N-GLYCOSYLATION OF PEPTIDES AND PROTEINS
A process for the production of a glycoconjugate by N-glycosylation of a protein or peptide comprising the sequence D/E-X—N—X—S/T, wherein each X is the same or different and is any natural amino acid other than proline, wherein the process comprises reacting the protein or peptide with a polyisoprenyl pyrophosphate of formula (I), or a salt thereof, in the presence of PglB: (I) to produce the glycoconjugate comprising the protein or peptide having a saccharide [SI] linked to the asparagine in the sequence D/E-X—N—X—S/T. Polyisoprenylpyrophosphates used as substrates in the biocatalytic process are also provided, as well as certain glycoconjugates.
1 . A process for the production of a glycoconjugate by N-glycosylation of a protein or peptide comprising the sequence D/E-X—N—X—S/T, wherein each X is the same or different and is any natural amino acid other than proline,
wherein the process comprises reacting the protein or peptide with a polyisoprenyl pyrophosphate of formula (I), or a salt thereof, in the presence of PglB:
wherein n is 1, 2, 3 or 4; and
[S1] is a monosaccharide or polysaccharide;
to produce the glycoconjugate comprising the protein or peptide having a saccharide [S1] linked to the asparagine in the sequence D/E-X—N—X—S/T.
2 . A process according to claim 1 , wherein the protein or peptide comprising the sequence D/E-X—N—X—S/T is an antibody.
3 . A process according to claim 1 , wherein n is 1, 2 or 3.
4 . A process according to claim 1 , wherein [S1] is a mono- or disaccharide.
5 . A process according to claim 1 , wherein the saccharide [S1] is modified at one or more positions.
6 . A process according to claim 1 , wherein [S1] is GlcNAc or GlcNAc-GlcNAc, and wherein [S1] is optionally further modified at one or more additional positions.
7 . A process according to claim 1 , wherein [S1] is modified to carry one or more groups R1, wherein R1 comprises a reactive group.
8 . A process according to claim 7 , wherein the reactive group is an azide, alkene or alkyne group.
9 . A process according to claim 7 , wherein R1 comprises the reactive group and a linker moiety L.
10 . A process according to claim 9 , wherein L is selected from an alkylene moiety, an oxy-alkylene- moiety and a moiety of formula —NH—C(═O)-alkylene-.
11 . A process according to claim 9 , wherein R1 is a group of formula —(C 1 -C 6 alkylene)-Y, —O—(C 1 -C 6 alkylene)-Y, —NH—C(═O)—(C 1 -C 6 alkylene)-Y, —NH—C(═O)—O—(C 1 -C 6 alkylene)-Y, —O—C(═O)—(C 1 -C 6 alkylene)-Y or —O—C(═O)—O—(C 1 -C 6 alkylene)-Y, wherein Y is a reactive group.
12 . A process according to claim 1 , which process further comprises reacting the glycoconjugate with a saccharide [S2] to produce a modified glycoconjugate comprising the protein or peptide having an extended saccharide [S1-S2] linked to the asparagine in the sequence D/E-X—N—X—S/T.
13 . A process according to claim 12 , wherein the saccharide [S2] is a mono- or polysaccharide modified to carry a group R2, wherein R2 comprises a reactive group capable of reacting with R1 or with OH.
14 . A process according to claim 13 , wherein [S2] is further modified to carry one or more labelling groups.
15 . A process according to claim 14 , wherein the labelling group is a radioactive label selected from 18-F, 64-C and 131-I.
16 . A polyisoprenyl pyrophosphate of formula (I) or a salt thereof
wherein n is 1, 2 or 3 and [S1] is a mono saccharide or polysaccharide.
17 . A process for the production of a glycoconjugate by N-glycosylation of a protein or peptide comprising the sequence D/E-X—N—X—S/T, wherein each X is the same or different and is any natural amino acid other than proline,
wherein the process comprises reacting the protein or peptide with a polyisoprenyl pyrophosphate of formula (I), or a salt thereof, in the presence of PglB:
wherein n is 1 to 7; and
[S1] is a monosaccharide or polysaccharide which is modified to carry one or more groups R1, wherein R1 comprises a reactive group;
to produce the glycoconjugate comprising the protein or peptide having a saccharide [S1] linked to the asparagine in the sequence D/E-X—N—X—S/T.
18 . A process according to claim 17 , which process further comprises reacting the glycoconjugate with a saccharide [S2] to produce a modified glycoconjugate comprising the protein or peptide having an extended saccharide [S1-S2] linked to the asparagine in the sequence D/E-X—N—X—S/T.
19 . A process according to claim 6 , wherein [S1] is modified to carry one or more groups R1, wherein R1 is a group of formula —(C 1 -C 6 alkylene)-Y, —O—(C 1 -C 6 alkylene)-Y, —NH—C(═O)—(C 1 -C 6 alkylene)-Y, —NH—C(═O)—O—(C 1 -C 6 alkylene)-Y, —O—C(═O)—(C 1 -C 6 alkylene)-Y or —O—C(═O)—O—(C 1 -C 6 alkylene)-Y, wherein Y is a reactive group.