Journal of Molecular Structure 738 (2005) 9–14 www.elsevier.com/locate/molstruc
Tautomerism of thymine on gold and silver nanoparticle surfaces: surface-enhanced Raman scattering and density functional theory calculation study Kwang-Hwi Choa, Jaebum Choob, Sang-Woo Jooc,* a
Department of Bioinformatics and Life Science, CAMDRC (Computer Aided Molecular Design Research Center), Soongsil University, Seoul 156-743, South Korea b Department of Applied Chemistry, Hanyang University, Ansan 425-791, South Korea c Department of Chemistry, CAMDRC (Computer Aided Molecular Design Research Center), Soongsil University, Seoul 156-743, South Korea Received 4 July 2004; revised 1 November 2004; accepted 1 November 2004 Available online 25 January 2005
Abstract Tautomerism of thymine on gold and silver nanoparticle surfaces has been comparatively analyzed by means of surface-enhanced Raman scattering (SERS). The intensities of the ring breathing mode of thymine indicated that the N3-deprotonated tautomer should be about 10 times more abundant than the N1-deprotonated tautomer on Ag surfaces whereas almost no N1-deprotonated tautomer was observed for Au under our experimental condition. The density functional theory (DFT) calculation was performed at the levels of B3LYP and MP2 to estimate the energetic stability of the N3 and N1-deprotonated tautomers on the surfaces. The N3-deprotonated tautomer was predicted to be more favorable on Au than on Ag from the DFT calculation as consistent with our SERS spectra. q 2004 Elsevier B.V. All rights reserved. Keywords: Thymine; Tautomerism; Au; Ag; SERS; DFT calculation
1. Introduction There has been increasing interest in estimating the stabilities of various geometric forms of nucleic acid bases due to its potential importance in genetics and its related fields [1,2]. Ab initio calculations have been currently employed to predict the structure and energetics of nucleic acid bases [3]. Thymine, one of the simplest pyrimidine base, has been a frequent subject of theoretical studies due to its biochemical importances [4,5]. Tautomers of thymine anions in solution could be differentiated by Raman spectroscopy in changing the dielectric constants of the medium [6]. For uracil, differently from its neutral state in the gas phase, the N3-deprotonated tautomer was found to be more favorable on Ag than the N1-deprotonated tautomer from a Raman spectroscopy and DFT calculation study [7]. * Corresponding author. Tel.:/fax: C82 2 8200434. E-mail address:
[email protected] (S.-W. Joo). 0022-2860/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2004.11.001
Adsorption of thymine on metal surfaces have been studied by means of various spectroscopic tools [8–11]. FT-IR and Raman spectra of thymine in its polycrystalline surfaces states have been reported from 50 to 3500 cmK1 [8]. Thymine adsorbed on Ag island films was investigated by means of surface enhanced vibrational spectroscopy [9]. Energetics of thymine–gold interactions was recently investigated by temperature programmed desorption and reflection absorption FT-IR spectroscopy [10]. Adsorption of single stranded DNA consisted of thymine bases was investigated by means of X-ray photoelectron and FT-IR spectroscopy [11]. Optical properties of metal nanoparticles have attracted both scientific and technological interest in the past two decades due to its potential applications in many areas [12]. Although gold colloidal dispersions have been studied ever since the report by Faraday, their physicochemical characterization were not performed systematically until lately [13]. Surface-enhanced Raman scattering (SERS) on
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metal surfaces has shown great potential for use in biomedical analysis [14]. The analysis of spectral features has provided detailed information on surface reactions and the geometry of adsorbates [15]. SERS has been recently used to monitor analyte-surface binding interactions as biosensors [16,17]. Numerous cases have been reported indicating that the adsorption behavior depends on the metal substrates [18–21]. The detailed origins of the different adsorption characteristics on Ag and Au have not been fully clarified, however. In this work, we performed an SERS study of thymine on gold and silver nanoparticles in a comparative way to aim at understanding tautomerism of the simplest pyrimidine base upon adsorption on the metal surfaces. The density functional theory (DFT) was conducted to calculate the tautomeric stability at the levels of B3LYP and MP2.
