Enzymatic Conversion of Unnatural Amino Acids by Yeast D‐Amino Acid Oxidase

Unnatural amino acids, particularly synthetic α‐amino acids, are becoming crucial tools for modern drug discovery research. In particular, this application requires enantiomerically pure isomers. In this work we report on the resolution of racemic mixtures of the amino acids d,l ‐naphthylalanine and d,l ‐naphthylglycine by using a natural enzyme, D ‐amino acid oxidase from the yeast Rhodotorula gracilis. A significant improvement of the bioconversion is obtained using a single‐point mutant enzyme designed by a rational approach. With this D ‐amino acid oxidase variant the complete resolution of all the unnatural amino acids tested was obtained: in this case, the bioconversion requires a shorter time and a lower amount of biocatalyst compared to the wild‐type enzyme. The simultaneous production of the corresponding α‐keto acid, a possible precursor of the amino acid in the L ‐form, improves the significance of the procedure.     

Identification and Characterization of D‐Succinylase,and a Proposed Enzymatic Method for D‐Amino Acid Synthesis

Chiral amino acids are important intermediates for the pharmaceutical industry. We have developed a novel one‐pot enzymatic method for D ‐amino acid synthesis by the dynamic kinetic resolution of N‐succinyl‐dl ‐amino acids using D ‐succinylase (DSA) and N‐succinylamino acid racemase (NSAR, EC 4.2.1.113). The DSA from Cupriavidus sp. P4‐10‐C, which hydrolyzes N‐succinyl‐D ‐amino acids enantioselectively to their corresponding D ‐amino acids, was identified for the first time by screening soil microorganisms. Subsequently, the DSA gene was cloned and overexpressed in Escherichia coli. DSA was shown to comprise two subunits with molecular masses of 26 kDa and 60 kDa. Additionally, the NSAR gene from Geobacillus stearothermphilus NCA1503, which racemizes N‐succinylamino acids, was also cloned and overexpressed in E. coli. The highly purified DSA and NSAR prepared from each recombinant E. coli were characterized and used for D ‐amino acid synthesis. A one‐pot enzymatic method converted 100 mM N‐succinyl‐dl ‐phenylalanine to D ‐phenylalanine in 91.1% conversion with 86.7% ee. This novel enzymatic method may be useful for the industrial production of many D ‐amino acids.

     

Cationic peptides that increase the thermal stabilities of 2'-O-MeRNA/RNA duplexes but do not affect DNA/DNA melting

Several different cationic nonapeptides have been synthesized and investigated with respect to how they can influence the thermal melting of 2′‐O‐methylRNA/RNA and DNA/DNA duplexes. Each peptide has a C‐terminal L ‐phenylalanine unit and is otherwise uniformly composed of a sequence of a specific basic D ‐amino acid that in most cases will be largely charged at neutral pH. These N‐terminal octamer stretches are composed variously of the amino acids D ‐lysine, D ‐diaminobutyric acid (D ‐Dab), D ‐diaminopropionic acid (D ‐Dap), or D ‐histidine. None of the peptides substantially affected the thermal melting of DNA/DNA duplexes, which was in sharp contrast with their effects on 2′‐O‐methylRNA/RNA duplexes. In particular, the peptides based on diaminopropionic and diaminobutyric acid units had strong positive effects on the melting temperatures of the 2′‐O‐methylRNA duplexes (up to 16 °C higher with 1 equivalent of peptide) at pH 7, whereas at pH 6 the effect was even more drastic (ΔT_m up to +25 °C). The shorter R groups of the Dap and Dab groups appear to have a better length than lysine for enhancement of the thermal melting of the 2′‐O‐methylRNA/RNA duplex, an effect that is more pronounced at lower pH but substantial even at pH 7, although the Dap derivative is not likely to be fully protonated. The dramatic difference between the influence, or lack thereof, on the 2′‐O‐methylRNA/RNA and the DNA/DNA thermal meltings suggest that, although electrostatic interactions probably play a role, there is another major and structurally dependent component influencing the properties of the duplexes. This is also seen in the observation that the oligo‐Dap and oligo‐Dab peptides give greater melting point enhancements than both the lysine peptide (with a longer side chain) and a β‐linked Dap peptide with a shorter side chain and a longer backbone.     

