SOLID PHASE PEPTIDE SYNTHESIS VIA SIDE CHAIN ATTACHMENT

SYNTHESE DE PEPTIDE EN PHASE SOLIDE A L'AIDE D'ATTACHEMENT DE CHAINE LATERALE

English Abstract

The present application discloses peptides and peptaibols of high purity may
be obtained by
solid phase peptide synthesis using as the starting resin hydroxy amino acids,
hydroxy amino
acid amides, hydroxy amino alcohols or small peptides containing hydroxy amino
acids
attached to polymers through their side chain.

Claims

What is claimed is:
1. A resin conjugate of the formula I or II, wherein:
\
H
Y I V Y
Pi '-Hya-A PP-Hya-A
Formula I Formula II
Hya is the residue of a hydroxy amino acid;
Pr' is H, or an amino protecting group which is orthogonal to the resin and to
other
protecting groups;
A is a hydroxyl group or a acid sensitive hydroxyl protecting group selected
from tBu,
Trt, Clt, or Nlele wherein le and le are H or C1_10 alkyl, or peptide ester or
peptide amide
or a peptaibol containing 0-30 amino acids;
X, Y, Z and V are each independently a substituent on the ortho, meta or para
positions
and is selected from H, CI, F, Ci_io alkyl or Ci_io alkoxy; and
P is an insoluble solid support or an insoluble linker-resin conjugate
suitable for the
solid phase synthesis of peptides.
2. A resin conjugate of the formula III-VI, wherein:
k,Z
H
-1)
Y 0 V 0 V
R4 R3 R4 R3
Pri¨N7\,-0-Pr2 Pr1¨N
Formula III Formula IV
H
V
0 0
O-Pr2
Prr Prr
Formula V Formula VI
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Pr' is H or an amino protecting group, which is orthogonal to the resin and to
other
protecting groups;
Pr2 is H or a hydroxyl protecting group which is orthogonal to the resin;
R3 and R4 are each independently H or C1-10 alkyl;
X, Y, Z and V are each independently on the ortho, meta or para positions
selected from
H, CI, F, Ci_io alkyl or C1_10 alkoxy; and
P is an insoluble solid support or an insoluble linker-resin conjugate
suitable for the
solid phase synthesis of peptides.
3. A method for the preparation of the resin conjugates of the formulae I-
VI comprising
the following steps:
preparing a hydroxyl containing amino acid or amino alcohol or peptide
derivative that
is unprotected on at least one of the side chains of the contained hydroxyl
amino acids or of
the contained amino alcohol or selectively deprotecting the hydroxyl amino
acid or the
hydroxyl amino alcohol or a peptide derivative at the side chain of the
hydroxyl amino acid
or the hydroxyl amino alcohol and then attaching it to a suitable resin by its
reaction with a
resin halide, wherein the resin is selected from the group of the trityl type
resins and linkers
or the benzhydryl type resins or the benzyl-type resins; and
an alcohol or thioalcohol is added to mask any unreacted resin halide.
4. A process for the preparation of monoalkylated Fmoc-amino di-alcohols,
the process
comprising the reaction of the Fmoc-di-amino alcohol with alkylhalide in the
presence of a
base such as a tertiary amine base in an organic solvent, such as
dichloromethane; and
wherein alkyl is a triarylmethyl selected from trityl, 4-methyltrityl, 4-
methoxytrityl and 2-
chlorotrityl.
5. A process for the solid phase synthesis of biologically active free or
partially protected
peptides, cyclic peptides and peptaibols, wherein the process comprises the
use of the resin
conjugates of Claim 1 or Claim 2 as functionalized resins in the solid phase
peptide
synthesis.
6. A peptide of the formula E-D-2-Nal-Cys(A)-Tyr(C)-D-Trp(F)-Lys(E)-Val-
Cys(A)-
Thr(Resin)-NH2 wherein:
D- designates the chirality of the amino acid that follows as a D-amino acid;
8116434
Date Recue/Date Received 2023-01-12

each A is independently a thiol protecting group selected from Trt, Mmt, Acm
or StBu;
C is a hydroxy protecting group selected from Clt, Trt or tBu;
F is H or Boc;
E is an amino protecting group selected from Mtt, Mmt, Trt or Boc; and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
7. The peptide of Claim 6, wherein the peptide is a resin-bound lanreotide.
8. A process for preparing lanreotide comprising:
treating the peptide resin conjugate of Claim 7 with a mild acid selected from
solutions
of trifluoracetic acid optionally containing scavengers; and
oxidizing the resulting peptide using an oxidizing agent selected from air,
hydrogen
peroxide, DMSO or iodine.
9. A process for preparing lanreotide comprising treating the peptide resin
conjugate of
Claim 7 with a mild acid such as a solution of trifluoracetic acid containing
iodine.
10. A peptide of the formula E-Phe-Val-Asn(A)-Gln(A)-His(A)-Leu-Cys(B)-Gly-
Ser(C)-
His(A)-Leu-Val-Glu(C)-Ala-Leu-Tyr(C)-Leu-Val-Cys(A)-Gly-Glu(C)-Arg(D)-Gly-Phe-
Phe-
Tyr(C)-Thr(C)-Pro-Lys(E)-Thr(Resin)-0-C wherein:
A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt or
Mmt;
B is a Cys protecting group selected from Mmt, Trt, Acm or StBu;
C is a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or
Clt;
D is a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt,
Mmt or
Clt; and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
11. The peptide of Claim 10, wherein the peptide is human insulin B-chain.
12. A process for preparing an insulin B-chain, wherein the process
comprises contacting
the resin-bound peptide of Claim 10 with an acid solution, such as
trifluoroacetic acid
solutions in dichloromethane, optionally containing scavengers.
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13. A peptide of the formula E-Ser(C)-His(A)-Leu-Val-Glu(C)-Ala-Leu-Tyr(C)-
Leu-Val-
Cys(B)-Gly-Glu(C)-Arg(D)-Gly-Phe-Phe-Tyr(C)-Thr(C)-Pro-Lys(E)-Thr(Resin)-0-
wherein:
A is a carboxamido or imidazol protecting group selected from Trt, Mtt or Mmt;
B is a Cys protecting group selected from Mmt, Trt, Acm or StBu;
Each C is independently a hydroxy, carboxyl or phenoxy protecting group
selected
from tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt,
Mmt or
Clt; and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
14. The peptide of Claim 13, wherein the peptide corresponds to the
partially protected 9-
30 fragment of human insulin B-chain.
15. A process for the preparation of insulin B-chain, the process
comprising:
condensation of the peptide of Claim 13 with a 1-8 protected insulin fragment
Boc-Phe-
Val-Asn(A)-Gln(A)-His(A)-Leu-Cys(B)-Gly-OH, wherein;
each A is independently a carboxamido or imidazol protecting group selected
from Trt, Mtt and Mmt; and
B is a Cys protecting group selected from Mmt, Trt, Acm or StBu.
16. .. A peptide of the formula E-Cys(B)-Ser(C)-Asn(A)-Leu-Ser(C)-Thr(C)-
Cys(B)-Val-
Leu-Gly-Lys(E)-Leu-Ser(C)-Gln(A)-Glu(C)-Leu-His(A)-Lys(E)-Leu-Gln(A)-Thr(C)-
Tyr(C)-
Pro-Arg(D)-Thr(C)-Asn(A)-Thr(C)-Gly-Ser(C)-Gly-Thr(Resin)-Pro-NH2, wherein:
each A is independently a carboxamido or imidazol protecting group selected
from Trt,
Mtt or Mmt;
each B is independently a Cys protecting group selected from Mmt, Trt, Acm or
StBu;
each C is independently a hydroxyl, carboxy or phenoxy protecting group
selected from
of tBu, Trt or Clt;
D is a guanidine protecting group selected from Pbf or Pmc;
each E is independently H or an amino protecting group selected from Fmoc,
Boc, Trt,
Nps, Mtt, Mmt or Clt; and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
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17. The peptide of Claim 16, wherein the peptide is salmon calcitonin.
18. A process for producing salmon calcitonin, the process comprising:
treating the peptide of Claim 16 with a mild acid such as a solution of
trifluoracetic acid
optionally containing scavengers;
oxidizing the obtained peptide solution using a suitable oxidizing agent
selected from
air, hydrogen peroxide, DMSO or iodine;
deprotecting the peptide;
purifying the peptide by chromatography; and
lyophilizing the peptide.
19. A process for producing salmon calcitonin, the process comprising:
treating the peptide resin conjugate of Claim 16 with a mild acid such as a
solution of
trifluoracetic acid;
oxidizing the peptide with iodine;
deprotecting the peptide by treatment with acids such as a solution of
trifluoroacetic or
hydrochloric acid optionally containing scavengers; and
purifying by chromatography and lyophilizing the salmon calcitonin peptide.
20. A process for the preparation of the peptide of Claim 17, the process
comprises
condensing the partially protected 11-32 fragment of salmon calcitonin of the
formula H-
Lys(E)-Leu-Ser(C)-Gln(A)-Glu(C)-Leu-His(A)-Lys(E)-Leu-Gln(A)-Thr(C)-Tyr(C)-Pro-

