COMPOSITION AND METHOD FOR THE RELEASE OF PEPTIDES PROTECTED FROM A RESIN BACKGROUND OF THE INVENTION The present invention relates to the synthesis of peptides and, in particular, to the synthesis of peptides in solid phase, or a combination of solid phase peptide synthesis. and liquid. Many methods for the synthesis of peptides are described in the literature (for example, see US Pat. No. 6,015,881; Mergler et al., (1988) Tetrahedron Letters 29: 4005-4008; Mergler et al., (1988) Tetrahedron Letters 29: 4009-4012; Kamber et al., (Eds), Peptides, Chemistry and Biology, ESCOM, Leiden (1992) 525-526; Riniker et al., (1993) Tetrahedron Letters 49: 11065-11133; and Andersson et al., (2000) ) Biopolymers 55: 227-250). In the synthesis of solid phase peptides (SPPS), an amino acid group or peptide is bound to a solid support resin. Successive amino acids adhere to the peptide attached to the support until the peptide of interest is formed. After the desired peptide is formed, it is separated from the resin. This requires separating the binding between the peptide and the resin and subsequently recovering the separated peptide using a suitable recovery technique. The amino acids from which the peptides are synthesized tend to have reactive side groups as well as reactive terminal ends. When
Ref. 196301 a peptide is synthesized, it is important that the amino group in one peptide reacts with the carboxyl group in another peptide. Undesirable reactions of side groups or at the wrong terminal end of a reagent produce undesirable byproducts. To minimize side reactions, it is common practice to block the side groups and terminal ends of the reagents to ensure that the desired reaction takes place. For example, a typical solid phase synthesis scheme involves the binding of a first amino acid to the support resin through the carboxyl moiety of the first amino acid (although some synthesis schemes link the first amino acid through the amino group). This allows the amino group of the amino acid attached to the resin to be coupled with another amino acid. Accordingly, the carboxyl moiety of a novel amino acid reacts with the free amino group of the material bound to the resin. To avoid side reactions involving the amino group of the new amino acid, said amino group is blocked with a protecting group during the coupling reaction. Two well-known amino protecting groups are the tert-butyloxycarbonyl group (BOC) and the 9-fluoroenylmethyl carbamate group (FMOC). Many others have also been described in the literature. After coupling, the protecting group (generally BOC or FMOC) can be removed at the N-terminus of the peptide bound to the resin, allowing the binding of additional amino acids to the growing chain in a similar manner. The reactive groups of the side chains of the reactive amino acids and the peptide bound to the resin can also be blocked with side chain protecting groups and remain blocked throughout the synthesis. After synthesis, some or all of the side chain protecting groups of the peptide product can be removed. When virtually all protective groups are removed, this is called global deprotection. The overall deprotection can occur simultaneously with the separation or be carried out later if the peptide is to be further processed, modified, coupled to other peptides or other material, etc. Some separation reagents not only separate the peptide from the resin, but also cause the overall deprotection to take place at the same time. For example, strongly acidic separation reagents associated with the chemical reaction type of BOC tend to cause overall deprotection at the time of separation. However, using the FMOC strategy allows the separation of the peptide from the resin while allowing the side chain protecting groups to remain so that other reactions can take place., like the condensation of fragments, without a significant interference of the groups of the side chains. In this way, the peptide is separated in the protected state. Usually, the yield of a peptide synthesized by solid phase peptide synthesis decreases as the length of the peptide chain increases, i.e., the longer the chain of the peptide, the more likely