Acylated glucagon analogues

A method of producing a compound having the formula:

• R1—P1—P2—R2
• wherein
• R1 is H, C1-4 alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;
• R2 is OH or NH2;
• P1 is a peptide having the sequence:
• His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-X24-WLE-X28-A
• wherein:
• X2 is selected from Aib, Ac3c, Ac4c and Ac5c;
• X3 is selected from Gln and His;
• X15 is selected from Asp and Glu;
• X16 is selected from Glu and ψ;
• X17 is selected from Arg and ψ;
• X18 is selected from Ala and Arg;
• X20 is selected from Lys and His;
• X24 is selected from Glu and ψ;
• and
• X28 is selected from Ser and ψ;
• and P2 is absent or is a sequence of 1-20 amino acid units independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn;
• wherein the compound contains one and only one ψ,
• and wherein said ψ is a residue of Lys, Arg, Orn or Cys in which the side chain is conjugated to a substituent having the formula —Z2Z1;
• —Z1 is a fatty chain having a polar group at one end of the chain and a connection to Z2, —X— at the end of the chain distal from the polar group,
• wherein the polar group comprises a carboxylic acid or a carboxylic acid bioisostere, a phosphonic acid, or a sulfonic acid group;
• and —X— is a bond, —CO—, —SO—, or —SO2—;
• —Z2— is a spacer of formula:
• wherein:
• each Y is independently —NH, —NR, —S or −0, where R is alkyl, a protecting group or forms a linkage to another part of the spacer Z2;
• each X is independently a bond, CO—, SO—, or SO2—;
• with the proviso that when Y is —S, X is a bond;
• each V is independently a bivalent organic moiety linking Y and X;
• and n is 1-10;
• the method comprising expressing a precursor peptide sequence from a nucleic acid construct that encodes the precursor peptide, recovering the expression product, and modifying the precursor peptide to yield the compound.