Enzymatic Synthesis of a CCK-8 Tripeptide Derivative

The enzymatic synthesis of CCK-8 tripeptide derivative Phac-Met-Asp(OMe)-Phe-NH2 is reported. Starting with Phac-Met-OCam, we have successfully synthesized the target tripeptide with three free or immobilized enzymes, ?chymotrypsin, papain and thermolysin in reasonable yields. The key steps in this synthesis were the coupling of Phac-Met-OCam and H-Asp(OMe)2 to form Met-Asp peptide bond catalyzed by ?chymotrypsin and the selective hydrolysis of -ester of Phac-Met-Asp(OMe)2 catalyzed by papain.

Chemoenzymatic Synthesis of Ramosetron Hydrochloride

A new method for the synthesis of ramosetron hydrochloride, the most wildly used 5-HT3 receptor antagonist, was reported. The intermediate 8 was obtained in a high e.e. value via lipase catalyzed hydrolytic kinetic resolution. Then enantiomerically pure ramosetron hydrochloride was synthesized in two steps with excellent yield （the total yield is 24.6%）. The method provides a new environment friendly and atom economy way to synthesize enantiomerically pure ramosetron.

Enzymatic Synthesis of Methyl-Galactoaldehyde

Methyl-galactosides were oxidized at room temperature by galactose oxidase in a one-step reaction and afforded methyl-galactoaldehyde in excellent yield and high purity. The resulting galactoaldehyde as a useful intermediate can be directly used in glycopeptide synthesis.

THE STUDY OF ENZYMATIC SYNTHESIS OF MACROCYCLIC LACTONES

The effects of the chain length and stereo-factor of substrates and solvents on the lipase catalysed lactonization of ω-hydroxy-ester were studied, And also the catalytic effects of various enzymes were compared.

Enzymatic synthesis of nucleosides by nucleoside phosphorylase co-expressed in Escherichia coli

Nucleoside phosphorylase is an important enzyme involved in the biosynthesis of nucleosides. In this study, purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase were co-expressed in Escherichia coli and the intact cells were used as a catalyst for the biosynthesis of nucleosides. For protein induction, lactose was used in place of isopropyl β-D-l-thiogalactopyranoside （IPTG）. When the concentration of lactose was above 0.5 mmol/L, the ability to induce protein expression was similar to that of IPTG. We determined that the reaction conditions of four bacterial strains co-expressing these genes （TUD, TAD, DUD, and DAD） were similar for the biosyntheses of 2,6-diaminopurine nucleoside and 2,6-diaminopurine deoxynucleoside. When the substrate concentration was 30 mmol/L and 0.5% of the recombinant bacterial cell volume was used as the catalyst （pH 7.5）, a greater than 90% conversion yield was reached after a 2-h incubation at 50℃. In addition, several other nucleosides and nucleoside derivatives were efficiently synthesized using bacterial strains co-expressing these recombinant enzymes.

Synthesis of Cholecystokinin Peptide CCK-4 Exclusively by Enzymatic Methods

The synthesis of CCK - 4 (H - Trp- Met- Asp- Phe- NH2 ) by using enzym es exclusively was described.As protection group for the amino group we used the Phenylacetyl group (Phac) which had been cleaved at the end of the synthesis with Penicillin G Amidase (PGA ) without affecting the peptide bonds.Thus,beginning with Phac- Trp- OH we had successfully synthesized the target peptide with following4 enzymes,α- Chym otrypsin,Papain,Therm olysin and PGA in four reac- tion steps.All reactions were carried out in aqueous buffer in reasonable yields(>6 5 % ) .FAB- MS or FD- MS verified the correct molecular mass of all peptides.

Enzymatic Synthesis of Dipeptide Derivatives Containing Non-coded Amino Acids in Organic Solvents

A series of dipeptide derivatives containing non-coded amino acids, N-Boc-4-X-Phe-Ala-NHNHPh (X = Cl, Br, I, NO2), were synthesized by using thermoase in organic solvents. The physical data were consistent with the same samples prepared by 3-(diethoxyphosphoryloxy)-1, 2,3-benzotriazin-4 (3H)-one (DEPBT). Influence of different substituted groups of the noncoded amino acids and different organic solvents on the enzymatic peptide synthesis was studied.

Enzymatic Synthesis of a CCK-8 Tripeptide Fragment

A process for the synthesis of CCK-8 tripeptide H-Gly-Trp-Met-OH catalyzed by immobilized enzyme was re-ported. Enzymes were used for the formation of peptide bonds and the removal of protecting group. Starting with phenylacetyl (PhAc) glycin, N-protected dipeptide PhAc-Gly-Trp-OMe was obtained by coupling PhAc-protected glycine carboxamidomethyl ester (OCam) with Trp-OMe catalyzed by immobilized papain in buffered ethyl acetate. Then the condensation between PhAc-Gly-Trp-OMe and Met-OEtHCl was carried out by immobilized -chy-motrypsin catalysis in solvent free system. Basic hydrolysis was followed getting PhAc-Gly-Trp-Met-OH. The PhAc-group was removed with penicillin G amidase and H-Gly-Trp-Met-OH was obtained in an overall yield of 43.9%. The reaction conversion of tripeptide in solvent free system was strongly affected by the system of basic salts added. The influence of the support materials used to deposit enzymes and structures of acyl donor and nu-cleophile on the reaction was also investigated.

