Molecular recognition of cationic phenothiazinium and phenoxazinium dyes with π-extended 2'-deoxyadenosine nucleotides

Article abstract of DOI:10.1039/b913315a

N⁶-(N'-Arylcarbamoyl)-2'-deoxyadenosine-H-phosphonates displayed molecular recognition towards cationic phenothiazinium and phenoxazinium dyes in aqueous solutions; studies have shown that binding is driven mainly by aromatic interactions and t

Full text of DOI:10.1039/b913315a

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Molecular recognition of cationic phenothiazinium and phenoxazinium
dyes with p-extended 20-deoxyadenosine nucleotidesw
´ ´
Karina Mondragon-Vasquez and Hugo Morales-Rojas*
Received (in Austin, TX, USA) 6th July 2009, Accepted 15th September 2009
First published as an Advance Article on the web 6th October 2009
DOI: 10.1039/b913315a
N6-(N0-Arylcarbamoyl)-20-deoxyadenosine-H-phosphonates dis-
played molecular recognition towards cationic phenothiazinium
and phenoxazinium dyes in aqueous solutions; studies have
shown that binding is driven mainly by aromatic interactions
and that size and shape-complementarity of the aromatic rings in
host and guest provides selectivity.
Non-covalent interactions between aromatic species play a
prominent role in molecular recognition phenomena in
chemistry, biology and materials sciences.1,2 Stacking of the
aromatic bases is the dominant contribution to the stability of
the DNA duplex, and the incorporation of natural and non-
natural nucleosides as dangling end residues generally provides
an extra stabilization due to the interaction of the aromatic
moiety with the neighboring base pair.3 For example,
N6-(N0-naphthylcarbamoyl)-20-deoxyadenosine (Scheme 1) as a
dangling end residue produced one of the largest stabilities of
the DNA duplex.4 Sugimoto and co-workers suggested that
co-planarity of the adenine and the naphthyl group, possibly
aided by an intramolecular hydrogen bond, virtually extends
the available p surface and enhances the stacking interaction.4
For aromatic guest molecules only a few water-soluble host
species are available and these are mostly macrocycles.1,5,6 For
Scheme 2
controversy as to the corresponding contribution by other
effects such as electrostatics and charge-transfer among
others.1a,c,e Therefore, novel water-soluble aromatic hosts in
which, size, shape and electronic properties can be tailored
to suit a particular guest are still needed. Compounds like
N6-(N0-arylcarbamoyl)-20-deoxyadenosine (1a–g, Scheme 2)
are synthetically accessible and may serve as water-soluble
receptors for aromatic species. Using 20-deoxyadenosine as a
scaffold also allows the incorporation of additional recognition
sites on the hydroxyl groups and possibly to further elongate
the mononucleoside to di- or trinucleotides employing DNA
synthetic methodologies.3,4 We now report on the molecular
recognition properties of H-phosphonate nucleotides 1a–g
that were synthesized as triethylammonium salts starting from
20-deoxyadenosine with excellent yields, offering a simple route
to incorporate different aromatic residues (Scheme S1 ESIw).
Cationic phenothiazinium and phenoxazinium salts were chosen
as the target aromatic guest. Among these are well-known
biological indicator probes such as methylene blue (MB), azur
C (AC), toluidine blue (TB), Nile blue (NB) and oxazine-170
(OX) (Scheme 2).7 They are also used as antitumorals,
antibacterials, DNA triplex stabilizers, and antimalarials,8
and in most cases their biological activities have been related
to their p-stacking capability.
instance, receptors derived from charged tetrakis-aryl-
porphyrins have shown selectivity to bind aromatic guests in
aqueous solutions.6 The systematic analysis of these supra-
molecular porphyrin complexes with ligands containing 6, 10
and 14 p-electrons showed that the association energy is well
described by constant free energy increments depending on the
number of participating p-electrons.6a Although dispersive
interactions frequently represent a significant portion of the
net attractive effect between two aromatic systems, there is still
Scheme 1
Centro de Investigaciones Quı´micas, Universidad Auto´noma del
Estado de Morelos, Av. Universidad 1001, C.P. 62209 Cuernavaca,
´
Mexico. E-mail: hugom@uaem.mx; Fax: +52 777 3297997;
Tel: +52 777 3297997
w Electronic supplementary information (ESI) available: Synthetic
procedures and characterization data, UV-vis titrations, 19F NMR
spectra, modeling, and thermodynamic data. See DOI: 10.1039/b913315a
In order to test the binding capacities of nucleotides 1a–g
towards the cationic dyes, association constants were
ꢀc
This journal is The Royal Society of Chemistry 2009
6726 | Chem. Commun., 2009, 6726–6728

View Article Online
Fig. 2 Equilibrium geometry for complex 1a–MB. (Bottom) Side
view showing co-planarity for adenine and phenyl rings and stacking
of the dye. (Top) View from MB face showing complementarity of
aromatic surfaces. (see ESIw).
