A simple method for the synthesis of ceramides and radiolabeled analogues.

A simple method has been developed for the coupling of amines to carboxylic acids. N-Fatty-acyl-sphingosine, cerebroside, and GM3, as well as their respective [14C] analogues, were synthesized using diethylphosphoryl cyanide as a potent coupling agent in the presence of triethylamine. The reaction procedure is rapid, racemization-free, and utilizes acids without derivatization. The desired ceramide products were obtained in 85-90% yield within one hour. The facile method presented here can also be used to synthesize [3H]-N-acyl-labeled, as well as [14C] [3H] double-labeled, ceramides.

Plasmalopsychosine of human brain mimics the effect of nerve growth factor by activating its receptor kinase and mitogen-activated protein kinase in PC12 cells. Induction of neurite outgrowth and prevention of apoptosis.

Plasmalopsychosine, a characteristic fatty aldehyde conjugate of beta-galactosylsphingosine (psychosine) found in brain white matter, enhances p140trk (Trk A) phosphorylation and mitogen-activated protein kinase (MAPK) activity and as a consequence induces neurite outgrowth in PC12 cells. The effect of plasmalopsychosine on neurite outgrowth and its prolonged activation of MAPK was similar to that of nerve growth factor (NGF), and the effect was specific to neuronal cells. Plasmalopsychosine was not capable of competing with cold chase-stable, high affinity binding of NGF to Trk A, indicating that plasmalopsychosine and NGF differ in terms of Trk A-activating mechanism. Tyrosine kinase inhibitors K-252a and staurosporine, known to inhibit the neurotrophic effect of NGF, also inhibited these effects of plasmalopsychosine, suggesting that plasmalopsychosine and NGF share a common signaling cascade. Plasmalopsychosine prevents apoptosis of PC12 cells caused by serum deprivation, indicating that it has "neurotrophic factor-like" activity. Taken together, these findings indicate that plasmalopsychosine may play an important role in development and maintenance of the vertebrate nervous system.

Effect of lactose derivatives on metastatic potential of B16 melanoma cells.

The effects of various sugars and sugar derivatives on lung colonization (i.e., metastatic deposition) of the highly metastatic BL6 clone of B16 mouse melanoma cells in syngeneic mice were studied, based on the assumption that carbohydrate structures, particularly those with a Gal terminus, play a crucial role in defining the metastatic potential of B16 cells. After incubation with sugar compounds (usually at 0.1 M concentration), tumor cells were injected via the tail vein into 8-week old female mice. Mice were sacrificed after 18-21 days, and tumor cell colonies in lung were counted under a dissecting microscope. Only methyl beta-D-lactoside and lacto-N-tetraose caused significant reduction (35-45% and 36%, respectively) of metastatic deposition compared to controls. Methyl beta-D-lactoside did not exhibit a growth inhibitory effect on BL6 tumor cells, as determined by several methods: in vitro [3H]thymidine incorporation assay, in vitro plating in RPMI-1640 medium culture under physiological conditions followed by cell counting, and in vivo subcutaneous inoculation of age-matched C57/BL mice followed by tumor measurement. These results indicate that the inhibitory effect of methyl beta-D-lactoside on tumor deposition was not related to its effect on tumor cell growth.

Synthesis of a heptasaccharide hapten related to a biantennary glycan chain of human chorionic gonadotropin of a choriocarcinoma patient. A convergent approach.