2. Experimental section 2.1. Preparation of samples Thymine (99%) was purchased from Sigma Aldrich. The citrate stabilized gold and silver sols were synthesized by following the recipes in the literature [22]. For Au, 133.5 mg of KAuCl4 (Aldrich) was initially dissolved in 250 mL of water, and the solution was brought to boiling. A solution of 1% sodium citrate (25 mL) was then added to the KAuCl4 solution under vigorous stirring, and boiling was continued for ca. 20 min. The resulting Au solution was stable for several weeks. For Ag, 90.0 mg of AgNO3 in 500 mL of water was brought to boiling. A solution of 1% sodium citrate (10 mL) was then added to the AgNO3 solution under vigorous stirring. All the chemicals otherwise specified were reagent grade and triply distilled water, of resistivity greater than 18.0 MU cm, was used in making aqueous solutions.
band of a silicon wafer at 520 cmK1 was used to calibrate the spectrometer, and the accuracy of the spectral measurement was estimated to be better than 1 cmK1. The Raman spectrometer was interfaced with an IBM-compatible PC, and the spectral data were analyzed using Renishaw WiRE software v. 1.2 based on the GRAMS/32C suite program (Galactic). The same excitation wavelength at 632.8 nm together with the almost identical sol medium produced by sodium citrate should alleviate the error in comparing the two SERS spectra. 2.3. Calculation methods All ab initio molecular orbital calculations were carried out using the GAUSSIAN 98 package with consideration of basis set superposition errors [23]. The geometry optimization of thymine and Ag/Au complex were carried out at the levels of MP2 and B3LYP. The LANL2DZ basis sets have been used for both cases. The two tautomeric forms were considered among many possibilities [4]. The geometry optimization for the tautomers was carried out starting from many possible orientations. During the geometry optimization, no geometric constraints such as planarity were used for the present calculation. Raman spectra frequencies are calculated at the B3LYP/LANL2DZ level of theory with the IntZFineGrid option. The calculated wavenumbers at the B3LYP level were scaled down by a single factor of 0.98.
3. Results and discussion 3.1. Raman spectra Fig. 1 shows the Raman solid spectrum of thymine, the Ag and Au SERS spectra at high bulk concentration of
2.2. Raman measurements Raman measurements were summarized in the previous reports [20,21] using a Renishaw Raman system model 2000 spectrometer equipped with an integral microscope (Olympus BH2–UMA). The 632.8 nm from a 17 mW air-cooled HeNe laser (Spectra Physics Model 127) were used as the excitation sources for the Ag and Au SERS experiments. A concentrated aqueous solution of uracil was added dropwise to w0.1 mL of Ag and Au solution to a final concentration of w10K3 M using a micropipet. An appropriate holographic notch filter was set in the spectrometer depending on the excitation source. Raman scattering was detected with 1808 geometry using a peltier cooled (K70 8C) CCD camera (400!600 pixels). A glass capillary (Chase Scientific) with an outer diameter of 1.1 mm was used as a sampling device. Data acquisition times used in the SERS experiments were 90 s. The holographic grating (1800 grooves/mm), and the slit allowed the spectral resolution to be 1 cmK1. The Raman
Fig. 1. (a) Raman solid spectrum of thymine and SERS spectra in (b) aqueous silver nanoparticle solutions and (c) aqueous gold nanoparticle solutions. All spectra were taken using the He–Ne laser at the 632.8 nm radiation. The spectral region between 2800 and 1800 cmK1 was omitted due to the lack of any information.
