bond, may overlap canonical backbone conformers observed for alpha-amino acids. Therefore, Substance P (SP) was used as a model peptide to analyse the structural and biological consequences of the substitution of Phe7 and Phe8 by (R)-beta2-HPhe and of Gly9 by HGly (R)-beta2-HAla or (S)-beta3-HAla. [(R)-beta2-HAla9]SP has pharmacological potency similar to that of SP while [HGly9]SP and [(S)-beta3-HAla9]SP show a 30- to 50-fold decrease in biological activities. The three analogues modified at position 9 are more resistant to degradation by angiotensin converting enzyme than SP and [Ala9]SP. NMR analysis of these SP analogues suggest that a beta-amino acid insertion in position 9 does not affect the overall backbone conformation. Altogether these data suggest that [HGly9]SP, [(S)-beta3-HAla9]SP and [(R)-beta2-HAla9]SP could adopt backbone conformations similar to that of SP, [Ala9]SP and [Pro9]SP. In contrast, incorporation of beta2-HPhe in position 7 and 8 of SP led to peptides that are almost devoid of biological activity. Thus, a beta-amino acid could replace an alpha-amino acid within the sequence of a bioactive peptide provided that the additional methylene group does not cause steric hindrance and does not confine orientations of the side chain to regions of space different from those permitted in the alpha-amino acid.     

Total synthesis and antifungal activity of (2S,3R)-2-aminododecan-3-ol

We report the total synthesis of (2S,3R)-2-aminododecan-3-ol has been achieved starting from commercially available 10-undecenoic acid. The key steps involved are Sharpless asymmetric epoxidation, Miyashita's boron-directed C-2 regioselective azidolysis, generated the asymmetric centers and in situ detosylation and reduction of azido tosylate. The antifungal activity of the synthesized (2S,3R)-2-aminododecan-3-ol was evaluated on several Candida strains and was comparable to miconazole, a standard drug.     

基于多酶级联协调表达策略高效催化合成 (S)-2-羟基丁酸

2-羟基丁酸 (2-hydroxybutyric acid,2-HBA) 是合成生物可降解材料和各种药物的重要中间体,化学法合成的外消旋2-HBA需要去消旋才能获得光学纯对映异构体,应用于工业。文中通过在大肠杆菌Escherichia coli BL21(DE3) 中共表达苏氨酸脱氨酶 (Threonine deaminase,TD)、l-乳酸脱氢酶 (l-lactate dehydrogenase,LDH)和甲酸脱氢酶 (Formate dehydrogenase,FDH),构建 (S)-2-HBA的合成途径及其辅因子NADH的循环系统,实现了基于三酶级联反应催化底物l-苏氨酸合成 (S)-2-HBA。为了解决多酶级联催化反应中中间产物2-酮丁酸的生成速率和消耗率不匹配的问题,文中通过启动子工程策略来调控TD和FDH的表达水平,获得了多酶催化速率平衡的重组大肠杆菌P21285FDH-T7V7827。在5 L发酵罐水平,全细胞催化反应16 h,(S)-2-HBA的最高产量为143 g/L,摩尔转化率为97%,为迄今报道的最高产量的1.83倍,使其具有较强的工业化应用潜力。此外,结果表明,在单细胞中构建可调节的多酶协调表达系统对生物催化制备羟基酸类化合物具有重要意义。     

Stereospecific synthesis of (2S)-2-methyl-3-(2',6'-dimethyl-4'-hydroxyphenyl)-propionic acid (Mdp) and its incorporation into an opioid peptide

To examine the effect of replacing the N-terminal amino group in opioid peptides with a methyl group on biological activity, a stereospecific synthesis of the tyrosine analogue (2S)-2-methyl-3-(2',6'-dimethyl-4'-hydroxyphenyl)-propionic acid (Mdp) was performed. The enkephalin analogue (2S)-Mdp-D-Ala-Gly-Phe-Leu-NH2 turned out to be a quite potent delta opioid antagonist and a somewhat less potent mu antagonist, indicating that a positively charged N-terminal amino group is not a conditio sine qua non for the binding of opioid peptides to delta and mu receptors but may be required for signal transduction.     

Preparation of single-enantiomer biofunctional molecules with (S)-2-methoxy-2-(1-naphthyl)propanoic acid

(RS)-beta-Ionol and (RS)-2-methyl-4-octanol were resolved by using (S)-2-methoxy-2-(1-naphthyl)propanoic acid [(S)-MalphaNP acid]. The specific stereochemistry of each MalphaNP ester was elucidated by 2D NMR analyses, and shielding by the 1-naphthyl group was observed in both the 1H- and 13C-NMR spectra. Solvolysis of the individual (S)-MalphaNP esters gave four single-enantiomer alcohols. The normal-phase HPLC elution order of each MalphaNP ester is also discussed.     

Synthesis of enantiopure (S)-2-hydroxyphenylbutanoic acid using novel hydroxy acid dehydrogenase from Enterobacter sp. BK2K

Enterobacter sp. BK2K, screened from soil samples, can enantioselectively reduce 2-oxo-4-phenylbutanoic acid into (S)-2-hydroxy-4-phenylbutanoic acid. alpha-Hydroxy acid dehydrogenase (HADH) (specific activity 62.6 U/mg) was purified from the crude extract of Enterobacter sp. BK2K, and its gene was cloned and functionally expressed in E. coli BL21. The optimal pH and temperature for the HADH activity were 6.5 and 30 degrees C, respectively. The purified enzyme catalyzes the reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (S)-2-hydoxy carboxylic acids using NADH as cofactor. For example, the Km and kcat/Km for 2-oxo-4-phenylbutaonoic acid in the presence of 2 mM NADH were 6.8 mM and 350 M-1 min-1, respectively. For practical applications, a NADH recycle system employing the recombinant formate dehydrogenase from E. coli K12 was coupled with HADH in E. coli BL21. Using the recombinant HADH (110 U of 11 U/mg crude cell extract) and formate dehydrogenase (670 U of 67 U/mg crude cell extract) in 10 mL of 500 mM phosphate buffer (pH 6.5), 96 mM of (S)-phenyllactic acid (> 94% ee) and 95 mM of (S)-2-hydroxy-4-phenylbutanoic acid (> 94% ee) were produced in quantitative yields from 100 mM of phenylpyruvate and 2-oxo-4-phenylbutanoic acid.