Synthesis and photophysical evaluation of new fluorescent 7-arylethynyl-7-deazaadenosine analogs

Article abstract of DOI:10.1139/cjc-2018-0303

Three new fluorescent 7-deaza-2′-deoxyadenosine analogs were synthesized via the Sonogashira cross-coupling reaction of 7-iodo-7-deaza-2′-deoxyadenosine with 1-ethynylpyrene, 2-ethynyl-6-methoxynaphthalene, and 9-ethynylphenanthrene. The spectral properties of these analogs were evaluated in dioxane, EtOH, and H₂O to determine their potential for use as environmentally sensitive fluorescent probes. All three analogs displayed large solvatofluorochromicity in H₂O, relative to their emission wavelengths in dioxane or EtOH. Moreover, all three analogs exhibited microenvironmental sensitivity of their fluorescence emission intensity, being moderate to high quantum yields in dioxane and EtOH and significantly lower in H₂O. Various attempts to perform domino cross-coupling and annuation reactions on 7-deaza-7-alkynyladenine derivatives to form a new fused tricyclic adenine analog were unsuccessful.

Full text of DOI:10.1139/cjc-2018-0303

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Synthesis and Photophysical Evaluation of New Fluorescent 7-Arylethynyl-
7-Deazaadenosine Analogs
Augusto Matarazzo, Justin Brow and Robert H. E. Hudson*
Department of Chemistry, The University of Western Ontario, London ON, N6A 5B7, Canada
*Corresponding author, email: rhhudson@uwo.ca; tel: 519-661-2111, ext. 86349; fax: 519-661-
3022
Dedicated to the memory of Professor J. Peter Guthrie
1

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ABSTRACT
Three new fluorescent 7-deaza-2´-deoxyadenosine analogs were synthesized via the Sonogashira
cross-coupling reaction of 7-iodo-7-deaza-2´-deoxyadenosine with 1-ethynylpyrene, 2-ethynyl-6-
methoxynaphthalene, and 9-ethynylphenanthrene. The spectral properties of these analogs were
evaluated in dioxane, EtOH, and H2O to determine their potential for use as environmentally
sensitive fluorescent probes. All three analogs displayed large solvatofluorochromicity in H2O,
relative to their emission wavelengths in dioxane or EtOH. Moreover, all three analogs exhibited
microenvironmental sensitivity of their fluorescence emission intensity, being moderate to high
quantum yields in dioxane and EtOH and significantly lower in H2O. Various attempts to perform
domino cross coupling/annuation reactions on 7-deaza-7-alkynyladenine derivatives to form a new
fused tricyclic adenine analog were unsuccessful.
KEYWORDS
fluorescence, environmentally sensitive fluorescence, solvatochromicity, 7-deazaadenine,
Sonogashira cross-coupling
2

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INTRODUCTION
Fluorescent nucleosides and analogs have long interested chemists and biochemists
involved in the study of nucleic acids. Due to the relative scarcity of naturally occurring fluorescent
nucleosides, there has been increasing focus on the synthesis of analogs.1,2,3,4 2-Aminopurine
nucleoside, a highly fluorescent isomer of adenosine, has been one of the most utilized adenosine
analogs for the past forty years.5,2 However, this compound does have some limitations for
studying nucleic acids. For instance, 2-aminopurine nucleoside (2-AP), suffers dramatically
reduced emission once incorporated into single-stranded oligonucleotides and further yet upon
duplex formation.6,2 This leaves relatively weak emission for studying the structure and dynamics
of duplex species. Therefore recent developments have sought to overcome the limitations of 2-
AP.7
Although adenosine analogs may be substituted at various positions1 (Figure 1), our interest
is specifically focused on adenosines which are capable of Watson-Crick base pairing and are
substituted at the 7-position with alkynes that engender fluorescence. The benefit of substitution
at the 7-position of the 7-deazapurine is that large groups can be accommodated in the major
groove of resultant duplexes.8
INSERT Figure 1
Seela and co-workers have synthesized a library of fluorescent 7-deazaadenosine and the
related 7-deaza-8-aza-2’-deoxyadenosine analogs via attachment of various alkynes to the 7-
position via the Sonogashira reaction.9,10,11,12 Fluorescence quantum yields (F) in H2O of the
various 7-substituted-7-deazaadenosine analogs reported by Seela et al. range from relatively weak
3

