Peptide precipitation method

Abstract

Methods for peptide synthesis are provided. The methods include a specific precipitation step that facilitates peptide filtration.

Description

Peptide precipitation method Methods for peptide synthesis are provided. The methods include a specific precipitation step that facilitates peptide filtration. Peptides, both in solution and in solid phase, are generally synthesized from the C-terminus to the N-terminus by condensation of the carboxyl group of one amino acid to the amino group of another. However, the peptides and amino acids from which peptides are synthesized tend to have reactive side-chain functional groups. Therefore, when synthesizing a peptide, it is important to ensure that the respective amino group reacts selectively with the carboxyl group without any side reactions at these side-chain functional groups, as this could severely impair the yield or even spoil the product being synthesized from a practical standpoint. To minimize such side reactions, the conventional practice is to appropriately mask the reactive side groups and terminal ends of the reactants to help ensure that the desired reaction occurs. Since the respective amino protecting groups (temporary protecting groups) are removed several times during synthesis, their removal must therefore be carried out under mild conditions. Meanwhile, the reactive side-chain groups of the amino acid and peptide reactants, including the resin-bound peptide material and any additional material to be added to the growing chain, typically remain masked by the side-chain protecting groups throughout the synthesis (permanent protecting groups) and are not only removed in the final stage of the synthetic process. Well-known temporary protecting groups are the ferc-butyloxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc) groups, which are cleaved by weak acids or weak bases. The cleavage of permanent protecting groups, as well as the cleavage of the resin, is generally influenced by strong acids or bases, optionally in the presence of a scrubber and a solvent. After cleavage, the peptide must be recovered from the cleavage solution. Typical methods for peptide recovery involve the use of acid / salt chemistry. For example, the peptide can be precipitated in aqueous salt (such as sodium chloride), and the solids can then be collected (e.g., by vacuum filtration), washed, and dried. However, such methods present problems for commercial-scale production, including high levels of impurities. Other recovery methods involve precipitating the peptide by adding a solvent, such as, in particular, an ether solvent, followed by collection of the precipitated peptide by filtration. Precipitation with an ether solvent, however, often results in peptide precipitates that clog the filtration apparatus (e.g., when the precipitate is too fine) and / or are sticky and thus complicate or even prevent filtration (e.g., when the precipitate is gelatinous).International patent application WO 2005 / 063793 discloses that precipitation of peptides using alcohol improves filtration capacity. For large-scale peptide production, these issues can therefore greatly influence the viability of the peptide synthesis scheme. Consequently, there is a constant need for peptide synthesis processes capable of producing commercially valuable peptide materials in large batch quantities. Recovery of peptide material after synthesis, for example, by precipitation and subsequent filtration, is an aspect of synthesis that requires further improvement, as conventional methodologies often still require lengthy filtration times, which significantly impact production costs. Surprisingly, it has now been discovered that peptide precipitates prepared according to the methods of the present invention can be filtered much faster than precipitates prepared according to conventional methods in the art. Consequently, the methods of the present invention can be advantageously used to provide improved commercial-scale processes for the synthesis and recovery of peptides. Therefore, in a first embodiment, the present invention relates to a method for obtaining a completely unprotected peptide, said method comprising the consecutive steps of a.) synthesize a peptide intermediate having at least one acid-unstable protecting group b.) removing said protecting group or groups using a cleavage reagent composed of an acid and optionally a scrubber and / or an aprotic solvent to form a solution of cleaved peptide (I), followed by c.) provide a solvent selected from the chloroform, dichloromethane or ester solvent group of formula (I) wherein R1 and R2 independently of each other are a linear or branched C1-C10 alkyl group or C3-C6 cycloalkyl group to the cleaved peptide solution (I) to form a cleaved peptide solution (II), followed by d.) provide a precipitating agent selected from the group consisting of at least one ether solvent and at least one hydrocarbon solvent, as well as mixtures thereof in a sufficient quantity to precipitate the peptide from the cleaved peptide solution (II) and e.) collect the peptide. In another aspect, the invention provides a