Synthesis and biological evaluation of novel biotin-iminoalditol conjugates.

Biotin-iminosugar conjugates of different configuration such as D-gluco, D-galacto, L-ido as well as a furanoid representative in the D-manno configuration have been synthesised and exhibit powerful inhibition of beta-glucosidase from Agrobacterium sp. with K(i) values in the range of the respective parent compounds. Such molecular probes have potential for activity-based protein profiling taking advantage of the biotin-(strept)avidin interaction.

Development of idraparinux and idrabiotaparinux for anticoagulant therapy.

Idraparinux is an analogue of fondaparinux binding with high affinity to antithrombin. It was designed for weekly, rather than daily, administration, with an exceptionally long half-life. One potential problem with small heparin-like fragments of this type is the difficulty of neutralising excessive activity in the case of side-effects or overdose. The efficacy of idraparinux was was proven in clinical studies with patients suffering from venous thromboembolism (VTE) or atrial fibrillation. Due to major bleeding events during treatment for more than six months the development of idraparinux was stopped. Idrabiotaparinux has an attached biotin moiety at the non-reducing end unit, which allows its neutralisation with avidin, an egg-derived protein with low antigenicity. This compound is currently investigated in clinical trials for prevention of recurrent VTE in patients with acute pulmonary embolism. The future of idrabiotaparinux depends also on the safety and efficacy of avidin.

A novel anionic modification of N-glycans on mammalian endothelial cells is recognized by activated neutrophils and modulates acute inflammatory responses.

We previously reported an unusual carboxylated modification on N:-glycans isolated from whole bovine lung. We have now raised IgG mAbs against the modification by immunization with biotinylated aminopyridine-derivatized glycans enriched for the anionic species and screening for Abs whose reactivities were abrogated by carboxylate neutralization of bovine lung glycopeptides. One such Ab (mAb GB3.1) was inhibited by carboxylated bovine lung glycopeptides and other multicarboxylated molecules, but not by glycopeptides in which the carboxylate groups were modified. The Ab recognized an epitope constitutively expressed on bovine, human, and other mammalian endothelial cells. Stimulated, but not resting, neutrophils bound to immobilized bovine lung glycopeptides in a carboxylate-dependent manner. The binding of activated neutrophils to immobilized bovine lung glycopeptides was inhibited both by mAb GB3.1 and by soluble glycopeptides in a carboxylate-dependent manner. The Ab also inhibited extravasation of neutrophils and monocytes in a murine model of peritoneal inflammation. This inhibition of cell trafficking correlated with the increased sequestration but reduced transmigration of leukocytes that were found to be adherent to the endothelium of the mesenteric microvasculature. Taken together, these results indicate that these novel carboxylated N:-glycans are constitutively expressed on vascular endothelium and participate in acute inflammatory responses by interaction with activated neutrophils.

New biotinylating reagent utilizing carbodiimide function.

A biotin derivative having a carbodiimide function (fig.1 Carbo-biotin) was synthesized and reacted with DNAs. This compound was used to label biotin to DNAs, by reaction of carbodiimide group and nucleotides as they form stable adducts. Our experiments showed that it is able to bind to both single and double stranded DNAs. The DNA oligomer labeled with this reagent was shown to hybridize to complementary DNA chain. From our results, this compound is expected to be a useful biotinylating reagent for non-radioactive detection.

A novel biotinylated adenylate analogue derived from pyrazolo[3,4-d]pyrimidine for labeling DNA probes.

A novel dATP analogue, 3-[5-[(N-biotinyl-6- amiocaproyl)amino]pentyl]-1-(2-deoxy-beta-D-erythro-pentofuranosyl )-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5'-triphosphate (9, bio-13-dAPPTP), which is modified at the 3-position with a flexible linker arm bearing a terminal biotin moiety, has been synthesized. This nucleotide is readily incorporated into DNA probes by nick translation. These probes hybridize to complementary targets as well as probes labeled with bio-dUTP, as judged by slot blot. When incorporated into oligonucleotides, they do not cause the loss of hybridization efficiency that an N-6-substituted adenine nucleotide does when incorporated into the same sites in the oligonucleotide.