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to moderate (F = 0.002 – 0.27).9-12 Although many of these modified nucleobases exhibit fairly
large Stoke shifts, a drawback associated with these fluorescent analogs is that their absorption
wavelength maxima range from 270 – 320 nm. The shorter wavelengths substantially overlaps
with the excitation wavelength of the natural nucleobases which implies that selective excitation
of the modified nucleobases may not be possible in all cases and there exists the hazard of
photochemical damage to the nucleic acids.
In 2004, Saito and co-workers developed a tethered pyrene adenosine analog, 7-deaza-7-
(1-pyrenecarboxamido)propyl-2’-deoxyadenosine (Apy).13 The authors did not report the
spectroscopic properties of the monomer, rather they showed that this fluorescent modification
exhibited
a
fluorescence quantum yield of 0.064 in the single strand 5’-
d(CGCAATApyTAACGC)-3’. The corresponding Apy-C, Apy-G and Apy-A mismatched duplexes
showed  F = 0.097, 0.098 and 0.054 respectively.13 Interestingly, the fluorescence quantum yield
of the fully complementary Apy-T duplex was significantly lower, F = 0.006, than the single
strand or the mismatched duplexes. The authors hypothesized that the observed fluorescence
quenching of the matched duplex was due to intercalation of pyrene into the duplex, which was
supported by molecular modelling studies.13
Later, in 2008, Hocek and co-workers reported on the synthesis and photophysical properties of 7-
deaza-2’-deoxyadenosine derivatives bearing bipyridine ligands and their Ru(II)-complexes.14,15
These Ru(II)-complexes showed rather weak red luminescence (MeCN, F = 0.021 - 3 x 10-4),14
but nonetheless one compound was transformed into the triphosphate and incorporated into
oligonucleotides that were used for SNP detection.15
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Saito and co-workers, in 2011, synthesized a novel push-pull fluorescent 7-deazaadenosine
analog based on an electron-withdrawing 4-cyanophenyl group (acceptor) and 7-deaza-2’-
deoxyadenosine (donor) bridged by a pyrene via rigid alkyne linkers.16 The nucleoside analog
showed strong emission in chloroform (ΦF = 0.448; Ex. = 416 nm,  Em. = 470 nm) and markedly
weaker emission 416 nm in DMF (ΦF = 0.089; Ex. = 416 nm,  Em. = 530 nm) but displayed
considerable solvatofluorochromicity (Δλ = 60 nm). More recently, Saito and co-workers
reported a structurally similar, but smaller, environmentally sensitive fluorescent 7-deaza-2’-
deoxyadenosine derivative possessing a 7-cyanonaphthylethyne substitution. The cyano-
naphthylethynylated 7-deaza-2’-deoxyadenosine exhibited large solvatochromatic properties
(CHCl3: Ex. = 345 nm,  Em. = 423 nm; MeCN: Ex. = 345 nm,  Em. = 494 nm), and, in the context
of oligonucleotide probes, signaled the difference between a perfectly matched complementary
strand versus a mismatch by a change in emission wavelength and intensity.17
Although there exists a variety of 7-deaza-7-substituted adenine fluorophores, at the time
of planning this work,18 there was still a need to develop brighter and more responsive probes for
use in nucleic acids research. Initially, we were interested in the synthesis of a novel fluorescent
tricyclic adenine analog by possibly extending the substrate scope of the Sonogashira cross-
coupling/heteroannulation chemistry that has been exploited in our group for cytosine analogs19 to
adenine analogs, Figure 2. Heteroannulation of nucleosides via the formation and reaction of 5-
pyrimidine- or 7-deazapurine-biaryl nucleoside adducts was first described by Matteucci and
coworkers. They demonstrated the formation of a carbazaole analog of 2’-deoxycytidine by an
intramolecular nucleophilic substitution on a suitably derivatized C4-position of a pyrimidine
5

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yielding a fused 5-membered ring. Matteucci’s work mainly focused on the base-pairing and helix
stabilizing properties of modified nucleosides once incorporated into oligonucleotides and not on
their photophysical properties. Much later, Hudson and Suchy recapitulated the synthesis of a
carbazole nucleobase analog and reported its bright blue, environmentally responsive
fluorescence.20 Following on their work with cytosine analogs, Matteucci showed that a similar
approach was feasible to produce tetracyclic adenine analogs.21 Since its first report, Wilhelmsson
and collaborators have extended the variety of fused aromatic ring adenine analogs greatly and
have shown that they are bright and tunable fluorophores and have employed them in FRET-based
probes.22,23,24,25 Given the structural similarity between the carbazole cytosine analog (a
benzofused pyrrolocytosine) and pyrrolocytosine (Figure 2b), which is obtained via Larock-type
cyclization26 of 5-alkynylcytosine derivatives, we reasoned that a tricyclic adenine analog derived
from the 7-deaza-7-alkynyladenine may possess useful and interesting fluorescence. This approach
was attractive because such adenine analogs are derived from readily available and diverse
alkynes.
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INSERT FIGURE 2
However, our efforts toward the annulation of 7-deaza-7-ethynyladenine analogs were
unsuccessful, yet nonetheless are described herein. The photophysical characterization of three
new 7-deazaadenosine analogs substituted at the 7-position with 9-ethynylphenanthrene, 2-
ethynyl-6-methoxynaphthalene and 1-ethynylpyrene are reported. The photophysical properties
of these analogs were evaluated in three different solvents, H2O, EtOH and 1,4-dioxane in order
to judge their microenvironment responsiveness.
EXPERIMENTAL
General experimental procedures
Reagents were commercially available and all solvents were HPLC grade except for water (18.2
MΩ•cm-1 millipore water) and DMF (dried over Al2O3 in a solvent purification system). Solvents
were removed under reduced pressure in a rotary evaporator, aqueous solutions were lyophilised
and organic extracts were dried over Na2SO4. Flash column chromatography (FCC) was carried
out using silica gel (SiO2), mesh size 230 - 400 Å and basic alumina (Al2O3), mesh size 80 - 200
Å. Thin-layer chromatography (TLC) was carried out on Al backed silica gel plate with
compounds visualised by I2 vapours, 5% ninhydrin stain, phosphomolybdic acid stain, and UV
light, as appropriate for the compounds under study. NMR spectra were recorded on either 600
1
MHz or 400 MHz spectrometers for H NMR spectra, as noted; δ values were referenced to
residual proton signal in the solvents as follows: CDCl3 (7.27 ppm); CD3OD (4.80, 3.30 ppm);
DMSO-D6 (2.49 ppm); D2O (4.75 ppm). Proton decoupled NMR for 13C were measured at 125
7

