Walther Caminati

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)

Pure rotational spectrum and model calculations of indole–water

The molecular beam Fourier transform microwave spectra of two isotopomers of the 1:1 complex between indole and water have been measured. The water molecule has been reliably located in the complex from these experimental data. The complex has a Cs symmetry with an N–H⋯O hydrogen bond and the plane of the H2O molecule perpendicular to the indole plane. The two-dimensional potential energy surface of the internal rotation and inversion of water in the complex, evaluated with B3LYP/6-31G** or MP2/6-31G** quantum chemical calculations, suggests the tunneling motion of water to take place with the contribute of both motions. The experimental evidence combined with flexible model calculations, indicate, however, that the tunneling motion is mainly an internal rotation of water around its C2 symmetry axis.

How water links to cis and trans peptidic groups: the rotational spectrum of N-methylformamide–water

We investigated the Fourier transform microwave spectra of the hydrated forms of N-methylformamide (NMF) in a supersonic expansion and assigned the rotational spectra of two mono-hydrated species. The conformation of each molecular complex was reliably determined on the basis of the values of the rotational constants, of the relative intensities of mu(a)- and mu(b)-type transitions, and of the features of the (14)N quadrupole hyperfine structure of the rotational transitions. In both complexes water acts as a proton donor and NMF has a trans configuration of the peptidic group. In the most stable of these conformers, water is also weakly bound to the methyl group.

Local anesthetics in the gas-phase: The rotational spectrum of butamben and isobutamben

MONTSERRAT VALLEJO-LÓPEZ, Physical Chemistry, University of the Basque Country, Leioa Bilbao, Spain; PATRICIA ECIJA, Physical Chemistry Department, Universidad del Paı́s Vasco, Bilbao, Spain; WALTHER CAMINATI, Dep. Chemistry ’Giacomo Ciamician’, University of Bologna, Bologna, Italy; JENS-UWE GRABOW, Institut für Physikalische Chemie und Elektrochemie, Gottfried-Wilhelm-LeibnizUniversität, Hannover, Germany; ALBERTO LESARRI, Departamento de Quı́mica Fı́sica y Quı́mica Inorgánica, Universidad de Valladolid, Valladolid, Spain; EMILIO J. COCINERO, Physical Chemistry Department, Universidad del Paı́s Vasco, Bilbao, Spain.

SOLVING THE TAUTOMERIC EQUILIBRIUM OF PURINE THROUGH THE ANALYSIS OF THE COMPLEX HYPERFINE STRUCTURE OF THE FOUR 14N NUCLEI

EMILIO J. COCINERO, ICIAR URIARTE, PATRICIA ECIJA, Physical Chemistry Department, Universidad del Paı́s Vasco (UPV/EHU), Bilbao, Spain; LAURA B. FAVERO, Istituto per lo Studio dei Materiali Nanostrutturati, Consiglio Nazionale delle Ricerche (ISMN-CNR), Bologna, Italy; LORENZO SPADA, CAMILLA CALABRESE, WALTHER CAMINATI, Dep. Chemistry ’Giacomo Ciamician’, University of Bologna, Bologna, Italy.