Interactions of cytidine with N2-functionalized guanosines and cytidine - Cytidine exchange involving a GC pair - NMR and fluorescence spectroscopic study

Article abstract of DOI:10.1139/V10-040

Two N²-functionalized guanosines by diphenylaminobiphenyl and di(2-pyridyl)aminobiphenyl have been found to act as the effective probes for G-C interactions in organic media. Because of the highly emissive nature of the N²-functional

Full text of DOI:10.1139/V10-040

524
Interactions of cytidine with N2-functionalized
guanosines and cytidine – cytidine exchange
involving a GC pair — NMR and fluorescence
spectroscopic study
Sanela Martic, Gang Wu, and Suning Wang
Abstract: Two N2-functionalized guanosines by diphenylaminobiphenyl and di(2-pyridyl)aminobiphenyl have been found to act
as the effective probes for G–C interactions in organic media. Because of the highly emissive nature of the N2-functionalized
guanosines in the visible region, the GC base pair formation event accompanied by distinct fluorescence quenching can be
readily monitored by fluorescence spectroscopy. NMR and fluorescence results confirm that the N2-arylguanosines form
H-bonded pairs with cytidine, selectively. An unusual exchange pathway between non-bound cytidine and bound cytidine,
in the GC pair, has been identified and extensively studied by NMR methods.
Key words: N2-guanosine, GC pair and cytidine exchange, fluorescence, NMR, hydrogen bonding.
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Resume : On a trouve que deux guanosines fonctionnalisees en N par des groupes diphenylaminobiphenyle et di(2-pyri-
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dyl)aminophenyle peuvent agir comme sondes efficaces pour les interactions G–C dans des milieux organiques. En raison
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du grand pouvoir emetteur des guanosines fonctionnalisees en N dans la region visible, il est tres facile par le biais de la
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spectroscopie de fluorescence de detecter la fluorescence distincte de piegeage qui accompagne la formation d’une paire
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de base GC. Les resultats de la RMN et de la fluorescence confirment que les N -arylguanosines forment selectivement
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des paires avec la cytidine, par le biais de liaisons hydrogenes. Faisant appel a des etudes par RMN, on a identifie et etu-
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die une voie d’echange inhabituelle entre la cytidine non liee et la cytidine liee, dans la paire GC.
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Mots-cles : N -guanosine, paire GC, echange de cytidine, fluorescence, RMN, liaison hydrogene.
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[Traduit par la Redaction]
combination of fluorescence, CD, and NMR spectroscopic
methods.4 In addition, the complementary base for guanine
in Watson–Crick interactions is cytosine; hence, various
functionalized guanosines or cytidines have been used previ-
ously to study the formation of the GC base pair and the
charge-transfer phenomenon via the GC pair between fluo-
rescent donor and acceptor.5 To our best knowledge, how-
ever, there have been no reports on GC base pairs involving
fluorescent N2-functionalized guanosines.6 To examine the
impact of N2-functionalization of guanosine on GC pair for-
mation, we investigated the interactions of the native cyti-
dine with G1 and G2 by both NMR and fluorescent
spectroscopic methods. However, because G1 and G2 have
poor solubilities in organic solvents and are sparingly solu-
ble in water or the mixture of water and alcohols, which
also compete for H bonds with guanosine and cytidine lead-
ing to complex H-bond patterns, no meaningful data could
be obtained. To overcome this problem, we converted G1
and G2 to lipophilic guanosines by protecting OH moieties
with acetyl groups, which greatly improved their solubility.