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MHz; chemical shift values (δ, ppm) were referenced to the solvent signals as follows: CDCl3
(77.0 ppm); CD3OD (49.0 ppm); DMSO-D6 (39.5 ppm). High resolution (HR) mass spectra (MS)
were obtained using electron impact (EI), chemical ionization (CI) or electrospray ionisation (ESI).
4-chloro-7H-pyrrolo[2,3-d]pyrimidine (1): 7H-pyrrolo[2,3-d]pyrimidin-4-ol (4.01 g, 29.7
mmol) was suspended in 45 mL of POCl3 and refluxed for 1 hour. The solution was cooled to
room temperature and excess POCl3 was removed by rotary evaporation. A mixture of
dichloromethane (600 mL) and ice water (200 mL) was then added to the dark-brown residue and
stirred for 24 hours, or until the dark-brown residue was completely dissolved. The aqueous layer
was then neutralized by adding increments of a saturated sodium bicarbonate solution (3 × 300
mL). The organic layer was collected, dried over MgSO4, and gravity filtered. The product was
1
dried by rotary evaporation, producing a pale yellow solid (2.35 g, 52%). H-NMR ( DMSO-d6,
400 MHz): δ = 12.57 (br s, 1 H), 8.59 (s, 1 H), 7.69 (d, J = 2.3 Hz, 1 H), 6.61 (d, J = 2.3 Hz, 1 H);
13C-NMR (DMSO-d6, 125 MHz): δ = 151.75, 150.41, 150.26, 128.35, 116.52, 98.76; HRMS (EI):
calcd. for C6H3ClN3[M]+ 153.0094, found 153.0090.
4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (2): Compound 1 (2.33 g, 15.2 mmol) was
dissolved in 54 mL of DMF, followed by the addition of N-iodosuccinimide (NIS, 3.58 g, 15.9
mmol). The reaction was stirred at room temperature for 2 hours. The solvent was removed by
rotary evaporation and 60 mL of ice water was added to the orange residue. The precipitate was
then collected by vacuum filtration, washed with ice water (3 × 15 mL), and concentrated in vacuo.
The product was obtained as a pale brown solid (3.75 g, 88%). 1H-NMR (DMSO-d6, 400 MHz): δ
13
= 12.94 (br s, 1 H), 8.59 (s, 1 H), 7.93 (s, 1 H). C-NMR (DMSO-d6, 125 MHz): δ = 151.95,
8

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151.18, 150.93, 134.31, 116.23, 52.12; HRMS (EI): calcd. for C6H3ClIN3[M]+ 278.9060, found
278.9052.
(2R,3S,5R)-5-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((4-
chlorobenzoyloxy)methyl)tetrahydrofuran-3-yl 4-chlorobenzoate (3): Compound 2 (1.00 g,
3.60 mmol) was dissolved in 28 mL of anhydrous acetonitrile, followed by the addition of sodium
hydride (0.15 g, 6.6 mmol). The reaction was stirred at room temperature under an atmosphere of
nitrogen for 30 minutes. Next, 1-chloro-2-deoxy-3,5-di-O-p-chlorobenzoyl-α-D-ribose (1.57 g,
3.6 mmol) was added to the reaction and the mixture was stirred at 50 °C for 2 hours. The solvent
was removed by rotary evaporation and the crude product was purified by flash chromatography
eluting with 2 % acetone in toluene to afford compound 3 as a pale yellow solid (1.36 g, 57%).
1H-NMR (CDCl3, 400 MHz): δ = 8.61 (s, 1H), 8.10 – 7.90 (m, 4H), 7.55 (s, 1H), 7.47 (m, 4H),
6.77 (t, J = 4.0 Hz, 1H), 5.76 (m, 1H), 4.80 – 4.50 (m, 3H), 2.90 – 2.70 (m, 2H). 13C-NMR (CDCl3,
125 MHz): δ = 165.0, 164.9, 152.8, 150.9, 150.4, 140.1, 139.9, 131.2, 131.0, 130.5, 128.9, 128.8,
127.5, 127.2, 117.4, 84.3, 82.3, 75.0, 63.9, 52.9, 37.9. HRMS (EI): calcd. for C25H17Cl3IN3O5[M]+
670.9278, found 670.9546.
(2R,3S,5R)-5-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-
(hydroxymethyl)tetrahydrofuran-3-ol (4): Compound 3 (0.55 g, 0.80 mmol) was added to a
thick-walled pressure vessel and suspended in 50 mL of methanol. The suspension was cooled to
0 oC followed by the addition of ammonia, which was bubbled into the suspension for 30 minutes.
o
The reaction vessel was tightly sealed and the reaction was stirred at 100 C for 24 hours. The
solvent was removed by rotary evaporation and the crude product was purified by flash
chromatography on SiO2, eluting with 10 % methanol in dichloromethane. The product was
9