it is that undesirable side products will be produced together with the desired peptide. Accordingly, for particularly long peptides, the final peptide product is produced in fragments, which are then combined to form the desired peptide product. For example, a hypothetical peptide of 75 amino acids can be synthesized in three peptide fragments, each fragment synthesized separately by synthesis of peptides in solid phase. Fragments consisting of 1 to 25 amino acids, 26 to 50 amino acids and 51 to 75 amino acids can be synthesized separately, and then combined in the fragment condensation step to form the complete 75 amino acid final peptide product. Prior art methods of separating a peptide from the resin support in a protected state usually result in byproducts having carboxylic acids. The carboxylic acids will interfere with the subsequent reaction of fragment condensation, originating undesirable byproducts. The prior art methods solved this problem by including an additional step following the separation to remove the undesirable carboxylic acids, generally by evaporation of the separation solution. This extra step and the necessary solvents consume more time, increase the cost and produce waste that must be eliminated, causing an additional expense. Accordingly, there is a need for a method of separating a peptide from a resin that is easier, cheaper and more effective by evading the production of by-products with undesirable carboxylic acids and, thereby, bypassing the subsequent steps that remove the acids carboxylic BRIEF DESCRIPTION OF THE INVENTION The present invention provides a composition and method for separating a peptide from a solid support resin. Hydrochloric acid is used in an organic solvent miscible with water to separate the peptide-resin bond. Optionally, trifluoroethanol or hexafluoroisopropanol can be added to the separation composition to improve the results. When the separation composition of the present is used, an evaporation step or another step is not necessary to remove the carboxylic byproducts following the separation reaction. After the resin is filtered out of the separation mixture, the peptide can be precipitated immediately with water. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a composition and method for separating a peptide from a solid support resin that dispenses from the need for a subsequent step to remove the carboxylic acids from the separation mixture prior to the condensation of fragments of peptides, as required when using the compositions and methods of the prior art. The present invention can provide a shorter processing time, increases in yield and purity, lower amounts of reagents, starting materials, solvents, debris, as well as other improvements in relation to the synthesis of peptides both small and large scale. The peptides produced according to the present invention can be synthesized by methods well known in the art and the present invention is not limited to any particular synthesis method. Any peptide according to the present invention can be produced. An advantage of the FMOC synthesis strategy is that the synthesized peptide can be separated from the solid support resin in an almost fully protected state, ie, the side chain protecting groups remain in the peptide. This is due to the acid sensitive binding of the peptide to the resin compared to the relatively strong binding of the side chain protecting groups, which requires a stronger acid to eliminate them from the peptide. Accordingly, a relatively low concentration of the acid can be used to separate the peptide from the resin, while allowing the side chain protecting groups to remain, since the acid solution is not strong enough to separate these groups. Usually, the 2-chlorotrityl chloride resin is used to facilitate these advantages, since the bond between the 2-chlorotryl chloride resin and the peptide is relatively sensitive to acids. However, other resins can be used. Although the present invention is described in connection with the peptide synthesis strategy with FMOC, other solid phase peptide synthesis strategies and systems may be employed in combination with the present