DE102010053416A1 - Synthesizing cysteine-containing peptides comprises native chemical ligating and/or oxidizing linear, reduced peptide sequences in biocompatible, non-toxic liquid and taking non-viscous ionic liquid for reacting - Google Patents

Synthesizing cysteine-containing peptides comprises native chemical ligating and/or oxidizing linear, reduced peptide sequences in biocompatible, non-toxic liquid and taking non-viscous ionic liquid for reacting Download PDF

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DE102010053416A1
DE102010053416A1 DE102010053416A DE102010053416A DE102010053416A1 DE 102010053416 A1 DE102010053416 A1 DE 102010053416A1 DE 102010053416 A DE102010053416 A DE 102010053416A DE 102010053416 A DE102010053416 A DE 102010053416A DE 102010053416 A1 DE102010053416 A1 DE 102010053416A1
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cysteine
peptides
ionic liquid
native chemical
containing peptides
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Dr. Imhof Diana
Dr. Stark Annegret
Alesia A. Miloslavina
Toni Kühl
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Friedrich Schiller Universtaet Jena FSU
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C07K1/026—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution by fragment condensation in solution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification

Abstract

Synthesizing cysteine-containing peptides comprises native chemical ligating and/or oxidizing at least one linear, reduced peptide sequences in a biocompatible, non-toxic liquid at room temperature and taking non-viscous ionic liquid independent of peptide chain length in a concentration of at least 3 mM as well as without requiring cooling or limiting to room temperature for reacting.

Description

Die Erfindung betrifft ein Verfahren zur Herstellung von Cystein-haltigen Peptiden, insbesondere Oligopeptiden, durch Native Chemische Ligation und/oder Oxidation in Ionischen Flüssigkeiten, insbesondere zur Darstellung von Peptiden mit mehreren Disulfidbrücken und längerkettigen Peptiden/Miniproteinen. Damit wird deren Verfügbarkeit in Mengen, die eine vollständige biologische und pharmakologische Charakterisierung erlauben, sowie deren weitere Verwendung in Therapie und Diagnostik ermöglicht.The invention relates to a process for the preparation of cysteine-containing peptides, in particular oligopeptides, by native chemical ligation and / or oxidation in ionic liquids, in particular for the preparation of peptides with a plurality of disulfide bridges and longer-chain peptides / miniproteins. Thus, their availability in amounts that allow a complete biological and pharmacological characterization, and their further use in therapy and diagnostics is possible.

Verschiedene Klassen von Peptiden, wie Zyklotide in Pflanzen, Hormone in Menschen sowie die Gifte der Kegelschnecken, Skorpione und Schlangen zählen zu Substanzen mit wichtiger pharmakologischer Wirkung (beispielsweise S. Beckerand H. Terlau: Toxins from cone snails: properties, applications and biotechnological production, Appl Microbiol Biotechnol. 79, 2008, 1–9 ). Aufgrund ihrer Aktivität besitzen diese Verbindungen das Potential, als Liganden zur Untersuchung von physiologischen und pathophysiologischen Funktionen ( M. A. Grant, X. J. Morelli and A. C. Rigby: Conotoxins and structural biology: a prospective paradigm for drug discovery, Curr. Protein. Pept. Sci. 5, 2004, 235–248 ).Various classes of peptides, such as cyclotides in plants, hormones in humans, as well as the toxins of cone snails, scorpions, and snakes, belong to substances with important pharmacological activity (for example S. Beckerand H. Terlau: Toxins from cone snails: properties, applications and biotechnological production, Appl Microbiol Biotechnol. 79, 2008, 1-9 ). Due to their activity, these compounds have the potential to act as ligands for the study of physiological and pathophysiological functions ( MA Grant, XJ Morelli and AC Rigby: Conotoxins and Structural Biology: A Prospective Paradigm for Drug Discovery, Curr. Protein. Pept. Sci. 5, 2004, 235-248 ).

Die vorgenannten Gifte bestehen aus einer Mischung von ca. 200 Peptiden, die unterschiedliche Wirkungen auf transmembranale Proteine, wie Innenkanäle, Rezeptoren und Transporter haben ( H. Terlau and B. M. Olivera: Conus venoms: A rich source of novel ion channel-targeted peptides, Physiol. Rev. 84, 2004, 1–68 ; R. J. Lewis and M. L. Garcia: Therapeutic potential of venom peptides, Nat. Rev. Drug. Discov. 2, 2003, 90–802 ).The aforementioned poisons consist of a mixture of approximately 200 peptides which have different effects on transmembrane proteins, such as internal channels, receptors and transporters ( H. Terlau and BM Olivera: Conus venoms: A rich source of novel ion channel-targeted peptides, Physiol. Rev. 84, 2004, 1-68 ; RJ Lewis and ML Garcia: Therapeutic potential of venom peptides, Nat. Rev. Drug. Discov. 2, 2003, 90-802 ).

Die meisten der Peptide stellen Cystein-haltige Strukturen dar, die verschiedene posttranslationale Modifikationen aufweisen. Die Ausbildung von Disulfidbrücken führt zur spezifischen Anordnung von Cysteinresten, beispielsweise in sogenannten „Cystinknoten” (cystine knots), die für die Ausbildung und Stabilisierung der dreidimensionalen Proteinstruktur und die Wirkung der Verbindungen verantwortlich sind ( N. L. Daly, K. J. Rosengren and D. J. Craik: Discovery, structure and biological activities of cyclotides, Adv. Drug. Deliv. Rev. 61, 2009, 918–930 ).Most of the peptides are cysteine-containing structures that have various post-translational modifications. The formation of disulfide bridges leads to the specific arrangement of cysteine residues, for example in so-called "cystine knots" (cystine knots), which are responsible for the formation and stabilization of the three-dimensional protein structure and the action of the compounds ( NL Daly, KJ Rosengren and DJ Craik: Discovery, structure and biological activities of cyclotides, Adv. Drug. Deliv. Rev. 61, 2009, 918-930 ).

In letzter Zeit ist eine große Zahl von neuen Arzneistoffen entwickelt worden, die ursprünglich durch Isolierung aus Giften gewonnen wurden. Dazu zählen beispielsweise das 25 Aminosäuren enthaltende Peptid ω-MVIIA mit dem Namen Prialt (Ziconotide) u. a. (D. J. Ellis, G. P. Miljanich and D. E. Shields: Pharmaceutical formulation comprising ziconotide, US-Patent 7524812B2 , 2009; I. Vetter and R. J. Lewis: Characterization of endogenous calcium responses in neuronal cell lines, Biochem. Pharmacol. 79, 2010, 908–920 ).Recently, a large number of new drugs have been developed which were originally obtained by isolation from poisons. These include, for example, the 25 amino acid-containing peptide ω-MVIIA with the name Prialt (Ziconotide) and others (DJ Ellis, GP Miljanich and DE Shields: Pharmaceutical formulations comprising ziconotide, U.S. Patent 7524812B2 , 2009; I. Vetter and RJ Lewis: Characterization of endogenous calcium responses in neuronal cell lines, Biochem. Pharmacol. 79, 2010, 908-920 ).

Die Gewinnung von nativen Toxinen aus der Natur ist nur in sehr geringen Mengen möglich. Deshalb ist es von besonderem Interesse, diese synthetisch herzustellen. Dadurch kann eine ausreichend hohe Menge erhalten werden, die es erlaubt die Liganden genauer zu charakterisieren, was die spätere Anwendung als Pharmaka ermöglicht. Die Darstellung der Verbindung kann mittels an sich bekannter rekombinanter Methoden (beispielsweise: N. Zilberberg, D. Gordon, M. Pelhate, M. E. Adams, T. M. Norris, E. Zlotkin and M. Gurevitz: Functional expression and genetic alteration of an alpha scorpion neurotoxin. Biochemistry 35 (31), 1996, 10215–22 , O. Froy, N. Zilberberg, D. Gordon, M. Turkov, N. Gilles, M. Stankiewicz, M. Pelhate, E. Loret, D. A. Oren, B. Shaanan and M. Gurevitz: The putative bioactive surface of insect-selective scorpion excitatory neurotoxins. J. Biol. Chem., 274 (9), 1999, 5769–5776 ), aber auch mittels ebenfalls an sich bekannter chemischer Festphasenpeptidsynthese (SPPS) (beispielsweise: T. Kimura: Oxidative refolding of multiple-cysteine peptides. In Houben-Weyl, Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics (M. Goodman, A. Felix, L. Moroder and C. Toniolo eds.), Thieme-Verlag, Stuttgart, 2002, 142–161 ) erfolgen. Kostenintensive und zeitaufwendige Arbeitsschritte sind bei den besagten rekombinanten Methoden große Nachteile. Weiterhin stellen dabei die posttranslationalen Modifikationen ein großes Problem dar, weil im Falle der Expression in nicht-eukaryotischen Systemen diese fehlen.The extraction of native toxins from nature is possible only in very small quantities. Therefore, it is of particular interest to synthesize them. As a result, a sufficiently high amount can be obtained, which allows to characterize the ligands more accurately, which allows for later use as pharmaceuticals. The representation of the compound can be determined by recombinant methods known per se (for example: N. Zilberberg, D. Gordon, M. Pelhate, ME Adams, TM Norris, E. Zlotkin, and M. Gurevitz: Functional expression and genetic alteration of an alpha scorpion neurotoxin. Biochemistry 35 (31), 1996, 10215-22 . O. Froy, N. Zilberberg, D. Gordon, M. Turkov, N. Gilles, M. Stankiewicz, M. Pelhate, E. Loret, DA Oren, B. Shaanan and M. Gurevitz: The putative bioactive surface of insect Selective scorpion excitatory neurotoxins. J. Biol. Chem., 274 (9), 1999, 5769-5776 ), but also by means of known chemical solid-phase peptide synthesis (SPPS) (for example: T. Kimura: Oxidative refolding of multiple-cysteine peptides. In Houben-Weyl, Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics (Goodman M., A. Felix, L. Moroder and C. Toniolo eds.), Thieme Verlag, Stuttgart, 2002, 142-161 ) respectively. Costly and time-consuming steps are in the said recombinant methods major disadvantages. Furthermore, the post-translational modifications represent a major problem because, in the case of expression in non-eukaryotic systems, these are absent.

