Chunjun Qin

Vaccines are the most effective method for preventing bacterial infections. Identification of the best hapten is an essential step in the development of third-generation Haemophilus influenzae type b (Hib) conjugate vaccines. A comprehensive understanding of the relationship between Hib oligosaccharide chain length and immunological activities remains elusive. A series of Hib capsular polysaccharide (CPS) oligosaccharide derivatives of different lengths were synthesized to establish carbohydrate structure-function relationships. Glycan microarray screening and immunological evaluation of oligosaccharides revealed that tetramer 4 , comprising four repeated disaccharide units, is the key immunogenic epitope of CPS. The investigation into the influence of carrier proteins, adjuvants, and vaccination dose on immunogenicity demonstrated that an equivalent dose of glycoconjugate 4 -TT without adjuvant can induce a similar immune response as a licensed Hib vaccine (MINHAI Hib). Adjuvant-free 4 -TT glycoconjugates can induce both a humoral and a cellular immune response as efficiently as licensed Hib vaccine. This formulation exhibited protective efficacy in a mouse septic infection model through effective pathogen clearance from the circulation to the liver-resident macrophage Kupffer cells and sinusoidal endothelial cells in a mouse septic infection model. In conclusion, a well-defined semisynthetic glycoconjugate vaccine candidate 4 -TT was developed with a more straightforward and adaptable manufacturing process and demonstrated excellent efficacy comparable to licensed Hib vaccine, providing a rationale for further evaluation in clinical trials.

The industrial production of biologically active glycans is pivotal to the development and manufacture of glycan-based medicinal products. Chemical synthesis is critical to acquiring well-defined glycans for glycobiology investigations and the advancement of glycan-enabled applications. Efforts toward industrial chemical glycan synthesis (ICGS) are hampered by unique challenges associated with their structural complexity and diversity. The major bottleneck in ICGS are regio- and stereoselective glycosylations. In this Perspective, we review the process intensification of glycosylations based on innovative methodologies and devices, and highlight its contributions to ICGS. Five topics including optimization of glycan assembly sequence, modulation of glycosylation reactivity, adjustment of environmental parameters, development of an automated chemical glycan synthesizer, and application of artificial intelligence (AI) to glycan synthesis will be discussed. We also present prospects for accelerating ICGS through process intensification of glycosylation, including the integration of AI techniques, the development of glycosylation-oriented special large-scale reactors and flow chemistry platforms.

The emergence of drug-resistant strains of Vibrio cholerae , coupled with the current limitations of oral vaccines, underscores the urgent need for the development of new vaccines. The O-antigen of V. cholerae serotype O100 has emerged as a promising candidate for vaccine development. To investigate the absolute configuration of 3,5-dihydroxyhexanoyl (dHh) and to evaluate the structure-activity relationship of the O-antigen trisaccharide repeating unit, we completed total synthesis of four potential trisaccharide isomers, along with 11 additional oligosaccharide fragments of the O-antigen. Stereoselective reduction was used for the synthesis of dHh, and the efficient assembly of dHh and ( R )-3-hydroxybutanoyl ( R Hb) was achieved through a post-glycosylation modification strategy. Through NMR analysis, the absolute configuration of dHh was assigned 3 S ,5 S . Glycan microarray screening indicated that R Hb is essential for the antigenicity of O-antigen. The nonreducing end disaccharide 59 may serve as the minimal antigenic epitope. These findings are an important step toward the design of semi-synthetic carbohydrate vaccines against V. cholerae .

Here we report the chemical synthesis of conjugation-ready trisaccharides, representing biological repeating units of Pseudomonas aeruginosa serotype 10 and 19 O-antigens. The α-d-QuiN₃ glycosidic bond was stereoselectively synthesized through TMSI─Ph₃P═O mediated 1,2-cis glycosylation. Selective oxidation of the C6-OH group at the disaccharide stage allowed for benzylidene-promoted construction of the α-l-GalN₃ glycosidic bond and simplification of the postglycosylation process at the trisaccharide stage. The low reaction temperature and neighboring electron-donating effect facilitated the efficient synthesis of the trisaccharide.