method for precipitating a peptide comprising the consecutive steps of: (a) providing a composition consisting essentially of a peptide intermediate having at least one acid-unstable protecting group and a cleavage reagent composed of an acid and optionally a scrubber and / or a solvent, followed by (b) providing a solvent selected from the group of chloroform, dichloromethane, or an ester solvent of formula (I) as defined above to the composition to obtain a cleaved peptide solution (II), followed by (c) providing a precipitating agent selected from the group consisting of an ether solvent and a hydrocarbon solvent, as well as mixtures thereof, to the cleaved peptide solution (II), followed by (d) collecting the peptide. The expression "consisting essentially of", as used according to the present invention, means that the total amount of the listed ingredients ideally sums to 100% by weight. However, the presence of small amounts of impurities or additives is not excluded, provided that the total amount of such impurities or additives is preferably less than 3% by weight, more preferably less than 2% by weight, and most preferably less than 1% by weight. In step b), the addition of the selected organic solvent from the chloroform, dichloromethane, or ester solvent group (I) may cause initial precipitation of the peptide. The amount of solvent, however, is adjusted so that, in the event of such precipitation, the peptide will re-solubilize to form a solution of the cleaved peptide. The amount of organic solvent can be easily adjusted by someone skilled in the art and may vary slightly depending on the peptide sequence and the temperature applied in the dilution / re-solubilization step b). Preferably, step b.) is carried out at room temperature, i.e., at a temperature ranging from 18 to 25 °C. Incorporating step b.) into the cleaved peptide isolation step provides drastically reduced filtration times compared to previous isolation techniques that omit the dilution / resolubilization step as illustrated in the examples. The expression "peptide intermediate having at least one acid-unstable protecting group," as used herein, refers to a peptide comprising at least one acid-unstable protecting group. Peptide intermediates according to the invention can therefore be fully protected, i.e., all reactive side-chain groups, as well as appropriate terminal amino acids, include acid-unstable protecting groups (including N-terminus and C-terminus protecting groups). In other respects, however, the invention can also be used to recover peptides where only all reactive side-chain groups or only the terminal amino acids include acid-unstable protecting groups. In the case of solid-phase synthesis, the resin itself could also be acid-unstable and would therefore be considered an acid-unstable protecting group.Preferably, the peptide intermediates according to the present invention, however, comprise at least one acid-unstable side-chain protecting group. An acid-unstable (side-chain) protecting group refers to a chemical residue attached to a peptide, preferably to the side chain (i.e., the R group in the general amino acid formula H₂N-C(R)(H)-COOH) of an amino acid. This group helps prevent unwanted side reactions, such as preventing a portion of the side chain from reacting with chemicals used in peptide synthesis, processing, and similar steps. These protecting groups can be removed with an acid, optionally in the presence of a scrubber. The choice of a suitable acid-unstable (side-chain) protecting group may depend on several factors, including the type of synthesis being performed, the processing the peptide will undergo, and the desired intermediate or final product. The specific acid-unstable (side-chain) protecting group also depends on the nature of the amino acid itself.In general, an acid-unstable (side-chain) protecting group is chosen so that it will not be removed during the deprotection of the α-amino groups during synthesis. Therefore, the α-amino protecting group and the side-chain protecting group are typically not the same. Examples of suitable acid-unstable protecting (side-chain) groups according to the present invention include ferc-butyl (tBu), triphenylmethyl (trityl, trt), dimethoxytrityl (DMT), 2-chlorotrityl (2-CITrt), 1-cyclopropyl-1-methylethyl (Dmcp), tetrahydropyranyl, t-butoxycarbonyl (BOC), methoxytrimethylbenzene sulfonyl- (Mtr-), 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl- (Pmc-), 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl- (Pbf-), adamantyloxycarbonyl, xanthyl (Xan), benzyl, 3-ethyl 3-pentyl, and 5-butyl 5-nonyl and t-butyl ester, benzyloxycarbonyl (Z) , 2-chlorobenzyloxycarbonyl (2-CI-Z) and t-amyloxycarbonyl (Aoc) without limitation. Preferred acid-unstable side-chain protecting groups in all embodiments according to the present invention include the t-Bu group for the amino acid residues tyrosine, threonine, serine and aspartic acid; the trt group for the amino acid residues histidine, glutamine and asparagine; and the Boc group for the amino acid residues lysine, DAB and tryptophan and the Mtr-, Pmc-, Bis-Boc or Pbf- group for the amino acid residue