The enzymatic synthesis of the cholecystokinin octapeptide (CCK-8) is reported. The target octapeptide CCK-8 is the minimum active sequence with the same biological activity as naturally occurring cholecystokinin and is a potential therapeutic agent in the control of gastrointestinal function as well as a drug candidate for the treatment of epilepsy and obesity. The protected CCK-8 was obtained by incubation of Bz-Arg-Asp(OEt)-Tyr-Met-OAl and Gly-Trp-Met-Asp(OMe)-Phe-NH2 with immobilized alpha-chymotrypsin. The Bz-Arg group was used as an N-terminal protecting group in the synthesis of the tripeptide fragment. The protected CCK-8 was treated with trypsin to remove the Bz-Arg group successfully. Free or immobilized enzymes were used as catalysts. The effect of the acyl donor ester structure, the C(alpha) protecting group of the nucleophile, reaction media, enzyme, and the carrier of the enzymes on the outcome of the coupling reaction was studied.

Two enzymatically synthetic strategies of the tripeptide derivative PhAc-Asp(OMe)-Tyr-Met-OAl are reported. The second strategy gains the advantage of more economical starting materials, less reaction steps and a higher overall isolated yield of this tripeptide fragment over the first strategy. The effect of the acyl-donor ester concentration and structure, the C-alpha protecting group of the nucleophile, reaction media, enzyme and the carrier on the tripeptide derivative synthesis were studied. This tripeptide selected is a fragment of the cholecystokinin C-terminal octapeptide (CCK-8), a potential therapeutic agent in the control of gastrointestinal function and also a drug candidate for the treatment of epilepsy.

The reversal of the Proteolytic cleavage of the p tide bond by proteases leads to the enzymatic peptide synthesis. In contrast to the chemical synthesis enzymes or, the peptide bond without the danger of racemisation. The coupling can be conducted in aqueous buffer, no hazardous chemicals are necessary. Quite often side chain protection of the amino acids can be omitted, thus simplifying the synthetic procedure. Enzymatic peptide synthesis is a very good example of green chemistry.Application of immobilised enzymes has several advantages in comparison to free enzymes in the synthesis of peptides; as therapeutical drugs. Here the Principles at enzymatic peptide synthesis are discussed and their application in our peptide synthetic work with immobilised enzymes is presented.

This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH2 of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt.HCl by alpha-chymotrypsin/Celite 545 in a solvent free system. For the 3+2 coupling PhAc-Gly-Trp-Met-OEt had to be converted into its OCam ester. The other fragment H-Asp(OMe)-Phe-NH2 resulted from the coupling of Cbo-Asp(OMe)-OH with H-Phe-NH2.HCl and thermolysin as catalyst, followed by catalytic hydrogenation. Finally PhAc-Gly-Trp-Met-Asp-Phe-NH2 was obtained in a smooth reaction from PhAc-Gly-Trp-Met-OCam and H-Asp(OMe)-Phe-NH2 with alpha-chymotrypsin/Celite 545 in acetonitrile, followed by basic hydrolysis of the beta-methyl ester. The PhAc-group is removed with penicillin G amidase and CCK-5 is obtained in an overall isolated yield of 19.6%.

process for the synthesis of CCK-8 tripeptide H-Gly-Trp-Met-OH catalyzed by immobilized enzyme was reported. 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-OEt.HCl was carried out by immobilized a-chymotrypsin 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 nucleophile on the reaction was also investigated.

OBJECTIVE:To synthesize a tripeptide derivative Phac-Met-Asp(OMe)-Phe -NH2, which is a fragment of the gastrin C-terminal tetrapeptide CCK-4, by enzymatic reaction.METHODS:Three free enzymes, alpha-chymotrypsin, papain and thermolysin from acyl donor Phac-Met-OCam was involved in three steps. The choice of appropriate enzymes and solvents was selected.RESULTS:All enzymatic reactions were obtained in reasonable yields(63%-92%). FAB-MS and FD-MS verified the correct molecular mass of the peptides.CONCLUSION:Studies on the alpha-chymotrypsin catalyzed coupling reaction between Phac-Met-OCam and H-Asp(OMe)2 have focused on the low water content media. By papain catalyzed saponification of Phac-Met-Asp(OMe)2, alpha-methyl ester of aspartic acid is selectively hydrolyzed to retain beta-methyl ester, and Phac-Met-Asp(OMe)-OH and H-Phe-NH2 can be coupled efficiently by thermolysin.

