Biocatalytic Asymmetric Phosphorylation Catalyzed by Recombinant Glycerate-2-Kinase.

The efficient synthesis of pure d-glycerate-2-phosphate is of great interest due to its importance as an enzyme substrate and metabolite. Therefore, we investigated a straightforward one-step biocatalytic phosphorylation of glyceric acid. Glycerate-2-kinase from Thermotoga maritima was expressed in Escherichia coli, allowing easy purification. The selective glycerate-2-kinase-catalyzed phosphorylation was followed by 31 P NMR and showed excellent enantioselectivity towards phosphorylation of the d-enantiomer of glyceric acid. This straightforward phosphorylation reaction and subsequent product isolation enabled the preparation of enantiomerically pure d-glycerate 2-phosphate. This phosphorylation reaction, using recombinant glycerate-2-kinase, yielded d-glycerate 2-phosphate in fewer reaction steps and with higher purity than chemical routes.

Synthesis and Characterization of Dioctanoyl Glycerate as Water-soluble Trypsin Inhibitor.

Glyceric acids (GAs) esterified with long acyl chains (> C16) exhibit antitrypsin activity (Folia Microbiol. 46, 21-23 (2001)). However, their hydrophobic nature, derived from the long acyl chains, has limited the number of studies on their physical and biological properties. To improve the water solubility of diacyl GAs, GA was esterified with octanoyl groups (C8), and its physical properties were investigated. Synthesized dioctanoyl GA was not water-soluble, whereas its sodium salt was. Surface tension measurements of dioctanoyl GA sodium salt (diC8GA-Na) in water revealed that the critical micelle concentration (CMC) was 0.82 mM, and surface tension at the CMC was 25.5 mN/m. Additionally, diC8GA-Na inhibited casein digestion by trypsin to a greater extent than dioleoyl GA. These data suggest that water-soluble diacyl GAs may have utility as surfactants and bioactive compounds.

Synthesis and biological evaluation of enantiomerically pure glyceric acid derivatives as LpxC inhibitors.

Inhibitors of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represent a promising class of novel antibiotics, selectively combating Gram-negative bacteria. In order to elucidate the impact of the hydroxymethyl groups of diol (S,S)-4 on the inhibitory activity against LpxC, glyceric acid ethers (R)-7a, (S)-7a, (R)-7b, and (S)-7b, lacking the hydroxymethyl group in benzylic position, were synthesized. The compounds were obtained in enantiomerically pure form by a chiral pool synthesis and a lipase-catalyzed enantioselective desymmetrization, respectively. The enantiomeric hydroxamic acids (R)-7b (Ki=230nM) and (S)-7b (Ki=390nM) show promising enzyme inhibition. However, their inhibitory activities do not substantially differ from each other leading to a low eudismic ratio. Generally, the synthesized glyceric acid derivatives 7 show antibacterial activities against two Escherichia coli strains exceeding the ones of their respective regioisomes 6.

Chiral resolution through stereoselective transglycosylation by sucrose phosphorylase: application to the synthesis of a new biomimetic compatible solute, (R)-2-O-α-D-glucopyranosyl glyceric acid amide.

Sucrose phosphorylase catalysed glycosylation of glyceric acid amide with complete regio- and diastereo-selectivity is studied. (R)-2-O-α-D-Glucopyranosyl glyceric acid amide was obtained in high yield from single-step transformation of racemic glyceric acid amide and sucrose. Non-productive binding of (S)-glyceric acid amide appeared to underlie strict enantiodiscrimination by the enzyme, thus supporting chiral resolutions based on stereoselective transglycosylation.

Stepwise synthesis of 2,3-O-dipalmitoyl-D-glyceric acid and an in vitro evaluation of its cytotoxicity.