arginine. Suitable acids in cleavage reagents are all acids that can completely deprotect acid-unstable side-chain protecting groups. The selection of an appropriate acid depends on the respective acid-unstable protecting groups used and can be readily selected by someone skilled in the art. Exemplary acids include carboxylic acids such as formic acid, acetic acid, and trifluoroacetic acid; sulfonic acids such as methanesulfonic acid (MSA), trifluoromethanesulfonic acid (triflic acid), benzenesulfonic acid, and p-toluenesulfonic acid; hydrogen bromide; trimethylsilyl trifluoromethanesulfonate (TMSOTf); trimethylsilyl bromide (TMSBr); and mixtures thereof. In all embodiments of the present invention, however, the use of acetic acid, trifluoroacetic acid, and methanesulfonic acid, as well as mixtures thereof, as the acid in step b) is preferred.The most preferred option is the use of trifluoroacetic acid and / or methanesulfonic acid. The total amount of one or more acids in the cleavage reagent is preferably selected in the range of 0.1 to 1000 mol-equivalents with respect to the number of acid-unstable protecting groups present in the peptide intermediate, more preferably from 10 to 500 mol-equivalents, even more preferably from 20 to 400 mol-equivalents, and most preferably from 25 to 250 mol-equivalents. Suitable scrubbers for use in the cleavage reagent according to the present invention are all scrubbers commonly used in peptide synthesis, such as phenols, for example, anisole, phenol; trialkylsilanes (more preferably C1-4-trialkylsilanes), for example, trimethylsilane, triethylsilane, or triisopropylsilane; thiols, for example, aliphatic thiols (alkylthiols), for example, ethanedithiol, dodecanethiol, or aromatic thiols, for example, thioanisole and thiophenol; and water, as well as mixtures thereof. In all embodiments of the present invention, however, the use of triisopropylsilane, dodecanethiol, and water, as well as mixtures thereof, is preferred. The total amount of one or more scrubbers in the cleavage reagent is preferably selected in the range of 0.1 to 500 mol-equivalents with respect to the number of acid-unstable protecting groups present in the peptide intermediate, more preferably from 1 to 100 mol-equivalents, even more preferably from 2 to 50 mol-equivalents, and most preferably from 3 to 40 mol-equivalents. In a preferred embodiment, the cleavage reagent according to the present invention necessarily comprises a scrubber, even more preferably the cleavage reagent comprises a scrubber selected from the group consisting of water, triethylsilane, triisopropylsilane or dodecanethiol, most preferably water or dodecanethiol. Suitable aprotic solvents used in the cleavage reagent in all embodiments according to the present invention include liquid aromatic hydrocarbons (i.e., aromatic hydrocarbons that are liquid at room temperature (i.e., 20 °C)) or C1-6 haloalkanes, such in particular as fluoro- and / or C1-6 chloroalkanes, for example, chloroform, dichloromethane, dichloroethane, 1,1-difluoroethane, as well as mixtures thereof. In all embodiments of the present invention, however, the use of toluene or dichloromethane is preferred. Preferably, an aprotic solvent is comprised solely in the cleavage reagent when the scrubber does not form a single phase with the acid alone, as in the case of triisopropylsilane or dodecanethiol. More preferably, an aprotic solvent is obligatorily present when the scrubber is triisopropylsilane. If present, the amount of aprotic solvent in the cleavage reagent is selected so that the peptide to be deprotected is solubilized in it at a temperature selected in the range of 10 °C to 40 °C, preferably from 0 °C to 30 °C, and even more preferably from 10 to 25 °C. Preferably, if present, the amount of aprotic solvent in the cleavage reagent is selected in the range of 0.25 to 10 mL / g of peptide intermediate, more preferably in the range of 0.5 to 5 mL / g of peptide intermediate, and most preferably in the range of 0.75 to 1.5 mL / g of peptide intermediate. Unless otherwise stated, the term peptide intermediate always refers to the fully protected peptide. The total amount of cleavage reagent is appropriately selected in the range of 0.5 to 50 ml / g of peptide intermediate, more preferably in the range of 1 to 50 ml / g of peptide intermediate, most preferably in the range of 2.5 to 20 ml / g of peptide intermediate (where the expression peptide intermediate refers to the fully protected peptide). Suitable ester solvents of formula (I) in all embodiments according to the present invention include linear or branched C1-C10 alkyl acetates, propionates, isopropionates, butyrates and the like. Preferred ester solvents according to the present invention are linear or branched C1-C4 alkyl acetates such as, for example, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, n-butyl acetate and ferc-butyl acetate, linear or branched C1-C4 alkyl propionates such as, for example, methyl propionate, ethyl propionate, propyl propionate, n-butyl propionate, ferc-butyl propionate, as well as C1-C4 alkyl butyrates such as, for example, methyl butyrate, ethyl butyrate, propyl