Process of making abaloparatide

1. A process for the preparation of abaloparatide comprising:

a) providing a peptide bound to a solid resin and having an initial N-terminus, wherein said bound peptide is NH 2 -Asp(OtBu)-Leu-Arg(Pbf)-Arg 20 (Pbf)-Arg(Pbf)-Glu(OtBu)-Leu-Leu-Glu 25 (OtBu)-Lys(Boc)-Leu-Leu-Aib-Lys 30 (Boc)-Leu-His(Trt)-Thr(tBu)-Ala 34 -Rink Amide MBHA resin (SEO ID NO: 2);

b) coupling a carboxyl terminus of Fmoc-(Trt)Gln 16 -OH to the initial N-terminus of the bound peptide, wherein coupling comprises adding to a suspension of the bound peptide in dimethylformamide from about 1.05 to about 2 molar equivalents of Fmoc-(Trt)Gln 16 -OH, relative to the bound peptide, in the presence of benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 1-Hydroxybenzotriazole (HOBt), N,N′-diisopropylcarbodiimide (DIC), and diisopropylethylamine (DIEA), and allowing the mixture to react for a period of time;

c) adding to the mixture a further from about 1.05 to about 2 molar equivalents of Fmoc-(Trt)Gln 16 -OH, relative to the bound peptide, in the presence of additional PyBOP, HOBt, DIC, and DIEA;

d) selectively cleaving the Fmoc group with a solution comprising an amine base to provide a peptide bound to a solid resin and having a new N-terminus.

2. The process of claim 1 , wherein the amine base is piperidine.

3. The process of claim 2 , wherein the piperidine is present as an approximately 20% solution by weight in DMF.

4. The process of claim 1 , further comprising

e) coupling a carboxyl terminus of Fmoc-Ile 15 -OH to the new N-terminus in the presence of a coupling reagent;

f) selectively cleaving the Fmoc group with a solution comprising an amine base to provide a peptide bound to the solid resin and having a new N-terminus; and

g) repeating steps e) and f), wherein each step e) is performed, in sequential order, substituting the Fmoc-Ile 15 -OH with the following Fmoc-protected amino acids: Ser(tBu) 14 -OH, Lys(Boc) 13 -OH, Gly 12 -OH, Lys(Boc) 11 -OH, Asp 10 (OtBu)-OH, His(Trt) 9 -OH, Leu 8 -OH, Leu 7 -OH, Gln(Trt) 6 -OH, His 5 (Trt)-OH, Glu(OtBu) 4 -OH, Ser(tBu) 3 -OH, Val 2 -OH, and Ala 1 -OH, to form a thirty-four amino acid peptide sequence bound to the solid resin.

5. The process of claim 4 , wherein the coupling reagent comprises PyBOP, HOBt, DIC, and DIEA.

6. The process of claim 4 , wherein each coupling step e) is performed using from about 1.05 to about 2 molar equivalents of the Fmoc-amino acid, relative to the bound peptide.

7. The process of claim 4 , wherein the amine base is piperidine.

8. The process of claim 7 , wherein the piperidine is present as an approximately 20% solution by weight in DMF.

9. The process of claim 4 , further comprising:

performing a Kaiser and/or Chloranil test after each step e) but before each step f) to confirm complete incorporation of each Fmoc-protected amino acid; and

performing a recoupling reaction if a positive Kaiser or Chloranil test result is obtained.

10. The process of claim 9 , wherein performing the recoupling reaction comprises performing a coupling reaction with the Fmoc-protected amino acid one or more additional times, using from 0.15 to 1.5 molar equivalents of the respective Fmoc-amino acid relative to the bound peptide, repeating said coupling until a negative Kaiser or Chloranil test result is obtained.

11. The process of claim 4 , further comprising:

treating the thirty-four amino acid peptide sequence bound to the solid resin with a solution comprising trifluoroacetic acid to provide crude abaloparatide.

12. The process of claim 11 , wherein the crude abaloparatide contains less than about 1% of des-Gln 16 -abaloparatide by weight, based on the total weight of the crude abaloparatide.

