Enzyme-catalyzed peptide synthesis in biphasic aqueous-organic systems

A preparative protease-catalyzed peptide synthesis in biphasic aqueous-organic systems is described. This approach using both free as well as immobilized proteases as catalysts provides high yields and includes the possibility to re-utilize the enzymes.     

Enzyme-catalyzed cascade synthesis of hydroxyiminoacetamides

In order to synthesize N-(3-azido-1-phenylpropyl)-2-hydroxyiminoacetamide, a key compound for the preparation of acetylcholinesterase (AChE) reactivators of the N-substituted 2-hydroxyiminoacetamide type, it was necessary to develop a method for forming an amide bond between an ethyl glyoxylate oxime and an amine. Using Candida antarctica lipase B (CAL-B) in a cascade enzyme-BOP catalyzed reaction, the efficient synthesis of the target hydroxyiminoacetamide was achieved.     

Enzyme-catalyzed synthesis of furanosyl nucleotides

A bacterial alpha-d-glucopyranosyl-1-phosphate thymidylyltransferase was found to couple four hexofuranosyl-1-phosphates, as well as a pentofuranosyl-1-phosphate, with deoxythymidine 5'-triphosphate, providing access to furanosyl nucleotides. The enzymatic reaction mixtures were analyzed by electrospray ionization mass spectrometry and NMR spectroscopy to determine the anomeric stereochemistry of furanosyl nucleotide products. This is the first demonstration of a nucleotidylyltransferase discriminating between diastereomeric mixtures of sugar-1-phosphates to produce stereopure, biologically relevant furanosyl nucleotides.     

Enzyme-catalyzed synthesis of natural and unnatural polysaccharides

Synthesis of natural and unnatural polysaccharide was achieved via “enzymatic polymerization” by utilizing a glycoside hydrolase as catalyst. Particularly, hyaluronan, chondroitin, and their derivatives belonging to glycosaminoglycans have been prepared using sugar oxazoline monomers designed on the basis of the concept “transition-state analogue substrate”. The oxazoline derivatives of N-acetylhyalobiuronate [GlcAβ(1→3)GlcNAc] and N-acetylchondrosine [GlcAβ(1→3)GalNAc], which have the repeating disaccharide structures of hyaluronan and chondroitin, respectively, were successfully polymerized by the catalysis of hyaluronidase, giving rise to synthetic hyaluronan and chondroitin. Their 2-substituted oxazoline derivatives were also polymerized to the corresponding N-acylated hyaluronan and chondroitin derivatives. Furthermore, N-acetylchondrosine oxazoline derivatives sulfated at the C4, the C6, and both the C4 and C6 of the GalNAc unit were catalyzed by hyaluronidase; the monomer sulfated at the C4 was polymerized to chondroitin 4-sulfate with well-defined structure, whereas the other two monomers were exclusively hydrolyzed to the corresponding disaccharides. These different kinds of natural and unnatural polysaccharides having relatively high molecular weights were produced in all cases by the catalysis of hyaluronidase. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 5014–5027, 2006     

Enzyme-catalyzed polyester synthesis in organic medium: Ring-chain equilibrium

Enzyme-catalyzed synthesis of aliphatic, unsaturated and semi-aromatic polyesters from methyl diesters and diols was carried out in toluene at 60°C in the presence of supported lipases. Whatever the monomer structure, rings were formed concurrently to linear chains excepted when dimethyl fumarate was used. The yield of rings depends on several parameters such as the monomer structure and the initial concentration of the reactive functions and strongly influences the average molar masses of the unfractionated samples. For aliphatic, unsaturated and semi-aromatic polyesters, the molar cyclization equilibrium constants (K) were determined and it would appear that the molar distribution of the ringed species obeys Jacobson-Stockmayer equation: LnK = LnB − 5/2 Lnn, where B and n represent a constant and the number of monomer units of each type in the ring, respectively.     