2,3-O-Dipalmitoyl-D-glyceric acid (PA2-DGA) was synthesized from D-glyceric acid calcium salt and palmitoyl chloride with improved yield. Direct condensation between the D-glyceric acid calcium salt and palmitoyl chloride produced PA2-DGA with a yield of <10%, whereas stepwise synthesis yielded this compound at up to 24% of overall yield. PA2-DGA was then subjected to a cytotoxic test using normal human dermal fibroblasts and primary normal human dermal microvascular endothelial cells. This compound had no toxic effects on human cells in vitro at concentrations up to 34 µM.

Atmospheric fate of methacrolein. 2. Formation of lactone and implications for organic aerosol production.

We investigate the oxidation of methacryloylperoxy nitrate (MPAN) and methacrylicperoxy acid (MPAA) by the hydroxyl radical (OH) theoretically, using both density functional theory [B3LYP] and explicitly correlated coupled cluster theory [CCSD(T)-F12]. These two compounds are produced following the abstraction of a hydrogen atom from methacrolein (MACR) by the OH radical. We use a RRKM master equation analysis to estimate that the oxidation of MPAN leads to formation of hydroxymethyl-methyl-α-lactone (HMML) in high yield. HMML production follows a low potential energy path from both MPAN and MPAA following addition of OH (via elimination of the NO(3) and OH from MPAN and MPAA, respectively). We suggest that the subsequent heterogeneous phase chemistry of HMML may be the route to formation of 2-methylglyceric acid, a common component of organic aerosol produced in the oxidation of methacrolein. Oxidation of acrolein, a photo-oxidation product from 1,3-butadiene, is found to follow a similar route generating hydroxymethyl-α-lactone (HML).

Synthesis of dilinoleoyl-D-glyceric acid and evaluation of its cytotoxicity to human dermal fibroblast and endothelial cells.

A novel derivative of glyceric acid (GA), dilinoleoyl-D-glyceric acid (LA2-DGA), was synthesized from D-GA calcium salt and linoleoyl chloride and evaluated for cytotoxicity. The D-GA calcium salt was first reacted with 4-methoxybezylchloride, and the resulting compound was esterified with linoleoyl chloride. This reaction was followed by hydrolysis of the 4-methoxybenzyl moiety, yielding LA2-DGA. LA2-DGA was then subjected to cytotoxicity testing using normal human dermal fibroblasts and primary normal human dermal microvascular endothelial cells. LA2-DGA showed no significant toxic effects in either type of cell.

Bioprocessing of glycerol into glyceric Acid for use in bioplastic monomer.

Utilization of excess glycerol supplies derived from the burgeoning biodiesel industry has recently become very important. Glyceric acid (GA) is one of the most promising glycerol derivatives, and it is abundantly obtained from glycerol by a bioprocess using acetic acid bacteria. In this study, a novel branched-type poly(lactic acid) (PLA) was synthesized by polycondensation of lactide in the presence of GA. The resulting branched PLA had lower crystallinity and glass transition temperatures than the conventional linear PLA, and the peak associated with the melting point of the branched PLA disappeared. Moreover, in a blend of the branched polymer, the crystallization of the linear PLA occurred at a lower temperature. Thus, the branched PLA containing GA synthesized in this study could potentially be used as a novel bio-based modifier for PLA.

Synthesis and evaluation of dioleoyl glyceric acids showing antitrypsin activity.

Previously, Lesová et al. reported the isolation and identification of metabolite OR-1, showing antitrypsin activity, produced during fermentation by Penicillium funiculosum. The structure of OR-1 was a mixture of glyceric acid (GA), esterified with C(14)-C(18) fatty acids, and oleic acid (C18:1) as the most predominant fatty acid (Folia Microbiol. 46, 21-23, 2001). In this study, dioleoyl D-GA and dioleoyl L-GA were synthesized via diesterification with oleoyl chloride, and their antitrypsin activities were evaluated using both a disk diffusion method and spectral absorption measurements. The results show that both compounds and their equivalent mixtures possess antitrypsin activities; however, their IC(50) values (approximately 2 mM) are much higher than that of OR-1 (4.25 µM), suggesting that dioleoyl GA does not play a major role in the OR-1 antitrypsin activity.