invention. The strategy with FMOC is simply the preferred way to synthesize the peptides on a large scale. Typically, prior to the present invention, when the desired peptide was synthesized in a solid support resin, the peptide was separated from the resin using a solution of acetic acid (AcOH) or trifluoroacetic acid (TFA) in a solvent such as dichloromethane ( DCM). However, the use of AcOH or TFA to separate the peptide produces carboxylic acids as by-products in the separation mixture. If they are not removed, these carboxylic acids will interfere with subsequent reactions, such as fragment condensation reactions, where two or more peptide fragments are combined. Accordingly, an additional step in the synthesis process is necessary to remove the carboxylic acids. This is generally done by evaporation of the separation mixture followed by reconstitution. This additional step requires more time, solvents, waste, increases spending and can decrease yields and purity. What's more, carboxy acids can not be completely eliminated. Accordingly, there will always be small amounts of residues present, which can decrease the purity of the final peptide product. The present invention practically eliminates the production of the carboxylic acids byproducts and the costly and slow step of the removal of said carboxylic acids which is required in the prior art methods by the use of a reagent and new separation method. In one embodiment, the separation reagent is a relatively low concentration of hydrochloric acid (HC1) in an organic solvent miscible with water. Examples of organic solvents miscible with water are dimethylformamide (DMF), N-methylpyrrolidone (N P), dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF) and dioxane. However, many other organic solvents miscible with water are known in the art. A large range of HCl concentrations will effectively separate a peptide from the resin and all are within the scope of the present invention. However, the best results were achieved with a particular range, preferably between about 0.05 N and about 0.5 N HCl, more preferably about 0.1 N HCl. In another embodiment of the present invention, the separation reagent of the invention further comprises a fluorinated alcohol including but not limited to trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP). The fluorinated alcohol is preferably in the range of about 1% to about 12%, more preferably from about 5% to about 10% and most preferably is about 10% of the separation reagent. The inventors found that between about 2 ml and 22 ml of reagent per gram of resin, preferably between about 4 ml and 10 ml of reagent per gram of resin and more preferably between about 4 ml and 6 ml of reagent per gram of resin are suitable to separate the peptide from the resin. However, other amounts of the separation reagent will likely effectively remove the peptide from the resin. As discussed earliersince virtually no carboxylic acid is produced as a by-product when a peptide is separated using the reagent and the separation method of the present invention, an evaporation step is not necessary to remove the carboxylic acids byproducts. Accordingly, several fragments of intermediate peptides can be formed using the present invention and then combined in a fragment condensation step. One embodiment of the present invention is characterized by the absence of an evaporation step following the separation. After separation, the separation mixture can be filtered to remove the resin and optionally washed with solvent. The filtrate obtained in this way is simply treated with water to precipitate the peptide fragment, which can then be filtered and optionally washed with water. Preferably cold water is used to precipitate the peptide, preferably in the temperature range from about 0 ° C to about 25 ° C, most preferably at about 0 ° C. However, higher water temperatures will also effectively precipitate the peptide from the separation filtrate. The inventors found that at least about 4 ml of water per gram of resin-peptide will effectively precipitate the separated peptide, although other amounts will probably also do so.
