HPLC FREE PURIFICATION OF PEPTIDES BY THE USE OF NEW CAPPING AND CAPTURE REAGENTS

Abstract

The present disclosure relates to the use of a capping and capture reagent in solid phase peptide synthesis. The present disclosure further relates to a method of solid phase peptide synthesis, wherein a capping and capture reagent according to the present disclosure is used. The present disclosure further relates to a method for purification of a (full-length) synthetic peptide via use of a capping and capture reagent according to the present disclosure. The present disclosure also relates to a kit comprising a capping and capture reagent according to the present disclosure and an amino oxy resin or a hydrazine resin and the use of the kit.

Claims

1. Use of a capping and capture (cap-capt) reagent comprising a capping moiety and a capture moiety in solid phase peptide synthesis, wherein said capping moiety is a carboxylic acid or an activated carboxylic acid and wherein said capture moiety is a protected carbonyl group.

2. Use of a capping and capture reagent according to claim 1, wherein the capping and capture (cap-capt) reagent comprises one capping moiety and one capture moiety.

3. Use of a capping and capture reagent according to claim 1, wherein the capping and capture reagent has Formula (I): ##STR00007## wherein R1 is hydrogen or an optionally substituted alkyl, R2 is OR5 or a halogen, each R3 and R4 is independently an optionally substituted alkyl, or form together with the oxygen atoms to which they are attached an optionally substituted 5-membered or 6-membered ring, R5 is selected from H, anhydride, succinimide, benzotriazole, pentafluorphenyl, and ##STR00008## and n is independently 0, 1, 2, 3 or 4.

4. Use of a capping and capture reagent according to claim 1, wherein the capping and capture reagent is selected from the group consisting of: ##STR00009## wherein R6, R7, R8 and R9 are each independently selected from hydrogen and alkyl and R10 is optionally substituted alkyl.

5. Use of a capping and capture reagent according to claim 4, wherein R6, R7, R8 and R9 are each independently selected from hydrogen and C.sub.1-C.sub.3 alkyl, and R10 is methyl or ethyl.

6. Use of a capping and capture reagent according to claim 1, wherein R1 is independently hydrogen, methyl or ethyl.

7. Use of a capping and capture reagent according to claim 1, wherein R2 is independently selected from the group consisting of O-succinimide, O-pentafluorophenyl, O-benzotriazole, anhydride and ##STR00010##

8. Use of a capping and capture reagent according to claim 1, wherein the capping and capture reagent is selected from ##STR00011##

9. A method for solid phase peptide synthesis (SPPS) wherein individual amino acids are coupled to each other via Fmoc-chemistry and, wherein after at least one coupling step a capping and capture reagent according to claim 1 is used for capping a failure peptide sequence.

10. Method according to claim 9, wherein the capping moiety of the capping and capture reagent is a carboxylic acid, further comprising the step of: activating the capping moiety, thus preparing an activated carboxylic acid with an activating agent, wherein the activating agent corresponds to the activating agent of Fmoc-chemistry.

11. A method for purification of a (full-length) synthetic peptide comprising the steps of: performing a solid phase peptide synthesis according to claim 9, cleaving the compounds thus synthesized from the solid phase, deprotecting the capture moiety, and binding the deprotected capture moiety to a solid resin support thereby binding the at least one failure peptide sequence to the solid resin support and separating the full-length synthetic peptide from the solid resin support comprising the failure peptide.

12. Method according to claim 11, wherein the solid resin support is an amino oxy resin, or hydrazine resin.

13. Method according to claim 11, wherein the separating of the full-length peptide from the solid resin support comprising the failure peptide is carried out by centrifugation or by filtration.

14. Kit comprising at least one capping and capture reagent according to claim 1 and at least one amino oxy resin, or hydrazine resin.

15. Use of a kit according to claim 14 for purification of compounds synthesized on a solid support, preferably for the purification of peptides.

Description

FIGURES

[0076] FIG. 1 illustrates the general reaction scheme for the synthesis of capping and capture reagents 1, or (1′), respectively, and 2 as well as of amino-oxy PEGA resin 3. Ethyl 2,2-diethoxyacetate and ethyl-2-methyl-1,3-dioxolane-2-acetate (fructone) were chosen as starting point for the synthesis of an aldehyde and a ketone containing capping moiety: the esters were hydrolyzed with NaOH (step a) and subsequently transformed to the corresponding symmetric anhydrides by treatment with dicyclohexylcarbodiimide (DCC) (step b).

