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CN-107109454-B - Method for modifying glycoprotein with beta- (1, 4) -N-acetylgalactosamine transferase or mutant thereof

CN107109454BCN 107109454 BCN107109454 BCN 107109454BCN-107109454-B

Abstract

The present invention relates to a method for modifying glycoprotein with beta- (1, 4) -N-acetylgalactosamine transferase or mutants thereof. The method comprises the step of contacting a glycoprotein comprising glycans containing terminal GlcNAc moieties with a non-natural sugar derivative nucleotide in the presence of a beta- (1, 4) -N-acetylgalactosamine transferase or a mutant thereof. The non-natural sugar derivative nucleotide is shown as a formula (3): Wherein A is selected from the group consisting of-N 3 ;-C(O)R 3 ;-C≡C-R 4 ;-SH;-SC(O)R 8 ;-SC(V)OR 8 , wherein V is O or S, -X, wherein X is selected from the group consisting of F, cl, br and I, -OS (O) 2 R 5 , optionally substituted C 2 -C 24 alkyl, optionally substituted terminal C 2 -C 24 alkenyl, and optionally substituted terminal C 3 -C 24 allenyl.

Inventors

  • A - A - Wasil
  • F.L. Van Delft
  • S S van Beaucaire

Assignees

  • 西纳福克斯股份有限公司

Dates

Publication Date
20260505
Application Date
20150804
Priority Date
20140804

Claims (14)

  1. 1. A method of modifying a glycoprotein comprising the step of contacting a glycoprotein comprising glycans containing terminal GlcNAc moieties with sugar derivative nucleotides Su (A) -Nuc in the presence of a beta- (1, 4) -N-acetylgalactosamine transferase, wherein: (i) The glycan comprising a terminal GlcNAc moiety is represented by formula (1) or (2): Wherein: b is 0 or 1; d is 0 or 1; e is 0 or 1, and G is a monosaccharide or a linear or branched oligosaccharide comprising from 2 to 20 saccharide moieties, and (Ii) The sugar derivative nucleotide Su (A) -Nuc is shown in a formula (3): Wherein: a is 0 or 1; Nuc is a nucleotide; U is [ C (R 1 ) 2 ] n , wherein n is 0,1, 2, or 3;R 1 independently selected from H, F, cl, br and I; T is a (hetero) arylene group selected from the group consisting of phenylene, pyridylene, pyridinylene, pyrimidinylene, imidazolylene, imidazoliylene, pyrrolylene, furanylene, and thiophenylene, wherein the (hetero) arylene group is optionally substituted with one or more substituents R 2 , wherein R 2 is independently selected from the group consisting of-F, -Cl, -Br, -CN, -NO 2 , methyl, and methoxy, and A is selected from: (a) -N 3 (b) -C(O)R 3 Wherein R 3 is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl; (c) -C≡C-R 4 wherein R 4 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl; (d) -SH (e) -SC(O)R 8 wherein R 8 is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl; (f) -SC(V)OR 8 wherein V is O or S, and R 8 is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl; (g) -X Wherein X is selected from F, cl, br and I; (h) -OS(O) 2 R 5 Wherein R 5 is selected from methyl, ethyl, phenyl or p-tolyl; (i) R 11 Wherein R 11 is ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl; (j) R 12 Wherein R 12 is selected from the group consisting of-C (H) =CH 2 、-CH 2 -C(H)=CH 2 and-CH 2 -CH 2 -C(H)=CH 2 , and (k) R 13 Wherein R 13 is selected from the group consisting of-C (H) =C=CH 2 and CH 2 -C(H)=C=CH 2 , and (Iii) Wherein the beta- (1, 4) -N-acetylgalactosamine transferase is a sequence selected from the group consisting of SEQ ID NO: 2, 3, 4,5,6, 7, 8, 9, 10, 11, 12, 13 and 14 or a sequence selected from the group consisting of SEQ ID NO: 25, 26, 27, 28, 29, 35, 36, 43, 44, 45, 49, 50, 51, 52, 53 and 71.
  2. 2. The method of claim 1, wherein the β - (1, 4) -N-acetylgalactosamine transferase is derived from caenorhabditis elegans, ascariasis suis, noctuid or drosophila melanogaster.
  3. 3. The method of claim 1, wherein n is 1,2 or 3.
  4. 4. The method of claim 1, wherein n is 0.
  5. 5. The method of claim 1, wherein the sugar derivative nucleotides Su (a) -Nuc are represented by formula (9) or (10): wherein Nuc, a, U and T are as defined in claim 1.
  6. 6. The method of claim 1, wherein the sugar derivative nucleotide is represented by formula (11), (12), (13), (14), (15), (16), (34) or (35): Wherein: a is as defined in claim 1, and M is an integer from 0 to 4; R 2 is independently selected from the group consisting of-F, -Cl, -Br, -CN, -NO 2 , methyl and methoxy, and R 6 is H or methyl.
  7. 7. The method of claim 1, wherein the nucleotide is UDP.
  8. 8. The method of claim 1, wherein the sugar derivative nucleotide is represented by formula (17), (18), (19), (20), (21) or (22): Wherein: x is F, cl, br or I; R 8 is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl; V is O or S; r is 0 or 1; s is 2 or 3; t is 1, 2 or 3, and U is 1 or 2.
  9. 9. The method of claim 1, wherein the sugar derivative nucleotide is represented by formula (23), (24) or (25): Wherein X is F, cl, br or I.
  10. 10. The method of claim 8 or 9, wherein the sugar derivative nucleotide is as shown in formula (17), (18), (19), (23) or (24), wherein (17), (18) and (19) are as defined in claim 8, wherein (23) and (24) are as defined in claim 9, and wherein X is Cl or F.
  11. 11. The method of claim 1, wherein the sugar derivative nucleotides Su (a) -Nuc are Su (a) -UDP as shown in formula (31): u, T, A and a are as defined in claim 1.
  12. 12. The process of claim 1, wherein the glycans containing a terminal GlcNAc moiety are represented by formula (1), (26) or (27): Wherein: b is as defined in claim 1.
  13. 13. The method of claim 1, wherein the glycoprotein comprising glycans with terminal GlcNAc moieties is represented by formula (7) or (8): Wherein: b. d, e and G are as defined in claim 1; y is an integer from 1 to 24, and Pr is protein.
  14. 14. The method of claim 1, wherein the glycoprotein comprising glycans with terminal GlcNAc moieties is an antibody.