Synthesis of the heptasaccharide hapten 8-methoxycarbonyloctyl O-beta-D-galactopyranosyl-(1----4)-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1----2)-O-[beta-D-galactopyranosyl-(1----4)-O-(2-acetam ido-2- deoxy-beta-D-glucopyranosyl)-(1----4)]-O-alpha-D-mannopyranosyl-(1----3) -O- [alpha-D-mannopyranosyl-(1----6)]-beta-D-mannopyranoside is described, by use of the known, protected glycosyl acceptor 8-ethoxy-carbonyloctyl O-(2,3,4,6-tetra-O-benzyl-alpha-D-mannopyranosyl)-(1----6)-2,4-di-O-benz yl-beta - D-mannopyranoside, and the key glycopentaosyl donors O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1----4)-O-(2-acetami do-3,6- di-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-(1----2)-O-[(2,3,4,6-tetra-O- acetyl-beta-D-galactopyranosyl)-(1----4)-O-(2-acetamido-3,6-di-O-acetyl- 2- deoxy-beta-D-glucopyranosyl)-(1----4)]-3,6-di-O-benzyl-alpha-D-mannopyra nosyl trichloroacetimidate (5) and the corresponding fluoride 7, which, in turn, were prepared in 5 steps from allyl 3,6-di-O-benzyl-alpha-D-mannopyranoside in 35 and 22% overall yields, respectively. In model experiments, the key glycosyl donors 5 and 7 were also treated with the simple glycosyl acceptor 8-ethoxycarbonyloctanol, to give 8-methoxycarbonyloctyl O-beta-D-galactopyranosyl-(1----4)-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1----2)-O-[beta-D-galactopyranosyl-(1----4)-O-(2-acetam ido-2- deoxy-beta-D-glucopyranosyl)-(1----4)]-alpha (and beta)-D-mannopyranoside.

Synthesis of a pentasaccharide hapten related to a monoantennary glycan chain of human chorionic gonadotropin.

A stereocontrolled synthesis of a pentasaccharide hapten, namely, 8-ethoxy-carbonyloctyl O-beta-D-galactopyranosyl-(1----4)-O-(2-acetamido-2-deoxy-beta-D-gluc opy ranosyl) -(1----2)-O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D-mannopyranosyl- (1----6)]-alpha-D-mannopyranoside, is described employing a trihexosyl glycosyl-donor, O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1----4)-O-(2-acet amido-3,6- di-O-acetyl-2-deoxy-beta- D-glucopyranosyl)-(1----2)-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl trichloroacetimidate, and a mannobiosyl glycosyl-acceptor, 8-ethoxycarbonyl-octyl O-(2,3,4,6-tetra-O-benzyl-alpha-D-mannopyranosyl)-(1----6)-2,4-di-O-benz yl-beta - D-mannopyranoside, as the key intermediates.

Synthesis of a heptasaccharide hapten related to an anomalous biantennary glycan-chain of human chorionic gonadotropin of a patient with choriocarcinoma. A stepwise approach.

A stereocontrolled synthesis of a heptasaccharide hapten, 8-methoxycarbonyloctyl 3-O-[2,4-di-O-(2-acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-beta-D- glucopyranosyl)-alpha-D-mannopyranosyl]-6-O-alpha-D-mannopyranosyl-beta- D-mannopyranoside (2), is described employing the lactosaminyl donor 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4, 6-tetra-O-acetyl-beta-D-galactopyranosyl)-beta-D-glucopyranosyl bromide and the mannotriosyl glycosyl acceptor 8-ethoxycarbonyloctyl 2,4-di-O-benzyl-3-O-(3,6-di-O-benzyl-alpha-D-mannopyranosyl)-6-O-(2,3, 4,6-tetra-O-benzyl-alpha-D-mannopyranosyl)-beta-D-mannopyranoside, the reaction of which gave the biantennary structure 8-ethoxycarbonyloctyl 2,4-di-O-benzyl-3-O-(3,6- di-O-benzyl-2,4-di-O-[3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3, 4,6-tetra-O-benzyl-alpha-D-mannopyranosyl)-beta-D-mannopyranoside (9), as well as a monoglycosylated product, 8-ethoxycarbonyloctyl 2,4-di-O-benzyl-3-O-(3,6-di-O-benzyl-4-O-[3,6- di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6- tetra-O-benzyl-alpha-D-mannopyranosyl)-beta-D-mannopyranoside. The diglycosylated product 9 was transformed into 2, and the structure was confirmed by 1H-n.m.r. data.