Microbiological Systems in Organic Synthesis: Preparative-Scale Resolution of (RS)-Glaucine by Fusarium solani and Stereospecific Oxidation of (R)-(-)-Glaucine by Aspergillus flavipes

The destructive resolution of (6aR,S)-glaucine (Ic) was accomplished by oxidation of the (6aS)-(+)-enantiomer (Ia), using Fusarium solani ATCC 12823 to yield the unnatural alkaloid (6aR)-(-)-glaucine (Ib). Eighteen cultures were examined for their ability to metabolize the (6aR)-(-)-enantiomer (Ib), and Aspergillus flavipes ATCC 1030 was found to catalyze the stereoselective oxidation of this substrate to didehydroglaucine. Thus, it has been demonstrated that "R" and "S" organisms exist with regard to the oxidation of aporphines to didehydroaporphines.     

Metabolic Engineering of Escherichia coli for Production of Enantiomerically Pure (R)-(−)-Hydroxycarboxylic Acids

A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.     

Microbiological systems in organic synthesis: 1. The stereospecific oxidation of glaucine by Fusarium solani

Fusarium solani stereospecifically and quantitatively oxidizes S-(+)-glaucine to dehydroglaucine. R-(?)-glaucine was not metabolized, and R,S-glaucine was oxidized to the extent of 50%. The reaction proceeds without production of complicating side products. The conversion represents a form of microbial destructive resolution of an alkaloidal racemate.     

Optical Resolution by Preferential Crystallization of (RS)-2-Amino-3-(2-carboxyethylthio)propanoic Acid

Electrophilic additions of DL- and L-Cys to propenoic acid afforded (RS)- and (R)-2-amino-3-(2-carboxyethylthio)propanoic acids [(RS)- and (R)-ACE], respectively. (RS)-ACE was found to exist as a conglomerate based on its melting point, solubility, and infrared spectrum. (RS)-ACE was optically resolved by preferential crystallization to yield (R)- and (S)-ACE. The obtained (R)- and (S)-ACE were efficiently recrystallized from water, taking account of the solubility of (RS)-ACE, to give them in optically pure form.     

(1′R,2′S,5′R)-Menthyl-(5R)-acetoxy-1,3-oxathiolan-(2R)-carboxylate: Synthesis and isomeric purity determination by HPLC

Chemoselective reduction of one isomer of the 1-menthylester of 1,3-oxathiolan-5-one-2-carboxylic acid produces a mixture of four lactol diastereomers from which the title compound was isolated after acylation. The isomeric purity and absolute stereochemistry were determined by spectroscopic methods, chiral HPLC techniques, and conversion to (?)-2′-deoxy-3′-thiacytidine (Lamivudine, 3TC^TM). © 1994 Wiley-Liss, Inc.     

Synthesis of (R)- and (S)- muscone

(R)-(-)-Muscone (3-methylcyclopentadecanone, 1) the key perfumery component isolated from the male musk deer, Moschus moschiferus,* was synthesized from the easily available chiral building block, (R)-3-tert-butoxycarbonyl-2-methylpropanoic acid (2), by employing ring-closing olefin metathesis (RCM). Antipode (+)-1 was also synthesized in a similar manner from tert-butyl (S)-3-methoxycarbonylbutanoate (10). *(a) Walbaum, H. J. J. Prakt. Chem., 73, 488 (1906); (b) Ruzicka, L., Further considerations on the constitution of muscone. Helv. Chim. Acta, 9, 715, 1008-1017 (1926).     

Resolution of (R,S)‐ibuprofen catalyzed by immobilized Novozym40086 in organic phase

The enantioselective esterification of ibuprofen catalyzed by Novozym40086 was successfully conducted in organic solvent. Removing‐water reagent was added into the reaction mixture to remove water produced in the esterification. The effects of temperature, n‐hexanol concentration, ibuprofen concentration, and loading of enzymes were investigated. Under the condition of equilibrium, the thermodynamic equilibrium constant (K) of 7.697 and enantioselectivity (E) of 8.512 were obtained. The esterification reaction achieved its equilibrium in approximately 30 hours with conversion of 56% and ee_S of 93.78%. The predicted values of X and ee_S were 67.90% and 95.60%, respectively. The experimental value is approximately equal to the theoretical value, which indicates the feasibility of ideal models.     