butyrate, isopropyl butyrate and ferc-butyl butyrate.In all embodiments of the present invention, however, the use of ethyl acetate, ethyl propionate, propyl acetate, and diisopropyl acetate, as well as mixtures thereof, is preferred. The total amount of the organic solvent selected from the group of dichloromethane, chloroform, or an ester solvent of formula (I) may vary depending on the respective peptide intermediate and can be easily adjusted by a person skilled in the art. If the peptide intermediate initially precipitates upon addition of the organic solvent, the amount of organic solvent is selected so that the peptide re-solubilizes. Preferably, in all embodiments of the present invention, the amount of organic solvent is selected in the range of 0.5 to 100 mL / g of peptide intermediate, more preferably in the range of 1 to 50 mL / g of peptide intermediate, and most preferably in the range of 5 to 35 mL / g of peptide intermediate (where the term "peptide intermediate" refers to the fully protected peptide). Additional suitable ranges include 10 to 30 mL / g of peptide intermediate or 15 to 30 mL / g of peptide intermediate. Suitable ether solvents for use as precipitating agents in all embodiments according to the present invention include cyclopentyl methyl ether, dibutyl ether, diethyl ether, diisopropyl ether, ethyl ferc-butyl ether, methyl ferc-butyl ether, and mixtures thereof. In all embodiments of the present invention, the use of diethyl ether and methyl ferc-butyl ether, as well as mixtures thereof, is preferred. Suitable hydrocarbon solvents for precipitation according to the present invention include pentane, hexane, heptane, and petroleum ether, as well as mixtures thereof. In all embodiments of the present invention, the use of petroleum ether and hexane, as well as mixtures thereof, is preferred. If a mixture of ether and hydrocarbon solvents is used as the precipitating agent, the total amount of hydrocarbon solvent in the precipitating agent should not exceed 35% by volume based on the ether solvent used. In a particular advantageous embodiment according to the present invention, however, only an ether solvent is used as the precipitating agent, as this leads to particular advantageous results. The precipitating agent is added to the cleaved peptide (II) solution in a sufficient amount to precipitate the peptide from the solution at a temperature selected in the range of -10 °C to 40 °C, preferably 0 °C to 30 °C, and even more preferably 10 to 25 °C. This temperature can be easily adjusted by someone skilled in the art. Preferably, the amount of precipitating agent is chosen to provide improved purity of the peptide precipitate. In some embodiments, the precipitating agent is added in an amount of at least approximately 0.25 volumes or in the range of 0.25 to 8 volumes per volume of the cleaved peptide (II) solution. In alternative embodiments, the precipitating agent is added in an amount in the range of 0.5 to 5 volumes per volume of the cleaved peptide (II) solution. In an advantageous embodiment of the present invention, the cleavage reagent essentially consists of an acid, most preferably trifluoroacetic acid, most preferably 95% trifluoroacetic acid with all the definitions and preferred as given herein. In another advantageous embodiment of the present invention, the cleavage reagent essentially consists of at least one acid, most preferably trifluoroacetic acid and / or MSA, dodecanethiol, and toluene, with all definitions and preferences as given herein. The precipitate can be collected by methods known to those skilled in the art, such as filtration or centrifugation. Preferably, the precipitate is collected by filtration, and even more preferably by filtration through a frit (fritted glass). The methods described in this document are particularly well-suited for improving aspects of scaled-up peptide synthesis. In preferred embodiments, these innovative methods can provide improvements such as reduced processing (synthesis) time, increased product yield, and enhanced product purity. The methods of the present invention can be used in connection with the synthesis of peptides of any suitable length and / or sequence. It is understood that the peptides of the invention can be synthesized or prepared by techniques well known in the art, such as solid-phase or solution synthesis. See, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman and Co., NY, or M. Bodanszky, Principles of Peptide Synthesis, 1984, Springer Science & Business Media, as well as references cited herein. In an advantageous aspect according to the present invention, the methods according to the present invention are used to recover peptides incorporating from approximately 2 to approximately 20, or from approximately 2 to approximately 15, or from approximately 3 to 12 residues of one or more amino acids, or from approximately 3 to approximately 6 residues of one or more amino acids. Optionally, residues of one or more monomeric, oligomeric, and / or polymeric constituents may be incorporated into a peptide. Non-peptide linkages may also be present. These non-peptide linkages may be between amino