Screening for Amidases: Isolation and Characterization of a Novel D‐Amidase from Variovorax paradoxus

Using racemic tert‐leucine amide as sole nitrogen source in minimal medium, 162 strains were isolated by enrichment techniques and shown to contain amidase activity. Among these isolates three D ‐amidase producers were found and identified as Variovorax paradoxus (two strains) and Klebsiella spec. The D ‐amidase from Variovorax paradoxus was purified to homogeneity by three chromatographic steps. With dl ‐Tle‐amide as substrate Michaelis Menten kinetics were observed with a K_M of 0.74 mM, a K_I of 640 mM and a V_max of 1.4 U/mg. The amidase has a broad pH‐optimum between 7 and 9.5 and a temperature optimum at 47–49 °C. The amidase hydrolyzed amino acid amides as well as carboxamides and 2‐hydroxy acid amides. The stereoselectivity of the reaction was variable, however. Hydrolyzing dl ‐Tle‐amide the enantiomeric ratio E was >200 resulting in D ‐Tle with an ee of >99% and up to 47% conversion. Similar results were obtained with dl ‐Leu‐amide and dl ‐Val‐amide while dl ‐Phe‐amide was hydrolyzed with an enantiomeric ratio E of only 5.     

Glutathione production by glutathione‐degrading enzymes displayed on proteoliposomes reconstituted from bovine kidney brush border membranes

Glutathione (L ‐γ‐glutamyl‐L ‐cysteinylglycine) is physiologically synthesized through two ATP‐dependent reactions catalyzed by γ‐glutamylcysteine synthase and glutathione synthase. The present study was designed to produce glutathione without the aid of ATP by using glutathione‐degrading enzymes, γ‐glutamyl transpeptidase and aminopeptidase M, in reverse: intrinsically the former enzyme catalyzes the cleavage of glutathione to give L ‐cysteinylglycine and a γ‐glutamyl moiety and the latter hydrolyzes the peptide linkage of L ‐cysteinylglycine. Both enzymes were simultaneously displayed on proteoliposomes, which were reconstituted from bovine kidney brush border membranes by a cholate dialysis method. The kinetic analysis using artificial substrates, L ‐γ‐glutamyl‐p‐nitroanilide for γ‐glutamyl transpeptidase and L ‐leucine‐p‐nitroanilide for aminopeptidase M, revealed that the proteoliposome reconstitution significantly increased the enzyme activities: for both the enzymes the maximum reaction rates were increased and Michaelis constants with the respective substrates were decreased. When the proteoliposomes were incubated with the amino acids glycine, L ‐cysteine, and L ‐glutamate (or L ‐glutamine) at 37 °C, a new product was determined on HPLC analyses using ODS and cation‐exchange columns, coinciding in retention time with authentic glutathione. This product was identified to be glutathione by LC–MS and ¹H‐NMR, after being purified by gel filtration using Sephadex G10 and HSKgel Toyopearl HW‐40F in succession. When the incubation mixture contained acivicin and bestatin, specific inhibitors for γ‐glutamyl transpeptidase and aminopeptidase M, respectively, glutathione was not produced at all. These results indicated that glutathione was produced by two‐step reversible reactions of aminopeptidase M and γ‐glutamyl transpeptidase from its constituent amino acids. The equilibrium glutathione concentration obtained with L ‐glutamine as a glutamyl donor substrate was about 3.5 times higher than that obtained with L ‐glutamate. The maximum pH for the glutathione production was 7.0–7.5, reflecting pH dependence of the activities of the enzymes. Copyright © 2004 Society of Chemical Industry     

Characterization of a novel glutamate decarboxylase from Streptococcus salivarius ssp. thermophilus Y2

