d-Glucose- and d-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-d-glucoses and -d-mannoses

Modified d-glucose and d-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of d-glucose and d-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-d-glucal.     

Guinea pig serum l-asparaginase: Purification, and immunological relationship to liver l-asparaginase and serum l-asparaginases in other mammals

l-asparaginase, an enzyme used in the treatment of acute lymphocytic leukemia, is found in the serum of only a few mammalian groups, including the guinea pig and its close relatives in the superfamily Cavioidea. This report describes the purification and characterization of l-asparaginase from guinea pig serum. Antiserum against the purified enzyme cross-reacted with sera from other Cavioidean species but not with mouse serum. Relatively weak cross-reaction with unpurified l-asparaginase in guinea pig liver indicates a significant degree of evolutionary divergence.     

Construction and co-expression of polycistronic plasmid encoding d-hydantoinase and d-carbamoylase for the production of d-amino acids

d-Hydantoinase and d-carbamoylase genes from Agrobacterium radiobacter TH572 were cloned by polymerase chain reaction (PCR). The plasmid pUCCH3 with a polycistronic structure that is controlled by the native hydantoinase promoter was constructed to co-express the two genes and transformed into Escherichia coli strain JM105. To obtain the highest level of expression of the d-carbamoylase and avoid intermediate accumulation, the d-carbamoylase gene was cloned closer to the promoter and the RBS region in the upstream of it was optimized. This resulted in high active expression of soluble d-hydantoinase and d-carbamoylase that is obtained without any inducer. Thus, by the constitutive recombinant JM105/pUCCH3, d-p-hydroxyphenylglycine (d-HPG) was obtained directly with 95.2% production yield and 96.3% conversion yield.     

Single-crystal and powder X-ray diffraction and solid-state C NMR of p-nitrophenyl glycopyranosides, the derivatives of d-galactose, d-glucose, and d-mannose

The X-ray diffraction patterns, ¹³C CP MAS NMR spectra, and powder X-ray diffraction analyses were obtained for selected p-nitrophenyl glycosides: α- and β-d-galactopyranosides (1 and 2), α- and β-d-glucopyranosides (3 and 4), and α- and β-d-mannopyranosides (5 and 6). In X-ray diffraction analysis of 1 and 2, characteristic shortening and lengthening of selected bonds were observed in the molecules of 1 due to anomeric effect, and in the crystal lattice of 1 and 2, hydrogen bonds of complex network were detected. In the crystal asymmetric unit of 1 there were two independent molecules, whereas in 2 there was one molecule. For 1 and 3–6 the number of resonances in solid-state ¹³C NMR spectra exceeded the number of the carbon atoms in the molecules, while for 2 there were distinct singlet resonances in its solid-state NMR spectrum. Furthermore, the powder X-ray diffraction (PXRD) performed for 1–3 and 5 revealed that 1, 3, and 5 existed as single polymorphs proving that the doublets observed in appropriate solid-state NMR spectra were connected with two non-equivalent molecules in the crystal asymmetric unit. On the other hand 2 existed as a mixture of two polymorphs, one of them was almost in agreement with the calculated pattern obtained from XRD (the difference in volumes of the unit cells), and the subsequent unknown polymorph existed in small amounts and therefore it was not observed in solid-state NMR measurements.     

l-Xylose and l-lyxose production from xylitol using Alcaligenes 701B strain and immobilized l-rhamnose isomerase enzyme

Xylitol was used as a raw material for production of l-xylose and l-lyxose using Alcaligenes 701B strain and immobilized l-rhamnose isomerase enzyme. Alcaligenes 701B converted xylitol to l-xylulose with a yield of 34% in the bioreactor. l-Xylulose was converted to l-xylose and l-lyxose using immobilized l-rhamnose isomerase enzyme. The final equilibrium between l-xylulose, l-xylose and l-lyxose was 53:26:21. The enzyme assays indicated that Alcaligenes 701B strain has an NAD-dependent xylitol dehydrogenase enzyme responsible for l-xylulose production. Furthermore, NAD(P)H-dependent l-xylulose reductase enzyme was active during conversion of xylitol to l-xylulose. The highest l-xylulose production rate corresponded with the highest growth rate. The Alcaligenes 701B strain used d-xylose for biomass growth, but xylitol was used only for l-xylulose production during conversion phase.     

Growth of Rhodopseudomonas capsulata on l- and d-malic acid

d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.     

Enzymatic epimerization of d-erythro-dihydroneopterin triphosphate to l-threo-dihydroneopterin triphosphate

An enzyme has been discovered in Escherichia coli that catalyzes the conversion of the triphosphate ester of 2-amino-4-hydroxy-6-(d-erythro-1′,2′,3′-trihydroxypropyl)-7,8-dihydropteridine, (i.e. d-erythro-dihydroneopterin triphosphate) to an epimer of this compound, l-threo-dihydroneopterin triphophate. The enzyme, which is here named “d-erythro-dihydroneopterin triphosphate 2′-epimerase,” needs a divalent cation (Mg²⁺ or Mn²⁺ is most effective) for maximal activity. Its molecular weight is estimated at 87 000–89 000. Little or no activity can be detected if either the monophosphate or the phosphate-free form of the substrate is incubated with the enzyme. Evidence is presented to establish that all three phosphate residues of the substrate are retained in the product and that the product is of the l-threo configuration.     