acid residues, between an amino acid and a non-amino acid residue, or between two non-amino acid residues. These alternative non-peptide linkages may be formed using reactions well known to those skilled in the art and may include, but are not limited to, imino, ester, hydrazide, semicarbazide, azo, and similar linkages. The amino acids from which peptides are derived can be naturally occurring amino acid residues, non-naturally occurring amino acid residues, or combinations thereof. The twenty common naturally occurring amino acid residues are as follows: A (Ala, alanine); R (Arg, arginine); N (Asn, asparagine); D (Asp, aspartic acid); C (Cys, cysteine); Q (Gln, glutamine); E (Glu, glutamic acid); G (Gly, glycine); H (His, histidine); I (Ile, isoleucine); L (Leu, leucine); K (Lys, lysine); M (Met, methionine); F (Phe, phenylalanine); P (Pro, proline); S (Ser, serine); T (Thr, threonine); W (Trp, tryptophan); Y (Tyr, tyrosine); and V (Val, valine). Uncommon amino acids of natural origin are also considered, including, for example, selenocysteine ​​and pyrrolysine. Non-natural amino acids include organic compounds that have a structure and reactivity similar to those of naturally occurring amino acids and include, for example, D-amino acids, beta-amino acids, omega-amino acids (such as 3-aminopropionic acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid (DAB), 4-aminobutyric acid, and the like), gamma amino acids, cyclic amino acid analogues, propargylglycine derivatives, 2-amino-4-cyanobutyric acid derivatives, but are not limited to them. The peptides according to the present invention may be substituted at the terminal α-amino group with a lipophilic moiety connected to the peptide by an ester, amide, N-alkyl, N-alkenyl, sulfonyl, urethane, or urea linkage. In particular, the peptide substituted with a lipophilic moiety refers to N-acyl derivatives thereof, such as, most particularly, N-palmitoyl, N-myristyl, N-stearoyl, N-benzoyl, N-acetyl, or N-tetradecylaminocarbonyl derivatives thereof. Particularly preferred peptide derivatives in all embodiments of the present invention are N-benzoyl or N-tetradecylaminocarbonyl derivatives thereof. The methods of the invention, which relate to precipitating peptides, can be integrated into any peptide synthesis procedure, such as solid-phase synthesis, liquid-phase synthesis, hybrid synthesis, or recombinant synthesis. When using solid-phase peptide synthesis (SPPS), the synthesized peptide can be cleaved from the solid support (such as a resin) prior to the use of the innovative methods described herein. Alternatively, the synthesized peptide can be cleaved from the solid support simultaneously with the removal of protecting groups using the cleavage reagent according to the present invention. The cleavage reagents that allow the peptide to be cleaved from the resin, as well as the protecting groups to be removed, are well known to those skilled in the art. For example, 0.5% to 1% solutions of trifluoroacetic acid (TFA) in dichloromethane or toluene, or a combination of 0.5% to 1% TFA in dichloromethane and toluene, can be used to cleave the peptide from the resin without removing the protecting groups. Alternatively, HBr in acetic acid (HOAc), 50% TFA in dichloromethane, or 95% TFA with 5% water, or other available scrubbers, can be used to cleave the peptide and simultaneously remove the protecting groups. The specific cleavage reagent, solvents, and cleavage time selected will depend on the particular peptide being cleaved and the protecting groups to be removed. These parameters fall within the scope of the relevant technique. In a particular preferred embodiment of the present invention, the peptide intermediate is a peptide intermediate comprising at least one side-chain protected arginine with Mtr-, Pmc- or Pbf- and / or at least one side-chain protected lysine with Boc and / or 2,3-diaminopropionic acid and / or 2,4-diaminobutyric acid (Dab) with all definitions and preferences as given herein. In a particular preferred embodiment of the present invention, the peptide intermediate is a peptide intermediate (I) comprising at least one arginine side-protected with Mtr-, Pmc- or Pbf- and at least one amino acid bearing an electron-rich aromatic side chain. The preferred electron-rich side chains in said peptide intermediates (I) are "C1-C6 arylalkyl groups" and / or "C1-C6 heteroarylalkyl groups". The expression "C1-C6 arylalkyl group," as used herein, refers to a C1-C6 alkyl aryl group (i.e., a C1-C6 alkyl group that is substituted with an aryl group, i.e., the point of attachment is the alkyl group), wherein the term "aryl" refers to an aromatic substituent containing 5 to 15 carbon atoms and containing a single aromatic ring or multiple aromatic rings that are fused together, directly joined, or indirectly joined (so that the different aromatic rings are attached to a common group such as a methylene or ethylene group). Particularly advantageous aryl groups according to the present invention contain 6 to 12 carbon atoms and contain a single aromatic ring or multiple aromatic rings that are fused together or directly joined. The most preferred