Convergent synthesis and properties of photoactivable NADPH mimics targeting nitric oxide synthases

Article abstract of DOI:10.1039/c6ob01533f

A new series of photoactivable NADPH mimics bearing one or two O-carboxymethyl groups on the adenosine moiety have been readily synthesized using click chemistry. These compounds display interesting one- or two-photon absorption properties. Their fluorescence emission wavelength and quantum yields (Φ) are dependent on the solvent polarity, with a red-shift in a more polar environment (λ_max,em = 460-467 nm, Φ > 0.53 in DMSO, and λ_max,em = 475-491 nm, Φ < 0.17 in Tris). These compounds show good binding affinity towards the constitutive nNOS and eNOS, confirming for the first time that the carboxymethyl group can be used as a surrogate of phosphate. Two-photon fluorescence imaging of nanotriggers in living cells showed that the presence of one carboxymethyl group (especially on the 3′ position of the ribose) strongly favors the addressing of nanotriggers to eNOS in the cell context.

Full text of DOI:10.1039/c6ob01533f

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Convergent synthesis and properties of
photoactivable NADPH mimics targeting
Cite this: DOI: 10.1039/c6ob01533f
nitric oxide synthases†
N.-H. Nguyen,a N. Bogliotti,a R. Chennoufi,b E. Henry,b P. Tauc,b E. Salas,c
L. J. Roman,c A. Slama-Schwok,d E. Deprezb and J. Xie*a
A new series of photoactivable NADPH mimics bearing one or two O-carboxymethyl groups on the
adenosine moiety have been readily synthesized using click chemistry. These compounds display interest-
ing one- or two-photon absorption properties. Their fluorescence emission wavelength and quantum
yields (Φ) are dependent on the solvent polarity, with a red-shift in a more polar environment (λmax,em
=
460–467 nm, Φ > 0.53 in DMSO, and λmax,em = 475–491 nm, Φ < 0.17 in Tris). These compounds show
good binding affinity towards the constitutive nNOS and eNOS, confirming for the first time that the
carboxymethyl group can be used as a surrogate of phosphate. Two-photon fluorescence imaging of
nanotriggers in living cells showed that the presence of one carboxymethyl group (especially on the 3’
position of the ribose) strongly favors the addressing of nanotriggers to eNOS in the cell context.
Received 18th July 2016,
Accepted 9th September 2016
DOI: 10.1039/c6ob01533f
www.rsc.org/obc
reductase domain with binding sites for the flavins and
NADPH, connected by a linker containing a Ca2+/calmodulin
Introduction
Nitric oxide (NO) is involved in numerous biological processes, binding site.2 The biological effect of NO is highly dependent
from neurotransmission, hormone secretion, platelet aggrega- on its concentration, site and dosage.3 Various strategies have
tion and adhesion to vasodilation, the immune response, been developed for therapeutic proposes based on lowering
infection and cancer.1 NO synthase (NOS) is a heme- and (through inhibition of NOS by targeting the oxygenase or the
flavin-containing enzyme that catalyzes the synthesis of NO reductase domain)4 or enhancing the concentration of NO
through two serial monooxygenase reactions. Electrons are (with NO-donors),5 or by combining NO-donors with drugs.6
transferred from NADPH, through the flavins FAD and FMN, A light-activated reaction should allow a spatiotemporal modu-
to the heme iron, where molecular oxygen is activated, allow- lation of NO concentration. In this context, both photoactive
ing formation of NO and L-citrulline from L-arginine. There are NO donors7 and caged NOS inhibitors8 have been recently
three isoforms in mammals: neuronal NOS (nNOS) and endo- developed for various biomedical applications.
