Enzymatic cyclization of dioxidosqualene to heterocyclic triterpenes

Oxidosqualene cyclases normally produce triterpenes from 2,3-(S)-oxidosqualene (OS) but also can cyclize its minor companion (3S,22S)-2,3:22,23-dioxidosqualene (DOS). We explored DOS cyclization in plant triterpene synthesis using a recombinant lupeol synthase (LUP1) heterologously expressed in yeast. Incubation of LUP1 with 3S,22S-DOS gave epoxydammaranes epimeric at C20 and a 17,24-epoxybaccharane in a 4:2:3 ratio. The products reflected a new mechanistic paradigm for DOS cyclization. The structures were determined by NMR and GC-MS, and recent errors in the epoxydammarane literature were rectified. Some DOS metabolites are likely candidates for regulating triterpenoid biosynthesis, while others may be precursors of saponin aglycones. Our in vivo experiments in yeast generated substantial amounts of DOS metabolites in a single enzymatic step, suggesting a seminal role for the DOS shunt pathway in the evolution of saponin synthesis. Quantum mechanical calculations revealed oxonium ion intermediates, whose reactivity altered the usual mechanistic patterns of triterpene synthesis. Further analysis indicated that the side chain of the epoxydammarenyl cation intermediate is in an extended conformation. The overall results establish new roles for DOS in triterpene synthesis and exemplify how organisms can increase the diversity of secondary metabolites without constructing new enzymes.     

Enzymatic synthesis of cyclic amino acids by N-methyl-l-amino acid dehydrogenase from Pseudomonas putida

A new enzymatic system for the synthesis of enantiomerically pure cyclic amino acids (CAA) from the corresponding diamino acids or racemic CAA is described. α,ω-Diamino acids were oxidized to α-keto acids with amino acid oxidases (AAO). The α-keto acids were spontaneously transformed into cyclic imino acids in the reaction medium. The resulting imines were reduced to the l-form CAA with N-methyl-l-amino acid dehydrogenase (NMAADH) from Pseudomonas putida ATCC12633 using NADPH as a cofactor. l-Form CAA were also obtained from racemic CAA using d-amino-acid oxidase and NMAADH. Using this method, a new compound [1,4]-thiazepane-3-carboxylic acid (Fig. 1) was synthesized from aminopropylcystein.     

Enzymatic synthesis of monolaurin

The aim of this study was to produce monolaurin utilizing a commercial immobilized lipase (Lipozyme IM-20; Novo Nordisk, Bagsvaerd, Denmark) through the direct esterification of lauric acid and glycerol in a solvent-free system. The influence of fatty acid/glycerol molar ratio, temperature, and Lipozyme (IM-20) concentration on the molar fraction of monolaurin were determined using an experimental design. The best conditions employed were 55°C, lauric acid/glycerol molar ratio of 1.0, and 3.0% (w/w) enzyme concentration. The final product, obtained after 6 h of reaction, was 45.5% monolaurin, 26.8% dilaurin, 3.1% trilaurin, and 24.6% lauric acid. The reusability of the enzyme was also studied.     

Enzymatic synthesis of prebiotic oligosaccharides

Prebiotic oligosaccharides are nondigestible carbohydrates that can be obtained by enzymatic synthesis. Glucosyltransferases can be used to produce these carbohydrates through an acceptor reaction synthesis. When maltose is the acceptor a trisaccharide composed of one maltose unit and one glucose unit linked by an alpha-1,6-glycosidic bond (panose) is obtained as the primer product of the dextransucrase acceptor reaction. In this work, panose enzymatic synthesis was evaluated by a central composite experimental design in which maltose and sucrose concentration were varied in a wide range of maltose/sucrose ratios in a batch reactor system. A partially purified enzyme was used in order to reduce the process costs, because enzyme purification is one of the most expensive steps in enzymatic synthesis. Even using high maltose/sucrose ratios, dextran and higher-oligosaccharide formation were not avoided. The results showed that intermediate concentrations of sucrose and high maltose concentration resulted in high panose productivity with low dextran and higher-oligosaccharide productivity.     

生物催化在非天然寡糖合成中的应用

酶已经成为现代有机合成中一种不可或缺的工具。近年来人们在克隆技术，微生物学以及蛋白纯化方面的研究成果为我们提供了越来越多的生物催化剂，酶最初用于天然产物的合成，而今其在非天然化合物中的应用更引起了生物化学家的广泛关注。酶催化反应可以使不加保护的高官能团化底物进行条件温和，环境友好地转化，得到具有高度化学、区域和立体选择性的产物。本文对近年来糖基转移酶在糖生物学中的应用，特别是其在利用Leloir方法合成非天然寡糖中的应用作一简要回顾。     

爱滋病病毒中肽段的酶促合成

为了进一步研究酶促合成在多肽合成中的实际应用,选择合成了爱滋病病毒(人类免疫缺损病毒,HIV-I)的gp41中氨基酸序列598-609的三个肽段,该部分是HIV-I中的2个抗原决定簇部分,H-Leu-Glg-Leu-Trp-Glg-cgs-Ser-Glg-Lgs-Leu-Ile-Cgs-OH可以作为抗原来检测HIV抗体.     

