Regioselective enzymatic acylation of N-acetylhexosamines

A careful choice of the reaction conditions (solvent, enzyme, acylating agent) allowed an efficient regioselective acylation of N-acetylhexosamines. 6-O-Acyl derivatives of 2-acetamido-2-deoxy- -glucopyranose (GlcNAc), 2-acetamido-2-deoxy- -galactopyranose (GalNAc) and 2-acetamido-2-deoxy- -mannopyranose (ManNAc) have been isolated from regioselective esterifications catalysed by the protease subtilisin in CH₃CN–DMSO 8:2 in good yields.     

New 6-butylamino-6-deoxycellulose and 6-deoxy-6-pyridiniumcellulose derivatives with highest regioselectivity and completeness of reaction

This paper investigates the nucleophilic substitution (S(N)) reactions of tosylcellulose with butylamine and pyridine, respectively. The S(N) reactions of tosylcellulose 1 (DS(Total) 2.02; DS(C-6) 1.0) with butylamine carried out at 25, 50, 75 and 100 degrees C in both dimethyl sulfoxide (DMSO) and pure butylamine showed that the regioselectivity of substitution at C-6 of cellulose is temperature dependent: the highest regioselectivity at C-6 can be reached at 25 and 50 degrees C; substitution at C-2 also occurred at 75 and 100 degrees C. The substitution speed in pure butylamine is greater than that in the presence of DMSO. A complete and regioselective substitution at C-6 with a DS of 1.0 was obtained under the conditions of 50 degrees C, 40 h in butylamine. The substitution reactions of 1 with pyridine carried out at 25, 50, 75 and 100 degrees C for 24h in DMSO did not occur. In contrast to this the S(N) reactions done in pure pyridine showed that a temperature- and steric-dependent, regioselective substitution took place at C-6 at temperatures from 25 to 145 degrees C. The highest regioselectivity and completeness at C-6 can be obtained at 100 degrees C for 90 h, whereas at 145 degrees C substitution also occurs at C-2. The results were proved by 1H NMR and 13C NMR spectroscopy.     

Exclusive and complete introduction of amino groups and their N-sulfo and N-carboxymethyl groups into the 6-position of cellulose without the use of protecting groups

A new regioselective synthesis of 6-amino-6-deoxycellulose with a DS 1.0 (degree of substitution) at C-6, and its 6-N-sulfonated and its 6-N-carboxymethylated derivatives, without using protecting groups is described in this paper. The reaction conditions were optimized for preparing cellulose tosylate with full tosylation at C-6 and partial tosylation at C-2 and C-3. The nucleophilic substitution (S(N)) reaction of the tosyl group by NaN(3) at low temperature of 50 degrees C in Me(2)SO was achieved completely at C-6, whereas the tosyl groups at C-2 and C-3 were not displaced. In contrast to this, at 100 degrees C the tosyl groups at C-6, and also those at C-2 and C-3, were replaced by azido groups. This regioselective reaction that depends on temperature makes it possible to reach a selective and quantitative S(N) reaction at C-6 at low temperatures. In the subsequent reduction step with LiAlH(4), the azido group at C-6 was reduced to the amino group, and the tosyl groups at C-2 and C-3 were simultaneously completely removed. Also reported is a temperature-dependent, regioselective and complete iodination by nucleophilic substitution of the tosyl group at C-6 at 60 degrees C. At higher temperatures from 75 to 130 degrees C, substitution is also observed to occur at C-2. The selective iodination at 60 degrees C was employed to confirm the complete tosylation at C-6 of cellulose. The reaction products were identified by four different independent quantitative methods, namely 13C NMR, elemental analysis, ESCA, and fluorescence spectroscopy. 6-N-Sulfonated and 6-N-carboxymethylated cellulose derivatives were also synthesized. The new derivatives are potent candidates for structure-function studies, e.g., studies in relation to regioselectively 2-N-sulfonated and 2-N-carboxymethylated chitosan derivatives.     