Arg(D)-Thr(C)-Asn(A)-Thr(C)-Gly-Ser(C)-Gly-Thr(Resin)-Pro-NH2 with a 1-10
partially
protected fragment of salmon calcitonin of the formula E-Cys(B)-Ser(C)-Asn(A)-
Leu-
Ser(C)-Thr(C)-Cys(B)-Val-Leu-Gly-OH; wherein
each A is independently a carboxamido or imidazol protecting group selected
from Trt,
Mtt or Mmt;
each B is independently a Cys protecting group selected from Mmt, Trt, Acm or
StBu;
each C is independently a hydroxyl, carboxy or phenoxy protecting group
selected from
tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
each E is independently H or an amino protecting group selected from Fmoc,
Boc, Trt,
Nps, Mtt, Mmt or Clt; and
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Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
21. A partially protected peptide of the formula:
CH3
LO-Resin
A-D-Phe-Cys(B)-Phe-D-Trp(C)-Lys(A)-Phe-Thr(E)-Cys(B)-NH
O-E
wherein:
A is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt or
Mmt;
D- designates the chirality of the amino acid that follows as a D-amino acid;
B is a thiol protecting group selected from Trt, Mmt, Acm or StBu;
C is H or Boc;
E is a hydroxy protecting group selected from Clt, Trt or tBu; and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
22. The peptide of Claim 21, wherein the peptide is a resin bound
octreotide,
23. A process for preparing octreotide, the process comprising:
treating the peptide resin conjugate of Claim 22 with a mild acid, such as a
solution of
trifluoracetic acid optionally containing scavengers; and
oxidizing the obtained peptide solution using a suitable oxidizing agent
selected from
air, hydrogen peroxide, DMSO or iodine.
24. A process for preparing octreotide comprising:
treating the peptide resin conjugate of Claim 22 with a mild acid such as a
solution of
trifluoracetic acid containing iodine;
deprotecting, purifying and lyophilizing and obtaining octreotide, wherein the

octreotide has a purity of >99%.
25. A partially protected peptide of the formula E-His(A)-Gly-Glu(C)-Gly-
Thr(C)-Phe-
Thr(C)-Ser(C)-Asp(C)-X-Lys(E)-Gln(A)-Met-Glu(C)-Glu(C)-Glu(C)-Ala-Val-Arg(D)-
Leu-
Phe-Ile-Glu(C)-Trp(F)-Leu-Lys(E)-Asn(A)-Gly-Gly-Pro-Ser(C)-Ser(C)-Gly-Ala-Pro-
Pro-
Pro-Ser(Resin)-NH2 wherein:
X is Leu-Ser(tBu) or Leu-tPSer;
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each A is independently H or a carboxamido or imidazol protecting group
selected from
Trt, Mtt or Mmt;
each C is independently a hydroxy, carboxyl or phenoxy protecting group
selected from
tBu, Trt or Clt;
D is H or a guanidine protecting group elected from Pbf or Pmc;
each E is independently H or an amino protecting group selected from Fmoc,
Boc, Trt,
Nps, Mtt, Mmt or Clt;
F is H or Boc; and
Resin is an acid labile resin suitable for solid-phase peptide synthesis.
26. The peptide of Claim 25, wherein the peptide is exenatide.
27. A partially protected peptide Y-Glu(C)-Glu(C)-Ala-Val-Arg(D)-Leu-Phe-
Ile-Glu(C)-
Trp(F)-Leu-Lys(E)-Asn(A)-Gly-Gly-Pro-Ser(C)-Ser(C)-Gly-Ala-Pro-Pro-Pro-
Ser(Resin)-
NH2 that is the exenatide fragments 12-39, 13-39, 14-39, wherein:
Y is H, E-Glu(C), E-Met-Glu(C), E-Gln(A)-Met-Glu(C) or E-Lys(E')-Gln(A)-Met-
Glu(C); wherein:
each A is independently H or a carboxamido protecting group selected from Trt,
Mtt
and Mmt;
E and E' are each independently H or an amino protecting group selected from
Fmoc,
Boc, Trt, Nps, Mtt, Mmt or Clt;
F is H or Boc;
each C is independently a hydroxy or carboxyl protecting group selected from
tBu, Trt
or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc; and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
28. A partially protected peptides that is E-His(A)-Gly-Glu(C)-Gly-Thr(C)-
Phe-Thr(C)-
Ser(C)-Asp(C)-X-Y in particular sequences corresponding to the exenatide
fragments 1-11,
1-13, 1-14 and 1-15, wherein:
X is Leu-Ser(tBu) or Leu-tPSer;
E is an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt and
Clt;
A is H, a carboxamido or imidazol protecting group selected from Trt, Mtt or
Mmt;
8116434
Date Recue/Date Received 2023-01-12