13. The process of claim 11 , wherein the crude abaloparatide contains less than about 0.5% des-Gln 16 -abaloparatide by weight, based on the total weight of the crude abaloparatide.

14. The process of claim 11 , wherein the crude abaloparatide contains less than about 0.3% des-Gln 16 -abaloparatide by weight, based on the total weight of the crude abaloparatide.

15. The process of claim 4 , wherein providing the peptide bound to the solid resin comprises:

anchoring Fmoc-protected alanine (Fmoc-Ala 34 -OH) to Rink amide 4-methylbenzhydrylamine (MBHA) resin;

capping the resin obtained in the anchoring step by acetylation;

selectively cleaving the Fmoc group with a solution comprising an amine base to provide an N-terminus;

coupling a carboxyl terminus of Fmoc-Thr(tBu) 33 -OH to the N-terminus in the presence of a coupling reagent;

performing a Kaiser and/or Chloranil test after each coupling step to confirm complete incorporation of each Fmoc-protected amino acid, and performing a recoupling reaction if a positive Kaiser or Chloranil test result is obtained;

selectively cleaving the Fmoc group with a solution comprising an amine base to provide an N-terminus; and

repeating sequentially each of the coupling, performing a Kaiser and/or Chloranil test, and selectively cleaving the Fmoc group steps, in each instance replacing the Fmoc-Thr(tBu) 33 -OH with the following series of Fmoc-protected amino acids: His(Trt) 32 -OH, Leu 31 -OH, (Boc)Lys 30 -OH, Aib 29 -OH, Leu 28 -OH, Leu 27 -OH, (Boc)Lys 26 -OH, (OtBu)Glu 25 -OH, Leu 24 -OH, Leu 23 -OH, (OtBu)Glu 22 -OH, (Pbf)Arg 21 -OH, (Pbf)-Arg 20 -OH, (Pbf)Arg 19 -OH, Leu 18 -OH, and (OtBu)Asp 17 -OH.

16. The process of claim 15 , wherein the coupling reagent comprises PyBOP, HOBt, DIC, and DIEA.

17. The process of claim 15 , wherein the amine base is piperidine.

18. The process of claim 17 , wherein the piperidine is present as an approximately 20% solution by volume in DMF.

19. The process of claim 15 , wherein each coupling step is performed using from about 1.05 to about 2 molar equivalents of Fmoc-amino acid, relative to the bound peptide.

20. The process of claim 15 , wherein performing the recoupling reaction comprises performing a coupling reaction with the Fmoc-protected amino acid one or more additional times, using from 0.15 to 1.5 molar equivalents of the respective Fmoc-amino acid relative to the bound peptide, and repeating said coupling until a negative Kaiser or Chloranil test result is obtained.

21. A process for the preparation of abaloparatide comprising:

a) anchoring Fmoc-protected alanine (Fmoc-Ala 34 -OH) to Rink amide 4-methylbenzhydrylamine (MBHA) resin;

b) capping the resin obtained in step a) by acetylation;

c) selectively cleaving the Fmoc group with a solution comprising piperidine to provide an N-terminus;

d) coupling a carboxyl terminus of Fmoc-Thr(tBu) 33 -OH to the N-terminus in the presence of a coupling reagent comprising benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 1-Hydroxybenzotriazole (HOBt), N,N′-diisopropylcarbodiimide (DIC), and diisopropylethylamine (DIEA), performing a Kaiser and/or Chloranil test after each step d) to confirm complete incorporation of each Fmoc-protected amino acid, and repeating said step d) if a positive Kaiser or Chloranil test result is obtained;

e) selectively cleaving the Fmoc group with a solution comprising piperidine to provide an N-terminus;