thelial NOS (eNOS) which are constitutive and modulated by
Directly targeting the NADPH binding site of NOS with a
intracellular Ca2+/calmodulin (CaM), and inducible NOS NADPH analog whose redox potential can be modulated upon
(iNOS). nNOS and eNOS control neuron function and blood light absorption is another way to photocontrol the biosynthesis
pressure, respectively, while iNOS is associated with the of NO. Replacing the nicotinamide moiety of NADPH by dienyl
immune response. NOSs are functional dimers, with each chromophore substituted by two donor groups had led to a
monomer containing an N-terminal oxygenase domain with photoactivable molecule (nanotrigger NT1 Fig. 1) designed to
binding sites for arginine and tetrahydrobiopterin, and a fit in the NADPH site of nNOS that is triggered by one- or two-
photon excitation to donate electrons synchronously to nNOS
or eNOS, resulting in NO formation.9 By linking the adenosine
nucleotide to a push–pull 4-(4-nitrostyryl)anilinyl chromo-
phore, which is unable to provide the electrons required for
NOS catalysis, a NOS inhibitor (nanoshutter NS1, Fig. 1) has
been generated that also imaged eNOS upon two-photon exci-
tation.10 These interesting results led to the elaboration of a
straightforward route for the synthesis of new suitable nano-
triggers for time-resolved studies of the NOS catalytic mechan-
ism. CuI-catalyzed Huisgen 1,3-dipolar cycloaddition (CuAAC)
aPPSM, ENS Cachan, CNRS, Université Paris-Saclay, Cachan, 94235 France.
E-mail: joanne.xie@ens-cachan.fr
bLBPA, ENS Cachan, CNRS, Université Paris-Saclay, Cachan, 94235 France
cDepartment of Biochemistry, University of Texas Health Science Center, San
Antonio, Texas 78384-7760, USA
dUniversité Paris Saclay, INRA UR 892, Jouy en Josas, 78350, France
†Electronic supplementary information (ESI) available: Absorption and fluo-
rescence titration spectra, immuno-staining of eNOS on fixed cells, 1H and 13C
NMR spectra of all compounds. See DOI: 10.1039/c6ob01533f
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Scheme 1 Synthesis of chromophores 13 and 14. (1) Propargyl
bromide, K2CO3, MeCN, 95% for 8; 52% for 14 (from 19). (2) POCl3, DMF,
90 °C, 82%. (3) i. tributyl(1,3-dioxolan-2-ylmethyl)phosphonium
bromide, NaH, THF, 0 °C to r.t.; ii. AcOH–H2O, CH2Cl2, 60% 10, 20% 11.
(4) NaH, THF, 45%. (5) MeI, NaH, THF, 94%. (6) Boc2O, THF, reflux, 90%.
(7) NaH, THF, 56%. (8) MeI, NaH, THF, 90%. (9) NaOH, EtOH, reflux, 76%.
Fig. 1 Structure of NADPH and its photoactivable mimics.
has been proven versatile for the generation of libraries of bio-
active compounds including nucleoside and nucleotide deriva-
tives,11 as the formed 1,2,3-triazole ring is resistant to
hydrolysis, oxidation and reduction. We have designed a novel
generation of photoactivable NADPH mimics with three modi-
fications: (a), replacement of the 2′-phosphate by a carboxy-
methyl group on the 2′-, 3′- or both the 2′- and 3′-positions to
determine whether the carboxymethyl group could be used as
a surrogate for phosphate and to study the influence of the
substitution position on its recognition by NOS, since the
phosphate group on the 2′-position of NADPH is in equili-
brium with 3′-O-phosphate in solution; (b), introduction of a
methyl group on the terminal amino function to investigate its
impact on NT photophysical properties in comparison with
the amine as a donor group; (c), use a triazole for linking the
photoactive chromophore moiety with the adenosine moiety
targeting the NADPH recognition site (Fig. 1).
Scheme 2 Synthesis of photoactivable NADPH mimics 1–7.
(1) BrCH2CO2tBu, NaH, DMF, −45 °C to r.t. (2) CuSO4·5H2O, Na ascor-
bate, DMF–H2O. (3) AcCl, MeOH.