Page 10 of 38
obtained as a white solid in 80 % yield. 1H NMR (DMSO-d6, 400 MHz): δ = 8.09 (s, 1H), 7.65 (s,
1H), 6.48 (m, 1H), 5.25 (d, J = 6.0 Hz, 1H), 5.02 (t, J = 6.0 Hz, 1H), 4.32 (m, 1H), 3.80 (m, 1H),
13
3.56 (m, 2H), 2.47 (m, 1H), 2.17 (m, 1H); C NMR (DMSO-d6, 125 MHz): δ = 157.3, 152.1,
150.0, 126.9, 103.3, 87.5, 83.1, 71.1, 62.0, 52.0, C(2’) overlapped with DMSO; HRMS (EI): m/z
calcd for C11H13 IN4O3: 376.0032; found: 376.0025.
7-(2-ethynyl-6-methoxynaphthalene)-7-deaza-2’-deoxyadenosine Amon (5): Compound 4 (85
mg, 0.20 mmol) was dissolved in dry, deoxygenated DMF (1.8 mL) to which CuI (8 mg, 0.042
mmol), Et3N (0.060 mL, 0.43 mmol), 2-ethynyl-6-methoxynaphthalene (163 mg, 0.80 mmol) and
tetrakis(triphenylphosphine) palladium (Pd(PPh3)4, 24 mg, 0.021 mmol) were sequentially added.
o
The reaction mixture was stirred in the dark at 60 C for 24 hours. The solvent was removed in
vacuo and the residue diluted with 15 mL of dichloromethane. The organic layer was washed with
3 % EDTA solution (3 x 5 mL) and brine (3 x 5 mL). The organic layer was dried and concentrated
in vacuo. The crude product was purified by FCC eluting with 10 % methanol in dichloromethane
to afford compound 5 in 33 % yield. 1H-NMR (DMSO-d6, 600 MHz): δ 8.16 (s, 1H), 8.12 (s, 1H),
7.89 (s, 1H), 7.90 -7.82 (m, 1H), 7.60 (dd, J1 = 12.0 Hz, J2 = 6.0 Hz, 1H), 7.55-7.37 (m, 1H), 7.22
(dd, J1 = 12.0 Hz, J2 = 6.0 Hz, 1H), 6.53 (m, 1H), 5.29 (m, 1H), 5.07 (t, J = 6.0 Hz, 1H), 4.37 (m,
1H), 3.88 (s, 3H), 3.83 (m, 1H), 3.59 (m, 1H), 3.57 (m, 1H), 2.23 (m, 1H); 13C NMR (DMSO-d6,
125 MHz): δ = 158.2, 157.7, 149.5, 138.5, 133.9, 129.4, 128.2, 119.5, 117.4, 102.2, 95.0, 91.8,
87.9, 82.7, 71.08, 62.0, 51.5, 50.0, C(2’) overlapped with DMSO; HRMS (EI): m/z calcd for
C24H22N4O4: 430.1641; found: 430.1636.
7-(9-ethynylphenanthrene)-7-deaza-2’-deoxyadenosine Aphen (6): Compound 4 (100 mg, 0.27
mmol) was dissolved in dry, deoxygenated DMF (2.0 mL) to which CuI (10 mg, 0.054 mmol),
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Et3N (0.075 mL, 0.52 mmol), 9-ethynylphenanthrene (215 mg, 1.08 mmol) and Pd(PPh3)4 (31 mg,
0.026 mmol) were sequentially added. The reaction mixture was stirred in the dark at 60 oC for 24
hours. The solvent was removed in vacuo and the residue diluted with 15 mL of dichloromethane.
The organic layer was washed with 3 % EDTA solution (3 x 5 mL) and brine (3 x 5 mL). The
organic layer was dried and concentrated in vacuo. The crude product was purified by FCC eluting
with 10 % methanol in dichloromethane to afford compound 6 in 55 % yield. 1H-NMR (DMSO-
d6, 600 MHz): δ 8.93-8.86 (m, 2H), 8.45 (m, 1H), 8.31 (s, 1H), 8.20 (s, 1H), 8.08 (s, 1H), 8.04 (d,
J = 6.0 Hz, 1H), 7.81-7.70 (m, 4H), 6.56 (m, 1H), 5.30 (d, J = 6.0 Hz, 1H), 5.10 (t, J = 6.0 Hz,
13
1H), 4.39 (m, 1H), 3.87 (m, 1H), 3.63 (m, 1H), 3.57 (m, 1H), 2.57 (m, 1H), 2.26 (m, 1H); C
NMR (DMSO-d6, 125 MHz): δ = 157.6, 152.8, 149.5, 132.3, 131.7, 131.4, 130.7, 130.5, 130.2,
129.6, 129.5, 128.5, 127.9, 127.5, 127.3, 122.9, 102.1, 94.6, 89.0, 87.6, 83.2, 70.9, 61.8, 30.7,
C(2’) overlapped with DMSO; HRMS (EI): m/z calcd for C27H22N4O3: 450.1692; found: 450.1685.
7-(1-ethynylpyrene)-7-deaza-2’-deoxyadenosine Apyr (7): Compound 4 (100 mg, 0.26 mmol)
was dissolved in dry, deoxygenated DMF (2.0 mL) to which CuI (10 mg, 0.052 mmol), Et3N
(0.075 mL, 0.52 mmol), 1-ethynylpyrene (240 mg, 1.04 mmol) and Pd(PPh3)4 (31 mg, 0.026
mmol) were sequentially added. The reaction mixture was stirred in the dark at 60 oC for 24 hours.
The solvent was removed in vacuo and the residue diluted with 15 mL of dichloromethane. The
organic layer was washed with 3 % ethylene diamine tetraacetic acid disodium salt solution
(EDTA, 3 x 5 mL) and brine (3 x 5 mL). The organic layer was dried and concentrated in vacuo.
The crude product was purified by FCC eluting with 10 % methanol in dichloromethane to afford
compound 7 in 33 % yield. 1H-NMR (DMSO-d6, 600 MHz): δ 8.59 (d, J = 12.0 Hz, 1H), 8.49 –
8.00 (m 10H), 6.58 (m, 1H), 5.31 (d, J = 6.0 Hz, 1H), 5.11 (t, J = 6.0 Hz, 1H), 4.41 (m, 1H), 3.88
13
(m, 1H), 3.75 - 3.50 (m, 2H), 2.58 (m, 1H), 2.27 (m, 1H). C-NMR (DMSO-d6, 125 MHz): δ
11

