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

Abstract

The task was to produce cysteine-containing and cysteine-containing, disulfide-bridged oligopeptides universally synthesizable, less effort, biocompatible, less polluting, if possible without disruptive side reactions and with high yield and quality. According to the invention, one or more linear, reduced peptide sequences (1) are used in a biocompatible, non-toxic, ionic liquid (IL) which is liquid at room temperature and is not highly viscous, regardless of its peptide chain length in a concentration of at least 3 mM and without the need for cooling or limitation to room temperature Brought reaction.

Description

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.

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 ).

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 ).

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 ).

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 ).

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.

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.

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.

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 ).

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 ).

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 ).

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 ).

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 ).

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.

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.

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.

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 ).

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 ).

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.

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.

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 ).

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.

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 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 ).

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 ).

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 ).

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 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 ).

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 ).

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 ).

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 ).

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 ).

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.

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.

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.

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.

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.

Furthermore, it is possible to dispense with the addition of additives or solvents, because the reaction proceeds exclusively through the influence of atmospheric oxygen.

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.

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.

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.

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.

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.

The invention will be explained below with reference to exemplary embodiments illustrated in the drawing.

Show it:

1 : Oxidative folding of the peptide δ-EVIA in an ionic liquid under the influence of atmospheric oxygen

2 : Native chemical ligation of two peptide segments in an ionic liquid, which in the overall sequence yield the natural product tridegin

3 : Selected Ionic Liquids Used to Investigate the Synthesis of Oxidative Folding and / or Native Chemical Ligation

4 : HPLC analysis of δ-EVIA forms A) and B) oxidized form, C) reduced form

• 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 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 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 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 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.

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 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.

LIST OF REFERENCE NUMBERS

1

Linear peptide δ-EVIA (reduced form)

2

Cyclic peptide δ-EVIA (oxidized form)

3

Peptide segment (thioester) of the natural product tridegin

4

Peptide segment (N-terminal cysteine) of the natural product tridegin

5

tridegin

IL

Ionic liquid

*

Peak of the IL

QUOTES INCLUDE IN THE DESCRIPTION

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.

Cited patent literature

• US 7524812 B2 [0005]
• US 5910554 A [0018]
• US 5656707A [0018]
• US 2010/0093975 A1 [0031]

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Claims (10)

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. 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. 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. 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. 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. A method according to claim 1, characterized in that the ionic liquid has a water content of up to 10%, preferably less than 5%. 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. 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. 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. 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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