Andrew Durgan Department of Chemistry & Biochemistry Gonzaga University September 24, 2009

Introduction – Carbon-Deuterium Labeled Amino Acids – Methods: Vibrational Techniques – Infrared Spectroscopy (IR) IR versus Raman Spectroscopy IR Sampling Results – IR of C  -D Proline – 13 C NMR of Proline – Calculations of Proline Conformations Conclusion/Future Research

-C-D-C-D O-H, C-H, N-H Amide bands & C=C, C=O, C=N C-D frequency shifted away from unlabeled vibrations Site-specific isotopic labeling improves structural resolution C-D bonds report on their local environment C  -D bonds report on ψ/Φ dihedral changes Deuterium labeling produces essentially no perturbation Protein IR Spectrum

Unique ring structure imposes backbone conformational constraints Proline appears in many neuropeptides cis-trans Amide isomerization is hypothesized to play a role in neuropeptide selectivity D CαCα

Raman Pro/Con Does not cause local heating or photodecomposition of the sample Can cause local heating or photodecomposition of the sample More accessible instrumentation Less accessible instrumentation due to laser requirement Con/Pro Spectral observations can be obscured by H 2 O (g) and CO 2 (g) absorptions Spectral observations are not obscured by H 2 O (g) and CO 2 (g) absorptions. Conclusion: Both spectroscopic techniques can be useful to C-D labeled amino acid investigations. Infrared (IR)

Potassium Bromide Pellets 2 mg of sample were homogenized with 80 mg of KBr Transmission Spectra 20 µL of aqueous samples (0.050 – 1.0 M) were dispensed between CaF 2 plates with a 75  m spacer. Scan Parameters The sample chamber was purged with N 2 and 8000 scans at 4 cm -1 resolution were collected using an MCT detector.

At least four absorptions related to Pro-a-d 1 were readily observed at approximately 2245, 2202, 2140, and 2060 cm -1

Similar to solid sampling, at least four absorptions related to Pro-  -d 1 were observed at approximately 2240, 2199, 2135, and 2050 cm -1 Solid: Solution:

D-C α -C′-OH Dihedral D-C α -C β -H syn Dihedral CβCβ CαCα H syn D C' B3LYP/6-31G* calculations with anharmonic corrections (Gaussian 03) were used to explore frequency changes Protonated proline was used as a model The D-C  -C  -H syn and D-C  -C'-OH dihedrals capture the ring flip and CO 2 H rotation respectively

CO 2 H rotation ring flip 5.2 kcal/mol 2225 cm -1 0 kcal/mol 2244 cm kcal/mol 2246 cm kcal/mol 2239 cm -1 Relative electronic energies and C-D stretching frequencies are presented. For zwitterionic Pro, only two conformations might be expected due to symmetry of the carboxylate group.

CH 2 / NH 2 twisting and H-O-C' bend 1220 cm -1 CH 2 / NH 2 twisting and D-C  -N bend 1035 cm -1 A combination band at 2255 cm -1 was predicted to be in resonance with the C  -D stretch for all four conformations. These calculations predict possible resonances associated with CH 2 / NH 2 twisting and/or CH 2 / NH 2 twisting overtones.

Pro ring flipping is faster than the NMR timescale but not the IR timescale, which is consistent with the observation of multiple IR absorptions and only one set of NMR signals. Calculations predict that the observed absorption bands arise from Pro ring flipping, resonances associated with CH 2 / NH 2 twisting, and possible CH 2 / NH 2 twisting overtones. Specific assignments are in progress.

Trp & Phe show absorptions at 2310 / 2280 & 2275 cm -1 Future work includes incorporating C-D labeled amino acids (Pro, Phe, & Trp) into neuropeptides (e.g. Try-Pro-Trp-Phe).

Dr. Matthew Cremeens, Gonzaga University Christy Watson, Gonzaga University Matt Deslman, Gonzaga University Dr. Steven Corcelli, Notre Dame Gonzaga Science Research Program Howard Hughes Medical Institute