2D NMR Methods for Structural Delineation of Copper(II) Complexes of Penicillin and Pilocarpine

A method was developed for delineating the structure of paramagnetic metal complexes. The selective disappearance of cross-peaks in proton-carbon shift correlated 2D NMR maps was shown to uniquely depend upon the scalar and/or dipolar interaction between ligand nuclei and the unpaired electron(s), thus providing a means of identifying binding sites. Copper(II) was shown to form metal complexes with both Penicillin (PNC) and Pilocarpine (PLC) and the structure of the two 1:2 complexes in water solution at physiological pH were determined.


INTRODUCTION
Metal ions are being given growing interest by the pharmacologist" not only several metallorganic and metalloinorganic molecules have been shown to possess wide ranges of biopharmaceutical activities, but also the effects of several drugs have been demonstrated to be modulated by the presence of selected metal ions.
However, the following problem has received poor, if any, Volume 1, Noa: [2][3]1994 2D NMR Method'.[or Structural Delineation of Copper(ll) Complexes of Penicillin andPilocarpine attention. Most drugs are flexible ligands that, in solution, give rise to conformational equilibria among several arrangments of similar conformational energy; it is on the contrary agreed that the drug bound to its receptor assumes a unique conformation that does not necessarily corresponds to the solid state structure. Thus binding to the receptor and, hence, eliciting the biological action also depends on a process of conformational selection that is likely to involve a role of the ubiquitous metal ions, such as calcium, zinc, copper etc. Comprehension of metal-ligand equilibria therefore plays a major role in determining the base for a rationale drug design.
NMR methods provide a great piece of information on metalligand structures and equilibria, especially when the metal ion is a paramagnetic one. In such a case the upor down-field shifts of nuclear resonances or the large enhancements of nuclear relaxation rates provide a unique tool for delineating the structure of metal complexes, as thoroughly reviewed elsewhere (1)(2)(3)(4). The main drawback of these methods is that a well resolved NMR spectrum is required and also that the theory can be applied only in situations where the metal complex is much less abundant than the free ligand and in a restricted range of exchange rates (5). In fact structural information can only be gained when R lb >> kb, where R lb is the nuclear spin-lattice relaxation rate in the metal-complexed environment and kb is the kinetic constant for the ligand leaving the metal coordination sphere. Here we present a novel NMR method that yields a direct structural elucidation of paramagnetic metal complexes by using common 2D techniques, such that resolution limits can be overcome. The structures of the complexes formed by copper(II) ions with pilocarpine, a cholinomimetic agent, and penicillin G are delineated as an example of the potential of the method. All NMR measurements were performed on a Varian VXR-200 spectrometer by using standard procedured for obtaining homoand heteronuclear shift correlated 2D spectra (6,7). The intensity of diagonal and cross peaks in the 2D contour maps was measured by the integral of the peak as obtained by the 1D traces.

THEORY
In order to ascertain the effect of paramagnetic ions on the appearance of 2D shift correlated contour maps, the simple 13C-1H AX spin system is considered. Under spin-decoupling conditions the hetero-COSY and the HOESY spectra both yield identical contour maps with a unique peak correlating the 13C and the H resonances.
The difference in the two pulse sequences (the HOESY experiment has an additional 90 pulse and an additional mixing time) determines that scalar and dipolar connectivities are respectively shown in the COSY and HOESY spectra. The intensity of the crosspeak in the HOESY spectrum can be calculated by (8)  If the same spin system is considered in a paramagnetic metal complex, the occurrence of a strong source of "external" relaxation determines the fast disappearence of the cross peak at increasing concentration of the paramagnetic ion. In fact, as shown in Table 2, it can be easily calculated that the cross-peak almost disappears for an "external" contribution of ca. 20 s-1. E. Gaggelli and 1780 s-1 respectively.
The conclusion is that there is not any chance of detecting cross-peaks in the 2D maps obtained with Table 2. Intensity of the crosspeak in a HOESY spectrum as a function of the "external contribution" to the spin-lattice relaxation However in situations where between the free bulk state and the being this last environment much less the ligand can exchange metal coordination sphere, abundant than the other one, the intensity of the cross-peak is determined by the eigenvalues of either the spectral or the exchange matrices; the consequence is that closeness to the paramagnetic ion selectively affects the volume of correspondent cross peaks in COSY as well as in HOESY spectra (10).

RESULTS AND DISCUSSION
The HOESY map obtained with pilocarpine 100 mM in deuterium oxide buffered at pH 5.5 and at T = 300 K is shown in Figure 1. Any cross-peak in the map represents a one-bond 13C-1H dipolar connectivity and belongs to the 13C_IH spin systems numbered perchlorate linewidths peaks 2D NMR Methods for Stntctural Delineation of Copper(ll) Complexes o.f Penicillin and Pilocarpine as in the molecular formula. The addition of copper gave rise to selective decrease of the cross-peaks' and intensities such that at [Cu++] 0.13 mM the crossboxed in Figure 1 had almost disappeared.
. c H3C-H2C-HC These data supports the evidence of an axclusive involvement of the pyridine-type imidazole nitrogen in metal-binding. Since pilocarpine hydrochloride was shown to preferentially occur in water solution in a conformation characterized by the absence of folding (11), the following conclusions could be drawn: (i) copper binds to pilocarpine (ii) binding of copper occurs at the imidazole protonation site (iii) binding of copper does not result in any evident change of conformation of the ligand. The hereto-COSY map obtained for penicillin G 10 mM in [2H6]-DMSO at T=300 K is shown in figure 2. In this case the crosspeaks represent the one bond 13C-1H scalar connectivities of the different 13C-1H spin systems. Addition of copper ions only affected the linewidths in a selective way such that at [Cu2+]=0.022 mM the boxed cross-peak in the figure had been widened beyond detection limits. Consideration of the possible sites for metal complexation led to conclude that copper is bound to the carboxyl group and to the thiazolidine tertiary nitrogen. Any involvement of the side chain in metal binding was excluded by the absence of any paramagnetic effect upon the C6-H6 or C10-H 10 spin systems.
The examples reported herein for very simple molecules should be taken as to demonstrate the potential of 2D NMR methods in delineating the structure of paramagnetic metal complexes. From this point of view, scalar or dipolar 13C-1H shift correlated 2D NMR contour maps offer similar opportunities of easy and direct detection of metal binding sites. At a deeper level of knowledge it must be however stressed that a comparison of paramagnetic effects on cross-peaks in hetero-COSY and HOESY maps even allows to discriminate between dipolar and scalar interactions with the unpaired electron spin (10).