Total enzymatic synthesis of the cholecystokinin octapeptide (CCK 26-33)

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Dokumentart: Dissertation
Date: 2006
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Sonstige - Chemie und Pharmazie
Advisor: Eckstein, Heiner
Day of Oral Examination: 2006-07-10
DDC Classifikation: 540 - Chemistry and allied sciences
Keywords: Organische Chemie , Peptidsynthese
Other Keywords:
Enzymatic peptide synthesis , immobilized enzymes , Cholecystokinin octapeptide , solvent free system , organic solvent system
License: Publishing license including print on demand
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Die Vorteile der enzymatischen Peptidsynthese gegenüber klassischen Methoden und der Festphasensynthese können bei den durchgeführten Synthesen voll ausgenutzt werden. Die Reaktionen verlaufen stereo- und regiospezifisch, können einfach kontrolliert und gesteuert werden und Seitenketten benötigen keinen oder nur minimalen Schutz. Ziel dieser Arbeit ist, einen vollenzymatischen Syntheseweg für das Cholecystokininoctapeptid (CCK26-33) zu finden. Die Phenylacetylgruppe (Phac-) dient als Aminoschutzgruppe, weil es durch Penicillin G Amidase (PGA) enzymatisch hydrolysiert werden kann. Es gibt bei der enzymatischen Peptidsynthese kein allgemeines Syntheseprotokoll. Jeder einzelne Kupplungsschritt muss optimiert werden. In dieser Arbeit wurden die Strategie, Struktur und Konzentration von den Substraten, der Reaktionsmedia und Temperatur optimiert. Für die Synthese von Phac-Asp(OMe)-Tyr-Met-OAl wurden zwei Synthesestrategien untersucht. Die zweite Strategie ist besser, da weniger Reaktionsschritte nötig sind, bei insgesamt höherer Ausbeute (45%) und einfach zugänglichen Ausgangsmaterialien. Das Pentapeptid Gly-Trp-Met-Asp(OMe)-Phe-NH2 wird schrittreise synthetisiert. Für jeden Kupplungsschritt können die immobiliserten Enzyme Papain, alpha-Chymotrypsin, Thermolysin und Pencillin G Amidase verwendet werden. Das Octapeptid Phac-Asp(OMe)-Tyr-Met-Gly-Trp-Met-Asp(OMe)-Phe-NH2 wird aufgebaut aus Phac-Asp(OMe)-Tyr-Met-OAl und Gly-Trp-Met-Asp(OMe)-Phe-NH2 mit immobilisiertem alpha-Chymotrypsin im Lösungsmittelsystem mit geringem Wassergehalt mit 11.5% HPLC Ausbeute.


Enzymes can be used often favorably in organic syntheses, because they can be applied at room or slightly elevated temperature and in aqueous phase. Therefore these enzymatically catalyzed reactions are economically and environmentally superior to classical organic reactions. The objective of this thesis is to develop a synthetic path to the cholecystokinin octapeptide CCK-8 using exclusively enzymatic methods. The fragment CCK-8 has nearly the full biological activity of cholecystokinin and its therapeutic potential against type 2 diabetes, obesity and epilepsy is studied intensively. During the coupling process, many side reactions observed in the chemical peptide synthesis can be avoided in the enzymatic peptide synthesis. However, each coupling reaction has to be optimized. That means the optimal strategy, substrate structure and concentration, reaction media and temperature, type of the protease and its support for enzyme deposition. In the synthesis of the N-terminal tripeptide fragment Phac-Asp(OMe)-Tyr-Met-OAl, two strategies (Scheme 3.5, Scheme 3.6) with stepwise peptide chain elongation from the N-terminus to the C-terminus were investigated. Because of less reaction steps, higher overall yield and more economic starting materials, the second strategy was superior to be integrated in the final CCK-8 assembly. Compared to the reported synthesis of the tripeptide fragment Phac-Asp(OBut)-Tyr-Met-OAl (Fite et al., 2002) in the second strategy two reaction steps could be avoided to synthesize an OCam ester and an overall yield of 45% of Phac-Asp(OMe)-Tyr-Met-OAl could be obtained. The pentapeptide fragment CCK-5 was synthesized by a stepwise coupling strategy from the N-terminus to the C-terminus using the enzymes papain, alpha-chymotrypsin, thermolysin and penicillin G amidase. In comparison with earlier developed syntheses of CCK-5 in our laboratory (Xiang et al., 2004) and in another group (Fite et al., 2002) several chemical reaction steps could be circumvented. But still one OCam ester was necessary. In the coupling with Met-OEt·HCl as nucleophile in a solvent free system, the OMe ester of Phac-Gly-Trp-OMe could be used directly as acyl-donor. Thus three reaction steps, otherwise necessary for the preparation of an OCam ester as reported (Capellas et al., 1996), could be avoided. Except of the Asp-Phe coupling using free thermolysin, all other peptide couplings and the cleavage of the Phac-group at the end of the synthesis could be performed with covalently immobilized enzymes. This is important for the large scale synthesis of peptides and for therapeutical applications. According to the literature, it is necessary that acyl-donors need to carry an ester group to form the acyl-enzyme with serine or cysteine proteases. However, in this work and also in earlier investigations in our group we observed that quite often peptide fragments with free carboxyl groups are good acyl-donors for couplings with papain and alpha-chymotrypsin. For the elongation of Phac-Gly-Trp-Met-OH to the tetrapeptide Phac-Gly-Trp-Met-Asp(OMe)-OH, the free carboxyl group of the acyl-donor was necessary. Neither an alkyl ester nor the usually most reactive OCam ester was a useful substrate for papain. The final fragment condensation of the tripeptide Phac-Asp(OMe)-Tyr-Met-OAl and the pentapeptide Gly-Trp-Met-Asp(OMe)-Phe-NH2 could be achieved with alpha-chymotrypsin/Eupergit C. According to LC-ESIMS the condensation reaction was quite effective. The protected CCK-8 peptide was formed in the HPLC yield of 11.5%. A by-product was an octapeptide with 32 mass units less. Most likely one of the beta-esters of the aspartic acid residue underwent a ring closure reaction, quite often observed as one of the most common side reactions of aspartic acid moieties under basic conditions. Another by-product with 64 mass units less was probably formed when both aspartic acid residues underwent ring closure reactions. It will need further investigation to avoid these side reactions.

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