Bei der SPPS dagegen werden im ersten Schritt lineare Vorläufer der Peptide synthetisiert. Die Faltung der linearen Struktur erfolgt durch Oxidation der Thiolgruppen der Cysteinreste unter Ausbildung von Disulfidbrücken. Das Auftreten von Nebenreaktionen bei der Synthese, wie Oligomerisierung, Dimerisierung und Fehlfaltung, resultiert in der Inaktivität der Peptide.In the SPPS, on the other hand, linear precursors of the peptides are synthesized in the first step. The folding of the linear structure is carried out by oxidation of the thiol groups of the cysteine residues to form disulfide bridges. The occurrence of side reactions in the synthesis, such as oligomerization, dimerization and misfolding, results in the inactivity of the peptides.

Bei Strukturen mit zwei Cysteinen innerhalb der Sequenz können als mögliche Nebenreaktionen intermolekulare Thiolverknüpfung und Oligomerisierung auftreten. Dabei kann die Oligomerisierung durch eine starke Verdünnung des Peptides umgangen werden.For structures with two cysteines within the sequence, intermolecular thiol linkage and oligomerization may occur as possible side reactions. In this case, the oligomerization can be avoided by a strong dilution of the peptide.

Ein größeres Problem stellt allerdings die Zyklisierung von Peptiden mit drei und mehr Disulfidbrücken dar. Die Zahl der auftretenden Disulfid-verbrückten Isomere steigt dabei enorm auf 15 (6 Cys), 105 (8 Cys) bzw. 945 (10 Cys). Der Einsatz weiterer reaktiver Reagenzien für die oxidative Faltung, kann die Anzahl von unerwünschten Nebenprodukten erhöhen ( V. Agoston, M. Cemazar, L. Kajan and S. Pongor: Graphrepresentation of oxidative folding pathways, BMC Bioinformatics. 6 (19), 2005 , C. B. Anfinsen and H. A. Scheraga: Experimental and theoretical aspects of protein folding, Adv Protein Chem. 29, 1975, 205–300 ; T. Kimura: Oxidative refolding of multiple-cysteine peptides. In Houben-Weyl, Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics (M. Goodman, A. Felix, L. Moroder and C. Toniolo eds.), Thieme-Verlag, Stuttgart, 2002, 142–161 ).A major problem, however, is the cyclization of peptides with three or more disulfide bridges. The number of disulfide-bridged isomers increases enormously to 15 (6 Cys), 105 (8 Cys), and 945 (10 Cys), respectively. The use of other reactive reagents for oxidative folding can increase the number of undesirable by-products ( V. Agoston, M. Cemazar, L. Kajan, and S. Pongor: Graph representation of oxidative folding pathways, BMC Bioinformatics. 6 (19), 2005 . CB Anfinsen and HA Scheraga: Experimental and theoretical aspects of protein folding, Adv. Protein Chem. 29, 1975, 205-300 ; T. Kimura: Oxidative refolding of multiple-cysteine peptides. In Houben-Weyl, Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics (Goodman M., A. Felix, L. Moroder and C. Toniolo eds.), Thieme Verlag, Stuttgart, 2002, 142-161 ).

Heute sind auch verschiedene Methoden bekannt, die bei der oxidativen Faltung von Peptiden zum Einsatz kommen. Diese können in zwei Gruppen aufgeteilt werden, einerseits in die spontane Oxidation in Lösung und andererseits in die gezielte Oxidation am polymeren Träger ( H. J. Musiol, F. Siedler, D. Quarzago and L. Moroder: Redox-active bis-cysteinyl peptides. I. Synthesis of cyclic cystinyl peptides by conventional methods in solution and on solid supports, Biopolymers 34, 1994, 1553–1562 ).Today, various methods are also known which are used in the oxidative folding of peptides. These can be divided into two groups, on the one hand in the spontaneous oxidation in solution and on the other hand in the targeted oxidation of the polymeric support ( HJ Musiol, F. Siedler, D. Quarzago and L. Moroder: Redox-active bis-cysteinyl peptides. I. Synthesis of cyclic cystinyl peptides by conventional methods in solution and on solid supports, Biopolymers 34, 1994, 1553-1562 ).

Mittlerweile ist auch die Kombination beider Gruppen in der Literatur beschrieben ( A. M. Steiner and G. Bulaj: Optimization of oxidative folding methods for cysteine-rich peptides: A study of conotoxins containing three disulfide bridges, J. Pept. Sci, 2010 ; A. S. Galanis, F. Albericio and M. Grotli: Development of new synthetic strategies for synthesis of alpha-conotoxin-MII, Amino Acids 33, 2007, XIV–XXV ).In the meantime, the combination of both groups has also been described in the literature ( AM Steiner and G. Bulaj: Optimization of oxidative folding methods for cysteine-rich peptides: A study of conotoxins containing three disulfide bridges, J. Pept. Sci, 2010 ; AS Galanis, F. Albericio and M. Grotli: Development of new synthetic strategies for synthesis of alpha-conotoxin MII, Amino Acids 33, 2007, XIV-XXV ).

Die erstgenannte und meist verwendete Methode der spontanen Oxidation erfolgt unter Verwendung von atmosphärischem Sauerstoff. Dieser Prozess verläuft üblicherweise in wässriger Lösung oder in Mischungen aus Wasser und organischen Lösungsmitteln bei pH 7.5–8.5 und sehr niedrigen Konzentrationen von 10–4–10–5 M. Es können Zusatzreagenzien zugegeben werden, wie z. B. redoxaktive Agenzien (Glutathion reduziert/oxidiert, Cysteamin/Cystamin) oder die Aggregation minimierende Agenzien, wie Harnstoff oder Guanidiniumhydrochlorid, ( F. Siedler, D. Quarzago, S. Rudolph-Bohner and L. Moroder: Redox-active bis-cysteinyl peptides. II. Comparative study on the sequence-dependent tendency for disulfide loop formation, Biopolymers 34, 1994, 1563–1572 ; R. DeLa Cruz, F. G. Whitby, O. Buczek and G. Bulaj: Detergent-assisted oxidative folding of delta-conotoxins, J. Pept. Res. 61, 2002, 202–212 ).The first and most commonly used method of spontaneous oxidation is by using atmospheric oxygen. This process usually proceeds in aqueous solution or in mixtures of water and organic solvents at pH 7.5-8.5 and very low concentrations of 10 -4 -10 -5 M. Additional reagents may be added, such as. B. redox-active agents (glutathione reduced / oxidized, cysteamine / cystamine) or aggregation minimizing agents such as urea or guanidinium hydrochloride, ( F. Siedler, D. Quarzago, S. Rudolph-Bohner and L. Moroder: Redox-active bis-cysteinyl peptides. II. Comparative study on the sequence-dependent tendency for disulfide loop formation, Biopolymers 34, 1994, 1563-1572 ; R. DeLa Cruz, FG Whitby, O. Buczek and G. Bulaj: Detergent-assisted oxidative folding of delta-conotoxins, J. Pept. Res. 61, 2002, 202-212 ).

Die Ionenstärke und die Temperatur der Reaktionslösung sind von großer Bedeutung ( S. Kubo, N. Chino, T. Kimura and S. Sakakibara: Oxidative folding of omega-conotoxin MVIIC: effects of temperature and salt, Biopolymers, 38, 1996, 733–744 ).The ionic strength and the temperature of the reaction solution are of great importance ( S. Kubo, N. Chino, T. Kimura and S. Sakakibara: Oxidative folding of omega-conotoxin MVIIC: effects of temperature and salt, Biopolymers, 38, 1996, 733-744 ).

Die Optimierung der Reaktionsbedingungen ist für jedes zu oxidierende Peptid erforderlich. Durch eine hohe Hydrophobizität der Peptide ist mitunter die Verwendung organischer Lösungsmittel essentiell. Aber dennoch können solche stark aggregierenden Sequenzen nur in sehr kleinen Ausbeuten oxidiert werden. Andere Methoden der Oxidation mit z. B. Wasserstoffperoxid, Jod/Ethyljodid ( B. Kamber, A. Hartmann, K. Eisler, B. Riniker, H. Rink, P. Sieber and W. Rittel: The Synthesis of Cystine Peptides by lodine Oxidation of S-Trityl-Cysteine and S-Acetamidomethyl-Cysteine Peptides, Helvetica Chimica Acta. 63, 1980, 899–915 ) oder Thallium(III)-trifluoracetat ( N. Fujii, A. Otaka, S. Funakoshi, K. Bessho, T. Watanabe, K. Akaji, and H. Yajima Studies on peptides. CLI. Syntheses of cystine-peptides by oxidation of S-protected cysteine-peptides with thallium(III)trifluoroacetate, Chem. Pharm. Bull. 35, 1987, 2339–2347 ) werden ebenfalls in wässrigen Lösungen oder unter Zusatz von organischen Lösungsmitteln durchgeführt. Das Problem in diesen Fällen ist eine unerwünschte Oxidation sensitiver Aminosäuren wie z. B. Methionin und Tryptophan. Bei letzterer Methode sind außerdem die hohe Toxizität und die schwierige quantitative Abtrennung von Thalliumverbindungen sehr nachteilig.The optimization of the reaction conditions is required for each peptide to be oxidized. High hydrophobicity of the peptides sometimes makes the use of organic solvents essential. Nevertheless, such strongly aggregating sequences can only be oxidized in very small yields. Other methods of oxidation with z. As hydrogen peroxide, iodine / ethyl iodide ( B. Kamber, A. Hartmann, K. Eisler, B. Riniker, H. Rink, P. Sieber and W. Rittel: The Synthesis of Cystine Peptides by iodine Oxidation of S-Trityl Cysteine and S-Acetamidomethyl Cysteine Peptides, Helvetica Chimica Acta. 63, 1980, 899-915 ) or thallium (III) trifluoroacetate ( N. Fujii, A. Otaka, S. Funakoshi, K. Bessho, T. Watanabe, K. Akaji, and H. Yajima Studies on peptides. CLI. Syntheses of cystine peptides by oxidation of S-protected cysteine peptides with thallium (III) trifluoroacetate, Chem. Pharm. Bull. 35, 1987, 2339-2347 ) are also carried out in aqueous solutions or with the addition of organic solvents. The problem in these cases is an unwanted oxidation of sensitive amino acids such. As methionine and tryptophan. In the latter method, the high toxicity and the difficult quantitative separation of Thalliumverbindungen are also very disadvantageous.

