Solid phase synthesis of acylated peptides

Abstract

The present invention relates to methods and compounds for the solid phase synthesis of peptides carrying a substituent at an amino group of an amino acid side chain.

Claims

1. A method for the full chemical synthesis of a semaglutide peptide, the method involving: a) providing a first solid-phase bound peptide sequence of the formula (i) ##STR00010## wherein: X is an amino protecting group R.sub.1 or is an amide bonded 8-amino-3,6-dioxa-octanoic acid moiety having an R.sub.1 protected amino group (AEEAc-R.sub.1); B is a solid phase functionalized with any suitable linker structure L; and PG1 through PG5 independently are absent or are side-chain protecting groups, which may be the same as or different from any of each other; b) removing the protecting group R.sub.1 so as to expose a free amino group; c) acylating said free amino group so as to obtain a second solid-phase bound peptide sequence of the formula (ii), wherein R.sub.5 and R.sub.6 are the same or different and are H or a carboxyl protecting group moiety; ##STR00011## d) removing the Alloc protecting group from the N-terminus of the solid phase bound peptide of formula (ii), so as to expose a free N-terminal alpha amino group; e) coupling, by stepwise solid-phase peptide synthesis, the amino acid sequence R.sub.7-His(PG19)-Aib-Glu(PG18)-Gly-Thr(PG17)-Phe(PG16)-Thr(PG15)-Ser(PG14)-Asp(PG13)-Val-Ser(PG12)-Ser(PG11)-Tyr(PG10)-Leu-Glu(PG9)-Gly-Gln(PG8)-Ala-Ala- to said free N-terminal alpha amino group so as to obtain a solid-phase bound semaglutide peptide, wherein: PG8 to PG19 independently are absent or are side-chain protecting groups, which may be the same as or different from any of each other, any of PG1 to PG7, or both; and R.sub.7 is H or an amino protecting group, which may be the same as or different from R.sub.1; and f) cleaving the semaglutide peptide from the solid phase.

2. The method according to claim 1, wherein step d) involves incubation of the solid-support bound peptide obtained in step c) with a solution comprising [Pd(PPh.sub.3).sub.4] and one or more scavengers.

3. The method according to claim 2, wherein the scavengers are selected from the group consisting of morpholine, dimethylamine borane-complex, and phenylsilane.

4. The method according to claim 1, further wherein at least one of step a), step c), or steps a) and c) involve(s) stepwise solid phase peptide synthesis.

5. The method according to claim 1, wherein each of steps a), c) and e) involves solid phase peptide synthesis.

6. The method according to claim 1, wherein solid phase peptide synthesis is performed using building blocks, which comprise a free carboxyl group or an activated ester of a carboxyl group, an R.sub.1 protected amino group, and optionally further protecting groups, and wherein the steps of: i) coupling a building block, which comprises an R.sub.1 protected amino group, to a free amino group generated in a previous step; and ii) removal of R.sub.1 to expose a free amino group are carried out in repeated cycles.

7. The method according to claim 1, wherein R.sub.1 is a 9-fluorenyl-methoxycarbonyl (Fmoc) group.

8. The method according to claim 7, wherein step b) is performed by incubating the compound of formula (i) with a mixture selected from the group consisting of 5-50% (v/v) piperidine or 4-methyl piperidine in N,N-dimethylformamide (DMF), 5-50% (v/v) piperidine or 4-methyl piperidine in N-methylpyrrolidone (NMP), 1-5% (v/v) 3,5 diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF, and 50% (v/v) morpholine in DMF.

9. The method according to claim 7, wherein step c) is performed by stepwise solid-phase synthesis using Fmoc-AEEAc-OH, Fmoc-Glu-OtBu, and octadecanedioic acid or octadecanedioic acid mono-t-butyl ester as building blocks.

10. The method according to claim 7, wherein each building block used in step a), step e) or steps a) and e) is selected independently from the group consisting of: 1) derivatives of alpha-amino acids, wherein the alpha amino group is protected by Fmoc, optionally further wherein the amino acid side chain carries a protecting group; 2) Alloc-Lys(Fmoc)-OH; 3) dipeptide derivatives, which comprise an Fmoc or Boc protected amino group, optionally further wherein at least one amino acid side chain carries a protecting group; and 4) a combination of two or all of 1) to 3).