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157.7, 152.8, 149.5, 131.0, 130.8, 130.7, 130.4, 129.5, 128.7, 128.2, 127.3, 127.2, 126.3, 126.0,
125.9, 125.8, 124.7, 123.6, 123.4, 117.1, 102.1, 94.9, 89.9, 88.9, 87.6, 83.2, 70.9, 61.9, 54.9, C(2’)
overlapped with DMSO; HRMS (EI): m/z calcd for C29H22N4O3: 474.1692; found: 474.1697.
Methods of photophysical analysis
All photophysical measurements were made in 1 cm × 1 cm quartz cuvettes (ℓ = 1 cm)
using spectroscopic grade EtOH, dioxane, and H2O (18.2 MΩ•cm-1 millipore water). All
absorption experiments were carried out using a CARY 300 UV-Visible spectrophotometer and
all fluorescence experiments were carried out using a PTI-QM-4-SE spectrofluorometer. Molar
extinction coefficients (ε) of Amon, Aphen and Apyr were calculated from plots using Beer-Lambert’s
law (A = εcℓ) from five different solutions of known concentration. Error in the molar extinction
coefficients was calculated based on a 95% confidence interval. Due to insufficient solubility of
Amon, Aphen and Apyr in water, they were first dissolved in 0.20 mL of spectroscopic grade DMSO,
prior to dilution with water. However, after dilution [DMSO] < 1% and was considered to be
negligible.
The relative fluorescence quantum yield (ΦF) for Amon, Aphen and Apyr in EtOH, dioxane,
and H2O were calculated using five solutions of different concentrations for both the analogue and
its corresponding fluorescence standard. The samples used for analysis were diluted such that their
absorbance was below 0.1, in order to minimize re-absorption effects. Excitation was done at the
wavelength of maximum absorption for each analog in the corresponding solvent (Table 1).
Integrated fluorescence intensities were then plotted against the absorbance for each sample, for
both the analog and its corresponding fluorescence standard. The slopes of these two plots were
then used in conjunction with the known fluorescence quantum yield of the fluorescence standard
12

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(ΦSTD), in order to determine the fluorescence quantum yield of the compound of interest. These
calculations are based on the following equation:
푆푙표푝푒퐹
휂퐹2
eq. 1
Φ퐹 = Φ푆푇퐷
(
)
(
2
)
푆푙표푝푒푆푇퐷
휂푆푇퐷
In order to determine the fluorescence quantum yields of Amon, Aphen and Apyr the fluorescence
standard’s quantum yield in the solvent of interest must be known. However, there were no
established fluorescence quantum yields in the literature for some of the fluorescent standards in
the solvents of interest. Thus, the fluorescence quantum yields of these standards were measured
relative to a known fluorescence quantum yield of the same standard in a different solvent. The
difference in solvent was accounted for by including a ratio of the refractive indices in the
calculation, as seen in eq. 1. The fluorescence quantum yields that were used for analysis are
presented in Table S1. Quinine sulfate in 0.5 M H2SO4 was used as the fluorescent standard for
the determination of the quantum yields of Amon, Aphen and Apyr in H2O.
RESULTS and DISCUSSION
The syntheses of the 7-deaza-7-alkynyladenine derivatives began with chlorination of
commercially available 7-deaza-6-hydroxypurine using POCl3,27 to afford the desired compound
1 in reasonable yield (Scheme 1). Iodination of 1 at C7 was accomplished by treatment with NIS
in DMF to obtain compound 2 in high yield.28 Glycosylation was effected by deprotonation of 2
(NaH in acetonitrile) followed by treatment with 1-chloro-2-deoxy-3,5-di-O-p-chlorobenzoyl-α-
D-ribose29 to afford exclusively the β-anomer, compound 3, in 57 % yield. Conversion of the 6-
chloro to 6-amino group and removal of the ester protecting groups was accomplished in one step
13