GABAA agonists: Resolution and pharmacology of (+)- and (−)-isoguvacine oxide

(3SR,4RS)-3,4-Epoxypiperidine-4-carboxylic acid (isoguvacine oxide) is a potent and specific GABA_A receptor agonist. Isoguvacine oxide, originally designed as a potentially alkylating agonist, turned out to interact with the GABA_A receptor in a fully reversible manner. The protected form of isoguvacine oxide, benzyl (3SR,4RS)-1-(benzyloxycarbonyl)-3,4-epoxypiperidine-4-carboxylate ( 1 ) (Scheme 1), has now been resolved by chiral chromatography using cellulose triacetate as a chiral stationary phase. The enantiomers of 1 (ee ≥ 98.8%) were subsequently deprotected by hydrogenolysis. Whereas both enantiomers of isoguvacine oxide were inactive as inhibitors of the binding of [³H]GABA to GABA_B receptor sites (IC₅₀ > 100 μM), (+)-isoguvacine oxide (IC₅₀ = 0.20 ± 0.03 μM) and (?)-isoguvacine oxide (IC₅₀ = 0.32 ± 0.05 μM) showed comparable potencies as inhibitors of the binding of [³H]GABA to GABA_A receptor sites. Furthermore, (+)-isoguvacine oxide (EC₅₀ = 6 μM; 33% relative efficacy) and (?)-isoguvacine oxide (EC₅₀ = 5 μM; 38% efficacy relative to 10 μM muscimol) were approximately equipotent and equiefficacious as stimulators of the binding of [³H]diazepam to the GABA_A receptor-associated benzodiazepine site. This latter effect is an in vitro estimate of GABA_A agonist efficacy. These pharmacological data for isoguvacine oxide and its enantiomers do not seem to support our earlier conception of the topography of the GABA_A recognition site(s), derived from extensive structure—activity studies on GABA_A agonists. Thus, the model of the GABA_A recognition site(s) comprising a narrow cleft or pocket, in which the anionic moiety of the zwitterionic GABA_A agonists is assumed to be embedded during receptor activation, may have to be revised. © 1995 Wiley-Liss, Inc.     

Synthesis of (+)-(R)- and (−)-(S)-trans-8-hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)] aminotetralin: New 5-HT1A receptor ligands

(R,S)-trans-8-Hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)amino]tetralin 7 , a new radioiodinated ligand based on 8-OH-DPAT, was reported as a potential ligand for 5-HT_1A receptors. The optically active (+)-(R)- and (?)-(S)- 7 were prepared to investigate the stereoselectivity of (R,S)- 7 . Racemic intermediate 8-methoxy-2-N-n-propyltetralin was reacted with the acyl chloride of (?)-(R)-O-methylmandelic acid to form a mixture of (S,R)- and (R,R)-diastereoisomers, which were separated by flash column chromatography. After removing the N-acyl group from the diastereoisomers, the desired (+)-(R)-or (?)-(S)- 7 was obtained by adding an N-iodopropenyl group. In vitro homogenate binding studies showed the stereoselectivity of this new compound for 5-HT_1A receptors. (+)-(R)- 7 isomer displayed 100-fold higher affinity than the (?)-(S)- 7 isomer. Biochemical study indicated that (+)-(R)- 7 potently inhibited forskolin-stimulated adenylyl cyclase activity in hippocampal membranes (E_max and EC₅₀ were 24.5% and 5.4 nM, respectively), while (?)-(S)- 7 showed no effect at 1 μM. The radioiodinated (+)-(R)- and (?)-(S)-[¹²⁵I] 7 were confirmed by coelution with the resolved unlabeled compound on HPLC (reverse phase column PRP-1, acetonitrile/pH 7.0 buffer, 80/20). The active isomer, (+)-(R)-[¹²⁵I] 7 , displayed high binding affinity to 5-HT_1A receptors (K_d = 0.09 ± 0.02 nM). In contrast, the (?)-(S)- 7 isomer displayed a significantly lower affinity to the 5-HT_1A receptor (K_d > 10 nM). Thus, (+)-(R)-[¹²⁵I]trans-8-OH-PIPAT, (+)-(R)- 7 , an iodinated stereoselective 5-HT_1A receptor agonist, is potentially useful for study of in vivo and in vitro function and pharmacology of 5-HT_1A receptors in the central nervous system. © 1995 Wiley-Liss, Inc.     

