Papain-catalyzed Synthesis Research Articles

Papain-Catalyzed Synthesis of Oligolysine in Low-Water Organic Reaction Media

Papain-Catalyzed Synthesis of Oligolysine in Low-Water Organic Reaction Media

Efficacy of L-Cysteine as an Anti-Oxidant in Papain Catalyzed Synthesis of Oligopeptides in Organic Solvent System

Enzymatic peptide synthesis has drawn considerable attention for synthesis of high by-pass oligopeptide feed supplements in animal nutrition. A hardy protease, (papain) with a cysteine moiety at the active site requires the presence of an anti-oxidant in the reaction medium to ensure that the thiol group remains intact. Free cysteine has been the antioxidant of choice for papain-catalyzed synthesis of oligopeptides in aqueous systems. However, due to limited solubility of cysteine in organic solvents, it is generally not a suitable antioxidant for the synthesis of oligopeptides in biphasic solvent systems; instead, mercaptoethanol is often used. Lysine and Methionine is couple of well-known limiting amino acids that find application in cattle feed and poultry. Synthesis of co-oligopeptides of Lysine and Methionine has generally been attempted in bi-phasic solvent systems with Mercaptoethanol as an anti-oxidant. The inherent toxicity of mercaptoethanol when present even in trace amounts make its use undesirable during synthesis of oligopeptides to be used as feed supplements. Use of non-toxic antioxidants such as Cysteine would make the end product more amenable as a nutritional supplement for animal feed. Therefore efficacy of L-cysteine as an anti-oxidant was investigated during papain catalyzed oligomerization of Lys, Arg, Glu and Asp in two organic systems: a three phase micro-aqueous media consisting of n-octane, DFP and water, as well as homogeneous ACN/water mixtures. Reactions were also carried out under an argon atmosphere in the presence and absence of anti-oxidants. The results of the experiments showed that L-cysteine facilitated oligomer synthesis in both the three phase system and the ACN/water mixture. The overall oligomer yields were found to be better than 75% in the presence of L-Cysteine. Oligopeptide yields obtained through reactions carried out under the argon atmosphere were less than 20%.

Papain-Catalyzed Synthesis of Polyglutamate Containing a Nylon Monomer Unit.

Peptides have the potential to serve as an alternative for petroleum-based polymers to support a sustainable society. However, they lack thermoplasticity, owing to their strong intermolecular interactions. In contrast, nylon is famous for its thermoplasticity and chemical resistance. Here, we synthesized peptides containing a nylon unit to modify their thermal properties by using papain-catalyzed chemoenzymatic polymerization. We used l-glutamic acid alkyl ester as the amino acid monomer and nylon 1, 3, 4, and 6 alkyl esters as the nylon unit. Papain catalyzed the copolymerization of glutamic acid with nylon 3, 4, and 6 alkyl esters, whereas the nylon 1 unit could not be copolymerized. Other proteases used in this study, namely, bromelain, proteinase K, and Candida antarctica lipase (CALB), were not able to copolymerize with any nylon units. The broad substrate specificity of papain enabled the copolymerization of l-glutamic acid with a nylon unit. The peptides with nylon units demonstrated different thermal profiles from that of oligo(l-glutamic acid). Therefore, the resultant peptides with various nylon units are expected to form fewer intermolecular hydrogen bonds, thus altering their thermal properties. This finding is expected to broaden the applications of peptide materials and chemoenzymatic polymerization.

Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides

Di-, tri-, and tetra-tyrosine peptides with angiotensin I-converting enzyme inhibitory activity were synthesized by papain-catalyzed polymerization of l-tyrosine ethyl ester in aqueous media at 30°C. Varying the reaction pH from 6.0 to 7.5 and the initial concentration of the ester substrate from 25 to 100mM, the highest yield of oligo-tyrosine peptides (79% on a substrate basis) was produced at pH 6.5 and 75mM, respectively. In the reaction initiated with 100mM of the substrate, approx. 50% yield of insoluble, highly polymerized peptides accumulated. At less than 15mM, the reaction proceeded poorly; however, from 30mM to 120mM a dose-dependent increase in the consumption rate of the substrate was observed with a sigmoidal curve. Meanwhile, each of the tri- and tetra-tyrosine peptides, even at approx. 5mM, was consumed effectively by papain but was not elongated to insoluble polymers. For deacylation of the acyl-papain intermediate through which a new peptide bond is made, l-tyrosine ethyl ester, even at 5mM, showed higher nucleophilic activity than di- and tri-tyrosine. These results indicate that the mechanism through which papain polymerizes l-tyrosine ethyl ester is as follows: the first interaction between papain and the ester substrate is a rate-limiting step; oligo-tyrosine peptides produced early in the reaction period are preferentially used as acyl donors, while the initial ester substrate strongly contributes as a nucleophile to the elongation of the peptide product; and the balance between hydrolytic fragmentation and further elongation of oligo-tyrosine peptides is dependent on the surrounding concentration of the ester substrate.