Page 14 of 38
via treatment of compound 3 with a saturated NH3/MeOH solution30 to afford compound 4 in good
yield. Lastly compound 4 was reacted with various alkynes under Sonogashira conditions19 to
obtain the desired 7-substituted-7-deazaadenosine nucleosides 5 – 7, Scheme 1. No annulated
product was observed to accompany the formation of the cross-coupled product, which is a
departure from the chemistry displayed by the analogous cytosine system.
INSERT Scheme 1
PHOTOPHYSICAL EVALUATION
The spectral properties of Amon, Aphen and Apyr were evaluated in dioxane, ethanol, and
water in order to determine the influence of solvent polarity on the fluorescence properties. Based
on the solvent’s dielectric constant (ε), dioxane represents a non-polar, aprotic solvent (ε = 2),
EtOH represents a polar, protic solvent (ε = 24), and water represents a highly polar, protic solvent
(ε = 80).31 The results obtained are presented in Table 1 and selected excitation-emission profiles
are shown in Figure 3 and Figure S15-17.
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INSERT Table 1
INSERT Figure 3
All three adenosine analogs were found to have absorption bands a longer wavelengths than
the naturally occurring nucleobases that roughly correlated with the number of polyaromatic rings
such that Apyr had the most red-shifted absorption wavelengths (λab ~ 365 nm), followed by Aphen
(λab ~ 334 nm), and Amon (λab ~ 318 nm). Similarly, in the three solvents examined (dioxane,
ethanol, water), Apyr exhibited the most red-shifted fluorescence emission (λEm = 429, 432, 474
nm), followed by Aphen (λEm = 390, 392, 444 nm), and Amon (λEm = 370, 370, 435 nm).
Furthermore, the emission wavelengths in water for all three analogs were significantly red-shifted
compared to emission wavelengths in ethanol and dioxane. Thus, all three analogs show
solvatofluorochromicity in water, relative to ethanol and dioxane. This result suggests that highly
polar aqueous solvents stabilize the excited states to a greater extent than organic solvents of both
high and low polarity. In addition, this result also indicates that polyaromatic substituents
containing conjugated electron-donating groups do not enhance the solvatofluorochromicity of 7-
deaza-2´-deoxyadenosines, as was found with conjugated electron-withdrawing groups.17
Due to the stabilizing effect of solvent polarity on the excited states of fluorophores, all
three analogs had Stokes shifts that increased with solvent polarity. In addition, the Stokes shifts
were found to increase with the number of aromatic rings present, such that Apyr had the largest
Stokes shifts (62, 66, 110 nm), followed by Aphen (54, 60, 109 nm), and Amon (51, 52, 118 nm).
However, one interesting exception to this trend was that Amon displayed a larger Stokes shift in
water than both Apyr and Aphen. This effect is believed to be a direct consequence of the presence
15

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of the methoxy group, since fluorophores of higher polarity are more sensitive to the stabilizing
effects of polar solvents.32
Due to the high fluorescence quantum yield of pyrene (ΦF in ethanol = 0.65),33 Apyr was
expected to display a high fluorescence quantum yield, which it did (dioxane, ΦF = 0.52, and
ethanol, ΦF = 0.43). Conversely, Apyr had a dramatically reduced fluorescence quantum yield in
water, ΦF = 0.01. These large differences in quantum yield are attractive because they enable Apyr
to be used as an environmentally sensitive fluorescent probe across all three solvents. In addition,
when the molar extinction coefficients of Apyr are taken into consideration, its high brightness
values (16.9, 14.1, 0.2) indicate the potential of Apyr as a sensitive fluorescence probe. Similarly,
due to the moderate fluorescence quantum yield of phenanthrene (ΦF in ethanol = 0.13),32 Aphen
was expected to display useful fluorescence properties. Overall, Aphen displayed modest
fluorescence quantum yield differences across dioxane ΦF = 0.14, ethanol ΦF = 0.08, and water ΦF
= 0.02. Although it was expected that Aphen would display lower quantum yields than Apyr, it was
interesting to find that Aphen also displayed lower fluorescence quantum yields than Amon, despite
the inherent benefit of additional size of the aromatic system. These moderate fluorescence
quantum yields significantly impacted the brightness values determined for Aphen (2.3, 1.2, 0.1
×103 M-1 cm-1), despite having high molar absorptivity. Lastly, due to previous reports that
conjugated electron-donating groups can be used to increase fluorescence quantum yields, Amon
was expected to show a marked increase in quantum yields, relative to the 2-ethynylnaphthalene
derivative.13 Amon exhibited its largest fluorescence quantum yield in ethanol ΦF = 0.24, followed
by dioxane ΦF = 0.15, and water, ΦF = 0.01. These moderately high fluorescence quantum yields
had a positive effect on fluorophore brightness (2.0, 1.9, 0.04 ×103 M-1 cm-1), compensating for
the compound’s relatively low molar absorptivity. Thus, Amon displays fair potential for use as an
16