Enzyme-catalyzed synthesis of nucleoside triphosphates from nucleoside monophosphates

Syntheses of adenosine 5′-triphosphate (ATP) from adenosine 5′-monophosphate (AMP) and ribavirin 5′-triphosphate (RTP) from ribavirin 5′-monophosphate (RMP) (1) were performed using enzymes as catalysts. Synthesis of ATP is based on acetyl phosphate as the phosphate donor, and acetate kinase (Bacillus stearothermophilus, EC 2.7.2.1), adenylate kinase (porcine muscle, EC 2.7.4.3), and inorganic pyrophosphatase (yeast, EC 2.6.1.1) as the catalysts. Three reactions on a 150-mmol scale provided ATP as its barium salt in 82% yield and 67% purity. Synthesis of RTP used phosphoenol pyruvate (PEP) as the phosphate donor, and pyruvate kinase (rabbit muscle, EC 2.7.1.40) and adenylate kinase (rabbit muscle) as the catalysts. A gram-scale reaction provided RTP as its barium salt in 93% yield and 97% purity. This work demonstrates the utility of the autoxidationresistant acetate kinase fromB. stearothermophilus, the value of pyrophosphatase in controlling the level of pyrophosphate in the reactions and the ability of adenylate kinase to accept at least one substrate other than a derivative of adenosine.     

Enzyme-catalyzed synthesis of a novel thermosensitive polyester with pendant ketoprofen

A novel thermosensitive polyester with pendant ketoprofen was synthesized by copolymerization of PEG 800 with 2‐ketoprofendimethylmalate using CAL‐B at 70 °C. The products were characterized by NMR spectra and GPC. The in vitro study demonstrated that the polyester could release ketoprofen in physiological conditions, which indicates that the polyester could be a promising prodrug.

     

酶催化聚合研究进展

酶催化聚合是最近发展起来的一种新型聚合方法，能在温和的条件下，高效率、高选择性地完成聚合反应，将对未来工业聚合方法产生深远影响。较详细地综述了近年来酶催化聚合，特别是缩聚反应和开环聚合的研究进展。     

Enzyme-catalyzed organic synthesis of sucrose and trehalose with in situ regeneration of UDP-Glucose

This paper describes cell-free enzymatic syntheses of sucrose and trehalose using partially-purified preparations of sucrose and trehalose synthetase. The coupling of the regeneration of uridine-5′-diphosphoglucose (UDP-Glc) with synthesis of the disaccharide offers a practical route to millimol quantities of these carbohydrates. The syntheses used pyruvate kinase, UDP-Glc pyrophosphorylase, and inorganic pyrophosphatase, and the regenerated UDP-Glc was cycled approximately 10 times. This work was supported by the NIH through several grants, most recently GM 30367.     

Enzymatic polyesterification in organic media. II. Enzyme-catalyzed synthesis of lateral-substituted aliphatic polyesters and copolyesters

Enzymatic polymerization of hydroxyesters in organic media has been the subject of a previous and ongoing research in our laboratory. As part of that study, four ?-substituted-?-hydroxyesters were synthesized, and then polymerized using crude porcine pancreatic lipase (PPL) in n-hexane. These lateral-substituted hydroxyesters polymerized enantioselectively to produce optically active oligomers and a resolved optically active unreacted monomer. It was found that with increasing bulkiness of the lateral substituent, in the order Me >Et >Ph, the enzymatic reaction becomes slower, yet the enantioselectivity is higher. The lateral-substituted hydroxyesters were also copolymerized enzymatically with the more reactive linear methyl ?-hydroxyhexanoate. Optically active copolymers were obtained, higher in molecular weight than the analogous homopolymers. © 1993 John Wiley & Sons, Inc.     

Enzyme-catalyzed,gas-phase reactions

UV-49, the mutant ofPenicillium funiculosum, showed higher production of cellulase although the specific activity with regards to filter paper activity was unaltered (0.7 U/mg). Fractionation of culture filtrates by isoelectric focusing indicated the presence of three endoglucanases and two β-glucosidases in the parent strain, whereas one endoglucanase and one β-glucosidase for UV-49. The thermostability of activity towards filter paper, CM-cellulose and ρ-nitrophenyl-β-D-glucoside of the parent strain was about 30–50% more than the corresponding activities of the mutant. The saccharification of bagasse with the enzymes from parent (66%) and mutant (62%) was comparable. However, the recovery of enzyme from residual cellulose was 10–20% less in case of the mutant. The formation of higher oligosaccharides in the hydrolysates of UV-49 on prolonged incubation indicated the presence of transglycosylation activity in the enzyme complex. In contrast the parent strain produces glucose; the desired end product of the practical saccharification.     

