Enzymatic production of glycosylated synthons
The present invention relates to a method for producing a glycosylated synthon or a monomer. Said method includes at least one step of placing at least one glycan-saccharase in the presence of at least one hydroxylated synthon and at least one saccharose. The invention also relates to a method for producing a glyco(co)polymer, including polymerizing at least two monomers separately obtained from the enzymatic glycosylation method according to the invention, and to a method for producing a glyco(co)polymer, preferably a block glyco(co)polymer, including coupling at least two monomers separately obtained from the enzymatic glycosylation method according to the invention.
1. A process for manufacturing a glycosylated synthon, or monomer, comprising at least one step of placing at least one glycan-saccharase in contact with at least one hydroxylated synthon and at least one sucrose, in which:
(A) said hydroxylated synthon is chosen from the group constituted of:
(i) (meth)acrylate/(meth)acrylamide synthons of formula (I):
in which:
R 1 represents a hydrogen atom or a C 1 -C 3 alkyl;
R 2 represents a C 1 -C 20 alkylene group; or a group (C 2 H 4 O) n , with n being an integer between 1 and 10; and
X 1 represents —(O)—, —(NH)—, —(S)— or —(NR′ 2 (OH))—, with R′ 2 representing a C 1 -C 20 alkylene group; or a group —(C 2 H 4 O) m —, with m being an integer between 1 and 10;
(ii) styrene-based synthons of formula (II):
in which R 3 represents a covalent bond; a C 1 -C 20 alkylene group; or a group (C 2 H 4 O) n , with n being an integer between 1 and 10;
(iii) N-carboxyanhydride (NCA) synthons of formula (III):
in which R 4 represents a covalent bond; a C 1 -C 20 alkylene group; or a group (C 2 H 4 O) n , with n being an integer between 1 and 10;
(iv) lactone/lactam/thiolactone synthons of formula (IV):
in which:
R 5 represents a covalent bond; a C 1 -C 20 alkylene group; or a group (C 2 H 4 O) m , with m being an integer between 1 and 10;
n represents an integer between 1 and 20;
X 2 represents —(O)—, —(NH)— or —(S)—; and
R 6 represents a hydrogen or a C 1 -C 20 alkyl group;
(v) synthons of formula (V):
in which R 7 represents a C 1 -C 20 alkylene group; and
(vi) synthons of formula (VI):
in which R 8 represents a C 1 -C 20 alkylene group; and
(B) said glycan-saccharase is selected from the group consisting of
a sequence having at least 80% identity with SEQ ID NO: 1,
a sequence having at least 80% identity with SEQ ID NO: 1 mutated once at any one of the positions R226, I228, F229, A289, F290, I330, V331, D394 and R446;
a sequence having at least 80% identity with SEQ ID NO: 11;
a sequence having at least 80% identity with SEQ ID NO: 12;
a sequence having at least 80% identity with SEQ ID NO: 13;
a sequence having at least 80% identity with SEQ ID NO: 14;
a sequence having at least 80% identity with SEQ ID NO: 15;
a sequence having at least 80% identity with SEQ ID NO: 16;
a sequence having at least 80% identity with SEQ ID NO: 17; and
a sequence having at least 80% identity with SEQ ID NO: 18.
2. The process as claimed in claim 1 , in which the sequence having at least 80% identity with SEQ ID NO: 1 mutated once at any one of the positions R226, I228, F229, A289, F290, I330, V331, D394 and R446 is chosen from:
a sequence having at least 80% identity with SEQ ID NO: 2, said sequence having an amino acid X 1 representing an amino acid chosen from the group constituted of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W and Y;
a sequence having at least 80% identity with SEQ ID NO: 3, said sequence having an amino acid X 2 representing an amino acid chosen from the group constituted of A, C, D, E, F, G, H, L, M, N, P, Q, R, S, T, V, W and Y;
a sequence having at least 80% identity with SEQ ID NO: 4, said sequence having an amino acid X 3 representing an amino acid chosen from the group constituted of A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;
a sequence having at least 80% identity with the sequence SEQ ID NO: 5, said sequence having an amino acid X 4 representing an amino acid chosen from the group constituted of C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;
a sequence having at least 80% identity with the sequence SEQ ID NO: 6, said sequence having an amino acid X 5 representing an amino acid chosen from the group constituted of A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;
a sequence having at least 80% identity with the sequence SEQ ID NO: 7, said sequence having an amino acid X 6 representing an amino acid chosen from the group constituted of A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W and Y;
a sequence having at least 80% identity with SEQ ID NO: 8, said sequence having an amino acid X 7 representing an amino acid chosen from the group constituted of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, 5, T, W and Y;
a sequence having at least 80% identity with SEQ ID NO: 9, said sequence having an amino acid X 8 representing an amino acid chosen from the group constituted of A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and
a sequence having at least 80% identity with SEQ ID NO: 10, said sequence having an amino acid X 9 representing an amino acid chosen from the group constituted of A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, V, W and Y.
