3-Methoxycarbonyl-5-nitrophenyl boronic acid: high affinity diol recognition at neutral pH Hormuzd R. Mulla, Nicholas J. Agard and Amit Basu* Chemistry Department, Brown University, Box H, Providence, RI 02912, USA Received 13 August 2003; revised 29 September 2003; accepted 15 October 2003 Abstract—Several boronic acids were screened for their ability to bind to diols. 3-Methoxycarbonyl-5-nitrophenyl boronic acid bound to both a catechol dye as well as fructose with a comparable affinity to that of an ortho-methylamino substituted boronic acid. This work suggests a greater role for appropriately functionalized electron deficient boronic acids in diol and carbohydrate recognition. # 2003 Elsevier Ltd. All rights reserved. In this paper, we report that a phenyl boronic acid suitably substituted with a nitro group can bind to a sugar in aqueous solution at neutral pH with reasonable affinity. Boronic acids are commonly used as carbohy- drate recognition motifs because they react with some 1,2 and 1,3 diols to form boronate esters. 1 À4 Although the boronate esters can form in aqueous solution, the equilibrium constant for their formation is generally not very large at neutral pH. Higher association constants can be obtained under basic conditions (generally pH=10), where the population of the tetrahedral bor- onate form is greater. 1,5 In order to obtain higher association constants at neu- tral pH, an ortho aminomethyl substituent is often introduced, as seen in compound 2. Introduction of the Lewis basic aminomethyl group facilitates boronate ester formation due to the donation of the nitrogen lone pair into the empty boron p-orbital. 6 The benzylic amine functionality is most often introduced by reduc- tive amination of the corresponding benzaldehyde or by nucleophilic displacement of a benzylic bromide. How- ever, once formed, the purification and subsequent syn- thetic manipulation of these types of compounds is often problematic due to the zwitterionic and ampho- teric character of the B–N moiety. As part of an on- going project for the development of oligosaccharide sensors, we sought an alternative boronic acid scaffold that would retain an appreciable affinity for carbohy- drates at physiological pH, but without recourse to the prosthetic benzyl amino functionality. The introduction of electron withdrawing groups onto the ring of a phenyl boronic acid stabilizes the boronate form of the acid and lowers the pK a value, which in turn favors ester formation at a lower pH. 7 We reasoned that an electron deficient aryl boronic acid may have an appreciable affinity for a carbohydrate at neutral pH. 8 Therefore, we screened several commercially available boronic acids (compounds 3–9) which varied in the location and identity of electron withdrawing groups on the ring. Phenyl boronic acid (PBA) 1 and the N,N-di- ethyl aminomethyl boronic acid 2 were included for comparison. 9 We used a fluorimetric assay recently reported by Springsteen and Wang to initially determine the binding constants of the boronic acids 1–9 with the catechol dye Alizarin Red S (ARS, 10). 10 The fluorescence of ARS increases significantly upon binding, providing a con- 0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2003.10.017 Bioorganic & Medicinal Chemistry Letters 14 (2004) 25–27 * Corresponding author. Tel.: +1-401-863-9897; fax: +1-253-540- 0698; e-mail: amit_basu@brown.edu