Controlling Glycosyl Bond Conformation of Guanine Nucleosides

Feb 8, 2012 - Department of Applied Chemistry, Faculty of Engineering, Aichi Institute of Technology, 1247 Yachigusa, Yakusa Cho, Toyota 470-0392, Jap...
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Controlling Glycosyl Bond Conformation of Guanine Nucleosides: Stabilization of the anti Conformer in 5′-O-Ethylguanosine Hiroya Asami,† Shu-hei Urashima,† Masaki Tsukamoto,*,‡ Ayaka Motoda,‡ Yoshihiro Hayakawa,§ and Hiroyuki Saigusa*,† †

Graduate School of Bio- and Nanosystem Sciences, Yokohama City University, Yokohama 236-0027, Japan Graduate School of Information Science, Nagoya University, Chikusa, Nagoya 464-8601, Japan § Department of Applied Chemistry, Faculty of Engineering, Aichi Institute of Technology, 1247 Yachigusa, Yakusa Cho, Toyota 470-0392, Japan ‡

S Supporting Information *

ABSTRACT: Nucleosides that consist of base and sugar moieties can adopt two main conformations, syn and anti, about the glycosidic bond. We have investigated the conformational properties of guanine nucleosides in the gas phase by using laser desorption combined with IR−UV double resonance spectroscopy. In guanosine, syn conformation is preferred as a result of internal hydrogen bonding between the 5′-OH group of the sugar and the N3 site of the guanine moiety. We have therefore employed a chemically modified nucleoside 5′-O-ethylguanosine, in which possible glycosyl bond conformations are restricted upon ethylation of the 5′-OH group. The result shows that anti conformer is stabilized by the formation of hydrogen bonding involving the 2′-OH group. SECTION: Kinetics, Spectroscopy (180 ± 90°) conformations.1 The anti conformation usually dominates due to less steric hindrance between the bulky parts of the sugar and base. However, this stereochemical preference can be relieved by chemical modifications of each component, if properly chosen, or by forming a hydrogen bond between two moieties. Crystal structure analyses of chemically modified purine nucleosides showed that the syn conformation is stabilized as a result of internal hydrogen bonding between the sugar and base.3 This conformational change is often accompanied by sugar puckering motions. In this study, we employ the technique of laser desorption combined with IR−UV double resonance spectroscopy to identify the conformational properties of guanine nucleosides (Chart 1). By using this technique, nonvolatile molecules can be brought into the gas phase as neutrals without serious degradation. Subsequent supersonic jet cooling enables us to isolate low-energy conformers under the low-temperature condition. The neutral gas-phase molecules are ionized by resonance-enhanced two-photon ionization (R2PI), thus allowing for conformational assignment based on the IR−UV double resonance scheme. A previous result for guanosine (Gs) obtained by a similar technique showed that it adopts the syn conformation, which is stabilized by the formation of internal hydrogen bonding between the 5′-OH group with the N3 site

T

he three-dimensional structure of DNA and RNA is intimately related to the conformational properties of nucleotides that consist of a base, sugar, and phosphate.1,2 It is characterized by several stable conformations associated with the structure of the sugar−phosphate backbone and the orientation of the base moiety relative to sugar. Among them, the orientation about the N-glycosidic bond is restricted by two main conformers called syn and anti. The anti conformation can be observed in typical DNA and RNA, while the syn conformation is adopted in Z-DNA. This conformational preference is affected by various factors. For purine nucleosides, the dihedral angle χ defined as that of O4′−C1′−N9−C4 in Chart 1 is used to distinguish between syn (0 ± 90°) and anti Chart 1. Structures and Atom Numbering for Guanine Nucleosidesa

Received: January 20, 2012 Accepted: February 8, 2012 Published: February 8, 2012

a The torsional angle χ is defined as the dihedral angle O4′−C1′−N9− C4.