A multi-enzymatic cascade reaction for the synthesis of bioactive C-oligosaccharides

C-Oligosaccharides are rare in nature and possess diverse bioactivities.However,their chemical synthesis faces many challenges.In this work,enzymatic introduction of C-linked sugar chains to target aglycones was successfully achieved by multi-enzymatic cascade reactions.A C-glycosyltransferase from Aloe barbadensis was employed to introduce the first C-linked glucose and then a cyclomaltodextrin glucanotransferase from Bacillus licheniformis was used to extend the sugar chain.A total of twenty C-oligosaccharides with 2-6 sugars were synthesized from scale-up reactions and exhibited good water solubility and sodium-dependent glucose transporter 2(SGLT2)inhibitory activity.Furthermore,a glucoamylase was used to control the length of the sugar chain and the C-maltosides were efficiently synthesized.These findings not only expanded the structural diversity of C-oligosaccharides,but also provided a strategy for the modification of C-glycoside drugs to improve the druggability.

Regioselective enzymatic acylation of troxerutin in nonaqueous medium

A series of monosubstituted troxerutin esters have been synthesized by enzyme-catalyzed regioselective acylation of troxerutin in nonaqueous medium.Using divinyl dicarboxylates(CH_2=CH-OOC-(CH_2)_n-COO-CH=CH_2,n = 2,3,4,7,8,11) featuring different chain length as acyl donors and alkaline protease from Bacillus subtilis as catalyst,troxerutin was regioselective acylated at B ethoxyl group.The results indicated that the regioselectivity of the enzyme-catalyzed acylation was not affected by the chain length o...

Kinetically Controlled Carboxypeptidase-Catalyzed Synthesis of Novel Antioxidant Dipeptide Precursor BOC-Tyr-Ala

Recently, enzymatic peptide synthesis has drawn increasing attention due to its eco-friendly reagents and mild conditions, as compared to traditional chemical peptide synthesis. In this study, we successfully produced an important antioxidant dipeptide precursor, BOC-Tyr-Ala, via a kinetically controlled enzymatic peptide synthesis reaction, catalyzed by the recombinant car- boxypeptidase Y （CPY） expressed in P. pastoris GS 115. In this reaction, the enzyme activity was 95.043 U/mL, and we used t-butyloxycarbonyl-L-tyrosine-methyl ester （BOC-Tyr-OMe） as the acyl donor and L-alanine （L-Ala） was the amino donor. We optimized the reaction conditions to be： 30 ℃, pH 9.5, organic phase （methanol）/aqueous phase = 1：20, BOC-Tyr-OMe 0.05 mol/L, Ala 0.5 mol/L, and a reaction time of 12 h. Under these conditions, the dipeptide yield reached 49.84%. Then, we established the kinetic model of the synthesis reaction in the form of Michaelis-Menten equation according to the con-centration-time curve during the process and the transpeptidation mechanism. We calculated the apparent Michaelis constant K^（app）mand the apparent maximum reaction rate r^（app）max to be 2.9946 x 10^-2 mol/L and 2.0406 x 10.2 mmol/（mL h）, respectively.

Cytotoxic Activity of Betulinic Acid Derivatives Synthesized Using Enzymes

Betulinic acid, a triterpenoid found in many plant species, has attracted attention due to its important pharmacological properties, such as anti-cancer and anti-HIV activities. In order to obtain derivatives potentially useful for detailed pharmacological studies, betulinic acid derivatives were synthesized by reaction of betulinic acid with benzoyl chloride and with acetic anhydride using lipase as catalyst. Enzyme-catalyzed of betulinic acid with benzoyl chloride converted betulinic acid into 3β-benzoil-lup-20（29）-ene-28-oic acid ester （BCL） whereas with acetic anhydride converted betulinic acid into 3β-acetoxy-lup-20（29）-ene-28-oic acid ester （BAA）. The BAA then underwent further reaction with l-decanol to produce 3β-acetoxy-lup-20（29）-ene-28 decanoate （BAAD）. Betulinic acid derivatives prepared were tested for cytotoxic activity on three cancer cell lines in vitro： all tested compounds showed stronger cytotoxic activity than betulinic acid,

Efficient synthesis of nigerose by a novel nigerose phosphorylase from Anaerosporobacter mobilis