each C is independently a hydroxy or carboxyl protecting group selected from
tBu, Trt
or Clt; and
Y is OZ, Lys(E)-G1n(A)-0Z, Lys(E)-Gln(A)-Met-OZ, Lys(E)-G1n(A)-Met-G1u(C)-0Z,
wherein each Z is independently H or a group which activate electrophilically
carboxyl
groups selected from Bt, Su, Pfp, Tcp or Pnp.
29. A process for the production of exenatide comprising:
condensing of one fragment of the Claim 27 with one fragment of Claim 28 to
form the
partially or resin-bound protected exenatide sequence;
deprotecting or cleaving from the resin; and
deprotecting, chromatographic purification and lyophilization of exenatide.
30. A partially protected peptide of the formula E-Lys(E)-Cys(B)-Asn(A)-
Thr(C)-Y-
Cys(B)-Y-Gln(A)-Arg(D)-Leu-Ala-Asn(A)-Phe-Leu-Val-His(A)-X-Asn(A)-Asn(A)-Phe-
Gly-Pro-Ile-Leu-Pro-Pro-Thr(C)-Asn(A)-Val-Gly-Ser(C)-Asn(A)-Thr(C)-Tyr(Resin)-
NH2
wherein X = Ser(tBu)-Ser(tBu) or Ser(tBu)-'PSer, wherein:
Y is Ala-Thr(tBu) or Ala-tPThr;
each A is independently H, a carboxamido or imidazol protecting group selected
from
Trt, Mtt or Mmt;
each B is independently a Cys protecting group selected from Mmt, Trt, Acm or
StBu;
each C is independently hydroxyl, carboxy or phenoxy protecting group selected
from
tBu, Trt or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt,
Mmt or
Clt; and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
31. The peptide of Claim 30 that is protected or partially protected
pramlintide.
32. A protected or partially protected peptide with the sequence Z-Asn(A)-
Phe-Leu-Val-
His(A)-X-Asn(A)-Asn(A)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(C)-Asn(A)-Val-Gly-
Ser(C)-
Asn(A)-Thr(C)-Tyr(Resin)-NH2 in particular the protected or partially
protected 10-38, 11-
38, 12-38 and 14-38 partially protected pramlintide fragments wherein:
Z is H or E-Gln(A)-Arg(D)-Leu-Ala, E-Arg(D)-Leu-Ala, E-Leu-Ala or E-Ala;
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X is Ser(tBu)-Ser(tBu) or Ser(tBu)-'PSer;
A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt or
Mmt;
C is a hydroxyl, carboxy or phenoxy protecting group selected from tBu, Trt or
Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt,
Mmt and
Clt; and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
33. A process for the preparation of a partially protected and oxidized
pramlintide
fragments, comprising:
1 I
E-Lys(E)-Cys-Asn(A)-Thr(C)-Y-Cys-Y-Gln(A)-Arg(D)-Leu-Ala-OZ
I I
E-Lys(E)-Cys-Asn(A)-Thr(C)-Y-Cys-Y-Gln(A)-Arg(D)-0Z
I I
E-Lys(E)-Cys-Asn(A)-Thr(C)-Y-Cys-Y-Gln(A)-0Z
I I
E-Lys(E)-Cys-Asn(A)-Thr(C)-Y-Cys-Y-OZ
assembling protected peptides on an acid sensitive resin, such as a 2-CTC-
resin;
cleaving the protected peptide from the resin; and
oxidizing the resin-bound peptides by contacting the resin-bound peptides with
mild
acid, such as dilute trifluoroacetic acid in an organic solvent, such as
dichloromethane,
wherein the solvent comprises a 2-200 molar excess of iodine over the
fragment.
34. A process for the production of pramlintide, where a fragment of Claim
33 is
condensed in solution or on solid phase with one of the fragments of Claim 32,
Claim 33 or
one of the fragments of the formula E-Lys(E)-Cys(B)-Asn(A)-Thr(C)-Y-Cys(B)-Y-
Gln(A)-
Arg(D)-Leu-Ala-OZ, E-Lys(E)-Cys(B)-Asn(A)-Thr(C)-Y-Cys(B)-Y-G1n(A)-Arg(D)-0Z,
E-
Lys(E)-Cys(B)-Asn(A)-Thr(C)-Y-Cys(B)-Y-G1n(A)-0Z, E-Lys(E)-Cys(B)-Asn(A)-
Thr(C)-
Y-Cys(B)-Y-OZ, wherein:
each Y is independently Ala-Thr(tBu) or Ala-tPThr;
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Z is H or a group selected from Bt, Su, Pfp, Tcp or Pnp;
each A is independently H or a carboxamido or imidazol protecting group
selected from
Trt, Mtt or Mmt;
each C is independently a hydroxy, carboxyl or phenoxy protecting group
selected from
tBu, Trt or Clt;
each D is independently H or a guanidine protecting group selected from Pbf or
Pmc;
each E is independently H or an amino protecting group selected from Fmoc,
Boc, Trt,
Nps, Mtt, Mmt or Clt;
each B is independently a Cys protecting group selected from Mmt, Trt, Acm or
StBu
and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
35. A protected or partially protected peptide of the formula E-D-Phe-Pro-
Arg(D)-Pro-Gly-
Gly-Gly-Gly-Asn(A)-Gly-Asp(C)-Phe-Glu(C)-Glu(C)-Ile-Pro-Glu(C)-Glu(C)-
Tyr(Resin)-
Leu-O-C, wherein:
A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt or
Mmt;
each C is independently a hydroxyl or carboxyl protecting group selected from
tBu, Trt
or Clt;
D is H or a guanidine protecting group selected from Pbf or Pmc;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt,
Mmt or
Clt; and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
36. The protected peptide of Claim 36, wherein the protected peptide is
bivalirudin.
37. A protected or partially protected fragment of the formula E-X-
Tyr(Resin)-Leu-O-C
wherein:
X is a bivalirudin peptide sequence;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt,
Mmt or
Clt;
C is a hydroxyl carboxyl protecting group selected from tBu, Trt or Clt; and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
38. A process for the preparation of bivalirudin comprising:
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condensing a bivalirudin fragment of the formula E-X-Tyr(Resin)-Leu-O-C of
Claim
37 in solution or on solid phase with a bivalirudin fragment of the formula E-
D-Phe-Y-OZ to
give E-D-Phe-Y-X-Tyr(Resin)-Leu-O-C;
deprotection or cleavage from the resin and deprotection provides bivalirudin;
purification of bivalirudin by chromatography;
lyophilizing to provide bivalirudin in >99% purity; wherein;
X and Y are independently bivalirudin peptide sequences;
D- designate the chirality of the amino acid that follows as being a D-amino
acid;
C is a hydroxy protecting group selected from Clt, Trt or tBu;
E is an amino protecting group selected from Fmoc, Mtt, Mmt, Trt, Boc or Nps;
and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
39. A protected or partially protected peptide with the formula E-Ser(C)-
Tyr(C)-Ser(C)-
Met-Glu(C)-His(A)-Phe-Arg(D)-Trp(F)-Gly-Lys(E)-Pro- Val-Gly-Lys(E)-Lys(E)-
Arg(D)-
Arg(D)-Pro-Val-Lys(E)-Val-Tyr(Resin)-Pro-O-C, wherein:
A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt or
Mmt;
each C is independently a hydroxy, carboxyl or phenoxy protecting group
selected from
tBu, Trt or Clt;
each D is independently H or a guanidine protecting group selected from Pbf or
Pmc;
each E is independently H or an amino protecting group selected from Fmoc,
Boc, Trt,
Nps, Mtt, Mmt or Clt;
F is H or Boc and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
40. The protected or partially protected peptide of Claim 39 that is a
ACTH(1-24) peptide.
41. A protected or partially protected fragment with the formula E-X-
Tyr(Resin)-Pro-O-C
wherein:
X is a ACTH(1-24) sequence;
E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt,
Mmt or
Clt;
C is a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt or
Clt; and
Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
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42. A process for the production of ACTH(1-24) comprising:
condensing a ACTH fragment of the formula E-X-Tyr(Resin)-Pro-O-C of Claim 37
in
solution or in solid phase with a ACTH fragment of the formula E-Y-OZ to give
E-Y-X-
Tyr(Resin)-Pro-O-C;
deprotecting or cleaving from the resin and deprotection provides ACTH(1-24)
which
is purified by chromatography and lyophilized to provide ACTH(1-24) of >9 9%
purity,
wherein;
X and Y are each independently ACTH sequences;
C is a hydroxy protecting group selected from Clt, Trt or tBu;
E is an amino protecting group selected from Fmoc, Mtt, Mmt, Trt, Boc or Nps;
and
Resin represents H or an acid labile resin suitable for solid-phase peptide
synthesis.
3 0
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Date Recue/Date Received 2023-01-12

Description

SOLID PHASE PEPTIDE SYNTHESIS
VIA SIDE CHAIN ATTACHMENT
SUMMARY:
[0001] Peptides and peptaibols of high purity were obtained by solid phase
peptide synthesis
using as the starting resin hydroxy amino acids, hydroxy amino acid amides,
hydroxy amino
alcohols or small peptides containing hydroxy amino acids attached to polymers
through their
side chain.
Definitions and Abbreviations:
[0002] "Hya" or "hydroxyl amino acid(s)" means amino acids that contain a
hydroxyl (-OH)
group.
[0003] N-terminus or amino terminus is the first amino acid in a peptide
chain.
[0004] C-terminus or carboxy terminus is the last amino acid in the peptide
chain as shown
below.
amino terminal carboxy terminal
or N-terminal or C-terminal
amino acid amino acid
A
r A __ , r ,
NH2-CHR-CO-Aai-AA2 ................................ AA-NH-CHR-CO2H
peptide
[0005] "P" or "solid support" or "resin" means an insoluble material
containing a functional
group(s) suitable to react and link with an amino acid or peptide. The solid
support or resins
are well known in the art.
[0006] "Alkyl" such as Ci_malkyl or C1_6alkyl, means a branched or unbranched
fully saturated
acyclic aliphatic hydrocarbon group (i.e. composed of carbon and hydrogen
containing no
double or triple bonds). In some embodiments, alkyls may be substituted or
unsubstituted.
Alkyls may include, but are not limited to methyl, ethyl, propyl, isopropyl,
butyl, isobutyl, tert-
butyl, pentyl, hexyl, and the like, and in some embodiment, each of which may
be optionally
substituted. Non-exclusive alkyl substituents may include C1_3alkoxy, halo (F,
Cl, Br or I),
nitro, amino, -SH and -OH.
[0007] "Attachment" means the linking of an amino acid or a peptide or peptide
derivative to
an insoluble support.
[0008] "Hse" means homoserine; "Hnv" means hydroxylnorvaline.
[0009] "SPPS" or "solid phase peptide synthesis" means the synthesis of a
peptide with the use
of a resin as described herein.
1
8116434
Date Recue/Date Received 2023-01-12

