Fluorescent Sensing of Guanine and Guanosine Monophosphate with Conjugated Receptors Incorporating Aniline and Naphthyridine Moieties

Article abstract of DOI:10.1021/acs.orglett.6b00311

Ethyne-linked naphthyridine-aniline conjugated molecules are selective sensors of decylguanine in dichloromethane and guanosine monophosphate in water (K_ass = 16 000 M^-1). The 2-acetamido-1,8-naphthyridine moiety binds with guanine in a DAA-ADD triply hydrogen-bonded motif. The aniline moiety enhances an electron-donating effect, and the substituent is tuned to attain extra hydrogen bonds, π-π stacking, and electrostatic interactions. The proposed binding modes are supported by a Job plot, ESI-MS, ¹H NMR, UV-vis, and fluorescence spectral analyses.

Full text of DOI:10.1021/acs.orglett.6b00311

Letter
pubs.acs.org/OrgLett
Fluorescent Sensing of Guanine and Guanosine Monophosphate
with Conjugated Receptors Incorporating Aniline and Naphthyridine
Moieties
Shao-Hung Lu,†,§ Riping Phang,†,§ and Jim-Min Fang*,†,‡
†Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
‡The Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
S
* Supporting Information
ABSTRACT: Ethyne-linked naphthyridine−aniline conju-
gated molecules are selective sensors of decylguanine in
dichloromethane and guanosine monophosphate in water (Kass
= 16 000 M−1). The 2-acetamido-1,8-naphthyridine moiety
binds with guanine in a DAA−ADD triply hydrogen-bonded
motif. The aniline moiety enhances an electron-donating
effect, and the substituent is tuned to attain extra hydrogen
bonds, π−π stacking, and electrostatic interactions. The
proposed binding modes are supported by a Job plot, ESI-
1
MS, H NMR, UV−vis, and fluorescence spectral analyses.
ational design of new DNA-binding agents provides an
Reffective approach for development of novel chemo-
therapeutic anticancer drugs.1 In nature, cytosine (C) binds
with guanine (G) to form a CG pair through the triply
hydrogen-bonded DAA−ADD motif, wherein A represents a
hydrogen acceptor and D represents a hydrogen donor. Cytosine
analogues have been designed as effective molecular receptors of
guanine and guanosine.2 In addition to triple hydrogen bonds, a
cytosine-based ditopic receptor is devised to contain terminal
amine groups for enhanced binding with guanosine 5′-mono-
Figure 1. Ethyne-linked naphthyridine−aniline molecules 1−5 as
phosphate (GMP) through electrostatic interactions of the basic
fluorescent sensors of 9-decylguanine and GMP. The substituents (R) of
amine with the phosphoric group.3
It is noted that 2-amido-1,8-naphthyridine also possesses a
aniline provide additional interactions to enhance the DAA−ADD triply
hydrogen-bonded motif.
DAA motif with a geometric fit to guanine in the formation of a
DAA−ADD triply hydrogen-bonded complex similar to the C
G pair.4−6 A single guanine bulge in duplex DNA is found to bind
The 2-acetamido-1,8-naphthyridine moiety is an electron-
withdrawing group (EWG), and the aniline moiety is an electron-
selectively with 2-amido-1,8-naphthyridine derivatives by fully
donating group (EDG). The ethynyl linker between naphthyr-
idine and aniline elongates the conjugate system in this sensor
complementary hydrogen bonds.7−9 Thus, dimeric and tetra-
meric 2-amido-1,8-naphthyridine compounds are further de-
molecule to make a better intramolecular charge transfer (ICT)
system upon binding with guanine derivatives,15,16 so that the
absorption and fluorescence wavelengths of the host−guest
complexes can fall in the visible region for naked-eye detection.
We have shown that the receptor molecule 1 forms a 1:1
complex with 9-decylguanine (G10) in CH2Cl2 solution with a
signed to detect the G−G mismatches in duplex DNA and
telomeres.10−12 Silica gel modified by covalent linkage of 2-
amido-1,8-naphthyridine units is also a suitable stationary phase
for 2′,3′,5′-tri-O-acetylguanosine in HPLC analysis.13 A Gd3+
complex of 7-acetamido naphthyridine−tetraazocyclodecane has
been utilized as a magnetic resonance imaging contrast for
detection of GMP.14
We have previously devised a series of ethyne-linked
naphthyridine−aniline receptor molecules, such as compound
1 (Figure 1), to monitor the binding events with guanine
derivatives.5
binding constant of (9.1
0.2) × 104 M−1 by fluorescence
titration at 535 nm.5 We conceive that the N-methylaniline
moiety of 1 can be further modified to attain extra interactions
Received: January 30, 2016
© XXXX American Chemical Society
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with the purine ring of a guanine derivative. Along this line, we
designed the new push−pull receptor molecules 2−4 (Figure 1)
that incorporate appropriate R substituents at the aniline site to
increase the binding affinity with a guanine derivative. The
hydroxyethyl and aminoethyl substituents in the aniline moieties
of compounds 2 and 3 are expected to provide hydrogen
bondings with G10, while the pyrene group of 4 may offer
hydrophobic and π−π stacking interactions with the purine ring.