3. The process as claimed in claim 1 , in which the sequence having at least 80% identity with SEQ ID NO: 1 mutated once at any one of the positions R226, I228, F229, A289, F290, I330, V331, D394 and R446 is chosen from:
a sequence having at least 80% identity with SEQ ID NO: 2, said sequence having an amino acid X 1 representing an amino acid chosen from the group constituted of C, H, K, M, N, Q, S, T and V;
a sequence having at least 80% identity with SEQ ID NO: 3, said sequence having an amino acid X 2 representing an amino acid chosen from the group constituted of H, L, T, V, W and Y;
a sequence having at least 80% identity with SEQ ID NO: 4, said sequence having an amino acid X 3 representing an amino acid chosen from the group constituted of C, D, E, G, H, I, K, M, N, P, Q, V, W and Y;
a sequence having at least 80% identity with the sequence SEQ ID NO: 5, said sequence having an amino acid X 4 representing an amino acid chosen from the group constituted of C, D, E, F, M, N, P, Q, S, T, V and W;
a sequence having at least 80% identity with the sequence SEQ ID NO: 6, said sequence having an amino acid X 5 representing an amino acid chosen from the group constituted of A, C, D, E, G, H, I, K, L, M, P, Q, S, T, V and W;
a sequence having at least 80% identity with the sequence SEQ ID NO: 7, said sequence having an amino acid X 6 representing an amino acid chosen from the group constituted of A, C, D, E, F, G, H, K, L, M, N, Q, S, V and Y;
a sequence having at least 80% identity with SEQ ID NO: 8, said sequence having an amino acid X 7 representing an amino acid chosen from the group constituted of A, C, D, E, F, G, H, I, K, L, N, Q, R, S, T, W and Y;
a sequence having at least 80% identity with SEQ ID NO: 9, said sequence having an amino acid X 8 representing an amino acid chosen from the group constituted of A, E, F, G, H, I, K and L;
a sequence having at least 80% identity with SEQ ID NO: 10, said sequence having an amino acid X 9 representing an amino acid chosen from the group constituted of A, C, G, K, L, M, N, and S.
4. The process as claimed in claim 1 , in which the hydroxylated synthon is chosen from the group consisting of 2-(hydroxy)ethyl methacrylate (HEMA), N-(hydroxy)methylacrylamide (NHAM), N-(hydroxy)ethylacrylamide (HEAA), 4-vinylphenol (VP), 4-vinylbenzyl alcohol (VBA), 4-(hydroxy)methyloxazolidine-2,5-dione (HMNCA), a-(hydroxy)methylcaprolactone (AHMCL), (±)-mevalonolactone (MVL), 2-mercaptoethanol (BME), 2-propen-1-ol (allyl alcohol) and N,N-bis(2-hydroxyethyl)acrylamide (NNHEA).
5. The process as claimed in claim 1 , in which the hydroxylated synthon is chosen from the group consisting of 2-(hydroxy)ethyl methacrylate (HEMA), N-(hydroxy)methylacrylamide (NHAM), N-(hydroxy)ethylacrylamide (HEAA), 4-vinylbenzyl alcohol (VBA), 2 propen-1-ol (allyl alcohol), 2-mercaptoethanol (BME) and N,N-bis(2-hydroxyethyl)acrylamide (NNHEA).
6. The process as claimed in claim 1 , in which the hydroxylated synthon is glycosylated or fructosylated at the end of the process.
7. A process for manufacturing a glyco(co)polymer, comprising the polymerization of at least two monomers obtained, independently, on conclusion of the process as claimed in claim 1 .
8. The process as claimed in claim 7 , comprising, in the following order, the following steps:
a) polymerization of two monomers obtained, independently of each other, at the end of the process as claimed in claim 1 , making it possible to obtain a chain of two monomers;
b) polymerization of the monomer chain obtained at the end of the preceding step with a monomer obtained at the end of the process as claimed in claim 1 ; and then
c) one or more successive, and independent, steps consisting in polymerizing the monomer chain obtained at the end of the preceding step with a monomer obtained at the end of the process as claimed in claim 1 .
9. The process as claimed in claim 8 , comprising, independently:
at least one step a′) between steps a) and b);
at least one step b′) between steps b) and c); and/or
at least one step c′) after any one of the steps c), in which the monomer chain obtained on conclusion of the preceding step is polymerized with at least one non-glycosylated synthon.
10. A process for manufacturing a glyco(co)polymer, comprising the coupling of at least two monomers obtained, independently, at the end of the process as claimed in claim 1 .
11. A process for manufacturing a glycol(co)polymer, comprising the coupling of a least two monomers obtained, independently, from synthons of Formula (V) and/or (VI) as defined in claim 1 .