Surprising metal binding properties of phytochelatin-like peptides prepared by protease-catalysis

Phytochelatins (PCs) consist of alternating glutamic acid and cysteine residues ([γGlu–Cys]–Xaa) and are responsible for binding to heavy metals for cellular metal homeostasis and detoxification. This paper describes the papain-catalyzed synthesis of cysteine-rich peptides as potential PC mimics. By adjusting the feed ratio of L-cysteine ethyl ester (L-Et-Cys) and L-glutamic acid diethyl ester (L-(Et)2-Glu) followed by de-esterification, α-linked oligo(L-Glu-co-47%L-Cys) was prepared that closely matches the 1 : 1 L-Glu-to-L-Cys molar ratio of PC peptides. Plots of absorbance difference as a function of total metal-to-peptide ([M]total–[P]total) molar ratio were constructed for titrations with Zn(II), Cd(II), Co(II) and Ni(II). A series of equations were generated to evaluate dissociation constants and the number of metal ions per peptide molecule in metal–peptide complexes. All of the four complexes have on average two oligo(L-Glu-co-47%L-Cys) molecules per divalent cation. The binding of metals to oligo(L-Glu-co-47%L-Cys) weakens in the order of Cd(II) > Zn(II) > Ni(II) > Co(II), the same trend observed for [(γGlu–Cys)4]–Gly. The peptide quantity required to sequester a given amount of Zn(II) and Cd(II) when using oligo(L-Glu-co-47%L-Cys) is only about twice as much as the quantity for perfect sequence peptide (γGlu–Cys)2–Gly. Furthermore, when [M]total is low, random oligo(L-Glu-co-47%L-Cys) sequesters much higher fractions of Co(II) and Ni(II) than does (γGlu–Cys)2–Gly. Results from this work provide the first evidence that, in some cases, uniform peptides synthesized by tedious solid or liquid phase peptide synthetic methods, can be replaced with peptide mixtures prepared by facile protease-catalyzed peptide synthetic methods without substantial loss in product performance.

High level production of bioactive di- and tri-tyrosine peptides by protease-catalyzed reactions

Papain-catalyzed polymerization of l-tyrosine ethyl ester in aqueous media was efficient for synthesis of oligo-tyrosine peptides having angiotensin I-converting enzyme inhibitory activity. Di-, tri-, and tetra-tyrosine accumulated in the soluble fraction of reaction mixture. On the other hand, the peptide products with degree of polymerization from 5 to 10 were insoluble, yields of which were influenced by initial concentrations of the ester substrate. The precipitated products could be used as substrates for α-chymotrypsin in DMSO/buffer systems producing soluble oligo-tyrosine peptides. In the reaction media containing DMSO at 40–50% (v/v), most of the precipitates were converted to soluble peptides. The two-step enzymatic reaction, that is papain-catalyzed synthesis of Tyr-polymers from l-tyrosine ethyl ester followed by their hydrolytic cleavage by α-chymotrypsin, is expected to be a potent procedure for synthesis of biologically active di- and tri-tyrosine peptides in good yield.

Papain-catalyzed synthesis of aspartame precursor: A comparison with thermolysin

A preliminary study was made on the feasibility of using papain as a catalyst in the synthesis of aspartame precursor (Z-Asp-OH + Phe-OMe→Z-Asp-Phe-OMe). The synthetic reaction was carried out by an equilibrium-controlled process using three different systems: precipitation, dissolved-state, and biphasic. A comparative study was made with thermolysin-catalyzed aspartame precursor synthesis in each reaction system. Papain was useful only when the biphasic system was employed for synthesis, while thermolysin was efficient in all the systems tested. Thermolysin appeared to be superior to papain even in the biphasic system; the reason for this was ascribed to the absence of esterase activity. The yield of Z-Asp-Phe-OMe in the biphasic system was increased when papain was used in a reaction mixture containing high concentrations of the starting materials. The likely reason for this is discussed.