aryl residues in all embodiments of the present invention are phenyl, naphthyl, and biphenyl.Particularly advantageous C1-C6 arylalkyl groups in all embodiments of the present invention are C1-C2 arylalkyl groups such as, in particular, phenyl(m)ethyl or naphthyl(m)ethyl. The expression "C1-C6 heteroarylalkyl group" refers to a C1-C6 alkyl-heteroaryl group (i.e., a C1-C6 alkyl group that is substituted with a heteroaryl group, i.e., the point of attachment is the alkyl group), where the term "heteroaryl" refers to a 5-, 6-, or 7-membered aromatic ring containing one or more heteroatoms, namely N, O, or S; these heteroaromatic rings can be fused to other aromatic systems. Particularly preferred heteroaromatic rings include imidazole, indole, pyridine, and quinoline. The aryl and heteroaryl residues may be, independently of each other, unsubstituted or substituted with one or more substituents. In all embodiments of the present invention, these substituents are preferably selected from halogen, hydroxy, nitro, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 alkanoyloxy. More preferably, in all embodiments of the present invention, the aryl and heteroaryl residues are, independently of each other, unsubstituted or substituted with one or two substituents selected from the group consisting of F, Cl, hydroxy, cyano, C1-C3 alkyl, C1-C3 alkoxy, and C1-C3 alkanoyloxy, such as, in particular, unsubstituted or substituted with one or two substituents selected from the group consisting of F or hydroxy. More preferably, in all embodiments of the present invention, the aryl and heteroaryl residues are unsubstituted or substituted with F, hydroxy, or nitro. In all peptide intermediates (I), the particularly advantageous C1-C6 arylalkyl group is a C1-C2 arylalkyl group, which is preferably unsubstituted or substituted with one or two substituents selected from the fluoro (F), hydroxy (OH), or nitro (NO2) group. The most preferred C1-C6 arylalkyl group in all embodiments of the present invention is phenyl(m)ethyl, 4-hydroxyphenyl(m)ethyl, 3-nitro-4-hydroxyphenyl(m)ethyl, or naphthyl(m)ethyl, such as, in particular, phenylmethyl, 4-hydroxyphenyl(m)ethyl, 3-nitro-4-hydroxyphenylmethyl, or 1- or 2-naphthylmethyl. In all peptide intermediates (I), the most preferred C1-C6 heteroarylalkyl group is a C1-C2 heteroarylalkyl group such as (1H-indol-3-yl)(m)ethyl, (1H-imidazol-4-yl)(m)ethyl, (pyridin-2-yl)(m)ethyl, (pyridin-3-yl)(m)ethyl, (quinolin-2-yl)(m)ethyl, and (quinolin-3-yl)(m)ethyl, which are preferably unsubstituted or substituted with one or two substituents selected from the F(fluoro), hydroxy (OH), or nitro (NO2) group. The most preferred in all embodiments of the present invention are (1H-indol-3-yl)(m)ethylene or (1H-imidazol-4-yl)(m)ethylene. In all peptide intermediates (I), the most preferred amino acids bearing an electron-rich aromatic side chain are selected from the group of D / L-histidine, D / L-tryptophan, 6-fluorotryptophan, 5-hydroxytryptophan, D / L-phenylalanine, D / L-tyrosine, D / Lm-nitrotyrosine, D / L-ethyltyrosine, and D / L-3-(2-naphthyl)-alanine. Particularly advantageous amino acids bearing an electron-rich aromatic side chain are selected from the group of D / L-histidine, D / L-phenylalanine, D / L-tyrosine, and D / L-tryptophan, as well as C1-C6 O-alkylated D / L-tyrosine; m / p-Cl / Br / ID / L-phenylalanine; and 4 / -5- / 6- / 7-OH / F / Cl / Br / methyl-D / L-tryptophan. The most preferred electron-rich side chains in all embodiments of the present invention are those of D / L-histidine, D / L-tryptophan, D / L-tyrosine, D / L-tryptophan, 5-hydroxy D / L-tryptophan, and 6-fluoro D / L-tryptophan. It is well understood that the term D / L encompasses the respective D-amino acids, L-amino acids, as well as mixtures thereof. In all embodiments of the present invention, most preferably the peptide intermediate (I) comprising an arginine side-protected with Mtr-, Pmc- or Pbf- also contains at least one amino acid bearing an electron-rich aromatic side chain selected from the group of D / L-histidine, D / L-tryptophan, tyrosine, D / L-6-fluoro-tryptophan and D / L-5-hydroxy-tryptophan. Even more preferably, the peptide intermediate (I) is a di- to a hexapeptide, i.e., a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, or a hexapeptide. In a further preferred embodiment, the Mtr-, Pmc-, or Pbf--protected arginine side chain and the at least one electron-rich aromatic side chain-bearing amino acid in the peptide intermediate (I) are separated by at most three amino acids, preferably by at most two amino acids, and most preferably by at most one amino acid. In all embodiments of the present invention, however, the electron-rich amino acid is very preferably directly adjacent to the Mtr-, Pmc-, or Pbf-protected arginine side chain. Even more preferably, in all embodiments of the present invention, the peptide intermediate (I) is a peptide intermediate (II) comprising at least one Pbf-protected arginine moiety and at least one amino acid moiety selected from the group of tyrosine, histidine (preferably T rt-protected), and / or tryptophan, wherein the histidine, tyrosine, and / or