Eine weitere Möglichkeit ist die Oxidation in wässriger Lösung unter Zusatz von Dimethylsulfoxid ( T. J. Wallace and J. J. Mahon: Reactions of Thiols with Sulfoxides. 3. Catalysis by Acids and Bases, J. Org. Chem. 30, 1965, 1502–1506 ). Diese Methode hat zwar den Vorteil die Reaktionsbedingungen in einem breiteren pH-Bereich (pH 1,0–8,0) variieren zu können ( J. P. Tam, C. R. Wu, W. Liu and J. W. Zhang: Disulfide Bond Formation in Peptides by Dimethyl-Sulfoxide – Scope and Applications, JACS 113, 1991, 6657–6662 ), aber mit steigender Konzentration an Dimethylsulfoxid (DMSO) im Reaktionsansatz ist die nachfolgende Abtrennung dieses Lösungsmittels von den Reaktanden erschwert.Another possibility is the oxidation in aqueous solution with the addition of dimethyl sulfoxide ( TJ Wallace and JJ Mahon: Reactions of Thiols with Sulfoxides. 3. Catalysis by Acids and Bases, J. Org. Chem. 30, 1965, 1502-1506 ). Although this method has the advantage of being able to vary the reaction conditions in a broader pH range (pH 1.0-8.0) ( JP Tam, CR Wu, W. Liu and JW Zhang: Disulfide Bond Formation in Peptides by Dimethyl Sulfoxide - Scope and Applications, JACS 113, 1991, 6657-6662 ), but with increasing concentration of dimethyl sulfoxide (DMSO) in the reaction mixture, the subsequent separation of this solvent from the reactants is difficult.

Die oxidative Faltung unter Verwendung von Azodicarbonsäure ( E. M. Kosower and H. Kanety-Londoner: JACS 98, 1976, 3001 ) oder einer Chlorsilan/-sulfoxid Mischung ( K. Akaji, T. Tatsumi, M. Yoshida, T. Kimura, Y. Fujiwara and Y. Kiso: Disulfide Bond Formation Using the Silyl Chloride Sulfoxide System for the Synthesis of a Cystine Peptide, JACS 114, 1992, 4137–4143 ; T. Koide, A. Otaka, H. Suzuki and N. Fujii: Selective Conversion of S-Protected Cysteine Derivatives to Cystine by Various Sulfoxide Silyl Compound/Trifluoroacetic-Acid Systems, Synlett, 1991, 345–346 ) sind ebenfalls publiziert, aber bisher nur für einfachere Peptide mit einer Disulfidbrücke. Nachteile der letztgenannten Methode sind die hohe Entzündlichkeit und Korrosivität der Reaktionsverbindungen.Oxidative folding using azodicarboxylic acid ( EM Kosower and H. Kanety-Londoner: JACS 98, 1976, 3001 ) or a chlorosilane / sulfoxide mixture ( K. Akaji, T. Tatsumi, M. Yoshida, T. Kimura, Y. Fujiwara and Y. Kiso: Disulfide Bond Formation Using the Silyl Chloride Sulfoxide System for the Synthesis of a Cystine Peptide, JACS 114, 1992, 4137-4143 ; T. Koide, A. Otaka, H. Suzuki, and N. Fujii: Selective Conversion of S-Protected Cysteine Derivatives to Cystine by Various Sulfoxide Silyl Compound / Trifluoroacetic Acid Systems, Synlett, 1991, 345-346 ) are also published, but so far only for simpler peptides with a disulfide bridge. Disadvantages of the latter method are the high flammability and corrosivity of the reaction compounds.

Bei der gezielten Oxidation am polymeren Träger werden die unterschiedlich geschützten Cysteinpaare schrittweise entschützt und miteinander verknüpft. Dabei ist jedoch die Ausbeute im Vergleich zur Lösungsoxidation viel geringer. Die Limitationen dieser Methode liegen weitgehend in der Notwendigkeit der Verwendung selektiv abspaltbarer Schutzgruppen an den in der Sequenz enthaltenen Cysteinresten, was eine sehr vorsichtige und fein aufeinander abgestimmte Wahl an Reaktionsbedingungen für die selektive Abspaltung des jeweiligen Cysteinpaares erfordert. Diese Methode ist nicht kompatibel mit der Oxidation unter Sauerstoffzufuhr sowie unter Zusatz von DMSO. Neben der Oligomerisierung und Polymerisierung treten hier aber auch vermehrt Nebenreaktionen auf ( K. Akaji and Y. Kiso: Synthesis of cysteine peptides. In Houben-Weyl, Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics (M. Goodman, A. Felix, L. Moroder and C. Toniolo eds.), 2002, 101–141, Thieme-Verlag, Stuttgart ).In the case of targeted oxidation on the polymeric support, the differently protected pairs of cysteines are gradually deprotected and linked to one another. However, the yield is much lower compared to the solution oxidation. The limitations This method is largely due to the need to use selectively cleavable protecting groups on the cysteine residues contained in the sequence, which requires a very cautious and finely tuned choice of reaction conditions for the selective cleavage of the respective cysteine pair. This method is not compatible with oxidation with oxygen supply and with the addition of DMSO. In addition to the oligomerization and polymerization but also occur more side reactions ( K. Akaji and Y. Kiso: Synthesis of cysteine peptides. In Houben-Weyl, Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics (M. Goodman, A. Felix, L. Moroder and C. Toniolo eds.), 2002, 101-141, Thieme-Verlag, Stuttgart ).

Kombinationen der beiden Strategien der Lösungs- und Festphasensynthese oxidativ gefalteter Peptide wurden auch unter Verwendung des polymeren Trägers CLEAR-OX beschrieben. Dieser Träger besteht aus dem CLEAR-Harz und einem Derivat des Ellman's Reagenz ( G. L. Ellman: Tissue Sulfhydryl Groups, Arch. Biochem. Biophys. 82, 1959, 70–77 ; US 5,910,554 A ; US 5,656,707 A ; US Patent Application 11/165609 (June 23, 2005); B. R. Green and G. Bulaj: Oxidative folding of conotoxins in immobilized systems, Prot. Pept. Lett. 13, 2006, 67–70 ; K. Darlak, D. W. Long, A. Czerwinski, M. Darlak, F. Valenzuela, A. F. Spatola and G. Barany: Facile preparation of disulfide-bridged peptides using the polymer-supported oxidant CLEAR-OX (TM), J. Pept. Res. 63, 2004, 303–312 ). Die Methode wurde erfolgreich für Peptide mit einer bis zwei Disulfidbrücken angewandt. Vorteilhaft war dabei, dass auch oxidationsempfindliche Aminosäuren nicht negativ durch die Oxidation beeinflusst wurden. Ein Vergleich der oxidativen Faltung in Puffer mit redoxaktiven Agenzien, der Verwendung von DMSO als Zusatz und durch die CLEAR-OX-Methode wurde kürzlich vorgestellt ( A. M. Steiner and G. Bulaj: Optimization of oxidative folding methods for cysteine-rich peptides: A study of conotoxins containing three disulfide bridges, J. Pept. Sci., 2010 ).Combinations of the two strategies of solution and solid phase synthesis of oxidatively-folded peptides have also been described using the polymeric carrier CLEAR-OX. This carrier consists of the CLEAR resin and a derivative of Ellman's reagent ( GL Ellman: Tissue Sulfhydryl Groups, Arch. Biochem. Biophys. 82, 1959, 70-77 ; US 5,910,554 A ; US 5,656,707 A ; US Patent Application 11/165609 (June 23, 2005); BR Green and G. Bulaj: Oxidative folding of conotoxins in immobilized systems, Prot. Pept. Lett. 13, 2006, 67-70 ; K. Darlak, DW Long, A. Czerwinski, M. Darlak, F. Valenzuela, AF Spatola and G. Barany: Facile preparation of disulfide-bridged peptides using the polymer-supported oxidant CLEAR-OX (TM), J. Pept. Res. 63, 2004, 303-312 ). The method was successfully applied to peptides with one to two disulfide bridges. It was advantageous that also oxidation-sensitive amino acids were not adversely affected by the oxidation. A comparison of oxidative folding in buffers with redox-active agents, the use of DMSO as an additive, and the CLEAR-OX method was recently presented ( AM Steiner and G. Bulaj: Optimization of oxidative folding methods for cysteine-rich peptides: A study of conotoxins containing three disulfide bridges, J. Pept. Sci., 2010 ).

