alpha-Chymotrypsin as the catalyst for peptide synthesis.

alpha-Chymotrypsin (EC 3.4.21.1)-catalysed syntheses of peptides were performed with various N-acylated amino acid or peptide esters as donors, and amino acid derivatives, peptides or their derivatives as acceptors. Under optimal conditions the synthesis was almost quantitative. As acceptor nucleophiles, free amino acids or the ester derivatives were inadequate, but amino acid amides or hydrazides, di- or tri-peptides, or the amides, hydrazides and esters of the peptides were useful. The nucleophile specificity for synthesis was markedly similar to the leaving-group specificity in hydrolysis; hydrophobic or bulky amino acid residues were most effecient at both P1' and P2' positions [notation of Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162], but L-proline as well as D-amino acid residues were the worst choices. The synthesis was further dependent on the solubility of the products synthesized; a higher yield of products was expected with lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible by using N-acylated peptide esters and various peptides. The present study suggested that alpha-chymotrypsin may become a useful tool for peptide synthesis.     

Peptide bond synthesis catalyzed by alpha-chymotrypsin.

alpha-Chymotrypsin [EC 3.4.21.1] catalyzed the syntheses of peptide bonds with various N-acylated amino acids or peptides having aromatic or hydrophobic amino acid residues at the C-terminal position as carboxyl components, and amino acid derivatives, peptides or their derivatives as amine components. A neutral pH was most efficient and quite high concentrations of alpha-chymotrypsin and starting materials were required for synthesis. Four amine components, hydrophobic or bulky amino acid residues were useful at the N-terminal position. Stereospecificity was also observed at the N-terminal position of amine components. Peptide synthesis was not usually seen when the products were soluble in the reaction mixture. This could be partly overcome by increasing the concentration of either the carboxyl or the amine component to more than ten times that of the other.     

Specificity of Proteinase K from Tritirachium album Limber for Synthetic Peptides

The specificity of proteinase K from Tritirachium album Limber was determined using various synthetic peptide substrates. The esterase activity against N-acylated amino acid esters indicated that the enzyme is primarily specific against aromatic or hydrophobic amino acid residues at the carboxyl side of the splitting point. Secondary interaction for hydrolysis was also studied using peptide esters or others, which showed that the enzyme activity is markedly promoted by elongating the peptide chain to the N-terminal from the splitting point. Thus, peptide chloromethyl ketone derivatives such as Cbz-Ala-Gly-PheCH₂Cl inactivated the enzyme activity markedly.     

Enzymatic synthesis of chromogenic substrates for Glu,Asp-specific proteinases.

Glu,Asp-specific endopeptidases represent a new subfamily of chymotrypsin-like proteolytic enzymes. These enzymes prefer Glu or Asp residues in the P1 position of the substrates. p-Nitroanilides of N-acylated di-, tri- and tetrapeptides with C-terminal glutamic or aspartic acid residues have been obtained. Acyl peptide p-nitroanilides were synthesized via acylation of glutamic or aspartic acid p-nitroanilides using methyl esters of the respective N-acylated peptides, generally with good yields. The reactions were performed in organic solvents using subtilisin 72 sorbed on silica as a catalyst. The kinetic parameters for the hydrolysis of these p-nitroanilides with proteinases from Bacillus intermedius and Bacillus licheniformis were determined.     

Determination of enzyme specificity in a complex mixture of peptide substrates by N-terminal sequence analysis.

