Different Conformers and Protonation States of Dipeptides Probed by Polarized Raman, UV-Resonance Raman, and FTIR Spectroscopy Guido Sieler, † Reinhard Schweitzer-Stenner,* ,† Janet S. W. Holtz, ‡ Vasil Pajcini, ‡ and Sanford A. Asher ‡ FB1-Institut fu ¨ r Experimentelle Physik, UniVersita ¨ t Bremen, 28359 Bremen, Germany, and Department of Chemistry, UniVersity of Pittsburgh, Pittsburgh, PennsylVania 15260 ReceiVed: June 8, 1998; In Final Form: October 5, 1998 We have measured the polarized nonresonance and resonance Raman as well as FTIR spectra of the model peptides glycylglycine and N-acetylglycine in H 2 O and D 2 O at pH/pD values between 1.5 and 12.0 with visible, near UV, and far UV excitation wavelengths. The spectra were self-consistently analyzed to obtain reliable spectral parameters of even strongly overlapping bands. Additionally, we have analyzed the polarized nonresonance and preresonance Raman spectra of glycylglycine single crystals. The most important result of this analysis is that for glycylglycine all amide bands as well as the symmetric carboxyl stretch band at ca. 1400 cm -1 are doublets. As shown in an earlier study (Sieler, G.; Schweitzer-Stenner, R. J. Am. Chem. Soc. 1997, 119, 1720) the amide I doublet results from vibrational coupling of the delocalized H 2 O bending mode with internal coordinates of the amide I mode. The amide III doublet is interpreted to result from vibrational coupling between the twisting mode of the C R methylene group and internal coordinates which normally give rise to the amide III vibration (i.e., CN and C R1 C stretching). In contrast, the amide II and carboxylate subbands are assigned to different conformers with respect to the torsional coordinate of the carboxylate group. While the higher frequency subband of the amide II and carboxylate bands may reflect a parallel orientation of the latter with respect to the peptide, which could be stabilized by hydrogen bonding to NH, the lower frequency band may reflect different orientations in which the carboxylate is hydrogen bonded to water. For N-acetylglycine we also observe two subbands underlying amide I and the carboxyl symmetric stretch band, which again reflects vibrational mixing with water and multiple rotational substates of the carboxylate, respectively. Introduction The simplest molecule containing a single peptide group which can serve as a model for investigating the amide linkage in proteins and peptides is N-methylacetamide (NMA, Figure 1). Numerous spectroscopic studies have examined its vibra- tional dynamics, 1 but a detailed understanding of this simple molecule only recently emerged by combining UV-Raman, visible, and FT-Raman as well as FTIR spectroscopies with a normal coordinate analysis based on force constants obtained from ab initio calculations. 2,3 These studies revealed that the amide II frequency depends significantly on the orientation of the methyl group attached to the peptide nitrogen. 2b,3c Even more important, a thorough spectral analysis and normal coordinate calculations on NMA-(H 2 O) 2 complexes revealed that the amide I band of aqueous NMA is a doublet due to vibrational mixing between this mode and the bending mode of surrounding water molecules. 2a,b Finally, the above studies revealed that the electronic transition from the highest occupied amide π orbital into the lowest occupied π* orbital involves bond length changes, not only of the carbonyl and CN bonds but also for the adjacent CC and NC bonds. This shows that the π f π* transition is much more delocalized than earlier anticipated. The advantage of using NMA as a model peptide stems to a significant extent from the fact that it does not contain any charged groups. Thus, however, NMA is of limited use for many peptides because Coulomb interactions between charged ter- minal groups significantly affect the structure as well as the dynamics of small peptides containing up to five amino acids. 4 Simple dipeptides such as glycylglycine (DGL: diglycine, Figure 1), which are zwitterions at physiological pH, are ideal model systems for exploring the influence of negatively and positively charged groups on the peptide’s vibrational dynamics as well as on its electronic and structural properties. A variety of techniques have been employed so far to characterize the ground-state structure of dipeptides. X-ray and neutron diffrac- tion data have shown that their carboxylate and amide planes are not parallel. 5 The angle between them, however, strongly depends on the crystal structure. Hence, these crystallographic data are of limited use for determining the solution structure of small peptides. NMR studies on the dipeptide isomers glycyl- L-alanine and L-alanylglycine by Beeson and Dix utilized methylene proton resonance to obtain conformational informa- tion. 6 Their results led them to suggest that the Coulomb interaction between the terminal groups favors rotamers in which the NH 3 + -carboxylate distance is minimized. The authors also performed molecular mechanics calculations based on a CHARMM force field, which yielded seven different rotamers with similar ground-state energies. While some of them meet the requirements for maximal Coulomb interactions between the terminal groups, the lowest energy conformer was a rotamer with a larger end to end distance. Thus, the role of electrostatic interactions in determining the structure of dipeptides remains unresolved. * To whom all correspondence should be addressed. Phone: **49-421- 218-2509. Fax: **49-421-218-7318. E-mail: stenner@theo.physik.uni- bremen.de. † Universita ¨t Bremen. ‡ University of Pittsburgh. 372 J. Phys. Chem. B 1999, 103, 372-384 10.1021/jp9825462 CCC: $18.00 © 1999 American Chemical Society Published on Web 12/23/1998