Results and discussion
Synthesis of photoactivable NADPH mimics
The second generation of nanotriggers was then synthesized 22–24 (Scheme 2). The chromophore 13 was prepared from the
by CuAAC between the alkyne-functionalized dienyl chromo- N-methylaniline, through N-propargylation and formylation
phore 13 or 14 (Scheme 1) and 5′-azido adenosine derivatives reactions to afford aldehyde 9 (Scheme 1). Wittig reaction of 9
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with tributyl[(1,3-dioxolan-2-yl)methyl]phosphonium bromide
gave, after mild acidic hydrolysis, the desired α,β-unsaturated
aldehyde 10 (60%) as well as the depropargylated compound
11 (20%) which might be formed by hydrolysis of ynamine, the
rearrangement product of N-propargyl amine in the presence
of NaH.12 Horner–Emmons–Wadsworth reaction of aldehyde
10 with the phosphonate 12 afforded the diene 13 which was
N-methylated to give the compound 14. The diene 14 could
also be prepared from the aldehyde 11 through N-Boc protec-
tion to α,β-unsaturated aldehyde 15 followed by reaction with
the phosphonate 16, N-methylation and removal of benzoyl
group. The Boc-protecting group in 13 could be readily
removed by AcCl in methanol to deprotected dienyl chromo-
phore 20. O-Alkylation of 5′-azido adenosine 2113 with NaH
and tert-butyl α-bromoacetate led to a mixture of O- and
N-alkylated products 22–26, depending on the quantity of
reagents (Scheme 2). Optimization with 1.1 equiv. NaH and
1 equiv. BrCH2CO2tBu gave 22 (18%), 23 (18%) and 24 (6%),
separable by column chromatography. The position of
O-alkylation in 23 and 24 has been determined by comparison
with 13C NMR data in the literature.14,15 Then, click reaction of
alkyne 13 or 14 with azido adenosines 21–24 afforded com-
pounds 27–33 in good yields. The target compounds 1–7 were
readily obtained after acidic deprotection.
Fig. 2 Representative fluorescence emission and two-photon exci-
tation spectra of compound 3 (20 μM). Top, fluorescence emission
spectra in Tris buffer (grey) or DMSO (black). Excitation wavelengths
were 370 and 390 nm in Tris and DMSO, respectively. Excitation and
emission slits were 5 nm and the PMT (photomultiplier tube) voltage
was 600 V. Bottom, two-photon excitation spectrum in DMSO (laser
power, 50 mW).
Photophysical studies
The absorbance and fluorescence properties of compounds
1–7 were next characterized in two different solvents: DMSO
and Tris buffer (20 mM, pH 7.4). All NTs show similar absorp-
tion (Fig. S1†) and emission spectra (Fig. 2), and the main
parameters are summarized in Table 1. For all NTs, the
maximum wavelength for one-photon absorption (λmax,abs) was
found to be strongly dependent on the solvent condition, with
a systematic red-shifted λmax,abs in DMSO compared to Tris
(about 20 nm). Moreover, in each solvent, NTs with the elec-
tron donating group NHMe (compounds 1, 3, 5) are character-
ized by a slight but significant red-shifted λmax,abs compared to
other NT compounds having the electron donating group NH2
(compounds 2, 4, 6, 7), with λmax,abs = 391–392 nm and
387–388 nm, respectively, in DMSO (370–372 nm and
367–368 nm, respectively, in Tris). This is consistent with the
expected lower HOMO–LUMO energy gap with NHMe as a
better electron donating group than NH2. Regarding the molar
extinction coefficients (ε), systematically higher ε values are
observed in Tris as compared to DMSO for compounds 1 and 3
while the opposite tendency is noticed for compounds 2, 4, 6
and 7, suggesting a differential interaction between the elec-
tron donor group and the solvent. To note, it has previously
been shown that the electron accepting nitro group of NS1 at
this terminal position is involved in probing solvation in its
41–68 GM range in DMSO) of NTs remain compatible with the
characteristic values found for NS110 or NT1.9 The
fluorescence emission wavelength (λmax,em) was dependent on
the solvent with an expected red-shift in a more polar environ-
ment (λmax,em = 460–467 nm in DMSO depending on the com-
pound and 475–491 nm in Tris). For all NT compounds, the
fluorescence quantum yields (Φ) were dramatically different in
Tris buffer and DMSO (Φ < 0.17 and Φ > 0.53, respectively),
suggesting that the binding of NTs to NOS should be associ-
ated with a substantial NT fluorescence enhancement (con-
firmed thereafter). As found for one-photon absorption
parameters, fluorescence emission of the NTs is dependent
on the nature of the electron donating group, especially their
Φ values, with the NHMe group leading to higher Φ values
than NH2 (Φ = 0.84–0.91 and 0.53–0.79, respectively, in
DMSO).