Description

Method for modifying glycoprotein with beta- (1, 4) -N-acetylgalactosamine transferase or mutant thereof Technical Field The present invention relates to a method for enzymatically modifying glycoproteins. More specifically, the present invention relates to a method for modifying glycoprotein with sugar derivative nucleotide using β - (1, 4) -N-acetylgalactosamine transferase or mutant thereof, and to β - (1, 4) -N-acetylgalactosamine transferase mutant. Background Glycosyltransferases constitute a superfamily of enzymes involved in the synthesis of complex carbohydrates present on glycoproteins and glycolipids. The basic role of glycosyltransferases is to transfer the glycosyl moiety of a nucleotide derivative to a specific sugar receptor. Beta-1, 4-galactosyltransferase (beta 4 Gal-T) (EC 2.4.1.38) constitutes one of the subfamilies of the glycosyltransferase superfamily, which contains at least seven members of Gal-T1 to Gal-T7, which catalyze the transfer of galactose (Gal) from UDP-Gal to different sugar receptors. The common motif produced by galactosyltransferases on terminal GlcNAc residues is the lactosamine sequence galβ4glcnac-R (LacNAc or LN) which is subsequently modified in various ways by the addition of other sugar and sulfate groups. The most common and important glycostructure of membrane glycoconjugates is poly-N-acetyllactosamine (poly-LN), which is linked to proteins (or lipids), plays an important role in cell communication, adhesion and signaling, and is an important molecule in the regulation of immune responses. Another common terminal sequence present in vertebrate and invertebrate glycoconjugates is the GalNAcβ4GlcNAc-R (LaCdiNAc or LDN) sequence. LDN motifs are present in mammalian pituitary glycoprotein hormones in which terminal GalNAc residues are 4-O-sulfated and serve as recognition markers for endothelial cell Man/S4GGnM receptor clearance. However, non-pituitary mammalian glycoproteins also contain LDN determinants. In addition, modification of LDN and LDN sequences is a common epitope in many parasitic nematodes and trematodes. The biosynthesis of LDN involves the transfer of GalNAc to terminal GlcNAc, a process performed by highly specific GalNAc transferases. For example, reported by Miller et al, J.biol.chem.2008,283, page 1985, incorporated herein by reference, two closely related beta 1, 4-N-acetylgalactosamine transferases-beta 4GalNAc-T3 and beta 4 GalNAc-T4-were considered to cause the protein-specific addition of beta 1,4 linked GalNAc to Asn-linked oligosaccharides on many glycoproteins including the glycoproteins Luteinizing Hormone (LH) and carbonic anhydrase-6 (CA 6). Beta- (1, 4) -acetylgalactosamine transferase (beta- (1, 4) -GalNAcT) has been identified in a range of organisms including human, caenorhabditis elegans (Caenorhabditis elegans) (Kawar et al, j.biol. Chem.2002,277,34924, incorporated herein by reference), drosophila melanogaster (Drosophila melanogaster) (Hoskins et al, science 2007,316,1625, incorporated herein by reference) and Trichoplusia ni (trichlorsia ni) (Vadaie et al, j.biol. Chem.2004,279,33501, incorporated herein by reference). Finally, in addition to GalT and GalNAcT involved in N-glycoprotein modification, an unrelated class of enzymes called UDP-N-acetylgalactosamine transferase (also called PPGALNACT) is responsible for the biosynthesis of mucin-type ligation (GalNAc-1-O-SeR/ThR). These enzymes transfer GalNAc from the sugar donor UDP-GalNAc to serine and threonine residues, forming the typical alpha anomeric bond in O-glycoproteins. Although PPGALNACT catalytic functions appeared simple, only 24 unique PPGALNACT human genes were estimated based on computer analysis. Because O-linked glycosylation proceeds stepwise, the addition of GalNAc to serine or threonine represents the first key step in mucin biosynthesis. Although this appears to be simple, multiple PPGALNACT family members appear to be necessary for complete glycosylation of their protein substrates. It has been shown that beta-1, 4-galactosyltransferase 1 (. Beta.4Gal-T1) is capable of transferring a range of unnatural galactose derivatives to acceptor GlcNAc substrates in addition to its natural substrate UDP-Gal. In particular, as reported by ramakrishenan et al j.biol. Chem.2002,23,20833 (incorporated herein by reference), the mutation of TyR residue 289 to Leu289 in bovine β4gal-T1 creates a cavity in the catalytic pocket of the enzyme that can accommodate UDP-Gal molecules carrying a chemical handle at C2, such as 2-keto-Gal. The mutant enzyme β4galt (Y289L) has been used to detect the presence of O-GlcNAc residues on proteins or terminal GlcNAc moieties on cell surface glycans of normal and malignant tissues in vitro by a two-step process involving first transferring a non-native galactose moiety followed by attachment of an oxime to the C-2 handle. For example Khidekel et al, J.am.chem.Soc.2003,125,16162 (incorporated herein by reference) discloses the chemosele