[0010] "pNA" means 4-nitro anilide.
[0011] "DME" means dimethoxy ethane.
[0012] "Acid sensitive resin" means an insoluble material or resin containing
a functional
group(s) suitable to react and link with an amino acid or peptide, which may
be cleaved from
the peptide by acidic treatment.
[0013] "Acid sensitive protecting group" means a protecting group which may be
cleaved from
the amino acid or peptide or peptide derivative by acidic treatment or under
acidic condition.
[0014] "Peptaibol" means a peptide which contain at its C-terminal position an
amino alcohol
instead of an amino acid or an amino acid amide.
[0015] "Step-by-step" means the method of peptide synthesis where any of the
amino acids
contained in the peptide chain is introduced individually and sequentially.
The method may or
may not involve an intermediate purification step.
[0016] "Protected peptide" means the peptide with all functional groups
blocked or protected
by protecting groups.
[0017] "Partially protected peptide" means the peptide which contains at least
one functional
group blocked or protected by a protecting group.
[0018] Solid phase peptide synthesis is traditionally performed by the
attachment of the C-
terminal amino acid through its a-carboxyl function on a suitable solid
support and elongating
the peptide chain towards the amino terminal of the peptide by adding
sequentially the amino
acid residues in the gradually growing peptide chain. Several hundred
thousands of
publications and patents describe this methodology and its application for the
production of
peptide pharmaceuticals.
[0019] In contrary to the attachment of the C-terminal carboxyl function,
attachment of amino
acids and peptides through an amino acid side chain on suitable resins and
their application in
SPPS is described very briefly, in particular in less than 30 publication and
patents. Most of
these publications describe the attachment of the amino acids through a side
chain carboxyl
function of Asp and Glu. To our knowledge, the reports of the side chain
attachment of amino
acids through a side chain hydroxyl function and application in peptide
synthesis are limited:
The side chain attachment of Fmoc-Hya-pNA (Formula Al) [A. Bernhardt, M.
Drewello and
M. Schutkowski, The solid-phase synthesis of side-chain-phosphorylated peptide-
4-
nitroanilides J. Peptide Res. 50, 1997. 143-1521 and their use for the
synthesis of short
nitroanilide substrates, the synthesis of Fmoc-Hya-Oallyl esters (Formula A2
[L. Rizzi, K.
Cendic, N. Vaiana, S. Romeo, Alcohols immobilization onto 2-
chlorotritylchloride resin under
microwave irradiation, Tetrahedron Letters 52 (2011) 2808-28111) on 2-
chlorotrityl resin
2
8116434
Date Recue/Date Received 2023-01-12

with the aid of microwaves for application in the preparation of cyclic
peptides and the
synthesis of Fmoc-Tyr-O-methyl ester (Formula A3 [C. Cabrele, M. Langer and A.
G. Beck-
Sickinger, Amino Acid Side Chain Attachment Approach and Its Application to
the Synthesis of
Tyrosine-Containing Cyclic Peptides, .1 Org. Chem. 1999, 64, 4353-43611),
attached on resins
of the benzyl-type by the Mitsunobu redox-alkylation of the Tyr-phenoxy
function and their
application for the synthesis of short cyclic peptides. To our knowledge, the
side chain
attachment of Hse and Hyp have never been disclosed. In addition, the
application of side
chain attached Hya on acid sensitive resins for the solid phase synthesis of
protected peptides,
protected peptide fragments and of protected peptide amides and peptaibols
have not been
reported.
'P
0
Al A2 A3
CI CI *
0 P 0 P 0
ill X
0 Fmoc-HN Fmoc-HN 0 Fmoc-HN CO2CH3
HN . NO2 0¨-
Solid Phase Peptide Synthesis
[0020] In one embodiment, there is provided an improved synthesis of peptide
acids, peptide
amides, and peptaibols of pharmaceutical interest.
P P
(
X \
/ %z X
\ H
\d- 0 -1)
Y I V Y I
Pr'-Hya-A Pr'-Hya- A
[0021] Formula I Formula ll
[0022] In one aspect of the present application, the peptides were produced
very efficiently in
high yield and purity by attaching a hydroxy amino acid through its amino acid
side chain, or a
small peptide which contain in its sequence a hydroxy amino acid on a resin of
the trityl or
benzhydryl-type, resulting in amino acid-resin conjugates or peptide resin
conjugates of
Formula I-TV, wherein P is a solid-phase support selected from the supports
used in solid phase
peptide synthesis, Pr' is H or an amino protecting group selected from Fmoc,
Boc, Trt, Dde
and Alloc, wherein pe is an acid sensitive hydroxyl protecting group selected
from Trt, Clt,
Mmt, Mtt, Dpm and tBu, wherein Hya is a hydroxy amino acid selected from D- or
L-Ser, Thr,
Tyr, Hse, Hyp, Hnv etc ., and A is OH, an acid sensitive alkoxy group selected
from OTrt,
0C1t, OMmt, OW ODpm and OtBu, NH2, NHR1, NR1Ic-r.2 wherein RI- and R2 are
3
8116434
Date Recue/Date Received 2023-01-12

independently an alkyl group a protected or semi protected peptide containing
1-10 amino
acids in its sequence.
[0023] In another embodiment, we disclose that peptaibols, such as octreotide,
were obtained
by solid phase synthesis using the resin-bound amino alcohols of the Formula
III-VI selected
from amino alcohols which are derived from the naturally occurring hydroxy
amino acids,
wherein P. X, V. Z and Pr' are as defined above, wherein R3, le are alkyl,
aryl or aralkyl
groups, and Pr' is an acid sensitive protecting group of the trityl,
benzhydryl or benzyl type.
X
/ %Z
-1=1
Y 0 V 0 V
R4 R3 R4 R3
Prl¨N Prl¨N
Formula III Formula IV
X
I-
-1=7
H
V V
0 0
Pr' Prr
Formula V Formula VI
[0024] In addition we disclose for the first time that peptides prepared with
the application of
resins of the Formula I-TV, where the peptides are attached through the side
chain hydroxyl
function of a hydroxyamino acid on resins of the trityl type, may be cleaved
from the resin by
mild acidic treatment and wherein the side chain protecting groups of the tBu
and benzyl-type
remain intact. In one aspect, the cleavage from the resin occurs by the
treatment with 1-3 %
acid solutions, such as TFA, diluted HC1 solutions, optionally adding
scavengers, in a solvent.
In another aspect, the cleavage may be performed in a solvent such as DCM or
acetone. Such
partially protected peptides have been found to be useful in the synthesis of
longer peptides by
fragment condensation in solution or on solid phase. The present method
expands the
versatility of the application of the resins described herein, and also
results in significantly
improving the purity of the resulting pharmaceutical peptides, and at the same
time,
substantially reducing the cost of their synthesis.
4
8116434
Date Recue/Date Received 2023-01-12