To probe GMP in water under physiological conditions, we also
designed a receptor molecule 5 (Figure 1) containing a terminal
guanidine group to form a stable ion pair with the negatively
charged phosphate ion via electrostatic interactions.17
Scheme 1 shows the synthetic routes to molecules 1−5 using
the Sonogashira coupling reaction to construct the naphthyr-
Scheme 1. Synthesis of Receptor Molecules 1−5
Figure 2. Titration of receptor 2 (1 × 10−5 M) upon incremental
additions of 9-decylguanine (1 × 10−2 M) in CH2Cl2 solution. (A) UV−
vis curves; (B) fluorescence curves (λex = 396 nm).
Similar to compound 2, formation of the 3−G10 complex in
1:1 stoichiometry was indicated by the Job plot based on the
absorption changes in the titration experiment (Figure S13 in
SI). The 3−G10 complex showed a signal at m/z 637.3727
attributable to the protonated molecular ion [M + H]+ in the
ESI-MS spectrum (Figure S14 in SI). Based on the changes of
fluorescence intensity at the highest point of each titration curve
(528−545 nm), the association constant of the 3−G10 complex
in CH2Cl2 solution was determined to be (2.3 0.4) × 105 M−1,
which was close to the value of the 2−G10 complex. Thus,
receptors 2 and 3 exhibited an affinity ∼2.5-fold stronger than
that of 1 toward the G10 molecule, presumably because the
terminal OH or NH2 group in receptors 2 and 3 provided extra
hydrogen bondings. In CH2Cl2 solution, the affinity of 2 to 9-
decyladenine (A10) [Kass = (4.5 0.7) × 103 M−1] was 50-fold
weaker than that to G10, and the binding of 3 with A10 was too
weak to induce any apparent change in the UV−vis and
fluorescence titrations. The triply hydrogen-bonded mode of
acetamido naphthyridine receptors 1−3 with guanine accounted
for the selective binding of G10 over A10.
In comparison with the structure of 3, compound 4 had an
additional pyrene group at the terminal end of the aniline site.
The UV−vis spectrum of 4 in CH2Cl2 solution (Figure S21 in SI)
showed the absorption maximum at 397 nm for the
naphthyridine−ethyne−aniline chromophore. The fluorescence
emitted at 523 nm upon excitation at 400 nm. Such a large
Stokes’ shift (Δλ = 126 nm) was attributable to an ICT process
that occurred by delocalization of the n,π-electrons from the
electron-donating aniline group to the electron-withdrawing
idine−ethyne−aniline scaffold. The azido compound 10 was
reduced by Ph3P in H2O−THF to give the receptor molecule 3
that bears a terminal amino group for the subsequent elaboration
to compounds 4 and 5 by reductive amination with pyrene-1-
carbaldehyde and guanidination, respectively. Attempts to
remove the Fmoc groups from the guanidination product by
piperidine under conventional reaction conditions (CHCl3
solution, rt, 3 h) unexpectedly caused a concomitant
deacetylation of the 2-acetamido substituent. Fortunately, the
Fmoc protecting groups were selectively removed by treatment
with tetrabutylammonium fluoride (0.1 M solution in DMF) at
room temperature for 10 min,18 giving the desired receptor
molecule 5 in 90% yield.
The photophysical properties of receptor molecules 2−5 were
studied in various solvents, and the binding events were
monitored by UV−vis and fluorescence spectroscopy. Upon
titration of 2 with incremental amounts of G10 in CH2Cl2
solution (Figure 2), the absorption band at λmax 392 nm gradually
shifted to 404 nm. The fluorescence also showed a bathochromic
shift from 528 to 539 nm, albeit in lower intensity. The decreased
intensity was related to the electron transfer from decylguanine
and the energy loss in the relaxation process. A Job plot based on
the absorption changes in the titration curves was established to
reveal the formation of a 2−G10 complex in 1:1 stoichiometry
(Figure S10 in Supporting Information (SI)). According to the
nonlinear least-squares curve-fitting method,19 the association
constant (Kass) of the 2−G10 complex was determined to be (2.2
0.2) × 105 M−1 at 298 K in CH2Cl2 solution.
