1. 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

2. 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

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

4. 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

5. 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

6. Experimental: CP-FTMW + Laser Ablation A commercial sample of uridine (m.p.: 163-167ºC) 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) 91–96 6-12GHz 120000 fids

7. Uridine: CP-FTMW rotational spectra Uracil 2 12  1 01 HC 3 N PHOTOFRAGMENTATION PRODUCTS 120000 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?

8. Uridine: CP-FTMW rotational spectra 10 19  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 1341012497973.81400 135812577977.91480 1331012398265.73370 150 141 8302.35480 150 140 8305.31120 14312133118454.27510 14411134108587.26500 1451013598591.03200 145913588612.29900 14212132118694.26730 150 141 8302.35480 160 151 8843.46960 161 151 8844.42330 160 150 8845.13850 161 150 8846.09220 8457348886.52800 14311133108913.66000 1561014699188.03220 156914689190.89320 15412144119196.13840 15213142129233.66550 1551014599254.50330 16215 1149295.98810 170 161 9384.15530 171 160 9385.63350 15411144109438.03790 9468359440.89890 7536429491.01450 7526439491.63440 15312143119544.75400 180 171 9924.60250 85474310095.35790 85374410097.55130 163131531210156.34540 95584410696.12500 95484510702.75310 105694511290.59790 1157104611874.58040

9. 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 886.0063 (24) d 901.2935.8790.0799.7925.5 B /MHz 335.59351 (71) 340.6308.4352.6330.6300.4 C /MHz 270.10754 (36) 276.6266.6261.4262.9264.0  μ a  /D Observed 2.11.63.14.52.3  μ b  /D Observed 3.72.81.50.92.3  μ c  /D Observed 0.90.70.81.30.2 14 N(1)  aa /MHz - 1.501.821.481.461.82  bb /MHz - 1.430.731.711.81-0.72  cc /MHz - -2.93-2.56-3.19-3.27-1.11 14 N(3)  aa /MHz - 1.742.031.781.621.98  bb /MHz - 1.110.471.341.51-0.75  cc /MHz - -2.85-2.50-3.12-3.13-1.23 NcNc 66 - ---- ΔE /cm -1 -0383664701751 1 Ab initio calculations MP2-6-311++G(d,p) below 1000 cm -1 NOT CONCLUSIVE IDENTIFICATION What about the quadrupole constants? The high resolution of LA-MB-FTMW is needed

10. Experimental: narrowband LA-MB-FTMW FT-MW Fabry-Pérot Resonator Picosecond Laser LA-MB-FTMW at GEM, Valladolid 14 N 10 19  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

11. 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 885.98961 (14) d 901.2935.8790.0799.7925.5 B /MHz 335.59720 (35) 340.6308.4352.6330.6300.4 C /MHz 270.11270 (20) 276.6266.6261.4262.9264.0 D J /kHz 0.0115 (10) -----  μ a  /D Observed 2.11.63.14.52.3  μ b  /D Observed 3.72.81.50.92.3  μ c  /D Observed 0.90.70.81.30.2 14 N(1)  aa /MHz 1.540 (42) 1.501.821.481.461.82  bb /MHz 1.456 (72) 1.430.731.711.81-0.72  cc /MHz -2.996 (72) -2.93-2.56-3.19-3.27-1.11 14 N(3)  aa /MHz 1.719 (40) 1.742.031.781.621.98  bb /MHz 1.261 (70) 1.110.471.341.51-0.75  cc /MHz -2.979 (70) -2.85-2.50-3.12-3.13-1.23 NcNc 47 ΔE /cm -1 0383664701751 1 Ab initio calculations MP2-6-311++G(d,p) below 1000 cm -1 CONCLUSIVE IDENTIFICATION

12. Intramolecular hydrogen bonding O3HO2HO2 C2HO5 Anti/C2’-endo g+ C6HO5 Intramolecular hydrogen bonding network: 1 Weak interactions: 2.97 1. Distances in Å

13. 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

14. ACKNOWLEDGMENTS Grants CTQ 2010- 19008, AYA 2009-07304 and AYA 2012-32032 CSD 2009-00038 Molecular Astrophysics Grants VA070A08 and CIP13/01 Grupo de Espectroscopia Molecular (GEM) Laboratorios de Espectroscopia y Bioespectroscopia, Unidad Asociada CSIC, UVa,Valladolid, Spain