[0077] FIG. 2 illustrates the capping and deprotection with reagents 1 and 2. A short peptide (WEGSKYA) was synthesized by standard Fmoc-based SPPS. After 5 couplings part of the resin was removed from the vessel, and treated with reagent 1 or reagent 2 (in DMF with a stoichiometric amount of lutidine). To deprotect and cleave these peptides from the resin a cleavage cocktail containing TFA, 5% anisole, 5% thioanisole and 5% water was used, because the use of triisopropylsilane has been described to cause reduction of aldehydes to alcohol. After 2 h treatment with the cleavage cocktail, the peptides were precipitated with cold diisopropyl ether and analysed by LCMS. This revealed that the capping with both reagents had occurred quantitatively, but while reagent 2 had been deprotected smoothly to yield the keto-capped peptide 5, reagent 1 was still quantitatively protected as acetal. The crude mixture was redissolved again in TFA and after 24 h at room temperature deprotection was finally observed.

[0078] FIG. 3 illustrates the capping of the peptide sequence with a cap-capt reagent according to the present invention.

[0079] FIG. 4 illustrates two HPLC chromatograms. FIG. 4a illustrates an HPLC chromatogram of a mixture of WEGSKYA (SEQ ID No. 1) 6 and keto-capped-GSKYA (SEQ ID No. 2) 5. FIG. 4b illustrates an HPLC chromatogram after 2 h incubation with amino-oxy resin 3.

[0080] FIG. 5 illustrates two HPLC chromatograms. FIG. 5a illustrates an HPLC chromatogram at 214 nm of a crude mixture of Angiotensin I and termination sequences after SPPS. FIG. 5b illustrates an HPLC chromatogram at 214 nm of Angiotensin I after incubation with amino-oxy resin 3. As model system the biological 10mer Angiotensin I (DRVYIHPFHL) (SEQ ID No. 3) was synthesized. To stress the conditions, single couplings with only one equivalent of amino acid per step were made. LCMS analysis of the crude material revealed a major termination after proline and further terminations after valine and tyrosine. All of them were quantitatively capped with reagent 2 (M+84) (FIG. 5a). The mixture was dissolved in acetate buffer at pH=4.5 and incubated on the amino oxy resin 3 for 2 h, obtaining full-length Angiotensin I (FIG. 5b) in good purity. Noteworthy is the complete removal of the keto-capped truncation Cap-PFHL (SEQ ID No. 4), which co-elutes with the main product.

[0081] FIG. 6 illustrates four HPLC chromatograms. As model peptide the 28mer des-octanoyl ghrelin (GSSFLSPEHQRVQQRKESKKPPAKLQPR) (SEQ ID No. 5) was synthesized in a 20 μmol scale. After SPPS, crude des-octanoyl ghrelin was obtained with a purity of 72%. The crude product was dissolved in acetate buffer (pH=4.5, 0.1 M) and analyzed by HPLC-MS (FIG. 6a). The crude product was divided in two equal aliquots. The first was purified by preparative HPLC (FIG. 6b), the second was incubated for two hours on the amino-oxy resin 3 (FIG. 6c). The HPLC purification gave a purity of 99% and yield (respective to the initial resin loading) of 25%. The strategy with the cap-capt reagent gave a purity of 92% and yield of 48% (FIG. 6c). FIG. 6d illustrates the HPLC chromatogram when the product is subjected to a preparative HPLC.

[0082] FIG. 7 is a schematic illustration of the use of a capping and capture reagent for HPLC free purification of peptides. During the acidic cleavage and deprotection of the peptides the acetal containing capping moiety is transformed into a ketone, which can be chemoselectively captured by an amino-oxy resin. The full length peptide remains in solution as pure product.