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environmentally sensitive fluorescent probe across all three solvents. In addition, the 2-
ethynylnaphthalene derivative was synthesized and photophysically characterized by Saito and co-
workers.13 The authors reported similar spectral characteristics to Amon in ethanol, λab = 318 nm,
λEm = 371 nm, however the fluorescence quantum yield in ethanol was significantly lower, ΦF =
<0.01.13 This result confirms the ability of conjugated electron-donating groups to enhance the
fluorescence quantum yield of 7-deaza-2´-deoxyadenosines. Overall, the fluorescence properties
of these derivatives is consonant with the emission from monomeric species in solution from
similarly derivatized purine nucleosides.34
Attempted cyclization of 7-alkynyl derivatives
Initially, the approach to this work was envisioned as employing sequential cross-
coupling/annulation sequences to access tricyclic adenine analogs from commercially available
diaminopyrimidine, Scheme 2. The attractive feature to this approach was the possibility that
diverse groups could be installed at both the C8 position of the purine (R1) and on the new C7-N6
fused ring (R2).
INSERT Scheme 2
Once it became clear that the nucleosides smoothly cross-coupled without further
cyclization, we prepared a nucleobase derivative (N9-methylene carboxyl, Scheme 3), that is more
robust to vigorous treatment due to the absence of an N-glycoside linkage, to serve as a substrate
to test a variety of reaction conditions. Furthermore, compounds of this type could be used in the
synthesis of peptide nucleic acids making the efforts for their preparation even more worthwhile.
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Commercially available diaminopyrimidine hemisulfate hydrate was iodinated, cross-
coupled and cyclized to yield 7-deazaadenine 10 according to a literature procedure.35 Iodination
was performed similarly as to 6-chloropurine, described earlier, and then alkylation at N9 by either
ethyl bromoacetate to give 12, or by t-butyl bromoacetate to give 15. The exocyclic amino group
of compound 12 was benzoylated giving 13, in order to study its effect on the ability to cyclize
after installation of the alkyne. Our previous studies on the annulation of 5-alkynyl cytosines
indicated that acylation of N4 facilitated the annulation reaction,19 although cyclization is possible
under more forcing conditions in the absence of acylation, see for example 9 → 10. Sonogashira
cross-coupling was performed on both 13 and 15 to give the 7-phenylalkynyl derivatives 14 and
16, respectively, which served as substrates subjected to various condition designed to promote
annulation, Figure 3.
INSERT Scheme 3
Initial attempts to promote the tandem Sonogashira/cyclization reaction on the adenine
scaffold included carrying out the reaction at higher temperatures (90 ºC or 120 ºC) while holding
all other reaction conditions constant. However, the increased temperatures did not promote the
cyclization reaction and only the straight cross-coupled product was formed. In all cases, the
isolated product showed the presence of alkyne carbons at  = 92.8 ppm (13C-NMR analysis) and
an alkyne stretch (ṽ = 2150 cm-1) in the IR spectrum confirming the lack of cyclization.
Since the problematic step was determined to be the annulation reaction rather than the
Sonogashira reaction, compound 14 was used as the substrate for the cyclization reaction using
greater amounts of CuI catalyst. At 30 mol % CuI, reacting for 24 h at 80 ºC, only 14 was
18

Page 19 of 38
o
recovered; however at 50 mol % CuI and allowing the reaction to proceed for 48 hours (80 C)
there was extensive decomposition of the material as determined by TLC analysis. Next, the
possibility of promoting the annulation reaction of substrate 16, that lacked the N6-benzoyl group,
in the presence of other alkynophilic metals, namely silver36,37 and gold38 under prolonged or
forcing conditions was briefly examined. Unfortunately, treating compound 16 with silver nitrate
in refluxing acetone for up to eleven days resulted in unreacted starting material. Similarly,
o
treatment of 16 with auric acid (HAuCl4) with heating (70 C) in ethanol for four days, with
periodic monitoring, resulted in nothing more than the t-butyl to ethyl transesterification product.
To aid in understanding the difficulty encountered in the cyclization, we turned to a
computational examination of the structures, specifically comparing the 5-alkynylcytosine
scaffold that successfully undergoes a 5-endo-dig reaction and the 7-alkynyl-7-deazaadenine
which resisted 6-endo-dig cyclization. In general terms, both of these reactions are predicted to
be favourable by Baldwin’s rules;39,40 however, the outcome depends on the length and nature of
the linking chain to permit the terminal atoms to achieve the required trajectory for ring formation.
Transition metal catalysis of alkyne reactions are well-established,41,42 especially by late transition
metal species. The coordination of a metal Lewis acid to the alkyne can promote nucleophilic
addition but distortion of alkyne geometry is not severe. For example, X-ray crystallographic
studies on a homoleptic series of monomeric Cu(I), Ag(I), and Au(I)-alkyne complexes revealed
modest deviation from linearity of the alkyne, thus implying in the present case that for a first
approximation that closing the distance of the terminal ends for ring formation by metal
coordination could be neglected.
19

