Specific tritium labeling of gangliosides at the 3-position of sphingosines
GM1 and GD1a gangliosides, treated with 2,3-dichloro-5,6-dicyano benzoquinone (DDQ) in the presence of Triton X-100 and in a toluene medium were specifically oxidized at the 3-position of sphingosine. The maximum reaction yield (65%) was obtained after 40 hours at 37 degrees C with the following molar ratio of reactants: ganglioside-Triton X-100-DDQ 1:70:125. The formation of the 3-keto derivatives of GM1 and GD1a was demonstrated by: a) the appearance of a sharp peak at 1700 cm-1 and of a broad band at 1250 cm-1 (typical of allylic ketones and of carbonyl groups, respectively) in the infra-red spectrum; b) the appearance of an absorption maximum at 230 nm, identical to that featured by 3-keto-cerebrosides, in the ultraviolet spectrum; c) the degradation of long chain bases during the process of release from gangliosides and derivatization for analysis by gas-liquid chromatography (expected for long chain bases carrying a keto group in the 3-position); and d) the quantitative transformation of 3-keto-GM1 and 3-keto-GD1a to GM1 and GD1a, respectively, upon NaBH4 reduction. Reduction of 3-keto-GM1 and 3-keto-GD1a with (/sup 3/H)-NaBH4 produced /sup 3/H-labeled GM1 and GD1a. (/sup 3/H)GM1 and (/sup 3/H)GD1a maintained the same carbohydrate and fatty acid composition of the original GM1 and GD1a, and did not contain any saturated long chain bases. Direct proof that the label was at C-3 of long chain bases was given by reoxidation with DDQ, which completely removed the label, and by ozonolysis, after which label was retained on the oligosaccharide-containing fragment. More than 99% of incorporated radioactivity was carried by the long chain bases. The radiochemical purity of labeled gangliosides was greater than 95% and the specific radioactivity was 1.25 and 1.28 Ci/m mol for (/sup 3/H)GM1 and (/sup 3/H)GD1a, respectively.
- Research Organization:
- Department of Biological Chemistry, The Medical School, University of Milano, Italy
- OSTI ID:
- 5450791
- Journal Information:
- J. Lipid Res.; (United States), Vol. 22:8
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEAR CHEMISTRY
GANGLIOSIDES
LABELLING
TRITIUM COMPOUNDS
CHEMICAL PREPARATION
BIOCHEMICAL REACTION KINETICS
CHEMICAL REACTION YIELD
CARBOHYDRATES
KINETICS
LABELLED COMPOUNDS
LIPIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
REACTION KINETICS
SACCHARIDES
SYNTHESIS
YIELDS
550601* - Medicine- Unsealed Radionuclides in Diagnostics
550201 - Biochemistry- Tracer Techniques
551001 - Physiological Systems- Tracer Techniques
400702 - Radiochemistry & Nuclear Chemistry- Properties of Radioactive Materials