The resulting G1A and G2A guanosines display selective
H-bonding with cytidine in solvents such as CH2Cl2 that
Introduction
Among the nucleobases, guanine displays the most versatile
H-bonding patterns, including Hoogsteen or Watson–Crick
interactions, or combination thereof, and is thus a valuable
building block in supramolecular chemistry.1 In addition,
the hydrogen-bonding motifs of guanine, such as G-quartets,
are also known to have important and unique biological
functions.2 Hence, developing new functionalized guano-
sines to facilitate the study of various H-bonding patterns
and self-assembled structures involving guanine has been a
very active research area. We have shown recently that the
attachment of an aryl group, such as a non-emissive n-butyl-
phenyl group, at the N2 site of the guanosine does not dis-
rupt the formation of G-quartet and G-octamer via H-bonds
in the presence of alkaline or alkaline-earth metal ions.3
Building on this knowledge, we have reported recently a
class of new luminescent hydrophilic N2-arylguanosines in-
cluding G1 and G2 shown in Chart 1.4 Because of the
highly emissive nature of the N2-functionalized guanosines
and their strong absorptions in the visible spectra, we have
shown that they can be used effectively in studying interac-
tions of guanosines with metal ions such as Zn(II) via a
Received 3 December 2009. Accepted 28 February 2010. Published on the NRC Research Press Web site at canjchem.nrc.ca on 5 May
2010.
S. Martic, G. Wu, and S. Wang.1 Department of Chemistry, Queen’s University, Kingston, ON K7L 3N6, Canada.
1Corresponding author (e-mail: wangs@chem.queensu.ca).
Can. J. Chem. 88: 524–532 (2010)
doi:10.1139/V10-040
Published by NRC Research Press

Martic et al.
525
Chart 1.
using a variable mixing times of 0.08, 0.1, and 0.3 s and
the 3 s relaxation delay.
Synthesis of 2’,3’,5’-O-triacetyl-N2-(p-4,4’-
biphenyldiphenylamino)guanosine (G1A)
To a suspension of N2-(p-4,4’-biphenyldiphenylamino)-
guanosine, G1, (0.34 g, 0.57 mmol) and dimethylaminopyri-
dine (0.005 g, 0.04 mmol) in dry acetonitrile (10 mL),
freshly distilled triethylamine (0.30 mL, 2.21 mmol) was
added. After stirring for 5 min, freshly distilled acetic anhy-
dride (0.19 mL, 2.00 mmol) was added dropwise over
5 min, and the mixture was stirred for 2 h at room temper-
ature. The reaction mixture was quenched with methanol
(5 mL), and the organic solvents were removed to dryness.
The residue was treated using the chromatographic column
on CH2Cl2 and CH2Cl2/MeOH (95:5, v/v) as the eluents to
give G1A as the white solid. Yield: 0.17 g (43%). Mp >
300 8C. UV absorption lmax (CH2Cl2), nm (3 dm3mol–1cm–1):
can be monitored by both NMR and fluorescent spectra. The
key results are reported herein.
Experimental section
236 (24 040) and 343 (36 272). Fluorescence (CH2Cl2): lex
=
All reagents were purchased from Aldrich Chemical Co.
and used without further purification unless stated otherwise.
Acetic anhydride, triethylamine, and acetonitrile were
freshly distilled under N2 prior to acetylation reactions.
Low-resolution and high-resolution mass spectrometry ex-
periments were performed using the electrospray ionization
mode on QSTAR XL MS/MS Systems using Analyst QS
Method. Excitation and emission spectra were recorded on
a Photon Technologies International QuantaMaster Model
C-60 spectrometer. Molecular orbital and molecular geome-
try calculations were performed using Gaussian 03 program
suite. Calculations were carried out at the B3LYP level of
theory using 6–31G** as the basis set for all atoms.
