Preparation and Use of Microarrays Containing Synthetic Heparin Oligosaccharides for the Rapid Analysis of Heparin–Protein Interactions

Abstract

Heparin is a highly sulfated, linear polymer that participates in a plethora of biological processes by interaction with many proteins. The chemical complexity and heterogeneity of this polysaccharide can explain the fact that, despite its widespread medical use as an anticoagulant drug, the structure-function relationship of defined heparin sequences is still poorly understood. Here, we present the chemical synthesis of a library containing heparin oligosaccharides ranging from di- to hexamers of different sequences and sulfation patterns. An amine-terminated linker was placed at the reducing end of the synthetic structures to allow for immobilization onto N-hydroxysuccinimide activated glass slides and creation of heparin microarrays. Key features of this modular synthesis, such as the influence of the amine linker on the glycosidation efficiency, the use of 2-azidoglucose as glycosylating agents for oligosaccharide assembly, and the compatibility of the protecting group strategy with the sulfation-deprotection steps, are discussed. Heparin microarrays containing this oligosaccharide library were constructed using a robotic printer and employed to characterize the carbohydrate binding affinities of three heparin-binding growth factors. FGF-1, FGF-2 and FGF-4 that are implicated in angiogenesis, cell growth and differentiation were studied. These heparin chips aided in the discovery of novel, sulfated sequences that bind FGF, and in the determination of the structural requirements needed for recognition by using picomoles of protein on a single slide. The results presented here highlight the potential of combining oligosaccharide synthesis and carbohydrate microarray technology to establish a structure-activity relationship in biological processes.

Differential Adeno-Associated Virus Serotype-Specific Interaction Patterns with Synthetic Heparins and Other Glycans

“…Fluorophore-labeled rAAV vectors were analyzed for binding to chemically defined heparins on a glycan array. N-Hydroxysuccinimide (NHS)-activated CodeLink slides (SurModics, Inc., Eden Prairie, MN, USA) containing synthetic heparan sulfate (HS)/ heparin oligosaccharides and 5-kDa natural heparin were prepared as described previously (23,24). Briefly, heparin oligosaccharides bearing an aminopentyl linker at the reducing end were procured through a modular chemical synthesis approach.…”

Antigenic Potential of a Highly Conserved Neisseria meningitidis Lipopolysaccharide Inner Core Structure Defined by Chemical Synthesis

“…Hep building blocks 14 and 15 were prepared by using our previously developed synthetic procedures (Yang et al, 2012(Yang et al, , 2013. Hep building block 13 was accessed by introducing an N-benzyl-N-benzyloxycarbonylpentyl linker (Mong et al, 2003;Noti et al, 2006) on the reducing end of Hep intermediate 16 that was, in turn, obtained by de novo synthesis (Ohara et al, 2010). Hep intermediate 16 was converted into the corresponding N-phenyl trifluoroacetimidate Tao, 2001, 2002) 17 upon selective anomeric delevulinoylation and N-phenyl trifluoroacetimidate formation (78% yield over two steps).…”

Synthesis of a tetrasaccharide substrate of heparanase

“…Thus, O-sulfonation of 26 was achieved by treatment with SO 3 ÁEt 3 N in DMF at 55°C, followed by N-sulfonation using SO 3 Ápyridine in triethylamine-pyridine, 22 affording the completely sulfonated tetrasaccharide, which was unambiguously confirmed by ESI-MS analyses. The sulfonated tetrasaccharide was directly subjected to saponification to remove the Ac, Bz, methyl, and benzyl groups, followed by hydrogenolytic cleavage of the benzyl groups.…”