The enzymatic synthesis of a tetrapeptide Phac-Met-Gly-Trp-Met-OEt, a fragment of the cholecystokinin C-terminal octapeptide CCK-8, was reported. This fragment was synthesized by coupling Phac-Met-OEt with Gly-OMe, Trp-OMe and Met-OEf successively. These three steps were catalyzed by α-chymotrpsin, Papain and α-chymotrpsin respectively. The results of FAB-MS showed that all the products had the correct molecular mass.

The synthesis of CCK-4 (H-Trp-Met-Asp-Phe-NH2) by using enzymes 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 following 4 enzymes,α-Chymotrypsin, Papain, Thermolysin and PGA in four reaction steps. All reactions were carried out in aqueous buffer in reasonable yields (>65%). FAB-MS or FD-MS verified the correct molecular mass of all peptides.

The restriction endonuclease EcoRI binds and cleaves DNA containing GAATTC sequences with high specificity. According to the crystal structure, most of the specific contacts of the enzyme to the DNA are formed by the extended chain region and the first turn of alpha-helix alpha 4 (amino acids 137-145). Here, we demonstrate that a dodecapeptide (WDGMAAGNAIER), which is identical in the underlined parts of its sequence to EcoRI amino acids 137-145, specifically binds to GAATTC sequences. The peptide inhibits DNA cleavage by EcoRI but not by BamHI, BclI, EcoRV, HindIII, PacI, and XbaI. DNA cleavage by XbaI is slowed down at sites that partially overlap with EcoRI sites. The peptide inhibits cleavage of GAATTC sites by ApoI, which recognizes the sequence RAATTY. It interferes with DNA methylation by the EcoRI methyltransferase but not by the BamHI methyltransferase. It competes with EcoRI for DNA binding. Based on these results, the DNA binding constant of the peptide to GAATTC sequences was calculated to be 3 x 10(4) M-1. DNA binding is not temperature-dependent, suggesting that binding of the peptide is entropy-driven. As the peptide does not show any nonspecific binding to DNA, its DNA binding specificity is similar to that of EcoRI, in spite of the fact that the affinity is much smaller. These results suggest that contacts to the phosphate groups in EcoRI mainly provide binding affinity, whereas the specificity of EcoRI is based to a large extent on sequence-specific base contacts.

Abstract Polystyrene-polyoxyethylene craft copolymers have been used for step-wise peptide synthesis. After completion of synthesis the protecting groups are cleaved under acidic conditions, where the polymer-peptide bond is stable. These gels in comparison to polystyrene peptide gels, show better properties for applications in affinity chromatography as well as synthesis on solid supports, because the advantageous properties of polystyrene beads are combined with the excellent spacer behavior of polyoxyethylene chains (mobility, solvation by water and organic solvents). Peptide gels with polylysine sequences have been synthesized as highly selective stationary phases for the separation of the homologous oligo desoxyribonucleotides (dT)n with n = 1-3. The principal possibilities of these gels for affinity chromatography is demonstrated.

AbstractThe syntheses of models of histones are described. The lysine derivative Nε‐Boc‐lysine methylester, the protected peptides (Lys(Z))5,(Lys(Boc))5‐Pro‐OtBu and the protected sequence 17–27 of the histone H1 of rabbit thymus, Pro‐Ala‐(Lys(Boc))4‐Ala‐Ala‐(Lys(Boc))2‐Pro‐OMe, were transferred via three different ways into insoluble gels. The first synthetic way started with acrylamides, obtained by the reaction of the active benzotriazol‐1‐yl acrylate (3) with Nα‐amino groups of the lysine derivative and of derivatives of lysine peptides. The acrylic peptides were copolymerized with tetramethylene dimethacrylate and thus transferred into insoluble gels. In a second way partially protected peptides were coupled with carboxylic groups of cross‐linked polyacrylic gels. The third way followed the principle of the solid phase peptide synthesis. Cleavage of the protecting groups resulted in lysine and lysine peptide gels, which resemble different models of histones. The peptide gels can be used both for column chromatographic investigations of the peptide‐nucleotide interaction and for affinity‐chromatography.

