MAGNETIC RESONANCE IN CHEMISTRY Magn. Reson. Chem. 2002; 40: 123–132 Dynamic and magnetic susceptibility effects on the MAS NMR linewidth of a tetrapeptide bound to different resins Julien Furrer, 1 Karim Elbayed, 1 Maryse Bourdonneau, 2 J´ esus Raya, 1 David Limal, 3 Alberto Bianco 4 and Martial Piotto 2* 1 Institut de Chimie, UMR 7510 CNRS-Bruker, Universit ´ e Louis Pasteur, 67084 Strasbourg, France 2 UMR 7510 CNRS-Bruker, 67160 Wissembourg, France 3 Laboratoire de Chimie Macromol ´ eculaire, ENSCMu, 68093 Mulhouse, France 4 Institut de Biologie Mol ´ eculaire et Cellulaire, Immunologie et Chimie Th ´ erapeutiques, UPR 9021 CNRS, 67084 Strasbourg, France Received 26 July 2001; Revised 28 September 2001; Accepted 28 September 2001 Under magic angle spinning, the NMR spectrum of the tetrapeptide Ala-Ile-Gly-Met bound to a Wang resin, and swollen in DMF, exhibits proton and carbon linewidths that are sharp enough to allow the complete characterization of the peptide using classical liquid-state NMR methods. The proton linewidths of the bound peptide remain, however, about three times larger than those of the free peptide in solution. The residual NMR linewidth originates essentially from incompletely averaged magnetic susceptibility effects due to the Wang resin. Replacing the aromatic Wang resin with a PEGA or POEPOP resin removes this effect. To investigate the contribution to line broadening of the peptide dynamics, relaxation studies were performed on the peptide bound to Wang and POEPOP resins. Copyright  2001 John Wiley & Sons, Ltd. KEYWORDS: NMR; 13 C NMR; 1 H NMR; high-resolution magic angle spinning; solid-phase peptide synthesis; relaxation; magnetic susceptibility INTRODUCTION Over the past 5 years, high-resolution magic angle spinning (HRMAS) 1–3 NMR has become the most powerful method for the characterization of compounds obtained from solid- phase synthesis directly attached to their solid support. 4–12 The technique involves swelling the sample with an appro- priate solvent to reintroduce enough mobility, and then spinning it rapidly at the magic angle. The reason for the success of HRMAS is that MAS has the unique property of averaging the large magnetic field gradients present in such heterogeneous samples to zero. 3,13,14 Under such conditions, the sample behaves very much like a liquid, and standard liquid-state NMR experiments can be employed to identify the bound molecule. There are many applications of HRMAS and it is not limited to the study of compounds issued from solid-phase synthesis. Important current applications include the characterization of polymers, lipids 15 and biological tissues. 16,17 Nevertheless, even under HRMAS conditions, a bound molecule exhibits NMR linewidths that are larger than those of the corresponding free molecule in solution. This extra line broadening clearly makes the study of such solid-supported compounds more challenging than their free counterparts. Ł Correspondence to: M. Piotto, UMR 7510 CNRS-Bruker, 67160 Wissembourg, France. For the purpose of extending the molecular weight range of the biomolecules that can be studied by HRMAS, it is of prime importance to understand the origins of these effects. Intuitively, the extra line broadening observed upon binding can be explained using simple dynamics arguments. When a molecule is linked to a solid support, it loses its ability to rotate freely and isotropically, which leads to shorter transverse relaxation times and broader resonances. This loss of mobility is, however, not the only factor contributing to the extra line broadening. The effects of the heterogeneity of the sample and the presence of strong magnetic susceptibility gradients have to be considered in greater detail, even under MAS conditions. In a recent study, we showed that most of the proton line broadening of the bound tetrapeptide Ala-Ile-Gly-Met was due to some incomplete averaging of magnetic susceptibility effects. 18 In this paper, we present a complete study of the mechanisms of line broadening that occur when the tetrapep- tide Ala-Ile-Gly-Met is covalently bound to three different solid supports. Results obtained on the Wang, PEGA 19 and POEPOP 20 resins are presented. In the first part, the possible causes of line broadening in the sample are discussed and a brief theoretical background concerning the effects of mag- netic susceptibility gradients is presented. In the second part, the dynamic behavior of the bound and of the free peptide is analyzed in order to differentiate between relaxation and magnetic susceptibility effects. In the third part, results DOI: 10.1002/mrc.970 Copyright  2001 John Wiley & Sons, Ltd.