[0025] Several peptides of pharmaceutical interest were produced as
representative of the new
process described herein, either by the step-by-step procedure or by fragment
condensation in
solution and on solid phase; or a combination thereof. The examples below are
representative
and do not limit their application in any way to other peptides.
Lanreotide:
[0026] In one embodiment, Lanreotide was produced by solid phase synthesis
using resin-
bound Thr-amide as shown below:
P
= Br-Resin
Frnoc-Thr(Resin)-NH2 -4 DIPEA Fmoc-Thr-NH2 + H OCH3
Br
ISequential deprotection and
coupliing steps, 7 cycles
Boc-D-2-Nal-Cys(Trt)-Tyr(C1t)-D-Trp-Lys(Mtt)-Val-Cys(Trt)-Thr(Resin)-N H2
deprotection, cleavage from the resin
and simultaneous iodine oxidation
I I
H-D-2-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-N H2 Lanreotide
Human insulin B-chain:
[0027] Optionally the human insulin B chain was synthesized by SPPS. In one
aspect, the
synthesis begins from the resin-bound Thr-t-butyl ester as described in the
example, using the
4-methoxy benzhydryl resin. Optionally the synthesis may also be performed on
solid phase
by condensing the 1-8 partially protected Boc-Phe-Val-Asn(TrO-Gln(TrO-His(Trt)-
Leu-
Cys(Trt)-Gly-OH fragment with the resin-bound 9-30 fragment; or after the
selective cleavage
of the partially protected 9-30 fragment from the resin with condensation in
solution of the 1-8
and 9-30 fragments.
P
= Br-Resin
Fmoc-Thr(Resin)-0tBu . DIPEA Fmoc-Thr-OtBu + H OCH3
Br
1 Sequential deprotection and
coupliing steps, 22 cycles
H-Phel-Val-Asn(Trt)-Gln(Trt)-His(Trt)-Leu-Cys(Trt)-Gly-Ser(tBu)-His(Trt)-
Leu-Val-Glu(tBu)-Ala-Leu-Tyr(tBu)-Leu-Val-Cys(Trt)-Gly-Glu(tBu)-
Arg(Pbf)-Gly-Phe-Phe-Tyr(tBu)-Thr(tBu)-Pro-Lys(Boc)-Thr(Resin)3 -0tBu
1 deprotection and cleavage from the resin
H-Phel-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-

Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr3 -OH human Insulin B-Chain
8116434
Date Recue/Date Received 2023-01-12

Salmon Calcitonin:
[0028] Optionally salmon calcitonin may be produced starting the synthesis
from resin bound
Fmoc-Thr-Pro-NH2. The peptide chain is then elongated using Fmoc-amino acids.
P
= Br-Resin
Fmoc-Thr(Resin DIPEA)-Pro-NH, _.., Fmoc-Thr-Pro-NH2 + H
OCH,
1
Sequential deprotection and
coupliing steps, 22 cycles Br
11
H-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-
Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-
Thr(Resin)-
Pro-NH2 32
1
I I
Boc-Cys-Ser(tBu)-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys-Val-Leu-Gly-OH
1 10
1 I I
Boc-Cys-Ser(tBu)-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys-Val-Leu-Gly-Lys(Boc)-
Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-

Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Thr(Resin)-Pro-NH2
32
TFA/TES/H20
1 I I
H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-
His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2
32
Salmon Calcitonin
[0029] Optionally the resin-bound salmon calcitonin is produced by fragment
condensation on
the resin as shown above, for example, or in solution as shown below using 2-4
fragments.
P
= Br-Resin
...
Fmoc-Thr(Resin)-Pro-NH2 _DIPEAFmoc-Thr-Pro-NH2 + H OCH3
1
Sequential deprotection and
coupliing steps, 22 cycles Br
11
H-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-
Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-
Thr(Resin)-
Pro-NH2 32
2% TFA/TES
11
H-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-
Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Thr-
Pro-
NI-1 32
I I
Boc-Cys-Ser(tBu)-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys-Val-Leu-Gly-OH
1 In
v
1 I I
Boc-Cys-Ser(tBu)-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys-Val-Leu-Gly-Lys(Boc)-
Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Tr)-Thr(tBu)-
Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(Trt)-Gly-Thr-Pro-NH2
32
TFA/TES/H20
1 I I
H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-
His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2
32
Salmon Calcitonin
6
8116434
Date Recue/Date Received 2023-01-12

Octreotide:
[0030] In another embodiment, octreotide was efficiently synthesized by the
attachment of
Fmoc-threoninol-OTrt to the 4-methoxybenzhydryl resin through the side chain
of threoninol
as shown below, followed by the octreotide chain assembly using Fmoc-amino
acids and
finally cleaving octreotide from the resin with subsequent or simultaneous Cys-
oxidation.
Fmoc-threoninol-OTrt is much easier to be produced than the Fmoc-Thr(tBu)-ol
which may be
attached onto the resin through the hydroxymethyl group of threoninol on a
suitable resin.
This is because H-Thr(OtBu)-ol, used as the starting material for the
production of Fmoc-
Thr(tBu)-ol, is much more difficult to be produced than Fmoc-threoninol-OTrt
used in the
attachment of threoninol through its side chain onto the resin.
OCH3
= Resin-Br
OH OH Fmoc-HN H P 0-ResIn
Br + Trt CI DIPEA Frn. FINCH, CH,
_________________________________________________ Fmoc-HN
¨OH O-Trt DIPEA O-Trt
0-Resin
CF13
H D Phe Cys(Mmt) Phe D Trp Lys(Mtt) Phe Thr(Trt)-Cys(Mmt) ¨HN¨
0-Txt
ITFA/DCM/TIPS
OH
_CH3
H D Pile Cys Phe D Trp Lys Phe Thr Cys HN
1 oxidation OH
OH
HCH,
H-D-Phe-Cys-Phe-D-Trp-Lys-Phe-Thr-Cys ¨N Octreotide
OH
Exenatide:
[0031] In another example, Fmoc-Ser-NH2 was attached through its side chain on
trityl resin
and used for the synthesis of exenatide. The synthesis may be performed by the
step-by-step
manner or by fragment condensation in solution after cleavage a partially
protected exenatide
fragment from the resin by mild acidic treatment or on solid phase, as
described below.
According to this method, most impurities typically formed during the
synthesis of many Pro
and Gly residues containing peptides are completely avoided and peptides of
high purity are
obtained. The method also allows the complete avoidance of impurities
originating from the
cleavage of peptides from peptide amide linkers using other methods known in
the art, which
significantly reduce the yields and purity of the peptide.
7
8116434
Date Recue/Date Received 2023-01-12

OH 0-Resin
Fmoc-HN +
¨ DIPEA
______________________________________ Fmoc-HN
_
CI P = Fmoc-Ser(Resin)-NH2
" ___
NH2 NH2
o = Resin-CI 0
step-by-step
38 cycles
r
H-His(Trt)-Gly-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-X-Lys(Boc)-
Gln(Trt)-Met-Glu(tBu)-Glu(tBu)-
Glu(tBu)-Ala-Val-Arg(Pbe-Leu-Phe-Ile-Glu(tBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-
Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-
Gly-Ala-Pro-Pro-Pro-Ser(Resin)-NH2
X = Leu-Ser(tBu) or Leu-LIJSer
1 Deprotection
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-
Arg-Leu-Phe-Ile-Glu-Trp-Leu-
Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-N H2
Exenatide
[0032] In one aspect, exenatide may be produced by cleavage of the partially
protected peptide
12-39 from the resin and condensing it in solution as shown below with the
partially protected
1-11 fragment. Alternatively, the condensation to obtain protected exenatide
may be
performed with the fragments 1-13 and 14-39.
OH 0-Resin
Fmoc-HN + C P DIPEA Fmoc-HN
-.1_ = Fmoc-Ser(Resin)-N H2
I
NH2 NH2
0 I = Resin-CI 0
1step-by-step
H-Gln(Trt)-Met-Glu(tBu)-Glu(tBu)-Glu(tBu)-Ala-Val-Arg(Pbf)-Leu-Phe-Ile-
Glu(tBu)-Trp(Boc)-Leu-
Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-
N H2
1 Boc-His(Trt)-Gly-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-
Ser(tBu)-Asp(tBu)-X-Lys(Boc)-OH
condensation X= Leu-Ser(tBu) or Leu-N,Ser
Boc-His(Trt)-Gly-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-X-
Lys(Boc)-Gln(Trt)-Met-
Glu(tBu)-Glu(tBu)-Glu(tBu)-Ala-Val-Arg(Pbf)-Leu-Phe-Ile-Glu(tBu)-Trp(Boc)-Leu-
Lys(Boc)-Asn(Trt)-
Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-N H2
1 deprotection, cleavage
from the resin
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-
Arg-Leu-Phe-Ile-Glu-Trp-Leu-
Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-N H2
Exenatide
Pramlintide:
[0033] The method is also highly effective in the production of amylin
peptides. In one aspect,
the side chain attachment may be performed using one of the C-terminal Ser,
Thr or Tyr
residues of amylin or its derivatives such as pramlintide. The synthesis may
be performed in
the step-by-step manner or by fragment condensation in solution or on solid
phase. By
8
8116434
Date Recue/Date Received 2023-01-12