Novel and highly regioselective route for synthesis of 5-fluorouridine lipophilic ester derivatives by lipozyme TL IM

For the first time, lipozyme TL IM, an inexpensive lipase from Thermomyces lanuginosa, was successfully applied to the regioselective synthesis of lipophilic 5-fluorouridine ester derivatives. The ESI-MS and (13)C NMR analysis confirmed that the end products of the acylation were 5'-O-acyl 5-fluorouridines, more powerful anti-tumor drugs than 5-fluorouridine itself. Notably, the chain length of acyl donors had an obvious effect on the initial rate and the maximum substrate conversion of the regioselective acylation. The acylation of 5-fluorouridine with vinyl laurate was used as a model to explore the influence of various factors on the reaction with respect to the initial rate, the maximum substrate conversion and the regioselectivity. The optimum water activity, the molar ratio of vinyl laurate to 5-fluorouridine, reaction temperature and shaking rate were 0.07, 15/1, 45 degrees C and 200rpm, respectively, under which the maximum substrate conversion and the regioselectivity were as high as 98.4 and >99%, respectively, after a reaction time of around 6h.     

Cytochrome P450 3A4-catalyzed testosterone 6beta-hydroxylation stereochemistry, kinetic deuterium isotope effects, and rate-limiting steps

Testosterone 6beta-hydroxylation is a prototypic reaction of cytochrome P450 (P450) 3A4, the major human P450. Biomimetic reactions produced a variety of testosterone oxidation products with 6beta-hydroxylation being only a minor reaction, indicating that P450 3A4 has considerable control over the course of steroid hydroxylation because 6beta-hydroxylation is not dominant in a thermodynamically controlled oxidation of the substrate. Several isotopically labeled testosterone substrates were prepared and used to probe the catalytic mechanism of P450 3A4: (i) 2,2,4,6,6-(2)H(5); (ii) 6,6-(2)H(2); (iii) 6alpha-(2)H; (iv) 6beta-(2)H; and (v) 6beta-(3)H testosterone. Only the 6beta-hydrogen was removed by P450 3A4 and not the 6alpha, indicating that P450 3A4 abstracts hydrogen and rebounds oxygen only at the beta face. Analysis of the rates of hydroxylation of 6beta-(1)H-, 6beta-(2)H-, and 6beta-(3)H-labeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect ((D)k) of 15. The deuterium isotope effects on k(cat) and k(cat)/K(m) in non-competitive reactions were only 2-3. Some "switching" to other hydroxylations occurred because of 6beta-(2)H substitution. The high (D)k value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high (D)k in the non-competitive experiments implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high (D)k value was also seen with a slower P450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking.     

Concepts for improved regioselective placement of O-sulfo, N-sulfo, N-acetyl, and N-carboxymethyl groups in chitosan derivatives

In the present paper a new strategy has been studied to introduce solely or in combination N-sulfo, O-sulfo, N-acetyl, and N-carboxymethyl groups into chitosan with highest possible regioselectivity and completeness and defined distribution along the polymer chain. The aim was to generate compounds having lowest toxicity for determining the pharmacological structure function relationships among different backbone structures and differently arranged functional groups compared to those of heparin and heparan sulfate. The water-soluble starting material, chitosan, with a degree of acetylation (DA) of 0.14 and a molecular weight of 29 kD, allows one to apply most of the known reactions of chitosan as well as some reactions of heparin chemistry successfully and with improved regioselectivity and completeness. On the other hand, a number of these reactions were not successful by application to water-soluble high-molecular-weight chitosan (DA 0.45 and 150 kD). The starting material showed statistical N-acetyl (N-Ac) distribution along the polymer chain according to the rules of Bernoulli, with highest abundance of the GlcNAc-GlcNAc diad along with a lower abundance of triads, tetrads, and pentads. The space between the N-Ac groups was filled up in homogeneous reactions by N-sulfo and/or N-carboxymethyl groups, which also resulted in a Bernoulli statistical distribution. The N-substitution reaction showed highest regioselectivity and completeness with up to three combined different functional groups. The regioselectivity of the 3-O-sulfo groups was improved by regioselective 6-desulfation of nearly completely sulfated 3,6-di-O-sulfochitosan. By means of desulfation reactions, all of the possible intermediate sulfated products are possible. 6-O-Sulfo groups can also be introduced with highest regioselectivity and completeness, and a number of partially 6-desulfated products are possible.     