K.-H. Cho et al. / Journal of Molecular Structure 738 (2005) 9–14 Table 1 Spectral data and vibrational assignment of thymine on Ag and Au Raman solid
426m 477m 558w 614s
Ag SERS
Au SERS
Assignmenta
237m 324w 431w 494w 587m 630m
259s
Stretch metal-N Wagging (C–CH3) (o.p.)b Ring torsion (o.p.) Ring deformation bend (i.p.)c Ring deformation bend (i.p.) Deformation bend C2O7, C4O8 (i.p.) Ring breathing (i.p.) Ring breathing (i.p.) Ring deformation bend (i.p.) Wagging (NH) (o.p.) Wagging (CH3) (o.p.) Rocking (CH3) (i.p.) Stretch (C–CH3) (i.p.) Ring stretch (i.p.) Ring stretch (i.p.) CH3 or C6H deformation bend (i.p.) N1H deformation bend (i.p.) N3H deformation bend (i.p.) CH3 or C6H deformation bend (i.p.) Ring stretch (i.p.) Stretch C4O8, bend N1H, C6H (i.p.)
436w 490w 585m 646vw
738s 745m 803m 982m 1049vw 1152w 1212w 1243w 1256w 1367vs
762m 780vs 817m 996m 1043w 1177m 1220vs 1276m 1348vs
1342vs
1405m 1429w 1457w
1403s
1402s
1455m
1486w 1558m
1670vs 1703vw 2894w 2932m 2964w 2988w 3062w
788s 815w 996m 1022w 1174vw 1222s 1250m
1601s 1633m 1657s
1490vw 1585s
1652vs 1652vs 2893w
2929w 2986vw 3068w
2968vw 3016vw 3060w
11
documented in the literature that the presence of the ring C–H stretching band in an SERS spectrum is indicative of a vertical (or at least tilted) orientation of the aromatic ring moiety on a metal substrate [20,21]. Since the same excitation wavelength at 632.8 nm was employed in obtaining all the Ag and Au SERS spectra, the instrumental effect or laser mode should be almost identical. In the Raman solid spectrum, the ring-breathing mode appeared at 745 and 738 cmK1. These two bands could be ascribed to the N3 and N1-deprotonated tautomer, respectively [7]. The ring-breathing mode in the Raman solid spectrum exhibited a substantial blue shift both in the Au and Ag SERS spectra with the increase of its bandwidth as shown in Fig. 2. The blueshift of the ring-breathing mode may indicate a rather weak interaction of the aromatic rings on the gold and silver surfaces. Neither a substantial red shift nor a significant band broadening of the ring breathing modes was observed for the case of thymine on both Ag and Au where the molecule is assumed to have a standing orientation. Although the band positions of Ag and Au SERS spectra looked comparable, the Ag SERS spectrum showed more complicated features indicating the existence of
C4O8 ring stretch (i.p.) C2O7 ring stretch (i.p.) Symmetric stretch CH2 (i.p.) Asymmetric stretch CH2 (i.p.) Symmetric stretch CH3 (i.p.) Asymmetric stretch CH3 (i.p.) Stretch (C6H) (i.p.)
Unit in cmK1. Abbreviations: vs, very strong; s, strong; m, medium; w, weak; vw, very weak. a,b,cBased on Refs. [8,9]. b Out-of-plane. c In-plane.
w10K3 M. It seemed a rather straightforward to correlate the Raman solid bands with the Au SERS bands. Their peak positions are listed in Table 1 along with the appropriate vibrational assignments. Our assignment is mainly based on the previous literatures [8,9]. Like other pyrimidine bases [7], thymine appeared to adsorb on metal surfaces as an anionic form. The concentration of thymine in the aqueous solution was w10K3 M. This concentration should correspond to thymine on gold at the concentration above a fullcoverage limit by assuming that the adsorbate was oriented perpendicularly with respect to the colloidal surfaces [20,21]. Our spectrum on Ag appeared to be consistent with this report [9]. We have not yet obtained concentration dependent SERS spectra due to its poor spectra quality at low concentration under our experimental condition. As shown in Fig. 1, it is noteworthy that the ring C–H stretching bands were identified at w3060 cmK1, albeit weakly, in the Au SERS spectra. It has been well
Fig. 2. Ring breathing mode regions of (a) Raman solid spectrum of thymine and SERS spectra in (b) aqueous silver nanoparticle solutions and (c) aqueous gold nanoparticle solutions.
