J. Phys. Chem. 1993,97, 9519-9524
9519
Vibrational Analysis of the Hydrogen Bonding of Cytidine and Guanosine Derivatives Pedro Carmona,’ Marina Molina, and Aurora Lasagabaster lnstituto de Optica, CSIC, Serrano 121, 28006 Madrid, Spain
Rosario Escobar Departamento de QuImica Analitica, Facultad de Quimica, Universidad de Sevilla, 41 01 2 Sevilla, Spain
Aida Ben Altabef Departamento de Quimica Fisica, Universidad de Tucumhn, Tucumhn, RepQblica Argentina Received: March I I , 1993; In Final Form: May 24, 1993”
Hydrogen bonding between 2’-deoxy-3’,5’-bis(triisopropylsilyl)guanosine ( G ) and 2’-deoxy-3’,5’-bis(triisopropylsily1)cytidine ( C ) has been studied by vibrational spectroscopy in chloroform solution. Strong interactions occur between the two derivatives of guanosine and cytidine and between CG base pair and cytidine, whose association constants were first determined. CGC trimers involve cyclic hydrogen bonds through the N(3) acceptors of both nucleobases and the guanine N(2)H and cytosine N(4)H donors. The main spectral changes of CG dimer accompanying CGC trimer formation are intensity and frequency decreases of the Raman band of guanine base near 1570 cm-1 as well as downshifting of the 1534-cm-1 Raman band of the cytosine base that binds to the previously formed CG base pair. Similar spectral changes are observed for the infrared bands located near these frequencies. The 1483-cm-l band of guanine is sensitive to hydrogen bonding at the N(7) position. Since this band is unchanged when the CG dimer is converted to the CGC trimer, it is concluded that no binding at the G ( 7 ) position occurs in the trimer. Raman spectroscopy can, then, distinguish between hydrogen bonds involving guanine N(7) and N(3) acceptors and offers prospect for determining these specific interactions in polynucleotide triplexes and nucleic acid-protein recognition.
1. IntToduction The fundamental chemistry underlying the stability of nucleic acid associations determines the molecular biology of genetic regulation and expression. Selective hydrogen bonding in complementary nucleobases determinesthe fidelity of replication and transcription processes, and hydrogen bonds contribute to stabilization of nucleic acid secondary and tertiary structures. Concerningthe formation of triple-stranded nucleic acid helices, there is considerable potential in DNA for some biological mechanism at the level of the genome for control of gene expression by a third-strand binding mechanism,’ since natural DNA has significant frequency homopurinehomopyrimidine sequences2.3 such as are required for triplex formation. Some X-ray crystallographic studies4have shown that one of the two major types of homopolymer triplexes according to the base type (pyrimidine or purine) of the triplex-forming strand involves pyrimidine-purine-pyrimidine triplet units. Vibrational spectroscopy has been applied to numerous problems on nucleic acids and related compounds and is now one of most important tools in structural studies of nucleic acids. Concerning the relationships between hydrogen bonding states of base pairs and vibrational frequencies, they have not been studied systematically. Such knowledge is believed to be important for analyzing the spectra of nucleic acids and their derivatives in various structural states. In this report, we describe the use of vibrational spectroscopy to determine the association constant and enthalpy of the binding of cytosine to a CG WatsonCrick-type base pair in chloroform and to characterize the structure of CGC triplet units. In spite of some studies on the interaction of C with G,” no work has been done on the quantitative determination of the strength of the specific binding of C to CG base pair. This work has been carried out using Author to whom correspondence should be addressed. *Abstract published in Advance ACS Abstracts, August 15, 1993.
