Phospholipid-protein coatings for chiral capillary electrochromatography

A phospholipid-bovine serum albumin (BSA) coating was developed for chiral capillary electrochromatographic separation of d- and l-tryptophan. Temperature, liposome composition, and liposome-BSA mixing and extrusion were found to have critical effects on the chiral separation of d- and l-tryptophan in terms of resolution, separation efficiency, and migration times. A solution of 0.5mM phosphatidylcholine (PC)-1 mg/ml BSA performed better than a solution of 0.5mM PC/phosphatidylserine (PS) (80:20, mol%)-1 mg/ml BSA as capillary coating; baseline separation of the enantiomers with satisfactory resolution was then achieved. Temperature played a crucial role in the chiral separation, as demonstrated for phospholipid-coated capillaries immobilized with BSA and lysozyme. The d- and l-tryptophans showed a marked difference in separation efficiency on the PC-BSA-coated capillary; the theoretical plate number of l-tryptophan was above 500,000 m(-1), whereas that of d-tryptophan was only about 22,000 m(-1). Immobilized BSA (pI 4.7) showed better chiral separation selectivity for the enantiomers than did immobilized lysozyme (pI 10.5), alpha-chymotrypsin (pI 8.1-8.3), or avidin (pI 10.0-10.5); also resolution was better and analysis time was faster. Hydrophobic interactions played an important role in the BSA-immobilized phospholipid-coated capillaries. The importance of protein net charge and molar mass for its immobilization in phospholipid-coated capillaries is discussed.     

Chiral C3 epoxides and halohydrins: Their preparation and synthetic application

Epichlorohydrin, 3-chloro-1,2-propanediol and glycidol are versatile chiral C3 syntons in organic synthesis. For the production of these, more efficient and more practical methods are required and many studies have been reported. In this review, biological and synthetic preparations of their synthons and microbial dehalogenation of halohydrins connected with the biological methods are described. Several of their synthetic applications are also introduced.     

Acetylation of (R,S)-propranolol catalyzed by Candida antarctica lipase B: An experimental and computational study

The chemo- and enantioselectivity of the Candida antarctica lipase B (CalB)-catalyzed acetylation reaction of (R,S)-propranolol using vinyl acetate as acyl donor and toluene as organic solvent was studied. Because of the poor solubility of propranolol in toluene small quantities of methanol were added as cosolvent. The effects of the propranolol/vinyl acetate ratio, the enzyme purification procedure and the methanol concentration on the reaction were investigated. The reactions occurring in the system were quantitatively investigated using ¹H and ¹³C NMR spectroscopy. The major reactions were the hydrolysis and alcoholysis of vinyl acetate, as a consequence of the presence of residual water and methanol in the reaction medium. Furthermore, the NMR analysis confirmed that O-acetyl-propranolol was formed exclusively. The reaction was also found to be enantioselective favoring the faster transformation of the R-propranolol. In addition to the experiments, molecular modeling was used to study the formation of the reactive Michaelis complexes between propranolol and acetylated CalB, using a combined molecular docking and molecular dynamics (MD) procedure. Only for the O-acetylation we found binding modes of the substrate leading to formation of the product, which explains the experimentally observed chemoselectivity of CalB.     

Immobilized Baliospermum montanum hydroxynitrile lyase catalyzed synthesis of chiral cyanohydrins