Pseudomonas aeruginosa bacteria are becoming increasingly resistant against multiple antibiotics. Therefore, the development of vaccines to prevent infections with these bacteria is an urgent medical need. While the immunological activity of lipopolysaccharide O-antigens in P. aeruginosa is well-known, the specific protective epitopes remain unidentified. Herein, we present the first chemical synthesis of highly functionalized aminoglycoside trisaccharide 1 and its acetamido derivative 2 found in the P. aeruginosa serotype O5 O-antigen. The synthesis of the trisaccharide targets is based on balancing the reactivity of disaccharide acceptors and monosaccharide donors. Glycosylations were analyzed by quantifying the reactivity of the hydroxyl group of the disaccharide acceptor using the orbital-weighted Fukui function and dual descriptor. The stereoselective formation of 1,2-cis-α-fucosylamine linkages was achieved through a combination of remote acyl participation and reagent modulation. The simultaneous S_N2 substitution of azide groups at C2' and C2″ enabled the efficient synthesis of 1,2-cis-β-linkages for both 2,3-diamino-D-mannuronic acids. Through a strategic orthogonal modification, the five amino groups on target trisaccharide 1 were equipped with a rare acetamidino (Am) and four acetyl (Ac) groups. Glycan microarray analyses of sera from patients infected with P. aeruginosa indicated that trisaccharides 1 and 2 are key antigenic epitopes of the serotype O5 O-antigen. The acetamidino group is not an essential determinant of antibody binding. The β-D-ManpNAc3NAcA residue is a key motif for the antigenicity of serotype O5 O-antigen. These findings serve as a foundation for the development of glycoconjugate vaccines targeting P. aeruginosa serotype O5.

Bacterial nonulosonic acids (NulOs), which feature a nine-carbon backbone, are associated with the biological functions of bacterial glycans. Here, an orthogonally protected 5-amino-7-azido-3,5,7,9-tetradeoxy-d-glycero-l-gluco-2-nonulosonic acid related to Fusobacterium nucleatum ATCC 23726 NulO was synthesized from N-acetylneuraminic acid with sequential performance of C5,7 azidation, C9 deoxygenation, C4 epimerization, and N5,7 differentiation. The C5 azido group in the obtained 5,7-diazido-NulO can be regioselectively reduced to differentiate the two amino groups.

The bacterium Vibrio vulnificus can cause severe infections internally and externally. The mortality rate of V. vulnificus-associated gangrene and septicemia has increased due to antibiotic resistance and the absence of human vaccine. Bacterial carbohydrates have been widely used in the development of bacterial vaccines. Here, a rare l-GalpNAmA-containing disaccharide that exists in V. vulnificus biotype 2 serovar A O-antigen was synthesized from d-glucose and l-galactose. An investigation on the influence of different linkers on the disaccharide synthetic efficiency indicated that the amine linker has more practicality. Two α-glycosidic linkages were constructed with good stereoselectivity by using nonparticipating C2 azido group and solvent effect. An orthogonal protection strategy allowed introductions of carboxyl and acetamidino (Am) groups at the disaccharide stage. The synthesis of the amine-linked disaccharide will provide a basis for the preparation of the trisaccharide repeating unit and longer fragments of V. vulnificus biotype 2 serovar A O-antigen, as well as other complex glycans.

Glycans on the surface of bacteria have diverse and essential biological functions and have widely been employed for treating various bacterial infectious diseases. Furthermore, these glycans comprise various functional groups, such as O-, N-, and carboxyl-modified, which significantly increase the diversity of glycan structures. These functional groups are not only crucial for glycans’ structural identity but are also essential for their biological functions. Therefore, a clear understanding of the biological functions of these modified groups in corresponding bacterial glycans is crucial for their medical applications. Thus far, the activities of functional groups in some biomedical active carbohydrates have been elucidated. It has been shown that some functional groups are key constituents of biologically active bacterial glycans, while others are actually not essential and may even mask the functions of the glycans. This paper reviews the structures of naturally occurring side-chain functional groups in glycans located on the bacterial surface and their roles in immunological responses.