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Table 2 Relative enhancement factors of SERS bands in thymine ring modes Symmetry
Vibrational modea
Enhancement factorb Ag
In-plane A1
axx ayy azz
K1 c
Au
Ring torsion (w430 cm ) Ring deformation bend (w490 cmK1) Ring deformation bend (w580 cmK1) Deformation bend C2O7, C4O8 (w640 cmK1) Ring breathing (w780 cmK1) Ring deformation bend (w815 cmK1) CH3 or C6H deformation bend (w1340 cmK1) N1H deformation bend (w1400 cmK1) CO ring stretch (w1650 cmK1) Stretch (C6H) (w3060 cmK1)
1.00 0.771
1.00 1.07
2.30
2.91
0.917
0.401
8.73 2.43
3.98 1.46
1.71
1.62
11.1
10.7
27.4 1.41
72.6 3.56
Wagging (NH) (w996 cmK1) Wagging (CH3) (w1030 cmK1)
1.56 4.83
2.94 12.8
Out-of-plane A2 axy
Symmetry types corresponding to the C2 point group, Subscripts, i.e. x, y, and z, correspond to the conventional molecular axes [20,21]. a Based on Refs. [8,9]. b Relative intensity of each vibrational band (ISERS/IOR). c Number in the parenthesis designates the frequency position in Table 1.
the N1-deprotonated tautomer. The difference in the SERS spectral bands may result from the dissimilar population of the tautomers of thymine upon adsorption on Ag and Au. The relative intensities have been evaluated for several vibrational modes of thymine as summarized in Table 2. 3.2. DFT calculation results of Ag and Au metal complexes with tautomers of thymine To check our spectra behaviors, DFT calculations were performed for the Ag and Au complexes of the N3 and N1deprotonated tautomers. The N3-deprotonated Au complex was found to be more stable by 1.9 and 2.1 kcal/mol than the N1-deprotonated Au complex at the B3LYP and MP2 levels, respectively. From the Boltzmann population equation, the N3-deprotonated tautomer should be about 30 times more abundant on Au. This view is consistent with the spectral behavior that only the N3-deprotonated tautomer is observed for the ring-breathing mode. For the Ag complex, the N3-deprotonated tautomer was calculated to be stable by only 0.3 kcal/mol at the B3LYP level as similar to the case of uracil [7]. The MP2 result yields a slightly more stable value by 0.03 kcal/mol for the N1-deprotonated tautomer on Ag to the contrary. This discrepancy appears to be alleviated after considering the effect of the basis set superposition error (BSSE) [23], although the absolute energy values could not be exactly compared. The calculated energy values are summarized in Table 3. Our results suggest that the N1-deprotonated
Table 3 Stabilities of the N3 and N1-deprotonated thymine tautomer complexes with Ag and Au from the DFT calculation Energy differencea
Before BSSEb
After BSSE
Thymine
B3LYP
MP2
B3LYP
MP2
E(N3)–E(N1)/Ag E(N3)–E(N1)/Au
K0.3 K1.9
0.03 K2.1
K13.1 K14.8
K11.8 K14.2
Unit: kcal/mol. a E(N3), energy of the N3-deprotonated tautomer complex with the metal; E(N1), energy of N1-deprotaned tautomer complex with the metal. b Basis set superposition error (BSSE).
tautomer could exist on Ag as evidenced in Raman spectra of Figs. 1 and 2. In fact the intensity of the N3-deprotonated tautomer was found to be about 10 times larger than that of N1-deprotonated tautomer. The geometry optimization for the tautomers of thymine–metal complexes was carried out at the B3LYP and LANL2DZ levels starting from many possible structures. The geometry optimizations were converged to one structure for each tautomer even though they were started from many possible configurations. For both cases, the N3-deprotonated complexes are estimated to be more stable than the N1-deprotonated form with the DFT calculation at the level of B3LYP. Plausible structures of Ag–thymine and Au–thymine for the N3-deprotonated tautomers are drawn in Fig. 3 with their optimized geometric variables. Our notation is based on the literature from Ref. [24]. The bond angle of Ag–N3–C4 is 103.28. The O8 is predicted to be closer than O7 to the Ag atom. It is not absolutely certain that this result may lead to the interpretation that thymine may interact with the Ag surface via its oxygen atom O8.