Comparative toxicity of (+)-(R)- and (−)-(S)-1,2-dibromo-3-chloropropane

The haloalkane 1,2-dibromo-3-chloropropane (DBCP), an environmental pollutant that was widely used as a soil fumigant, is a carcinogen and a mutagen and displays target-organ toxicity to the testes and the kidneys. Because little is known about effects of stereochemistry on the metabolism and toxicity of halogenated alkyl compounds and because DBCP, which has a chiral center at C-2, may show enantioselectivity in its metabolism and/or toxicities, the optically pure enantiomers of DBCP were tested in vivo in rats for organ toxicity as well as for bacterial mutagenicity. Organ toxicity studies showed that (S)-DBCP was slightly more renal toxic than (R)-DBCP but was not significantly more toxic than the racemate, and that no significant differences were observed in the extents of testicular necrosis and atrophy caused by either enantiomer or the racemate. In contrast, (R)-DBCP was more mutagenic than either (S)-DBCP or the racemate to Salmonella typhimurium (S. typhimurium) strains TA 100 and TA104. However, there was little or no enantioselectivity in glutathione S-transferase (GST)-catalyzed conjugation reactions of glutathione with DBCP based on the lack of selectivity in the rates of disappearance of the enantiomers of DBCP in the presence of glutathione (GSH) and GSTs as monitored by chiral gas chromatography (GC). © 1995 Wiley-Liss, Inc.     

Diastereoselective synthesis of (R)-(alkyl)-beta-D-galactopyranoside by using beta-galactosidase (Aspergillus oryzae) in low-water media

A beta-galactosidase (from Aspergillus oryzae) preparation viz. EPRP (enzyme precipitated and rinsed with propanol), obtained by the removal of bulk water by precipitation with n-propanol, showed higher biological activity than the lyophilized powder. FT-IR study confirmed that EPRP had retained the alpha-helical content of the native structure better than the lyophilized form. Use of this formulation of beta-galactosidase under low water conditions (temperature 55 degrees C, reaction time of 4 h) gave enantioselectivity, E > 1000 for the stereoselective synthesis of (R)-(1-phenylethyl)-beta-D-galactopyranoside, starting from racemic 1-phenylethanol and D-galactose. For racemic 2-octanol also, EPRP worked better. Under similar conditions, (R)-(2-octyl)-beta-D-galactopyranoside was formed with an enantioselectivity, E = 38.     

Stereospecific oxidation of (R)- and (S)-1-indanol by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

A recombinant Escherichia coli strain which expresses naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized (S)-1-indanol to trans-(1S,3S)-indan-1,3-diol (95.5%) and (R)-3-hydroxy-1-indanone (4.5%). The same cells oxidized (R)-1-indanol to cis-1,3-indandiol (71%), (R)-3-hydroxy-1-indanone (18.2%), and cis-1,2,3-indantriol (10.8%). Purified NDO oxidized (S)-1-indenol to both syn- and anti-2,3-dihydroxy-1-indanol.     

Synthesis of α-(S)- acylamino-n-(hydroxydioxocyclobutenyl)- γ-lactams

Monocyclic γ - lactams 2, activated by a hydroxycyclobutenedione moiety have been prepared from (L)-N^tBoc-glutamine, as potential antibacterial agents.     

Microbiological hydroxylation of estradiol: formation of 2- and 4-hydroxyestradiol by Aspergillus alliaceus

Microorganisms known to hydroxylate alkaloids, amino acids, and aromatic substrates were examined for their potential to hydroxylate 17 beta-estradiol and estrone. Thin-layer chromatography of fermentation extracts revealed a wide range of steroid products. Aspergillus alliaceus (UI 315) was the only culture capable of producing good yields of catechol estrogens with 17 beta-estradiol. The organism also transformed estrone but not to catechol products. Analytical experiments with high-performance liquid chromatography revealed that A. alliaceus formed 4- and 2-hydroxyestradiol with yields of 45 and 16%, respectively. A preparative-scale incubation was conducted in 2 liters of medium containing 1 g of 17 beta-estradiol as substrate. 4-Hydroxyestradiol was isolated and identified by proton nuclear magnetic resonance and high-resolution mass spectrometry. Ascorbic acid was added to microbial reaction mixtures as an antioxidant to prevent the decomposition of unstable catechol estrogen metabolites. The microbial transformation of 17 beta-estradiol by A. alliaceus provides an efficient one-step method for the preparation of catechol estrogens.     

Synthesis and cytotoxicity of (R)‐ and (S)‐ricinoleic acid amides and their acetates

An environment‐friendly, free of solvent, process for the synthesis of (R )‐ and (S )‐ricinoleic acid amides has been developed. Starting from methyl ricinoleates and pyrrolidine or ethanolamine, the corresponding amides were obtained with yields ranging from 83–88%. Among 12 synthesized derivatives of ricinoleic acid, including the starting methyl esters, amides, and their acetates, nine compounds were obtained and tested for the first time. Studies on ricinoleic acid derivatives cytotoxicity showed that methyl esters were the least cytotoxic compounds and modification of their structure resulted in increasing cytotoxicity of the obtained products against both cancer cells and normal lymphocytes. Both enantiomers of the ethanolamine‐derived amides showed the most promising anticancer potential.     