FeCl3 mediated arylidenation of carbohydrates

Glycosides and thioglycosides based on monosaccharides in reaction with benzaldehyde dimethylacetal or p-methoxybenzaldehyde dimethyl acetal undergo FeCl₃-catalyzed (20 mol %) regioselective 4,6-O-arylidenation producing the corresponding acetals in high yields. FeCl₃ also mediates acetalation of glycosides and thioglycosides of cellobiose, maltose, and lactose affording the corresponding 4′,6′-O-benzylidene acetals, which were isolated after their acetylation in situ with acetic anhydride and pyridine. The combined yields (two steps) of these final products are also high (61–84%). The procedure is applicable to a wide variety of functional groups including –OBn.     

谷氨酰胺转氨酶研究进展

谷氨酰胺转氨酶是一种催化酰基转移反应的转移酶,它可使蛋白分子间和分子内产生共价交联,从而改变蛋白的功能特性,在食品、医药、化妆品、纺织等领域具有重要的潜在应用价值。研究表明,谷氨酰胺转氨酶广泛存在于生物组织中。为了更好的研究开发这一资源,对谷氨酰胺转氨酶的来源、理化特性、作用机制、功能以及工业生产的现状进行了综述,其中重点探讨了不同来源的谷氨酰胺转氨酶在理化特性以及底物特异性方面的差异。     

Regioselective hydroxylation of cholecalciferol,cholesterol and other sterol derivatives by steroid C25 dehydrogenase

Steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S is a molybdenum oxidoreductase belonging to the so-called ethylbenzene dehydrogenase (EBDH)-like subclass of DMSO reductases capable of the regioselective hydroxylation of cholesterol or cholecalciferol to 25-hydroxy products. Both products are important biologically active molecules: 25-hydroxycholesterol is responsible for a complex regulatory function in the immunological system, while 25-hydroxycholecalciferol (calcifediol) is the activated form of vitamin D₃ used in the treatment of rickets and other calcium disorders. Studies revealed that the optimal enzymatic synthesis proceeds in fed-batch reactors under anaerobic conditions, with 6–9 % (w/v) 2-hydroxypropyl-β-cyclodextrin as a solubilizer and 1.25–5 % (v/v) 2-methoxyethanol as an organic co-solvent, both adjusted to the substrate type, and 8–15 mM K₃[Fe(CN)₆] as an electron acceptor. Such thorough optimization of the reaction conditions resulted in high product concentrations: 0.8 g/L for 25-hydroxycholesterol, 1.4 g/L for calcifediol and 2.2 g/L for 25-hydroxy-3-ketosterols. Although the purification protocol yields approximately 2.3 mg of pure S25DH from 30 g of wet cell mass (specific activity of 14 nmol min⁻¹ mg⁻¹), the non-purified crude extract or enzyme preparation can be readily used for the regioselective hydroxylation of both cholesterol and cholecalciferol. On the other hand, pure S25DH can be efficiently immobilized either on powder or a monolithic silica support functionalized with an organic linker providing NH₂ groups for enzyme covalent binding. Although such immobilization reduced the enzyme initial activity more than twofold it extended S25DH catalytic lifetime under working conditions at least 3.5 times.

     

Design and synthesis of carbocyclic versions of furanoid nucleoside phosphonic Acid analogues as potential anti-hiv agents

Novel 5'-norcarbocyclic adenine and guanine phosphonic acid analogues with 6',6'-difluorine moiety were designed and synthesized from commercially available epichlorohydrin 5. A regioselective Mitsunobu reaction successfully proceeded from an allylic functional group 16b at low reaction temperature in polar cosolvent to give purine phosphonate analogues 17 and 24, respectively. The purine nucleoside phosphonate and phosphonic acid analogues were subjected to antiviral screening against HIV-1. Adenine analogue 21 and its SATE prodrug 29 show significant anti-HIV activity in MT-4 cell lines.     