Binding assay
direct vicinity.10 The influence of the solvent or the electron The apparent binding affinities of the NTs with nNOS and
donating group was much less pronounced on the optimal eNOS were evaluated by the increase in the NT intrinsic fluo-
wavelength for two-photon absorption (λmax,2-PE = 750 nm, rescence at 460 nm (excitation 420 nm) that was blue shifted
regardless of the compound or the solvent), with however a upon binding to protein, as previously reported with NT19d
higher two-photon absorption cross-section (σ2) in DMSO com- (Fig. S2,† Table 2).16 Compounds 1 to 6 bound to both nNOS
pared to Tris. Except for compound 7, the σ2 values (in the and eNOS, showing slightly greater affinity to eNOS over nNOS.
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Table 2 App Kd values in μM ( standard error) for 1–7 with nNOS and
eNOS
nNOS
eNOS
1
2
3
4
6
7
38.3 3.1
21.5 0.7
26.8 2.1
32.6 2.7
30.0 3.8
58.0 14.5
19.5 1.1
14.7 1.1
19.5 1.1
28.6 4.6
24.1 1.9
>80
NT19a
NS110
7
3
4.3 0.6
Compound 1 bearing two carboxymethyl groups shows the
greatest selectivity for eNOS over nNOS. Although the binding
affinities of compounds 1–6 were lower than those of NT1 or
NS1, these results confirmed that the carboxymethyl group
could be used as a surrogate of phosphate. Compound 7
without carboxymethyl group bound poorly to nNOS and very
poorly to eNOS. Furthermore, no significant difference exists
between mono- or di-O-carboxymethyl compounds (3,4,6 vs.
1,2), between 2- or 3-O-carboxymethyl derivatives (6 vs. 3,4),
and amino- or N-methylamino derivatives (2,4,6 vs. 1,3).
Nevertheless, an effect of charge on binding to eNOS is
observed in that the apparent affinity of compounds 2 > 4 ≈
6 ≫ 7.
Cellular imaging studies
Thanks to relatively good σ2 and Φ, the two-photon fluo-
rescence brightnesses of the 2nd-generation NTs with values
spanning from 15 to 54 GM are compatible with imaging
studies using multiphoton microscopy. Taking into account
the significant fluorescence enhancement of NT compounds
when they are bound to eNOS, we next addressed the
question of NT fluorescence brightness in the cell context.
Two-photon fluorescence imaging of NTs in living cells was
then performed in two distinct cell lines, endothelial HUVEC
cells (Human Umbilical Vein Endothelial Cells) and HeLa
cells (cervical, cancer) that express and do not express eNOS,
respectively.17–20 In a previous study, we confirmed that
HUVECs efficiently express eNOS by using immuno-staining
of eNOS.10 Here, by using the same immuno-staining pro-
cedure, we confirmed that no expression of eNOS occurs in
HeLa cells (Fig. S3†). HUVEC and HeLa cells were incubated
with the different NT compounds for varying times and
imaged using λex = 760 nm (Fig. 3). No detectable NT fluo-
rescence was obtained in HeLa cells whereas, in sharp con-
trast,
a two-photon emission is observed in HUVECs,
however to different extent depending on the compound.
Among the different compounds, 3 and 4 displayed the
highest intracellular fluorescence intensities in HUVECs and
were characterized by a rapid cellular uptake (less than
5 min). Weaker fluorescence intensities were detected with 1
and 7 and very weak or no signal was detected with 2 and 6.
This hierarchy (3,4 ≫ 1,7 > 2,6) is clearly different than the
hierarchy established for two-photon brightness values as
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Fig. 3 Two-photon fluorescence images of NT compounds (1–4, 6, 7) in living cells. HUVEC (A) or HeLa (B) cells were treated for varying times
with NT compounds (final concentration, 5 μM). Compound 5 was not studied for stability reason (see text). Imaging was performed at fixed time
intervals (explicitly mentioned below the images). Excitation was 760 nm and the emission slit setting was 450–550 nm. Only fluorescence images
corresponding to t = 120 min are shown for HeLa cells and only DIC (differential interference contrast) transmission images corresponding to t =
120 min are shown for HUVEC and HeLa cells.
