Vibrational spectroscopy is a classical technique used to examine molecular structure and dynamics.1 The major challenge often is assigning vibrational modes and interpreting their frequencies and intensities in terms of the molecular coordinates. This is often simplified if group vibrations occur which are associated with molecular coordinates of interest.2 Functional group vibrations within polymers can couple if they are located close to one another to give collective polymer vibrations. The observation of coupled vibrations should be more common in IR spectroscopy because IR selects for vibrations with large dipole moment changes. Vibrations with large dipole moment changes can couple through space by transition dipole coupling. In contrast, resonance Raman spectroscopy (RR) selects through criteria independent of dipole moment changes. Thus, vibrations observed by RR are less likely to be coupled. Here we examine coupling between amide vibrations in tripeptides and in derivatives with adjacent amide groups and ask whether the vibrations observed by RR result from vibrations localized within individual amide peptide bonds, or whether these vibrations are delocalized and result from coupled motion of adjacent amide peptide bonds. This work is part of a research program where we are developing UVRR for studying biological structure and function.3 We recently demonstrated that RR excitation within the amide π f π* transitions enhanced amide R bands.4 The resulting amide RR spectra quantitatively determine peptide and protein secondary structure.5,6 In addition, we have used UVRR to probe the first steps in the folding and unfolding of peptides due to ns temperature jumps.7,8 We determined the extent of vibrational coupling between amide groups by measuring the RR spectra of linked amides. We compared the spectra of natural isotopomers to those formed by exchanging the labile N-H groups by N-D in D2O. We then measured these derivatives in a mixed H2O/D2O solution, where N-H groups were only partially deuterated. Replacement of NH by ND dramatically alters the normal mode, since N-H motion no longer couples to C-N motion. The hypothesis follows: if normal modes of the linked amides couple, deuteration of an amide would perturb the frequencies and RR cross sections of the linked nondeuterated amide. Thus, UVRR could not be modeled as a sum of pure deuterated and nondeuterated derivatives.9 Figure 1 shows the RR spectra of the linked amide derivatives N-acetyl-N′-methylglycinamide (AcGNMe), N-acetyl-N′-methylL-alanylamide (AcANMe), triglycine (G3), and trialanine (A3) in water and pure D2O. We observe a more complex spectrum in H2O, where the linked amides give overlapping AmI, AmII, and AmIII bands. The AmII and AmIII bands are characteristically described as involving coupled C-N stretching and N-H inplane bending. However, as noted by others, the AmIII vibration has a more complex composition which depends on the exact molecular structure.10-12 The spectra considerably simplify in D2O because the AmII and III modes disappear and are replaced by very intense AmII′ modes, which are mainly C-N stretching.13 The AmI′ modes of the deuterated derivatives shift relative to those of the hydrogenated derivative. Thus, the N-H spectra and N-D spectra differ dramatically. AcGNMe, which is the simplest model peptide containing two adjacent amide groups, shows a spectrum similar to N-methylacetamide (NMA).13 The AmIII, AmII, and AmI bands occur at ∼1305, ∼1573, and ∼1640 cm-1. Obviously, bands from the two amide groups overlap. The AIII low-frequency shoulder (1255 cm-1) originates from a CH2tw mode, which also contains CNs. The ∼1380 cm-1 band is mainly due to (C)CH3 sb.13 The ND derivative of AcGNMe (AcGNMeD) shows a spectrum identical to that of NMAD. It is dominated by the 1490 cm-1 AmII′ band, with a weaker ∼1640 cm-1 AmI′ band. The spectra of AcGNMe and AcGNMeD are almost identical to those of AcANMe and AcANMeD. * To whom correspondence should be addressed. Phone: 412-624-8570. Fax: 412-624-0588. E-mail: asher+@pitt.edu. † University of Pittsburgh. § Universität Bremen. ‡ University of Puerto Rico. (1) Schrader, B., Ed. Infrared and Raman Spectroscopy; VCH Publishers, Inc.: New York, 1995. (2) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic Press: San Diego, CA, 1991. (3) (a) Asher, S. A. Anal. Chem. 1993, 65, 59A-66A. (b) Asher, S. A. Anal. Chem. 1993, 65, 201A-210A. (4) Chi, Z.; Chen, X. G.; Holtz, J. S. W.; Asher, S. A. Biochemistry 1998, 37, 2854-2864. (5) Chi, Z.; Asher, S. A. Biochemistry 1998, 37, 2865-2872. (6) Chi, Z.; Asher, S. A. Biochemistry 1999, 38, 8196-8203. (7) Lednev, I. K.; Karnoup, A. S.; Sparrow, M. C.; Asher, S. A. J. Am. Chem. Soc. 1999, 121, 8074-8086. (8) Lednev, I. K.; Karnoup, A. S.; Sparrow, M. C.; Asher, S. A. J. Am. Chem. Soc. 1999, 121, 4076-4077. (9) Sieler, G.; Schweitzer-Stenner, R. J. Am. Chem. Soc. 1997, 119, 1720. (10) Oboodi, M. R.; Alva, C.; Diem, M. J. Phys. Chem. 1984, 88, 501. (11) Qian, W.; Bandekar, J.; Krimm, S. Biopolymers 1991, 31, 193-210. (12) (a) Lee, S. H.; Krimm, S. Biopolymers 1998, 46, 283-317. (b) Cheam, T. C.; Krimm, S. J. Mol. Struct. (THEOCHEM) 1989, 188, 15-43. (13) Chen, X. G.; Asher, S. A.; Schweitzer-Stenner, R.; Mirkin, N. G.; Krimm, S. J. Am. Chem. Soc. 1995, 117, 2884-2895. (14) Chen, X. G.; Schweitzer-Stenner, R.; Asher, S. A.; Mirkin, N. G.; Krimm, S. J. Phys. Chem. 1995, 99, 3074-3083. Figure 1. 206.5-nm excited UVRR in H2O and D2O of N-acetyl-N′methylglycinamide (1.5 mM, pH/pD ∼ 5, AcGNMe), N-acetyl-N′methylalanylamide (3.0 mM, pH/pD ∼ 5, AcANMe), triglycine (1.5 mM, pH/pD ∼ 5, G3), and trialanine (1.5 mM, pH/pD ∼ 5, A3). 9028 J. Am. Chem. Soc. 2000, 122, 9028-9029