© 2012 American Chemical Society

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of the guanine moiety.4,5 Likewise, a modified guanine nucleotide, diethyl guanosine 5′-monophosphate, was found to exist in the syn conformation as a result of internal hydrogen bonding between the PO group of phosphate and the amino group of the guanine moiety.6 Here, it is demonstrated that the syn/anti conformation of guanine nucleoside can be controlled by chemical substitution of the sugar. In order to investigate the preference of the anti conformer, we have employed a nucleoside substituted at the 5′-OH group, 5′-O-ethylguanosine (5′-OEtGs, Chart 1). The lowest-energy structure of 5′-OEtGs corresponds to the anti conformation about the glycosyl bond with the guanine moiety either in the enol or keto form. In addition, there are two rotamers for the enol tautomer depending on the orientation of the enol group with respect to the guanine moiety (Figure S1 in Supporting Information). Among these lowest-energy anti conformers, the enol tautomer with its OH bond directed against the base (anti-enol-syn, Figure 1a) is

Supporting Information). The energy calculation also indicates that for each tautomeric structure, there are two additional lowenergy conformers associated with the orientation of the ethyl group. These conformers labeled as ethyl-2 and ethyl-3 (Figure S1c, Supporting Information) are 9−10 kJ/mol higher in energy than the lowest-energy conformer (ethyl-1) shown in Figure 1a. The most stable syn conformer about the glycosyl bond in 5′OEtGs (Figure 1a) is calculated to be ∼18 kJ/mol higher than the anti conformer. This conformer reveals a dihedral angle of 69.9° with the sugar puckering mode in the C3′-endo conformation (syn-C3′-endo). This is in contrast to the case of Gs where the anti conformer with its 2′-OH group hydrogen bonded to the N3 site is 8.7 kJ/mol higher than the most stable syn conformer (Figure 1b). Figure 2a shows the UV spectrum of 5′-OEtGs recorded by the R2PI method. The lowest-energy transition assignable to its

Figure 2. UV spectra of (a) 5′-OEtGs and (b) Gs, obtained by R2PI. Peaks assignable to the respective electronic origins are marked by asterisks.

electronic origin is observed at 34487 cm−1 followed by weaker peaks at irregular intervals. These low-frequency modes are assigned to mutual motions of the sugar and guanine moiety. This vibronic activity appears to be less prominent than that of Gs shown in Figure 2b, suggesting that the base−sugar conformation in 5′-OEtGs is not significantly affected by the electronic excitation. It is also noted that the sharp peaks are accompanied by an underlying background across this energy range. As discussed later, we assign the sharp spectral features as originating from a single conformer about the glycosyl bond, while the broad background is due to other species, presumably clusters or hydrates, which exist in different glycosyl bond conformations and dissociate into monomer ions upon ionization. The UV spectrum of Gs shown in Figure 2b, redshifted from that of 5′-OEtGs, was assigned to the lowestenergy syn conformer with the guanine moiety in the enol form (Figure 1b).4,5 The IR−UV double resonance spectrum obtained by fixing the UV laser to the most intense peak at 34487 cm−1 is shown in Figure 3a. The two peaks located at 3467 and 3582 cm−1 correspond well to those of Gs at 3457 and 3576 cm−1 in Figure 3b, which have been already assigned to the symmetric and antisymmetric NH2 (sNH2 and aNH2) stretch transitions of the amino group.4 The peak at 3589 cm−1 also matches the

Figure 1. Lowest-energy syn and anti conformers of (a) 5′-OEtGs and (b) Gs. In both cases, the most stable enol-syn tautomer is displayed. The relative energies are shown in kJ/mol, while the dihedral angles χ(O4′−C1′−N9−C4) are in degrees. The dotted lines indicate the formation of internal hydrogen bonding. The bond distances are in angstroms.

found to be most stable. The enol-anti and keto tautomers are 1.4 and 2.3 kJ/mol higher, respectively, analogous to the case of Gs.4,5 The dihedral angle χ in these anti conformers is 170.7°, and the sugar puckering mode is in the C2′-endo conformation (anti-C2′-endo). In this major puckering mode, the C2′ atom is displaced on the same side as the C5′ atom with respect to the approximate plane defined by other non-hydrogen atoms.1 The corresponding conformer with the C3′-endo puckering mode (anti-C3′-endo), in which the C3′ atom is on the same side as the C5′ atom, is less stable by 19.8 kJ/mol (Figure S1b, 572

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Figure 3. IR−UV double resonance spectra of (a) 5′-OEtGs, (b) Gs, and (c) 9MG. The ordinate scale corresponds to the logarithm of the depletion yield (see Experimental Methods).