tryptophan moiety is preferably at most two, and more preferably at most one, such as being directly adjacent to the protected arginine moiety. The particularly preferred amino acids tyrosine, histidine, and / or tryptophan are histidine, tyrosine, tryptophan, 5-hydroxytryptophan, and 6-fluorotryptophan, as well as mixtures thereof. It is well understood that, as mentioned above, the Pbf-protected arginine moiety, as well as the amino acid moiety, may be in the L or D configuration, or be mixtures of both configurations (within the peptide). Particularly preferred (I) peptide intermediates, respectively (II) according to the present invention are Boc-Trp-Arg (Pbf) -OH, Ac-Arg (Pbf) -His (Trt) -Phe-OH, TFA*H-Tyr (tBu) -Arg (Pbf) -Pro-OH, Boc-G (Pbf) -Glu (OtBu) -OH, Boc-Tyr (Et) -Arg (Pbf) -Ala-Phe-OH, Ac-5- (OH) Trp-Ala-Arg (Pbf) -Ser (tBu) -Leu-Phe-OH, Boc-Arg (Mtr) -Tyr (tBu) -Boc-Phe-N-2-Arg (Pbf) -Phe-OH, Ac-Arg (Pbf) -Met-m (NO2) Tyr-Pro-OH y Bz-Gly-His (Trt) -D-Phe-Arg (Pbf) -D-Trp-N (Pr) 2, Bz-Gly-His-D-Phe-Arg (Pbf) -D (Trt) -D-Phe-Arg (Pbf) -D-Trp (Boc) -NPr2. The most preferred peptide intermediate (I), respectively (II) is Bz-Gly-His (Trt) -D-Phe-Arg (Pbf) -Trp-NPr2. In another particular preferred embodiment of the present invention, the peptide intermediate is a peptide intermediate (III) comprising, alongside the side chain, at least one lysine protected with Boc and / or 2,3-diaminopropionic acid and / or 2,4-diaminobutyric acid (Dab) and / or at least one valine or leucine or isoleucine. Even more preferably, said peptide intermediates (III) are peptide intermediates (IV) consisting solely of a lysine side chain protected with Boc or 2,4-diaminobutyric acid and valine. Even more preferably, the peptide intermediate (III) and (IV) is a di- to a hexapeptide, i.e., a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, or a hexapeptide. The most preferred peptide intermediates (III) and (IV) according to the present invention are TDAC-Dab (Boc) -Val-Dab (Boc) -OH, TDAC-Dab (Boc) -Val-Dab (Boc) -O-resin and Ac-Lys (Boc) -Val-Lys (Boc) -Val-Lys (Boc) -Val-OH. The invention is further illustrated by reference to the following non-limiting examples, in which all percentages are by weight based on the total weight unless otherwise indicated. Example Abbreviations Acetyl acyl Arg arginine Boc tert-butyloxycarbonyl Bz benzoyl DCM dichloromethane D-Phe D-phenylalanine EtOAc ethyl acetate EtOPr ethyl propionate Et2O diethyl ether Glycine iPropOAc isopropyl acetate minute minute MSA methanesulfonic acid MTBE methyl tert-butyl ether Pbf 2, 2, 4, 6, 7-pentamethyl-dihydrobenzofuran-5-sulfonyl ProOAc propyl acetate TFA trifluoroacetic acid tryptophan Trt trityl Val valine Conventional (reference) method: The respective protected peptide (800 mg) as summarized in Tables 1 and 2 was placed in a flask. After this, the cleavage reagent (ref 1: 0.8 ml toluene, 387 ml dodecanethiol, 245 ml MSA and 4.15 ml 100% TFA; ref 2 and 3: 5.0 ml 95% TFA) was added to the peptide and the resulting reaction mixture was shaken for 1 h to form the cleaved peptide solution (I). Next, the cleaved peptide solution (I) was transferred to 43 mL of MTBE, causing precipitation of the cleaved peptide. The resulting peptide suspension was stirred at room temperature for approximately 15 min. Afterward, the peptide suspension was transferred to a G4 pore-size sintered frit. The liquid level was marked, and a reduced pressure of 30 kPa (300 mbar) was applied to the frit. After the liquid level dropped to 2 cm (measured from the top of the frit), the filtration time was recorded until all the liquid had been removed from the frit. Method according to the invention: The respective protected peptide (800 mg) was placed in a flask. The cleavage reagent (I-1 to I-10: 0.8 mL toluene, 387 g / L dodecanethiol, 245 g / L methanesulfonic acid, and 4.15 mL 100% TFA; I-11 and I-1: 5.0 mL 95% TFA) was then added to the peptide, and the resulting reaction mixture was stirred for 1 h to form the cleaved peptide solution (I). The cleaved peptide solution (I) was then transferred to an organic solvent as summarized in Tables 1 and 2 (which, in the case of I-5, I-6, I-7, I-11, I-12, and I-13, caused an initial precipitation of the peptide, which was subsequently re-solubilized). Stirring was continued at room temperature (approximately 20 °C) producing a cleaved peptide (II) solution (approximately 15 min). The precipitating agent was then added to the cleaved peptide (II) solution and the resulting peptide suspension was stirred for another 15 min.The peptide suspension was then transferred to a G4 pore size sintered frit. The liquid level was marked, and a reduced pressure of 30 kPa (300 mbar) was applied to the frit. After the liquid level dropped to 2 cm (measured from the top of the frit), the filtration time until all the liquid was removed from the frit was recorded. T l 1: D rin Bz- l -Hi Tr -D-Ph -Ar P f -Tr -NPr __________________________________________________________________________ Table 2: Deprotection of TDAC-Dab (Boc) -Val-Dab (Boc) -OH (R-2 and I-11 and I-12) respectively Ac-Lys (Boc) -Val-L s Boc -Val-L s Boc -Val-OH R-3 and I-13