derived from in vitro experiments (2 > 3 > 1 > 6 > 4 > 7 in 3′ position of the ribose, while no detectable NT fluorescence
Table 1). Note that compound 5 was discarded from the was obtained in HeLa cells.
imaging study because of its particular chemical instability
in culture medium. The differential fluorescence emission in
HUVEC and HeLa cells correlates well with the expression
level of eNOS. The imaging study shows that the presence of
one carboxymethyl group (especially on the 3′ position of the
Experimental
General
ribose as in compounds 3 and 4) strongly favors the addres- All commercial available reagents purchased from Sigma-
sing of NTs to eNOS in the cell context. Finally, the similar Aldrich, TCI Chemical and Carlo-Erba were used without
behavior of compounds 3 and 4 indicates that the nature of further purification. Column chromatography was performed
the electron donating group on the chromophore (NH2 vs. on Silica gel 60 (40–60 μm). The solvents for column chrom-
NHMe) does not play an important role for eNOS addressing. atography were used without purification. The reactions
carried out under anhydrous conditions are performed under
argon in glassware previously dried in an oven. Methanol is
dried over molecular sieve 3 Å. THF, DMF, dichloromethane,
acetonitrile and toluene were previously dried through
Conclusions
alumina cartridge using
a solvent purificator MBRAUN
In summary, we have synthesized a series of photoactive SPS-800. Reactions were monitored by TLC on Silica Gel
NADPH mimics bearing one or two O-carboxymethyl groups 60F-254 plates with detection by UV (254 nm or 365 nm) or
on the adenosine moiety. These compounds display good by spraying with 10% H2SO4 in EtOH and heating about 30 s
binding affinity towards the constitutive nNOS and eNOS, con- at 400–600 °C. Melting points were determined with a Kofler
firming that the carboxymethyl group can be used as a surro- melting point apparatus. Optical rotations were measured
gate of phosphate. Two-photon fluorescence imaging of NTs in using a Jasco P-2000 polarmeter at room temperature in a
living cells showed a two-photon emission in HUVECs, with 10 cm, 1 mL cell. NMR spectra were recorded on a JEOL
the highest intracellular fluorescence intensities displayed by ESC-400 spectrometer in CDCl3, CD3OD or DMSO-d6 solution.
compounds 3 and 4 bearing one carboxymethyl group on the Chemical shift was given in units of parts per millon related
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to TMS or solvent protons as internal reference. High-resolu- 600 mg, 15.0 mmol). After one night reaction at r.t., the reac-
tion mass Spectra (HRMS) were recorded on a Q-TOF MaXis tion mixture was neutralized with a saturated solution of
using standard conditions or Bruker Microflex™ MALDI-TOF NH4Cl. After solvent evaporation, the residue was extraited
mass spectrometry.
with CH2Cl2 (3 × 100 mL). The combined organic layers were
dried over MgSO4, filtered and concentrated. The obtained
acetal was then dissolved in CH2Cl2 (25 mL) and treated with
General procedure for the deprotection of Boc
AcCl (40 equiv.) was added slowly to MeOH at 0 °C under Ar. a mixture of CH3CO2H/H2O (0.15 mL/0.75 mL) during 2 days
After stirring 5 min at 0 °C, the Boc-protected substrate was at r.t., neutralised with a saturated solution of NaHCO3. After
added. The solvent was evapored and the residue co-evapored solvent evaporation, the residue was extracted with AcOEt
with MeOH (3×) after completion of reaction (2 h).
(3 × 100 mL). The combined organic layers were dried over
MgSO4, filtered, evaporated and purified by column chrom-
atography over silica gel (CH2Cl2/AcOEt: 10/0.1) to afford the
N-Methyl-N-(prop-2-yn-1-yl)aniline (8)
To a une solution of N-methylaniline (1.66 mL, 15.2 mmol) in title compound 10 as an orange solid (598 mg; 60%) and the
acetonitrile (30 mL) at 0 °C under Ar, were added K2CO3 depropargylated compound 11 as
a red brown powder
(3.154 g, 22.8 mmol) and propargyl bromide (80% in toluene, (173 mg; 20%).