11. The method according to claim 1, wherein one or more R.sub.1-protected pseudoproline dipeptide derivative(s) is/are used in step e).

12. The method according to claim 11, wherein the R.sub.1-protected pseudoproline dipeptide derivative(s) is/are selected from Fmoc-Gly-Thr(Psi(Me,Me)pro)-OH, Fmoc-Thr(tBu)-Ser(Psi(Me,Me)pro)-OH, Fmoc-Val-Ser(Psi(Me,Me)pro)-OH, Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH, and Fmoc-Phe-Thr(Psi(Me,Me)pro)-OH.

13. The method according to claim 1, wherein all of steps a), d), and e) are carried out by means of Fmoc solid-phase synthesis.

14. The method according to claim 1, wherein at least one of steps a), c), and e) comprises activating the building blocks used by means of one or more coupling reagent mixtures comprising reagents selected for each coupling reaction independently from the group consisting of: (A) (benzotriazolyl)tetramethyluronium tetrafluoroborate (TBTU) plus diisopropylethylamine (DIPEA); (B) diisopropylcarbodiimide (DIC) plus cyano-hydroxyimino-acetic acid ethyl ester (Oxyma); (C) 3-(diethoxy-phosphoryloxy)-3H-benzo[d][1,2,3]triazin-4-one (DEPBT) plus DIPEA; (D) DIC plus hydroxybenzotriazole (HOBt); (E) N-[(7-Aza-1H-benzotriazol-1-yl) (dimethylamino)-methylene]-N-methylmethanaminium tetrafluoroborate N-oxide (TATU) plus DIPEA; (F) 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) plus DIPEA; and (G) N-[(7-Aza-1H-benzotriazol-1-yl) (dimethylamino)-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) plus DIPEA.

15. The method according to claim 1, further wherein protecting groups are cleaved from the semaglutide peptide.

16. The method according to claim 1, wherein all side chain protecting groups, protecting groups bound to the alpha carboxyl group or both of the building blocks used are orthogonal to R.sub.1 and Alloc.

17. The method according to claim 1, wherein the side chain protecting groups, protecting groups bound to the alpha carboxyl group or both of the building blocks used are independently selected from the group consisting of 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), 2,3,6-trimethyl-4-methoxybenzenesulfonyl (Mtr), t-butyl (tBu), trityl (Trt), 4-methoxytrityl (Mmt), 4-methyltrityl (Mtt), t-butyl ester (OtBu), 3-methylpent-3-yl ester (OMpe), 2-phenyl isopropyl (OPp), t-butoxycarbonyl (Boc), 2-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) isovaleryl (ivDe).

Description

EXAMPLES

(1) General Methods

(2) Analytical HPLC

(3) Samples were analyzed on a C18 stationary phase using a mobile phase comprising 0.05% TFA (v/v) and acetonitrile. The acetonitrile gradient was from about 30% to about 60% (v/v) within 30 min. This gradient was designed to resolve impurities with elution characteristics similar to the product, which may become challenging during work-up of the crude peptide.

(4) Solid Phase Peptide Synthesis (SPPS) of the Peptide Backbone

(5) The synthesis was performed on preloaded H-Gly-2-chlorotrityl resin using a fully automated peptide synthesizer. Coupling reactions were carried out applying standard Fmoc-amino acid derivatives and Fmoc-dipeptide derivatives with DIC/Oxyma or TBTU/DIPEA as coupling reagents/additives in DMF as solvent. Fmoc-deprotection was carried out using a solution of 20% piperidine in DMF. DMF and IPA were used as solvents for washing steps. After each coupling, an acetylation step was performed routinely. The peptide was cleaved from the resin by incubation with TFA/H2O/TIPS (90:5:5, v/v/v) or TFA/EDT/water (90:5:5, v/v/v) separated from the resin by filtration, and precipitated on cold IPE.

(6) Removal of Alloc Protecting Group

(7) The Alloc-deprotection was performed for 30 to 360 min at room temperature in a manual SPPS reactor using 12.0-18.0 eq. dimethylamine borane-complex and/or 4.0-6.0 eq. morpholine as scavenger(s) with 0.02 eq.-1.15 eq. [Pd(PPh.sub.3).sub.4] as catalyst and DCM as solvent under N.sub.2 atmosphere. Washing steps included MeOH, DMF and IPA. Alternatively, 15-20 eq. phenylsilane may be used as a scavenger with [Pd(PPh.sub.3).sub.4] as catalyst and DCM as solvent under N.sub.2 atmosphere.