Fig. 1 Comparison of association constants (Kas) for nucleotides
1a–d with phenothiazinium and phenoxazinium dyes. (See ESIw).
determined by UV-vis absorption titrations in MOPS
buffer (4-morpholinepropanesulfonic acid, pH 7, 25 1C and
0.010 M NaCl). Qualitative information on the binding modes
was obtained by 1H and 19F NMR spectroscopy and molecular
modeling. The titrations were monitored by the decay of the
charge-transfer bands that regularly appear in the 530–680 nm
region for the dyes. In some cases the absorbance decay was
concomitant with the emergence of a new red-shifted band
featuring an isosbestic point. Nucleotides without further aryl
residue such as AdNH2 and AdNHEt, only produced a marginal
change in the absorbance within the concentration range used
for 1a or 1b (Fig. S9 ESIw). Table S1 (ESIw) summarizes the
average binding constants obtained from the titration experiments
and the data for derivatives 1a–1d are depicted in Fig. 1.z For
the phenyl derivative 1a the binding constants for the
complexation of phenothiazinium dyes were in the order
of B104–105 Mꢁ1 following the trend TB 4 MB 4 AC.
The same tendency was observed for the 1-naphthyl
derivative 1b, with a constant gain in the free binding energy
of DDGo B ꢁ2.5 kJ molꢁ1 ongoing from 1a to 1b, thus
indicating that the size of the aromatic residue is of importance
for the binding strength. In order to assess the contribution
from ion-pair interactions, monoanionic 1-naphthyl compounds
1c and 1d, in which one H-phosphonate at positions 50 or 30 is
present, were also tested.y These monoanionic nucleotides
reported lower binding energies in comparison to the 30,50-
bis-H-phosphonate salt 1b. In some cases the difference in
energy was approximately 5–6 kJ molꢁ1, which matches
the value expected per salt bridge in aqueous solutions
(Table S2 ESIw).1a,6 Further insight in the ion-pair interaction
came from studying the binding dependence on the ionic
strength for complex 1b–TB. The binding constant for this
complex decreased concomitant to the increase of saline
concentration from 5 ꢂ 10ꢁ3 to 8 ꢂ 10ꢁ2 M NaCl giving an
overall 7.5-fold drop (Fig. S10 ESIw). This behavior is
typical for host–guest complexes in which ion-pair interactions
contribute to the overall affinity.1a,6
guanines and 2,6-diaminopurines that display an anti/syn
conformational equilibrium in non-aqueous solutions.9,10
Generally, conformations with N–Hꢃ ꢃ ꢃNarom interactions are
favored. Assuming that this configuration holds in the
complexes, an adequate contact between the aromatic surfaces
can be found for the three ring-systems of the phenothiazinium
dyes and nucleotides 1a–1d, as exemplified for 1a–MB in Fig. 2.
To shed light on the mechanism driving the recognition between
the aromatic groups, the estimated free energy contribution
from salt bridges was subtracted from the observed free binding
energies. For complex 1a–MB, after subtraction of two salt
bridges (10 kJ molꢁ1),1a,6 the remaining free binding energy was
ꢁ18.2 kJ molꢁ1. This value is in excellent agreement with the
value previously reported for condensed three ring systems with
14 p-electrons (DGdispersion = ꢁ18.5 kJ molꢁ1).6a In the case of
1b–MB, their salt bridge contributions can be estimated more
accurately from the DDGo values obtained in the comparison with
complexes 1c–MB (+6 kJ molꢁ1) and 1d–MB (+6.7 kJ molꢁ1
)
(see Table S2 ESIw). After subtracting these values from
DGo = ꢁ30.6 kJ molꢁ1 (for 1b–MB) the remaining free binding
energy was ꢁ17.9 kJ molꢁ1, which also agrees well with the
DGdispersion indicated above. Thus, for complexes in which host
and guest aromatic surface fits well, the association energy can be
explained from aromatic and ion-pair interactions, underscoring
dispersion as the main mechanism in aromatic recognition.
Preliminary thermodynamic data were acquired for 1b–TB
by measuring the binding constants in the temperature range
from 5 to 45 1C, and then fitting the data to the van0t Hoff
equation (see Fig. S13 ESIw). An enthalpy of DH1 = ꢁ29.8 ꢄ
3 kJ molꢁ1 was thus obtained. For comparison, a value of
DGo = ꢁ33.6 kJ molꢁ1 was measured for 1b–TB at 298 K,
pointing out that the association is enthalpically driven, which
further supports the dominance of aryl–aryl interactions.