EXAMPLES

Example 1

[0083] General procedure for the ester hydrolysis (see also FIG. 1) (see Tartaggia, S.; Fogal, S.; Motterle, R.; Ferrari, C.; Pontini, M.; Aureli, R.; De Lucchi, O., Chemoenzymatic Synthesis of δ-Keto β-Hydroxy Esters as Useful Intermediates for Preparing Statins. European Journal of Organic Chemistry 2016, 2016 (19), 3162-3165.): To a solution of methyl 2,2-dimethoxyacetate, ethyl 2,2-diethoxyacetate, or fructone (75 mmol) in EtOH (100 mL), respectively, was added NaOH (2 M aqueous solution, 50 mL). After stirring the reaction at r.t. for 4 h, EtOH was removed under reduced pressure. The aqueous phase was then washed with diisopropyl ether (2×20 mL), acidified with 2M HCl and extracted with EtOAc (3×100 mL). The combined organic fractions were dried over Na.sub.2SO.sub.4 and the solvent was removed under reduced pressure affording the desired product as slightly yellow oils (2,2-dimethoxyacetic acid (1a′); 2,2-diethoxyacetic acid (1a): 85%; 2a: 79%, respectively). I.e., compound 1 was obtained in a yield of 85% and compound 2 was obtained with a yield of 79%. This means that no chromatographic purification, but only an extraction in the first step and a filtration in the second step were necessary to obtain the capping reagents in good yield and purity. Scale up to up to 100 g anhydride in a single synthesis run was easily feasible. As basis for the capture resin a PEG based polymer (AminoPEGA® resin) was used, suitable for working with both organic and aqueous solvents. Boc-amino-oxy-acetic acid was installed on the resin (step c) and deprotected with TFA (step d) (see also FIG. 1).

[0084] The products were used for the next step without further purification.

[0085] General procedure for the anhydride synthesis (see also FIG. 1): To a solution of 2,2-dimethoxyacetic acid (1a′), 2,2-diethoxyacetic acid (1a) or 2-(2-methyl-1,3-dioxolan-2-yl)acetic acid (2a) (20 mmol) in CH.sub.2Cl.sub.2 at 0° C. was slowly added N, N′-dicyclohexylcarbodiimide (10 mmol). The reaction was allowed to warm up to room temperature under vigorous stirring. After 3 h the reaction was filtrated, the solid removed and the solvent of the filtrate removed under reduced pressure affording the two anhydrides, respectively, in quantitative yield as slightly yellow resins, which were used without further purification.

[0086] 2,2-dimethoxyacetic anhydride (1′):

[0087] .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 3.45 (s, 12H) 4.93 (s, 2H) 2,2-diethoxyacetic anhydride (1):

[0088] .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.10 (t, 12H) 3.50 (q, 8H) 4.93 (s, 2H) 2-(2-methyl-1,3-dioxolan-2-yl)acetic anhydride (2):

[0089] .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.50 (s, 6H) 2.82 (s, 4H) 3.98 (s, 8H)

[0090] Synthesis of the amino-oxy PEGA resin (3) (see also FIG. 1): Amino-PEGA-resin (Novabiochem, 1.00 g, loading 0.37 mmol/g) was washed with DMF (5×10 mL). Boc-amino-oxy-acetic acid (212 mg, 1.11 mmol), HATU (380 mg, 1.00 mmol) and DIPEA (383 μL, 2.2 mmol) were mixed in DMF (10 mL) and added to the resin. After 30 minutes shaking at room temperature the resin was washed with DMF, and a Kaiser test confirmed the absence of residual amino groups. Treatment of the resin with TFA+2% H.sub.2O for 1 h and subsequent washing with acetate buffer (0.1 M, pH=4.5) afforded the desired amino oxy resin.

Example 2

[0091] Peptide synthesis: Automated Fmoc based solid-phase peptide synthesis was performed by using a Syro I synthesizer and Rink-Amide Tentagel resin (loading 0.19 mmol/g). Fmoc deprotection was performed by treatment of the resin with 20% piperidine in DMF, 2×5 minutes. The successive Fmoc-protected amino acid (0.25 M in NMP+HOAt 0.25 M) was coupled on the resin using HCTU (0.8 M) and N-methyl morpholine (NMM, 3 M in DMF) for 15 min at room temperature. For each coupling, if not otherwise stated 5 equivalents amino acid (respectively to the resin loading) were used and the coupling repeated twice. Capping of residual free amino functions was performed by treatment of the resin with 2-(2-methyl-1,3-dioxolan-2-yl)acetic anhydride (2) and lutidine (both 0.5 M in DMF; 20 equivalents, respectively, to the resin loading). Cleavage from the resin was performed using TFA/thioanisole/anisole/H.sub.2O (85:5:5:5). The resin was incubated for 30 minutes with this cocktail and then the cleavage solution was kept for 1-2 hours at room temperature. After reduction of the volume of the solution under nitrogen stream, crude peptides were obtained by precipitation with diisopropyl ether and centrifugation.