Nigerose is a kind of rare disaccharide connected by anα-1,3 glucosidic bond,which is a potential probiotic due to its antidigestive properties and beneficial functions.This study identified and characterized a novel GH65 glycoside phosphorylase derived from Anaerosporobacter mobilis(AmNP).This new protein could specifically catalyze the phospholysis of nigerose to generate glucose and glucose-1-phosphate in the presence of phosphate,indicating it was a typical nigerose phosphorylase.Compared to the previously reported nigerose phosphorylases,AmNP exhibited lower affinity towards nigerose in phosphorolysis reaction and higher affinity towards glucose in reverse phosphorolysis reaction,which indicated that AmNP might be superior in the synthetic capability of disaccharide.Then AmNP was employed to synergize with maltose phosphorylase from Lactobacillus brevis(LbMP)to catalyze the synthesis of nigerose using maltose as the substrate.After optimization of reaction conditions,the highest nigerose yield reached 132.0 g/L with a 66.3%conversion rate,which was higher than ever reported cases using the same reaction pathway to our knowledge.These findings on AmNP in this work were expected to provide a new candidate for large-scale enzymatic synthesis of nigerose and have important theoretical significance for studying nigerose phosphorylase.

Red yeast catalyzed animation of olefinic bonds and synthesis of optically pure S-amino acids

By using red yeast (Rhodotorula rubra AS 2.166) resting cells which possess the phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity as biocataiyst, enantiomerically pure S-3-(2-chloro-phenyl) alanine (2a) and S-3-(3-hydroxyphenyl) alanine (2d) have been synthesized from the corresponding 3-aryl substituted acrylic acid (1a and 1d) and ammonia.

Green Synthesis of Wax Ester by Immobilized Lipase

1 Results Enzyme catalysis is most attractive for the synthesis and modification of biologically relevant classes of fine organic compounds, which are difficult to prepare and to handle by conventional means[1]. In this study, commercial immobilized lipase from Candida antarctica (Novozym 435) was used in the preparation of fine organic compound with excellent properties and application as raw material for cosmetic formulation - oleyl palmitate. The effect of various reaction parameters were optimized c...

High yield synthesis of nigerooligosaccharides by transglycosylation catalyzed by α-glucosidase TaAglA from Thermoplasma acidophilum

Nigerooligosaccharides (NOS) is a new functional oligosaccharide containing α-1,3 glucosidic bond with good anti-digestive properties and intestinal probiotics.Transglycosylation catalyzed by α-glucosidase is an effective method for the preparation of oligosaccharides.However,there are few reports on the enzymatic synthesis of nigerooligosaccharides by α-glucosidase at present.This study was aimed to investigate the transglycosylation property of the GH31 α-glucosidase from Thermoplasma acidophilum,TaAglA,and also evaluate its application performance in the preparation of NOS.It was found that TaAglA exhibited selectivity for α-1,3 and α-1,4 linkages when catalyzing hydrolysis,but for α-1,3 and α-1,6 linkages when catalyzing transglycosylation.Using 10% glucose and 20% maltose as substrates,TaAglA yielded 88.5 g/L NOS under the condition of pH 6.0,80 ℃ and 1 U/mL enzyme addition,which was the highest level to our knowledge.In addition,components with higher polymerization degrees,i.e.nigerotriose and nigerosyl-glucose,occupied 53.6% proportion of the total NOS products,giving it better probiotic functions.Futhermore,after purification by glucoamylase digestion and yeast culture,the final yield of NOS was 26.1%,and the purity of the product was 93%.These findings on TaAglA were expected to provide a new candidate for large-scale enzymatic synthesis of NOS,and also have important theoretical significance for the study of GH31 α-glucosidase.

Enzymatic production of N‑acetylneuraminic acid:advances and perspectives

N-Acetyl-d-neuraminic acid(NeuAc),a well-known and well-studied sialic acid,is found in cell surface glycolipids and glycoproteins,where it performs a variety of biological functions.The use of NeuAc as a nutraceutical for infant brain development and as an intermediate for pharmaceutical production demands its production on an industrial scale.Natural extraction,chemical synthesis,enzymatic synthesis,and biosynthesis are the methods used for NeuAc production.Among these methods,enzymatic synthesis using N-acetyl-glucosamine(GlcNAc)2-epimerase(AGE)for epimerization and N-acetyld-neuraminic acid lyase(NAL)for aldol condensation,has been reported to produce NeuAc with high production efciency.In this review,we discuss advances in the two-step enzymatic synthesis of NeuAc using pyruvate and GlcNAc as substrates.The major challenges in producing NeuAc with high yield are highlighted,including multiple parameter-dependent processes,undesirable reversibility,and diminished solubility of AGEs and NALs.Further,diferent strategies applied to overcome the limitations of the two-step enzymatic production are discussed,such as pyruvate concentration and temperature shift during the process to increase conversion yield,use of mathematical and computational simulations for process optimization,enzyme engineering to make enzymes highly efcient,and the use of tags and chaperones to increase enzyme solubility.We suggest future directions and the strategies that can be followed to improve enzymatic synthesis of NeuAc.