An improved method for the preparation of 3-O-benzyl-6-O-pivaloyl-alpha-D-glucopyranose 1,2,4-orthopivalate

The synthetic route to 3-O-benzyl-6-O-pivaloyl-alpha-D-glucopyranose 1,2,4-orthopivalate (1), which was previously established, was shortened by introducing two novel reactions, regioselective pivaloylation with dibutyltin oxide in toluene for the regioselective activation of hydroxyl groups, and intramolecular orthoesterification with benzenesulfonyl chloride and triethylamine in dichloromethane. Compound 1 was obtained in 58.8% overall yield from commercially available 1,2:5,6-di-O-isopropylidene-alpha-D-glucopyranose (2) via four reaction steps.     

Application of selectively acylated glycosides for the α-galactosidase-catalyzed synthesis of disaccharides

4-Nitrophenyl alpha-D-galactopyranosyl-(1-->3)-6-O-acetyl-alpha-D-galactopyranoside was prepared in a transglycosylation reaction catalyzed by alpha-D-galactosidase from Talaromyces flavus using 4-nitrophenyl alpha-D-galactopyranoside as a glycosyl donor and 4-nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside as an acceptor. 4-Nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside and 4-nitrophenyl 6-O-acetyl-beta-D-galactopyranoside were prepared in a regioselective enzymic transesterification in pyridine-acetone catalyzed by the lipase PS from Burkholderia cepacia. A series of water-miscible organic solvents (acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, 2-methoxyethanol, pyridine, 2-methylpropan-2-ol, tetrahydrofuran, propargyl alcohol) were used as co-solvents in this enzymic reaction. Their influence on the activity and stability of the alpha-galactosidase from T. flavus was established. 2-Methylpropan-2-ol and acetone (increasing the solubility of the modified substrate acceptors and displaying the minimum impairment of the activity and stability of the enzyme) were used as co-solvents in transglycosylation reactions.     

Novozym 435-catalyzed regioselective benzoylation of 1-β-d-arabinofuranosylcytosine in a co-solvent mixture of C4MIm·PF6 and pyridine

Regioselective acylation of 1-β-d-arabinofuranosylcytosine (ara-C), using vinyl benzoate (VB) as acyl donor and Novozym 435 as catalyst, was carried out in various reaction media including pure organic solvents, organic solvent mixtures, and ionic liquid (IL)-containing systems. Although the reaction was highly regioselective in all the media assayed, remarkable enhancement of substrate conversion was achieved with a co-solvent mixture of 1-butyl-3-methylimidazolium hexafluorophosphate (C₄MIm·PF₆) and pyridine as the reaction medium, compared with other media tested. Additionally, the results demonstrated that the anions of ILs had a significant effect on the initial rate and substrate conversion. To better understand the reaction performed in IL-containing system, several variables were examined. The optimum molar ratio of VB to ara-C, initial water activity, temperature and shaking rate were 25:1, 0.11, 40°C and 250rpm, respectively. Under these optimum reaction conditions, the initial rate, substrate conversion, and regioselectivity were 0.49mMmin^?1, 99.4 and 99%, respectively. The product of the lipase-catalyzed reaction was characterized by ¹³C NMR and was shown to be 5′-O-benzoyl ara-C.     

Engineered,highly productive biosynthesis of artificial,lactonized statin side-chain building blocks: The hidden potential of Escherichia coli unleashed

