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.

Natural Killer (NK) cells recognize and destroy tumors and virus-infected cells in an antibody-independent manner. The regulation of NK cells is mediated by activating and inhibiting receptors on the NK cell surface. One important family of activating receptors is the natural cytotoxicity receptors (NCRs) which include NKp30, NKp44 and NKp46. The NCRs initiate tumor targeting by recognition of heparan sulfate on cancer cells. This study aims to elucidate heparan sulfate structural motifs that are important for NCR binding. Microarray and surface plasmon resonance experiments with a small library of heparan sulfate/heparin oligosaccharides helped to clarify the binding preferences of the three NCRs. We demonstrate that the NCRs interact with highly charged HS/heparin structures, but differ in preferred modification patterns and chain lengths. The affinity of NKp30 and NKp44 for synthetic HS/heparin is approximately one order of magnitude higher than the affinity of NKp46. We further show the relevance of synthetic HS/heparin for the binding of NCRs to tumor cells and for NCR-mediated activation of natural killer cells. In conclusion, NCRs recognize different microdomains on heparan sulfate with different affinities.

Glycosaminoglycans (GAGs), such as heparin or heparan sulfate, are required for the in vivo function of chemokines. Chemokines play a crucial role in the recruitment of leukocyte subsets to sites of inflammation and lymphocytes trafficking. GAG-chemokine interactions mediate cell migration and determine which leukocyte subsets enter tissues. Identifying the exact GAC sequences that bind to particular chemokines is key to understand chemokine function at the molecular level and develop strategies to interfere with chemokine-mediated processes. Here, we characterize the heparin binding profiles of eight chemokines (CCL21, IL-8, CXCL12, CXCL13, CCL19, CCL25, CCL28, and CXCL16) by employing heparin microarrays containing a small library of synthetic heparin oligosaccharides. The chemokines differ significantly in their interactions with heparin oligosaccharides: While some chemokines, (e.g., CCL21) strongly bind to a hexasaccharide containing the GlcNSO3(6-OSO3)-IdoA(2-OSO3) repeating unit, CCL19 does not bind and CXCL12 binds only weakly. The carbohydrate microarray binding results were validated by surface plasmon resonance experiments. In vitro chemotaxis assays revealed that dendrimers coated with the fully sulfated heparin hexasaccharide inhibit lymphocyte migration toward CCL21. Migration toward CXCL12 or CCL19 was not affected. These in vitro homing assays indicate that multivalent synthetic heparin dendrimers inhibit the migration of lymphocytes toward certain chemokine gradients by blocking the formation of a chemokine concentration gradient on GAG endothelial chains. These findings are in agreement with preliminary in vivo measurements of circulating lymphocytes. The results presented here contribute to the understanding of GAG-chemokine interactions, a first step toward the design of novel drugs that modulate chemokine activity.

We present the first preparation of microarrays containing synthetic heparin oligosaccharides in order to elucidate the heparin-protein interactions involved in a variety of biological processes. For this purpose, we have developed a novel linker strategy that is compatible with the protecting-group manipulations required for the synthesis of the highly sulfated oligosaccharides and can also be extended to an automated solid phase approach. Strategic placement of the orthogonally protected amine linker was key to the success of the array construction. These heparin chips allow for the high-throughput screening of oligosaccharides by using approximately picomoles of protein. The potential of the new method was demonstrated by probing the carbohydrate affinity of two heparin-binding growth factors, FGF-1 and FGF-2, that are implicated in the development and differentiation of several tumors.

Heparin, the drug of choice for the prevention and treatment of thromboembolic disorders, has been shown to interact with many proteins. Despite its widespread medical use, little is known about the precise sequences that interact with specific proteins. The minimum heparin binding sequence for FGF1 and FGF2 necessary to promote signaling was investigated. A characteristic pentasaccharide sequence, DEFGH, is required to accelerate the inhibition of thrombin and factor Xa in the blood-coagulation cascade. The first synthetic heparin pentasaccharide drug has been approved in Europe and the US and is sold under the trade name Arixtra. Other oligosaccharides with different composition are under clinical investigation. The enormous interest in the assembly of heparin oligosaccharides will stimulate the development of new synthetic approaches. Heparin-oligosaccharide-synthesis automation similar to that of DNA or peptide synthesis will play an important role.