Die aktuellste Neuentwicklung auf diesem Gebiet ist das Konzept der ”Integrativen oxidativen Faltung”. Hier wird der Ersatz einer Disulfidbrücke durch eine Diselenidbrücke kombiniert mit dem Einsatz (15N/13C)-markierter Disulfidbrücken, wodurch sowohl die Herstellung als auch die Strukturanalyse mit Hilfe der NMR-Spektroskopie erleichtert wird. Jedoch erfordert diese Methode die Verwendung selektiv-geschützter Selenocysteinreste, was eine Modifikation im Vergleich zu den in der Natur vorkommenden Cysteinen darstellt ( M. Muttenthaler, S. T. Nevin, A. A. Grishin, S. T. Ngo, P. T. Choy, N. L. Daly, S. H. Hu, C. J. Armishaw, C. I. Wang, R. J. Lewis, J. L. Martin, P. G. Noakes, D. J. Craik, D. J. Adams and P. F. Alewood: Solving the alpha-conotoxin folding problem: efficient selenium-directed on-resin generation of more potent and stable nicotinic acetylcholine receptor antagonists. JACS 132, 2010, 3514–3522 ; A. Walewska, M. M. Zhang, J. J. Skalicky, D. Yoshikami, B. M. Olivera and G. Bulaj: Integrated oxidative folding of cysteine/selenocysteine containing peptides: improving chemical synthesis of conotoxins, Angew. Chem. Int. ed. 48, 2009, 2221–2224 ). Außerdem macht die Verwendung sehr teurer, markierter Aminosäurederivate und Selenocysteinderivate diese Methode sehr kostenintensiv und unrealistisch für die Herstellung größerer Mengen dieser Peptide.The most recent development in this field is the concept of "Integrative Oxidative Folding". Here, the replacement of a disulfide bond by a diselenide bridge is combined with the use of ( 15 N / 13 C) -labeled disulfide bridges, which facilitates both the preparation and the structural analysis by means of NMR spectroscopy. However, this method requires the use of selectively-protected selenocysteine residues, which is a modification compared to the naturally occurring cysteines ( M. Muttenthaler, ST Nevin, AA Grishin, ST Ngo, PT Choy, NL Daly, SH Hu, CJ Armishaw, CI Wang, RJ Lewis, JL Martin, PG Noakes, DJ Craik, DJ Adams and PF Alewood: Solving the Alpha congenital congenital problem: efficient selenium-directed on-resin generation of more potent and stable nicotinic acetylcholine receptor antagonists. JACS 132, 2010, 3514-3522 ; A. Walewska, MM Zhang, JJ Skalicky, D. Yoshikami, BM Olivera and G. Bulaj: Integrated oxidative folding of cysteine / selenocysteine containing peptides: improving chemical synthesis of conotoxins, Angew. Chem. Int. ed. 48, 2009, 2221-2224 ). In addition, the use of very expensive, labeled amino acid derivatives and selenocysteine derivatives makes this method very costly and unrealistic for the production of larger amounts of these peptides.

Zusammenfassend kann bezüglich der Darstellung von Cystein-haltigen Peptiden durch Oxidation festgehalten werden, dass je höher die Anzahl an Cysteinen in der Struktur ist, desto schwieriger wird auch die Synthese des entsprechenden Peptids (K. Akaji and Y. Kiso: Synthesis of cysteine peptides. In Houben-Weyl, Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics ( M. Goodman, A. Felix, L. Moroder and C. Toniolo eds.), Thieme-Verlag Stuttgart, 2002, 101–141 ). Die Entwicklung von neuen Strategien ist demnach ein Erfordernis, um den Zugang zu Cystein-reichen Peptiden zu vereinfachen bzw. wirtschaftlich überhaupt erst zu ermöglichen.In conclusion, the higher the number of cysteines in the structure, the more difficult it is to synthesize the corresponding peptide (K. Akaji and Y. Kiso: Synthesis of cysteine peptides. In Houben-Weyl, Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics ( M. Goodman, A. Felix, L. Moroder and C. Toniolo eds.), Thieme-Verlag Stuttgart, 2002, 101-141 ). The development of new strategies is therefore a requirement to simplify the access to cysteine-rich peptides or economically even in the first place.

In jüngster Zeit wird insbesondere für die Synthese längerer Peptide (> 60 AS) die Methode der „Nativen Chemischen Ligation” (native chemical ligation) angewandt. Dies wird durch die Verknüpfung von zwei Fragmenten ermöglicht, wobei das N-terminale Segment als Thioester vorliegt und sich im C-terminalen Peptid N-terminal ein Cysteinrest befinden muss. Über ein Thioester-gebundenes Intermediat und einen S→N-Acyl Shift wird die Peptidbindung an der Stelle des Cysteins gebildet. Dadurch konnten Peptide aus bis zu 200 Aminosäuren hergestellt werden ( P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. Kent: Synthesis of proteins by native chemical ligation, Science 266, 1994, 776–779 ; S. B. Kent: Total chemical synthesis of proteins, Chem. Soc. Rev. 38, 2009, 338–351 ).Recently, the method of "native chemical ligation" has been used, in particular for the synthesis of longer peptides (> 60 aa). This is made possible by the linking of two fragments, wherein the N-terminal segment is present as a thioester and must be in the C-terminal peptide N-terminal cysteine. Via a thioester-bound intermediate and an S → N-acyl shift, the peptide bond is formed at the site of the cysteine. This enabled peptides of up to 200 amino acids to be prepared ( PE Dawson, TW Muir, I. Clark-Lewis and SB Kent: Synthesis of proteins by native chemical ligation, Science 266, 1994, 776-779 ; SB Kent: Total chemical synthesis of proteins, Chem. Soc. Rev. 38, 2009, 338-351 ).

Die Effizienz der Nativen Chemischen Ligation kann durch Ausnutzen spezifischer Charakteristika des Reaktionsverlaufes beachtlich erhöht werden. Häufig bedingt das z. B. das Hinzufügen ausgewählter Zusatzstoffe („Additive”) zum Ligationsansatz.The efficiency of native chemical ligation can be significantly increased by exploiting specific characteristics of the reaction process. Often the z. For example, adding selected additives ("additives") to the ligation mixture.

Eine dieser ausgenutzten Eigenschaften ist, dass die Thiol-Umlagerung den geschwindigkeitsbestimmenden Schritt dieser Reaktion darstellt. Eine Möglichkeit, so auf die Reaktion Einfluss zu nehmen, ist daher die Zugabe eines Thiols, das zur Aktivierung des synthetisierten Peptidthioesters durch einen der Ligation vorgelagerten Thiol-Thioester-Austausch beitragen kann. Dabei wird eine bessere Abgangsgruppe am Peptidthioester generiert, die den Schritt der reversiblen Thiol-Umlagerung zu Gunsten des Thioester-verknüpften Intermediats aus den beiden Segmenten beschleunigt ( P. E. Dawson, M. J. Churchill, M. R. Ghadiri and S. B. Kent: Modulation of reactivity in native chemical ligation through the use of thiol additives, JACS 119, 1997, 4325–4329 ). Es wurden bereits zahlreiche solcher Thiole auf deren Anwendbarkeit untersucht und charakterisiert, um so Rückschlüsse auf die Effizienz und den Katalysemechanismus für die gewünschte Umsetzung ziehen zu können ( E. C. Johnson and S. B. Kent: Insights into the mechanism and catalysis of the native chemical ligation reaction, JACS 128, 2006, 6640–6646 ).One of these exploited features is that the thiol rearrangement is the rate-limiting step in this reaction. One possibility for influencing the reaction is therefore the addition of a thiol, which can contribute to the activation of the synthesized peptide thioester by a thiol-thioester exchange upstream of the ligation. A better leaving group is generated on the peptide thioester, which is the step of the reversible thiol rearrangement in favor of the thioester-linked intermediate from the accelerated both segments ( PE Dawson, MJ Churchill, MR Ghadiri and SB Kent: Modulation of reactivity in native chemical ligation through the use of thiol additives, JACS 119, 1997, 4325-4329 ). Numerous such thiols have already been investigated and characterized for their applicability in order to be able to draw conclusions about the efficiency and the catalytic mechanism for the desired reaction ( EC Johnson and SB Kent: Insights into the Mechanism and Catalysis of the Native Chemical Ligation Reaction, JACS 128, 2006, 6640-6646 ).

In weiteren Untersuchungen zur Nativen Chemischen Ligation wurde vor allem bei Cystein-reichen Peptidsegmenten die nachteilige Eigenschaft beobachtet, dass diese im Ligationsansatz zu Dimerbildung neigen ( S. E. Escher, E. Klüver and K. Adermann: Fmoc-based synthesis oft he human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation, Lett. Pept. Sci. 8, 2002, 349–357 ). Daher kann als weiteres Additiv zur Ligationsreaktion Tris(2-carboxyethyl)phosphin (TCEP) gewählt werden, von dem bekannt ist, dass es ähnlich wie DTT die Ausbildung von Disulfidbrücken unterdrücken kann ( E. B. Getz, M. Xiao, T. Chakrabarty, R. Cooke and P. R. Selvin: A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry, Anal. Biochem. 273, 1999, 73–80 ). So soll vermieden werden, dass vorzeitige Oxidation der Cysteine, insbesondere des für die NCL-Reaktion notwendigen Cysteins, auftritt ( E. C. Johnson and S. B. Kent: Insights into the mechanism and catalysis of the native chemical ligation reaction, JACS 128, 2006, 6640–6646 ; E. B. Getz, M. Xiao, T. Chakrabarty, R. Cooke and P. R. Selvin: A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry, Anal. Biochem. 273, 1999, 73–80 ).In further investigations on native chemical ligation, especially with cysteine-rich peptide segments, the disadvantageous property was observed that these tend to form dimers in the ligation mixture ( SE Escher, E. Klüver and K. Adermann: Fmoc-based synthesis often he human CC chemokines CCL14 / HCC-1 by SPPS and native chemical ligation, Lett. Pept. Sci. 8, 2002, 349-357 ). Thus, as a further ligation reaction additive, tris (2-carboxyethyl) phosphine (TCEP), which is known to be capable of suppressing disulfide bond formation similar to DTT, can be chosen ( EB Getz, M. Xiao, T. Chakrabarty, R. Cooke and PR Selvin: A comparison between the sulfhydryl reductants tris (2-carboxyethyl) phosphines and dithiothreitol for use in protein biochemistry, Anal. Biochem. 273, 1999, 73-80 ). Thus, it is to be avoided that premature oxidation of the cysteines, in particular of the cysteine necessary for the NCL reaction, occurs ( EC Johnson and SB Kent: Insights into the Mechanism and Catalysis of the Native Chemical Ligation Reaction, JACS 128, 2006, 6640-6646 ; EB Getz, M. Xiao, T. Chakrabarty, R. Cooke and PR Selvin: A comparison between the sulfhydryl reductants tris (2-carboxyethyl) phosphines and dithiothreitol for use in protein biochemistry, Anal. Biochem. 273, 1999, 73-80 ).

Neben der Wahl der Additive spielt aber auch die Wahl der C-terminalen Aminosäure des N-terminalen Segments eine entscheidende Rolle für die Geschwindigkeit der Umsetzung. So wurden vor allem Aminosäuren, die β-verzweigt sind, als ungünstigere Reaktionspartner für die Umsetzung mit dem Cystein des C-terminalen Segments ermittelt, da hier die Reaktion durchschnittlich länger dauert ( T. M. Hackeng, J. H. Griffin and P. E. Dawson: Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology, Proc. Nat. Acad. Sci. USA. 96, 1999, 10068-10073 ).In addition to the choice of additives, however, the choice of the C-terminal amino acid of the N-terminal segment plays a decisive role in the speed of the reaction. For example, amino acids that are β-branched were found to be less favorable reaction partners for the reaction with the cysteine of the C-terminal segment, since the reaction takes on average longer ( TM Hackeng, JH Griffin and PE Dawson: Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology, Proc. Nat. Acad. Sci. USA. 96, 1999, 10068-10073 ).