Example: The following example briefly describes the synthesis of a peptide using the present invention. Although the example describes the synthesis of enfuvirtide, the principles described can be applied to any peptide, preferably any protected peptide that is synthesized on a labile support against acid such as chlorotryril chloride resin (CTC), a resin Sieber or a resin Rink. Non-limiting examples of said peptides include pramlintide, exenatide, enfuvirtide, calcitonin and PYY-3-36. The following example is simply a preferred method of preparation of enfuvirtide and is not intended to limit the present invention in any way. FMOC-amino acid loading in the CTC resin: 1. General method A solution of FMOC-amino acid (0.8 to 1.5 mols eq.) In DCM or DMF + DCM (4: 1) containing DIEA (1 to 1.7 mols eq) was added. .) to the previously swollen CTC resin (1 mol eq.) and stirred for 2 hours under a stream of nitrogen. It was drained and stirred with MeOH + DIEA mixture (9: 1) for 20 to 30 minutes to destroy the excess active chloride in the resin. The resin was filtered, washed with DMF (1 x 3 min), DCM (1 x 3 min), IPA (2 x 3 min) and dried to a constant weight. The substitution density of the charged amino acid was determined by the weight gain method and the DBU analysis method. 2. Synthesis of fragment 1: AC-AA (1-16) -CTR Synthesis was carried out manually in a 250 ml reactor starting with 17.5 g of FMOC-Gln-CTR (sust = 0.60 mm / g) and using the SPPS based on FMOC. The FMOC group was eliminated with piperidine in 20% NMP (2 x 20 min.) And the coupling of all the FMOC-amino acids was carried out by the HBTU / HOBT method in the presence of DIEA (1.5 eq. Each) in NMP + DCM (3: 1) except for FMOC-Gln (Trt) at position 15, which was carried out using 2.5 mols equivalent of the reagents. All the amino acids were incorporated by means of a single coupling except Asn (trt) 14, Gln (trt) 13 and Ser (tBu) 12 where a double coupling was required. After removal of the FMOC group from the last amino acid, the resin was treated with 5 mols equivalents of acetic anhydride and pyridine in NMP for one hour to incorporate the acetyl group at the N-terminus. The fully protected peptide resin yield was 34.9 g (72.8%) compared to a theoretical yield of 48 g. According to HPLC, the purity of the peptide was > 89.1% (at 262 nm). 3. Synthesis of fragment 2: FMOC-AA (17-26) -CTR Synthesis was started with 25 g of FMOC-Leu-CTR
(sust = 0.8 mm / g) using the coupling method of HBTU / HOBT (1.5 mols eq.). All amino acids (1.5 mols eq.) Were incorporated by a single coupling using NMP + DCM (3: 1) as coupling solvent and DIEA (1.5 mols eq.) As the base. The completion of the coupling was monitored by the Kaiser test. The elimination of the FMOC group was carried out with piperidine in 20% DMC (2 x 20 min). The yield of resin-peptide was protected was 58.7g (93.4%) compared to a theoretical yield of 62.9g. According to HPLC, the purity of the peptide was > 97.1% (at 262 nm). 4. Synthesis of fragment 3: FMOC-AA (27-35) -CTR Synthesis was started with 37.5 g of FMOC-Trp
(BOC) -CTR (sust = 0.7 mm / g) using the coupling method of HBTU / HOBT in NMP + DCM (3: 1) as solvent. All amino acids were coupled by a single coupling except the last amino acid FMOC-Asp (otBu) which was coupled twice (2 x 2 hours) followed by acetylation. A 1.5-fold excess of amino acids and reagents was used for coupling and the termination of the coupling was monitored by the Kaiser method. The resin-peptide yield gone was 73 g (89.3%) compared to a theoretical yield of 81.8 g. According to HPLC, the purity of the peptide was > 80.65% (at 220 nm). 