New class of 8-aryl-7-deazaguanine cell permeable fluorescent probes

Article abstract of DOI:10.1016/j.bmcl.2015.08.054

A one step synthesis of fluorescent 8-aryl-(7-deazaguanines) has been accomplished. Probes exhibit blue to green high quantum yield fluorescence in a variety of organic and aqueous solutions, high extinction coefficients, and large Stokes shifts often abo

Full text of DOI:10.1016/j.bmcl.2015.08.054

Bioorganic & Medicinal Chemistry Letters 25 (2015) 4593–4596
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New class of 8-aryl-7-deazaguanine cell permeable fluorescent
probes
b
a
a
Ilirian Dhimitruka a, , Timothy D. Eubank , Amy C. Gross , Valery V. Khramtsov
⇑
a Dorothy M. Davis Heart & Lung Research Institute, and Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, United States
b Department of Microbiology, Immunology, and Cell Biology, West Virginia University Health Sciences, Morgantown, VW 26506, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A one step synthesis of fluorescent 8-aryl-(7-deazaguanines) has been accomplished. Probes exhibit blue
to green high quantum yield fluorescence in a variety of organic and aqueous solutions, high extinction
coefficients, and large Stokes shifts often above 100 nm. The probes are highly cell permeable, and exhibit
stable bright fluorescence once intracellular; therefore are suited to the design of biosensors.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 27 July 2015
Accepted 20 August 2015
Available online 21 August 2015
Keywords:
8-Aryl-7-deazaguanine
Fluorescent probes
2,4-Diamino-6-hydroxypyrimidine
Phenacyl bromides
Cellular permeability
7-Deazaguanine, nomenclature name 2-amino-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-one, is an important modification of
nucleotide base guanine where N7 is replaced by carbon.1 Natu-
rally occurring 7-deazaguanines such as nucleotide Q, preQ, preQ0,
and archaeosine serve as substrates of TGT enzymes that mediate
their introduction into select tRNA of both eukaryotic and prokary-
otic cells in place of guanine.2 This tRNA modification is thought to
have significant impact on cellular proliferation, aging, and tumor
progression. Applications of synthetic 7-deazaguanines in nucleic
acid hybridization probes are important and well documented.3–5
Prominently chemotherapeutic drugs based on 7-deazaguanine
are capable of a multitude of interactions with intracellular
enzymes of the folic acid cycle that are indispensable to cellular
proliferation such as TS, DHFR, GARF, and cellular membrane trans-
(Scheme 1).15 We implemented the above protocol with minor
modifications. 8aryl7DGs were obtained as single products in high
yields by reacting equivalent molar amounts of commercially
available 2,4-diamino-6-hydroxypyrimidine
2 and substituted
phenacyl bromides 1(a–l). We found that using Et3N in 1,4-dioxane
instead of the usual sodium acetate in THF–water mixture led to
simple and efficient purification by precipitation in water. Due to
mild reaction conditions, a wide range of substrates is tolerated.
The SN2 reaction between nucleophilic C5 of pyrimidine 2 and
phenacyl bromides 1 determines the regiochemistry of the final
products, of which there was no ambiguity. Our spectral data were
compared, and are substantially different from, published data on
7aryl7DG regioisomers.16 This synthetic work adds to an already
rich literature on 7DG. There is interest in efficient syntheses of
8aryl7DG as intermediates of pharmacologically active lead com-
pounds.17 A similar synthesis of 8aryl7DGs was recently reported
in literature without any experimental or spectroscopic details.18
Our protocol was developed independently prior to this
publication.19
porters such as folate receptors (FRa
, FRb, RFC, PCFT).6–8 In light of
the importance of this motif in the biochemistry of living cells, we
investigated the design of fluorescent probes around 7-deazagua-
nine. In terms of precedent, fluorescent 8-aryl-7-deazaguanines
(
8aryl7DGs) were not described in literature. However, extension
of the
p
-system of guanine itself at the 8-position with various aro-
Three other 8aryl7DGs were produced from 3c, according to liter-
ature protocol, as shown in Scheme 2.16
matic motifs results in highly fluorescent probes.9 Therefore, we
hypothesized that 7-deazaguanine analogs would exhibit similar
fluorescence emission.10–14 Data presented here far exceeded our
expectations. Specific experimental procedures for the synthesis
of 8aryl7DGs were not published. Often 7-deazaguanines are
obtained using the addition–condensation reaction by Secrist III
8aryl7DGs are freely soluble in DMSO; therefore, samples were
prepared from DMSO stock solutions diluted in the appropriate
solvent to less than 2% DMSO. Fluorescence emission and absorp-
tion spectra were measured in DMSO, diluted aqueous solutions,
and 10% fetal bovine serum (FBS) in phosphate buffer, which are
most likely to be used in biological applications. Optical properties
are summarized Tables 1–3. Detailed spectra in various organic
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2015.08.054
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