A method has been developed to determine preferred residue substitutions in the P' position of peptide substrates for proteolytic enzymes. The method has been validated with four different enzymes; the angiotensin I-converting enzyme, atrial dipeptidyl carboxyhydrolase, bacterial dipeptidyl carboxyhydrolase, and meprin A. A mixture of N-acylated potential peptide-substrates for each of the enzymes was prepared in a single synthesis procedure on the same solid-phase synthesis resin. The peptides were identical in all residue positions except the P' position to be studied, into which numerous amino acid residues were incorporated on a theoretical equimolar basis. After cleavage and extraction of the peptides from the resin, no attempt was made to purify them individually; the exact concentration of each peptide in the mixture was determined by quantitative amino acid analysis. Incubation of an enzyme with its peptide-substrate mixture at [S] much less than Km yielded peptide hydrolytic products with newly exposed N-termini. The identity and amount of each hydrolysis product was determined by automated N-terminal sequence analysis. One cycle of sequencing revealed preferred amino acid substitutions in the P'1 position, two cycles the P'2 position, and so forth. Comparison of the rates of production of the various products indicates the preferred substitution in that particular P' position. New information on the substrate specificities of each of the enzymes tested was obtained and it is clear that this approach can be applied to any protease with a defined (or suspected) point of cleavage in a peptide substrate.     

Synthesis and solubility properties of peptide fragments of human hemoglobin alpha-chain (123-136)

The solubility prediction method for protected peptides was successfully applied to relatively small peptide fragments of human hemoglobin alpha-chain (123-136) which contained various polar amino acid residues such as Asp(OBzl), Glu(OBzl), Lys(Z), Ser(Bzl), and Thr(Bzl). As reported previously for hydrophobic peptides and human proinsulin C-peptide fragments, solubility data indicated that the insolubility of protected peptides having a mean value of Pc value below 0.90 appeared to begin at the octa- or nonapeptide sequence level and that beta-sheet structure played an important role in the insolubility of peptides. When a peptide has a beta-sheet structure in the solid state, we can clearly determine the critical chain length for peptide insolubility, the solubility dependence on solvent properties, and the solubility independence of amino acid compositions of peptides.     

Protease-catalysed peptide synthesis in solvent-free system

Summary A new method of enzymatic peptide synthesis without any liquid added has been proposed. The method is based on the admixing of N-acylamino acid (peptide) esters and nucleophiles (amides or tert.-butylesters of amino acids or peptides, peptides) with various proteolytic enzymes such as α-chymotrypsin, trypsin, proteinase K, subtilisin, elastase and papain in the presence of Na₂CO₃. 10H₂O. In most instances, acylating components were completely converted within a few hours giving satisfactory yields of desired peptide products.     

Amino acid pyridoxyl esters in peptide synthesis

Pyridoxyl residue was suggested to be used as a multifunctional protective and modifying group in peptide synthesis. The modification was carried out by introducing the pyridoxyl residue in free or partially protected peptides or by the addition of amino acid pyridoxyl esters by the methods of conventional peptide synthesis without the removal of the pyridoxyl group at the terminal stages of the synthesis (the second approach is more convenient). Pyridoxyl residue was also used as a spacer in solid phase peptide synthesis. It was attached to the polymer by the alkylation of the hydroxyl groups or of the pyridine ring of the pyridoxyl derivatives with the chloromethylated styrene-divinylbenzene copolymer (the standard Merrifield resin). Potentials for the use of pyridoxyl derivatives in the synthesis of linear, multiplet, and cyclic peptides are discussed.     

Peptide enzymatic microsynthesis, using carboxypeptidase Y as the catalyst: application to stepwise synthesis of Leuenkephalin

In order to develop the use of carboxypeptidase Y (CPD-Y, EC 3.4.12.1) as a catalyst for radioactive hormonal synthesis, the stepwise synthesis of a pentapeptide, Leu-enkephalin, was studied on a microscale. Each peptide bond was formed by enzymatic catalysis, using microquantities of the precursors (amino acid or peptide esters as acyl-components and amino acid ester or amide as nucleophiles). The high condensation yields obtained suggests that CPD-Y can be a useful tool for preparation of radioactive hormonal peptides.     

