A Novel Probe for Determination of Association Constants between Barbituric Acid Derivatives and Their Receptors

Shin-ichi Kondo, Katsuhiro Utsumi, and Yumihiko Yano*

Department of Chemistry, Gunma University, Kiryu, Gunma 376, Japan

e-mail: yano@cc.gunma-u.ac.jp

Tel: +81-277-30-1230

Fax: +81-277-30-1234

Received 29 May 1997

Accepted 10 June 1997

Published 11 June 1997

Copyright © 1997 Internet Journal of Science - Biological Chemistry

Abstract: 5-Anthryl-5-butylbarbituric acid was synthesized as a probe for spectral determination (UV-vis and fluorescence) of association constants for a barbiturate receptor via six hydrogen bonds in organic solvents.

Introduction

Artificial receptors for biologically active molecules have attracted considerable attention from a viewpoint of molecular recognition. For example, Hamilton et al. reported that a molecule containing two 2,6-diaminopyridine moieties linked by an isophthalate spacer binds strongly barbiturates via six hydrogen bonds, [1] and employed this combination in the binding sites of enzyme models.[2] Melamine derivatives could be used instead of 2,6-diaminopyridine moieties, where introduction of plural functional groups is easier for the triazine ring than for the pyridine ring. [3] Thus, it is desired to exploit a general and convenient method to determine the association constants between a barbiturate and a bis-melamine derivative in various organic solvents. In this paper, we wish to report that a barbiturate bearing an anthryl group (1) is quite useful for determination of the association constants for a bis-melamine receptor (2) by UV-vis or fluorescence spectroscopies (Scheme 1).

Since an anthryl group is well known as a fluorophore and chromophore,[4] 5-anthryl-5-butyl barbiturate (1) was synthesized and employed for association study with a receptor (2). Synthetic routes are outlined in Schemes 2 and 3.[5]

It should be noted that the association constant between 5,5-diethyl barbiturate and 2 could not be determined by UV-vis and ¹H NMR spectral titrations due to almost no spectral changes for the former, and broadening of NH signals for the latter upon addition of the barbiturate in chloroform (or deuterated). On the contrary, absorption spectra of 1 were found to change, passing through isosbestic points, on addition of 2 in chloroform as shown in Fig. 1.

The association constant (K_ass) for 12 was determined to be 50,000 ± 1,500 M^-1 by the non-linear least squares curve fitting of the titration curve at 404.4 nm. Fluorescence spectral changes of 1 excited at 340 nm on addition of 2 in chloroform are shown in Fig. 2.

Similarly, K_ass was determined to be 50,000 M^-1 from the titration curve at 442 nm, which is in good agreement with that obtained by the absorption spectra. The 1 : 1 stoichiometry for the complex formation was confirmed by the Job plot. Furthermore the K_ass value is a little larger than that of the corresponding bis-2,6-diamidopyridine receptor (K_ass = 20,800 M^-1).[1a] These results imply the complex formation via six hydrogen bonds as depicted in Scheme 1.

It is useful to know the solvent effect on formation of six hydrogen-bonded complex for design of artificial enzymes, in which such a strong noncovalent association could be employed as a binding element between a catalyst and a substrate.[6] Although hydrogen bonding is strengthened in a nonpolar solvent, choice of solvent systems is restricted by solubility of the compounds to be used. Thus, solvent effect on the association constants for 12 was examined. As can be seen in Table 1, the increase of CH₃CN content in CHCl₃ decreases gradually K_ass. The complex formation is still observed even in 100 % CH₃CN (K_ass =420 M^-1). However, hydrogen-bond acceptor solvents [7] such as DMF and DMSO dramatically decrease K_ass. In the 1 : 1 solvent system, no complex formation was observed for both solvents. We consider that these results of solvent effects on formation of six hydrogen-bonded complex would be helpful for choice of the solvents in investigation of noncovalently assembled catalytic systems.

In summary, we demonstrated that 5-anthryl barbiturate is a useful probe for determination of association constants with a receptor molecule by UV-vis or fluorescence spectroscopies. We believe that the probe is widely applicable for barbiturate-receptor complexation.

References and Notes

1. (a) Chang, S.-K.; Hamilton, A. D. J. Am. Chem. Soc., 1988, 110, 1318. (b) Chang, S.-K.; Engen, D. V.; Farr, E.; Hamilton, A. D. J. Am. Chem. Soc., 1991, 113, 7640. (c) Valenta, J. N.; Dixon, R. P.; Hamilton, A. D. Anal. Chem., 1994, 113, 2397. (d) Hamilton, A. D. Bioorganic Chemistry Frontiers, 1991, 2, 115.

2. Tecilla, P.; Jubian, V.; Hamilton, A. D. Tetrahedron, 1995, 51, 435.

3. (a) Clark-Lewis, J. W.; Thompson, M. J. J. Chem. Soc., 1957, 442. (b) Markees, D. G.; Dewey, V. C.; Kidder, G. W. J. Med. Chem., 1968, 11, 126.

4. Czarnik, A. W. Acc. Chem. Res., 1994, 27, 302.

5. 1; mp 267 – 268 8C. ¹H NMR of 1 (CDCl₃) d 0.92 (t, 3H, J = 7 Hz, CH₃), 1.10 – 1.48 (m, 4H, CH₂), 2.41 – 2.50 (m, 2H, COCCH₂), 4.93 (s, 22H, Anth- CH₂), 7.41 – 7.49 (m, 4H, Anth-2CH, 3CH, 6CH, and 7CH), 7.94 – 7.99 (m, 2H, Anth-4CH and 5CH), 8.19 – 8.24 (m, 2H, Anth-1CH, 8CH), 8.39 (s, 1H, Anth-10CH). Anal. Calcd for C₂₃H₂₂N₂O₃: C, 73.78; H, 5.92; N, 7.48. Found: C, 73.96; H, 5.92; N, 7.24).

6. (a) Ohshima, S.; Tamura, N.; Nabeshima, T.; Yano, Y. J. Chem. Soc., Chem. Commun., 1993, 712. (b) Tamura, N.; Mitsui, K.; Nabeshima, T.; Yano, Y. J. Chem. Soc., Perkin Trans. 2, 1994, 2583. (c) Tamura, N.; Kajiki, T.; Nabeshima, T. Yano, Y. Chem. Lett., 1997, 75.

7. Reichardt, C. Solvents and solvent effects in Organic Chemistry; VCH Weiheim, 1988, p.16.