Figure 4. Harmonic vibrational spectra for (a) anti and (b) syn conformers of 5′-OEtGs calculated at the B3LYP/6-311++G(d,p) level and scaled by 0.957. Each spectrum is calculated for the most stable tautomer with the guanine moiety in the enol-syn form. IR spectra calculated for the anti conformers in the enol-anti and keto forms are shown in Figure S2 (Supporting Information).

enol−OH stretch transition at 3587 cm−1 in the spectrum of Gs, which confirms that the guanine moiety is in the enol form. This assignment is consistent with the result for 9methylguanine (9MG) shown in Figure 3c. On the contrary to the result obtained for unsubstituted guanine in helium droplets,7 only one rotamer of the enol form, either enol-syn or enol-anti, was observed for Gs4 and 9MG8 isolated in the gas phase, although they are calculated to be nearly isoenergetic. The failure to observe the other enol rotamer and keto tautomer could be explained by the use of R2PI, in which species having short excited-state lifetimes are not ionized with high efficiency as demonstrated for guanine.9,10 We tentatively assign the observed enol rotamer to the enol-syn form on the basis of the relative stability by 1.4 kJ/mol with respect to the enol-anti rotamer (Figure S1a, Supporting Information). In contrast, the possibility of an anti rotamer was suggested for the R2PI spectrum of 9MG by comparing the vibrational frequency of the enol−OH stretch (3592 cm−1)8 with that of bare guanine (3591 cm−1).7 As can be expected, the 5′-OH stretch transition of Gs at 3249 cm−1 disappears upon ethyl substitution. Moreover, the sharp transition due to 2′-OH at 3688 cm−1 is replaced with a weak, broad peak appearing at 3386 cm−1. The observation of the less red shifted transition for the bound 2′-OH stretch than that for the 5′-OH transition of Gs points to weaker hydrogen bonding in the anti conformation. This occurs as a result of a geometric restriction for this conformer, in which the O2′−H bond axis is significantly distorted from that of the hydrogen bond to N3, as shown in Figure 1a. In addition, the hydrogen bond distance of 2.128 Å is substantially longer than the corresponding value in the syn conformer of Gs (1.860 Å, Figure 1b). It should also be noticed that the transition of the 3′-OH stretch is apparently absent from the spectrum. The corresponding transition of Gs is observed at 3604 cm−1, which was explained by the occurrence of a weak hydrogen-bonding interaction with 2′-OH,4 as shown in Figure 1b (left). The harmonic vibrational frequencies calculated for the most stable anti and syn conformers (Figure 1a) are shown in Figure 4. On the basis of the bridged 2′-OH band position, the experimental IR spectrum is assigned to the anti conformer of 5′-OEtGs. Furthermore, the computed frequency for the 3′-OH

stretch transition is nearly identical to that of the enol−OH stretch transition. We therefore explain that these two transitions are nearly overlapped around 3589 cm−1 and thus cannot be separated at the spectral resolution employed in this study (∼1.5 cm−1). This explanation is supported by the observation that its relative intensity to the amino stretch transition at 3582 cm−1 appears to be stronger than the corresponding intensity in Gs. The red shift of the 3′-OH transition with respect to that of Gs (3604 cm−1) is presumably due to the presence of the hydrogen bond between the 2′-OH and N3 site in the anti conformation, which is expected to reinforce the interaction with the 3′-OH group. The bridged 2′-OH stretch band possesses a weak shoulder at 3405 cm−1, split from the 2′-OH transition by 19 cm−1. One plausible explanation is to ascribe this shoulder to the lowestfrequency mode involving a mutual motion of the base and sugar. The calculated frequency of this mode is 17 cm−1, being substantially lower than that in Gs (43 cm−1) owing to the presence of ethyl group. This prediction is consistent with the observation that the corresponding peak of Gs is observed at 3295 cm−1 with a splitting of 46 cm−1 from the 5′-OH transition (Figure 3b). In order to confirm that we observe only a single conformer about the glycosyl bond in the UV spectrum of 5′-OEtGs, we have employed the technique of IR-purified UV spectroscopy.11,12 This method was shown to be effective when UV spectra consist of sharp features with an underlying broad background,12 as in the case of Figure 2a. In this method, the IR laser is used to pump the bridged 2′-OH stretch transition at 3386 cm−1, which is the signature of the anti conformer, and depopulate this conformer in the ground state. Then, the UV laser delayed by 100 ns is scanned across the relevant energy region. If there exist other species that do not absorb at this IR frequency, then the UV signal will not be depleted. The result is shown in Figure 5. It can be seen that in the IR-purified spectrum in Figure 5b, the sharp peaks observed in the UV spectrum are nearly suppressed, while the underlying background remains intact. Moreover, the difference spectrum in 573