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I. Dhimitruka et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4593–4596
O
N
H
O
O
Et3N, 1,4-dioxane
reflux 24-92 hrs
C7
R
H
NH
NH2
Br
C5 NH
+
R
N9
H
H2N
N
2
NH2
Aryl
3
1
O
O
O
N
R
NH
N
NH
NH
NH2
R
N
N
H
N
NH2
N
NH2
H
H
3j
3h R=OMe
3a R=H
3i R=NO2
3b R=phenyl
3c R=CN
3d R=Br
3e R=OMe
3f R=NO2
3g R=CF3
O
O
NH
NH2
NH
N
N
N
H
H
N
NH2
3l
3k
Scheme 1. One step syntheses of hydrophobic 8aryl7DG.
O
O
NH
NH2
NaOH, water-1,4-dioxane
reflux, >99 %
NH
HOOC
NC
N
N
N
N
NH2
H
H
3c
3m
O
N
O O
NH
NH
NH2
1. NMM, CDMT, H-Glu(OtBu)-OtBu
2. CF3COOH, 30 % overall
HO
HO
3m
N
H
3n
O
O
O
O
O
NH
NMM, CDMT
NH
NH2
+
3m
N
N
N
99 %
N
NH2
H
O
O
3o
Scheme 2. Syntheses of hydrophilic 8aryl7DG.
Table 1
Fluorescence properties,
e
(MÀ1 cmÀ1), k (nm), measured in DMSO
3a
3b
3c
3d
3e
3g
3h
3j
3k
3l
3m
3n
3o
kabs
kem
328
401
73
355
470
115
382
474
92
340
403
63
319
402
83
350
432
82
329
399
70
342
474
132
384
530
146
344
546
202
365
410
45
358
468
100
359
467
108
D
k
e
U
20,634
0.96
22,772
0.70
21,227
0.84
18,243
0.04
20,615
0.55
11,640
0.78
15,204
0.80
21,188
0.69
11,906
0.09
29,422
0.08
18,224
0.52
13,231
0.85
12,503
0.13
and aqueous solvents for each probe are included in SI. With the
exception of nitro substituted, all 8aryl7DGs exhibit above average
to excellent quantum yields in DMSO. All scenarios of fluorescence
modulation are observed: moderate changes in fluorescence in all
mediums (3b, 3h, 3j), several fold increase in fluorescence intensity
from DMSO to bovine serum solutions (3k, 3l), as well as the oppo-

