Glycoconjugations of Biomolecules by Chemical Methods

Abstract

Bioconjugations under benign aqueous conditions have the most promise to covalently link carbohydrates onto chosen molecular and macromolecular scaffolds. Chemical methodologies relying on C-C and C-heteroatom bond formations are the methods of choice, coupled with the reaction conditions being under aqueous milieu. A number of methods, including metal-mediated, as well as metal-free azide-alkyne cyclo-addition, photocatalyzed thiol-ene reaction, amidation, reductive amination, disulfide bond formation, conjugate addition, nucleophilic addition to vinyl sulfones and vinyl sulfoxides, native chemical ligation, Staudinger ligation, olefin metathesis, and Suzuki-Miyaura cross coupling reactions have been developed, in efforts to conduct glycoconjugation of chosen molecular and biomolecular structures. Within these, many methods require pre-functionalization of the scaffolds, whereas methods that do not require such pre-functionalization continue to be few and far between. The compilation covers synthetic methodology development for carbohydrate conjugation onto biomolecular and biomacromolecular scaffolds. The importance of such glycoconjugations on the functional properties is also covered.

Keywords: biomolecules; carbohydrates; conjugations; glycoconjugations; lipids; nucleic acids; peptides; proteins.

Scheme 1

Amidation of: (A) a trisaccharide with BSA protein; (B) 2-amino-2-deoxy sugar with amino acids; (C) carboxylic acid derivative of a nucleobase using a glycosyl amine.

Scheme 2

Glycoconjugation of: (A) a Brucella cell wall component tetrasaccharide with BSA using bis-squaramide; (B) two types of squarate diesters; (C) LacNAc-containing tetrasaccharide on to BSA.

Scheme 3

(A) Allyl glycoside as a precursor for ozonolysis and conjugation with BSA through reductive amination. (B) Molecular structure of MBr1 (globo-H) hexasaccharide-KLH glycoconjugate. (C) Reductive amination of protein BSA using p-formylphenyl glycoside.

Scheme 4

(A) Reductive amination of linker-tethered N-acetylglucosamine oligomers to BSA. (B) Oxidation of primary hydroxy groups in a mannan polysaccharide and subsequent conjugation with a peptide through reductive amination. (C) Zwitterionic polysaccharide conjugation with Tn-based glycosyl hydroxyamine.

Scheme 5

(A) Oxidative cleavage at the non-reducing end of capsular polysaccharide CPS of Campylobacter jejuni, followed by reductive amination with carrier protein diphtheria toxin mutant CRM197. (B) Reductive amination of Kdo by allylamine and 2-(4-aminophenyl)ethylamine. (C) NBS-mediated cleavage of the product of reductive amination. Pathways (a) or (b) are dependent on the nature of the substituent R.

Scheme 6

(A) Native chemical ligation involving the reaction of a C-terminus thioester with N-terminus cysteine to afford a new peptide bond retained with a free cysteine. (B) Full length erythropoietin synthesis involving C-terminus thioester and N-terminus cysteine in EPO fragment pairs (i) 98–124 and 125–166; (ii) 60–97 and 98–166; (iii) 29–59 and 60–166; (iv) 1–28 and 29–166, respectively, undergoing NCL, combined with necessary deprotections, desulfurizations, and the final step of folding through cysteine-cystine redox reaction.

Scheme 7

(A) NCL on mucin 1 peptide and photocleavable auxiliary containing glycopeptide fragments to afford defined mucin 1 glycopeptides. (B) NCL and thiol-ene coupling to prepare a glycopeptide. (C) Oxo-ester activation to an in situ generation of thioester intermediate and subsequent NCL reaction on a functionalized solid surface.

Scheme 8

(A) Synthesis of an N-glycosylated amino acid derivative through the Staudinger ligation. (B) Glycosyl amides formation with the aid of Staudinger reaction. (C) Traceless Staudinger reaction to prepare N-glycosyl amides. Key intermediates of the reaction are given in the bracket.