BACKGROUND: γ‐Aminobutyric acid with several well‐known physiological functions is biosynthesized via the irreversible α‐decarboxylation of L ‐glutamate catalysed by glutamate decarboxylase (GAD). Although Streptococcus salivarius ssp. thermophilus has been widely applied to the dairy, the characterization of its GAD has not been reported. In this paper, the purification and the characterization of S. salivarius ssp. thermophilus GAD were investigated. RESULTS: GAD was purified 22‐fold from crude protein extracts with a yield of 7.8% in five steps. The final preparation gave a single band on SDS‐PAGE. The molecular weight of GAD determined by SDS‐PAGE and gel filtration was 46.9 kDa and 103.6 kDa, respectively, indicating that the enzyme exists as a dimmer of homological subunits. The optimum temperature and pH of GAD was 55 °C and pH 4.0, respectively. The enzyme reacted only with L ‐glutamate among 19 α‐amino acids with apparent K_m at 2.3 mmol L^?1 and did not react with D ‐glutamic acid. Activity of the enzyme could significantly be activated by 5 mmol L^?1 of BaCl₂ and inhibited by FeSO₄, ZnSO₄, CuSO₄, MnSO₄, Na₂SO₄, AgNO₃, CoCl₂, LiCl and KCl, respectively. The N‐terminal amino acid sequence of GAD was NH₂‐MNEKLFREI. CONCLUSION: Both the characterization and the deduced amino sequence (ABI31651) showed the purified enzyme was a novel GAD. Copyright © 2008 Society of Chemical Industry     

Alanine Scan of the Peptide Antibiotic Feglymycin: Assessment of Amino Acid Side Chains Contributing to Antimicrobial Activity

The antibiotic feglymycin is a linear 13‐mer peptide synthesized by the bacterium Streptomyces sp. DSM 11171. It mainly consists of the nonproteinogenic amino acids 4‐hydroxyphenylglycine and 3,5‐dihydroxyphenylglycine. An alanine scan of feglymycin was performed by solution‐phase peptide synthesis in order to assess the significance of individual amino acid side chains for biological activity. Hence, 13 peptides were synthesized from di‐ and tripeptide building blocks, and subsequently tested for antibacterial activity against Staphylococcus aureus strains. Furthermore we tested the inhibition of peptidoglycan biosynthesis enzymes MurA and MurC, which are inhibited by feglymycin. Whereas the antibacterial activity is significantly based on the three amino acids D ‐Hpg1, L ‐Hpg5, and L ‐Phe12, the inhibitory activity against MurA and MurC depends mainly on L ‐Asp13. The difference in the position dependence for antibacterial activity and enzyme inhibition suggests multiple molecular targets in the modes of action of feglymycin.     

A thermal study on the use of immobilized penicillin G acylase in the formation of 7‐amino‐3‐deacetoxy cephalosporanic acid from cephalosporin G

Penicillin G acylase (PGA) is an important enzyme for the industrial production of 7‐amino‐3‐deacetoxy cephalosporanic acid (7‐ADCA) from cephalosporin G (Ceph‐G), and 6‐aminopenicillanic acid (6‐APA) from penicillin G (Pen‐G). These products are used for the manufacture of semi‐synthetic cephalosporins and penicillins. In this study, immobilized PGA was utilized to catalyze the conversion of Ceph‐G to 7‐ADCA. The optimal conditions were found to be an operating temperature of 45 °C, 0.2 M phosphate buffer, a substrate concentration of 30 mg cm^?3 and a catalyst particle concentration of 0.01 g cm^?3 (specific activity of 623.2 U g^?1). Up to 45 °C the reaction was characterized by an activation energy of 38.66 kJ mol^?1. Beyond 57.5 °C there was a sharp decline of activity, characterized by a deactivation energy of 235.88 kJ mol^?1. Copyright © 2004 Society of Chemical Industry     

Stereocontrolled Synthesis and Pharmacological Evaluation of Azetidine‐2,3‐Dicarboxylic Acids at NMDA Receptors

The four stereoisomers of azetidine‐2,3‐dicaroxylic acid (L ‐trans‐ADC, L ‐cis‐ADC, D ‐trans‐ADC, and D ‐cis‐ADC) were synthesized in a stereocontrolled fashion following two distinct strategies: one providing the two cis‐ADC enantiomers and one giving access to the two trans‐ADC enantiomers. The four azetidinic amino acids were characterized in a radioligand binding assay ([³H]CGP39653) at native NMDA receptors: L ‐trans‐ADC showed the highest affinity (K_i=10 μM ) followed by the D ‐cis‐ADC stereoisomer (21 μM ). In contrast, the two analogues L ‐cis‐ADC and D ‐trans‐ADC were low‐affinity ligands (>100 and 90 μM , respectively). Electrophysiological characterization of the ADC compounds at the four NMDA receptor subtypes NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D expressed in Xenopus oocytes showed that L ‐trans‐ADC displayed the highest agonist potency at NR1/NR2D (EC₅₀=50 μM ), which was 9.4‐, 3.4‐, and 1.9‐fold higher than the respective potencies at NR1/NR2A–C. D ‐cis‐ADC was shown to be a partial agonist at NR1/NR2C and NR1/NR2D with medium‐range micromolar potencies (EC₅₀=720 and 230 μM , respectively). A subsequent in silico ligand–protein docking study suggested an unusual binding mode for these amino acids in the agonist binding site.     

