Synthesis of low molecular weight mimetics of heparin

Natural hexosaminoglycan heparin remains the most commonly prescribed anticoagulant in hospitalized patients. However its administration could induce side clinical events, including thrombocytopenia and bleeding. This explaines the need of development of alternative anticoagulant drugs based on modified heparin and polyanionic oligo- and polysaccharide derivatives, such as sulfated glucans, phosphomannans and fucoidans. Here we review the works on the synthesis of oligosaccharides related to low molecular weight hepain fragments and their derivatives, as well as oligosaccharides, which imitate parts of heparin chain responsible for biological activity. These works were aimed to develop the pharmaceutical preparations lacking ofheparin disadvantages.     

Undersulfated, low-molecular-weight glycol-split heparin as an antiangiogenic VEGF antagonist

Vascular endothelial growth factor (VEGF) represents a target for antiangiogenic therapies in a wide spectrum of diseases, including cancer. As a novel strategy to generate nonanticoagulant antiangiogenic substances exploiting binding to VEGF while preventing receptor engagement, we assessed the VEGF-antagonist activity of a low-molecular-weight (LMW) compound (ST2184, Mw = 5800) generated by depolymerization of an undersulfated glycol-split heparin derivative. The parental compound was obtained by introducing regular sulfation gaps along the prevalently N-sulfated heparin regions, followed by glycol-splitting of all nonsulfated uronic acid residues (approximately 50% of total uronic acid residues). ST2184 was endowed with a negligible anticoagulant activity after S.C. injection in mice. ST2184 binds VEGF165 as evaluated by its capacity to retard 125I-VEGF165 electrophoretic migration in a gel mobility shift assay and to prevent VEGF165 interaction with heparin immobilized onto a BIAcore sensor chip. Unlike heparin, ST2184 was unable to present 125I-VEGF165 to its high-affinity receptors in endothelial cells and inhibited VEGF165-induced neovascularization in the chick embryo chorioallantoic membrane. Undersulfated, LMW glycol-split heparins may therefore provide the basis for the design of novel nonanticoagulant angiostatic compounds.     

Synthesis of alpha-helix mimetics with four side-chains

The synthesis of a heterocyclic piperazine-based scaffold that mimics the i, i+4, i+8, i+11 side-chain projections of an alpha-helix is described. The synthesis is designed to be modular to allow the targeting of a range of protein-protein interactions involving alpha-helices.     

New synthetic heparin mimetics able to inhibit thrombin and factor Xa.

Synthetic pentadeca-, heptadeca- and nonadecasaccharides, comprising an antithrombin III (AT III) binding pentasaccharide prolonged at the non-reducing end by a thrombin binding domain have been obtained. The pentadecasaccharide is the shortest oligosaccharide able to catalyse thrombin inhibition by AT III. The nonadecasaccharide is a more potent thrombin inhibitor than standard heparin.     

Immunological characterization of peptide mimetics of carbohydrate antigens in vaccine design strategies.

Targeting antigens which cannot be readily addressed by genetic vectors is a major challenge in vaccine design. The inter-conversion of carbohydrate antigens into peptide mimetic forms provides a means to broaden the immune response to carbohydrate antigens. Peptides that mimic carbohydrate antigens offer new possibilities to augment immune responses to such antigens that include inducing carbohydrate reactive T-cell responses. Peptide mimeotopes can be formulated in a variety of ways that include multiple antigen peptides (MAP) and as DNA vaccines that prime for different antibody isotypes. On the immunological side we observe that: (i) depending on the immunogen formulation peptide mimetics can be processed by either CD5+ or CD5-B cells; (ii) peptide mimeotope immunization can induce cross-reactive responses to multiple carbohydrate forms; (iii) priming with peptide mimeotopes can enhance carbohydrate immune responses upon boosting and (iv) immunization with peptide mimeotopes can induce carbohydrate reactive T cells.     

Synthesis of new polyether glycodendrons as oligosaccharide mimetics

Divalent and tetravalent glycomimetics based on polyether glycodendrons have been prepared. The branched scaffolds were decorated with galactose moieties on one hand and were elaborated into new glycodendrons of a 'mixed' type on the other, carrying both galactose and mannose moieties as biologically important sugar epitopes. All synthesized glycodendrons possess a focal point that can be employed for further derivatization and functionalization.     

