Method for inverse solid phase synthesis of peptides

1. A method for preparing an immobilized peptide derivative of formula (I):

Sub-[L]-[NH-A-C(O)] n+m —OH  (I)

comprising:

(a) reacting an immobilized compound of formula (II):

Sub-(L)-[NH-A-C(O)] n —OH  (II)

 with an amino acid ester or peptide derivative of formula (III):

H—[NH-A-C(O)] m —O(tBu)  (III)

 in the presence of a coupling agent to yield an immobilized peptide derivative of general formula (IV):

Sub-[L]-[NH-A-C(O)] n+m —O(tBu)  (IV);

(b) removing the t-Bu (t-butyl) group to produce an immobilized peptide derivative of general formula (I);

wherein n is a positive integer, m is 1, Sub is a solid support, L is a linker to the amino acid ester or peptide derivative, each NH-A-C(O) independently is the residue of an optionally sidechain-protected amino acid; or the structure —NH-A represents a heterocyclic group.

2. The method of claim 1 which further comprises, prior to step (a): forming an immobilized peptide derivative of general formula (VI):

Sub-[L]-[NH-A-C(O)] n —O(tBu)  (VI);

by reacting a functionalized solid support of general formula Sub[L]X with a compound of formula: H—[NH-A-C(O)] n —O(tBu), wherein X is a group that links L to NH 2 -A by elimination of XH; and

removing the t-Bu group to yield the immobilized peptide derivative of formula (II).

3. The method of claim 1 or 2 further comprising cleaving the immobilized peptide derivative of formula (IV) at the L-NH bond and removing the t-Bu group to yield a compound of formula (IX);

H- [HN-A-C(O)] n+m —OH  (IX).

4. The method of claim 1 further comprising, following step (b); carrying out, x times, the steps of reacting an immobilized peptide derivative of formula (I) with an amino acid ester derivative of formula (III) and removing the t-Bu group to yield an immobilized peptide derivative of formula (VIII):

Sub-(L)-[NH-A-C(O)] n+m(x+1) —OH  (VIII)

wherein x is an integer.

5. The method of claim 2 wherein [L]X is —OC(O)(CH 2 ) 2 CH(NHZ)CO 2 H, wherein Z is a protecting group.

6. The method of claim 2 wherein [L]X is HNC(O)(CH 2 ) 2 CH(NHZ)CO 2 H, wherein Z is a protecting group.

7. The method of claim 5 or 6 wherein Z is benzyloxycarbonyl (Cbz).

8. The method of claim 1 wherein each NH-A-C(O) is individually the optionally sidechain-protected residue of an α-amino acid.

9. The method of claim 1 wherein each NH-A-C(O) is individually the optionally sidechain-protected residue of a naturally occurring L-amino acid.

10. The method of claim 1 wherein the coupling agent is HATU.

11. The method of claim 1 or 2 wherein the t-Bu group is removed with TFA.

12. The method of claim 3 wherein the cleavage is carried out using TFMSA/TFA.

13. The method of claim 3 wherein Sub is a polymer comprising free NH 2 or OH groups.

14. The method of claim 13 wherein the polymer comprises polystyrene.

15. The method of claim 1 further comprising reacting an immobilized peptide derivative of formula (I) with H 2 N(alkyl)CH(OH)CF 3 (XV) to yield the corresponding amide of the immobilized peptide derivative of formula (I) and oxidizing the CH(OH) moiety to yield an immobilized peptide derivative of general formula (XII):

Sub-(L)-[NH-A-C(O)] n+m —NH(alkyl)C(O)CF 3   (XII).

16. The method of claim 15 further comprising cleaving the immobilized peptide derivative of formula (XII) to yield a compound of formula:

H-L-[NH-A-C(O)] n+m —NH(alkyl)C(O)CF 3 .

17. The method of claim 4 further comprising reacting the immobilized peptide derivative of formula (VIII) with compound (XV) to yield the corresponding amide of the immobilized peptide derivative of formula (VIII) and oxidizing the CH(OH) moiety to yield an immobilized peptide derivative of general formula (XVI):

Sub-(L)-[NH-A-C(O)] n+m(x+1) —NH(alkyl)C(O)CF 3   (XVI).

18. The method of claim 17 further comprising cleaving compound (XVI) from Sub to yield a compound of formula:

H-L-[NH-A-C(O)] n+m(x−1) —NH(alkyl)C(O)CF 3 .

19. The method of claim 1 further comprising reacting the immobilized peptide derivative of formula (I) with H 2 N(alkyl)OH (XVII) to yield the hydroxyalkylamide of (I) and oxidizing the terminal hydroxy group to yield an immobilized peptide derivative of formula (XIII):

Sub-(L)-[NH-A-C(O)] n+m —NH(alkyl)CHO  (XIII).

20. The method of claim 4 further comprising reacting the immobilized peptide derivative of formula (VIII) with H 2 N(alkyl)OH (XVII) to yield the hydroxyalkylamide of (VIII) and oxidizing the terminal hydroxyl group to yield the immobilized peptide derivative of formula (XVIII):

Sub-(L)-[NH-A-C(O)]n+m(x+ 1 )—NH(alkyl)CHO  (XVIII).

21. The method of claim 19 further comprising protecting the terminal CHO group of the immobilized peptide derivative of formula (XIII).

22. The method of claim 20 further comprising protecting the terminal CHO group of the immobilized peptide derivative of formula (XVIII).

23. The method of claim 21 or 22 wherein the CHO group of the immobilized peptide compound of formula XIII or of formula XVIII is converted into an ethylene glycol acetal, a propylene glycol acetal, an ethylene dithiol acetal or a 1,3-dithiane.

24. The method of claim 23 further comprising cleaving the immobilized peptide derivative wherein the CHO group is converted into an ethylene glycol acetal, a propylene glycol acetal, an ethylene dithiol acetal or a 1,3-dithiane to yield a compound of formula:

wherein each X is O or each X is S, and p is 0 or 1.

25. The method of claim 4 further comprising reacting an immobilized peptide derivative of formula (VIII) with a boroamine to yield an immobilized peptide derivative of formula (XIV):

Sub-[L]-[NH-A-C(O)] n+m(x+1) —NH(A)B(OR) 2   (XIV),

wherein R is (C 1 –C 6 )alkyl, phenyl, or the residue of a diol and wherein NH(A)B(OR) 2 is the residue of an O-protected aminoboronic acid.

26. The method of claim 1 further comprising reacting an immobilized peptide derivative of formula (I) with a boroamine to yield a compound of fonnula (XIX):

Sub-[L]-[NH-A-C(O)] n+m —NH(A)B(OR) 2   (XIX),

wherein R is (C 1 –C 6 )alkyl, phenyl, or the residue of a diol and wherein NH(A)B(OR) 2 is the residue of an O-protected aminoboronic acid.

27. The method of claim 25 further comprising cleaving compound (XIV) to yield a compound of formula:

H-[L]-[NH-A-C(O)] n+m(x+1) —NH(A)B(OH) 2 .

28. The method of claim 26 further comprising cleaving compound (XIX) to yield a compound of formula:

H-[L]-[NH-A-C(O)] n+m —NH(A)B(OH) 2 .

29. The method of claim 1 or 2 wherein n is 1.

30. The method of claim 4 wherein x is 1–10.

31. The method of claim 30 wherein x is 1–5.

32. The method of claim 2 wherein [L]X is —OC(O)(CH 2 ) 2 CO 2 H.

33. The method of claim 1 wherein at least one NH-A-C(O) comprises a residue of proline or hydroxyproline.