Hydroxynitrile lyase (HNL) catalyzed enantioselective CC bond formation is an efficient approach to synthesize chiral cyanohydrins which are important building blocks in the synthesis of a number of fine chemicals, agrochemicals and pharmaceuticals. Immobilization of HNL is known to provide robustness, reusability and in some cases also enhances activity and selectivity.We optimized the preparation of immobilization of Baliospermium montanum HNL (BmHNL) by cross linking enzyme aggregate (CLEA) method and characterized it by SEM. Optimization of biocatalytic parameters was performed to obtain highest % conversion and ee of (S)-mandelonitrile from benzaldehyde using CLEA-BmHNL. The optimized reaction parameters were: 20 min of reaction time, 7 U of CLEA-BmHNL, 1.2 mM substrate, and 300 mM citrate buffer pH 4.2, that synthesized (S)-mandelonitrile in ∼99% ee and ∼60% conversion. Addition of organic solvent in CLEA-BmHNL biocatalysis did not improve in % ee or conversion of product unlike other CLEA-HNLs. CLEA-BmHNL could be successfully reused for eight consecutive cycles without loss of conversion or product formation and five cycles with a little loss in enantioselectivity. Eleven different chiral cyanohydrins were synthesized under optimal biocatalytic conditions in up to 99% ee and 59% conversion, however the % conversion and ee varied for different products. CLEA-BmHNL has improved the enantioselectivity of (S)-mandelonitrile synthesis compared to the use of purified BmHNL. Nine aldehydes not tested earlier with BmHNL were converted into their corresponding (S)-cyanohydrins for the first time using CLEA-BmHNL. Among the eleven (S)-cyanohydrins syntheses reported here, eight of them have not been synthesized by any CLEA-HNL. Overall, this study showed preparation, characterization of a stable, robust and recyclable biocatalyst i.e. CLEA-BmHNL and its biocatalytic application in the synthesis of different (S)-aromatic cyanohydrins.     

A study on increasing enzymatic stability and activity of Baliospermum montanum hydroxynitrile lyase in biocatalysis

HNL catalysis is usually carried out in a biphasic solvent and at low pH to suppress the non-enzymatic synthesis of racemic cyanohydrins. However, enzyme stability under these conditions remain a challenge. We have investigated the effect of different biocatalytic parameters, i.e., pH, temperature, buffer concentrations, presence of stabilizers, organic solvents, and chemical additives on the stability of Baliospermum montanum hydroxynitrile lyase (BmHNL). Unexpectedly, glycerol (50 mg/mL) added BmHNL biocatalysis had produced >99% of (S)-mandelonitrile from benzaldehyde, while without glycerol it is 54% ee. Similarly, BmHNL had converted 3-phenoxy benzaldehyde and 3,5-dimethoxy benzaldehyde, to their corresponding cyanohydrins in the presence of glycerol. Among the different stabilizers added to BmHNL at low pH, 400 mg/mL of sucrose had increased enzyme’s half-life more than fivefold. BmHNL’s stability study showed half-lives of 554, 686, and 690 h at its optimum pH 5.5, temperature 20 °C, buffer concentration, i.e., 100 mM citrate-phosphate pH 5.5. Addition of benzaldehyde as inhibitor, chemical additives, and the presence of organic solvents have decreased both the stability and activity of BmHNL, compared to their absence. Secondary structural study by CD-spectrophotometer showed that BmHNL’s structure is least affected in the presence of different organic solvents and temperatures.     

Highly diastereoselective conjugate addition of nitroalkanes to α,β-unsaturated sugar lactones for the efficient synthesis of chiral 2-pyrrolidones

A series of 4,5-substituted chiral γ-lactams were synthesized through a highly diastereoselective addition—rearrangement approach from 2,3-unsaturated sugar lactones. The single-crystal X-ray structure of one product indicated that the sugar ring was attacked from the axial side. Partial reduction of the nitro group produced N-hydroxy-γ-lactams, which were further reduced with TiCl₃ to yield the 4,5-substituted chiral γ-lactams. The absolute configuration of C5 of the γ-lactam was determined by NOESY spectra.     

Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop

2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (R_P/S_P). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (k_on = 174 M^?1 s^?1) was ca. 8-fold more potent than the d-isomer (k_on = 21.5 M^?1 s^?1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be S_P. The S_P-isomers inhibited human GGT (k_on = 21.5–174 M^?1 s^?1), while the R_P-isomers were inactive even at concentrations of 0.1 mM.     