Efficient Preparation of (R)‐2‐chloromandelic Acid Via a Recycle Process of Resolution

Efficient preparation of (R)‐2‐chloromandelic acid (R)-1 based on a recycle process of resolution is described. In the process, the desired (R)-1 was obtained by coordination‐mediated resolution with D‐O,O'‐di‐(p‐toluoyl)‐tartaric acid in the presence of Ca²⁺. Meanwhile, the undesired (S)-1 could be racemized in the presence of sodium hydroxide and the product was suitable for further resolution. A carbanion mechanism for the racemization of (S)-1 is proposed. Chirality 27:281–285, 2015. © 2015 Wiley Periodicals, Inc.     

Synthesis of (R)-2,3-epoxypropyl(1-->3)-beta-D-pentaglucoside

The title pentasaccharide was synthesized via a 2+3 strategy. The disaccharide donor, 3-O-acetyl-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (8), was obtained by selective coupling of allyl 2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranoside with 3-O-acetyl-2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (4), followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor, allyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranoside (12), was prepared by coupling of allyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranoside with 4, followed by deacetylation. Condensation of 8 with 12, followed by epoxidation, and deprotection, gave the target pentaoside.     

Toxic doses of rac-, (−)-(S)- and (+)-(R)-propranolol in rats and rabbits

The contribution of the individual enantiomers ([+]-[R]- and [−]-[S]-propranolol) to rac-propranolol intoxication was studied in anaesthetized, spontaneously breathing (SB) rats and artificially ventilated (AV) rats and rabbits. In the SB rat, propranolol (30 mg.kg⁻¹.h⁻¹ i.v.) decreased heart rate and mean arterial blood pressure and caused hypoventilation, serious hypoxaemia, respiratory acidosis, and death by respiratory arrest. Survival time (ST) in the (+)-(R)-propranolol group (ST 91 ± 5 min) was significantly longer than in the rac-propranolol group (ST 68 ± 6 min). In AV rats and rabbits toxic doses of rac-, (−)-(S)- and (+)-(R)-propranolol, 30 mg.kg⁻¹.h⁻¹ and 15 mg.kg⁻¹.h⁻¹ i.v., respectively, induced comparable effects on haemodynamic variables as in the SB rat. Artificial ventilation lengthened ST by a factor of three to four in rats. In the AV rat, ST's were not significantly different between the rac-, (−)-(S)- and (+)-(R)-propranolol groups. In the rabbit, as in the SB rat, ST in the (+)-(R)-propranolol group was significantly longer than ST's in the rac- and (−)-(S)-propranolol groups. The acute respiratory acidosis in SB rats and the prolonged ST in AV rats suggest that respiratory failure is the primary and cardiovascular failure the secondary cause of death in propranolol intoxication. The potentiation of the toxic effect of the enantiomers observed after dosing the racemate instead of the pure enantiomers could not be explained by a stereoselective difference in plasma propanolol concentration. © 1996 Wiley-Liss, Inc.     

Stereoselective Synthesis of (R)‐3‐Methylthalidomide by Piperidin‐2‐one Ring Assembly Approach

A simple and stereoselective synthesis of 3‐methylthalidomide, a configurationally stable thalidomide analog, is presented. Herein we describe the synthesis of (R)‐3‐methylthalidomide starting from (S)‐alanine by piperidin‐2‐one ring assembly approach in high yield and enantiomeric purity without using a chiral auxiliary or reagent. Starting from (R)‐alanine, the corresponding (S)‐3‐methylthalidomide can be prepared using the same methodology. Chirality 27:619–624, 2015. © 2015 Wiley Periodicals, Inc.     

Microbiological hydroxylation of estradiol: formation of 2- and 4-hydroxyestradiol by Aspergillus alliaceus.

Microorganisms known to hydroxylate alkaloids, amino acids, and aromatic substrates were examined for their potential to hydroxylate 17 beta-estradiol and estrone. Thin-layer chromatography of fermentation extracts revealed a wide range of steroid products. Aspergillus alliaceus (UI 315) was the only culture capable of producing good yields of catechol estrogens with 17 beta-estradiol. The organism also transformed estrone but not to catechol products. Analytical experiments with high-performance liquid chromatography revealed that A. alliaceus formed 4- and 2-hydroxyestradiol with yields of 45 and 16%, respectively. A preparative-scale incubation was conducted in 2 liters of medium containing 1 g of 17 beta-estradiol as substrate. 4-Hydroxyestradiol was isolated and identified by proton nuclear magnetic resonance and high-resolution mass spectrometry. Ascorbic acid was added to microbial reaction mixtures as an antioxidant to prevent the decomposition of unstable catechol estrogen metabolites. The microbial transformation of 17 beta-estradiol by A. alliaceus provides an efficient one-step method for the preparation of catechol estrogens.