Enzymatic resolution of racemic secondary cyclic allylic alcohols

The resolutions of five racemic cyclic alcohols: 6,6-dimethylcyclohex-2-en-1-ol (±)-5, 4,4-dimethylcyclohex-2-en-1-ol (±)-7, 5,5-dimethylcyclohex-2-en-1-ol (±)-11 and isomeric trans-(±)-13 and cis-piperitols (±)-14 are presented. They were resolved by enzymatic esterification with vinyl esters or by enzymatic hydrolysis of their racemic esters in phosphate buffer. The following lipases were used as biocatalysts: Novozyme 435 (Candida antarctica), Amano PS (Burkholderia cepacia) and lipase from Candida cylindracea. All isomers of alcohols were obtained with at least 96% ee.     

Efficient synthesis of morolic acid and related triterpenes starting from betulin

Morolic acid (1) is a naturally occurring pentacyclic triterpene whose derivatives exhibit promising anti-HIV and other biological activities. An efficient synthesis of 1 has been accomplished in 11 steps with a total yield of 24% starting from betulin. Some related natural triterpenes including moradiol (4), acridocarpusic acid D (5), acridocarpusic acid E (6), and moronic aldehyde (7) have also been synthesized. Biological assay results showed that 1, 5, and 6 exhibited moderate inhibitory activity against glycogen phosphorylase.     

Enzymatic degradations of copolymers of L-lactide with cyclic carbonates

Syntheses and biodegradation of random copolymers of L -lactide (L -LA) with trimethylene carbonate (TMC), 1,1-dimethyltrimethylene carbonate (1,1-DTMC) and 2,2-dimethyltrimethylene carbonate (2,2-DTMC) were investigated at various monomer ratios using SmMe(C₅Me₅)₂THF as an initiator at 80 °C for 24 h in toluene. Enzymatic degradation of these polymers were performed using cholesterol esterase, lipoprotein lipase, and proteinase K. Poly(TMC) was effectively biodegraded by cholesterol esterase and lipoprotein lipase, while poly(2,2-DTMC) and all the copolymers were hardly degraded using these enzymes. Biodegradations of poly(L -LA-co-TMC) (97:3) and poly(L -LA-co-2,2,DTMC) (95:5) show rapid degradations using TES buffer, a compost and proteinase K. The physical properties of these copolymers were also examined.

Enzymatic degradation of L -LA/2,2-DTMC copolymers by proteinase K in Tricine buffer (pH 8.0) at 37 °C: a 98:2, b 82:18, c 100:0, d 66:34, e 34:66, f 0:100.     

Enzymatic synthesis of gallic acid esters

Esters of gallic acid were synthesized by enzymatic means using tannase fromAspergillus niger. Alcohols ranging from C₁ through C₁₂ and diols from C₃ through C₆ were tested and found to form esters in the presence of the enzyme.     

Enzymatic synthesis of flavin adenine dinucleotide

A new and simple enzymatic method for the synthesis of flavin adenine dinucleotide (FAD) from flavin mononucleotide by the transadenylylation reaction using microbial cells is described. Among various microorganisms tested,Artherobacter globiformis IFO 12138 and two soil bacteria were selected as useful enzyme sources. Under suitable reaction conditions, the amount of FAD synthesized was 2.25 μmol/mL with cells ofA. globiformis. The transadenylylation reaction could be coupled with the ATP supplying system through a glycolysis process with yeast.     

Solid-phase synthesis of cyclic imides

A cyclative cleavage strategy for the synthesis of cyclic imides on a polystyrene resin is described. After optimization of the cleavage conditions, a small array of succinimides and phthalimides was synthesized. The methodology was then applied to a drug discovery project, in which it was used to synthesize a new class of delta-opioid receptor ligand by both automated and manual methods.     

Microwave-enhanced synthesis of cyclic amidines

We present a simple and efficient microwave based protocol for the synthesis of heterocyclic amidines by PPE promoted cyclodehydration of N-aryl-N′-acylalkylenediamines. The method is general for five- to eight-membered heterocycles and affords high yields of the desired products in remarkably short reaction times.     

Solid-phase synthesis of cyclic polyamines

A method for the synthesis of cyclic polyamines based on solid-phase chemistry is shown. Linear polyamines are stepwise synthesized on solid support from the center by repetitive alkylations at benzylic N-atoms. Cyclizations at the resins were effected conventionally by direct intramolecular S_N2 reactions between sulfonyl-protected terminal amino groups and primary alkyl bromides or by intramolecular Mitsunobu reactions between sulphonamides and primary alcohols. Particularly the latter transformation proved to be powerful for the construction of medium- as well as large-sized rings.