1
362 nm and lem = 410 nm. H NMR (400 MHz, DMSO-d6,
298 K) d (ppm): 10.82 (1H, br s, N1H), 8.96 (1H, br s,
N2H), 8.00 (1H, s), 7.61–7.58 (6H, m, J = 5.8, 8.6 Hz, Ph),
7.32 (4H, t, J = 7.9 Hz, Ph), 7.05 (8H, m, J = 7.8, 8.5 Hz,
Ph), 6.02 (1H, d, J = 5.0 Hz, 1’-H), 6.00 (1H, t, J = 5.8 Hz,
2’-H), 5.37 (1H, t, J = 5.7 Hz, 3’-H), 4.28 (1H, dd, J = 4.4,
5.3 Hz, 4’-H), 4.20 (1H, m, J = 4.1, 12.1 Hz, 5’-H), 4.13
(1H, m, J = 4.1, 12.4 Hz, 5’-H), 2.07 (3H, s, CH3), 2.05
(3H, s, CH3), 1.84 (3H, s, CH3). 13C NMR (500 MHz,
MeOD) d (ppm): 171.2, 170.1, 169.9, 148.1, 138.1, 134.3,
129.6 (6C), 127.6 (2C), 127.1 (2C), 124.6 (6C), 124.1 (2C),
123.3 (2C), 122.3 (2C), 121.1, 115.6, 96.8 (2C), 87.5 (C1’),
80.1 (C4’), 72.6 (C2’), 70.9 (C3’), 63.1 (C5’), 19.6 (CH3), 19.5
(CH3), 19.4 (CH3). ESI-MS+ m/z: 729.2465 [M + H]+.
HRMS-EI+ m/z calcd. for C40H36N6O8: 728.2594, found:
728.2538.
Fluorescence experiments
Excitation and emission spectra were recorded on a Pho-
ton Technologies International QuantaMaster Model C-60
spectrometer. To the prepared solutions of G1A (2.5 ꢀ
10–5 mol/L) and G2A (2.5 ꢀ 10–5 mol/L) in CH2Cl2, the
solution of 4-C (6.0 ꢀ 10–3 mol/L) was added in 5 mL ali-
quots.
Synthesis of 2’,3’,5’-O-triacetyl-N2-(p-4,4’-
biphenyldipyridylamino)guanosine (G2A)
G2A was obtained using the same procedure as for G1A
with G2 as the starting material. Yield: 0.08 g (40%).
Mp 241–250 8C. UV absorption lmax (CH2Cl2), nm (3
dm3mol–1cm–1): 235 (24 003) and 319 (50 240). Fluores-
NMR experiments
All 1D and 2D NMR experiments were recorded on
Bruker Avance 400 MHz or 600 MHz spectrometer at 298
1
cence (CH2Cl2): lex = 344 nm and lem = 386 nm. H NMR
1
(400 MHz, DMSO-d6, 298 K) d (ppm): 10.83 (1H, br s,
N1H), 8.97 (1H, br s, N2H), 8.25 (2H, d, J = 3.7 Hz, Py),
8.02 (1H, s, 8-H), 7.72–7.65 (8H, m, J = 7.8, 8.4 Hz, Ph),
7.16 (2H, d, J = 8.4 Hz, Ph), 7.04 (2H, d, J = 5.3 Hz, Py),
6.99 (2H, d, J = 8.8 Hz, Py), 6.08 (1H, d, J = 5.3 Hz, 1’-H),
6.01 (1H, t, J = 5.8 Hz, 2’-H), 5.39 (1H, t, J = 5.7 Hz, 3’-H),
4.28 (1H, m, J = 4.3, 9.6 Hz, 4’-H), 4.22 (1H, m, J = 3.9,
12.4 Hz, 5’-H), 4.19 (1H, m, J = 3.9, 12.3 Hz, 5’-H), 2.09
(3H, s, CH3), 2.06 (3H, s, CH3), 1.85 (3H, s, CH3). 13C
NMR (500 MHz, MeOD) d (ppm): 170.9, 170.2, 170.1,
158.2, 150.4, 147.9 (3C), 144.1, 138.8 (3C), 138.4, 138.1,
137.6, 136.4, 127.9 (3C), 127.3 (2C), 127.2 (3C), 122.5
(2C), 119.0 (2C), 117.9 (2C), 87.6 (C1’), 80.0 (C4’), 72.5
(C2’), 70.8 (C3’), 62.8 (C5’), 19.3 (CH3), 19.2 (CH3), 19.1
(CH3). ESI-MS+ m/z: 731.8851 [M + H]+. HRMS-ESI+ m/z
calcd. for C38H34N8O8 + H: 731.2572, found: 731.2578.