Multi‐Enzymatic Synthesis of Optically Pure β‐Hydroxy α‐Amino Acids

A novel enzymatic production system of optically pure β‐hydroxy α‐amino acids was developed. Two enzymes were used for the system: an N‐succinyl L ‐amino acid β‐hydroxylase (SadA) belonging to the iron(II)/α‐ketoglutarate‐dependent dioxygenase superfamily and an N‐succinyl L ‐amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N‐succinyl aliphatic L ‐amino acids and produced N‐succinyl β‐hydroxy L ‐amino acids, such as N‐succinyl‐L ‐β‐hydroxyvaline, N‐succinyl‐L ‐threonine, (2S,3R)‐N‐succinyl‐L ‐β‐hydroxyisoleucine, and N‐succinyl‐L ‐threo‐β‐hydroxyleucine. LasA catalyzed the desuccinylation of various N‐succinyl‐L ‐amino acids. Surprisingly, LasA is the first amide bond‐forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L ‐leucine. By combining SadA and LasA in a preparative scale production using N‐succinyl‐L ‐leucine as substrate, 2.3 mmol of L ‐threo‐β‐hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β‐hydroxy α‐amino acids.

     

13-Hydroxy-9Z,15Z-Octadecadienoic Acid Production by Recombinant Cells Expressing Lactobacillus acidophilus 13-Hydratase

Recombinant Escherichia coli cells expressing linoleate 13‐hydratase from Lactobacillus acidophilus were permeabilized by treating with 0.2 M NaCl. The optimal conditions for the production of 13‐hydroxy‐9,15(Z,Z)‐octadecadienoic acid (13‐HODE) from α‐linolenic acid by permeabilized recombinant cells were pH 6.0, 40 °C, 7.5 % (v/v) methanol, 60 g/l permeabilized cells, and 15 g/l α‐linolenic acid. Under these conditions, permeabilized cells produced 7.5 g/l 13‐HODE after 6 h, with a conversion yield of 50 % (w/w) and a volumetric productivity of 1.25 g/l/h. These values were 161 and 160 % of those obtained by nonpermeabilized cells, respectively. To the best of our knowledge, this is the first report on the process optimization for the biotechnological production of 13‐HODE.     

Enhancement of Gluconobacter oxydans catalyzing D‐lactic acid production by coupling of synthesis and separation

BACKGROUND: Gluconobacter oxydans was applied for enantioselective oxidation of racemic‐1,2‐propanediol. Accumulation of product in the biotransformation reactor inhibited the activity and enantioselectivity of oxidative enzymes, and it also led to overproduction of lactaldehyde as an intermediate of incomplete oxidation. It was important to improve the enzymatic activity and ee value of the chiral product. RESULTS: The coupling of biotransformation and separation was studied with an in situ product removal method. Anion exchange resin was applied to remove D ‐lactic acid on line. The ee value achieved was 95% and product yield increased by 50%, compared with the control result without coupling system. CONCLUSION: Coupling of biotransformation and separation effectively decreased the free D ‐lactic acid concentration in the system. Product inhibition of enzyme activity and enantioselectivity was reduced, and the ee value and product yield were improved. Copyright © 2009 Society of Chemical Industry     

Structural Analysis Reveals the Substrate‐Binding Mechanism for the Expanded Substrate Specificity of Mutant meso‐Diaminopimelate Dehydrogenase

A meso‐diaminopimelate dehydrogenase (DAPDH) from Clostridium tetani E88 (CtDAPDH) was found to have low activity toward the D ‐amino acids other than its native substrate. Site‐directed mutagenesis similar to that carried out on the residues mutated by Vedha‐Peters et al. resulted in a mutant enzyme with highly improved catalytic ability for the synthesis of D ‐amino acids. The crystal structures of the CtDAPDH mutant in apo form and in complex with meso‐diaminopimelate (meso‐DAP), D ‐leucine (D ‐leu), and 4‐methyl‐2‐oxopentanoic acid (MOPA) were solved. meso‐DAP was found in an area outside the catalytic cavity; this suggested a possible two‐step substrate‐binding mechanism for meso‐DAP. D ‐leu and MOPA each bound both to Leu154 and to Gly155 in the open form of CtDAPDH, and structural analysis revealed the molecular basis for the expanded substrate specificity of the mutant meso‐diaminopimelate dehydrogenases.     