Synthesis and antimicrobial evaluation of farnesyl diphosphate mimetics

The synthesis and first antimicrobial evaluation of farnesyl diphosphate mimetics are described. Several analogues (10, 12, 13, and 20) are inhibitors of Candida albicans, Shizosaccharomyces pombe, and Saccharomyces cerevisiae. The activities of analogues 10, 12, and 13, which contain a omega-phenyl moiety and a diphosphate isostere, are not attributable to inhibition of sterol biosynthesis via squalene synthase. Two geranyl phenylsulphones (14 and 15) are potent inhibitors of Escherichia coli. Analogue 15 exhibits potent activity towards Salmonella typhimurium and Pseudomonas aeruginosa (MIC-2 microg/mL) and represents the first type of semi-synthetic terpenoid allylic sulphone active against these bacteria.     

Detection of low-molecular-weight heparin oligosaccharides (Fragmin) using surface plasmon resonance

During the last decades there has been a growing realization of the central biological role that oligosaccharides and oligosaccharide-protein interactions play. One of the most striking examples is the use of heparin and low-molecular-weight heparin oligosaccharides (Fragmin) to modify blood coagulation. Several monoclonal antibodies directed against glycosaminoglycan structures have been produced. However, their clinical use is limited by the difficulty of detection systems for oligosaccharides. In the present study we used a monoclonal antibody directed against heparin oligosaccharides prepared by partial nitrous acid deamination of heparin. Using a biosensor (BIAcore), purified antibody was immobilized on sensor surfaces and binding of oligosaccharide was measured by surface plasmon resonance. Using this technique, it was possible to quantitate low-molecular-weight heparin oligosaccharides in nanomolar concentrations.     

Synthesis of dehydroalanine fragments as thiostrepton side chain mimetics

Syntheses of dehydroalanine derivatives via a solid-support route, starting from selenocystein, and via conventional solution phase chemistry are described along with initial biological testing. The target compounds were designed as mimetics of the dehydroalanine side chain of the macrocyclic antibiotic thiostrepton that acts on the bacterial ribosome.     

Synthesis and properties of novel lipopeptides and lipid mimetics

Lipid mimetics, synthetic molecules that resemble natural lipids either structurally or functionally, have been developed as potential medicinal substances. They have been successfully applied in the development of drug and peptide delivery systems and for the development of inhibitors or lipid metabolizing enzymes. Phospholipase A2 is considered to be involved as the rate‒limiting step in the production of lipid mediators of inflammatory responses and, as such, it has been a target for drug design. A series of lipid mimetics including lipopeptides, amides and alcohols of lipidic α‒amino acids, have been tested by bulk and monolayer assay techniques. The findings suggested the direct interaction of the tested compounds with porcine pancreatic phospholipase A2. The inactivation of the enzyme occurred in a competitive manner. The most active compound 1 (2-amino-N-hexadecyl-L -hexanamide) showed an apparent IC₅₀ of 12 μ M and inhibitory power Z=13 in the monolayer assay. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Synthetic oligosaccharides having various functional domains: potent and potentially safe heparin mimetics.

A synthetic heptadecasaccharide, comprising an antithrombin III binding domain, a thrombin binding domain, and a neutral methylated hexasaccharide sequence, was obtained through a convergent synthesis. This compound displayed in vitro anticoagulant properties similar to that of standard heparin but, in contrast with heparin, escaped neutralization by platelet factor 4, a protein released by activated platelets.     

The effect of low-molecular-weight heparin in the survival of a rabbit congested skin flap