Biphasic electrochemical peptide synthesis

The large amount of waste derived from coupling reagents is a serious drawback of peptide synthesis from a green chemistry viewpoint. To overcome this issue, we report an electrochemical peptide synthesis in a biphasic system. Anodic oxidation of triphenylphosphine (Ph₃P) generates a phosphine radical cation, which serves as the coupling reagent to activate carboxylic acids, and produces triphenylphosphine oxide (Ph₃P O) as a stoichiometric byproduct. In combination with a soluble tag-assisted liquid-phase peptide synthesis, the selective recovery of desired peptides and Ph₃P O was achieved. Given that methods to reduce Ph₃P O to Ph₃P have been reported, Ph₃P O could be a recyclable byproduct unlike byproducts from typical coupling reagents. Moreover, a commercial peptide active pharmaceutical ingredient (API), leuprorelin, was successfully synthesized without the use of traditional coupling reagents.

The large amount of waste derived from coupling reagents is a serious drawback of peptide synthesis from a green chemistry viewpoint.     

Continuous-flow solid-phase peptide synthesis

We describe a simple manually operated synthesizer for solid-phase peptide synthesis (SPPS). The synthesis is performed on standard polystyrene-based resin in a flow reactor under low-pressure conditions. The usefulness of the present configuration of SPPS is exemplified on the synthesis of two octapeptide and one decapeptide amides.     

Use of enzymes in peptide synthesis

Recent experiments in several laboratories have emphasized the benefits of proteolytic enzymes as effective catalysts for the formation of peptide bonds for synthesis and semisynthesis. This review summarizes successful applications in both stepwise synthesis for small peptides and fragment condensation to produce large polypeptides and proteins.     

Microwave heating in solid-phase peptide synthesis

The highly refined organic chemistry in solid-phase synthesis has made it the method of choice not only to assemble peptides but also small proteins - mainly on a laboratory scale but increasingly also on an industrial scale. While conductive heating occasionally has been applied to peptide synthesis, precise microwave irradiation to heat the reaction mixture during coupling and N(α)-deprotection has become increasingly popular. It has often provided dramatic reductions in synthesis times, accompanied by an increase in the crude peptide purity. Microwave heating has been proven especially relevant for sequences which might form β-sheet type structures and for sterically difficult couplings. The beneficial effect of microwave heating appears so far to be due to the precise nature of this type of heating, rather than a peptide-specific microwave effect. However, microwave heating as such is not a panacea for all difficulties in peptide syntheses and the conditions may need to be adjusted for the incorporation of Cys, His and Asp in peptides, and for the synthesis of, for example, phosphopeptides, glycopeptides, and N-methylated peptides. Here we provide a comprehensive overview of the advances in microwave heating for peptide synthesis, with a focus on systematic studies and general protocols, as well as important applications. The assembly of β-peptides, peptoids and pseudopeptides are also evaluated in this critical review (254 references).     

Enzyme-catalyzed enantioselective diaryl ketone reductions

The synthesis of diarylmethanols via the reduction of a range of substituted benzophenone and benzoylpyridine derivatives with ketoreductase enzymes (KREDs) has afforded chiral products with high yield (>90%) and ee (up to >99%). Ortho, meta, and para substitutions with a variety of electron-donating, electron-withdrawing, and halogen groups were examined. Substitution at the ortho position and/or highly electronically dissymmetric molecules were not required for good selectivity, as is the case with conventional chemical catalyst reductions. [reaction: see text].     

Amphiphilic gels for peptide synthesis

A new class of “amphiphilic” copoly(styrene-acrylamide)s of potential interest for peptide synthesis is described. As examples of the new polymers, those carrying nitrophenol or piperazine functionality were used for model reactions in peptide synthesis, and were found to be substantially more efficient than similar reagents derived from polystyrene, polydimethylacrylamide or silica gel. Due to their amphiphilic structure, the new resins have general substrate compatibility and are permeated by all of the commonly used solvents, including toluene and ethyl acetate on the one hand, through dimethylformamide, to dimethylsulfoxide and water on the other. The improved performance of the new polymers is attributed to their alternating amphiphilic structure and favorable polymer–solvent–substrate interactions. © 1992 John Wiley & Sons, Inc.