Biodegradations of statistical copolymers composed of D,L‐lactide and cyclic carbonates

Syntheses and biodegradation of statistical copolymers of D ,L ‐lactide (D ,L ‐LA) with trimethylene carbonate (TMC), rac‐1‐methyltrimethylene carbonate (1‐MTMC) 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. Biodegradations of poly(D ,L ‐LA‐co‐racemo‐1‐MTMC) (95/5) and poly(D ,L ‐LA‐co‐2,2‐DTMC) (98/2) with a compost at 60 °C proceed rapidly. Enzymatic degradations of these polymers were also performed using cholesterol esterase, lipoprotein lipase and proteinase K. Only poly(D ,L ‐LA‐co‐TMC) was biodegraded with cholesterol esterase, while poly(TMC), poly(1‐MTMC), poly(2,2‐DTMC) and poly(D ,L ‐LA) were barely degraded with these enzymes. Biodegradations of poly(D ,L ‐LA‐co‐TMC) (87/13) and poly(D ,L ‐LA‐co‐racemo‐1‐MTMC) (95/5) are rapid using proteinase K. Physical properties of these copolymers were also described. © 2003 Society of Chemical Industry     

Glass transition and mechanical properties of PLLA and PDLLA‐PGA copolymer blends

The change of the glass transition temperatures (T_g) in the blend of poly(L ‐lactic acid) (PLLA) and the copolymers of poly(D,L ‐lactic acid) and poly(glycolic acid) (PDLLA‐PGA) with different D,L ‐lactic acid and glycolic acid composition ratio (50 : 50, 65 : 35, and 75 : 25) was studied by DSC. Dynamic mechanical measurement and tensile testing were performed at various temperatures around T_g of the blend. In the blend of PLLA and PDLLA‐PGA50 (composition ratio of PDLLA and PGA 50 : 50), T_g decreased from that of PLLA (about 58°C) to that of PDLLA‐PGA50 (about 30°C). A single step decrease was observed in the DSC curve around T_g between the weight fraction of PLLA (W(PLLA)) 1.0 and 0.7 (about 52°C) but two‐step changes in the curve are observed between W(PLLA) = 0.6 and 0.3. The T_g change between that of PLLA and that of PDLLA‐PGA and the appearance of two T_gs suggest the existence of PLLA rich amorphous region and PDLLA‐PGA copolymer rich amorphous region in the blend. A single step decrease of E′ occurs at around T_g of the pure PLLA but the two‐step decrease was observed at W(PLLA) = 0.6 and 0.4, supporting the existence of the PLLA rich region and PDLLA‐PGA rich region. Tensile testing for various blends at elevated temperature showed that the extension without yielding occurred above T_g of the blend. Partial miscibility is suggested for PLLA and PDLLA‐PGA copolymer blends. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 2164–2173, 2004     

Lipase‐catalyzed dynamic hydrolytic resolution of (R,S)‐2,2,2‐trifluoroethyl α‐chlorophenyl acetate in water‐saturated isooctane

The hydrolytic resolution of (R,S)‐2,2,2‐trifluoroethyl α‐chlorophenylacetate in water‐saturated isooctane containing Lipase MY(I) at 35 °C is selected as the best reaction condition for producing (R)‐α‐chlorophenyl acetic acid. The kinetic constants, and hence an enantiomeric ratio of 33.6, are estimated and employed for the modeling of time‐course conversions of both substrates by considering product inhibition and enzyme deactivation effects. A successful dynamic kinetic resolution is also achieved, giving the desired (R)‐α‐chlorophenylacetic acid of 93.0% yield and ee_P = 89.5% when 80 mmol dm^?3 trioctylamine acting as the racemization catalyst and enzyme activator is initially added. Copyright © 2006 Society of Chemical Industry     

Biochemical Characterization and Mechanistic Analysis of the Levoglucosan Kinase from Lipomyces starkeyi

Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6‐anhydro‐β‐d ‐glucopyranose) in the presence of Mg²⁺–ATP. For the Lipomyces starkeyi LGK, we show here with real‐time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α‐d ‐glucose‐6‐phosphate in a manner reminiscent of an inverting β‐glycoside hydrolase. Kinetic characterization revealed the Mg²⁺ concentration for optimum activity (20–50 mm ), the apparent binding of levoglucosan (K_m=180 mm ) and ATP (K_m=1.0 mm ), as well as the inhibition by ADP (K_i=0.45 mm ) and d ‐glucose‐6‐phosphate (IC₅₀=56 mm ). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d ‐glucose‐6‐phosphate and ADP was observed under a broad range of conditions. 6‐Phospho‐α‐d ‐glucopyranosyl fluoride and 6‐phospho‐1,5‐anhydro‐2‐deoxy‐d ‐arabino‐hex‐1‐enitol (6‐phospho‐d ‐glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip ⁴C₁→¹C₄ required for 1,6‐anhydro‐product synthesis from d ‐glucose‐6‐phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.     

Synthesis of silk sericin peptides–L‐asparaginase bioconjugates and their characterization

The natural silk sericin, recovered from Bombyx mori silk waste by degumming and degrading, is a water‐soluble peptide with different molecular masses, ranging from 20 to 60 kDa. It is composed of 15 sorts of amino acids, among which the polar amino acids with hydroxyl, carboxyl and amino groups such as aspartic acid, serine and lysine account for 72%. The covalent attachment of the silk sericin peptides to L ‐asparaginase (ASNase) produces silk sericin peptides–L ‐asparaginase (SS–ASNase) bioconjugates that are active, stable, have a lower immune response, and have extended half‐lives in vitro in human serum. The modified enzyme coupled with sericin protein retains 55.8% of the original activity of the native enzyme. The optimal pH of SS–ASNase derivatives shifts considerably, to 5.0 in comparison with pH 6.0–8.0 of the native form. The thermostability and resistance to trypsin digestion of the modified enzyme are greatly enhanced as compared with ASNase alone. The Michaelis constant (K_m) of SS–ASNase is 65 times lower than that of the enzyme alone. This suggests that the affinity of the enzyme to its substrate L ‐asparagine greatly increases when bioconjugated with silk sericin. The in vivo experiments also show that the silk sericin peptides have no immunogenicity, and the antigenicity of the enzyme is obviously decreased when coupled covalently with the silk sericin peptides. Copyright © 2005 Society of Chemical Industry     

Ring opening polymerization of aliphatic cyclic carbonates in the presence of natural amino acids

Poly(dimethyl trimethylene carbonate) (PDTC) and poly(trimethylene carbonate) (PTMC) were synthesized by ring‐opening polymerization (ROP) of dimethly trimethylene carbonate (DTC) and trimethylene carbonate (TMC) in the presence of five kinds of natural amino acids (L ‐alanine, L ‐valine, L ‐leucine, L ‐proline, and L ‐phenylalanine). PDTCs with number‐average molecular weight (M_n) from 6700 to 18,900 g/mol and PTMCs with M_n from 7200 to 17,800 g/mol were obtained at a feed ratio of [monomer]/[L ‐phenylalanine] ranging from 50 to 200. The results of ¹H nuclear magnetic resonance and titration proved amino acid connecting onto the polymer backbone. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Synthesis of poly (γ‐benzyl‐L‐glutamate) with well‐defined terminal structures and its block polypeptides with alanine,leucine and phenylalanine

The ring‐opening polymerization of γ‐benzyl‐L ‐glutamate N‐carboxyanhydride (BLG‐NCA) was initiated by n‐hexylamine in N,N‐dimethyformamide under normal pressure at 0 °C. The products were characterizated by gel permeation chromatography, matrix‐assisted laser desorption/ionization time of flight mass spectroscopy (MALDI‐TOF MS), nuclear magnetic resonance etc. MALDI‐TOF MS gave direct evidence that the side reactions during the polymerization of BLG‐NCA could be greatly reduced by decreasing the reaction temperature, e.g. from room temperature to 0 °C. As a result, over 90% of the products were amino‐terminated poly(γ‐benzyl‐L ‐glutamate) (PBLG) with low polydispersity index when the polymerization was carried out at 0 °C, which could be used to re‐initiate the polymerization of other NCAs. Then several well‐defined PBLG‐containing block copolypeptides were successfully synthesized in a convenient way. Copyright © 2012 Society of Chemical Industry