Weitere typische Stellgrößen für die Native Chemische Ligation stellen unter anderem die Parameter Temperatur, Menge des eingesetzten Additivs sowie wiederholte Zugaben des Additivs nach festgelegten Zeitintervallen dar ( S. E. Escher, E. Klüver and K. Adermann: Fmoc-based synthesis oft he human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation, Lett. Pept. Sci. 8, 2002, 349–357 ; C. Haase, H. Rohde and O. Seitz: Native chemical ligation at valine, Angew. Chem. Int Ed. 47, 2008, 6807–6810 ).Other typical manipulated variables for the native chemical ligation include the parameters temperature, amount of the additive used and repeated additions of the additive at specified time intervals ( SE Escher, E. Klüver and K. Adermann: Fmoc-based synthesis often he human CC chemokines CCL14 / HCC-1 by SPPS and native chemical ligation, Lett. Pept. Sci. 8, 2002, 349-357 ; C. Haase, H. Rohde and O. Seitz: Native chemical ligation at valine, Angew. Chem. Int Ed. 47, 2008, 6807-6810 ).

Seit der Entdeckung der Nativen Chemischen Ligation wurden bisher primär zwei unterschiedliche Puffersysteme für den erfolgreichen Ablauf der dabei stattfindenden Reaktionen etabliert: Phosphatpuffer ( T. M. Hackeng, J. H. Griffin and P. E. Dawson: Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology, Proc. Nat. Acad. Sci. USA 96, 1999, 10068–10073 ; J. Offer, C. N. Boddy and P. E. Dawson: Extending synthetic access to proteins with a removable acyl transfer auxiliary, JACS 124, 2002, 4642–4646 ) und Carbonatpuffer ( P. Thongyoo, N. Roque-Rosell, R. J. Leatherbarrow and E. W. Tate: Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides, Org. Biomol. Chem. 6, 2008, 1462–1470 ). In beiden Fällen handelt es sich um wässrige Systeme mit neutralem pH (6,8–7) zur Generierung physiologischer Bedingungen ( S. B. Kent: Total chemical synthesis of proteins, Chem. Soc. Rev. 38, 2009, 338–351 ). Eine Erhöhung des pH-Wertes (bis zu 8,5) wird nur in Betracht gezogen, wenn unmittelbar nach der Nativen Chemischen Ligation die Oxidation der Cysteine folgen soll und für diese Zwecke ein Thiol-Disulfid-Redoxpaar zusätzlich zu den Reagenzien der Nativen Chemischen Ligation ergänzt wird ( S. B. Kent: Total chemical synthesis of proteins, Chem. Soc. Rev. 38, 2009, 338–351 ; S. E. Escher, E. Klüver and K. Adermann: Fmoc-based synthesis of the human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation, Lett. Pept. Sci. 8, 2002, 349–357 ).Since the discovery of Native Chemical Ligation, two different buffer systems have so far been established for the successful completion of the reactions taking place: phosphate buffer ( TM Hackeng, JH Griffin and PE Dawson: Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology, Proc. Nat. Acad. Sci. USA 96, 1999, 10068-10073 ; J. Offer, CN Boddy and PE Dawson: Extending synthetic access to proteins with a removable acyl transfer auxiliary, JACS 124, 2002, 4642-4646 ) and carbonate buffer ( P. Thongyoo, N. Roque-Rosell, RJ Leatherbarrow and EW Tate: Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides, Org. Biomol. Chem. 6, 2008, 1462-1470 ). Both are aqueous systems with neutral pH (6.8-7) for the generation of physiological conditions ( SB Kent: Total chemical synthesis of proteins, Chem. Soc. Rev. 38, 2009, 338-351 ). An increase in pH (up to 8.5) is only considered if oxidation of the cysteines is to follow immediately after Native Chemical Ligation and, for these purposes, a thiol disulfide redox couple in addition to the Native Chemical Ligation Reagents is supplemented ( SB Kent: Total chemical synthesis of proteins, Chem. Soc. Rev. 38, 2009, 338-351 ; SE Escher, E. Klüver and K. Adermann: Fmoc-based synthesis of the human CC chemokines CCL14 / HCC-1 by SPPS and native chemical ligation, Lett. Pept. Sci. 8, 2002, 349-357 ).

In den letzten Jahren ist das Interesse an der umweltfreundlichen Durchführung chemischer Reaktionen durch den Einsatz von nicht traditionellen Lösungsmitteln oder Reagenzien für Reaktionen in der Organischen Chemie, u. a. Oxidationsreaktionen, enorm gestiegen ( P. Wasserscheid: Chemistry – Volatile times for ionic liquids, Nature 439, 2006, 797–797 ). Sogenannte „Ionische Flüssigkeiten” (ionic liquids, ILs) sind vor allem im Bereich der „Grünen Chemie” bekannt geworden und zeichnen sich durch ihre Eigenschaften, wie z. B. geringer Dampfdruck, Nichtbrennbarkeit, thermische Stabilität und Wiederverwendbarkeit aus ( P. Wasserscheid and A. Stark: Handbook of green Chemistry, In Green Solvents (P. T. Anastas ed.), 39, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim: Yale University, Center of Green Chemistry & Green Engineeging, 2010 ). Immer mehr wurden Ionische Flüssigkeiten aber auch für analytische und synthetische Methoden der Biochemie und insbesondere der Peptidchemie entdeckt. Einmalig an Ionischen Flüssigkeiten ist der Fakt, dass durch Modifikationen des jeweiligen Kations und Anions „Designer-ILs” entwickelt werden können, die die optimalen Eigenschaften für einen entsprechenden Einsatz besitzen ( Y. H. Moon, S. M. Lee, S. H. Ha and Y. M. Koo: Enzyme-catalyzed reactions in ionic liquids, Korean. J. Chem. Eng. 23, 2006, 247–263 ).In recent years, interest in environmentally friendly chemical reactions has increased tremendously through the use of non-traditional solvents or reagents for reactions in organic chemistry, including oxidation reactions. P. Wasserscheid: Chemistry - Volatile times for ionic liquids, Nature 439, 2006, 797-797 ). So-called "ionic liquids" (ILs) are mainly known in the field of "green chemistry" and are characterized by their properties such. Low vapor pressure, non-combustibility, thermal stability and reusability ( P. Wasserscheid and A. Stark: Handbook of Green Chemistry, In Green Solvents (PT Anastas ed.), 39, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim: Yale University, Center of Green Chemistry & Green Engineing, 2010 ). More and more ionic liquids have been discovered but also for analytical and synthetic methods of biochemistry and in particular of peptide chemistry. A unique feature of ionic liquids is the fact that modifications of the respective cation and anion can be used to develop "designer ILs", which are the most commonly used have optimal properties for a corresponding use ( YH Moon, SM Lee, SH Ha and YM Koo: Enzyme-catalyzed reactions in ionic liquids, Korean. J. Chem. Eng. 23, 2006, 247-263 ).

Das Interesse an Ionische Flüssigkeiten für den Einsatz zur oxidativen Faltung von Cystein-haltigen, langkettigen Peptiden resultierte aus der Erkenntnis, dass Kohlenhydrate, Oligonucleotide und Proteine sehr gute Löslichkeitseigenschaften in ILs aufweisen ( K. Fujita, D. R. MacFarlane and M. Forsyth: Protein solubilising and stabilising ionic liquids, Chem. Commun., 2005, 4804–4806 ; J. Kiefer, K. Obert, A. Bosmann, T. Seeger, P. Wasserscheid and A. Leipertiz: Quantitative analysis of alpha-D-glucose in an ionic liquid by using infrared spectroscopy, Chem. Phys. Chem. 9, 2008, 1317–1322 ; U. Kragl, M. Eckstein and N. Kaftzik: Enzyme catalysis in ionic liquids, Curr Opin Biotech. 13, 2002, 565–571 ; R. P. Swatloski, S. K. Spear, J. D. Holbrey and R. D. Rogers: Dissolution of cellose with ionic liquids, JACS 124, 2002, 4974–4975 ). Bisher wurden Ionische Flüssigkeiten in der Peptidsynthese als Reaktionsmedium für oxidative Prozesse ( N. Jiang and A. J. Ragauskas: Vanadium-catalyzed selective aerobic alcohol oxidation in ionic liquid [bmim]PF6, Tetr. Lett. 48, 2007, 273–276 ) und für die Synthese von kleinen Peptiden (bis zu fünf Aminosäuren) eingesetzt ( L. Chen, M. F. Zheng, Y. Zhou, H. Liu and H. L. Jiang: Ionic-liquid-supported total synthesis of sansalvamide a peptide, Synth. Commun. 38, 2008, 239–248 ).The interest in ionic liquids for the oxidative folding of cysteine-containing, long-chain peptides resulted from the finding that carbohydrates, oligonucleotides and proteins have very good solubility properties in ILs ( K. Fujita, DR. MacFarlane and M. Forsyth: Protein solubilising and stabilizing ionic liquids, Chem. Commun., 2005, 4804-4806 ; J. Kiefer, K. Obert, A. Bosmann, T. Seeger, P. Wasserscheid and A. Leipertiz: Quantitative analysis of alpha-D-glucose in an ionic liquid by using infrared spectroscopy, Chem. Phys. Chem. 9, 2008, 1317-1322 ; U. Kragl, M. Eckstein and N. Kaftzik: Enzyme catalysis in ionic liquids, Curr Opin Biotech. 13, 2002, 565-571 ; RP Swatloski, SK Spear, JD Holbrey and RD Rogers: Dissolution of cellosis with ionic liquids, JACS 124, 2002, 4974-4975 ). So far, ionic liquids have been used in peptide synthesis as a reaction medium for oxidative processes ( N. Jiang and AJ Ragauskas: Vanadium-catalyzed selective aerobic alcohol oxidation in ionic liquid [bmim] PF6, Tetr. Lett. 48, 2007, 273-276 ) and for the synthesis of small peptides (up to five amino acids) ( L. Chen, MF Zheng, Y. Zhou, H. Liu and HL Jiang: Ionic-liquid-supported total synthesis of sansalvamide a peptide, Synth. Commun. 38, 2008, 239-248 ).