Pyridoxyl amino acid esters in peptide synthesis

Pyridoxyl residue was suggested to be used as a multifunctional protective and modifying group in peptide synthesis. The modification was carried out by introducing the pyridoxyl residue in free or partially protected peptides or by the addition of amino acid pyridoxyl esters by the methods of conventional peptide synthesis without the removal of the pyridoxyl group at the terminal stages of the synthesis (the second approach is more convenient). Pyridoxyl residue was also used as a spacer in solid phase peptide synthesis. It was attached to the polymer by the alkylation of the hydroxyl groups or of the pyridine ring of the pyridoxyl derivatives with the chloromethylated styrene-divinylbenzene copolymer (the standard Merrifield resin). Potentials for the use of pyridoxyl derivatives in the synthesis of linear, multiplet, and cyclic peptides are discussed.     

Galactosylated derivatives of low-molecular-weight chitosan: production and properties

Chitosan, a binary heteropolysaccharide consisting of 2-acetamide-2-deoxy-beta-D-glucopyranose and 2-amino-2-deoxy-beta-D-glucopyranose residues linked in different proportions via beta-glycosidic bonds. The presence of a primary amino group in the chitosan structure allows for the synthesis of various derivatives. The procedure of obtaining activated N-hydroxysuccinimide esters with the use of lactobionic acid was applied to obtain galactosylated derivatives of low-molecular-weight chitosan with a substitution degree varying from 8 to 23%. The properties of these derivatives (viscosity, solubility, and biodegradability) were studied. These derivatives are well soluble at pH values greater than the acidity constant of amino groups of chitosan (6.5). Broadening the pH range towards increase and the presence of galactose residues allows these derivatives to be used in working with biological objects.     

The synthesis and some biological properties of N-(6-purinyl)peptides

The chemical synthesis and biological properties of N-(6-purinyl)peptides are described. N-(6-Purinyl)amino-acid derivatives were synthesized and condensed with amino acid esters and peptide esters using the dicyclohexylcarbodiimide/N-hydroxysuccinimide method. The products were isolated via gel filtration on Sephadex G-10 in 0.05M NH4HCO3 followed by either ion exchange chromatography on SP-Sephadex or by preparative HPLC. The methyl esters were saponified and the tert-butyl ester group was removed by treatment with trifluoroacetic acid without damaging the purinyl residue. N-(6-Purinyl)peptides were characterised by chromatographic and spectroscopic methods. Acid hydrolysis of N-(6-purinyl)-L-amino acids caused the racemization of the neighbouring L-amino acid. Model studies were performed with N-(6-purinyl)-L-alanine, N-(6-purinyl)-D-alanine, N-(6-purinyl)-L-alanyl-L-leucine and N-(6-purinyl)-D-alanyl-L-leucine. After acid hydrolysis the N-(6-purinyl)amino acids were totally racemized and the N-(6-purinyl)dipeptides formed 14% of the enantiomer of alanine. The N-(6-purinyl)-omega-amino acids and the N-(6-purinyl)peptides were screened in a limited number of tests as immunomodulators (antibody-secretion, phagocytosis, cytostatic activity of macrophages) and as cytotoxic agents.     

Galactosylated derivatives of low-molecular-weight chitosan: Obtaining and properties

Chitosan, a binary heteropolysaccharide consisting of 2-acetamide-2-deoxy-β-D-glucopyranose and 2-amino-2-deoxy-β-D-glucopyranose residues linked in different proportions via β-glycosidic bonds. The presence of a primary amino group in the chitosan structure allows for the synthesis of various derivatives. The procedure of obtaining activated N-hydroxysuccinimide esters with the use of lactobionic acid was applied to obtain galactosylated derivatives of low-molecular-weight chitosan with a substitution degree varying from 8 to 23%. The properties of these derivatives (viscosity, solubility, and biodegradability) were studied. These derivatives are well soluble at pH values greater than the acidity constant of amino groups of chitosan (6.5). Broadening the pH range towards increase and the presence of galactose residues allows these derivatives to be used in working with biological objects.     