incorporating pseudoprolines OP, see Mutter et al, Peptide Res. (1995 8, 145)
into the growing
peptide chain, the synthesis is accelerated and the purity of the peptide
obtained is improved.
Fmoc-Tyr(Resin)-NH2
step-by-step
Boc-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(tBu)-Y-Cys(Trt)-Y-Gin(Trt)-Arg(Pbf)-Leu-Ala-
Asn(Trt)-Phe-
Leu-Val-His(Trt)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr(tBu)-
Asn(Trt)-Val-Gly-
Ser(tBu)-Asn(Trt)-Thr(tBu)-Tyr(Resin)-N H2
Resin = 2-chlorotrityl resin
1%-TFA/DCM/12
I 1
Boc-Lys(Boc)-Cys-Asn(Trt)-Thr(tBu)-Y-Cys-Y-Gin(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-
Phe-Leu-Val-
His(Trt)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-
Gly-Ser(tBu)-
Asn(Trt)-Thr(tBu)-Tyr-NH2
deprotection
I I
H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gin-Arg-Leu-Ala-Asn-Phe-Phe-Leu-Val-His-
Ser-Ser-Asn-Asn-Phe-Gly-Pro-lle-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-N
H2
pramlintide
X = Ser(tBu)-Ser(tBu) or Ser(tBu)-LIJSer
Y = Ala-Thr(tBu) or Ala-q-/Thr
= pseudoprolin
[0034] Alternatively, the synthesis of pramlintide may be performed in liquid
phase with equal
success concerning the purity and the yield of the obtained pramlintide. In
one embodiment,
the protected peptide which is bound on the resin through the side chain of
Fmoc-Tyr-NH2 may
be quantitatively cleaved from the resin with the side chain protecting groups
of the tBu-type
remaining intact, using mild acidic treatment at various positions of the
peptide chain. In one
example, as shown below the partially protected 1-10 fragment prepared on the
2-chlorotrityl
resin in the step by step manner was condensed successfully with the partially
protected 11-37
fragment amide.
[0035] Pramlintide:
Fmoc-Tyr(Resin)-NH2
lir step-by-step
H-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-
Pro-lle-Leu-
Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr(tBu)-Tyr(Resin)-N H2
Resin = 2-chlorotrityl resin I %-TFA/DCM/TES
H-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-X-Asn(Trt)-Asn(Trt)-Phe-Gly-
Pro-lle-Leu-Pro-
Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tB ll )-Asn(Trt)-Thr(tB1-1)-Tyr-N H2
I I
X = Ser(tBu)-Ser(tBu) or Ser(tBu)--PSer Boc-Lys(Boc)-Cys-Asn(Trt)-Thr(tBu)-
Y-Cys-Y-Gln-OH
Y = Ala-Thr(tBu) or Ala-LliThr condensation
deprotection
pramlintide
9
8116434
Date Recue/Date Received 2023-01-12

Tetracosactide (ACTH 1-24):
[0036] In another example, ACTH 1-24 was effectively prepared starting from
resin-bound
Fmoc-Tyr-Pro-OtBu by the step by step procedure or by condensing the 1-10
partially
protected fragment in solution with the 11-24 fragment or with the resin-bound
11-24
fragment, as shown below.
Fmoc-Tyr-OH + H-Pro-OtBu coupling Fmoc-Tyr-Pro-OtBu =
CI-Resin
I Resin-CUDIPEA CI P
CI
Fmoc-Tyr(Resin)-Pro-OtBu
1 step-by-step
H-Lys(Boc)-Pro- Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-
Val-Tyr(Resin)-Pro-OtBu
Boc-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(tBu)-
His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-OH
condensation step-by-step then
deprotection and cleavage
from the resin
Boc-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(tBu)-His(Trt)-Phe-
Arg(Pbf)-Trp(Boc)-Gly-OH-Lys(Boc)-Pro- Val-Gly-Lys(Boc)-
Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(Resin)-Pro-
OtBu
deprotection, cleavage
from the resin .
H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-
Val-Lys-Val-Tyr-Pro-OH
Tetracosactide (ACTH 1-24)
Bivalirudin:
[0037] In another example, bivalirudin was produced in high yield and high
purity starting
from resin-bound Fmoc-Tyr-Leu-OtBu, extending the peptide chain in the step-by-
step manner
with Fmoc-amino acids and finally deprotecting and cleaving the peptide from
the resin as
shown below.
[0038] Alternatively, bivalirudin was obtained by the condensation of
protected fragments on
the resin or by cleaving a partially protected peptide which contain 4-15
amino acid residues
from the resin and condensing it in solution with a bivalirudin fragment which
contain 5-16
amino acids. The bivalirudin synthesis by fragment condensation on the resin
of the 1-10
partially protected bivalirudin fragment with the resin-bound 11-20 partially
protected
bivalirudin fragment is described below.
8116434
Date Recue/Date Received 2023-01-12

coupling
_,...
Fmoc-Tyr-OH + H-Leu-OtBu Fmoc-Tyr-Leu-OtBu = CI-Resin
Resin-Cl/DIPEA I
CI CI P
Fmoc-Tyr(Resin)-Leu-OtBu
1 step-by-step
H-Asp(tBu)-Phe-Glu(tBu)-Glu(tBu)-1Ie-Pro-Glu(tBu)-Glu(tBu)-Tyr(Resin)-Leu-OtBu
Boc-D-Phe-Pro-Arg(Pbf)-Pro-
Gly-Gly-Gly-Gly-Asn(Trt)-Gly-OH
condensation step-by-step then
deprotection and cleavage
from the resin
Boc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-
Asn(Trt)-Gly-Asp(tBu)-Phe-Glu(tBu)-Glu(tBu)-1Ie-
Pro-Glu(tBu)-Glu(tBu)-Tyr(Resin)-Leu-OtBu
1
deprotection, cleavage
from the resin 1'
H-D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-
Tyr-Leu-OH
Bivalirudin
EXAMPLES
Example 1:
[0039] Preparation of Fmoc-Thr(4-methoxybenzhydryl polystyry1)-0tBu
P
= Br-Resin
Fmoc-Thr-OtBu + H Br DIPEA OCH3 ,-- Fmoc-Thr(Resin)-0tBu
[0040] 30 mmol Fmoc-Thr-OtBu prepared from H-Thr-OtBu by its reaction with
Fmoc-OSu
following conventional methods were reacted with 20 g (30 mmol) of 4-
methoxybenzhydryl
polystyrene resin (product of CBL-Patras) and 60 mmol DIPEA in 250 ml THF for
10 h at RT.
To the mixture were then added 60 mmol methanol and the mixture was shaken for
additional
4 h. The resin was filtered and washed 3X with THF/Me0H/DIPEA (85:10:5), 6X
DMF, 4X
IPA, 3X DEE and dried in vacuum to constant weight. 29 g of resin-bound Fmoc-
Thr-OtBu
were obtained with a loading of 0.95 mmol/g resin.
Example 2
[0041] Fmoc-Thr(4-methoxybenzhydryl poly styry1)-0-Clt
P
= Br-Resin
Trt-Thr-OMe + H Br DIPEA OCH3 ..- Trt-
Thr(Resin)-0Me
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10/ TFA,. 1N-LiOH
______________________ H-Thr(Resin)-0Me __
i.- H-Thr(Resin)-OH
Fmoc-OSu Clt-CI . Fmoc-Thr(Resin)-OH > Fmoc-Thr(Resin)-0-Clt
DIPEA
[0042] 30 mmol Trt-Thr-OMe prepared from H-Thr-OMe by its reaction with Trt-
C1/Me3SiC1
and DIPEA following conventional methods were reacted with 20 g (30 mmol) of 4-
methoxy
4'-polystyryl benzhydryl bromide resin (product of CBL-Patras) and 60 mmol
DIPEA in 250
ml THF for 10 h at RT. To the mixture were then added 60 mmol methanol and the
mixture
was shaken for additional 4 h. The resin was filtered and washed 3X with
THF/Me0H/DIPEA
(85:10:5), 3X DCM, 3X 1% TFA in DCM, 4X THF, 3X 1N-LiOH in THF/Water/Methanol
(70:15:15), 3X THF/Water (75:25) 4X DMF and then reacted for 2 hat RT with 60
mmol
Fmoc-OSu and 30 mmol DIPEA, washed 3X DMF, 3X DCM and then reacted for 3h at
RT
with 50 mmol Trt-Cl and 50 mmol DIPEA, washed 4X DMF, 6X DEE and dried in
vacuum to
constant weight. 32.3 g of resin-bound Fmoc-Thr-OtBu were obtained with a
loading of 0.78
mmol/g resin.
Example 3
[0043] Fmoc-Throl(4-methoxy benzhydryl polystyry1)-0-Clt
[0044] A) Starting from Fmoc-threoninol
OCH3
= Resin-Br
OH OH H P 0-Resin
_CH3 +
DIPEA )¨CH, Br _CH3
Fmoc-HN at-CI Fmoc-HN_ Fmoc-HN
OH 0-CR DIPEA 0-Clt
[0045] 50 mmol commercial Fmoc-threoninol (CBL-Patras) in 350 ml DCM were
reacted with
55 mmol monomeric CU-CI and 55 mmol DIPEA for 4 h at RT. The obtained mixture
was
extracted as usual with water and the DCM phase was dried over anhydrous
sodium sulphate
and filtered. To the resulting solution 30 g of 4-methoxy, 4-polystyryl
benzhydryl bromide
(CBL-Patras) were added and 50 mmol DIPEA and the resulting mixture was
stirred for 4 h at
RT. The resin was filtered and washed 6XDMF, 4XIPA and 4X DEE and dried in
vacuum to
constant weight. 38.4 g of resin-bound Fmoc-threoninol were obtained with a
loading of 0.82
mmol/g.
[0046] B) Starting from Trt-Thr(Resin)-0Me
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Me 0-resin LiBH4 Me 0-resin 1% TFA Me 0-resin
OH H2NOH
0
Me 0-resin
Fmoc-OSu CR-CI Me 0-resin
Fmoc¨N DIPEA
[0047] 30 mmol Trt-Thr-OMe prepared from H-Thr-OMe by its reaction with Trt-
C1/Me3SiC1
and DIPEA following conventional methods were reacted with 20 g (30 mmol) of 4-
methoxy
4'-polystyryl benzhydryl bromide resin (product of CBL-Patras) and 60 mmol
DIPEA in 250
ml THF for 10 h at RT. To the mixture were then added 60 mmol methanol and the
mixture
was shaken for additional 4 h. The resin was filtered and washed 3X with
THF/Me0H/DIPEA
(85:10:5), 5X THF, and then reacted with 30 mmol LiBILI in THF. The resin was
then filtered
and washed 6X THF, 4X DCM, 6X 1% TFA in DCM, 3X with DMF/DIPEA (97:3) and then