Dabei können sie als Medium, in dem die Reaktion stattfindet (beispielsweise zur Stabilisierung von Proteinstrukturen), oder auch als Reaktand fungieren ( D. Constantinescu, H. Weingartner and C. Herrmann: Protein denaturation by ionic liquids and the Hofmeister series: a case study of aqueous solutions of ribonuclease A, Angewandte Chemie (International ed. 46, 2007, 8887–8889 ; S. Dreyer, P. Salim and U. Kragl: Driving forces of protein partitioning in an ionic liquid-based aqueous two-phase system, Biochem. Eng. J. 46, 2009, 176–185 ).They can act as a medium in which the reaction takes place (for example, to stabilize protein structures), or as a reactant ( D. Constantinescu, H. Weingartner and C. Herrmann: Protein denaturation by ionic liquids and the Hofmeister series: a case study of aqueous solutions of ribonuclease A, Angewandte Chemie (International ed. 46, 2007, 8887-8889 ; S. Dreyer, P. Salim and U. Kragl: Driving forces of protein partitioning in an ionic liquid-based two-phase aqueous system, Biochem. Closely. J. 46, 2009, 176-185 ).

Die Entwicklung von Ionischen Flüssigkeiten zur Darstellung von biologischen Oligomeren (Oligopeptiden, Oligosachariden und Oligonukleotiden) zeigt das große Potential, das Ionische Flüssigkeiten besitzen ( US 2010/0093975 A1 ).The development of ionic liquids for the preparation of biological oligomers (oligopeptides, oligosaccharides and oligonucleotides) shows the great potential of ionic liquids ( US 2010/0093975 A1 ).

Es konnte auch gezeigt werden, dass der Einsatz von 1-Ethyl-3-methylimidazolium-acetat ([C2mim][OAc]) zur oxidativen Faltung einer kleinen Auswahl bioaktiver Peptide (μ- und δ-Conotoxine) eine hervorragende Alternative zu konventionellen Methoden in Redoxpuffern darstellt ( A. A. Miloslavina, E. Leipold, M. Kijas, A. Stark, S. H. Heinemann and D. Imhof: A room temperature ionic liquid as convenient solvent for the oxidative folding of conopeptides, J. Pept. Sci. 15, 2009, 72–77 ).It has also been shown that the use of 1-ethyl-3-methylimidazolium acetate ([C 2 mim] [OAc]) for the oxidative folding of a small selection of bioactive peptides (μ- and δ-conotoxins) is an excellent alternative to conventional ones Represents methods in redox buffers ( AA Miloslavina, E. Leipold, M. Kijas, A. Stark, SH Heinemann and D. Imhof: A room temperature ionic liquid as convenient solvent for the oxidative folding of conopeptides, J. Pept. Sci. 15, 2009, 72-77 ).

Auch andere biokompatible Ionische Flüssigkeiten, wie z. B. [C2mim][OTs], [C2mim][DEP], [C2mim][OAc] und [C2mim][N(CN2)2], wurden zur Oxidation eines weiteren kardioaktiven Peptids, CCAP-vil, beispielhaft auf ihr Potential für diese Reaktion getestet. CCAP-vil stellt dabei ein einfaches, nur mit einer Disulfidbindung verbrücktes Peptid dar ( A. Miloslavina, C. Ebert, D. Tietze, O. Ohlenschläger, C. Englert, M. Görlach and D. Imhof: An unusual peptide from Conus villepinii: Synthesis, solution structure, and cardioactivity, Peptides. 31, 2010, 1292–1300 ). Überraschenderweise wurde eine unterschiedliche Effizienz und Reaktionsausbeute bei Verwendung der verschiedenen Ionischen Flüssigkeiten beobachtet.Other biocompatible ionic liquids such. B. [C 2 mim] [OTs], [C 2 mim] [DEP], [C 2 mim] [OAc], and [C 2 mim] [N (CN 2) 2], for the oxidation of a further cardioactive peptide , CCAP-vil, exemplified on their potential for this reaction. CCAP-vil is a simple peptide bridged with only one disulfide bond ( A. Miloslavina, C. Ebert, D. Tietze, O. Ohlenschläger, C. Englert, M. Görlach and D. Imhof: An unusual peptide from Conus villepinii: Synthesis, solution structure, and cardioactivity, Peptides. 31, 2010, 1292-1300 ). Surprisingly, a different efficiency and reaction yield was observed when using the various ionic liquids.

Der Erfindung liegt die Aufgabe zugrunde, Cystein-haltige und Cystein-haltige, Disulfid-verbrückte Oligopeptide in einem für alle Peptidarten universell anwendbaren Verfahren synthetisierbar, aufwandgeringer, bioverträglich, weniger umweltbelastend, möglichst ohne störende Nebenreaktionen sowie mit hoher Ausbeute und Qualität herzustellen.The invention is based on the object, cysteine-containing and cysteine-containing, disulfide-bridged oligopeptides in a universally applicable to all types of peptides synthesizable process, low, biocompatible, less polluting, if possible without interfering side reactions and produce high yield and quality.

Erfindungsgemäß wird die Aufgabe gelöst, indem ein oder mehrere lineare, reduzierte Peptidsequenzen in einer biokompatiblen, nicht toxischen, bei Raumtemperatur flüssigen und nicht hochviskosen Ionischen Flüssigkeit unabhängig von ihrer Peptidkettenlänge in einer Konzentration von mindestens 3 mM sowie ohne erforderliche Kühlung oder Begrenzung auf Raumtemperatur zur Reaktion gebracht werden.According to the invention the object is achieved by one or more linear, reduced peptide sequences in a biocompatible, non-toxic, liquid at room temperature and not highly viscous ionic liquid regardless of their Peptidkettenlänge in a concentration of at least 3 mM and without cooling or limitation to room temperature to the reaction to be brought.

Auf diese Weise gelingt für alle Peptide unabhängig von ihrer Größe, insbesondere für Oligopeptide, überraschend ein universell anwendbares Syntheseverfahren mit vergleichweise geringem Syntheseaufwand, hohen Ausbeuten sowie ohne störende Nebenreaktionen, so dass auch keine Nebenprodukte entstehen, welche im Vergleich zum bekannten Stand der Technik die Reinheit der Peptide beeinträchtigen können.In this way, for all peptides, regardless of their size, in particular for oligopeptides, surprisingly a universally applicable synthesis method with comparatively low synthesis effort, high yields and without interfering side reactions, so that no by-products are formed, which in comparison to the prior art, the purity may affect the peptides.

Die oxidative Faltung von Peptiden mit 2 bis 10 Cysteinresten in Ionischen Flüssigkeiten ist durch eine bessere Löslichkeit der Ausgangsstoffe (Möglichkeit der Lösung Schwer- oder unlöslicher Ausgangsstoffe) gegenüber der Oxidation in Puffern erleichtert und verbessert. Mit der Ionischen Flüssigkeit als Lösungsmittel und der vorschlagsgemäß hohen Konzentration der zu oxidierenden Peptide (in der Praxis weit größer als 3 mM) gelingt eine wesentliche Minimierung der Nebenproduktbildung und Aggregation, die Beschleunigung der Reaktion und damit Verkürzung der Reaktionszeit (die Reaktion erfolgt innerhalb von Minuten oder wenigen Stunden im Vergleich zu Tagen bei konventionellen Methoden), die Durchführbarkeit der Reaktion bei Temperaturen größer als 25°C (keine Kühlapparatur zur Einhaltung von 4°C notwendig) und letztlich erhebliche Steigerungen von Ausbeute (um mehr als mindestens 250%) und Qualität.The oxidative folding of peptides with 2 to 10 cysteine residues in ionic liquids is facilitated and improved by a better solubility of the starting materials (possibility of dissolving heavy or insoluble starting materials) over the oxidation in buffers. With the ionic liquid as a solvent and the proposed high concentration of peptides to be oxidized (in practice far greater than 3 mM), a substantial minimization of by-product formation and aggregation, acceleration of the reaction and thus shortening of the reaction time (the reaction takes place within minutes or a few hours compared to days with conventional methods), the feasibility of the reaction at temperatures greater than 25 ° C (no cooling apparatus to maintain 4 ° C necessary) and ultimately significant increases in yield (by more than at least 250%) and quality.

Des Weiteren kann auf die Zugabe von Additiven oder Lösungsmitteln verzichtet werden, weil die Reaktion ausschließlich durch den Einfluss von Luftsauerstoff abläuft.Furthermore, it is possible to dispense with the addition of additives or solvents, because the reaction proceeds exclusively through the influence of atmospheric oxygen.

Selbiger Effekt ist bei der Nativen Chemischen Ligation zu verzeichnen, bei welcher die Zugabe von Thiol-haltigen Additiven durch die Erfindung ebenfalls nicht zwingend erforderlich ist. Die Ionische Flüssigkeit erweist sich als höchst zweckmäßiges Medium für die Ligation zweier Peptidsegmente, unter anderem weil die Reaktion durch verbesserte Löslichkeit der Reaktanden in einer homogenen Lösung stattfindet. Neben der Verbesserung der Löslichkeit der Ausgangsstoffe kommen bei der Nativen Chemischen Ligation grundsätzlich dieselben und zur oxidativen Faltung besagten Vorteile zum Tragen.The same effect can be observed in the native chemical ligation, in which the addition of thiol-containing additives by the invention is also not mandatory. The ionic liquid proves to be a highly convenient medium for the ligation of two peptide segments, inter alia because the reaction takes place through improved solubility of the reactants in a homogeneous solution. In addition to improving the solubility of the starting materials, native phosphorylation basically has the same advantages and advantages mentioned for oxidative folding.