K, unless otherwise specified. H NMR titrations were per-
formed using the solutions of G1A (6.8 ꢀ 10–4 mol/L) or
G2A (1.6 ꢀ 10–2 mol/L) in CD2Cl2 with the solution of 4-C
in CD2Cl2 being added in 10 mL aliquots. DOSY NMR ex-
periments were carried out with Bruker Avance-600 MHz
spectrometer using the pulse sequence of longitudinal eddy
current delay (LED) with bipolar-gradient pulses. The diffu-
sion period was varied from 50 to 90 ms. Calibration of the
field gradient strength was achieved by measuring the value
1
of translational diffusion coefficient (Dt) for the residual H
signal in D2O, Dt = 1.91 ꢀ 10–9 m2/s. All NOESY spectra at
298 K were acquired using a mixing time of 0.3 or 0.4 s and
the 10 s recycling delay. Phase-sensitive NOESY experi-
ment at 195 K was performed using a mixing time of 0.1 s
and a 2 s recycling delay. Phase-sensitive ROESY NMR
spectra were recorded on 400 MHz spectrometer at 195 K
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Can. J. Chem. Vol. 88, 2010
Synthesis of 2’,3’,5’-O,N2-tetraacetyl-N2-(p-4,4’-
biphenyldipyridylamino)guanosine (G2B)
Chart 2.
G2B was isolated as a side product from the same reac-
1
tion for G2A. Yield: 0.03 g (18%). Mp 156–164 8C. H
NMR (400 MHz, CD3CN, 298 K) d (ppm): 12.98 (broad,
N1H, 1H), 8.24 (d, J = 4.5 Hz, 2H), 7.82 (d, J = 8.4 Hz,
2H), 7.73 (s, 1H, H8), 7.72 (d, J = 6.5 Hz, 2H), 7.65 (dt, J =
1.8, 9.1 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.21 (d, J =
8.5 Hz, 2H), 7.02 (m, 4H), 5.75 (d, J = 4.6 Hz, 1H, H1’),
5.57 (t, J = 4.9 Hz, 1H, H2’), 4.41 (t, J = 6.0 Hz, 1H, H3’),
4.05 (m, J = 5.1, 11.5 Hz, 1H, H4’), 3.71 (m, 2H, H5’, H5@),
2.03 (s, CH3, 3H), 1.94 (s, CH3, 3H), 1.88 (s, CH3, 3H), 1.86
(s, CH3, 3H). 13C NMR (400 MHz, CD3CN, 298 K) d
(ppm): 175.7, 170.2, 169.4, 169.3, 158.1 (2C), 155.3, 150.5,
148.3, 147.5 (2C), 145.4, 140.7, 139.4, 138.0 (2C), 137.7
(2C), 135.9, 129.7 (2C), 128.4 (2C), 127.9 (2C), 127.3
(2C), 121.9, 118.6 (2C), 117.3, 87.9 (C1), 79.4 (C4), 71.9
(C2), 70.7 (C3), 63.8 (C5), 25.8 (CH3), 19.8 (CH3),
19.6 (CH3), 19.5 (CH3). HRMS-ESI+ m/z calcd. for
C40H36N8O9ꢁH+: 773.2678, found: 773.2666.
where the H-donor site of the N2 group is blocked by an
acetyl group. Not surprisingly, G2B did not form a GC
dimer as confirmed by NMR (see Fig. S5 in the Supplemen-
tary data for NMR). Thus, the NMR data established un-
equivocally that the N2-functionalization by an aryl group
does not impede the ability of the guanosine to form a H-
bonded base pair with cytidine.
