Vanesa Vaquero

All Five Forms of Cytosine Revealed in the Gas Phase

The determination of preferred tautomers of nucleobases has been of interest since the structure of nucleic acid and its base pairs was first reported. Molecular-level understanding of their structure can provide important insight into the relationship that exists between the presence of tautomeric forms and spontaneous mutation in DNA. The best experimental approach to address the structural preferences of nucleobases is to place them under isolation conditions in the gas phase, cooled in a supersonic expansion. Under these conditions, the various tautomers/conformers can coexist and are not affected by the bulk effects of their native environments, which normally mask their intrinsic molecular properties. The main restriction to the gas-phase study of these building blocks is the difficulty in their vaporization owing to their high melting points (ranging from 316 8C for guanine to 365 8C for adenine) and associated low vapor pressures. We have shown previously that the use of molecular beam Fourier transform microwave (MB-FTMW) spectroscopy in conjunction with laser ablation (LA) enables these vaporization problems to be overcome and renders the study of the rotational spectra of coded amino acids accessible. The success of these LA-MBFTMW experiments prompted their application to nucleic acids, and our initial studies on uracil and thymine enabled the determination of the structures of their diketo forms present in the gas phase. A subsequent study of guanine led us to unequivocally identify the four most stable tautomers in the gas phase. The molecular system of cytosine (CY) is even more complex than that of guanine. Figure 1a shows the five most stable species, in order of stability according to theoretical calculations: enol–amino trans (EAt), enol– amino cis (EAc), keto–amino (KA), keto–imino trans (KIt), keto–imino cis (KIc). In 1988, Szczesniak et al. observed the infrared spectra of CY isolated in inert Ar and N2 matrices and showed that isolated cytosine exists under these conditions as a mixture of the KA and EA forms (they did not distinguish between EAt and EAc). Brown et al. reported the free-jet millimeterwave absorption spectra of three species, which were tentatively assigned as the KA, EAt, and KI forms. The identification was based on the values of the rotational constants alone. In contrast, Dong and Miller used infrared laser spectroscopy in helium nanodroplets to characterize EAt, EAc, and KA species. Nir et al. attributed two features observed in the vibronic spectra to the KA and EA forms. The electron diffraction pattern was interpreted in terms of a conformation mixture dominated by the EA forms. X-ray photoemission spectra provide spectral signatures of oxo and hydroxy populations. In recent experiments in an Ar matrix, photoisomerization processes induced by narrowband tunable near-infrared and UV light were interpreted in terms of the existence of various tautomers of CY. No conclusive experimental evidence for the coexistence of the five predicted forms has yet been reported. We took advantage of the capabilities of LA-MB-FTMW spectroscopy to investigate the rotational spectra of cytosine in the solvent-free environment of a supersonic expansion. In this technique, the solid samples are vaporized by laser ablation, and the molecules are seeded in a supersonic expansion, in which CY is ideally frozen and the most stable forms trapped in their energy minima. The rotational spectrum of each of these molecular forms can be analyzed separately by Fourier transform microwave spectroscopy. Figure 1b shows details of the five 11,1–00,0 transitions corresponding to five different rotamers of CY observed in the 5100–5300 MHz frequency range. Each rotational transition shows a very complex hyperfine structure composed of tens of quadrupole component lines owing to the presence of three N nuclei. This hyperfine structure arises from the coupling of the N nuclear-spin angular momenta (I = 1) to the overall rotational angular momentum through the interaction of the quadrupole moment of each N nucleus with the electric-field gradient created at the site of this nucleus by the rest of the molecular charges. Analysis of this hyperfine structure yields the nuclear quadrupole coupling constants cab (a,b = a, b, c), which are extremely sensitive to the electronic distribution around the quadrupolar nuclei N1, N3, and N8 (see Figure 1a for nitrogen-atom labeling) and can be used as a valuable tool for the unambiguous identification of tautomers of CY. [*] Prof. J. L. Alonso, Dr. V. Vaquero, Dr. I. PeÇa, Prof. J. C. L pez, S. Mata, Prof. W. Caminati Grupo de Espectroscop a Molecular (GEM), Edificio Quifima Laboratorios de Espectroscopia y Bioespectroscopia Parque Cient fico UVa, Universidad de Valladolid 47005 Valladolid (Spain) E-mail: jlalonso@qf.uva.es Homepage: http://www.gem.uva.es [] Present address: Dipartimento di Chimica “G. Ciamician” dell’Universit via Selmi 2, 40126 Bologna (Italy)

Observation of dihydrated glycine

The complex of glycine with two water molecules glycine-(H2O)2 has been generated by laser ablation in a supersonic expansion and characterised using rotational spectroscopy. The water molecules bind to the carboxylic group of glycine and to each other through three intermolecular hydrogen bonds, closing an eight-membered ring. In the complex, glycine adopts the conformation found to be the most stable for bare glycine.

Alanine Water Complexes

Two complexes of alanine with water, alanine-(H2O)n (n = 1,2), have been generated by laser ablation of the amino acid in a supersonic jet containing water vapor and characterized using Fourier transform microwave spectroscopy. In the observed complexes, water molecules bind to the carboxylic group of alanine acting as both proton donors and acceptors. In alanine-H2O, the water molecule establishes two intermolecular hydrogen bonds forming a six-membered cycle, while in alanine-(H2O)2 the two water molecules establish three hydrogen bonds forming an eight-membered ring. In both complexes, the amino acid moiety is in its neutral form and shows the conformation observed to be the most stable for the bare molecule. The microsolvation study of alanine-(H2O)n (n = 1,2) can be taken as a first step toward understanding bulk properties at a microscopic level.