f) repeating steps d) and e) sequentially, replacing the Fmoc-Thr(tBu) 33 -OH with the following series of Fmoc-protected amino acids: His(Trt) 32 -OH, Leu 31 -OH, (Boc)Lys 30 -OH, Aib 29 -OH, Leu 28 -OH, Leu 27 -OH, (Boc)Lys 26 -OH, (OtBu)Glu 25 -OH, Leu 24 -OH, Leu 23 -OH, (OtBu)Glu 22 -OH, (Pbf)Arg 21 -OH, (Pbf)-Arg 20 -OH, (Pbf)Arg 19 -OH, Leu 18 -OH, and (OtBu)Asp 17 -OH, and performing a Kaiser and/or Chloranil test after each step d) before each step e) to confirm complete incorporation of each Fmoc-protected amino acid, and repeating said step d) if a positive Kaiser or Chloranil test result is obtained;

g) coupling a carboxyl terminus of Fmoc-(Trt)Gln 16 -OH to the N-terminus, wherein coupling comprises adding to a suspension of the bound peptide in dimethylformamide from about 1.05 to about 2 molar equivalents of Fmoc-(Trt)Gln 16 -OH, relative to the bound peptide, in the presence of a coupling reagent comprising PyBOP, HOBt, DIC, and DIEA, and allowing the mixture to react for a period of time;

h) adding to the mixture a further from about 1.05 to about 2 molar equivalents of Fmoc-(Trt)Gln 16 -OH, relative to the bound peptide, in the presence of additional PyBOP, HOBt, DIC, and DIEA;

i) selectively cleaving the Fmoc group with a solution comprising piperidine to provide an N-terminus;

j) coupling a carboxyl terminus of Fmoc-Ile 15 -OH to the N-terminus in the presence of a coupling reagent comprising PyBOP, HOBt, DIC, and DIEA;

k) selectively cleaving the Fmoc group with a solution comprising piperidine to provide an N-terminus;

l) repeating steps j) and k) sequentially, replacing Fmoc-Ile 15 -OH with the following series of Fmoc-protected amino acids: Ser (tBu) 14 -OH, Lys (Boc) 13 -OH, Gly 21 -OH, Lys(Boc) 11 -OH, Asp(OtBu) 10 -OH, His(Trt) 9 -OH, Leu 8 -OH, Leu7-OH, Gln(Trt) 6 -OH, His (Trt) 5 -OH, Glu(OtBu) 4 -OH, Ser(tBu) 3 -OH, Val 2 -OH, and Ala 1 -OH;

m) performing a Kaiser and/or Chloranil test after each step j) before each step k) to confirm complete incorporation of each Fmoc-protected amino acid, and repeating said step j) if a positive Kaiser or Chloranil test result is obtained; and

n) treating the resulting anchored, protected abaloparatide with a solution comprising trifluoroacetic acid to provide crude abaloparatide.

22. The process of claim 21 , wherein:

each initial coupling reaction is performed using from about 1.05 to about 2 molar equivalents of the respective Fmoc-amino acid relative to the bound peptide; and

any repeated couplings performed following a positive Kaiser or Chloranil test are performed using from 0.15 to 2 molar equivalents of the respective Fmoc-amino acid.

23. The process of claim 21 , wherein in step f), the coupling of Fmoc-protected Aib 29 -OH is repeated once prior to cleavage of the Fmoc group, without regard to the Kaiser or Chloranil test, the repeated coupling using from about 1.05 to about 2 equivalents of the Fmoc-protected Aib 29 -OH.

24. The process of claim 21 , wherein in step f), the coupling of the Fmoc-protected Leu 28 -OH is repeated once prior to cleavage of the Fmoc group, without regard to the Kaiser or Chloranil test, the repeated coupling using from about 1.05 to about 2 equivalents of the Fmoc-protected Leu 28 -OH.

25. The process of claim 21 , wherein the piperidine is present as an approximately 20% solution by volume in DMF.

26. The process of claim 21 , wherein the obtained crude abaloparatide contains less than about 1% of des-Gln 16 -abaloparatide by weight, based on the total weight of the crude abaloparatide.

27. The process of claim 21 , wherein the obtained crude abaloparatide contains less than about 0.5% des-Gln 16 -abaloparatide by weight, based on the total weight of the crude abaloparatide.

28. The process of claim 21 , wherein the obtained crude abaloparatide contains less than about 0.3% des-Gln 16 -abaloparatide by weight, based on the total weight of the crude abaloparatide.