Enzymatic production of glycosylated synthons

Patent metadata

Field	Value
Publication number	US-10696995-B2
Application number	US-201615559193-A
Country	US
Kind code	B2
Filing date	Mar 17, 2016
Priority date	Mar 19, 2015
Publication date	Jun 30, 2020
Grant date	Jun 30, 2020

Abstract

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.

First claim

The invention claimed is:

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)methylcapr

Assignees

• Inst Nat De La Rech Agronomique Inra
• Institut Nat Des Sciences Appliquees De Toulouse Insat
• Centre Nat Rech Scient
• Univ Bordeaux
• Inst Polytechnique Bordeaux
• Institut National De Rech Pour Lagriculture Lalimentation Et Lenvironnement
• Institut Nat Des Sciences Appiquees De Toulouse Insat

Inventors

• Andre Isabelle
• Grelier Stephane
• Guieysse David
• Lafraya Alvaro
• Monsan Pierre
• Moulis Claire
• Peruch Frederic
• Remaud-Simeon Magali
• Vuillemin Marlene

Classifications

• C12P19/58

  having an oxygen atom of the saccharide radical directly bound through only acyclic carbon atoms to a non-saccharide heterocyclic ring, e.g. bleomycin, phleomycin · CPC title

• C12P19/44

  Preparation of O-glycosides, e.g. glucosides {(polysaccharides and not substituted disaccharides C12P19/04, C12P19/12)} · CPC title

• C12N9/24

  acting on glycosyl compounds (3.2) · CPC title

• C08F20/28

  containing no aromatic rings in the alcohol moiety · CPC title

• C08F20/58

  containing oxygen in addition to the carbonamido oxygen {, e.g. N-methylolacrylamide, N-acryloylmorpholine} · CPC title