Page 20 of 38
Using DFT methods (B3LYP/6-31+G**) implemented by Spartan14 (Figure 4), revealed
the distance between the exocyclic nitrogen and the alkyne carbon in the ground state structures.
The distance for the 7-deazaadenine substrate was calculated to be 3.84 Å, whereas the distance
between the exocyclic nitrogen and the alkyne carbon in the cytosine substrate was calculated to
be 3.64 Å. Perhaps the 0.2 Å greater distance between the nucleophile and electrophile in the 7-
deazaadenine substrate is the reason behind the difficulty in the annulation reaction, although
differences in the electronics of both systems may also influence the outcome.
INSERT Figure 4
A slightly less rigid system, N9-propargyl adenine was shown to undergo Ag(I) catalyzed
6-endo-dig cyclization with the N3 ring nitrogen,43 the main difference is the presence of a
methylene in the linker which permits the alkyne closer approach to the nucleophilic nitrogen. As
well, Furstner and Davis44 demonstrated the PtCl2 -catalyzed carboalkoxylation reaction of an o-
alkynylbenzoate giving the product of a 6-endo-dig cyclization in which all the linking atoms are
sp- or sp2-hybridized as is the case for the 7-deaza-7-alkynyladenine substrate; thus, all avenues
of investigation have not yet been exhausted.
CONCLUSIONS
Three new fluorescent 7-deaza-2´-deoxyadenosine derivatives connected to rigid,
polyaromatic chromophores via an ethyne bridging group were successfully synthesized
employing the Sonogashira cross-coupling reaction of 7-iodo-7-deaza-2´-deoxyadenosine (4) with
1-ethynylpyrene, 2-ethynyl-6-methoxynaphthalene, and 9-ethynylphenanthrene. These 7-deaza-
20

Page 21 of 38
2´-deoxyadenosine analogs were spectrally characterized in dioxane, EtOH, and H2O in order to
evaluate their potential for use as environmentally sensitive fluorescent probes.
Among them, Apyr showed the most promise, due to its red-shifted absorption wavelengths,
red-shifted emission wavelengths, large molar extinction coefficients, high fluorescence quantum
yields in dioxane and EtOH, and dramatic decrease in fluorescence quantum yield in H2O. Such
sizable differences in quantum yields and molar absorption coefficients allow for Apyr result in
relatively large brightness values. These characteristics, in conjunction with the benefit that large
substituents at the 7-position of the 7-deazapurine can be accommodated in the major groove of
resultant duplexes, suggests that Apyr has considerable potential for use as an environmentally
sensitive fluorescent probe. In addition, Amon and Aphen were found to display modest potential for
use as environmentally sensitive fluorescent probes. Both Amon and Aphen had red-shifted
absorption and emission wavelengths above those of the naturally occurring nucleobases.
However, the combination of lower quantum yields and lower molar absorptivity values result in
significantly lower brightness for these fluorophores. However, the fluorescence quantum yield
of the 2-ethynyl-6-methoxynaphthalene derivative (Amon) was found to be dramatically larger
(EtOH: ΦF = 0.24) than that of the 2-ethynylnaphthalene derivative (EtOH: ΦF = <0.01),13 thus
confirming the ability of polyaromatic substituents containing electron-donating groups to increase
the fluorescence quantum yields of 7-deaza-2´-deoxyadenosines.
The 7-alkynyl-7-deazaadenine analogs that were examined resisted undergoing annulation
reactions which are prevalent for 5-alkynylpyrimine derivatives. Reactions using archetypal
alkynophilic metals such Cu(I), Pd(0), Ag(I) or Au(III) to induce nucleophilic addition by the
exocyclic amino group failed to produce the desired products.
21

Page 22 of 38
ACKNOWLEDGEMENTS
We would like to thank the Natural Sciences and Engineering Research Council of Canada
(NSERC) for funding.
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Figure 1: 2´-deoxyadenosine with the heterocycle numbering scheme shown, its 7-deaza-7-
alkynyl-substituted analog.
Figure 2: Synthetic approaches to the annulation of nucleobase derivatives for cytosine
(left) and adenine (right): a) intramolecular nucleophilic aromatic substitution on biaryl
derivatives derived from Suzuki-Miyaura type reactions at the 5-position of pyrimindine
(left) or 7-position of 7-deazapurine (right); X = leaving group, b) 5-endo-dig
intramolecular cyclization of 5-alkynylcytosine (left) and the proposed 6-endo-dig of 7-
alkynyl-7-deazaadenine.
Figure 3: Excitation (blue) and emission (red) profiles of 5, Amon (left), 6, Aphen (middle)
and 7, Apyr (right) in EtOH.
Figure 4: Calculated equilibrium geometries of the N9- or N1-methyl 7-deazaadenine (left)
and cytosine (right) analog substrates for cyclization. The distance between the exocyclic
amine and distal alkyne carbon indicated by the double-headed arrow.
Scheme 1: Synthesis of 7-deaza-2’-deoxyadenosine analogs.
Scheme 2: Proposed approach to 7-deazaadenine analogs via sequential cross-coupling and
annulation reactions.
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Scheme 3: Synthesis of model compounds for cyclization attempts. See the supplemental
information for experimental details. Various conditions for the attempted annulation of
14 or 16 described in the text.
Table 1: Photophysical properties of fluorescent 7-deaza-2’-deoxyadenosines.
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Figure 1: 2´-deoxyadenosine with the heterocycle numbering scheme shown, its 7-deaza-7-alkynyl-
substituted analog.
110x46mm (300 x 300 DPI)