Dynamic exchange of non-bound and bound 4-C in the
GC pair
Although the GC base pair formation has been exten-
sively investigated, little is known about the interaction and
the exchange mechanism of a GC pair with non-bound G or
C. A previous NMR study on the interaction of a GC pair
with free guanosine has identified a GCG trimer.8 However,
the exact nature of the interactions between the GC pair and
free cytidine remains unclear. The GC pair formed by G1A
and G2A with 4-C provided us an opportunity to study such
interactions by NMR methods. As shown in Fig. 3, the addi-
tion of more than 1 equiv. of 4-C to the solution of G1A
results in the sharpening of the N1H and N2H (G1A) reso-
nances along with a slight downfield shift. Surprisingly, the
N4H (4-C) proton is shifted upfield (~3 ppm) and broad-
ened, indicating a dynamic process. The overall chemical
shift positions and the sharpness of the exchangeable G1A
protons indicate the complex formation by H-bonds. At
[G1A]:[4-C] ’ 1:4 ratio, the chemical shifts of the G1A
protons are similar to those of [G1A]:[4-C] = 1:1, while the
chemical shifts of 4-C exhibit significant changes. These
findings support that in the presence of excess amount of 4-C,
G1A remains involved in the H-bonding in the GC pair
while cytidine undergoes some dynamic exchange. Similar
trends were also observed for G2A when treated with exces-
sive 4-C (see Fig. S6 in the Supplementary data). DOSY
NMR study on [G2A]:[4-C] and [G2A]:[4-C]2 shows that
the diffusion coefficients Dt of these two species are similar
(see Fig. S7 in the Supplementary data), thus supporting that
the stable GCC trimer species is unlikely, but it may be in-
volved in a dynamic process.
Results and discussion
NMR measurements
GC base pair formation
The structure of the cytidine (4-C) used in the study and
the GC base pair with labels are shown in Chart 2. H NMR
1
spectra indicated that in the absence of 4-C, G1A and G2A
exist predominantly as monomers in CD2Cl2 at 298 K. The
addition of 4-C to the solution of G1A or G2A in CD2Cl2
results in upfield shifts of the imino and amino resonances
of the guanine and cytosine units, indicative of H-bonding.
As shown in Fig. 1, upon [G1A]:[4-C] base pair formation,
a slight change was observed for the phenyl protons (Ho) di-
rectly attached to the N2-site of guanine, while the H8 signal
of the guanine ring remains virtually unchanged. The most
significant change is observed for the H6 and H5 resonances
of 4-C, which shift ~ 0.1–0.3 ppm upfield. Upon cooling to
228 K, the [G1A]:[4-C] dimer can be clearly identified
through the sharp peaks at ~ 13.1, 12.2, and 10.7 ppm that
can be assigned to the N1H (G1A), N4H (4-C), and N2H
(G1A), respectively, as presented in Fig. 2. At 228 K, the
NOE cross peaks are of the same phase as the diagonal
peaks. The key NOE cross peaks between the N1H (G1A)
with N4H (4-C) in the GC base pair were observed in the
NOESY NMR spectrum, confirming the GC pair formation.
The GC pair formation was further supported by ESI-MS
data, with a peak at m/z 1140 identified as [G1A:4-C + H]+
(see Fig. S1 in the Supplementary data). Similar NMR and
ESI-MS data were also obtained for the [G2A]:[4-C] base
pair (see Figs. S2–S4 in the Supplementary data). The rela-
tive ribose stereogeometry of the GC base pair was assigned
by NOESY NMR and was found to be syn for both N2-
arylguanosines, owing to the strong NOE cross peak between
its H1 and H8, and anti for 4-C, judging from the strong
NOE between its H2 and H6 protons.7 An association con-
stant could not be determined by NMR dilution method be-
cause of the strong GC association. As a control study, we
also examined the interaction of 2’,3’,5’-O,N2-tetraacetyl-N2-
(p-4,4’-biphenyldipyridylamino)guanosine (G2B) with 4-C,
Variable temperature NMR experiments were performed
for [G1A]:[4-C]4 to further probe the chemical-exchange
process. As shown in Fig. 4, at temperatures below 208 K,
one type of G1A and two types of 4-C peaks (denoted as 4-C
and 4-C*) were observed. The sharp peaks between 10.5
and 13.4 ppm are assigned to N2H (G1A), N1H (G1A), and
N4H (4-C) of the GC pair. The broad resonance at 10.2 ppm
can be assigned to a N4H* of the non-bound 4-C*. Since the
N4H* proton remains broad and below 12 ppm, we can con-
clude that it does not belong to the typical [4-C]2 dimer.
