IS ARGININE ZWITTERIONIC OR NEUTRAL IN THE GAS PHASE? RESULTS FROM IR CAVITY RINGDOWN SPECTROSCOPY

It is commonly thought that amino acids and peptides exist in a neutral configuration (protonated carboxylic acid and deprotonated amine) in the gas-phase1,2 because zwitterionic charge separation is unfavorable in the absence of solvation. However, a recent report suggested that the most stable form of gaseous arginine is actually zwitterionic.3 In this proposed configuration, the guanidine in the side chain serves as an intramolecular proton acceptor, whereas the carboxylic acid serves as the donor (cf. Figure 1). We know of no previous direct experimental evidence that can support this intriguing claim and have accordingly performed a spectroscopic study of jet-cooled arginine using the novel technique of infrared cavity ringdown laser absorption spectroscopy (IR-CRLAS). Our results confirm that arginine indeed exists in the neutral configuration in a supersonic molecular beam. The characterization of isolated gas-phase zwitterions is an important objective that has thus far proved elusive. Detailed spectroscopic information would facilitate understanding of the structure and energetics of these systems, providing data that could be used to better parametrize biomolecular potential models.4 Moreover, such studies constitute an essential starting point for investigating salient details of biomolecular solvation. In infrared spectroscopy experiments, one could unambiguously identify the neutral form of arginine by the presence of carbonyl stretch bands at ca. 1700 cm-1 and the zwitterion by carboxylate asymmetric and symmetric stretches at ca. 1500-1600 cm-1. IR-CRLAS is a new ultrasensitive direct absorption technique that has recently been used to study a variety of molecular systems.5-7 The method employs a set of highly reflective mirrors that form an optical cavity into which an absorbing sample can be placed. The sample absorption is determined by monitoring the exponential intensity decay of laser radiation coupled into the cavity. Resonant absorption will attenuate more light on each pass than does the passive cavity, leading to a faster decay of the light intensity. By measuring the time constant of the exponential decay, one can directly extract the absolute absorbance of the sample. Tuning the light over a given frequency range will thus produce an absorption spectrum. Ultrahigh sensitivity results from the combination of a large path length and insensitivity to fluctuations in the total intensity. Gas-phase arginine was produced using a heated, pulsed, 4-inch-slit molecular beam source, described previously.8 The free base form of L-arginine (CAS 74-79-3) was obtained from Sigma. No additional sample preparation was used. Maintaining the source at a temperature ca. 170 °C optimized the arginine signal. After the experiment was run, a mass spectrum and FTIR spectrum (taken in a KBr pellet) of the sample remaining in the source as well as the residue collected on a microscope slide downstream of the source confirmed that the sample in the molecular beam did not undergo significant decomposition. The IR-CRLAS spectrum of arginine in the 1550-1750 cm-1 region revealed two peaks near 1700 cm-1 (cf. Figure 2; 1666 cm-1, 1693 cm-1), corresponding to a carbonyl stretch of a carboxylic acid and confirming that neutral arginine was present in our molecular beam. The fact that several peaks were observed in this region can be explained by the presence of several nearly isoenergetic conformers. Different structures led to distinct local environments and vibrational frequencies. Similar patterns have been found in matrix isolation studies of other amino acids.9 Scans near 1600 cm-1 showed no peaks. Consequently, a significant population of zwitterions cannot be present in our molecular beam, assuming that the carboxylate mode intensity of the zwitterionic form of arginine is comparable to the carbonyl mode intensity of the neutral. (1) Reva, I. D.; Plokhotnichenko, A. M.; Stepanian, S. G.; Ivanov, A. Yu.; Radchenko, E. D.; Sheina, G. G.; Blagoi, Y. P. Chem. Phys. Lett. 1995, 232, 141-148. (2) Locke, M. J.; McIver, R. T. J. Am. Chem. Soc. 1983, 105, 4226-4232. (3) Price, W. D.; Jockusch, R. A.; Williams, E. R. J. Am. Chem. Soc. 1997, 119, 11988-11989. (4) Gregurick, S. K.; Fredj, E.; Elber, R.; Gerber, R. B. J. Phys. Chem. B 1997, 101, 8595-8606. (5) Scherer, J. J.; Voelkel, D.; Rakestraw, D. J.; Paul, J. B.; Collier, C. P.; Saykally, R. J.; O’Keefe, A. Chem. Phys. Lett. 1995, 245, 273-280. (6) Paul, J. B.; Collier, C. P.; Saykally, R. J.; Scherer, J. J.; O’Keefe, A. J. Phys. Chem. A 1997, 101, 5211-5214. (7) Paul, J. B.; Saykally, R. J. Anal. Chem. 1997, 69, A287-A292. (8) Liu, K.; Fellers, R. S.; Viant, M. R.; McLaughlin, R. P.; Brown, M. G.; Saykally, R. J. ReV. Sci. Instrum. 1997, 67, 410-416. (9) Reva, I. D.; Stepanian, S. G.; Plokhotnihenko, A. M.; Radchenko, E. D.; Sheina, G. G.; Blagoi, Yu. P. J. Mol. Struct. 1994, 318, 1-13. Figure 1. Zwitterionic form of arginine. Notice that this is not the standard zwitterion associated with biomolecules. For this system, the guanidine in the side chain is the base, as opposed to the backbone amine of the amino acid.