Swelling and congestion of flaps are frequently seen postoperatively and can cause unexpected necrosis. According to previous reports, venous thrombosis seems to be a more frequent problem than arterial occlusion in both experimental and clinical surgery. Few satisfactory venous trauma models exist, and reports on experimental venous thrombosis are rare. The object of this study was to create a rabbit venous occlusion flap model and to evaluate the effect of low-molecular-weight heparin on this flap. Eight New Zealand rabbits were used in the pilot study, in which the ideal congested flap was investigated using a flap pedicle based on the central auricular artery with a skin pedicle 0, 1, 2, or 3 cm wide. The flap (3 x 6 cm) was designed on the central part of the left ear, and the central auricular vein and nerve, the former for venous return, were cut out at the base of the flap. The flaps with skin pedicles 0, 1, 2, or 3 cm wide showed mean necrosis length of 60.0, 9.3, 4.2, and 0.0 mm, respectively. The flaps with skin pedicles 0, 1, 2, or 3 cm wide showed mean necrosis of 100, 15.5, 7, and 0 percent, respectively. Therefore, the flap, based on a 1-cm-wide skin pedicle and the central auricular artery, was selected as an optimal congested flap model showing 15.5 percent necrosis. The congested flap was then elevated on the left ear of another 10 rabbits. Subcutaneous low-molecular-weight heparin (320 IU/kg) was administered immediately after surgery to five of the rabbits (the low-molecular-weight heparin group), and the remaining five were used as a control group. Fluorescein was injected 15 minutes after surgery to evaluate the circulatory territory of the flap, and the circulatory territory was measured 5 minutes after injection. The flaps were assessed 7 days after surgery by angiography, histology, and clinical findings. The circulatory territory was significantly greater in the low-molecular-weight heparin group (mean +/- SD, 39.2 +/- 3.0 mm) than the control group (mean +/- SD, 48.0 +/- 1.0 mm) (p < 0.001) assessed 7 days after surgery. The longest flap survival length in group A and group B ranged from 40 to 55 mm (mean +/- SD. 49.4 +/- 5.6 mm) and complete survival (mean +/- SD, 60.0 +/- 0.0 mm). The improvement in survival was statistically significant for group B compared with group A (p < 0.015). Histologic evaluation revealed moderate to severe venous congestion and inflammation in the control group, whereas there were minimal changes in the low-molecular-weight heparin group. Angiography of the flap revealed obvious venous occlusion in the periphery in the control group compared with the low-molecular-weight heparin group. The authors conclude that subcutaneous administration of low-molecular-weight heparin has a great potential to improve the survival length of a congested flap without major complications.     

Synthesis and biological evaluation of nonpeptide mimetics of omega-conotoxin GVIA

A benzothiazole-derived compound (4a) designed to mimic the C(alpha)-C(beta) bond vectors and terminal functionalities of Lys2, Tyr13 and Arg17 in omega-conotoxin GVIA was synthesised, together with analogues (4b-d), which had each side-chain mimic systematically truncated or eliminated. The affinity of these compounds for rat brain N-type and P/Q-type voltage gated calcium channels (VGCCs) was determined. In terms of N-type channel affinity and selectivity, two of these compounds (4a and 4d) were found to be highly promising, first generation mimetics of omega-conotoxin. The fully functionalised mimetic (4a) showed low microM binding affinity to N-type VGCCs (IC(50)=1.9 microM) and greater than 20-fold selectivity for this channel sub-type over P/Q-type VGCCs, whereas the mimetic in which the guanidine-type side chain was truncated back to an amine (4d, IC(50)= 4.1 microM) showed a greater than 25-fold selectivity for the N-type channel.     

Synthesis of aminoazalactams as cyclic mimetics of basic alkyl amino acids

Novel medium ring conformationaly constrained amino acid derivatives 2 for incorporation into collagenase inhibitors were prepared, utilising natural amino acids as starting materials, via a reductive intramolecular imine cyclisation.     

Synthesis of superabsorbent from carbohydrate waste

A mixture of acrylamide (AM) and acrylic acid (AA) monomers were grafted on germinated gelatinized wheat starch using potassium persulfate (KPS) as an initiator to yield superabsorbent. The effects of different parameters such as time, temperature, monomer feed (AM:AA), starch: monomers ratio and initiator concentration on graft add-on were studied, and the optimized values were found to be 2 h, 60 °C, 1:1 (w/w), 1:1(w/w) & 1% (with respect to starch powder), respectively. The product so formed was saponified with 0.1N NaOH, dried and finely powdered sample was characterized using FT-IR, TGA. Product showed maximum absorbency of 150 g/g.     

Probing the carbohydrate recognition domain of E-selectin: The importance of the acid orientation in sLex mimetics

The selectin–leukocyte interaction is the initial event in the early inflammatory cascade. This interplay proceeds via the terminal tetrasaccharide sialyl Lewis^x (sLe^x), present on physiological selectin ligands and E- and P-selectins located on the endothelial surface. Blocking this process is regarded as a promising therapeutic approach for inflammatory diseases where excessive leukocyte efflux is responsible for tissue damage. Selectin antagonists are generally based on sLe^x as lead structure, containing the essential pharmacophores pre-oriented in the bioactive conformation. In this work, we describe a set of competitive sLe^x mimetics possessing the carboxylic acid pharmacophore equipped with additional hydrophobic substituents as neuraminic acid (Neu5Ac) replacements. This small library of antagonists derived from Huisgen-1,3-dipolar cycloadditions allows to further probe the carbohydrate recognition domain of E-selectin.