Enantioselective synthesis and antioxidant activity of 3-(3,4-dihydroxyphenyl)-glyceric acid--basic monomeric moiety of a biologically active polyether from Symphytum asperum and S. caucasicum.

The racemic and enantioselective synthesis of a novel glyceric acid derivative, namely, 2,3-dihydroxy-3-(3,4-dihydroxyphenyl)-propionic acid as well as the antioxidant activities is described. The virtually pure enantiomers, (+)-(2R,3S)-2,3-dihydroxy-3-(3,4-dihydroxyphenyl)-propionic acid and (-)-(2S,3R)-2,3-dihydroxy-3-(3,4-dihydroxyphenyl)-propionic acid were synthesized for the first time via Sharpless asymmetric dihydroxylation of trans-caffeic acid derivatives using the enantiocomplementary catalysts, (DHQD)(2)-PHAL and (DHQ)(2)-PHAL. The determination of enantiomeric purity of the novel chiral glyceric acid derivatives was performed by high-performance liquid chromatographic techniques on the stage of their alkylated precursors. The novel glyceric acid derivatives show strong antioxidant activity against hypochlorite and N,N-diphenyl-N-picryl-hydrazyl free radical. Their antioxidant activity is about 40-fold higher than that of the corresponding natural polyether and three-fold higher of trans-caffeic acid itself.

Design of new enzyme stabilizers inspired by glycosides of hyperthermophilic microorganisms.

In response to stressful conditions like supra-optimal salinity in the growth medium or temperature, many microorganisms accumulate low-molecular-mass organic compounds known as compatible solutes. In contrast with mesophiles that accumulate neutral or zwitterionic compounds, the solutes of hyperthermophiles are typically negatively charged. (2R)-2-(alpha-D-Mannopyranosyl)glycerate (herein abbreviated as mannosylglycerate) is one of the most widespread solutes among thermophilic and hyperthermophilic prokaryotes. In this work, several molecules chemically related to mannosylglycerate were synthesized, namely (2S)-2-(1-O-alpha-D-mannopyranosyl)propionate, 2-(1-O-alpha-D-mannopyranosyl)acetate, (2R)-2-(1-O-alpha-D-glucopyranosyl)glycerate and 1-O-(2-glyceryl)-alpha-D-mannopyranoside. The effectiveness of the newly synthesized compounds for the protection of model enzymes against heat-induced denaturation, aggregation and inactivation was evaluated, using differential scanning calorimetry, light scattering and measurements of residual activity. For comparison, the protection induced by natural compatible solutes, either neutral (e.g., trehalose, glycerol, ectoine) or negatively charged (di-myo-inositol-1,3'-phosphate and diglycerol phosphate), was assessed. Phosphate, sulfate, acetate and KCl were also included in the assays to rank the solutes and new compounds in the Hofmeister series. The data demonstrate the superiority of charged organic solutes as thermo-stabilizers of enzymes and strongly support the view that the extent of protein stabilization rendered by those solutes depends clearly on the specific solute/enzyme examined. The relevance of these findings to our knowledge on the mode of action of charged solutes is discussed.

Synthesis and mesophases of glycerate surfactants.

In the quest to rationally design novel mesophase systems the challenge remains to deconvolute the relationship between structure, composition, and function. In the current study, novel glycerol-derived surfactants with high negative interfacial areas and a preference for inverse phase behavior have been targeted and synthesized. This has been achieved by application of the rule-of-thumb afforded by the critical packing parameter (CPP), namely, that inverse phase behavior is favored by wedge-shaped molecules with relatively small head group versus chain volume. Highly splayed hydrophobes with exaggerated cross section such as oleyl (cis-octadec-9-enyl) hexahydrofarnesyl (3,7,11-trimethyl-dodecyl) and phytanyl (3,7,11,15-tetramethyl-hexadecyl) were particularly successful for this purpose across many variations of head group. The phase behavior of the binary system in water of many of these surfactants is relatively simple. Typically, cubic or inverse hexagonal phases exist at the interface with water with the inverse micellar phases present at lower hydration. The inverse liquid crystalline phases were present for a broad range of temperatures and compositions.