Besonders vorteilhaft ist auch, dass die Erfindung die selektive Darstellung langkettiger, Disulfid-verbrückter Oligopeptide oder Miniproteine in Ionischen Flüssigkeiten durch Kombination der Nativen Chemischen Ligation mit der oxidativen Faltung ermöglicht. Beide Reaktionen können in ein- und demselben Medium stattfinden, was gegenüber dem Pufferwechsel und damit verbundenen Aufarbeitungsschritten nach der Nativen Chemischen Ligation wesentliche Verfahrenserleichterungen und -verbesserungen bringt. Zunächst läuft dabei die Native Chemische Ligation in der Ionischen Flüssigkeit unter Argonatmosphäre ab, im Anschluss daran wird die Reaktionsmischung in diesem Medium belassen und Luftsauerstoff zur Initiation der oxidativen Faltung zugeführt. Diese Reaktionsfolge ist mit konventionellen Methoden so nicht möglich, womit für diese Synthesen auch eine weitere Steigerung der Ausbeute ermöglicht wird, insbesondere aufgrund des ausbleibenden Verlustes an Reaktionsmischung, welcher bisher bei erforderlichen Aufarbeitungen in nunmehr entfallenden Zwischenstufen gegeben war.It is also particularly advantageous that the invention enables the selective presentation of long-chain, disulfide-bridged oligopeptides or miniproteins in ionic liquids by combining the native chemical ligation with the oxidative folding. Both reactions can take place in one and the same medium, which brings significant procedural simplifications and improvements over the buffer change and related work-up steps after the Native Chemical Ligation. First, the Native Chemical Ligation in the ionic liquid proceeds under an argon atmosphere, after which the reaction mixture is left in this medium and atmospheric oxygen is fed to initiate the oxidative folding. This reaction sequence is not possible with conventional methods, which also allows for these syntheses a further increase in the yield, in particular due to the lack of loss of reaction mixture, which was previously given necessary work-up in now attributable intermediates.

Die Durchführung der Nativen Chemischen Ligation und/oder oxidativen Faltung in einer Ionischen Flüssigkeit ist selektiv, mit einem geringeren Aufwand an Technik (Verzicht auf komplizierte Reaktions- und Kühlapparaturen) und Material (Verzicht auf zusätzliche Reagenzien und Lösungsmittel), Zeit (Verminderung der Reaktionszeit) und Kosten (geringerer Materialaufwand, weniger Aufarbeitungsschritte, geringe Kosten für Lösungsmittel) verbunden und stellt damit ein universell anwendbares, aufwandgeringes Verfahren zur Herstellung Cystein-haltiger oder Disulfid-verbrückter Oligopeptide oder Miniproteine dar.Performing native chemical ligation and / or oxidative folding in an ionic liquid is selective, with less engineering (no complicated reaction and cooling equipment) and material (no need for additional reagents and solvents), time (reduction in reaction time) and costs (less material, less processing steps, low cost of solvents) and thus represents a universally applicable, low-cost process for the preparation of cysteine-containing or disulfide-bridged oligopeptides or miniproteins.

Die vorgeschlagene Herstellung Cystein-haltiger Peptide durch oxidative Faltung und/oder Native Chemische Ligation ist durch die Verwendung von Ionischen Flüssigkeiten als Reaktionsmedium auch dadurch von Vorteil, dass schwer oder unlösliche Ausgangssubstanzen (z. B. reduzierte, lineare Vorläufermoleküle, Peptidsegmente) überhaupt zur Reaktion gebracht werden können, dass auf die Zugabe toxischer und/oder aggressiver Reagenzien sowie auf die Anwendung umweltschädlicher Lösungsmittel verzichtet werden kann und aufgrund der besagten Qualitäts- und Ausbeutesteigerung.The proposed production of cysteine-containing peptides by oxidative folding and / or native chemical ligation is also advantageous in that the use of ionic liquids as reaction medium means that difficultly or insoluble starting substances (eg reduced, linear precursor molecules, peptide segments) react at all can be made that can be dispensed with the addition of toxic and / or aggressive reagents and the use of environmentally harmful solvents and due to said quality and yield increase.

Die verwendbaren Ionischen Flüssigkeiten sind gut verfügbar, nicht toxisch und biokompatibel. Sie liegen bei Raumtemperatur bis 100°C flüssig vor und unterscheiden sich primär in der Art des Anions und damit in ihrer Fähigkeit, als Wasserstoffbrücken-Akzeptor zu fungieren. Aufgrund dieser Eigenschaften stellen sie hervorragende Reaktionsmedien dar, die unter anderem, wie erwähnt, die Umsetzung von Substanzen erlauben, welche mit Hilfe konventioneller Methoden schlecht oder gar nicht zur Reaktion gebracht werden können.The usable ionic liquids are readily available, non-toxic and biocompatible. They are liquid at room temperature to 100 ° C and differ primarily in the type of anion and thus in their ability to act as a hydrogen bond acceptor. Because of these properties, they are excellent reaction media, which, among other things, as mentioned, allow the reaction of substances that can be reacted with the aid of conventional methods poorly or not at all.

Die Erfindung soll nachstehend anhand von in der Zeichnung dargestellten Ausführungsbeispielen näher erläutert werden.The invention will be explained below with reference to exemplary embodiments illustrated in the drawing.

Es zeigen:Show it:

1: Oxidative Faltung des Peptides δ-EVIA in einer Ionischen Flüssigkeit unter dem Einfluss von Luftsauerstoff 1 : Oxidative folding of the peptide δ-EVIA in an ionic liquid under the influence of atmospheric oxygen

2: Native Chemische Ligation von zwei Peptidsegmenten in einer Ionischen Flüssigkeit, die in der Gesamtsequenz den Naturstoff Tridegin ergeben 2 : Native chemical ligation of two peptide segments in an ionic liquid, which in the overall sequence yield the natural product tridegin

3: Ausgewählte Ionische Flüssigkeiten, mit denen die Synthese der Oxidativen Faltung und/oder Nativen Chemischen Ligation untersucht wurde 3 : Selected Ionic Liquids Used to Investigate the Synthesis of Oxidative Folding and / or Native Chemical Ligation

4: HPLC-Analyse von δ-EVIA-Formen A) und B) oxidierte Form, C) reduzierte Form 4 : HPLC analysis of δ-EVIA forms A) and B) oxidized form, C) reduced form

5: MALDI-TOF MS-Spektren von Tridegin

  • a) NCL in [C2mim][OAc]
  • b) NCL in Phosphatpuffer (pH 7,8)
  • c) SPPS-Totalsynthese des Peptids
5 : MALDI-TOF MS spectra of tridegin
  • a) NCL in [C 2 mim] [OAc]
  • b) NCL in phosphate buffer (pH 7.8)
  • c) Total SPPS synthesis of the peptide

1 zeigt als Beispiel eine schematische Darstellung der oxidativen Faltung eines linearen Peptids 1 (Peptidtoxin δ-EVIA) in einer Ionischen Flüssigkeit IL unter dem Einfluss von Luftsauerstoff. Hierzu wird 1 mg (7,6 mM) des besagten linearen, reduzierten Peptids 1 in 40 μl der Ionischen Flüssigkeit [C2mim][OAc] gelöst und bei Raumtemperatur zwei Stunden gerührt. Die Reaktionsmischung hat währenddessen Kontakt zu atmosphärischem Sauerstoff. Anschließend wird das in der Ionischen Flüssigkeit IL befindliche Reaktionsprodukt als zyklisches Peptid 2 (δ-EVIA in oxidierte Form) mit 40% Acetonitril/Wasser aufgenommen und den nachfolgenden Aufarbeitungs- und Analyseschritten (wie chromatographische Reinigung, massenspektrometrische Analyse) zur Verfügung gestellt. Im Vergleich zu konventionellen Methoden (Ausbeute unter 10%) kann dieses Peptidtoxin mit Hilfe dieses einfachen Reaktionsansatzes und einer einfachen Apparatur aus Rührtisch und Reaktionsgefäß in einer Ausbeute von 25% rein erhalten werden bei gleichzeitigem Verzicht auf große Reaktionsgefäße, Kühlvorrichtung und Vorrichtung für Schutzgasatmosphäre (Argon). 1 shows by way of example a schematic representation of the oxidative folding of a linear peptide 1 (Peptide toxin δ-EVIA) in an ionic liquid IL under the influence of atmospheric oxygen. To this is added 1 mg (7.6 mM) of said linear, reduced peptide 1 dissolved in 40 μl of the ionic liquid [C 2 mim] [OAc] and two at room temperature Hours stirred. Meanwhile, the reaction mixture is in contact with atmospheric oxygen. Subsequently, the reaction product contained in the ionic liquid IL becomes a cyclic peptide 2 (δ-EVIA in oxidized form) taken with 40% acetonitrile / water and the subsequent work-up and analysis steps (such as chromatographic purification, mass spectrometric analysis) provided. In comparison to conventional methods (yield below 10%), this peptide toxin can be obtained pure with the aid of this simple reaction mixture and a simple apparatus from the stirring table and reaction vessel in a yield of 25% while dispensing with large reaction vessels, cooling apparatus and apparatus for protective gas atmosphere (argon ).

2 zeigt die schematische Darstellung der Nativen Chemischen Ligation von zwei Peptidsegmenten 3, 4 in der Ionischen Flüssigkeit IL, die nach Ligation die lineare, reduzierte Sequenz des Naturstoffs Tridegin 5 ergeben. Dazu werden die beiden Peptidsegmente 3 (N-terminales Segment) und 4 (C-terminales Segment) in einer Konzentration von jeweils 3 mM in der Ionischen Flüssigkeit [C2mim][OAc]) unter Argonatmosphäre bei Raumtemperatur zur Reaktion gebracht. Eine Kühlung der Reaktionsmischung auf 4°C sowie eine Reaktionszeit von 24 Stunden oder mehr sind nicht erforderlich. Die Reaktionsmischung wird zur oxidativen Faltung des ligierten, linearen Tridegin-Vorläufermoleküls in der Ionischen Flüssigkeit IL belassen. Durch Zufuhr von Luftsauerstoff (Entfernen der Argonatmosphäre) wird nach einer Reaktionszeit von 4 Stunden das oxidierte Tridegin 5 erhalten. Die Ionische Flüssigkeit IL wird vom Reaktionsprodukt 5 durch chromatographische Trennverfahren (z. B. HPLC) entfernt. 2 shows the schematic representation of the native chemical ligation of two peptide segments 3 . 4 in the ionic liquid IL, after ligation the linear, reduced sequence of the natural product tridegin 5 result. These are the two peptide segments 3 (N-terminal segment) and 4 (C-terminal segment) in a concentration of 3 mM in the ionic liquid [C 2 mim] [OAc]) under argon atmosphere at room temperature for reaction. Cooling of the reaction mixture to 4 ° C and a reaction time of 24 hours or more are not required. The reaction mixture is left to oxidatively fold the ligated, linear tridegin precursor molecule in the ionic liquid IL. By addition of atmospheric oxygen (removal of the argon atmosphere), the oxidized tridegin is formed after a reaction time of 4 hours 5 receive. The ionic liquid IL is the reaction product 5 removed by chromatographic separation methods (eg HPLC).