Claims

1. A method for obtaining a fully unprotected peptide, said method comprising the steps of a.) synthesizing a peptide intermediate having at least one acid-unstable protecting group; b.) removing said protecting group or groups using a cleavage reagent composed of an acid and optionally a scrubber and / or an aprotic solvent to form a cleaved peptide solution (I); followed by c.) providing an organic solvent selected from the chloroform, dichloromethane, or ester solvent group of formula (I) wherein R1 and R2 are independently of each other a linear or branched C1-C10 alkyl group or C3-C6 cycloalkyl group to the cleaved peptide solution (I) to form a cleaved peptide solution (II); followed by d.

1. Providing a precipitating agent selected from the group consisting of at least one ether solvent and at least one hydrocarbon solvent, as well as mixtures thereof, in an amount sufficient to precipitate the peptide from the cleaved peptide solution (II), and collecting the precipitate.

2. The method according to claim 1, characterized in that the acid in step b) is selected from the group of formic acid, acetic acid, trifluoroacetic acid, a sulfonic acid, hydrogen bromide, trimethylsilyl trifluoromethanesulfonate, trimethylsilyl bromide, as well as mixtures thereof, preferably acetic acid, trifluoroacetic acid, and methanesulfonic acid, as well as mixtures thereof. 3.The method according to claim 1 or 2, characterized in that the amount of acid is selected from the range of 0.1 to 1000 mol-equivalents with respect to the number of acid-unstable protecting groups present in the peptide intermediate, more preferably from 10 to 500 mol-equivalents, and even more preferably from 20 to 400 mol-equivalents.

4. The method according to any one of the preceding claims, characterized in that the scrubber is selected from the group of phenols, trialkylsilanes, thiols and water, as well as mixtures thereof, preferably triisopropylsilane, dodecanethiol and water, as well as mixtures thereof. 5.The method according to any one of the preceding claims, characterized in that the amount of the scrubber is selected in the range of 0.1 to 500 mol-equivalents with respect to the number of acid-unstable protecting groups present in the peptide intermediate, more preferably from 1 to 100 mol-equivalents, and even more preferably from 2 to 50 mol-equivalents.

6. The method according to any one of the preceding claims, characterized in that the aprotic solvent in the cleavage reagent is selected from the group of liquid aromatic hydrocarbons or C1-6 haloalkanes, as well as mixtures thereof. 7.The method according to any one of the preceding claims, characterized in that the amount of the aprotic solvent is selected from the range of 0.25 to 10 ml / g of peptide intermediate, more preferably from the range of 0.5 to 5 ml / g of peptide intermediate, and most preferably from the range of 0.75 to 1.5 ml / g of peptide intermediate.

8. The method according to any one of the preceding claims, characterized in that the amount of the cleavage reagent is selected from the range of 0.5 to 50 ml / g of peptide intermediate, more preferably from the range of 1 to 50 ml / g of peptide intermediate, and most preferably from the range of 2.5 to 20 ml / g of peptide intermediate.

9. The method according to any one of the preceding claims, characterized in that the amount of the organic solvent in step c.) is selected in the range of 0.5 to 100 ml / g of peptide intermediate, more preferably in the range of 1 to 50 ml / g of peptide intermediate, and most preferably in the range of 5 to 35 ml / g of peptide intermediate.

10. The method according to any one of the preceding claims, characterized in that the ester solvent of formula (I) is selected from the group of ethyl acetate, ethyl propionate, propyl acetate, and diisopropyl acetate, as well as mixtures thereof.

11. The method according to any one of the preceding claims, characterized in that the ether solvent in step d) is selected from the group of cyclopentyl methyl ether, dibutyl ether, diethyl ether, diisopropyl ether, ethyl ferc-butyl ether, methyl ferc-butyl ether, and mixtures thereof, preferably diethyl ether and methyl ferc-butyl ether, and mixtures thereof. 12.The method according to any one of the preceding claims, characterized in that the peptide incorporates from 2 to 20, preferably from 2 to 15, more preferably from 3 to 12, and most preferably from 3 to 6 residues of one or more amino acids.

13. The method according to any one of the preceding claims, characterized in that the peptide is substituted at the α-amino terminal group with a lipophilic moiety selected from the group of acetyl, benzoyl, or tetradecylaminocarbonyl moieties.

14. The method according to any one of the preceding claims, characterized in that the peptide intermediate comprises at least one arginine side-chain protected with Mtr-, Pmc-, or Pbf- and / or at least one lysine side-chain protected with Boc, 2,3-diaminopropionic acid, or 2,4-diaminobutyric acid. 15.The method according to any one of the preceding claims, characterized in that the peptide intermediate is selected from the group of Bz-Gly-His (Trt) -D-Phe-Arg (Pbf) -D-Trp-N (Pr) 2 , Bz-Gly-His-D-Phe-Arg (Pbf) -D-Trp-N (Pr) 2 , Bz-Gly-His-D-Phe-Arg (Boc) 2-D-Trp-N (Pr) 2 , Bz-Gly-His (Trt) -D-Phe-Arg (Boc) 2-D-Trp-N (Pr) 2 , TDAC-Dab (Boc) -Val-Dab (Boc) -OH, TDAC-Dab (Boc) -Val-Dab (Boc) -OfBu, TDAC-Dab (Boc) -Val-Dab (Boc) -O-resin, Ac-Lys (Boc) -Val-Lys (Boc) -Val-Lys (Boc) -Val-OH.