Fluoroaromatics have become the standard probe to
illustrate the relative importance of electrostatics in aromatic
interactions.2d,2e,11 Derivatives containing 4-fluorophenyl (1e),
3,5-difluorophenyl (1f) and pentafluorophenyl (1g) were also
examined. As shown in Fig. S14 and Table S1 (ESIw), no clear
trend can be delineated from the binding constants; for
example, by complexation of MB with 1e and 1f the binding
constants decreased by 2.5-fold in comparison to 1a, but in other
complexes the binding constant remained the same (i.e., 1e–NB) or
even increased by 2.3 and 4-fold for 1f–TB and 1f–AC,
The calculated equilibrium geometry for compounds 1a–1d
indicated molecular structures with intramolecular H-bonding
interactions between the purinic N1 nitrogen and the ureido
hydrogen having an (E,Z) conformation (see Fig. S11 ESIw);
these results are in qualitative agreement with the structures
observed for urea-functionalized pyridines, pyrimidines,
ꢀc
This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 6726–6728 | 6727

View Article Online
from aromatic and ion-pairing interactions. In fluorophenyl
derivatives polarity seems to be less important than size and
shape-complementarity. These findings indicate that dispersion
interactions contribute more significantly to aromatic recognition
in aqueous solution. Exploration of further host derivatives
and guest species is underway.
This work was supported by CONACyT J50827Q. We are
grateful to Dr Felipe Medrano for access to the modeling
programs.
Notes and references
z Binding constants were obtained from triplicate experiments and data
averaged from at least five wavelengths. Non-linear fit to a 1 : 1 binding
model in which concentrations of host and guest are of similar magnitude
was used. See H.-J. Schneider and A. K. Yatsimirsky, Principles and
Methods in Supramolecular Chemistry, Wiley, 2000, p. 142.
Fig. 3 (Top) 1H NMR spectra of 1e (1 ꢂ 10ꢁ3 M in D2O). (Bottom)
Changes upon addition of one molar equivalent of AC. Only the
region between 5.5 to 9.0 ppm is shown. R2 and R3 = PO2Hꢁ.
y 1-Naphthylcarbamoyl-20-deoxyadenosine nucleoside containing free
50,30-OH groups is not soluble enough to perform titration experi-
ments in buffer aqueous solutions.
Table 1 19F NMR complexation induced shifts (Dd) in titrations of
nucleotides 1e, 1f and 1g with phenothiazinium dyesa
1 (a) H.-J. Schneider, Angew. Chem., Int. Ed., 2009, 48, 3924;
(b) J.-H. Ryu, D.-J. Hong and M. Lee, Chem. Commun., 2008,
1043; (c) E. A. Meyer, R. K. Castellano and F. Diederich, Angew.
Chem., Int. Ed., 2003, 42, 1210; (d) M. L. Waters, Curr. Opin.
Chem. Biol., 2002, 6, 736; (e) C. A. Hunter, K. R. Lawson,
J. Perkins and C. J. Urch, J. Chem. Soc., Perkin Trans. 2, 2001,
651; (f) S. K. Burley and G. A. Petsko, Science, 1985, 229, 23.
2 For model systems with rotameric or conformational flexible
molecules: (a) F. Cozzi, M. Cinquini, R. Annunziata, T. Dwyer
and J. S. Siegel, J. Am. Chem. Soc., 1992, 114, 5729; (b) E. Kim,
S. Paliwal and C. S. Wilcox, J. Am. Chem. Soc., 1998, 120, 11192;
(c) S. L. Mckay, B. Haptonstall and S. H. Gellman, J. Am. Chem.
Soc., 2001, 123, 1244; (d) M. J. Rashkin and M. L. Waters, J. Am.
Chem. Soc., 2002, 124, 1860; (e) B. W. Gung, X. W. Xue and
Y. Zou, J. Org. Chem., 2007, 72, 2469; (f) W. R. Carroll,
P. Pellechia and K. D. Shimizu, Org. Lett., 2008, 10, 3547.
3 (a) K. M. Guckian, B. A. Schweitzer, R. X. F. Ren, C. J. Sheils,
D. C. Tahmassebi and E. T. Kool, J. Am. Chem. Soc., 2000, 122, 2213;
(b) J. Isaksson and J. Chattopadhyaya, Biochemistry, 2005, 44, 5390.
4 S. Nakano, Y. Uotani, S. Nakashima, Y. Anno, M. Fujii and
N. Sugimoto, J. Am. Chem. Soc., 2003, 125, 8086.
Dye
1e
1f
1gb
MB
AC
TB
ꢁ6.9
+0.472
+0.216
+0.171
+0.174, +0.128, +0.037
+0.081, +0.067, +0.026
+0.252, +0.207, +0.132
ꢁ3.16
ꢁ0.553
a
Dd measured in deuterated water at 25 1C using KF as reference.
ortho, meta and para F positions in 1g, respectively.
b
respectively. As depicted in Fig. 3, the 1H NMR spectra of the
nucleotide 1e in the regions corresponding to the purine
(H2, H8) and the 4-fluorophenyl (Ho, Hm) protons showed
upfield-shifts in the presence of one molar equivalent of the
dye AC, thus indicating that its aromatic surface stacks over
both rings of the nucleotide. Likewise the anomeric proton
(H1) showed the same tendency, supporting a preference of the
dye to stack over one of the faces of the nucleotide. It is worth
noting that only the 50-H-phosphonate was shifted towards
lower fields.