(8) Acylation of Side Chain Amino Groups

(9) Amino acid or peptide side chain amino groups were acylated using one of the two protocols below.

(10) Protocol a):

(11) After deprotection of the side chain amino group of the peptide resin, stepwise manual or automated SPPS was carried out using Fmoc-AEEAc-OH, Fmoc-Glu-OtBu, and either octadecanedioic acid or octadecanedioic acid mono-t-butyl ester. DIC/Oxyma or TBTU/DIPEA coupling chemistry was employed and the coupling time was 18-22 h or 2-16 h.

(12) After each coupling, an acetylation step was performed routinely. Fmoc was cleaved by incubation with 20% piperidine in DMF, followed by extensive washes with DMF and/or IPA.

(13) Protocol b):

(14) The protected side chain sequence fragment was synthesized by manual SPPS on a pre-loaded H-AEEAc-2-chlorotrityl resin, using essentially the same conditions as set out for protocol a). The fragment, e.g. mono-tBu-carboxyheptadecanoyl-γ-Glu(OtBu)-AEEAc-AEEAc-OH, was then cleaved using HFIP in DCM and concentrated before coupling using EDC, HOSu, and DIPEA in DMF. Alternatively, DIC/Oxyma or TBTU/DIPEA may be used as coupling reagents.

(15) Preparative HPLC

(16) The crude peptide was purified by two dimensional RP-HPLC using a C8 hydrocarbon bonded silica (10 micrometer particle diameter, pore size 100 Å). 0.1% H.sub.3PO.sub.4 aq. as eluent system containing ACN as organic modifier was used as a mobile phase in the first dimension, 0.1% TFA aq. containing ACN as organic modifier was used in the second dimension.

Example 1: Full Chemical Synthesis of Semaglutide

(17) SPPS of the semaglutide backbone sequence was performed on preloaded H-Gly-2-chlorotrityl resin using a fully automated peptide synthesizer. Coupling reactions were carried out applying Fmoc-AA derivatives (including Fmoc-Lys(Alloc)-OH) or Fmoc-dipeptide derivatives and DIC/Oxyma or TBTU/DIPEA as coupling reagents/additives in DMF as solvent. Fmoc-deprotection was carried out using a solution of 20% piperidine in DMF. DMF and IPA were used as solvents for washing steps. After each coupling, an acetylation step was performed. The SPPS was carried out on 30 mmol scale. Crude material obtained from a test cleavage showed 45.4% U-HPLC purity.

(18) Alloc-deprotection was performed as outlined in the general methods above for 30 min at room temperature in a manual SPPS reactor using morpholine and dimethylamine borane-complex as scavengers with [Pd(PPh.sub.3).sub.4] as catalyst and DCM as solvent under N.sub.2 atmosphere.

(19) The side chain amino group was acylated according to protocol a) above. Semaglutide was cleaved from the peptidyl resin by incubation with TFA/EDT/water (90:5:5), separated from the resin by filtration, and precipitated on cold IPE. The resulting crude material showed 36.1% HPLC purity determined with the above analytical protocol (or 45.5% U-HPLC purity). Liquid chromatography mass spectrometry revealed two species (relative peak area: 3.1% and 1.0%) with M+40, which are considered to correspond to (N.sup.ε-allyl)-Lys derivatives of semaglutide.

Example 2: Full Chemical Synthesis of Semaglutide

(20) The synthesis was performed on a preloaded H-Gly-2-chlorotrityl resin using a fully automated or manual peptide synthesizer and Fmoc-Lys(AEEAc-AEEAc-(γ-Glu-OtBu)-17-t-butoxycarboxyheptadecanoyl)-OH as a building block. This latter moiety may be synthesized according to protocol b) above. Coupling reactions were carried out applying Fmoc-amino acid derivatives or Fmoc-dipeptide derivatives and DIC/Oxyma or TBTU/DIPEA as coupling reagents/additives in DMF as solvent.

(21) Fmoc-deprotection was carried out using a solution of 20% piperidine in DMF (v/v). DMF and IPA were used as solvents for washing steps. After each coupling, an acetylation step was performed. The SPPS was carried out on 1.66 mmol scale. Semaglutide was cleaved from the peptidyl resin by incubation with TFA/EDT/water (90:5:5, v/v/v), separated from the resin by filtration, and precipitated on cold IPE. The resulting crude material showed −39.1% HPLC purity determined the above analytical protocol.