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The technique of IR-purified UV spectroscopy12 was employed for the separation and identification of possible isomers (conformers and tautomers). In this method, an IR laser with a high pulse energy of ∼5 mJ is tuned to a specific transition of a given isomer. As a result of depopulation in the vibrational ground state, its contribution to the UV signal is effectively suppressed. The resulting UV spectrum therefore consists of spectral features of the isomers that are not depopulated. Conversely, the UV spectrum of the IRdepopulated species can be obtained by recording the difference between the R2PI signals with IR laser on and off. Stable structures of 5′-OEtGs were optimized at the M062X/6-311++G(d,p) level. The zero-point vibrational energy correction was also performed at the same level. The harmonic vibrational frequencies were calculated at the B3LYP/6-311+ +G(d,p) level and scaled by a factor of 0.957.

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Figure 5. (a) UV and (b) IR-purified UV spectra of 5′-OEtGs. The spectrum in (b) was obtained upon IR purification for the 2′-OH stretch transition at 3386 cm−1. (c) Difference spectrum (b) − (a).

* Supporting Information Details of the synthesis, structures, relative energies, and calculated IR spectra of 5′-OEtGs. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 5c agrees with the sharp features of the UV spectrum, confirming that these peaks arise from the conformer that was purified by the IR irradiation, namely, the anti conformer. It should also be mentioned that anti conformers with the guanine moiety in the different tautomeric forms (Figure S1a, Supporting Information) or those with different orientations of the ethyl group (Figure S1c, Supporting Information) cannot be separated by this technique. In summary, we have investigated the conformational preference of guanine nucleosides, syn and anti conformers about the glycosyl bond, by the chemical modification of the sugar. A guanine nucleoside 5′-OEtGs in which the hydrogen of 5′-OH is substituted with an ethyl group is found to exist in the anti conformation, in contrast to the syn conformation found in Gs. The results of IR−UV double resonance measurements and quantum chemical calculations show that this anti conformation is stabilized as a result of internal hydrogen bonding formed between the 2′-OH group and the N3 site of the guanine moiety. The significance of this finding is that conformational preference of nucleosides can be controlled by introducing steric hindrance and hydrogen-bonding interactions via chemical modifications.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +81 52 789 4779. E-mail: [email protected]. jp (M.T.); Tel: +81 45 787 2179. Fax: +81 45 787 2413. Email: [email protected] (H.S.) Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Grant-in-Aid (20350012, 22008000, and 23009470) from JSPS and by the Grant-in-Aid (22018023) for Scientific Research in the priority area “Molecular Science for Supra Functional Systems” from MEXT. We thank Dr. Kin-ichi Oyama (Chemical Instrumentation Facility, Research Center for Materials Science, Nagoya University) for the FAB-MS measurements and Noritaka Suzuki (Nagoya University) for technical assistance.

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REFERENCES

(1) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag: New York, 1984. (2) Voet, D.; Voet, J. G.; Pratt, C. W. Fundamentals of Biochemistry: Life at the Molecular Level, 2nd ed.; Wiley: New York, 2006; Chapter 23. (3) Tavale, S. S.; Sobell, H. M. Crystal and Molecular Structure of 8Bromoguanosine and 8-Bromoadenosine, Two Purine Nucleosides in the syn Conformation. J. Mol. Biol. 1970, 48, 109−123. (4) Nir, E.; Hünig, I.; Kleinermanns, K.; de Vries, M. S. Conformers of Guanosines and Their Vibrations in the Electronic Ground and Excited States, as Revealed by Double-Resonance Spectroscopy and Ab Initio Calculations. ChemPhysChem 2004, 5, 131−137. (5) Abo-Riziq, A.; Crews, B. O.; Compagnon, I.; Oomens, J.; Meijer, G.; von Helden, G.; Kabelác,̌ M.; Hobza, P.; de Vries, M. S. The MidIR Spectra of 9-Ethyl Guanine, Guanosine, and 2′-Deoxyguanosine. J. Phys. Chem. A 2007, 111, 7529−7536. (6) Asami, H.; Tsukamoto, M.; Hayakawa, Y.; Saigusa, H. Gas-Phase Isolation of Diethyl Guanosine 5′-Monophosphate and Its Conformational Assignment. Phys. Chem. Chem. Phys. 2010, 12, 13918−13921. (7) Choi, M. Y.; Miller, R. E. Four Tautomers of Isolated Guanine from Infrared Laser Spectroscopy in Helium Nanodroplets. J. Am. Chem. Soc. 2006, 128, 7320−7328.