0022-365419312097-9519$04.00/0
bis(triisopropylsily1)ated nucleosideswhich have suitablesolubility in chloroform, with their triisopropylsilyl groups preventing the ribose hydroxyls from forming hydrogen bonds. 2. Experimental Section
Preparation of Nucleosides. 2’-Deoxy-3’,5’-bis(triisopropylsily1)guanosine and 2’-deoxy-3’,5’-bis(triisopropylsilyl)cytidine (Figure I), which will be referred to as G and C, respectively, were prepared through a modification of a published procedure.5 The preparation of these compounds simply requires dissolving each nucleoside, triisopropylsilyl chloride, and imidazole in dimethylformamide. The starting nucleosides were 2’-deoxyguanosine and 2‘-deoxycytidine hydrochloride, which were obtained from Fluka. The reactions were allowed to proceed under stirring at room temperature for 24 h, and the resulting bis(triisopropylsily1)ated nucleosides were obtained in yields of greater than 90%. The reaction products were purified by column chromatographyusing a solvent composed of chloroform-ethanol (8:2, v/v) to elute G and tetrahydrofuran for the elution of C. The resulting nucleosides were pure as determined by elemental analysis and 1H-NMR. Deuterochloroform (99.8 atom 3’% D) was purchased from Sigma and stored in the dark. The poly(C).poly(G) duplex was obtained from Sigma as well as poly(C) used to form poly(C+)-poly(G)-poly(C)triplex. Aqueous solutions of this triplex were prepared by mixing equimolar amounts of poly(C+) and poly(C).poly(G) duplex. Methods. To obtain the association constant and enthalpy of CGC trimer, we first measured the CC, GG, and CG association constants. A method used for hydrogen-bonded dimerization of cyclic cis-amides9 has been applied to the self-association of C and G extended to the determination of the heteroassociation constant of the CGC trimer. 0 1993 American Chemical Society
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The Journal of Physical Chemistry, Vol. 97, No. 37, 1993
(c
- Pr ) 3 Si 0
Carmona et al.
YY B
A
Figure 1. Nucleobase atomicnumbering in theguanosine(A) and cytidine (B) derivatives.
0.50r
i
1.30'
3.0
'
'
'
'
'
'
3.2
3.1
'
3.3
I 3.4
'
3.5
(+) x I 0 3
0.40
Figure 3. Plot of log KCX against 1/T for the self-association of C. Estimated correlation coefficient: 0.99.
Koa, over the concentration range 0.001-0.0045 infrared cell with 1-cm path length was used.
M,for which an
If two nucleosides, N and M, are mixed together in solution, the total concentration is
e,
td
0.201 0.040
d,
e,= I
0.050
1
c;
-
I
I
0.060
CN
+ 2 c N N + CNM
(5)
where CNis the concentration of monomer and CNNand CNMare the concentrations of NN and NM dimers, respectively, whose associations constants are defined as
AN
Figure 2. Plot of AN against &/A, for the self-association of C. Estimated correlation coefficient: 0.99.
If the nucleoside molecule, N, forms homodimers, the selfassociation constant KNNis defined as
where CN and CNNare the monomer and homodimer concentrations, respectively. The total concentration in moles of dissolved nucleoside per liter is
e,
e, = CN + 2cNN
(2)
Equations 3, 5 , and 6 lead to
where CY is K N Mcan be obtained from the slope and the intercept of the straight line given by a plot of AN versus G/ANif CY and KNNhave been previously measured.
AN = aNCNI (3) where is the absorption coefficient of the monomer band and I is the path length in centimeters. Equations 1, 2, and 3 then lead to
We have applied eq 7 to determine the association constant between CG dimer and C, since the association constant of CG dimer is very high, as described in the Results section, and an excess of C added to equimolar solutions of C and G does not lead to disruption of CG base pairs previously present in solution. The concentration ranges used were 0.0024.012 and 0.006-0.036 M for G and C, respectively, and the infrared cell used was 1 mm in path length.
A straight line should be obtained if AN is plotted against &/A,, from the slope and intercept of which K" can be calculated. The self-association constant of C, KCC,was determined over the concentration range 0.0094.03 M at several temperatures, and the plot of log K against 1 / T gave a straight line from which AHo could be determined (Figures 2 and 3). The infrared cell used was 1 mm in path length. The same procedure was followed to investigate the self-association constant of G,
If the heteroassociation constant for two nucleosides is very great, as occurs for CG dimer, and consequently the monomer bands are not measurable due to their weak intensity, then eq 7 cannot be applied. So, KCO was determined as follows. In equimolar solutions of C and G over the concentration range 0.002-0.01 M for each nucleobase, the concentration of C monomer is assumed to be - CCO, and C m being the total concentration of C and the concentration of the CG dimer, respectively. This is because the concentrations of C monomer in these solutions and KCCwere found to be so small that the concentrations of CC homodimer can be reasonably neglected.
On the other hand, the absorbance ANof a monomer band should be
e,
H Bonding of Cytidine/Guanosine Derivatives
The Journal of Physical Chemistry, Vol. 97, No. 37, 1993 9521 0
3600
3500
3400
3300
3200
cm-i Figure 4. CDCI3 solution infrared spectra of (upper) 1.3 mM isotopically dilute C containing 15% deuterium, path length 1.0 cm, and (lower) 14 mM C, path length 0.1 cm.
H
\
PN N
Therefore, it follows that
R/N