Further lowering of the temperature to <188 K results in the
Published by NRC Research Press

Martic et al.
527
1
Fig. 1. Partial H NMR spectra of (A) 4-C, (B) G1A, and (C) [G1A]:[4-C] (CD2Cl2, 298 K).
Fig. 2. Partial NOESY NMR spectrum of [G1A]:[4-C] (CD2Cl2, 228 K).
splitting of all non-exchangeable protons into 4-C and 4-C*
as well.
between N1H (G2A), N4H (4-C), and N4H* (4-C*) with
N4Ac (4-C) and N4Ac* (4-C*) indicate that these protons
are in close proximity to each other (see Fig. S8 in the Sup-
plementary data). In the presence of excessive cytidine, all
the cross peaks in the NOESY spectrum are of the same
phase as the diagonal peaks.
To confirm NOE correlations and to distinguish them
from chemical-exchange cross peaks, a 2D ROESY NMR
spectrum was recorded at 195 K (Fig. 6), which reveals
clear NOE cross peaks between the bound 4-C and non-
bound 4-C* (e.g., N4H (4-C)/N4Ac* (4-C*) and N4H* (4-
C*)/N4Ac (4-C)). In addition, the ribose protons associated
with 4-C in the [G2A]:[4-C] base pair are found to be in
close proximity to H6* (4-C*) proton and vice versa. Fur-
thermore, the H6 proton (4-C) exhibits a NOE cross peak with
the H5* (4-C*) proton. In addition, the chemical-exchange
To gain structural information about the dynamics of this
system and to establish the connectivity between the GC
pair and the non-bound 4-C*, COSY, NOESY, and ROESY
NMR experiments were performed on the mixture of
[G1A]:[4-C]2 and [G2A]:[4-C]2. The spectroscopic data for
G2A are presented here because of the higher resolution and
spectral quality compared with those for G1A. Complete
spectral assignment for G2A and 4-C resonances in
[G2A]:[4-C]2 was established using COSY. NOESY NMR
was performed at 195 K, allowing the identification of
closely related protons. Figure 5 shows the strong NOE sig-
nature cross peaks for [G2A]:[4-C]2 between N1H (G2A)/
N4H (4-C) protons indicative of Watson–Crick H-bonding.
It is also worth noting that the NOE cross peaks observed
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Can. J. Chem. Vol. 88, 2010
1
Fig. 3. H NMR spectral change of G1A with the addition of 4-C (CD2Cl2, 298 K, [G1A] = 6.8 ꢀ 10–4 mol/L).
Fig. 4. Partial variable temperature NMR spectra of [G1A]:[4-C] = 1:4, (CD2Cl2, [G1A] = 6.8 ꢀ 10–4 mol/L).
Fig. 5. Partial NOESY NMR spectrum of [G2A]:[4-C]2 (CD2Cl2, 195 K).
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Martic et al.
529
Fig. 6. Partial ROESY NMR spectra of [G2A]:[4-C]2 (CD2Cl2, 195 K, red: NOE cross peaks, black: chemical-exchange cross peaks).
interaction was observed between the proton pairs of H6 (4-C)/
H6* (4-C*) and H5 (4-C)/H5* (4-C*). The key protons that
are involved in GC base pair formation all undergo chemical
exchange with each other, which makes the complete as-
signment of the final structure difficult. However, on the ba-
sis of the relative chemical shifts of non-exchangeable
protons and the specific NOE interactions shown in Fig. 6,
it can be concluded that there are two types of cytidines,
non-bound 4-C* and bound 4-C with G2A, which undergo
chemical exchange with each other, leading to the averaging
of the 4-C signals at above 208 K. These findings are con-
sistent with the presence of p–p stacking interactions be-
tween 4-C in the [G2A]:[4-C] pair and non-bound 4-C*.