5. Separation of protected fragments from support a. Separation of fragment 1: Ac-AA (1-16) -OH 10.0 g (2 mm) of Ac-AA (1-16) -CTR was stirred with 100 ml of 0.1N HC1 in DMF for 4.5 hours at room temperature, it was filtered and then washed with DMF. The combined filtrate was added to stirring water at 0 ° C and the precipitated solid was filtered, then washed and dried to yield 5.62 g (78.5%) of the protected peptide, compared to a theoretical yield of 7.2 g. According to HPLC, the purity was >; 85.64% (IPA system). When the resin-peptide linkage was cut with 0.1 N HC1 in DMF containing 10% TFE, the yield of the protected peptide was 92% with a purity by HPLC > 96.86% (IPA system). b. Separation of fragment 2: FMOC-AA (17-26) -OH A sample of protected peptide-resin (5 g, 1.7 mm) was stirred with 50 ml of 0.1N HC1 in DMF for 4.5 hours, filtered and then washed with DMF. The filtrate was added to stirring water at 0 ° C and the precipitated solid was filtered, washed with water and dried to yield 2.9 g (74.2%) of the protected peptide. According to HPLC, the purity of the peptide was > 90.1% (ACN system) and > 81% (IPA system). When the separation of the resin-peptide linkage was performed with 0.1 N HC1 in DMF containing 10% TFE, the yield of the peptide was 91.6% and the purity by HPLC was> 0.05. 96.66% (IPA system). c. Separation of fragment 3: FMOC-AA (27-35) -OH A sample of 2.5 g (0.8 mm) of resin-peptide was stirred with 25 ml of 0.1 N HC1 in DMF including 10% TFE at room temperature for 4.5 hours and it leaked. The filtrate was added to stirring water at 0 ° C and the solid obtained was collected by filtration followed by washing with water. After drying overnight, yielded 72.4% (1.28 g) of the desired peptide with an HPLC purity of 84%. When the separation of the resin-peptide bond was made with 0.1 N HC1 in D F containing 5% TFE, the yield of the protected peptide was 67.9 g (1.2 g) and the purity was 88.2%. 6. Synthesis of enfuvirtide protected by condensation of fragments in solution a. Coupling fragment 3 with Phe-NH2 within FMOC-AA (27-36) -NH2 A mixture of fragment 3 (2.62 g, 1 eq.), Phe-NH2 (0.24 g, 1.2 eq.) And HOAT ( 0.2 g, 1.2 eq.) With 30 ml of DMF in the presence of DIEA (0.43 ml, 2.1 eq.) And the solution treated with HBTU (0.55 g, 1.2 eq.) At 0 ° C for 15 to 20 minutes and then at room temperature for 70 to 80 minutes. The progress of the reaction was monitored by TLC (CM-10) and HPLC. The reaction mixture was cooled to 0 ° C and treated with 20 to 30 ml of water, the separated colorless solid was filtered, then washed with water and dried to yield 2.76 g (98.6%) of FMOC-AA ( 27-36) -NH2. According to HPLC, the purity of the peptide was > 88.15%. The experiment was repeated several times and the yields obtained varied between 97% and 100% with a purity between 82.6% and 88.2%. b. Deprotection of FMOC-AA (27-36) -NH2 within H-AA (27-36) -NH2 fragment 4 A solution of FMOC-AA (27-36) -NH2 (1.16 g, 0.5 mm) was stirred in 5 ml. ml of piperidine in 5% DMA for 2 hours at room temperature and then diluted with stirring with 15 ml of water at 0 ° C. The separated colorless solid was filtered, then washed with water and dried. It was washed with ether and hexane (once each) to yield 0.91 g (86.7%) of the product with an HPLC purity of 85.85% (ACN system). c. Coupling of fragments 2 and 4 within F-AA (17-36) -NH2 [A] Method of HBTU / HOAT A solution of fragment 2 (1.80 g, 0.8 mm, 1 eq.), Fragment 4 (1.68) was stirred g = 0.8 mm), HBTU (0.3 g, 0.8 mm) and HOAT (0.16 g, 1.2 mm, 1.5 eq.) in 23 ml of DMF containing DIEA (0.2 ml, 1.2 mm) at 0-5 o for 15 to 20 minutes and at room temperature for 2 hours, and the progress of the reaction was monitored by TLC in CMA (90: 8: 2) and HPLC. It was treated with 23 ml of cold water at 0-5 ° C and after stirring for 30 minutes it was filtered, then washed with water and dried to yield 3.53 g (101.2%) of the product with a purity of 79.4%. After crystallization with 95% IPA / H20, the yield was 74.5% and the purity was 89.96%. It was contaminated with small amounts of fragment 2 (0.24%) and fragment 4 (0.15%) (IPA system). [B] TBTU / HOAT method The coupling reaction was performed in the DMA solvent using TBTU in the same proportion of mols mentioned above and the yield was 3.56 g (101.95%) with a purity of 70.2%. After crystallization with 95% IPA / H20, the yield was 74.5% (2.6 g) with an HPLC purity of 88.4% (IPA system). It was deblocked with piperidine (10 eq.) In DMA and isolated with water for a yield of 93.3% of the product with an HPLC purity of 85.3% (IPA system). d. Coupling fragment 1 with H-AA (17-36) -NH2 within protected enfuvirtide. Fragment 1 (0.4 g, 1 eq.), HOAT (0.03 g, 1.5 eq.) And DIEA (0.03 ml, 1.5 