O-N intramolecular acyl migration reaction in the development of prodrugs and the synthesis of difficult sequence-containing bioactive peptides

N-Ointramolecular acyl migration in Ser- or Thr-containing peptides is a well-known side reaction in peptide chemistry. It results in the mutual conversion of ester and amide bonds. Our medicinal chemistry study focused on the fact that the O-acyl product can be readily converted to the original N-acyl form under neutral or slightly basic conditions in an aqueous buffer and the liberated ionized amino group enhances the water solubility of O-acyl products. Because of this, we have developed a novel class of "O-N intramolecular acyl migration"-type water-soluble prodrugs of HIV-1 protease inhibitors. These prodrugs released the parent drugs via a simple chemical mechanism with no side reaction. In this study, we applied this strategy to important cancer chemotherapeutic agents, paclitaxel and its derivatives, to develop water-soluble taxoid prodrugs, and found that these prodrugs, 2'-O-isoform of taxoids, showed promising results with higher water solubility and proper kinetics in their parent drug formation by a simple pH-dependent chemical mechanism with O-N intramolecular acyl migration. These results suggest that this strategy would be useful in toxicology and medical economics. After the successful application of O-N intramolecular acyl migration in medicinal chemistry, this concept was recently used in peptide chemistry for the synthesis of "difficult sequence-containing peptides." The strategy was based on hydrophilic O-acyl isopeptide synthesis followed by the O-N intramolecular acyl migration reaction, leading to the desired peptide. In a model study with small, difficult sequence-containing peptides, synthesized "O-acyl isopeptides" not only improved the solubility in various media and efficiently performed the high performance liquid chromatography purification, but also altered the nature of the difficult sequence during SPPS, resulting in the efficient synthesis of O-acyl isopeptides with no complications. The subsequent O-N intramolecular acyl migration of purified O-acyl isopeptides afforded the desired peptides as precipitates with high yield and purity. Further study of the synthesis of a larger difficult sequence-containing peptide, Alzheimer's disease-related peptide (A beta 1-42), surprisingly showed that only one insertion of the O-acyl group drastically improved the unfavorable nature of the difficult sequence in A beta 1-42, and achieved efficient synthesis of 26-O-acyl isoA beta 1-42 and subsequent complete conversion to A beta 1-42 via the O-N intramolecular acyl migration reaction of 26-O-acyl isoA beta 1-42. This suggests that our new method based on O-N intramolecular acyl migration is an important method for the synthesis of difficult sequence-containing bioactive peptides.     

An approach to the differentiation of leucine and isoleucine residues in EI mass spectra of peptides

Residues of leucine and isoleucine cannot generally be distinguished in the electron impact (EI) generated mass spectra of N-acylated peptide esters. We have obtained the mass spectra of model peptide esters containing leucine or isoleucine in various positions and trifluoroacetyl perdeutero leucine as the N-terminal blocking group. The mass spectra of the peptide derivatives show a pair of peaks as a result of the elimination from the M⁺ ion of neutral fragment of perdeuterated isobutene (M⁺-64) from the leucine side chain of the N-terminal blocking group and isobutene or butene (M⁺-56) from leucine or isoleucine residues of the peptide. The ratios of the intensities of the peaks $\text{M}{}^{\mathrm{+}}\text{-56}\text{M}{}^{\mathrm{+}}\text{-64}$ show considerable variation with the position of leucine or isoleucine in the peptide chain and the length of the peptide, but for peptides which are identical except for the fact that one contains leucine and the other isoleucine in a given position the ratio is always smaller for the isoleucine containing peptide. The differences are sufficient to distinguish the isomeric residues if comparison spectra are available.     