Since G2A does not exhibit NOE with H6* (4-C*) and H5*
(4-C*) protons the possibilities of p–p interactions between
G2A in the [G2A]:[4-C] pair and 4-C* can be ruled out.
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Can. J. Chem. Vol. 88, 2010
Fig. 7. Fluorescence titrations of (A) G1A and (B) G2A with 4-C (CH2Cl2, 2.5 ꢀ 10–5 mol/L, lex = 362 nm (G1A) and 344 nm (G2A).
Inset: Stern–Volmer plots at lmax
.
Fig. 8. Fluorescence spectra of G1A in the presence of A (left) or T (right) nucleosides with or without 4-C (CH2Cl2).
Using the variable temperature NMR data, the exchange rate
kc and the activation free energy DG{ for the exchange pro-
cess were estimated to be 88 s–1 and 43 kJ mol–1, respec-
tively, at 208 K.
data), which, not surprisingly, does not show any fluorescent
quenching, thus further supporting the role of selective H-
bonding between G2A and 4-C in the fluorescence quench-
ing. The selectivity of the fluorescence response of G1A and
G2A toward cytidine was further confirmed by the competi-
tion experiments of 2’,3’,5’-O-triacetyladenosine (A) and
3’,5’-O-diacetylthymidine (T) with 4-C for binding with
G1A and G2A. As shown in Fig. 8, both A and T have no
impact at all on the fluorescent spectrum of G1A. Similar
results were also obtained for G2A (see Fig. S11 in the Sup-
plementary data).
Fluorescence measurement
In contrast to the non-functionalized guanosines, which
are weakly emissive in the UV region, G1A and G2A are
highly emissive in the visible region, thus making it possible
to monitor the G–C interactions by fluorescence spectro-
scopy as well. As shown in Fig. 7, the addition of 4-C to
the solution of G1A or G2A in CH2Cl2 results in quenching
of the fluorescence emission of the N2-arylguanosines. The
Stern–Volmer plots support the formation of a stable 1:1
species with 4-C (Fig. 6, inset), which is most likely respon-
sible for the quenching of the guanosine emission. The bind-
ing constants for the GC pairs were determined to be 5 ꢀ
105 M–1 and 2 ꢀ 106 M–1 for G1A and G2A, respectively
(see Fig. S9 in the Supplementary data).9 These binding
constants are in agreement with those reported in the litera-
ture for other GC pairs.10
Molecular orbital calculations
Our previous TD-DFT calculations have established that
the lowest electronic transitions and the fluorescence of
G1A and G2A are p–p* transitions centered on guanine
and the biphenyl-NAr2 group.4 To understand the origin of
the fluorescence quenching upon GC pair formation, DFT
calculations were performed for the [G1A]:[4-C] pair. The
ground-state structure of the [G1A]:[4-C] pair was fully op-
timized at a B3-LYP/6–31G** level of theory and is pre-
sented in Fig. 9 along with the diagrams of the HOMO and
LUMO levels.11 The HOMO level of [G1A]:[4-C] consists
As a control study, the fluorescence titration of G2B with
4-C was also carried out (see Fig. S10 in the Supplementary
Published by NRC Research Press

Martic et al.
531
Fig. 9. HOMO and LUMO orbitals of the [G1A]:[4-C] base pair.
dine molecules and a potential p-stacked intermediate have
been identified by NMR data.
Supplementary data
Supplementary data for this article are available on the
journal Web site (canjchem.nrc.ca).
Acknowledgement
We thank the Natural Sciences and Engineering Research
Council of Canada (NSERC) for financial support.
References
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