One of the benefits of beta-peptides as potential candidates for biological applications is their stability against common peptidases. Attempts have been made to rationalize this stability by altering the electron availability of a given amide carbonyl bond through the introduction of polar substituents at the alpha-position of a single beta-amino acid. Such beta-amino acids (beta-homoglycine, beta-homoalanine), containing one or two fluorine atoms or a hydroxy group in the alpha-position, were prepared in enantiopure form. A versatile method for preparing these alpha-fluoro-beta-amino acids by the homologation of appropriate alpha-amino acids and C-OH->C-F or C=O-->CF(2) substitution with DAST, is described. Consequently, a series of beta-peptides possessing an electronically modified residue at the N terminus or embedded within the chain was synthesized, and their proteolytic stability was investigated against a selection of enzymes. All ten beta-peptides tested were resilient to proteolysis. Introducing a polar, sterically undemanding group, into the alpha-position of beta-amino acids in a beta-peptide chain does not appear to facilitate localized or general enzymatic degradation.

Heparin is a highly sulfated polysaccharide that regulates a variety of cellular processes by interaction with a host of proteins. We report the preparation of synthetic heparin oligosaccharide glycodendrimers and their use as heparin mimetics to regulate heparin-protein interactions. The multivalent display of sugar epitopes mimics the naturally occurring glycans found on cell surfaces and enhances their binding capacity. Binding of the heparin dendrimers to basic fibroblast growth factor (FGF-2) was analyzed using heparin microarray experiments and surface plasmon resonance measurements on gold chips. Heparin-coated dendrimers bind FGF-2 significantly more effectively than monovalent heparin oligosaccharides. Dendrimer 1, which displays multiple copies of the sulfated hexasaccharide (GlcNSO(3)[6-OSO(3)]-IdoA[2-OSO(3)])3, was employed to promote FGF-2-mediated mitogen-activated kinase activation, demonstrating the utility of glycodendrimers to modulate heparin-protein interactions.

Nervous System Research, S-386-745, Novartis Pharma AG, CH-4002 Basel N-Acyl-b 2 /b 3 -dipeptide-amide somatostatin analogs, 5 ± 8, with b 2 -HTrp-b 3 -HLys (×natural× sequence) and b 2 -HLys-b 3 -HTrp (retro-sequence) have been synthesized (in solution). Depending on their relative configurations and on the nature of the terminal N-acyl and terminal C-amino group, the linear b-dipeptide derivatives have affinities for the human receptor hsst 4, ranging from 250 to > 10000 nanomolar (Fig. 3). Also, N-Actetrapeptide amides 9 and 10, which contain one a-and three b-amino acid residues (N-b-a-b-b-C), have been prepared (solid-phase synthesis), with the natural (Phe, Trp, Lys, Thr) and the retro-sequence (Thr, Lys, Trp, Phe) of side chains and with two different configurations, each, of the two central amino acid residues. The novel −mixed×, linear a/b-peptides have affinities for the hsst 4 receptor ranging from 23 to > 10000 nanomolar (Fig. 4), and, like −pure× b-peptides, they are completely stable to a series of proteolytic enzymes. Thus, the peptidic turn of the cyclic tetradecapeptide somatostatin (Fig. 1) can be mimicked by simple linear di-and tetrapeptides. The tendency of b-dipeptides for forming hydrogen-bonded rings is confirmed by calculations at the B3LYP/6-31G(d,p) level (Fig. 2). The reported results open new avenues for the design of low-molecularweight peptidic drugs. b-Peptides have recently emerged as a promising class of peptidomimetics for medicinal chemistry. The ability of b-peptides (for reviews, see [1]) to adopt secondary structures such as helices [1], sheets [2a] [3], and, especially, turns [3] suggests that these compounds might be structural and functional mimics of natural peptides. The potential of such mimics is evident from the facts that a) these secondary structures can be readily designed [1 ± 5], and that b) the b-peptides are completely stable against proteolytic degradation in vitro and in vivo [2a] [6].So far, b-peptides, designed to form amphiphilic helices, have been shown to inhibit an intestinal membrane-bound cholesterol-and lipid-transporting protein [5]. Also, similar peptides have been shown to possess antimicrobial, and sometimes, hemolytic activities [7].