Scheme 9

Staudinger ligation of: (A) two glycopeptide fragments to secure glyco-oligopeptide; (B) oligo/polypeptides on to carrier proteins tetanus taxoid and bovine serum albumin; (C) a peptide fragment with azidoethylphosphate-derivatized mannopyranosyl trisaccharide.

Scheme 10

(A) Metabolic engineering and Staudinger ligation at the Jurkat / HeLa cell surfaces. (B) Staudinger ligation of sialic acid derivative on HEK293 cells using photocleavable precursor.

Scheme 11

(A) Synthesis of a 1,2,3-triazole-linked 1,4-disubstituted glycopeptide conjugate. (B) A double click reaction at the C-2′ carbon of an oligonucleotide. (C) 1,4-Disubstituted [1,2,3]triazole linked complex biantennary N-glycan–HSA conjugate. (D) Double conjugation-based glycopeptide synthesis.

Scheme 12

(A) Double conjugation involving Cu(I)-catalyzed azide-alkyne click reaction and thia-Michael reaction. (B) Sialyl-lactose derivative conjugation with modified lipid A through Cu(I)-catalyzed azide-alkyne click reaction. (C) Metal-free strain-promoted azide-alkyne cycloaddition reaction occurring at the metabolically engineered cell surface. (D) Double conjugation of metal-free azide-alkyne click and 6π-electrocyclization reactions, permitting the conjugation of biantennary complex N-glycan with human serum albumin.

Scheme 13

(A) Thiyl radical generation from thiol and reaction with ene to afford thioether product. (B) Methods of preparation of glycosyl thiols. (C) Glycoconjugation of calix-4-arene with thiol-tethered nojirimycin.

Figure 1

Molecular structures of: (A) β-cyclodextrin-cored multivalent heteroglycocluster; (B) dual drug-loadable mannopyranoside-halloysite-CD conjugate; (C) octavalent heteroglycocluster possessing two different types of glycoconjugation linkages; (D) hyaluronan hexasaccharide with thiol tether conjugated with engineered thermostable lipase TTL, through a thiol-ene reaction.

Scheme 14

Glycoconjugation of: (A) BSA protein with an activated glycosyl moiety through mixed disulfide bond formation; (B) serine protease subtilisin Bacillus lentus mutant S156C (SBL-Cys156), modified with phenylselenylthio moiety, with a glycosyl thiol; (C) a 21-mer RNA sequence using glycosyl thiols.

Scheme 15

(A) Suzuki-Miyaura cross-coupling reaction of the functionalized SBL mutant protein with glucose-tethered vinylboronate. (B) Formation of biaryl core containing peptide and sugar segments.

Scheme 16

Cross metathesis reactions to the synthesis of: (A) glycosyl amino acid; (B) globotrioside (Gb3) conjugated amino acid; (C) glycosyl moieties incorporated SBL protein; (D) cholesterol moieties conjugated cellulose.

Scheme 17

(A) Synthesis of glucosyl vinyl sulfone. Glycoconjugation of: (B) hen egg white lysozyme with glucosyl vinyl sulfone; (C) vinyl sulfone-functionalised β-cyclodextrin with nanobody cAb-An33.

Scheme 18

(A) Ferrier reaction of 3,4,6-tri-O-acetyl glycal to afford 2,3-unsaturated thioglycoside and a subsequent conversion of thioglycoside to the O-glycoside. (B) Formation of sugar vinyl sulfoxide through Pummerer rearrangement and an oxidation. (C) Reaction of oxygen nucleophiles with sugar vinyl sulfoxide.

Scheme 19

Reactions of: (A) amines on sugar vinyl sulfoxides; (B) sugar vinyl sulfoxide with carbon and sulfur nucleophiles; (C) vinyl sulfoxide with 2-aminoethanol, serine and arginine.

Scheme 20

(A) Reaction of vinyl sulfoxide with lysine, di- and tripeptides. (B) Conjugation of vinyl sulfoxide with amine initially, followed by the alkoxide nucleophile. (C) Glycoconjugation of lysozyme with sugar vinyl sulfoxide.