2.54 mL, 22.8 mmol) drop by drop. After one night reaction at
Compound 10: Rf = 0.39 (CH2Cl2/AcOEt: 10/0.1); mp:
r.t., the mixture was filtrated on silica gel and the solvent eva- 89–91 °C; IR (ATR): 3318, 1644, 1582, 1530, 1388, 1138, 958,
pored under reduced pressure. The residue was then purified 805 cm−1; RMN 1H (CDCl3, 400 MHz): δ 9.60 (d, 1H, J =
by chromatography on silica gel (petroleum ether/AcOEt: 50/1) 7.8 Hz, CHO), 7.49–7.47 (m, 2Har), 7.39 (d, 1H, J = 15.6 Hz,
to afford the title compound as an orange oil (2.101 g, 95%). CHv), 6.82–6.80 (m, 2Har), 6.57 (dd, 1H, J = 7.8, 15.6 Hz,
Rf = 0.30 (petroleum ether/AcOEt: 50/1); IR (ATR): 3289, 1675, CHv), 4.11 (d, 2H, J = 2.3 Hz, CH2), 3.07 (s, 3H, CH3), 2.24 (t,
1599, 1504, 1337, 1243, 1199, 1114, 997, 754, 691 cm−1; RMN 1H, J = 2.3 Hz, CHu); RMN 13C (CDCl3, 100 MHz): δ 193.9
1H (CDCl3, 400 MHz): δ 7.29–7.25 (m, 2Har), 6.88–6.80 (m, (CHO), 153.6 (CH), 151.1 (Cq), 130.5 (CHar), 124.8 (CH), 123.5
3Har), 4.05 (d, 2H, J = 2.3 Hz, CH2), 2.98 (s, 3H, CH3), 2.17 (t, (Cq), 113.3 (CHar), 78.6 (Cq), 72.5 (CH), 41.9 (CH2), 38.5 (CH3);
1H, J = 2.3 Hz, CHu); RMN 13C (CDCl3, 100 MHz): δ 149.0 HRMS (ESI) m/z: Calcd for C13H14NO [M + H]+: 200.1075;
(Cq), 129.2 (CHar), 118.4, 114.3 (CHar); 79.4 (Cq), 72.1 (CH), Found: 200.1068.
42.5 (CH2), 38.6 (CH3).
Compound 11: Rf = 0.27 (CH2Cl2/AcOEt: 10/0.1); mp:
111–113 °C; IR (ATR): 3215, 1644, 1582, 1519, 1378, 1327,
4-[N-Methyl-N-(prop-2-yn-1-yl)amino]benzaldehyde (9)
1
1135, 927, 806, 730 cm−1; RMN H (CDCl3, 400 MHz): δ 9.59
To dry DMF (150 mL) under Ar at 0 °C, was added dropwise (d, 1H, J = 7.8 Hz, CHO), 7.43–7.41 (m, 2Har), 7.37 (d, 1H, J =
POCl3 (4.10 mL, 43.9 mmol). After 10 min stirring at 0 °C and 15.6 Hz, CHv), 6.60–6.58 (m, 2Har), 6.54 (dd, 1H, J = 15.6,
15 min at r.t., a solution of 8 (6.371 g, 43.88 mmol) in dry 7.8 Hz, CHv), 2.90 (s, 3H, CH3); RMN 13C (CDCl3, 100 MHz):
DMF (10 mL) was added dropwise at 0 °C. The reaction δ 193.9 (CHO), 154.4 (CH), 152.2 (Cq), 130.7 (CHar), 123.4 (CH),
mixture was stirred 1 h at r.t. and 4 h at 90 °C. After neutraliz- 122.4 (Cq), 112.0 (CHar), 30.0 (CH3); HRMS (ESI) m/z: Calcd for
ation with aqueous saturated solution of NaHCO3, the solvent C10H12NO [M + H]+: 162.0919; Found: 162.0910.