We have recently reported the development of an efficient, whole-cell process for chemoenzymatic production of key chiral intermediates in statin synthesis by employing high-density Escherichia coli culture with the overexpressed deoxyribose-5-phosphate aldolase (DERA). The optically pure, 6-substituted cyclic hemiacetals can be used for the synthesis of atorvastatin, rosuvastatin and pitavastatin using further chemical steps. All of the synthetic routes established to date begin with a regiospecific oxidation of these lactol intermediates into the corresponding lactones, followed by several steps yielding 6-substituted, open-chain or lactonized derivatives which can be coupled by various approaches with the heterocyclic part of the statin molecule. Here we report for the first time the use of PQQ-dependent glucose dehydrogenases for a highly efficient, regioselective oxidation of artificial, derivatized aldohexoses, more specifically, the statin lactol intermediates. First, PQQ-dependent dehydrogenases of both soluble and membrane-bound type were characterized for their activity toward various DERA-derived lactols. Further, we describe a highly productive whole-cell system for oxidation of these 2,4-dideoxyaldopyranoses using a PQQ-dependent glucose dehydrogenase (Gcd) overexpressed in E. coli while taking advantage of the respiratory chain as the mediator of the electron transfer to oxygen. Finally, a two-step artificial biosynthetic pathway was developed by unleashing the intrinsic genetic potential of E. coli. The combined overexpression of the endogenous DERA and the membrane-bound, PQQ-dependent glucose dehydrogenase, the latter being coupled to the respiratory chain, allows direct biosynthesis of 6-substituted lactones in a highly productive, high-yield, cost-effective and industrially scalable process.     

A study of n-pentenylorthoesters having manno, gluco and galacto configurations in regioselective glycosylations

Kochetkov's extensive investigations of glycosyl orthoester and their analogs as glycosyl donors revealed that the alkyl derivatives were plagued by competition between the departing alcohol and the incoming acceptor. n-Pentenyl orthoesters (NPOEs) obviate competition by sequestering the departing pentenyl alcohol as a 2-halomethyl tetrahydrofuran. Exquisitely regioselective glycosidations of diol acceptors can be carried out with NPOEs triggered specifically with Yb(OTf)(3)/NIS. However with Sc(OTf)(3), double glycosidation is the major reaction. manno, gluco and galacto NPOEs have been investigated. The latter two, which require a different experimental procedure for the manno counterpart, also give an excellent regioselectivity with Yb(OTf)(3), but the yields are much lower than with manno.     

翻译后修饰调控TRPV亚家族通道功能的研究进展

瞬时受体势(transient receptor potential,TRP)通道广泛分布于神经和非神经系统中,响应温度、化学和机械等多种刺激,在机体对外界环境的精确感知中发挥重要功能.根据蛋白质序列的相似性,哺乳动物中TRP通道家族的27个成员分属TRPA、TRPC、TRPM、TRPML、TRPP和TRPV 6个亚家...     

Chemoenzymatic Route for the Synthesis of (S)‐Moprolol,a Potential β‐Blocker

A biocatalytic route for the synthesis of a potential β‐blocker, (S)‐moprolol is reported here. Enantiopure synthesis of moprolol is mainly dependent on the chiral intermediate, 3‐(2‐methoxyphenoxy)‐propane‐1,2‐diol. Various commercial lipases were screened for the enantioselective resolution of (RS)‐3‐(2‐methoxyphenoxy)propane‐1,2‐diol to produce the desired enantiomer. Among them, Aspergillus niger lipase (ANL) was selected on the basis of both stereo‐ and regioselectivity. The optimized values of various reaction parameters were determined such as enzyme (15 mg/mL), substrate concentration (10 mM), organic solvent (toluene), reaction temperature (30 °C), and time (18 h).The optimized conditions led to achieving >49% yield with high enantiomeric excess of (S)‐3‐(2‐methoxyphenoxy)propane‐1,2‐diol. The lipase‐mediated catalysis showed regioselective acylation with dual stereoselectivity. Further, the enantiopure intermediate was used for the synthesis of (S)‐moprolol, which afforded the desired β‐blocker. Chirality 28:313–318, 2016. © 2016 Wiley Periodicals, Inc.     