eq.) In 8 ml of DMA were added to obtain a clear solution and then stirred at 0.5 ° C. . TBTU (0.04 g, 1 eq.) Was added and the solution was stirred at 0 ° C for 15 to 20 minutes and then a solution of H-AA (17-36) -NH2 (0.5 g, 1 eq.) Was added. in DMA and stirring was continued at 0 ° C for 30 minutes and then at room temperature for 2 hours. Cold water (~ 15 mL) was added at 0 ° C and the separated solid was filtered, then washed with water and dried to yield protected 97.5% (0.9 g) enfuvirtide yield with HPLC purity >; 66.93% (IPA system). After crystallization with 95% ACN / H20, the yield was 55.3% and the purity of the peptide was > 72.8%. (Reaction scheme 1) 7. In situ coupling of the fragments a. Release of F-AA (27-35) -OH from its CTC resin 5.0 g (1.62 mm) of FMOC-AA (27-35) -CTR was stirred with 25 ml of 0.1N HC1 in DMF for 4 hours, filtered and then washed once with 10 ml of DMF. Total volume of FMOC-AA (26-35) -OH in DMF = 35 ml (1.62 mm, of course). b. Preparation of FMOC-AA (27-36) -NH2? H. A (27-36-NH2) The above solution (from step No. 1) was stirred at 0 ° C and neutralized with DIEA until pH ~ 7. A 1.2-fold excess of Phe-NH2 (0.32 g, 1.94 mm) was added. ), HOAT (0.26 g), HBTU (0.74 g) and a 2.1-fold excess of DIEA (0.6 ml, 3.4 mm) and the mixture was stirred at 0 ° C for 0.5 hours and at room temperature for 2 hours. the reaction was monitored by TLC (CM-10) At that time, DBU (10 eq.) was added and stirring was continued for another 2 hours and the progress of the deblocking was monitored by TLC (CM-10) and HPLC. Release of FMOC-AA (17-26) from the support and its coupling with H-AA (27-36) -NH2 within FMOC-AA (17-36) -NH2? H.AA (17-36) -NH2 4.7 g (1.62 mm) of FMOC-AA (17-26) -CTR was stirred with 25 ml of 0.1N HC1 in DMF for 4 hours, filtered and then washed with 10 ml of DMF (total volume = 35) and the solution was stirred at 0 [deg.] C. It was treated with a solution of H-AA (27-36) -NH2 (step No. 2) and the pH of the mixture used to 7. HOAT, HBTU (1 mol eq. each) and DIEA (1.8 mols eq.) and the mixture was stirred at 0 ° C for 0.5 hours and at room temperature between 2 hours and overnight. The progress of the reaction was monitored by TLC (C-10) and HPLC. The mixture was then treated with DBU (10 eq.) For 2 hours to remove the N-terminal FMOC group (mixing volume = 70 ml). It was neutralized at pH ~7 at 0 ° C for use in the next reaction. d. Release of Ac-AA (1-16) -OH from the support and its coupling with H-AA (17-36) -NH2 within AC-AA (1-36) -NH2 8.3 g (1.62 mm) of AC was separated -AA (1-16) -CTR with
40 ml of HC1 0.1 N in DMF for 4 hours as described above and the pH of the filtered solution at 0 ° C was adjusted to 7. Then it was treated with HOAT (1.5 eq.), DIEA (1.5 eq.) And HBTU (1 eq.) In order and after stirring at 0 ° C for 15 to 20 minutes, the unblocked solution was added (step N ° 3 ) and stirring was continued for 0.5 hours at 0 ° C and between 2 hours and overnight at room temperature. At that time it was added to water in agitation (-300 mi) while precipitating out a solid. After stirring for one hour, the solid was filtered, then washed with water and dried. The dried solid was washed with hexane to yield 10.9 g (91.2%) of the product. According to HPLC, the purity of the product was only 32.71% and contained approximately 26% of unreacted fragment 1. Accordingly, it was reacted again using 50% of the amount of H-AA (17-36) -NH2, HOAT, DIEA and
HBTU and processed in the usual manner to produce 13.2 g (110%) of the product. According to HPLC, the purity of the peptide was 45.7%. When the stirring solution at 0 ° C of the fragment
1 isolated (0.98 g, 0.3 mm) in DMF was condensed with DBU to which the FMOC had been removed and a neutralized solution of FMOC-AA (17-36) -NH2 isolated (1.31 g, 0.3 mm) in DMF in the presence of 1.5 mols equivalent of HOAT, DIEA and 1 mol equivalent of HBTU, the isolated yield of the product was 91.9% (2 g) with a purity by HPLC > 62.1%. Similarly, when the isolated H- (17-36) -NH2 was coupled to fragment 1 without isolating, the yield and purity of the peptide were 95.7% and 33.6%, respectively. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.