I. Dhimitruka et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4593–4596
4595
Table 2
Fluorescence properties,
e
(MÀ1 cmÀ1), k (nm), measured in 10% FBS in phosphate buffer
3a
3b
3c
3d
3e
3g
3h
3j
3k
3l
3m
3n
3o
kabs
kem
318
385
77
343
431
88
354
466
112
326
405
79
310
381
71
336
413
77
316
390
86
337
436
99
378
496
118
339
504
165
341
455
114
343
487
144
344
484
140
D
k
e
U
16,696
0.12
18,940
0.40
19,093
0.19
15,764
0.03
19,592
0.017
9892
0.50
13,823
0.14
17,330
0.38
10,290
0.27
14,320
0.30
18,958
0.23
10,485
0.10
11,263
0.057
Table 3
Fluorescence properties,
e
(MÀ1 cmÀ1), k (nm), measured in distilled water
3aa
3b
3c
3d
3e
3g
3ha
3j
3k
3l
3m
3n
3o
kabs
kem
322
410
88
22,051
0.27
331
461
129
16,229
0.68
351
476
125
23,120
0.05
318
400
82
17,743
0.0015
306
403
97
19,403
0.006
327
416
89
10,842
0.32
325
415
80
14,278
0.55
329
482
153
17,525
0.21
—
—
—
—
—
—
—
—
—
—
336
466
110
19,110
0.18
342
491
149
12,829
0.05
340
509
169
11,405
0.006
D
k
e
U
a
Probes dissolved in 10 mM NaOH in distilled water.
site (3a, 3c, 3e). The probes can be thought of as refinements of
core 8Ph7DG (3a). Introducing methoxy group (3e, 3h) leads to high
quantum yield blue fluorescence. Introducing bromine leads to
poor fluorescence (3d). No change in kem ꢀ400 nm is observed
compared to 3a. Stokes shifts measure ꢀ80 nm (Fig. 1A). One can
fine-tune fluorescence properties compared to 3a to higher kem
ꢀ450–470 nm by extending the aryl system (3b, 3j) or introducing
groups such as CN (3c), CO2H (3m), and amide (3n). These probes
exhibit excellent green fluorescence, and large Stokes shift often
>100 nm (Fig. 1B). Attaching two known hydrocarbon fluorophores
such as pyrene and anthracene to 7DG results in probes 3k, 3l with
extraordinary large Stokes shifts in DMSO, 146 nm and 202 nm,
respectively (Table 1), and maximum kem ꢀ530 nm (Fig. 1C). All
probes exhibit equal or higher fluorescence emission intensities
in organic solvents such as DMF, CHCl3, acetonitrile, 1,4-dioxane
compared to DMSO (see SI, pg8). The highly hydrophobic 3k
and A549 lung cancer cells. The toxicity of the probes on both cell
lines is minimal at physiological levels of use. In repeated measure-
ments, we were unable to find an IC50 using the XTT assay in con-
centrations up to 20
l
M. Probes exited optimally at kex ꢀ350 nm
were imaged using a low resolution optical microscope equipped
with a DAPI fluorescence filter. Probe 3k (8pyr7DG) that is exited
optimally at kex = 405 nm was imaged using a high resolution con-
focal microscope. KB and A549 cells were stained at 37 °C, 5% CO2,
95% air, in RPMI 1640 growth medium containing probes at
1–20
l
M concentrations. No cellular uptake was observed for
M and 20 M concentra-
hydrophilic probes 3c, 3m and 3n at 5
l
l
tion. This result was expected based on sequestration by serum
proteins of probes, which are anionic under incubating conditions.
On the other hand, hydrophobic 3b, 3j, 3k, and 3l exhibited high
cellular uptake and stability inside cells. Perhaps most informative
are confocal microscopy images of cells stained with 1 lM solution
(
8pyr7DG) and 3l (8anthra7DG) exhibit a 2–4 fold increase in fluores-
of probe 3k (Fig. 2). Within minutes, intracellular fluorescence is
observed. Images were taken successively for 2 h. The complete
set of low and high resolution images is included in SI. Note that
probes remain largely cytosolic upon prolonged exposure with lit-
tle to no fluorescence observed in the nuclear compartments in low
and high resolution microscopy. This suggests receptor mediated
uptake as efflux mechanisms might be working in tandem with
free diffusion cellular uptake.
In conclusion, the diversity of the library examined suggests any
8-aryl-7-deazaguanine could be an above average fluorophore.
Bright fluorescence in the blue to green region and large Stokes
shifts are some of the qualities observed. It is important for future
biological applications that fluorescence properties are preserved
cence emission intensity in benzene, chloroform and 1,4-dioxane
compared to DMSO (Figs. SI-25 and SI-28). As expected, 8aryl7DGs
experience various degrees of fluorescence quenching in methanol
and aqueous solutions (SI, pg8). However, most probes exhibit ana-
lytically viable quantum yields UF >0.1 in 10% FBS solutions, and
often above average UF ꢀ0.2–0.5 (Table 2). Impressively, six out
of 13 8aryl7DGs retain high quantum yield fluorescence either in
pure water, or basic aqueous solutions, often in higher values com-
pared to serum (3a, 3b, and 3h). Fluorescence emission of all
probes is quenched in acidic solutions, except for probe 3b.
Next we proceeded to test the cellular permeability of 8aryl7DGs.
Two human cell lines were used: nasopharyngeal (KB) cancer cells
1.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
3g
3m
3k
3b
3c
3j
3n
3o
B
A
3h
3d
3e
3a
C
0.8
0.6
0.4
0.2
0.0
3l
200
300
400
λ (nm)
500
600
100
200
300
400
500
600
700
200
300
400
500
600
700
λ (nm)
λ (nm)
Figure 1. (A–C) Normalized fluorescence excitation (dashed lines) and emission spectra (solid lines) in DMSO.