reacted for 2 h at RT with 60 mmol Fmoc-OSu and 30 mmol DIPEA, washed 3X DMF,
3X
DCM and then reacted for 3h at RT with 50 mmol CU-CI and 50 mmol DIPEA, washed
4X
DMF, 6X IPA and 6X DEE and dried in vacuum to constant weight. 34.7 g of resin-
bound
Fmoc-Throl-O-Clt were obtained with a loading of 0.74 mmol/g resin.
Example 4
[0048] Fmoc-Ser(trityl resin)-NH2
[0049] 50 mmols of Fmoc-Ser-NH2, prepared according to standard procedures
known in the
art, were dissolved in 0.5 liter of DCM. To the suspension 30 g of Trityl
chloride resin (36
mmol) were added and 65 mmol DIPEA and the mixture was stirred for 6 h at RT.
Then and
then 25 ml methanol and 30 mmol DIPEA were added and the mixture was stirred
for
additional 2h at RT. The resin was then filtered and washed 3X with
DCM/Me0H/DIPEA
(90:5:5), 5X DMF, 4X IPA, 4X DEE and dried in vacuum to constant weight. 41.1
g of Fmoc-
Ser-NH2-containing resin were obtained with a loading of 0.71 mmol/g.
Example 5
[0050] Fmoc-Tyr(2-chlorotrityl resin)-NH2
[0051] Following the above procedure, 50 mmol Fmoc-Tyr-NH2 and 30 g 2-CTC
chloride
resin gave 43.7 g resin with a loading of 0.81 g Tyr/g resin.
Example 6
[0052] Fmoc-Hyp(4-methyl benzhydryl resin)-NH2
[0053] Following the above procedure 50 mmol Fmoc-Hyp-NH2 and 30 g 4-methyl
benzhydryl bromide resin gave 39.8 g resin with a loading of 0.49 g Hyp/g
resin.
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Example 7
[0054] Fmoc-Thr(4-methoxybenzhydryl resin)-Pro-N}12
[0055] 50 mmols of Fmoc-Thr-Pro-NI-12 prepared from coupling of Fmoc-Thr(tBu)-
OH with
H-Pro-NH2 according to standard procedures known in the art, were dissolved in
0.5 liter of
DME. To the resulting solution 30 g of 4-methoxy benzhydryl bromide resin (45
mmol) were
added and 65 mmol DIPEA and the mixture was stirred for 6 h at RT. Then 25 ml
methanol
and 50 mmol DIPEA were added and the mixture was stirred for additional 2h at
RT. The
resin was then filtered and washed 3X with DME/Me0H/DIPEA (90:5:5), 5X DMF, 4X
IPA,
4X DEE and dried in vacuum to constant weight. 44.5 g of Fmoc-Thr-Pro-NH2
containing
resin with a loading of 0.77 mmol/g was obtained.
Example 8
[0056] Fmoc-Tyr(2-chlorotrityl resin)-Pro-OtBu
[0057] 50 mmols of Fmoc-Tyr-Pro-OtBu were prepared according to standard
procedures
known in the art, were dissolved in 0.5 liter of DCM. To the resulting
solution 30 g of 2-
chlorotrityl chloride resin (48 mmol) were added and 65 mmol DIPEA and the
mixture was
stirred for 12 h at RT. Then 25 ml methanol and 50 mmol DIPEA were added and
the mixture
was stirred for additional 2h at RT. The resin was then filtered and washed 3X
with
DCM/Me0H/DIPEA (90:5:5), 5X DMF, 4X IPA, 4X DEE and dried in vacuum to
constant
weight. 44.5 g of Fmoc-Tyr-Pro-OtBu containing resin with a loading of 0.64
mmol/g was
obtained.
Example 9
[0058] Fmoc-Tyr(2-chlorotrityl resin)-Leu-OtBu
[0059] 50 mmols of Fmoc-Tyr-Leu-OtBu, prepared according to standard
procedures known in
the art, were dissolved in 0.5 liter of THF. To the resulting solution 30 g of
2-CTC chloride
resin (48 mmol) were added and 65 mmol DIPEA and the mixture was stirred for
12 h at 60 C.
Then 25 ml methanol and 50 mmol DIPEA were added and the mixture was stirred
for
additional 2 h at RT. The resin was then filtered and washed 3X with
DCM/Me0H/DIPEA
(90:5:5), 5X DMF, 4X IPA, 4X DEE and dried in vacuum to constant weight. 44.5
g of Fmoc-
Tyr-Leu-OtBu-containing resin with a loading of 0.64 mmol/g was obtained.
Example 10
[0060] Solid-phase synthesis of peptides and protected peptide segments.
General procedure.
[0061] Al. Preparation of loaded 2-chlorotrityl resins, general procedure
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[0062] 2-Chlorotrityl chloride resin (CTC-C1) (100 g; loading 1.6 mmol/g) of
CBL-Patras, is
placed in a 2 L peptide synthesis reactor and is swollen with 700 mL
dichloromethane
(DCM):dimethylformamide (DMF) 1:1 for 30 min at 25 C. The resin is filtered
and a solution
of 100 mmol Fmoc-amino acid and 300 mmol diisopropylethylamine (DIEA) in 500
mL DCM
is added. The mixture is stirred under nitrogen for 2 hours at 25 C. Then,
the remaining
active sites of the 2-CTC resin are neutralised by adding 10 mL of methanol
(Me0H) and
reacting for 1 hour. The resin is filtered and washed twice with 400 mL DMF.
The resin is
filtered and treated twice with 500 mL 25% by volume of piperidine in DMF for
30 min. The
resin is then washed four times with 500 mL DMF. The resin is deswelled with 3
washes with
500 mL of isopropanol (IPA). The resin is dried to constant weight. On the
resin was bound
the 70-95% of the mmol of the used amino acid.
[0063] B. Solid-phase synthesis, a general protocol
[0064] The solid-phase synthesis was performed at 24 C with 1.0 g amino acid
or peptide
esterified to the resin of the trityl or benzhydryl type or attached through
its side chain as
described in Part A or in the examples Example 1. The following protocol was
used in the
synthesis.
[0065] Bl. Swelling of the resin
[0066] The resin was placed in a 15 ml reactor and treated twice with 7 mL
NMP, followed by
filtration.
[0067] B2. Activation of the amino acid
[0068] The amino acid (3.0 equiv.) and 1-hydroxybenzotriazole (4.0 equiv.) was
weighted and
dissolved in a reactor with 2.5 their volume in NMP and cooled to 0 C. DIC
was then added
(3.0 equiv.) and the mixture was stirred for 15 min.
[0069] B3. Coupling
[0070] The solution which was prepared in B2 was then added to the B1 reactor.
The reactor
was washed once with one volume of DCM and was added to the reactor which was
stirred for
1-3 h at 25 -30 C. In a sample the Kaiser Test was performed to determine
the completion of
the reaction. If the coupling reaction was not completed after 3 h (positive
Kaiser Test), the
reaction mixture was filtered and recoupled with a fresh solution of activated
amino acid.
After completion of the coupling the reaction mixture was filtered and washed
4 times with
NMP (5 volumes per wash).
[0071] B4. Removal of the Fmoc-group
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[0072] The resulting resin in B3 was filtered and then treated for 30 min with
5 mL of a
solution which contained 25% by volume of piperidine. The resin is then washed
three times
with 5 mL NMP.
[0073] B5. Elongation of the peptide chain
[0074] After the incorporation of each amino acid the steps Bl-B5 were
repeated until the
completion of the peptide chain.
[0075] For the introduction of each individual amino acid the following Fmoc-
amino acids
were used: Fmoc-Ala-OH, Fmoc-Arg(Pb0-0H, Fmoc-Asn-OH, Fmoc-Asn(TrO-OH, Fmoc-D-
Cys(TrO-OH, Fmoc-Cys(TrO-OH, Fmoc-Gln-OH, Fmoc-Gln(TrO-OH, Fmoc-Glu(tBu)-0H,
Fmoc-Gly-OH, Fmoc-His(TrO-OH, Fmoc-Hyp(tBu)-0H, Fmoc-Ile-OH, Fmoc-Leu-OH,
Fmoc-Met-OH, Fmoc-D-Phe-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-0H, Fmoc-
Ser(TrO-OH, Fmoc-Thr(tBu)-0H, Fmoc-Ser(TrO-OH, Fmoc-D-Trp-OH, Fmoc-Trp-OH,
Fmoc-D-Trp(Boc)-0H, Fmoc-Trp(Boc)-0H, Fmoc-Tyr(tBu)-0H, Fmoc-Tyr(C10-0H, Fmoc-
Val-OH, Boc-D-Cys(TrO-OH, Boc-His(TrO-OH, Boc-Lys(Boc)-0H, Boc-D-2-Nal-OH, Boc-

