# US 9,040,480 B2 · Synthesis of glucagon-like peptide

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- Number: US 9,040,480 B2
- Document ID: US9040480B2
- Status: Granted
- Kind: B2
- Application number: 13/872,952
- Filed: 2013-04-29
- Published: 2013-12-05
- Granted: 2015-05-26
- Earliest claimed: 2006-02-08
- Assignees: Lonza Ltd.
- Applicants: Lonza AG
- Inventors: Oleg Werbitzky; Stéphane Varray; Matthieu Giraud; Carsten Meininghaus
- Web page: https://patentgrep.com/patent/US9040480B2

## CPC

- C07K 14/605: Glucagons
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

A new method of synthesizing GLP-1 peptide is devised.

## Claims

### Claim 1

Method of manufacturing a GLP-1 or GLP-1 agonist peptide, wherein said peptide comprises:
A-(R1)x-(R2)y-R3-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-R8-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-R4-R5-(R6)w-(R7)z-B (SEQ ID NO: 1)
or is of formula
A-(R1)x-(R2)y-R3-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-B (SEQ ID NO: 2)
or is of formula
A-(R1)x-(R2)y-R3-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-B (SEQ ID NO: 3)
or is of formula
A-(R1)x-(R2)y-R3-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-B (SEQ ID NO: 4)
or is of formula
A-(R1)x-(R2)y-R3-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-R8-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-R4-B (SEQ ID NO: 5)
or is of formula
A-(R1)x-(R2)y-R3-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-R8-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-R4-Gly-B (SEQ ID NO: 6)
wherein
B=—OH or —NH2;
A=H-, Ac-, Boc-, Fmoc-;
R1=His, D-His, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine or alpha-methyl-histidine;
R2=Ala, D-Ala, Val, D-Val, Gly, Aib (α-aminoisobutyric acid);
R3=Glu, Asp, Met, Leu;
R4=Lys or Arg;
R5=Gly, Aib, Ala, D-Ala;
R6=Arg, Lys or Gly;
R7=Arg, Lys or Gly;
R8=Gly or Aib
and wherein, independently, x, y, w, z are 0 or 1, with the proviso that y=1 where x=1 and with the proviso that w=1 where z=1, and further wherein individual amino acids may optionally carry protection groups, comprising the steps of
a) synthesizing the peptide on a solid phase by stepwise coupling of Fmoc-protected, amino acids or dipeptides comprising pseudoproline dipeptides, in a linear fashion, with the proviso that at one suitable sequence position, which is Val-Ser- or Val-Ser-Ser, a first pseudoproline dipeptide is coupled to the growing peptide chain,
wherein the pseudoproline dipeptide is selected from the group consisting of Fmoc-Val-Ser(ψMe,Mepro)-OH, Fmoc-Val-Ser(ψH,Hpro)-OH, Fmoc-Ser(P)-Ser(ψMe,Mepro)-OH and Fmoc-Ser(P)-Ser(ψH,Hpro)-OH, wherein P is an acid-cleavable protection group that is cleaved under strongly acidic condition of at least 80% TFA, and
b) cleaving the peptide from the solid phase, and deprotecting the peptide chain with at least 80% TFA.

### Claim 2 (depends on 1)

Method according to claim 1, characterised in that at least one glycidyl residue is additionally N-protected on its backbone Nα with N-(o,p-dialkoxy-benzyl) or with N-(o-hydroxy-p-alkoxy-benzyl) or with N-(o-acyloxy-p-alkoxy-benzyl) with the acyloxy and alkoxy being, independently, C1-C4-alkoxy and C1-C4-acyloxy, respectively, with the proviso that said glycidyl residue is not comprised in a sequence—Gly-Thr(ψMe,Mepro)- or -Gly-Thr(ψH,Hpro)- and is at least spaced apart by two intervening amino acid residues from a pseudoproline or Nα-protected glycidyl residue.

### Claim 3 (depends on 1)

Method according to claim 1, wherein a second pseudoproline dipeptide is spaced apart by at least four intervening amino acid residues from said first pseudoproline dipeptide.

### Claim 4 (depends on 1)

Method according to claim 1, wherein the first pseudoproline dipeptide is Fmoc-Val-Ser(ψMe,Mepro)-OH.