A NUCLEOSIDE UNDER OBSERVATION IN THE GAS PHASE: A ROTATIONAL STUDY OF URIDINE I. PEÑA, J.L. ALONSO Grupo de Espectroscopia Molecular. Unidad asociada CSIC Laboratorios de Espectroscopia y Bioespectroscopia Edificio Quifima. Parque Científico Universidad de Valladolid SPAIN

Introduction GLYCOSIDIC BOND URACIL RIBOSE A nucleoside is composed of a nitrogen base bound to either ribose or deoxyribose via a beta-glycosidic linkage. In particular, uridine is composed of uracil and ribose [1]J. Phys. Chem. A, 111, 3443 (2007) The jet-cooled rotational spectrum of uracil has been investigated using LA-MB-FTMW spectroscopy. 1 The diketo structure of uracil has been determined URIDINE PLAUSIBLE TAUTOMERS OF URACILDIKETO FORM Nucleosides bound to one or more phosphate groups (nucleotides) are considered the molecular building blocks of DNA and RNA

Introduction For D-ribose, eight different PYRANOSE FORMS have been recently characterized in gas phase using CP-FTMW spectroscopy + Laser Ablation

Uridine: the simplest nucleoside Why did nature choose the furanose and not the pyranose form of ribose in RNA???? The most stable form of URACIL + The most stable  and β forms of RIBOSE URIDINE Furanose form of ribose in RNA X

Uridine: the simplest nucleoside Some structural arguments could be based on the intramolecular interactions between the base and ribose moieties, which would stabilize the furanose form versus the pyranose form The aim of the present work is the observation of the isolated nucleoside uridine in the gas phase, free from the bulk effects of the native envinroment, to reveal the biologically important uracil/ribose intramolecular interactions

Experimental: CP-FTMW + Laser Ablation A commercial sample of uridine (m.p.: ºC) was vaporized using the third harmonic of a 20 ps Nd:YAG laser. Products of the laser ablation were supersonically expanded in Ne and probed by broadband CP-FTMW CP-FTMW + LA at GEM, Valladolid S. Mata I. Peña, et al. J. Mol. Spectr. 280(2012) 91– GHz fids

Uridine: CP-FTMW rotational spectra Uracil 2 12  1 01 HC 3 N PHOTOFRAGMENTATION PRODUCTS fids Photofragmentation products such as uracil, glyceraldehyde, ethylene glycol, acetic acid, oxoacetic acid, formaldehyde, acrolein and cyanoacetylene seem to be dominant in the spectrum Is neutral uridine present in the supersonic expansion in an appreciable amount to be detected?

Uridine: CP-FTMW rotational spectra  9 18 rotational transition Resultant spectrum without photofragmentation products Assignment of  a and  b -type R-branch progressions (with the quantum number J ranging from 4 to 17), as corresponding to one rotamer J’K -1 ’K’ +1 J’’K -1 ’’K +1 ’’ obs

Results EXPERIMENTTHEORY Parameter RotamerAnti/C2’endo g+ 1 Syn/C2’endo g+Anti/C3’endo g+Anti/C2’endo tSyn/C3’endo g+ A /MHz (24) d B /MHz (71) C /MHz (36)  μ a  /D Observed  μ b  /D Observed  μ c  /D Observed N(1)  aa /MHz  bb /MHz  cc /MHz N(3)  aa /MHz  bb /MHz  cc /MHz NcNc ΔE /cm Ab initio calculations MP G(d,p) below 1000 cm -1 NOT CONCLUSIVE IDENTIFICATION What about the quadrupole constants? The high resolution of LA-MB-FTMW is needed

Experimental: narrowband LA-MB-FTMW FT-MW Fabry-Pérot Resonator Picosecond Laser LA-MB-FTMW at GEM, Valladolid 14 N  9 18 rotational transition Narrowband LA-MB-FTMW Sub-Doppler resolution 3-10GHz Third harmonic of a 20 picosecond laser Microwave radiation pulse of 0.3  s duration Frequency accuracy better than 5 kHz and an estimated resolution of 7 kHz Broadband CP-FTMW I. Peña et al. JACS 134 (2012) 2305–2312

Results EXPERIMENTTHEORY Parameter RotamerAnti/C2’endo g+ 1 Syn/C2’endo g+Anti/C3’endo g+Anti/C2’endo tSyn/C3’endo g+ A /MHz (14) d B /MHz (35) C /MHz (20) D J /kHz (10)  μ a  /D Observed  μ b  /D Observed  μ c  /D Observed N(1)  aa /MHz (42)  bb /MHz (72)  cc /MHz (72) N(3)  aa /MHz (40)  bb /MHz (70)  cc /MHz (70) NcNc 47 ΔE /cm Ab initio calculations MP G(d,p) below 1000 cm -1 CONCLUSIVE IDENTIFICATION

Intramolecular hydrogen bonding O3HO2HO2 C2HO5 Anti/C2’-endo g+ C6HO5 Intramolecular hydrogen bonding network: 1 Weak interactions: Distances in Å

Conclusions  Uridine has been placed in the gas phase by laser ablation and the most stable Anti/C2’-endo g+ has been characterized by broadband CP-FTMW and narrowband LA-MB-FTMW 1. E. A. Green et al., Acta Cryst. (1975), B31, 102  The preference for furanoses in RNA might be due to the observed intramolecular hydrogen bond network and the weak interactions between uracil and ribose, which over stabilizes this species and prevents the generation of pyranoses.  The observation of this conformer is in accordance with ab initio results, but in relative contrast with those obtained by X-ray 1 where C3’endo/anti conformation has been observed. It has been shown that the C3’-endo configuration, which is in RNA, is favored when an explicit water molecule is introduced into the calculation. Nevertheless, previous calculations indicate that the change of the sugar pucker from C3’-endo to C2’-endo in single-stranded RNA is energetically possible

ACKNOWLEDGMENTS Grants CTQ , AYA and AYA CSD Molecular Astrophysics Grants VA070A08 and CIP13/01 Grupo de Espectroscopia Molecular (GEM) Laboratorios de Espectroscopia y Bioespectroscopia, Unidad Asociada CSIC, UVa,Valladolid, Spain