Formation of p-cresol:piperazine complex in solution monitored by spin–lattice relaxation times and pulsed field gradient NMR diffusion measurements Erika Martins de Carvalho, a Marcia Helena Rodrigues Velloso, a Luzineide Wanderley Tinoco, b and Jos  e Daniel Figueroa-Villar a, * a Departamento de Qu  ımica, Instituto Militar de Engenharia, Prac ßa General Tib urcio 22290-270, Rio de Janeiro, Brazil b Centro Nacional de Resson^ ancia Magn etica Nuclear, Departamento de Bioqu  ımica M edica, Universidade Federal de Rio de Janeiro, Brazil Received 2 January 2003; revised 3 July 2003 Abstract A study of the nature of the anthelmintic p-cresol:piperazine complex in chloroform solution has been conducted using different NMR techniques: self-diffusion coefficients using DOSY; NOE, NULL, and double-selective T 1 measurements to determine inter- molecular distances; and selective and non-selective T 1 measurements to determine correlation times. The experimental results in solution and CP-MAS were compared to literature X-ray diffraction data using molecular modeling. It was shown that the p-cresol:piperazine complex exists in solution in a very similar manner as it does in the solid state, with one p-cresol molecule hydrogen bonded through the hydroxyl hydrogen to each nitrogen atom of piperazine. The close correspondence between the X-ray diffraction data and the inter-proton distances obtained by NULL and double selective excitation techniques indicate that those methodologies can be used to determine inter-molecular distances in solution. Ó 2003 Published by Elsevier Inc. Keywords: Molecular complexes; Diffusion coefficients; Inter-molecular distances; Longitudinal relaxation; Selective T 1 1. Introduction The great importance of molecular recognition in biochemical processes has given impulse to research on inter-molecular interaction phenomena [1]. For exam- ple, understanding how supramolecular complexes are formed and how the forces that keep them together are affected by changes in the molecular environment is crucial for the rational design of pharmacologically ac- tive compounds [2]. Among the interactions that are involved in inter-molecular recognition, the hydrogen bond is one of the most important ones [1,3]. This kind of interaction is involved in several of the most impor- tant biochemical processes, such as base pairing and interactions in nucleic acids [4], substrate recognition by the active sites of several enzymes [5], cell recognition by antibodies [6], protein folding [7], etc. Clearly, under- standing how hydrogen bonds are affected by the chemical nature of the compounds involved and by the changes in the molecular environment (pH, viscosity, solvent nature, and salinity, etc.) is of great importance to have a deep insight of such biochemical processes and to be able to manipulate them. It is clear that the study of hydrogen bonding in biochemical processes, and any other similar interac- tions, would be better carried out in solution, since those conditions would be closest to physiological conditions. On the other hand, from a technical point of view, the most attractive features of NMR for inter-molecular interaction studies are three. First, the possibility of determination of inter-atomic distances and the topo- logy of the molecular recognition using techniques which are based on nuclear relaxation and related phe- nomena. Among those techniques are included non-se- lective, selective, and double selective longitudinal or spin–lattice relaxation rates (R 1 , R S 1 , and R DS 1 ), which allow for the determination of inter-proton distances Journal of Magnetic Resonance 164 (2003) 197–204 www.elsevier.com/locate/jmr * Corresponding author. Fax: +5521-2546-7059. E-mail address: d5figuer@epq.ime.be.br (J.D. Figueroa-Villar). 1090-7807/$ - see front matter Ó 2003 Published by Elsevier Inc. doi:10.1016/S1090-7807(03)00252-0