Covalent chromatography — The isolation of tryptophanyl containing peptides by novel polymeric reagents

Specific polymeric reagents for reversible covalent binding of tryptophan residues have been developed. Polymers bearing Aryl-SxCl groups (x=2–3) were prepared by binding thioaryl groups to cross-linked polyacrylamide, and subsequently reacting the products with an excess of S₂Cl₂. The resulting polymers were allowed to react with various mixtures of amino acids and peptides (excluding cysteine and its peptides) in acidic media. It was found that tryptophan as well as tryptophan-containing peptides were selectively bound to the polymer. Upon reduction with thiols (e.g. dithiothreitol), 2-thiotryptophan or its peptide derivatives were cleaved from the polymeric matrix. The proposed method is used for a one step isolation of tryptophanyl-containing peptides from peptide mixtures as well as for introducing thiol groups at the tryptophanyl residues.     

Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.     

Synthesis of N-carboxyalkyl and N-aminoalkyl functionalized dipeptide building units for the assembly of backbone cyclic peptides.

To improve the assembly of backbone cyclic peptides, N-functionalized dipeptide building units were synthesized. The corresponding N-aminoalkyl or N-carboxyalkyl amino acids were formed by alkylation or reductive alkylation of amino acid benzyl or tert-butyl esters. In the case of N-aminoalkyl amino acid derivatives the aldehydes for reductive alkylation were obtained from N,O-dimethyl hydroxamates of N-protected amino acids by reduction with LiAlH4. N-carboxymethyl amino acids were synthesized by alkylation using bromoacetic acid ester and the N-carboxyethyl amino acids via reductive alkylation using aldehydes derived from formyl Meldrums acid. Removal of the carboxy protecting group leads to free N-alkyl amino acids of very low solubility in organic solvents, allowing efficient purification by extraction of the crude product. These N-alkyl amino acids were converted to their tetramethylsilane-esters by silylation with N,O-bis-(trimethylsilyl)acetamide and could thus be used for the coupling with Fmoc-protected amino acid chlorides or fluorides. To avoid racemization the tert-butyl esters of N-alkyl amino acids were coupled with the Fmoc-amino acid halides in the presence of the weak base collidine. Both the N-aminoalkyl and N-carboxyalkyl functionalized dipeptide building units could be obtained in good yield and purity. For peptide assembly on the solid support, the allyl type protection of the branching moiety turned out to be most suitable. The Fmoc-protected N-functionalized dipeptide units can be used like any amino acid derivative under the standard conditions for Fmoc-solid phase synthesis.     

Optimization of solid-phase synthesis of difficult peptide sequences via comparison between different improved approaches

Summary. Different approaches are applied to avoid the strong aggregation of the difficult peptide sequences, which is considered as the main reason for incomplete acylation and deprotection reactions hindering the synthesis of these sequences. Temporary protection of amide nitrogen of peptide bond using 2-hydroxy-4-methoxybenzyl (Hmb) and 2,4,6-timethoxybenzyl (Tmob) amino acid derivatives, introduction of D-Ala or Pro residues in the peptide chain sequences and utilization of microwave energy are proved to be useful methods in the enhancement of solubility and in the hindrance of the aggregation during the solid-phase synthesis of oligoalanine. Oligoalanine is chosen to demonstrate the difficult sequences and to compare the efficiencies of these methods.     

Pepsin as a catalyst for peptide synthesis: formation of peptide bonds not typical for pepsin substrate specificity.

Porcine pepsin in water solutions containing 15-28% of dimethylformamide at pH 5 and 20-37 degrees C catalysed the formation of peptide bonds between Z-Ala-Ala-Phe-OH and various amino acid or peptide derivatives. Substrate binding subsite S1' of pepsin demonstrated broad specificity in these reactions but revealed a certain preference for hydrophobic amino acid residues, including non-proteinous homophenylalanine, p-nitrophenylalanine, S-methylcysteine, as well as for those that contained, in addition to the hydrophobic elements, a group capable of donating a hydrogen bond, e.g. o-nitrotyrosine. This observation increases the range of peptides that might be prepared by pepsin-catalysed synthesis.