Enzymatic synthesis of carboxyglutamic acid containing peptides in organic media

Kinetically-controlled papain-catalyzed synthesis of carboxyglutamic acid containing peptides is reported. The stereoselectivity of papain allowed to use the racemic Z- γ, γ′-di- tert-butyl-D,L-Gla-OMe intermediate as acyl donor ester to obtain diasteromerically pure peptide derivatives. As examples, several carboxyglutamic acid containing dipeptide derivatives were synthesized enzymatically in high chemical and enantiomeric yields.

Papain-catalyzed synthesis of peptide isosteres

The thiol protease papain catalyses the transfer of the Cbz-Gly moiety from the donor substrate Cbz-Gly-OMe to the amino group of various α-hydroxy-and α-keto-amines, providing a new route to peptide isosteres.

How to predict product yield in kinetically controlled enzymatic peptide synthesis

The published theory of kinetically controlled enzymatic peptide synthesis needed experimental verification. We carried out the measurement of the peptide yield and estimation of the key parameters alpha and beta for papain-catalyzed synthesis of Mal-L-Phe-L-Ala-LLeuNH(2) from Mal-L-Phe-L-AlaOMe and L-LeuNH(2). The experimental results demonstrate that this theory adequately describes the real process.

ChemInform Abstract: Stereoselective Papain‐Catalyzed Synthesis of Alafosfalin (I).

ChemInformVolume 21, Issue 28 Natural Products ChemInform Abstract: Stereoselective Papain-Catalyzed Synthesis of Alafosfalin (I). V. A. SOLODENKO, V. A. SOLODENKO Inst. Bioorg. Chem., Ukrainian Acad. Sci., Kiev 252660, USSRSearch for more papers by this authorV. P. KUKHAR', V. P. KUKHAR' Inst. Bioorg. Chem., Ukrainian Acad. Sci., Kiev 252660, USSRSearch for more papers by this author V. A. SOLODENKO, V. A. SOLODENKO Inst. Bioorg. Chem., Ukrainian Acad. Sci., Kiev 252660, USSRSearch for more papers by this authorV. P. KUKHAR', V. P. KUKHAR' Inst. Bioorg. Chem., Ukrainian Acad. Sci., Kiev 252660, USSRSearch for more papers by this author First published: July 10, 1990 https://doi.org/10.1002/chin.199028288AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume21, Issue28July 10, 1990 RelatedInformation

Papain-catalyzed fragment synthesis of protected cholecystokinin derivatives

Sodium salts of methyl esters of N-tert-butyloxycarbonyl-β-tert-butylaspartyl-O4-sulfotyrosine (II) or N-tert-butyloxycarbonyl-O4-sulfotyrosine (III) were condensed with amino components derived from peptide amides IVb-IVe and IVg (simulating the carboxy-terminal part of cholecystokinin) under catalysis with papain. Rates and yields of conversion of these peptides to the corresponding derivatives Ib-If were compared with the results reported previously for analogous papain-catalyzed fragment synthesis of the protected carboxy-terminal octapeptide of cholecystokinin Ia. In the condensation reactions with the individual amino components quantitative changes were observed in the ability of papain to catalyze the peptide bond synthesis which appeared as differences in the maximum yield (and the time required for achieving it) of the condensation reaction, monitored by HPLC. The observed differences are related not only with the distance of the amino acid substitution from the P'1 subsite in the given amino component but also with the side-chain structure of the substituting amino acid.

Stereoselective papain-catalyzed synthesis of alafosfalin

Papain in powdered form efficiently catalyzes alafosfalin synthesis in miscible aqueous-organic solvent involving L-aminophosphonate in peptide bond formation.

Papain-catalyzed synthesis of aspartame precursor in biphasic system

Papain-catalyzed synthesis of aspartame precursor in biphasic system

Papain-catalyzed synthesis of 2-naphthylamides of N-acylamino acids and dipeptides

2-Naphthylamides of several N-acylamino acids were prepared by papain-catalyzed condensation reaction in acidic medium. Under the same conditions, papain catalyzed the synthesis of peptide bond between benzyloxycarbonylglycine and phenylalanine 2-naphthylamide and between benzyloxycarbonylserine and tyrosine 2-naphthylamide. Phenylalanine 2-naphthylamide was also acylated with benzyloxycarbonyl glycine methyl ester in an alkaline medium. For comparison, papain-catalyzed condensations of benzyloxycarbonyl-S-benzylcysteine or benzyloxycarbonylalanine with aniline and its derivatives, benzylamine, phenylhydrazine, cyclohexylamine and 1-naphthylamine, were studied.