Homologues of deoxyriboadenylic and deoxyribothymidylic acid show different degrees of retardation in a pentalysine gel. This chromatographic retardation is directly related to the strength of the interaction between the oligonucleotides and the immobilized pentalysine peptide residues. The strength of this interaction is determined by the total negative charge of the oligonucleotide, and by the nature of the constituent base of the oligonucleotide. The specificity of the interaction depends upon differences in conformation of the oligonucleotides.

AbstractDie Synthese von ω‐(N‐Acryloyl)‐tryptophanpeptid‐Monomeren (8) und deren radikalische Copolymerisation mit Diacrylaten zu Tryptophanpeptid‐Gelen wird beschrieben. An einem Tryptophanpeptid‐Gel mit fünf Tryptophyl‐Resten wird die Peptid‐Nukleotid‐Wechselwirkung säulenchromatographisch untersucht. Hierbei werden Desoxyribonukleotide nur gering, Desoxyribonukleoside dagegen stärker retardiert. Deutlich von allen Verbindungen abgetrennt ist Adenosin. Aus den Trennergebnissen folgt, daß Peptidgele auf Polyacrylbasis zur Untersuchung der Peptid‐Nukleotid‐Wechselwirkung und Affinitätschromatographie geeignet sind. Darüber hinaus können diese Gele als spezifische Sorbentien zur Trennung von Nukleinsäurebausteinen auch in präparativem Maßstab verwendet werden.

AbstractDie Sequenz 81 – 94 des Myoglobins nimmt im nativen Zustand weitgehend helicale Konformation ein und trägt den proximalen Histidinrest in Position 93. — Das Hexapeptid 89 — 94 wurde aus 3 Fragmenten aufgebaut und mit dem aus 2 Fragmenten erhaltenen Hexapeptid 83 — 88 zum Dodecapeptid 83 — 94 gekuppelt. Das Hexapeptid 83 — 88 wurde durch das schwer zugängliche Di‐peptid Z‐His‐His‐N2H3 zum Octapeptid 81 — 88 verlängert. Obwohl bei beiden Teilsequenzen die Verknüpfungen 88 → 89 als auch 82 → 83 gelingen, ließ sich das Tetradecapeptid weder durch [2 + 12]‐ noch [8 + 6]‐Verknüpfung aufbauen. Die dafür verantwortlichen Gründe und Lösungsmöglichkeiten werden diskutiert.

AbstractDie Synthese von W‐(Benzyloxycarbonyl)histidin (4) durch direkte Acylierung von Histidin wird beschrieben. Im Vergleich zu anderen Methoden bietet sie einige Vorteile im Hinblick auf Gesamtausbeute, Zeitersparnis und Einfachheit der experimentellen Durchführung. Es wurden einige bereits beschriebene Derivate von 4 [z. B. Z‐His(Tos) und Z‐His(Ado)]1), teilweise in besserer Ausbeute, synthetisiert. Ihre bisher nicht veröffentlichten physikalischen Daten werden angegeben. Außerdem werden die Synthese von Z‐His(Boc) und dessen Eigenschaften beschrieben.

Article Eine Mikromethode zur Konfigurationsbestimmung von Histidin in Peptiden. Der Nachweis partieller Razemisierung bei der Peptidsynthese was published on January 1, 1976 in the journal Biological Chemistry (volume 357, issue 2).

AbstractDie Synthesen von mehreren Tryptophanpeptiden werden beschrieben. Durch geeignete Schutzgruppen konnten diese Peptide frei von Oxidationsprodukten der Tryptophanreste erhalten werden. Ihre UV‐Spektren zeigten beim Übergang von Trp zu Trp‐Trp und zu Trp‐Trp‐Trp charakteristische Unterschiede.