Chiral Recognition of Verapamil by Cyclodextrins Studied With Capillary Electrophoresis, NMR Spectroscopy, and Electrospray Ionization Mass Spectrometry BEZHAN CHANKVETADZE,† NAIRA BURJANADZE, GIORGIO PINTORE,‡ DIRK STRICKMANN, DIETER BERGENTHAL, AND GOTTFRIED BLASCHKE* Institute of Pharmaceutical Chemistry, University of Mu ¨nster, Hittorfstrasse 58-62, 48149 Mu ¨nster, Germany ABSTRACT Capillary electrophoresis (CE) allows the observation of the opposite affinities of the enantiomers of (±)-verapamil [2-isopropyl-2,8-bis(3,4-dimethoxyphenyl)- 6-methyl-6-azaoctannitrile, VP] toward -cyclodextrin (-CD) and heptakis(2,3,6-tri-O- methyl)--CD (TM--CD). In addition, in the presence of -CD in the background elec- trolyte, longer migration times and lower separation factors were observed compared to TM--CD. The binding constants of (+)- and (-)-VP with -CD and TM--CD determined using 13 C NMR spectroscopy explain the results observed in CE. Electrospray ionization mass spectrometry (ESI-MS) was used as an alternative technique for the characteriza- tion of VP-CD complexes. Chirality 11:635–644, 1999. © 1999 Wiley-Liss, Inc. KEY WORDS: chiral CE; NMR spectroscopy; ESI-MS; cyclodextrins; (±)-verapamil/ cyclodextrin complexes; stoichiometry; binding constants Capillary electrophoresis (CE) is rapidly being estab- lished as one of the major techniques for analytical-scale enantioseparations. 1 The most important advantage of this technique is the high peak efficiency that allows the detec- tion of very weak intermolecular solute/selector interac- tions or very low stereoselectivities in these interactions. Further advantages of CE include small amounts of samples, selectors and buffers, short analysis times, flex- ibility, low costs, etc. The immobilization of the selector is also not required in CE. On the other hand, CE does not provide direct information on the molecular mechanisms of chiral recognition. Other instrumental techniques, such as nuclear magnetic resonance (NMR) spectroscopy, mass- spectrometry (MS), and X-ray crystallography may complement CE well from this viewpoint. Cyclodextrins (CD) are the most widely used chiral se- lectors in CE. 1–4 Despite many efforts, 5–7 the study of their chiral recognition mechanisms still remains challenging. 8,9 One of the key issues in understanding how CDs work stereoselectively is the enantiomer recognition pattern. It is known that the affinity of the enantiomers of some chiral compounds is opposite toward differently modified CDs. 10 For instance, D-alanine naphthylamide is preferentially complexed with native -CD whereas the L-enantiomer is more strongly bound by heptakis(2,3-diacetyl)--CD. 11 -CD prefers D-oxamniquine as a guest, whereas the L- enantiomer is preferentially bound by randomly substi- tuted 2-hydroxypropyl--CD. 12 Further examples are de- scribed in the literature. 13–15 It is difficult to predict how a given chemical modification affects the multiple forces in- volved in the intermolecular interactions between CDs and their guests. This knowledge may contribute to the under- standing of the nature of the major forces contributing to solute/CD binding and those responsible for stereoselec- tivity. Chiral recognition of the enantiomers of the calcium channel blocker verapamil (VP, 2-isopropyl-2,8-bis(3,4- dimethoxyphenyl)-6-methyl-6-azaoctannitrile) by various cyclodextrins was studied in this work using CE, NMR spectroscopy, and ESI-MS spectrometry. MATERIALS AND METHODS Materials Racemic VP (Fig. 1) was obtained from Sigma-Aldrich Chemie (Deisenhofen, Germany). The enantiomers of VP were obtained by diastereomeric crystallization with opti- cally pure (+)- or (-)-1,1'-binaphthyl-2,2'-diyl hydrogen phosphate as described in Ref. 19. -, -, and -CD, car- boxymethyl--CDs (CM--CD) with average molecular substitution degrees (DS) of 2.1 and 3.5, succinyl--CD (SUC--CD) with a DS of 3.5, sulfoethyl--CD (SEE--CD) with a DS of 2.8, and methyl--CDs (ME--CD) with a DS of 4.2 and 12.6 were a gift from Wacker-Chemie (Munich, Germany). Heptakis(2,6-di-O-methyl)--CD (DM--CD), heptakis(2,3,6-tri-O-methyl)--CD (TM--CD), 2-hydroxy- propyl--CD (HP--CD) with a DS of 4.2, and 2-hydroxy- *Correspondence to: Prof. G. Blaschke. E-mail: blaschg@uni-muenster.de. †Permanent address: Department of Chemistry, Tbilisi State University, Chavchavadze Ave. 1, Tbilisi 380028, Georgia. ‡On leave from: Dipartimento Farmaco Chimico Tossicologico, Faculty of Pharmacy, University of Sassari, Via Muroni 23, I-07100 Sassari, Italy. Received for publication 20 October 1998; Accepted 22 February 1999 CHIRALITY 11:635–644 (1999) © 1999 Wiley-Liss, Inc.