3 zeigt beispielhaft eine Auswahl von Ionischen Flüssigkeiten mit ihrer jeweiligen Struktur und Bezeichnung, welche für die erfindungsgemäße Verwendung zur Erfüllung der Aufgabenstellung getestet wurden und besonders gut als Lösungsmittel zur Anwendung geeignet erscheinen. Die Verwendung von Ionischen Flüssigkeiten ist nicht auf die angegebene Auswahl beschränkt. 3 shows by way of example a selection of ionic liquids with their respective structure and name, which have been tested for the inventive use to fulfill the task and appear particularly suitable as a solvent for use. The use of ionic liquids is not limited to the specified range.

4 zeigt als Beispiel für die oxidative Faltung (vgl. auch 1) die HPLC-Analyse der Verbindung δ-EVIA in reduzierter (C) und oxidierter (A, B) Form. Die oxidative Faltung wurde dabei in (A) in Redoxpuffer unter Zusatz von reduziertem und oxidiertem Glutathion (1–2 mM) und in (B) in [C2mim][OAc] durchgeführt. Die Reaktionszeit betrug 1 Stunde. 4 shows as an example of the oxidative folding (see also 1 ) HPLC analysis of the compound δ-EVIA in reduced (C) and oxidized (A, B) form. The oxidative folding was carried out in (A) in redox buffer with the addition of reduced and oxidized glutathione (1-2 mM) and in (B) in [C 2 mim] [OAc]. The reaction time was 1 hour.

Die Bedingungen für die HPLC-Analyse sind wie folgt gewählt worden: 25–27% Eluent B in 30 min, wobei Eluent A 0,1% TFA in Wasser, Eluent B 0,1% TFA in Acetonitril und eine Flussrate von 1 ml/min gewählt wurden. Die Detektion erfolgte bei 220 nm.
(* Dieser Peak resultiert aus der Ionischen Flüssigkeit, die nahe dem Injektionspeak eluiert.)The conditions for the HPLC analysis were chosen as follows: 25-27% Eluent B in 30 min, whereby eluent A 0.1% TFA in water, eluent B 0.1% TFA in acetonitrile and a flow rate of 1 ml / min were selected. Detection was at 220 nm.
(* This peak results from the ionic liquid eluting near the injection peak.)

5 zeigt ein MALDI-TOF Massenspektrum der Reaktionsprodukte aus verschiedenen Synthesen des Naturstoffs Tridegin 5 (vgl. auch 2). Spektrum (a) repräsentiert das Ergebnis der Nativen Chemischen Ligation in [C2mim][OAc] und (b) in 0,1 M Phosphatpuffer (Na2HPO4, pH 7,8) und 1 M Guanidiniumhydrochlorid. Die Totalsynthese der Sequenz mit Hilfe der SPPS als Kontrolle ist in (c) dargestellt. Das Molekulargewicht tritt als [M-NH2+Na]+ in allen Spektren auf. 5 shows a MALDI-TOF mass spectrum of the reaction products from various syntheses of the natural product tridegin 5 (see also 2 ). Spectrum (a) represents the result of native chemical ligation in [C 2 mim] [OAc], and (b) in 0.1 M phosphate buffer (Na 2 HPO 4, pH 7.8) and 1 M guanidinium hydrochloride. The total synthesis of the sequence using SPPS as a control is shown in (c). The molecular weight occurs as [M-NH 2 + Na] + in all spectra.

BezugszeichenlisteLIST OF REFERENCE NUMBERS

11
Lineares Peptid δ-EVIA (reduzierte Form)Linear peptide δ-EVIA (reduced form)
22
Zyklisches Peptid δ-EVIA (oxidierte Form)Cyclic peptide δ-EVIA (oxidized form)
33
Peptidsegment (Thioester) des Naturstoffs TrideginPeptide segment (thioester) of the natural product tridegin
44
Peptidsegment (N-terminales Cystein) des Naturstoffs TrideginPeptide segment (N-terminal cysteine) of the natural product tridegin
55
Tridegintridegin
ILIL
Ionische FlüssigkeitIonic liquid
**
Peak der ILPeak of the IL

ZITATE ENTHALTEN IN DER BESCHREIBUNG QUOTES INCLUDE IN THE DESCRIPTION

Diese Liste der vom Anmelder aufgeführten Dokumente wurde automatisiert erzeugt und ist ausschließlich zur besseren Information des Lesers aufgenommen. Die Liste ist nicht Bestandteil der deutschen Patent- bzw. Gebrauchsmusteranmeldung. Das DPMA übernimmt keinerlei Haftung für etwaige Fehler oder Auslassungen.This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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Claims (10)

Verfahren zur Synthese von Cystein-haltigen Peptiden durch Native Chemische Ligation und/oder Oxidation, bei dem ein oder mehrere lineare, reduzierte Peptidsequenzen in einer biokompatiblen, nicht toxischen, bei Raumtemperatur flüssigen und nicht hochviskosen Ionischen Flüssigkeit unabhängig von ihrer Peptidkettenlänge in einer Konzentration von mindestens 3 mM sowie ohne erforderliche Kühlung oder Begrenzung auf Raumtemperatur zur Reaktion gebracht werden.A method of synthesizing cysteine-containing peptides by native chemical ligation and / or oxidation in which one or more linear, reduced peptide sequences in a biocompatible, non-toxic, liquid at room temperature and not highly viscous ionic liquid regardless of their peptide chain length in a concentration of at least 3 mM and without necessary cooling or limitation to room temperature to be reacted. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, dass zur oxidativen Faltung von Cystein-haltigen Peptiden eine lineare, reduzierte Peptidsequenz mit 2 bis 10 Cysteinresten in der Ionischen Flüssigkeit in einer Konzentration von größer als 10 mM gelöst wird.A method according to claim 1, characterized in that for the oxidative folding of cysteine-containing peptides, a linear, reduced peptide sequence having 2 to 10 cysteine residues in the ionic liquid is dissolved in a concentration greater than 10 mM. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, dass zur Nativen Chemischen Ligation von Cystein-haltigen Peptiden zwei lineare Peptidsequenzen mit wenigstens einem Cysteinrest im N-Terminus des C-terminalen Segmentes in der Ionischen Flüssigkeit gelöst werden.A method according to claim 1, characterized in that for the native chemical ligation of cysteine-containing peptides two linear peptide sequences having at least one cysteine residue in the N-terminus of the C-terminal segment in the ionic liquid are dissolved. Verfahren gemäß Anspruch 3, dadurch gekennzeichnet, dass die zwei linearen Peptidsequenzen in der Summe eine Anzahl von 2 bis 10 Cysteinresten enthalten.A method according to claim 3, characterized in that the two linear peptide sequences in the sum contain a number of 2 to 10 cysteine residues. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, dass zur Synthese der Cystein-haltigen Peptide durch Native Chemische Ligation und Oxidation zunächst die Reaktion der Nativen Chemischen Ligation in der Ionischen Flüssigkeit durchgeführt wird und dass anschließend in derselben Ionischen Flüssigkeit die Oxidation ohne erforderliche Zugabe von toxischen Additiven, Detergenzien und/oder anderen Lösungsmitteln erfolgt.A method according to claim 1, characterized in that for the synthesis of the cysteine-containing peptides by Native Chemical Ligation and oxidation, first the reaction of the Native Chemical Ligation is performed in the ionic liquid and then in the same ionic liquid, the oxidation without the necessary addition of toxic additives , Detergents and / or other solvents. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, dass die Ionische Flüssigkeit einen Wassergehalt von bis zu 10%, vorzugsweise weniger als 5% aufweist.A method according to claim 1, characterized in that the ionic liquid has a water content of up to 10%, preferably less than 5%. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, dass die Synthese bei höheren Temperaturen als Raumtemperatur, insbesondere für langkettige Peptide bei ca. 80°C, durchgeführt wird.A method according to claim 1, characterized in that the synthesis at higher temperatures than room temperature, in particular for long-chain peptides at about 80 ° C, is performed. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, dass als Lösungsmittel Ionische Flüssigkeiten mit 1-Ethyl-3-Methylimidazolium-Ionen als Kationen und vorzugsweise [OAc]–, [OTs]– oder [DEP]– als Anionen verwendet werden.A method according to claim 1, characterized in that as solvents ionic liquids with 1-ethyl-3-methylimidazolium ions as cations and preferably [OAc] - , [OTs] - or [DEP] - are used as anions. Verfahren gemäß Anspruch 2, dadurch gekennzeichnet, dass für die oxidative Faltung von Cystein-haltigen Peptiden je nach Peptidkettenlänge und Anzahl der zu bildenden Disulfidbrücken Reaktionszeiten von 30 min bis 6 h zur Anwendung kommen.Process according to Claim 2, characterized in that reaction times of 30 minutes to 6 hours are used for the oxidative folding of cysteine-containing peptides, depending on the peptide chain length and number of disulfide bridges to be formed. Verfahren gemäß Anspruch 3, dadurch gekennzeichnet, dass für die Native Chemische Ligation von Cystein-haltigen Peptiden Reaktionszeiten von weniger als 24 Stunden zur Anwendung kommen.A method according to claim 3, characterized in that for the native chemical ligation of cysteine-containing peptides reaction times of less than 24 hours are used.
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