Fig. 3. Plausible structures of (a) Ag–thymine (N3-deprotonated), (b) Ag– thymine (N1-deprotonated) (c) Au–thymine (N3-deprotonated), and (d) Au– thymine (N1-deprotonated) complexes from the ab initio calculations at the B3LYP level.
K.-H. Cho et al. / Journal of Molecular Structure 738 (2005) 9–14
In fact the vibrational band at w1633 cmK1 partially ascribed to the C2O7 stretching was shown to be relatively weaker in the Ag SERS spectrum. This band was not found for the Au SERS spectrum. Metal to the nitrogen of thymine appeared to be almost linear for Au. 3.3. Surface orientation of thymine on Ag and Au Since an unequivocal selection rule is not available for SERS, the exact tilt angle cannot be determined for thymine at present. According to the electromagnetic (EM) theory on the SERS selection rule [15], vibrations along the direction perpendicular to the surface are expected more enhanced than vibrations in the parallel direction. This rule suggested that the in-plane vibration modes should be more enhanced than the out-of-plane ones. For thymine, most ring modes were found to ascribe to the in-plane mode. The bands at w259 and w237 cmK1 could be ascribed to the surface-adsorbate Au–N and Ag–N vibration, respectively. This metal–N bond was observed higher on Au than on Ag. As predicted from our DFT calculation as shown in Fig. 3, the Au–N3 distance (0.203 nm) is expected to be shorter than that of the Ag–N3 bond (0.215 nm). Also the Au–N1 distance (0.204 nm) is calculated to be shorter than that of the Ag–N1 bond (0.223 nm). This may result in the higher stretching vibrational frequency for Au–N as shown in Fig. 1. More distinctive spectral feature for the several ring modes in the Au SERS spectra may relate to its closeness and the resulting stronger interaction to the Au metal surface for the N3-deprotonated tautomer. As shown in Fig. 2, the ring-breathing mode is found to split at 745 and 738 cmK1 due to the existence of the N3 and N1-deprotonated tautomer as consistent with the previous report. It is intriguing that the intensity of the N3deprotonated band at 780 cmK1 increased, whereas the N1-deprotonated band appeared as a shoulder peak at 762 cmK1 in the Ag SERS spectrum. This suggests that the N3-deprotonated tautomer should be more stable on Ag than the N1-deprotonated tautomer. On Au, only the band at 788 cmK1 corresponding to the N3-deprotonated tautomer was observed without a feature from the N1-deprotonated tautomer. This result seems consistent with our theoretical prediction that the N3-deprotonated tautomer should be more stable on Au than on Ag as listed in Table 3. Our calculations indicated that the N3-deprotonated tautomer appeared to be favored on Au than on Ag. Although the stableness of the N3-deprotonated tautomer on Au is not well understood, it can cause different spectral features from those on Ag as shown in Fig. 1. Additional bands at 1633 and 1389 cmK1 on Ag could be ascribed to the N1-deprotonated tautomer as in the case of the SERS spectrum of uracil [7]. On the other hand, almost all the SERS bands could be assigned to the N3-protonated tautomer for Au as listed in Table 1. It is not absolutely certain whether the predominance of the ring breathing mode at w780 cmK1 in the SERS spectra
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would suggest a more perpendicular stance on Ag. Also, it is intriguing that the ring modes between 1700 and 900 cmK1 became more prominent on Au than on Ag, if compared with the ring breathing mode found at w780 cmK1. The aromatic ring modes of thymine are expected more enhanced on Au presumably due to its closeness to the metal surface. It is not absolutely certain either whether the methyl vibration of thymine would have different orientations on Au and Ag. The relative enhancement factors on Ag and Au are summarized in Table 2. It was reported that the charge transfer (CT) mechanism could also significantly contribute the enhancement of the SERS intensities [15]. Although the SERS spectra feature could be roughly described by the electromagnetic (EM) mechanism, it is admitted that the CT mechanism may also contribute the SERS intensities of several vibrational bands of thymine on Au. In fact the different enhancement values for the same symmetry as listed in Table 2 could be better explained with a combination