Enolase and the arsonomethyl analogue of 2-phosphoglycerate.

(RS)-3-Arsono-2-(hydroxymethyl)propionic acid was synthesized by the action of alkaline arsenite on 3-bromo-2-(bromomethyl)propionic acid. It is a substrate for yeast enolase (EC 4.2.1.11) with a Km of 6.5 mM (for 2-phospho-D-glycerate Km = 0.08 mM). The catalytic constant of the enzyme with the arsonomethyl analogue is 230 times lower than with 2-phosphoglycerate.

Thermal synthesis and hydrolysis of polyglyceric acid.

Polyglyceric acid was synthesized by thermal condensation of glyceric acid at 80 degrees in the presence and absence of two mole percent of sulfuric acid catalyst. The acid catalyst accelerated the polymerization over 100-fold and made possible the synthesis of insoluble polymers of both L- and DL-glyceric acid by heating for less than 1 day. Racemization of L-glyceric acid yielded less than 1% D-glyceric acid in condensations carried out at 80 degrees C with catalyst for 1 day and without catalyst for 12 days. The condensation of L-glyceric acid yielded an insoluble polymer much more readily than condensation of DL-glyceric acid. Studies of the hydrolysis of poly-DL-glyceric acid revealed that it was considerably more stable under mild acidic conditions compared to neutral pH. The relationship of this study to the origin of life is discussed.

Oligoglyceric acid synthesis by autocondensation of glyceroyl thioester.

The autocondensation of the glyceroyl thioester S-glyceroyl-ethanethiol yielded oligoglyceric acid. The rates of autocondensation and hydrolysis of the thioester increased from pH 6.5 to pH 7.5 in 2,6-lutidine and imidazole buffers. Autocondensation and hydrolysis were much more rapid to imidazole buffers than in 2,6-lutidine buffers of the same pH. The efficiency of ester bond synthesis was about 20% for 40 mM S-glyceroyl-ethanethiol in 2,6-lutidine and imidazole buffers near neutral pH. The size and yield of the oligoglyceric acid products increased when the concentration of the thioester was increased. The relationship of these results to prebiotic polymer synthesis is discussed.

A method for the synthesis of D-3-phospho[U-14C]glycerate.

An enzymatic method for the synthesis of radioactive D-3-phosphoglycerate from commercially available D-[U-14C]fructose 1,6-diphosphate is described. The unique aspect of this procedure is the substitution of arsenate for phosphate in the glyceraldehyde-3-phosphate dehydrogenase reaction. The 1-arseno-3-phosphoglycerate formed spontaneously hydrolyzes to form the D-3-phosphoglycerate product. The methods detailed below for the synthesis, isolation, and analysis of the 3-phospho[U-14C]glycerate product are relatively easy.

Preparation and physiological properties of alkyl esters of 2,3-dihydroxypropionic acid.

In order to investigate the physiological behavior of alkyl esters of 2,3-dihydroxypropionic acid two such compounds have been synthesized. One of them, the 1-dodecylester of 2,3-ditetradecyloxypropionic acid was subjected to digestion by pancreatic lipase. The substance remained unaffected. For an in vivo experiment a doubly labelled homolog, the [1'-14C]decyl ester of 2,3-di[1'-3H]hexadecyloxypropionic acid was synthesized. This compound was fed by stomach tube to three groups of male albino rats. The experimental animals were killed after 2,4 and 6 h, those of the control groups after 6 h. Blood, urine, small intestines and livers were examined for radioactivity. From the recovery rates it could be derived that the molecule had been metabolized and absorbed. Obviously, the alkyl chain labelled with 14C was split off first and the alkyl chains labelled with 3H were split off thereafter. As the substance is metabolized in vivo it cannot be utilized as a 'non-fattening fat'.