Quantifying the Efficiency ofN-Phenyl-D-mannosamine to Metabolically Engineer Sialic Acid on Cancer Cell Surface

Abstract

A convenient method was developed for the quantification of sialic acids expressed by cells and used to analyze the efficiency of N-phenylacetyl-D-mannosamine (ManNPhAc) to metabolically glycoengineer SKMEL-28 cancer cell. For this purpose, ManNPhAc-cultured cells were treated with 2M acetic acid to release sialic acids, and the products were treated with 1,2-diamino-4,5-methylenedioxybenzene to form the corresponding derivatives that had strong UV absorptions. The reaction mixture was then applied to HPLC-UV analysis to determine the amounts and the ratios of natural sialic acid and its unnatural analog. It was confirmed that after incubation with ManNPhAc SKMEL-28 cell was effectively glycoengineered to express a significant amount of unnatural sialic acid.

A fully synthetic self-adjuvanting globo H-Based vaccine elicited strong T cell-mediated antitumor immunity

“…8 For example, inammatory responses at the injection site and systemic syndromes such as fever, arthralgias, and myalgias induced by QS21 have been reported during the clinical trials of KLH-globo H conjugates. [18][19][20] To overcome these problems associated with protein-TACA conjugates, a variety of new constructs of carbohydrate-based vaccines using non-protein carriers, e.g., dendrimers, 21,22 polysaccharides, 23 nanoparticles, 24 and lipids, [25][26][27][28][29][30][31] and vaccines with self-adjuvanting properties [32][33][34][35][36][37][38][39] have been designed and explored. Among them, lipid carrierbased fully synthetic glycoconjugate vaccines are particularly attractive as they possess homogeneous and dened chemical structures, which would not only streamline their characterization and quality control but also enable detailed immunological studies to gain insights into their functions, functional mechanisms, and structure-activity relationships to guide the design and further optimization of related vaccines.…”

Novel sialoglycan linkage for constructing adjuvant-protein conjugate as potent vaccine for COVID-19

“…Different concentrations of standard Neu5Ac samples were treated with DMB, and the UV absorbance at 370 nm was measured by HPLC to establish proper calibration curve (Fig. S1) [ 47 ]. The HPLC peak areas were in good linear relationship ( R 2 value = 0.9956) with the Neu5Ac concentrations.…”

“…As previously reported [ 47 ], a Neu5Ac standard sample (40, 20, 10, 5 or 2.5 μg) was mixed with the DMB reaction solution (200 μL; 6.9 mM DMB⸱2HCl, 18 mM Na 2 S 2 O 4 , 1.4 M acetic acid, 0.75 M β-mercaptoethanol in Milli-Q water). The mixture was stirred at 50 °C for 3 h in a thermomixer (300 rpm) in the dark.…”

Metabolic Remodeling of Cell‐Surface Sialic Acids: Principles, Applications, and Recent Advances

“…As a rule, functional groups with no branching are well accommodated by ManNAc analogues, whereas sialic acid analogues can deliver bulkier functional groups. Notably, exceptions to this rule, such as ManNAc analogues with branching or bulky groups, have been seen, but often show low incorporation efficiency …”