of the EM and CT mechanisms. The detailed origins in different adsorption characteristics of thymine on Au and Ag are not still clear yet. In our previous SERS studies [20], the band analysis on the basis of the electromagnetic surface selection rule indicated that 4-biphenylisocyanide should have a slightly more vertical orientation on silver than on gold. This weakness of the nas(CH2) stretching band at w2930 cmK1 in the SERS spectra of benzylisocyanide suggests a rather bent angle of C–NbC: on the gold surfaces [25]. On the other hand, a linear angle of C–NbC: appears to be more favorable on silver surfaces. Although the N3 and N1-deprotated anions should have different charge distribution, it seems, however, difficult to discuss the hybridization state of the nitrogen atom of thymine adsorbed on Ag and Au. Invoking the report that thymine is assumed to be the weakest base on gold [10], binding properties of other bases have been currently investigated on gold nanoparticle surfaces by means of SERS and UV–Vis absorption spectroscopy. We have recently performed an SERS and DFT study of uracil on Ag and Au [26]. The energetic stabilities of the N3-deprotonated tautomer is also predicted to be more stable for uracil on Au and Ag. The ring breathing mode was not found to split as in the case of thymine on Au. Our study indicates that N3 appears to be more acidic than N1 on gold and silver for thymine and uracil. Considering that N1 of thymine is a part of glycosidic bond to the pentose ring [11], our study seems to remain dealing with fundamental physical properties of bases on gold and silver surfaces instead of developing a methodology for solid phase nucleic acid synthesis. Energetic or kinetic factors may also result in different adsorption behaviors on Ag and Au. Since the experiment was conducted in the solution phase and the Raman spectra appeared to depend on the sol condition as well as the bulk concentration, we have to mention that our spectral data mainly provide information on the adsorption pathways
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occurring at the surface rather than on the electronic interaction between the adsorbate and substrate. It seems problematic to explain the differences in adsorption mechanism on Ag and Au merely by our spectral analysis. We plan to conduct electrochemical studies combined with concentration dependent SERS in order to firmly understand the adsorption characteristics and surface orientations of thymine on Ag and Au nanoparticles. Our study shall be helpful to understand the interaction of pyrimidine bases and metal nanoparticles.
4. Summary and conclusion The SERS spectral evidence in silver and gold nanoparticle solutions indicated that the population of thymine tautomers should be different depending on metal surfaces. On Au, most vibrational bands in the SERS spectra appear to ascribe to the N3-deprotonated thymine, whereas additional vibrational bands suggest the existence of the N1deprotonated tautomer on Ag. Our DFT calculations indicated that the energetic difference of the N3 and N1deprotonated tautomers on gold should be much larger than that on silver. On Ag, the N3-deprotonated tautomer was found to be about 10 times more abundant than N1deprotonated tautomer under our experimental condition. No band corresponding to the N1-deprotonated tautomer was observed on Au. We also attempted to compare the orientation of thymine on Au and Ag nanoparticle surfaces by analyzing the relative enhancement factors of each vibrational band. The metal–N bond distance was calculated to be shorter for Au. This should result in the higher vibrational frequency of the metal–N stretch vibration on Au upon adsorption of thymine.
Acknowledgements This work was supported by the ABRL program of Korean Science and Engineering Foundation (KOSEF Grant R-14-2002-004-01002-0). This work was also supported by grant No. R01-2002-000-00378-0 from the Basic Research Program of the Korea Science & Engineering Foundation. This work was also supported by the grant
from the Korean Ministry of Science and Technology (Korean Systems Biology Research Grant, M1030914000303B501400310). S.W.J would like to thank Prof Kwan Kim for introducing SERS studies of aromatic adsorbates on gold and silver.
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