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naphthyridine center along the ethynyl bridge. Upon titration of
4 with incremental amounts of G10 in CH2Cl2 solution (Figure
S22 in SI, λex = 417 nm), the fluorescence maximum gradually
shifted to longer wavelengths with decreased intensity. A Job plot
analysis of the 550 nm emission indicated that receptor 4 and
G10 formed a complex in 1:1 stoichiometry (Figure S23 in SI). A
signal at m/z 851.4509 in the ESI-HRMS spectrum was ascribed
to the [M + H]+ ion of the 4−G10 complex (Figure S24 in SI).
The association constant of the 4−G10 complex in CH2Cl2
solution was determined to be (1.4 0.2) × 106 M−1, about 6-
fold stronger than that in the 3−G10 complex. In contrast, the
binding between 4 and A10 was very weak according to the
fluorescence titration experiment (Figure S25 in SI).
anisotropic effect of the pyrene ring also rendered the upfield
shifts of the C8 aromatic proton (Hc) and N9-CH2 (He) in G10.
A possible binding mode of the 4−G10 complex is depicted in
Figure 3 to address the multiple hydrogen bonds and π−π
stacking interactions. The greater binding strength of 4−G10
compared to that of 2−G10 and 3−G10 complexes might be
attributable to the additional hydrophobic20 and π−π stacking21
interactions between pyrene and the decylpurine moiety. As
pyrene is known to be a DNA intercalating agent,1,22 compound
4 may be explored to serve as a fluorescent probe for detection of
a guanosine mutation by simultaneous fluorescence changes
from pyrene and the conjugated naphthyridine system.
A guanidine group was introduced to the naphthyridine−
aniline molecule 5 to improve its water solubility and acquire
binding with the phosphate group in GMP. The fluorescence of 5
appeared at 445 nm in water by excitation at 350 nm. Upon
incremental additions of GMP to the aqueous solution of 5, the
intensity of fluorescence decreased in appreciable amounts
(Figure S29 in SI). By monitoring the intensity of fluorescence at
450 nm, the Job plot was established to show the formation of a
5−GMP complex in 1:1 stoichiometry (Figure S30 in SI).
According to the fluorescence titration, the binding constant of
the 5−GMP complex in water was calculated to be (1.6 0.3) ×
104 M−1. The binding mode of 5−GMP (Figure 4) is proposed
The 1H NMR titrations of 2 and 4 were undertaken in CDCl3
solutions to gain more information on their binding modes with
G10 (Figure 3). Upon incremental additions of G10, all of the
Figure 4. Illustration of the binding mode of the 5−GMP complex.
to emphasize the strong electrostatic interactions between the
guanidinium ion of receptor 5 and the phosphate ion of GMP in
an aqueous system. In contrast, adenosine 5′-monophosphate
(AMP) and 3′,5′-cyclic GMP (cGMP) did not provide effective
complexation with compound 5 in water (Figures S31 and S32 in
SI) because AMP could not provide sufficient hydrogen
bondings and cGMP lacked the cooperative electrostatic
interaction. This study reveals that the guanidine-hinged
molecule 5 is highly selective for GMP over cGMP and AMP
in aqueous solution. Compound 5 may be useful for monitoring
the activity of phosphodiesterase in the hydrolysis of cGMP to
GMP because it strongly binds with GMP but is insensitive to
cGMP.
1
Figure 3. H NMR titration of compounds 2 and 4 at 2 mM with
incremental additions of 9-decylguanine in CDCl3 solution. The
diagnostic protons a−f are marked.
protons on the naphthyridine and aniline rings of compound 2
gradually shifted to lower fields. The downfield shifts were
related to the ICT system with multiple hydrogen bonds
between 2 and guanine. In contrast, addition of G10 to 4 had a
distinct pattern that showed the acetyl protons (Hf at δ 2.24)
downfield shifted and the naphthyridine C3 proton (Ha at δ
8.23) upfield shifted. The pyrene protons (Hb) and its adjacent
methylene protons (Hd) at δ 4.59 also shifted upfield, indicating
the anisotropic effect exerted by the purine ring of G10 through
the π−π stacking interactions. In a reciprocal manner, the
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.6b00311.
Experimental procedures, NMR, UV−vis, fluorescence,
and ESI-MS spectra, and Job plots of synthetic compounds
and host−guest complexes (PDF)
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AUTHOR INFORMATION
Corresponding Author
■
*E-mail: jmfang@ntu.edu.tw.
Author Contributions
§S.-H.L. and R.P. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the Ministry of Science and Technology for financial
support.
■
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