Isomerization of d-fructose by base: Liquid-chromatographic evaluation and the isolation of d-psicose

Several bases have been evaluated as catalysts for the production of d-psicose (d-ribo-2-hexulose) from d-fructose. The hexose levels in the isomerized mixtures were quantified by l.c. on a μBondapak/Carbohydrate column. The most effective and convenient base was found to be pyridine, and mixtures produced by boiling concentrated solutions (1 g/mL) of d-fructose in pyridine under reflux contained 12.4% of psicose, lesser proportions of glucose and mannose, and 25.8% of the starting material. Following removal of solvent, fermentation with bakers' yeast removed most hexoses other than d-psicose, which was isolated by chromatography on cellulose. The entire procedure required three days, and d-psicose was obtained in gram quantities in 6.8% of the theoretical yield.     

Synthesis and antitumor activity of new d-seco and d-homo androstane derivatives

Starting from 3β-hydroxy-17-oxo-16,17-secoandrost-5-ene-16-nitrile (1), the new 16,17-secoandrostane derivatives 4–9 were synthesized. On the other hand, 3β-hydroxy-17-oxa-d-homoandrost-5-ene-16-one (10) yielded the new d-homo derivatives 12, 13 and 15. In vitro antiproliferative activity of selected compounds against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER−, MDA-MB-231, prostate cancer AR−, PC-3, and normal fetal lung fibroblasts, MRC-5) was evaluated. Compounds 3 and 12 showed strong antiproliferative activity against PC-3 cells, the IC₅₀ values being 2 μM and 0.55 μM, respectively. Compounds 6 (10 μM) and 14 (9 μM) showed moderate activity against MDA-MB-231 cells. The synthesized compounds 1–3, 5–8, 10 and 12–15 were not toxic to normal fetal lung fibroblasts cells, MRC-5.     

d-Amino acid production by E. coli co-expressed three genes encoding hydantoin racemase, d-hydantoinase and N-carbamoyl-d-amino acid amidohydrolase

Three genes respectively encoding d-specific hydantoinase (DHHase), N-carbamoyl-d-amino acid amidohydrolase (DCHase) and hydantoin racemase (HRase) were co-expressed in E. coli in a system designed for the efficient enzymatic production of d-amino acids via a combination of hydantoin hydrolysis and hydantoin racemization. With the use of whole cells, the d-forms of eight amino acids – d-phenylalanine, d-tyrosine, d-tryptophan, O-benzyl-d-serine, d-valine, d-norvaline, d-leucine and d-norleucine – were efficiently converted from the corresponding dl-5-monosubtituted hydantoin compounds.     

Enzymatic synthesis of d-xylulose 5-phosphate from hydroxypyruvate and d-glyceraldehyde-3-phosphate

An enzymatic method for obtaining d-xylulose 5-phosphate has been developed, based on the irreversible reaction catalyzed by transketolase: hydroxypyruvate + d-glyceraldehyde-3-phosphate → d-xylulose 5-phosphate. The preparations of sodium d-xylulose 5-phosphate, obtained using this approach, were 88% pure and contained no aldehyde admixtures.     

Enzymatic conversion of N carbamoyl-d-amino acids to d-amino acids

An enzyme isolated from Agrobacterium radiobacter was shown to catalyse the following reaction: H₂O + N-carbamoyl-d-amino acid→ d-amino acid + NH₃ + CO₂ Some properties of this new enzyme, N-carbamoyl-d-amino acid amidohydrolase, are presented in this paper. The potential application of this enzyme for the preparation of some d-amino acids used as pharmaceutical intermediates is discussed.     

Determination of two neuropeptide growth factor antagonists, [d-Arg,d-Phe,d-Trp,Leu]-substance P and [Arg, d-Trp,N-MePhe]-substance P(6–11), by high-performance liquid chromatography with electrochemical detection

n-MePhe⁸]-substance P(6–11) (G) are currently undergoing preclinical evaluation as potential anticancer agents and clinical trials are planned for G in the near future. A reversed-phase high-performance liquid chromatographic separation has been developed which is both sensitive (limit of detection 250 pg/263 fmol for G; 500 pg/330 fmol for D) and selective, based on electrochemical detection of the two tryptophan residues present in each peptide. Two ion-pairing agents were included in the isocratic mobile phase to eliminate adsorption of the peptides onto the analytical column. Extensive sample clean-up procedures have been developed for plasma, tissue and tumour based on solid-phase extraction. Precision and accuracy of each assay was 91.3 ± 16.9% (between-day) for G and 99.3 ± 16.9% (between-day) for D. The assays were able to detect the intact peptides and a number of their metabolites in plasma, liver and the WX 322 SCLC human xenograft in nude mice for at least 6 hr after administration of therapeutic and pharmacological doses.     

A short synthesis of d-glycero-d-manno-heptose 7-phosphate

d-glycero-d-manno-Heptopyranose 7-phosphate—an intermediate in the biosynthesis of nucleotide-activated heptoses—has been prepared in good overall yield from benzyl 5,6-dideoxy-2,3-O-isopropylidene-α-d-lyxo-(Z)-hept-5-enofuranoside by a short-step synthesis. Phosphitylation using the phosphoramidite procedure followed by in situ oxidation afforded the corresponding 7-O-phosphotriester derivative in high yield. Subsequent osmylation proceeded in good diastereoselectivity (4:1) to furnish the d-glycero-d-manno-configured derivative, which was separated from the l-glycero-l-gulo-isomer by chromatography. Hydrogenolysis led to simultaneous removal of the benzyl and isopropylidene groups and afforded the target compound in high yield, which serves as a substrate of bacterial heptose 7-phosphate kinases.