Cisplatin-mediated selective hydrolytic cleavage of methionine-containing peptides with neighboring serine or histidine residues

The pH- and time-dependent reactions of the anticancer drug cisplatin, cis-[PtCl(2)(NH(3))(2)], with the peptides Ac-Gly-Met-Gly-OH, Ac-Ser-Met-OH and Ac-Met-His-OH (Gly=glycyl, Met=methionyl, Ser=seryl, His=histidyl) at 313 K have been investigated by high-performance liquid chromatography, nuclear magnetic resonance and mass spectrometry. In the major equimolar reaction pathway for Ac-Gly-Met-Gly-OH, rapid anchoring at the methionine sulphur (kappaS) is followed by successive metalations of the methionine N(M) and glycyl N(G1) amide nitrogens in N-terminal direction to afford bidentate kappa(2)S,N(M) and tridentate kappa(3)S, N(M),N(G1) complexes. Cleavage of acetic acid at the second upstream amide bond is observed after 10 h leading to slow formation of [Pt(H-Gly-MetH(-1)-Gly-OH-kappa(3)S,N(M),N(G1))(NH(3))](+) at pH<6. [Pt(H-Ser-MetH(-1)-OH-kappa(3)S,N(M),N(S))(NH(3))](+) results from an analogous cisplatin-mediated regioselective hydrolytic cleavage reaction for Ac-Ser-Met-OH in moderately acid solution (pH<4). After passing through a minimum at pH 4.4, the concentration of the cleavage product in the reaction mixture after 500 h increases steadily on raising the pH and release of acetic acid is effectively quantitative for 7pH9.5. A competing mechanism involving nucleophilic attack of the serine side chain on the acetyl function can be inferred for pH>6 by the HPLC detection of a second intermediate kappa(3)S,N(M),N(S) species. In striking contrast, the reaction of cisplatin with Ac-Met-His-OH leads to release of acetylmethionine and formation of a final histidine product cis-[PtCl(H-His-OH-kappa(2)N3,N(H)) (NH(3))](+) at pH<6 by a kappaS-->kappa(2)S, N3-->kappa(3)S, N(H),N3-->kappa(2)N3,N(H)(H-His-OH) pathway.     

Mechanistic and structural analysis of aminoglycoside N-acetyltransferase AAC(6')-Ib and its bifunctional, fluoroquinolone-active AAC(6')-Ib-cr variant

Enzymatic modification of aminoglycoside antibiotics mediated by regioselective aminoglycoside N-acetyltransferases is the predominant cause of bacterial resistance to aminoglycosides. A recently discovered bifunctional aminoglycoside acetyltransferase (AAC(6')-Ib variant, AAC(6')-Ib-cr) has been shown to catalyze the acetylation of fluoroquinolones as well as aminoglycosides. We have expressed and purified AAC(6')-Ib-wt and its bifunctional variant AAC(6')-Ib-cr in Escherichia coli and characterized their kinetic and chemical mechanism. Initial velocity and dead-end inhibition studies support an ordered sequential mechanism for the enzyme(s). The three-dimensional structure of AAC(6')-Ib-wt was determined in various complexes with donor and acceptor ligands to resolutions greater than 2.2 A. Observation of the direct, and optimally positioned, interaction between the 6'-NH 2 and Asp115 suggests that Asp115 acts as a general base to accept a proton in the reaction. The structure of AAC(6')-Ib-wt permits the construction of a molecular model of the interactions of fluoroquinolones with the AAC(6')-Ib-cr variant. The model suggests that a major contribution to the fluoroquinolone acetylation activity comes from the Asp179Tyr mutation, where Tyr179 makes pi-stacking interactions with the quinolone ring facilitating quinolone binding. The model also suggests that fluoroquinolones and aminoglycosides have different binding modes. On the basis of kinetic properties, the pH dependence of the kinetic parameters, and structural information, we propose an acid/base-assisted reaction catalyzed by AAC(6')-Ib-wt and the AAC(6')-Ib-cr variant involving a ternary complex.     

Asymmetric synthesis of apio fluoroneplanocin A analogs as potential AdoHcy hydrolase inhibitor

Apio fluoroneplanocin A (apio F-NPA, 3) and its uracil analogue 4 have been designed and asymmetrically synthesized starting from D-ribose. Introduction of fluoro group into vinylic position of 5 was accomplished successfully over 5 steps employing key reactions such as iodination according to an addition-elimination reaction mechanism, stereo- and regioselective reduction of alpha,beta-unsaturated ketone, and electrophilic fluorination. This methodology can be adapted to the synthesis of fluoro compounds extensively.