EXPERIMENTAL METHODS

The laser desorption/supersonic jet cooling/R2PI apparatus used in this study has been described elsewhere.13,14 The sample of 5′-OEtGs was synthesized according to Scheme S1 in the Supporting Information. A 10 mg sample of 5′-OEtGs was mixed with a graphite matrix (5%), which absorbs the 532 nm output of a YAG laser. The plume of desorbed molecules was entrained into a supersonic expansion of argon (5 atm) and ionized in the R2PI scheme using a frequency-tunable UV laser. The resulting ions were analyzed by a time-of-flight mass spectrometer. Mass-selected electronic spectra were recorded by probing the ion signal at a particular mass channel while scanning the UV frequency. The corresponding IR spectra were obtained in the 3100−3750 cm−1 region by the technique of IR−UV double resonance spectroscopy.15,16 The IR laser (LaserVision, bandwidth: 1.5 cm−1) was operated at 5 Hz, and the UV signal was probed at 10 Hz. The alternate UV signals measured with the IR laser turned on (I) and off (I0) were fed into a boxcar integrator and converted to the absorbance scale. 574

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(8) Chin, W.; Mons, M.; Piuzzi, F.; Tardivel, B.; Dimicoli, I.; Gorb, L.; Leszczynski, J. Gas Phase Rotamers of the Nucleobase 9Methylguanine Enol and Its Monohydrate: Optical Spectroscopy and Quantum Mechanical Calculations. J. Phys. Chem. A 2004, 108, 8237−8243. (9) Mons, M.; Piuzzi, F.; Dimicoli, I.; Gorb, L.; Leszczynski, J. NearUV Resonant Two-Photon Ionization Spectroscopy of Gas Phase Guanine: Evidence for the Observation of Three Rare Tautomers. J. Phys. Chem. A 2006, 110, 10921−10924. (10) Seefeld, K.; Brause, R.; Häber, T.; Kleinermanns, K. Imino Tautomers of Gas-Phase Guanine from Mid-Infrared Laser Spectroscopy. J. Phys. Chem. A 2007, 111, 6217−6221. (11) Mons, M.; Dimicoli, I.; Piuzzi, F.; Tardivel, B.; Elhanine, M. Tautomerism of the DNA Base Guanine and Its Methylated Derivatives as Studied by Gas-Phase Infrared and Ultraviolet Spectroscopy. J. Phys. Chem. A 2002, 106, 5088−5094. (12) Asami, H.; Urashima, S.; Saigusa, H. Structural Identification of Uric Acid and Its Monohydrates by IR−UV Double Resonance Spectroscopy. Phys. Chem. Chem. Phys. 2011, 13, 20476−20480. (13) Saigusa, H.; Tomioka, A.; Katayama, T.; Iwase, E. A Matrix-Free Laser Desorption Method for Production of Nucleobase Clusters and Their Hydrates. Chem. Phys. Lett. 2006, 418, 119−125. (14) Saigusa, H. Excited-State Dynamics of Isolated Nucleic Acid Bases and Their Clusters. J. Photochem. Photobiol. C 2006, 7, 197−210. (15) Saigusa, H.; Urashima, S.; Asami, H. IR−UV Double Resonance Spectroscopy of the Hydrated Clusters of Guanosine and 9Methylguanine: Evidence for Hydration Structures Involving the Sugar Group. J. Phys. Chem. A 2009, 113, 3455−3462. (16) Asami, H.; Urashima, S.; Saigusa, H. Hydration Structures of 2′Deoxyguanosine Studied by IR−UV Double Resonance Spectroscopy: Comparison with Guanosine. Phys. Chem. Chem. Phys. 2009, 11, 10466−10472.

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