D-Phe-OH, Boc-Ser(tBu)-0H.
[0076] C. General method for the acidic cleavage from the CTC- resin of
peptides and of
protected peptide segments, which contain Fmoc- or Boc-groups on their N-
terminus.
[0077] The resin-bound peptide or peptide segment which was produced as
described above in
B1-B5 was washed 4 times with 5 mL NMP, 3 times with 5 ml IPA and finally 5
times with 7
ml DCM to remove completely any residual NMP or other basic components. The
resin was
then cooled to 0 C, filtered from DCM and was treated twice with a solution
of 10 mL 1-2%
TFA/DCM at 5 C. The mixture is then stirred 20 min at 0 C and filtered. The
resin is then
washed three times with 10 mL DCM. Pyridine is then added to the filtrates
(1.3 equiv.
relative to TFA) to neutralize the TFA. The cleavage solution in DCM is then
mixed with an
equal volume of water. The resulting mixture is distilled at reduced pressure
to remove DCM
(350 ton at 28 C). The peptide or peptide segment precipitated after the
removal of DCM.
The resulting peptide is washed then with water and dried at 30-35 C under 15
Ton vacuum.
Example 11
[0078] Synthesis of resin-bound protected peptides by the condensation of an N-
terminal
protected fragment with a resin-bound C-terminal protected fragment.
General procedure
[0079] To a solution of 0.15 mmol/ml of an N-terminal protected peptide
fragment in
DMSO/DCM (95:5) are added 0.2 mmol HOBt and the resulting solution is cooled
to 5 C.
Then 0.14 mmol DIC were added and the mixture is stirred for 20 min at 15 C
and added then
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to 0.1 mmol of a resin-bound C-terminal fragment and stirred for additional 6
h at RT. The
completion of the condensation reaction is checked by the Kaiser test. In the
cases where the
Kaiser test remained blue a second condensation was performed in order to
drive the
condensation into completion.
Example 12
[0080] Synthesis of partially protected peptides by the condensation of an N-
terminal protected
fragment with a C-terminal protected fragment in solution.
General procedure
[0081] To a solution of 0.15 mmol/ml of an N-terminal protected fragment in
DCM are added
0.2 mmol HOBt and the resulting solution is cooled to 5 C. Then 0.15 mmol
EDAC were
added and the mixture is stirred for 20 min at 15 C and added then to 0.15
mmol of a C-
terminal protected fragment and stirred for additional 2-5 h at RT. The
completion of the
condensation reaction is checked by HPLC. In the cases where an incomplete
condensation
was observed an additional portion of 0.015 mmol EDAC was added and the
reaction was left
to proceed for an additional hour at RT.
Example 13
[0082] Deprotection and simultaneous cleavage from the resin of peptides.
General method
[0083] 1.00 g of the protected resin-bound peptide, produced as described
above is treated with
20 mL TFA/DTT/water (90:5:5) for 3 h at 5 C and for 1 h at 15 C. The resin
is then washed
3X with the cleavage solution and the combined filtrates are then concentrated
in vacuum and
crude peptide is precipitated by the addition of ether, washed several times
with ether and dried
in vacuum until constant weight over KOH.
Example 14
[0084] Peptide Deprotection
General method
[0085] 1.00 g of the protected peptide, produced as described above was
treated with 20 mL
TFA/DTT/water (90:5:5) for 3 h at 5 C and for 1 h at 15 C. The resulting
solution is
concentrated in vacuum and then the deprotected peptide was precipitated by
the addition of
diisopropylether and washed three times with 10 mL diisopropylether. The
resulting solid was
dried in vacuum (25 C, 15 Ton) until constant weight under KOH.
Example 15
[0086] Purification of crude peptides. Isolation of peptides.
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General procedure
[0087] The solution of the peptides obtained as described above was
concentrated in vacuum
and ice water and ether were added. After separation of the organic layer the
remaining water
solution of the peptide was extracted for additional two times with ether and
the resulting
solution was sparged with nitrogen or helium, filtered and directly loaded on
a semipreparative
column 10x25 cm, LichrospherTM 100, RP-18, 12 micron (Merck); Phase A
= 1%-TFA in
acetonitrile, phase B = 1%-TFA in water; or Kromasil. HPLC fractions
containing the purified
peptide were concentrated in vacuum to remove as much as possible the
contained acetonitrile
and lyophilized using a standard lyophilisation program. prepare the listed
compounds.
[0088] Examples 16 to 23, as noted below, were performed using the above
procedures to
prepare the listed compounds.
[0089] Example 16: Lanreotide
[0090] Example 17: Insulin B-chain
[0091] Example 18: Salmon calcitonin
[0092] Example 19: Octreotide
[0093] Example 20: Exenatide
[0094] Example 21: Pramlintide
[0095] Example 22: Tetracosactide (ACTH 1-24)
[0096] Example 23: Bivalirudin
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