Synthesis and characterization of poly-O-methyl-[n]-polyurethane from a d-glucamine-based monomer

Aminoalditol 1-amino-1-deoxy-d-sorbitol (1) was readily converted into 2,3,4,5-tetra-O-methyl derivative 5, a key precursor of a sugar-based [n]-polyurethane. For the polymerization, the free amino or primary hydroxyl groups of 5 were selectively activated and employed as starting monomers in two alternative procedures. Thus, the amino function of 5 was converted into the isocyanate derivative by treatment with di-tert-butyltricarbonate, and polymerized in situ in the presence of Zr(IV) acetylacetonate. The resulting poly(1-amino-1-deoxy-2,3,4,5-tetra-O-methyl-d-sorbitol)urethane (8) had a moderate molecular weight and showed the presence of urea units. The alternative synthesis of 8 involved the activation of the free hydroxyl group of 5 as the corresponding phenylcarbonate. The polymerization of this α-amino-ω-phenylcarbonate alditol monomer does not require a metal catalyst. The resulting material exhibited an improved molecular weight and higher purity than that obtained via the isocyanate. [n]-polyurethane 8 was highly soluble in water as well as in common organic solvents (chloroform, acetone, ethyl acetate, etc) and was obtained as an amorphous material which was characterized thermally and spectroscopically.     

A Relativistic Neutron Fireball from a Supernova Explosion as a Possible Source of Chiral Influence

We elaborate on a previously proposed idea that polarized electrons produced from neutrons, released in a supernova (SN) explosion, can cause chiral dissymmetry of molecules in interstellar gas-dust clouds. A specific physical mechanism of a relativistic neutron fireball with Lorentz factor of the order of 100 is assumed for propelling a great number of free neutrons outside the dense SN shell. A relativistic chiral electron–proton plasma, produced from neutron decays, is slowed down owing to collective effects in the interstellar plasma. As collective effects do not involve the particle spin, the electrons can carry their helicities to the cloud. The estimates show high chiral efficiency of such electrons. In addition to this mechanism, production of circularly polarized ultraviolet photons through polarized-electron bremsstrahlung at an early stage of the fireball evolution is considered. It is shown that these photons can escape from the fireball plasma. However, for an average density of neutrals in the interstellar medium of the order of 0.2 cm⁻³ and at distances of the order of 10 pc from the SN, these photons will be absorbed with a factor of about 10⁻⁷ due to the photoeffect. In this case, their chiral efficiency will be about five orders of magnitude less than that for polarized electrons.     

1,5-Anhydro-D-fructose; a versatile chiral building block: biochemistry and chemistry

There is a steadily increasing need to expand sustainable resources, and carbohydrates are anticipated to play an important role in this respect, both for bulk and fine chemical preparation. The enzyme alpha-(1-->4)-glucan lyase degrades starch to 1,5-anhydro-D-fructose. This compound, which has three different functional properties, a prochiral center together with a permanent pyran ring, renders it a potential chiral building block for the synthesis of valuable and potentially biologically active compounds. 1,5-Anhydro-D-fructose is found in natural materials as a degradation product of alpha-(1-->4)-glucans. The occurrence of lyases and the metabolism of 1,5-anhydro-D-fructose are reviewed in the biological part of this article. In the chemical part, the elucidated structure of 1,5-anhydro-D-fructose will be presented together with simple stereoselective conversions into hydroxy/amino 1,5-anhydro hexitols and a nojirimycin analogue. Synthesis of 6-O-acylated derivatives of 1,5-anhydro-D-fructose substituted with long fatty acid residues is carried out using commercially available enzymes. Those reactions lead to compounds with potential emulsifying properties. The use of protected derivatives of 1,5-anhydro-D-fructose for the synthesis of natural products is likewise reviewed. The potential utilization of this chemical building block is far from being exhausted. Since 1,5-anhydro-D-fructose now is accessible in larger amounts through a simple-enzyme catalyzed degradation of starch by alpha-(1-->4)-glucan lyase, the application of 1,5-anhydro-D-fructose may be considered a valuable contribution to the utilization of carbohydrates as the most abundant resource of sustainable raw materials.