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I. Dhimitruka et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4593–4596
cyanine dyes could lead to near infrared probes with kem
>600 nm which are better suited for optical spectroscopy and
imaging applications, in vivo. Hydrophobic 8aryl7DGs exhibited
excellent cellular permeability in low concentrations and no appar-
ent toxicity; therefore, are suited to the design of biosensors.
Acknowledgments
This work was supported by the Pulmonary Division of the
Department of Internal Medicine, the Ohio State University Medi-
cal College, and NIH Grant R03 EB016859. The authors thank Dr.
Sara Cole, assistant director of Campus Microscopy & Imaging
Facility (CMIF) of the Ohio State University.
Supplementary data
Supplementary data (synthetic procedures, NMR, HRMS, absor-
bance and fluorescence spectra) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2015.08.054.
References and notes
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in aqueous solutions, and in some instances enhanced in 10% FBS
in phosphate buffer. Functional groups introduced into the aryl
frame of 8Ph7DG (3a) have defined effects on fluorescence proper-
ties causing ꢀ80 nm redshift to kem ꢀ465–480 nm for phenyl, CN,
CO2H and amide groups, and kem ꢀ530 nm for larger aryl groups
such as pyrene or anthracene. Diversity-oriented synthesis of lar-
ger fluorophore libraries is possible because 8aryl7DG core is assem-
bled in one step from readily available starting materials. This
process will facilitate identification of probes for specific future
applications. Few fluorescent C8-arylguanine designs are used to
study the reaction of phenoxyl radical with DNA strands.11,20 C8-
arylguanines are reported to stabilize GG mismatches and to stabi-
lize the transition from B-DNA to Z-DNA as well.21,22 Therefore,
8aryl7DGs being isosteric might find immediate applications. It is
well known that groups appended to the 7-position of 7-deazagua-
nine are known to accommodate better within the major groove of
the DNA duplexes.3 Thus, designing an analogous fluorescent
7aryl7DG series to complement the available 8aryl7DG series is a
priority. Importantly, there is room for improving fluorescence
properties. Introducing polyene bridges into 7-deazaguanine,
and/or heterocyclic chromophores in designs which are similar to
22. Gannett, P. M.; Heavner, S.; Daft, J. R.; Shaughnessy, K. H.; Epperson, J. D.;
Greenbaum, N. L. Chem. Res. Toxicol. 2003, 16, 1385.