Kanamycin A: imine formation in aqueous solution Yanet Fuentes-Martínez a , Carolina Godoy-Alcántar a *, Felipe Medrano a and Alexander Dikiy b Imine formation in aqueous solution of kanamycin A with pyridoxal 5’-phosphate and other aldehydes was studied by potentiometry, NMR spectroscopy and computational chemistry. It was found that imines are formed with yields near 100 % at pH 7 in equimolar reactant ratio. In order to identify the kanamycin amino groups involved in the reaction, a NMR spectroscopic study was conducted. The structures of possible imines formed between kanamycin and FURAN or PLP were optimized by molecular mechanics with the OPLS-2005 force field. The 1 H NMR spectra were calculated at the DFT-GIAO B3LYP/6-31G(d) level of theory for all structures and compared with the experimentally observed spectra. From these results a probable structure of the imines was proposed. The results obtained in this work show that kana- mycin has the ability to form imine derivatives in high yields due to its anion recognition properties. Copyright © 2012 John Wiley & Sons, Ltd. Supporting information may be found in the online version of this paper. Keywords: aldehyde; binding constants; imine formation; kanamycin A INTRODUCTION In our previous studies the sites of stepwise protonation of kana- mycin A have been assigned by 1 H- 13 C-HSQC spectra at variable pH in D 2 O. [1] On the other hand, the interactions of the proton- ated forms of kanamycin A with nucleotides and several simple phosphate anions have been studied by potentiometric and NMR titrations. [2] The affinity of kanamycin A to anions is compa- rable to that observed with other aliphatic polyammonium receptors of similar charge, but it discriminates triphosphate nucleotides with different nucleobases giving binding constants in the order GTP>> CTPATP. Continuing with our studies on kanamycin A, in the present work we report its ability to form Schiff bases in aqueous solution at neutral pH. Reactions involving the amino groups of aminoglycosides have been reported previously, [3] and an important option is the forma- tion of Schiff bases with aldehydes in aqueous alcoholic media. [3] However, in the reactions of aminoglycosides with formaldehyde, some problems have been detected as the products correspond to different degrees of substitution depending on the reaction conditions. In other cases the amino-groups are hardly distinguish- able regarding their reactivity, and the chemical reaction involves simultaneously all groups. But in some cases it is possible to establish a sequence of reactivity for the amino groups, [3] or to obtain specific product, as it has been shown [4] for pyridoxal-5’- phosphate which forms a Schiff base by covalent binding to the amino groups of tobramycin in water at pH 7.4. Pharmacological studies indicated that the Schiff base formed protects rat kidneys from nephrotoxicity induced by free tobramycin. Vazquez and coworkers [5,6] carried out kinetic studies of the Schiff-base formation occurring between pyridoxal 5’-phosphate and dodecylamine [5] or leucine, using cationic surfactants. [6] They found that the formation rate constants are much larger than those reported for related systems and attributed their find- ings to the occurrence of local charge concentration phenomena in the vicinity of the carbinolamine, which stabilized the corresponding transition state of the dehydratation reactions. The hydrolysis rate constants for this system were considerably smaller as a result of the hydrocarbon chain acting as a bulky hy- drophobic rest thus protecting the imine bond from nucleophilic attack by water. The experimental observation that the carbinola- mine is stabilized by a charged environment was confirmed by DFT studies on the Schiff base formation of vitamin B 6 analogues. [7] These calculations included the description of the geometries of all intermediates and transition states along the reaction pathway: carbinolamine formation, dehydration and imine formation. The carbinolamine intermediate is formed via a zwitterionic state and the dehydratation is the limiting step of the reaction, in accordance with experimental evidence. During the carbinolamine formation, an auxiliary water molecule plays a key role for the nucleophilic at- tack of the incoming amine to the carbonyl group. Previous studies reported by our group have shown that the imine formation in aqueous solution is favored only with primary amines at high concentrations of in the 7-11 pH range. [8] Re- cently, Saggiomo and Lüning [9] investigated the contradictory results published in relation to the formation of imines in water: while only small amounts of imines were detected by NMR anal- ysis, [8] its extraction with dichloromethane afforded high yields * Correspondence to: C. Godoy-Alcántar, Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, C.P. 62209 Cuernavaca, Morelos, México. E-mail: cga@uaem.mx a Y. Fuentes-Martínez, C. Godoy-Alcántar, F. Medrano Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, C.P. 62209 Cuernavaca, Morelos, México b A. Dikiy Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway Research Article Received: 12 May 2012, Accepted: 6 September 2012, Published online in Wiley Online Library: 8 October 2012 (wileyonlinelibrary.com) DOI: 10.1002/poc.3057 J. Phys. Org. Chem. 2012, 25 1395–1403 Copyright © 2012 John Wiley & Sons, Ltd. 1395