On the use of carboxamidomethyl esters in the protease-catalyzed peptide synthesis

Carboxamidomethyl esters (CAM esters) ofZ- andBoc-protected alanine and phenylalanine were prepared in order to investigate their usefulness as substrates for α-chymotrypsin- and papain-catalyzed hydrolysis and peptide synthesis reactions. The easy removal of theCAM-C-protecting group under mild conditions and dependent on the enzyme specificity was demonstrated. Examples are given for the protease-catalyzed synthesis of various peptide derivatives usingCAM esters as C- and N-components in aqueous-organic media. Comparatively short reaction times were observed.

Papain-catalyzed synthesis of phenylhydrazides of N-acylamino acids

Phenylhydrazides of Nα-acyl derivatives of all coded amino acids, except proline, were prepared by papain (E.C. 3.4.22.2)-catalyzed synthesis. Comparison of yields affords information about the suitability of the amino acid in position P1 in papain-catalyzed syntheses of peptides.

Papain-catalyzed synthesis of methionine-enriched soy plasteins. Average chain length of the plastein peptides

Papain-catalyzed synthesis of methionine-enriched soy plasteins. Average chain length of the plastein peptides

Location and State of Methionine Residues in a Papain-synthesized

With the aid of papain, a plastein was synthesized from a 1:10 mixture of Lmethionine ethyl ester and a peptic hydrolysate of soybean protein. Dialysis of the whole reaction-product yielded a methionine-incorporated plastein (Met-plastein) as the nondialyzable fraction, its yield being 78.2% on a dry-matter basis, of the whole reaction-product. Methionine content in this Met-plastein was 7.22% on a weight basis, while its content in the material hydrolysate was only 1.25%. Carboxypeptidase A treatment of Met-plastein liberated methionine at an outstandingly rapid rate. A similar, but not so outstanding, rate was observed for the methionine liberation from Met-plastein by treatment with leucine aminopeptidase. Methyl isothiocyanate treatment and subsequent cyclization yielded a mixture of methylthiohydantoins from Met-plastein. Gas chromatographic analysis of this mixture after trimethylsilylation showed a result that methionine occupied 33.2%, on a molar basis, of the total N-terminal amino acids. Lithium borohydride reduction and 6 N hydrochloric acid hydrolysis of Met-plastein produced monomeric aminols, and their 2, 4-dinitrophenylation followed by thin-layer chromatography gave a result that methionine occupied 84.9%, on a molar basis, of the total C-terminal amino acids; the residues amounting to 14.4%, of the C-terminal methionine residues remained as an ethyl eater form. The selective degradation probe employing cyanogen bromide to generate free homoserine disclosed that the occurrence of the polymeric methionine-methionine sequence was little if any in Met-plastein. Based on the above experiments as well as an evaluation of the esterase activity against L-methionine ethyl ester, a possible mechanism was discussed of the papain-catalyzed synthesis of plastein in a system containing such an ester.

The papain-catalyzed synthesis of hippuryl anilide II. Variation of rate with substrate concentration and determination of the equilibrium constant

Summary 1. The kinetics of the papain-catalyzed condensation of hippuric acid and aniline to form hippuryl anilide were followed at pH 4.9 and at 39° by observing the initial disappearance of amine. Initial rate data satisfied a rate law deduced from a mechanism in which one substrate, hippuric acid, is in equilibrium with the enzyme. Kinetic theory was introduced to provide a method for distinguishing two substrate reactions in which one product is present at zero time. For the case of papain (EC 3.4.4.10) the “quasi-equilibrium” assumption was found to apply, and K m , equal to K s for hippuric acid, was found to be 0.82 M. The value of ks was found to be 60.2 M −1 · min −1 and it was suggested that this bimolecular rate constant is associated with the decomposition of the acyl-enzyme, hippurylpapain. 2. The apparent equilibrium constant, K ′ e , was found to be 0.094 indicating that hydrolysis is the thermodynamically favored process. Thermodynamic arguments were presented to suggest that one substrate, hippuric acid, reacts with papain only in the form of the conjugate base, hippurate ion.