Synthesis of Mannose‐6‐Phosphate Analogues and their Utility as Angiogenesis Regulators

Although carbohydrates are the most abundant natural products, their use as therapeutic agents has been limited. However, since carbohydrate binding proteins are involved in many biological processes, including cellular communication, the prospects for carbohydrate-based drugs seem bright. Here, we provide a synthetic route to bioactive mannose derivatives that serve as both positive and negative effectors of angiogenesis, thereby laying the groundwork for future drug development. The current, limited applications of carbohydrates as therapeutics may, in part, be related to the high complexity of interactions between carbohydrate and carbohydrate binding proteins. Carbohydrate oligomers are themselves complex; for example, four different monosaccharides can form 35 560 distinct tetrasaccharides—this large number reflects the multiple hydroxy attachment sites on each component sugar. Thus, a relatively small polysaccharide has an enormous capacity to encode biological information. When these polysaccharides are conjugated to proteins, the complexity further increases. To date, more than 80 carbohydrate binding proteins have been identified, and their binding specificities have been described (or are about to be). Among these proteins, the lectin family has been extensively studied and classified into subfamilies according to their cellular location and their carbohydrate binding specificities. For example, the P-type lectins recognize mannose-6-phosphate (M6P), the motivation behind efforts in the design and synthesis of new M6P analogues. P-type lectins encompass the 46 kD cation-dependent M6P receptor (CD-M6PR), the 300 kD cation-independent M6P receptor (CI-M6PR), and proteins harboring M6P homology domains. One major cellular function of the receptors is to help cargo M6P-containing proteins between various subcellular compartments. In addition, CI-M6PR is actually a large multipartner receptor. It binds insulin-like growth factor-2 (IGF-2) at the cell surface and internalizes this growth factor for degradation inside lyzozomes. The functions of other proteins harboring M6P residues are also dependent on their interactions with this receptor. For example, latent transforming growth factor-b (TGF-b) is converted into active TGF-b upon M6P interaction with the receptor. Leukemia inhibitory factor (LIF), a growth factor, is cleared from the extracellular medium via receptor interaction with M6P, and subsequent internalization prevents an excessive accumulation that is detrimental to health especially during development. Moreover, proliferindependent angiogenesis also requires CI-M6PR. The obvious importance of M6P in biological processes, including angiogenesis, prompted us to evaluate the activity of our new analogues in blood vessel formation. As will be shown, the analogues do indeed have substantial effect on angiogenesis. The monosaccharides synthesized and examined here include a variety of substituents at the mannose 6-position: azido (4), aminomethyl (6), carboxyl (7), malonate (8), sulfonate (9), carboxymethyl (10), and phosphonate (3). The replacement of the phosphate head group by other moieties, mostly bioisosteres of phosphate, are intended to provide enzymatically stable compounds that could be used as tools to better understand the chemical factors involved in the modulation of angiogenic activities. In the past, the development of carbohydrates for therapeutic purposes has been considered problematic due to the challenges in synthesizing carbohydrate mimics; however, recent progress in this area has allowed us to overcome these limitations. We now present a simple and efficient synthesis of seven M6P analogues using a cyclic sulfate intermediate. Current routes to M6P analogues exploit activating the C-6 position by a variety of methods, such as halogenation, sulfonation, epoxidation and the use of phosphonium salts followed by nucleophilic substitution. Homologation of mannose has been achieved previously by oxidation of the primary alcohol followed by a Wittig–Horner reaction on the resultant aldehyde. Recently, we introduced substitutions at the C-6 position of mannose via a Mitsunobu reaction. These approaches can be efficient, but a need for a more versatile method led us to access such compounds via the cyclic sulfate method previously developed by us to obtain, in just a few steps with the potential for scale-up, a wide variety of C-6 mannose derivatives (Scheme 1). Regioselective nucleophilic displacement of cyclic sulfate 2 is the key step to obtain all the described mannose derivatives. In the case of phosphonate 3, malonate 8, and sulfonate 9, the corresponding nucleophiles were prepared with the aid of a strong base such as n-butyllithium or sodium hydride. An [a] Dr. V. Barragan-Montero, A. Awwad, S. Combemale, Prof. J.-L. Montero Institut des Biomol! cules Max Mousseron (IBMM) UMR 5247 UM2-UM1-CNRS, ENSCM 8 rue de l’Ecole Normale, 34296 Montpellier cedex 5 (France) E-mail : veronique.montero@univ-montp2.fr [b] Dr. P. de Santa Barbara INSERM U1046, UM1 371 avenue du doyen G. Giraud, 34295 Montpellier Cedex 5, (France) [c] B. Jover CNRS-FRE3400 IURC, UM1 641 avenue du doyen G. Giraud, 34093 Montpellier Cedex 5 (France) [d] Dr. J.-P. Mol"s INSERM U1058, UM1, CHU Montpellier 15 avenue Charles Flahault, 34093 Montpellier Cedex 5 (France) E-mail : jean-pierre.moles@inserm.fr Supporting information for this article is available on the WWW under http://dx.doi. org/10.1002/cmdc.201100293.