Synthesis of reference standards to enable single cell metabolomic studies of tetramethylrhodamine-labeled ganglioside GM1

Abstract

Ganglioside GM1 and its seven potential catabolic products: asialo-GM1, GM2, asialo-GM2, GM3, Lac-Cer, Glc-Cer and Cer, were labelled with tetramethylrhodamine (TMR) to permit ultra-sensitive analysis using laser-induced fluorescence (LIF) detection. The preparation involved acylation of the homogenous C18 lyso-forms of GM1, Lac-Cer, Glc-Cer and Cer with the N-hydroxysuccinimide ester of a β-alanine-tethered 6-TMR derivative, followed by conversion of these labelled products using galactosidase, sialidase and sialyltransferase enzymes. The TMR-glycolipd analogs produced are detectable on TLC down to the 1 ng level by naked eye. All 8 compounds could be separated in under 4 minutes in capillary electrophoresis where they could be detected at the zeptomole (ca 1000 molecule) level using LIF.

A facile method for controlling the reaction equilibrium of sphingolipid ceramide N-deacylase for lyso-glycosphingolipid production

“…After 12 h of reaction time, 75 mg of pure lyso-GM1 was isolated using Sep-Pak tC18 cartridges (90% yield). The yield was much larger than has previously reported for the aqueous-organic biphasic system (62-72% yield with 2 weeks of reaction time) ( 22,36 ). In addition of 100 mM Ca 2+ enhanced the yield to 81.6% ( Fig.…”

“…These limitations make the aqueous-organic biphasic system ineffi cient for the preparation of lyso-GSLs, especially on a larger scale. As two examples, lyso-GM1 was prepared at the 10 to 20 mg scale using the biphasic system ( 22,36 ). After 2 weeks of reaction, the yield of lyso-GM1 was 62-72% ( 22,36 ), which is similar that produced by chemical catalysis ( 19 ).…”

“…TK4 (PS_SCD) and Shewanella alga G8 (SA_SCD) have been well characterized (33)(34)(35). Both enzymes have been used in the preparation of lyso-GSLs ( 22,36,37 ). Previously, we found that SA_SCD showed higher catalytic effi ciency and broader fatty acid specifi city, making it a better biocatalyst than the commercial PS_SCD ( 35 ).…”

“…Kurita et al (38) reported that GSL hydrolysis has been improved by an aqueous-organic biphasic system in which the fatty acids released from the hydrolysis reaction were diffused into the water-immiscible organic phase, enhancing GSL hydrolysis in the aqueous phase. However, the yield of the biphasic system is decreased in preparative-scale reactions ( 22,36 ), in which case the extraction of fatty acid is not so effi cient because of higher substrate concentration and larger reaction volume.…”

Application of Fluorescently-Labeled Glycosphingolipids to Metabolic Profiling in Single Cells Using Capillary Electrophoresis

“…The usefulness of BODIPY in lipid and glycolipid metabolism studies was first demonstrated in a set of studies by Pagano et al (22,23) where cellular uptake was observed. To facilitate the preparation of various fluorescent GSLs without protecting group manipulation on the sugar moiety, we utilized readily available glycolipids and modified them with commercial or recombinant enzymes (24,25).…”