Example 3

[0092] The peptide sequence H-SKSYS-Resin (SEQ ID No. 6) (10 μmol) was synthesized according to the standard method described above (but using acetic anhydride instead of 2 for capping). The resin was divided in 2 equal parts (each 5 μmol). The first aliquot was treated with a solution of 2 (0.5 M in DMF, 0.5 mL) and NMM (N-methylmorpholine) (3 M in DMF, 100 μL) for 15 minutes. The second aliquot (Aliquot 2) was treated with a solution of 2a (0.5 M in DMF, 0.5 mL), NMM (3 M in DMF, 100 μL) and HCTU (2-(6-Chlor-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium-hexafluorophosphat) (0.8 M in DMF, 250 μL) for 15 minutes. Cleavage from the resin was performed using TFA/thioanisole/anisole/H.sub.2O (85:5:5:5). Both capping methods showed full capping of the terminal amino group. (FIG. 3).

[0093] MS (ESI): 654.28 [M+H].sup.+

Example 4

[0094] The peptide sequence H-GSKYA-Resin (SEQ ID No. 7) (15 μmol) was synthesized according to the standard method described above (but using acetic anhydride instead of 2 for capping). As illustrated in FIG. 2, the resin was divided in 3 equal parts (each 5 μmol). The first aliquot (Aliquot 1) was treated with a solution of 1 (50 μmol) and 2,6-lutidine (50 μmol) in DMF (1 mL) for 15 minutes. The second aliquot (Aliquot 2) was treated with a solution of 2 (50 μmol) and 2,6-lutidine (50 μmol) in DMF (1 mL) for 15 minutes. Cleavage from the resin was performed using TFA/thioanisole/anisole/H.sub.2O (85:5:5:5). Both Aliquot 1 and Aliquot 2 showed full capping of the terminal amino group. While the acetal of Aliquot 2 was completely deprotected generating the corresponding ketone, the acetal protection of Aliquot 1 was still intact after the cleavage procedure. The remaining aliquot (Aliquot 3) was subjected to two further solid phase synthesis steps, with following cleavage from the resin, generating peptide H-WEGSKYA.

Example 5

[0095] Keto capped GSKYA (SEQ ID No. 2) (5) (1 μmol) and H-WEGSKYA (SEQ ID No. 1) (6) (1 μmol) were dissolved in acetate buffer (0.1 M, pH=4.5) and analyzed by HPLC-MS (FIG. 4a). Amino-oxy PEGA resin (3) was added to the mixture and after 2 h the supernatant was analysed by HPLC-MS, showing only 6 left in solution (FIG. 4b).

Example 6

[0096] Angiotensin I was synthesized according to the procedure described above with a modification in the amount of amino acid used for the couplings: instead of 2×5 equivalents Fmoc-amino acid, a single coupling with 1 equivalent amino acid was performed. The crude product was dissolved in acetate buffer (0.1 M, pH=4.5) and analyzed by HPLC-MS (FIG. 5a). Amino-oxy PEGA resin (3) was added to the mixture and after 2 h the supernatant was analysed by HPLC-MS, showing the successful removal of most impurities (FIG. 5 b).

Example 7

[0097] Des-octanoyl ghrelin (GSSFLSPEHQRVQQRKESKKPPAKLQPR) (SEQ ID No. 5) (20 μmol) was synthesized according to the general peptide synthesis procedure described above. The crude product was dissolved in acetate buffer (0.1 M, pH=4.5) and analyzed by HPLC-MS (FIG. 6a), and resulted to contain 72% of the desired product. The mixture was divided in 2 aliquots. The first was purified by preparative HPLC, affording the desired product in 99% purity and 25% yield (FIG. 6b). The second aliquot was incubated for 2 h with the Amino-oxy PEGA resin (3). The desired product was isolated in 92% purity and 48% yield (FIG. 6c). Subjecting this product to a preparative HPLC chromatography the product could be isolated with a final purity of 99% and overall 30% yield (FIG. 6d).

TABLE-US-00001 purification with a cap- HPLC capt reagent according to purification the present invention purity 99% 92% yield 25% 48% solvent 1200 mL 10 mL parallel purifications not possible possible