Example 3: Full Chemical Synthesis of Semaglutide

(22) The peptidyl moiety corresponding to amino acids 31-20 of the semaglutide backbone was synthesized on a preloaded H-Gly-2-chlorotrityl resin using standard Fmoc-amino acid derivatives and Alloc-Lys(Fmoc)-OH as starting materials. Coupling reactions were carried out applying Fmoc-amino acid derivatives and DIC/Oxyma or TBTU/DIPEA as coupling reagents/additives in DMF as solvent. Fmoc-deprotection was carried out using a solution of 20% piperidine in DMF (v/v).

(23) DMF and IPA were used as solvents for washing steps. After each coupling, an acetylation step was performed. Acylation of the epsilon amino group was performed by stepwise Fmoc-SPPS, essentially according to protocol a) above.

(24) After completion of the side-chain synthesis, the Alloc group was removed from the alpha amino group of the Lys moiety as set out in the general methods above.

(25) Subsequently, the sequence corresponding to amino acids 1-19 of the semaglutide sequence was added by SPPS. Coupling reactions were carried out applying Fmoc-AA derivatives or Fmoc-dipeptide derivatives and DIC/Oxyma as coupling reagents/additives in DMF as solvent. Fmoc-deprotection was carried out using a solution of 20% piperidine in DMF. DMF and IPA were used as solvents for washing steps. After each coupling, an acetylation step was performed. The peptide was cleaved from the resin as set out in the general methods above. The resulting crude material showed 42.1% HPLC purity determined with the above analytical protocol. Liquid chromatography mass spectrometry revealed one species (relative peak area 1.6%) with M+40, which is considered to correspond to a (N-allyl)-Lys derivative of semaglutide. More precisely, it is expected to be a (Nα-allyl)-Lys derivative of semaglutide.

(26) Upon two dimensional purification of the crude peptide by RP-HPLC, the impurity was reduced to trace amounts.

Example 4: Full Chemical Synthesis of Semaglutide

(27) The synthesis of the complete semaglutide backbone will be performed on preloaded H-Gly-2-chlorotrityl resin using a fully automated peptide synthesizer and Fmoc-Lys(AEEAc-Alloc)-OH as a building block. This latter moiety may be synthesized according to protocol b) above. Coupling reactions will be carried out applying Fmoc-amino acid derivatives or Fmoc-dipeptide derivatives and DIC/Oxyma or TBTU/DIPEA as coupling reagents/additives in DMF as solvent. Fmoc-deprotection will be carried out using a solution of 20% piperidine in DMF (v/v). DMF and IPA will be used as solvents for washing steps. After each coupling, an acetylation step will be performed.

(28) After removal of the Alloc group according to the general protocol above, the resulting free amino group will be reacted with Fmoc-AEEAc-OH, Fmoc-Glu-OtBu, and subsequently (second) with either octadecanedioic acid or octadecanedioic acid mono-t-butyl ester as set out in general protocol a) above. Semaglutide will be cleaved from the peptidyl resin, separated from the resin by filtration, and precipitated on cold IPE.

Example 5: Full Chemical Synthesis of Semaglutide

(29) The peptidyl moiety corresponding to amino acids 31-20 of the semaglutide backbone will be synthesized on a preloaded H-Gly-2-chlorotrityl resin using standard Fmoc-amino acid derivatives and Alloc-Lys(AEEAc-Fmoc)-OH as starting materials.

(30) Coupling reactions will be carried out applying Fmoc-amino acid derivatives and DIC/Oxyma or TBTU/DIPEA as coupling reagents/additives in DMF as solvent. Fmoc-deprotection will be carried out using a solution of 20% piperidine in DMF (v/v). DMF and IPA will be used as solvents for washing steps. After each coupling, an acetylation step will be performed.

(31) Next, acylation of the epsilon amino group will be performed by stepwise Fmoc-SPPS, according to protocol a) above.

(32) After completion of the side-chain synthesis, the Alloc group will be removed from the alpha amino group of the Lys moiety as set out in the general methods above. Subsequently, the sequence corresponding to amino acids 1-19 of the semaglutide sequence will be added by SPPS and the peptide will be cleaved from the resin as set out in the general methods above.