PAPER www.rsc.org/obc | Organic & Biomolecular Chemistry Polycationic calix[8]arenes able to recognize and neutralize heparin †‡ Tommaso Mecca, Grazia M. L. Consoli, Corrada Geraci, Rita La Spina and Francesca Cunsolo* Received 22nd June 2006, Accepted 10th August 2006 First published as an Advance Article on the web 30th August 2006 DOI: 10.1039/b608887b A mutual induced fit mechanism is responsible for the exceptional complexation performances exhibited by calix[8]arene polycations towards heparin. The recognition process was studied in comparison with two other heparin antagonists: protamine and polylysine. The arrangement of multiple functional groups on the flexible macrocyclic scaffold of calix[8]arene, with respect to the conformationally rigid protamine and low ordered polylysine, allowed a mutual adaptability between calixarene polycations and heparin, significantly enhancing the recognition performances. Fluorescence, NMR titration, and activated partial thromboplastin time (aPTT) experiments confirmed that these calixarene derivatives have a very high specificity and affinity towards heparin neutralization as in aqueous solution as in blood. Analogous results were obtained with low molecular weight heparin (LMWH) whose effect protamine is unable to completely reverse. Introduction Heparin, a sulfated polysaccharide, is known as one of the most powerful anticoagulant drugs, based on its ability to accelerate the rate at which antithrombin, a naturally occurring serine protease inhibitor, inactivates several coagulation factors such as thrombin and factor Xa, whose action is essential in the blood coagulation cascade. 1 Heparin also interacts with a number of proteins involved in many basic biological processes like angiogenesis, tumour growth and infectious attack by bacteria, protozoa and viruses. 2 To overcome the natural blood tendency to form clots, 3 systemic heparinization is the most common anticoagulation procedure in surgical practice and extracorporeal therapies such as heart–lung oxygenation and kidney dialysis. To avoid risk of bleeding, the excess of heparin needs to be balanced and, if necessary, carefully neutralized. Therefore, heparin, its analogues and inhibitors have attracted high interest in the therapeutic field. Heparin is a mixture of helical polysaccharides with chains of different lengths, mainly composed of repeating disaccharide units of 1→4-linked sulfated iduronic acid and sulfated glucosamine residues (Fig. 1); sulfur-containing and carboxyl groups are displayed at defined intervals and orientation along the flexible polysaccharide backbone, and provide the highest negative charge density of any known biological macromolecule. 2 For these reasons the key features to consider for heparin neutralization are anion–cation interactions and conformational flexibility. CNR-Istituto di Chimica Biomolecolare, Via del Santuario, 110, I-95028, Valverde (CT), Italy. E-mail: francesca.cunsolo@icb.cnr.it; Fax: +39 095 7212141; Tel: +39 095 7212136 †Electronic supplementary information (ESI) available: Spectral data of compounds 1a and 2a. Kinetic model for heparin–polycation com- plexation. Graphics of competitive titration experiments between 2a and protamine, and 1a and polylysine. Low molecular weight heparin NMR titration and activated partial thromboplastin time (aPTT) calibration curve for low molecular weight heparin. See DOI: 10.1039/b608887b ‡ Dedicated to Professor Mario Piatelli on the occasion of his 80th birthday. Fig. 1 Major heparin repeating unit. One of the most used heparin antagonists is protamine sulfate, a low molecular weight protein bearing a high positive charge density due to the numerous arginine residues (ca. 20). 4 However, since protamine often causes severe side effects, 5 the finding of safe and efficacious heparin antagonists is currently a goal of great clinical importance. With this aim, synthetic medium-sized peptides, 6 polypeptides (polylysine and polyarginine), 7–9 as well as low molecular weight protamine 10 and, very recently, foldamers 11 have been reported. Moreover, proteins such as lactoferrin, 12 histones 7 and antibodies 13 have been studied as heparin-neutralizing agents, but up to now, protamine, in spite of its well-known side effects, remains clinically the most extensively employed heparin antagonist. Our strategy for the design of polyvalent heparin inhibitors with improved complexation properties was based on the use of a calix[8]arene 14 molecular scaffold having a high degree of functionalization, a well defined non-polymeric structure and elevated conformational adaptability, in order to achieve high affinity towards heparin through a mutual induced fit recognition mechanism. In fact, mutual adaptability amplifies immeasurably the scope and efficiency of the molecular complementarity on which molecular recognition is based, ultimately playing an essential role in most, if not in all, chemical and biochemical processes. 15 We expected that the mutual induced fit complexation mechanism would give rise to an improvement in the neutralization This journal is © The Royal Society of Chemistry 2006 Org. Biomol. Chem., 2006, 4, 3763–3768 | 3763 Published on 30 August 2006. Downloaded by CNR on 22/08/2013 08:02:05. View Article Online / Journal Homepage / Table of Contents for this issue