Upon addition of phenothiazine dyes the 19F NMR spectra
of 1e–1g also changed. The induced shifts at 100% complexation
(CIS, Dd) are summarized in Table 1. The MB dye produced
large CIS values in the presence of 1e and 1f, but these changes
occurred in opposite directions displacing the para-F
substituent in 1e upfield, while the 3,5-meta-F in 1f were
shifted downfield. This indicates that the overlap of MB
relative to the substituted phenyl group in these complexes
differs as suggested by molecular modeling (see Fig. S17 ESIw).
The results with nucleotide 1g indicated that the pentafluoro-
phenyl ring does not lie on the same plane as the adenine
group due to steric hindrance of the ortho-F substituents and
the urea carbonyl group. Plausible complexes with 1g gave
interesting cleft-type structures (See Fig. S18 ESIw). The small
downfield displacements measured for complexes between 1g
and phenothiazine dyes (Table 1) are in agreement with this
model structure.
5 (a) D. B. Smithrud, T. B. Wyman and F. Diederich, J. Am. Chem.
Soc., 1991, 113, 5420; (b) C. Jasper, T. Schrader, J. Panitzky and
F.-G. Klarner, Angew. Chem., Int. Ed., 2002, 41, 1355;
(c) M. S. Cubberley and B. L. Iverson, J. Am. Chem. Soc., 2001,
123, 7560.
6 (a) H.-J. Schneider and M. Xue, J. Org. Chem., 1994, 59, 7464;
(b) T. Liu and H.-J. Schneider, Angew. Chem., Int. Ed., 2002, 41, 1368;
(c) M. Sirish and H.-J. Schneider, J. Am. Chem. Soc., 2000, 122, 5881.
7 (a) P. Mukerjee and A. K. Ghosh, J. Am. Chem. Soc., 1970, 92,
6419; (b) E. M. Tuite and J. M. Kelly, Biopolymers, 1995, 35, 419.
8 (a) J. L. Vennerstrom, M. T. Makler, C. K. Angerhofer and
J. A. Williams, Antimicrob. Agents Chemother., 1995, 39, 2671;
(b) J. Jose and K. Burgess, Tetrahedron, 2006, 62, 11021;
(c) G. Song, F. Xing, X. Qu, J. B. Chaires and J. Ren, J. Med.
Chem., 2005, 48, 3471; (d) K. Takasu, T. Shimogama, C. Satoh,
M. Kaiser, R. Brun and M. Ihara, J. Med. Chem., 2007, 50, 2281.
9 (a) C.-H. Chien, M.-K. Leung, J.-K. Su, G.-H. Li, Y.-H. Liu and
Y. Wang, J. Org. Chem., 2004, 69, 1866; (b) S. Rashdan,
M. E. Light and J. Kilburn, Chem. Commun., 2006, 4578.
10 (a) A. M. Martin, R. S. Butler, I. Ghiviriga, R. E. Giessert,
K. A. Abboud and R. K. Castellano, Chem. Commun., 2006,
4413; (b) K. Miyata, A. Kobori, R. Tamamushi, A. Ohkubo,
H. Taguchi, K. Seio and M. Sekine, Eur. J. Org. Chem., 2006,
3626; (c) H. C. Ong and S. C. Zimmerman, Org. Lett., 2006, 8, 1589.
11 (a) J. S. Lai, J. Qu and E. T. Kool, Angew. Chem., Int. Ed., 2003,
42, 5973; (b) S. L. Cockroft, J. Perkins, C. Zonta, H. Adams,
S. E. Spey, C. M. R. Low, J. G. Vinter, K. R. Lawson, C. J. Urchc
In conclusion, our results have shown that extending the
p-surface of 20-deoxyadenosine 30,50-H-phosphonate nucleotides
leads to flat aromatic based receptors that are suitable to
recognize selectively aromatic species in aqueous solutions.
Association constants can be explained by free energy increments
´
and C. A. Hunter, Org. Biomol. Chem., 2007, 5, 1062; (c) S. Perez-
Casas, J. Hernandez-Trujillo and M. Costas, J. Phys. Chem. B,
´
2003, 107, 4167; (d) K. Frey and S. A. Woski, Chem. Commun.,
2002, 2206.
ꢀc
This journal is The Royal Society of Chemistry 2009
6728 | Chem. Commun., 2009, 6726–6728