was evaporated and the residue extracted with CH2Cl2 (3 ×
500 mL). The combined organic layers were dried over MgSO4,
filtered, evaporated and purified by column chromatography
Diethyl 4-tert-butoxycarbonylamino-benzylphosphonate (12)
over silica gel (petroleum ether/AcOEt: 9/1.5) to give the alde- To a solution of diethyl 4-aminobenzylphosphonate (487 mg,
hyde 9 as a yellow solid (6.258 g, 82%). Rf = 0.33 (petroleum 2.00 mmol) in dry THF (3.5 mL) under Ar, was added Boc2O
ether/AcOEt: 9/1.5); mp: 74–76 °C; IR (ATR): 3246, 2821, 2748, (1.00 g, 4.58 mmol). After stirring at reflux during 4 h, the
1651, 1591, 1550, 1533, 1385, 1335, 1239, 1165, 998, 937, 816, solvent was evaporated and a saturated solution of NH4Cl
740, 680 cm−1; RMN 1H (CDCl3, 400 MHz): δ 9.76 (s, 1H, added. The aqueous layer was then extracted with CH2Cl2 (3 ×
CHO), 7.77–7.74 (m, 2Har), 6.82–6.80 (m, 2Har), 4.12 (d, 2H, J = 100 mL). The combined organic layers were dried over MgSO4,
2.3 Hz, CH2), 3.08 (s, 3H, CH3), 2.24 (t, 1H, J = 2.3 Hz, CHu); filtered, evaporated and purified by column chromatography
RMN 13C (CDCl3, 100 MHz): δ 190.5 (CHO), 153.2 (Cq), 131.9 over silica gel (petroleum ether/AcOEt: 1/4) to give 12 as a
(CHar), 126.5 (Cq), 112.3 (CHar), 78.3 (Cq), 72.6 (CH), 41.8 white solid (672 mg, 98%). Rf = 0.31 (petroleum ether/AcOEt:
(CH2), 38.4 (CH3); HRMS (ESI) m/z: Calcd for C11H12NO [M + 1/4); mp: 116–118 °C; IR (ATR): 3273, 2979, 1709, 1533, 1417,
1
H]+: 174.0919; Found: 174.0908.
1242, 1162, 1053, 1031, 965 cm−1; RMN H (CDCl3, 400 MHz):
δ 7.34–7.32 (m, 2Har), 7.21–7.18 (m, 2Har), 6.94 (br s, 1H, NH),
4.05–3.95 (m, 4H, 2CH2), 3.10 (d, 2H, J = 21.1 Hz, CH2), 1.51 (s,
9H, tBu), 1.24 (t, 6H, J = 6.9 Hz, 2CH3); RMN 13C (CDCl3,
100 MHz): δ 152.9, 137.5 (Cq); 130.3 (CHar), 125.8 (d, J = 8.0 Hz,
(2E)-3-{4-[N-Methyl-N-(prop-2-yn-1-yl)amino]phenyl}prop-2-
enal (10) and (2E)-3-[4-(N-methylamino)phenyl]prop-2-enal)
(11)
To a solution of 9 (869 mg, 5.00 mmol) in dry THF (25 mL) Cq), 118.7 (CHar), 80.4 (Cq), 62.2 (CH2), 33.0 (d, J = 137 Hz,
under Ar at 0 °C, were added tributyl(1,3-dioxolan-2-ylmethyl) CH2), 28.4, 16.4 (CH3); HRMS (ESI) m/z: Calcd for C16H27NO5P
phosphonium bromide (2.52 g, 6.51 mmol) and NaH (60%, [M + H]+: 344.1627; Found: 344.1623.
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4-[(1E,3E)-4-(4-tert-Butoxycarbonylaminophenyl)buta-1,3-
dienyl]-N-methyl-N-(prop-2-ynyl)aniline (13)
(2E)-3-{4-[N-Methyl-N-(tert-butoxycarbonyl)amino]phenyl}prop-
2-enal (15)
To a suspension of NaH (60% in oil, 2.271 g, 56.78 mmol) in To a solution of 11 (646 mg, 4.02 mmol) in dry THF (80 mL)
dry THF (15 mL) under Ar at 0 °C, were added dropwise a under Ar at 0 °C, was added Boc2O (3.509 g, 16.08 mmol).
solution of 12 (6.842 g, 18.93 mmol) in dry THF (20 mL) and After refluxing during 48 h, the solvent was evaporated.
a solution of 10 (1.875 g, 9.46 mmol) in dry THF (20 mL). Dichloromethane and a saturated solution of NH4Cl were then
After stirring 6 h at r.t., the reaction mixture was neutralised added. After layers separation, the aqueous layer was extracted
with a saturated solution of NH4Cl. After concentration, the with CH2Cl2 (3 × 100 mL). The combined organic layers were
residue was extracted with CH2Cl2 (3 × 250 mL). The combined dried over MgSO4, filtered, evaporated and purified by column
organic layers were dried over MgSO4, filtered, evaporated and chromatography over silica gel (petroleum ether/AcOEt: 4/1) to
purified by column chromatography over silica gel (petroleum afford 15 as a white solid (950 mg, 90%). Rf = 0.35 (petroleum
ether/CH2Cl2: 1/4) to afford 13 as a yellow solid (1.654 g, ether/AcOEt: 4/1); mp: 59–61 °C; IR (ATR): 2974, 2053, 1703,
45%). Rf
=
0.42 (petroleum ether/CH2Cl2: 1/4); mp: 1669, 1601, 1511, 1430, 1354, 1256, 1148, 1126, 1106, 969,
179–181 °C; IR (ATR): 3365, 3262, 1701, 1516, 1235, 1162, 813 cm−1; RMN 1H (CDCl3, 400 MHz): δ 9.68 (d, 1H, J = 7.8 Hz,
1110, 978, 799 cm−1; RMN H (CDCl3, 400 MHz): δ 7.35–7.25 CHO), 7.54 (d, 2H, J = 8.7 Hz, 2Har), 7.46 (d, 1H, J = 15.6 Hz,
1
(m, 6Har), 6.88–6.74 (m, 4H, 2Har, 2CHv), 6.59–6.51 (m, 2H, CHv), 7.35 (d, 2H, J = 8.7 Hz, 2Har), 6.68 (dd, 1H, J = 7.8, 15.6
2CHv), 4.05 (d, 2H, J = 2.3 Hz, CH2), 2.99 (s, 3H, CH3), 2.18 Hz, CHv), 3.30 (s, 3H, CH3), 1.49 (s, 9H, tBu); RMN 13C
(t, 1H, J = 2.3 Hz, CHu), 1.52 (s, 9H, tBu); RMN 13C (CDCl3, (CDCl3, 100 MHz): δ 193.8 (CHO), 154.2 (Cq), 152.2 (CHv),
100 MHz): δ 152.7, 148.5, 137.4, 132.9 (Cq); 132.4, 130.4, 128.8 146.4, 130.5 (Cq); 128.8 (CHar), 128.0 (CHv), 125.2 (CHar), 81.1
(CH); 127.8 (Cq), 127.5, 126.9 (CHar); 126.3 (CH), 118.7, 114.2 (Cq), 36.9, 28.3 (CH3). HRMS (ESI) m/z: Calcd for C15H20NO3
(CHar); 80.7, 79.2 (Cq); 72.2 (CH), 42.4 (CH2), 38.7, 28.5 (CH3); [M + H]+: 262.1438; Found: 262.1437.
HRMS (ESI) m/z: Calcd for C25H29N2O2 [M + H]+: 389.2229;
Found: 389.2222.
Diethyl 4-benzoylamino-benzylphosphonate (16)
To a solution of diethyl 4-aminobenzylphosphonate (1.95 g,
8.01 mmol) in toluene (80 mL), were added benzoyl chloride
(1.02 mL, 8.81 mmol) and Et3N (1.67 mL, 12.02 mmol). After
one night reaction at r.t., water was added. The aqueous layer
was extracted with CH2Cl2 (3 × 100 mL). The combined organic
layers were dried over MgSO4, filtered, evaporated and purified
by column chromatography over silica gel (petroleum ether/
AcOEt: 1/4) to afford 16 as a white solid (2.75 g, 99%). Rf = 0.16
(petroleum ether/AcOEt: 1/4); mp: 157–159 °C; IR (ATR): 3271,
3245, 2985, 1651, 1601, 1532, 1515, 1414, 1323, 1246, 1057,
N-Methyl-4-[(1E,3E)-4-(4-tert-butoxycarbonylaminophenyl)
buta-1,3-dienyl]-N-methyl-N-(prop-2-ynyl)aniline (14)
From 13: To a solution of 13 (530 mg, 1.37 mmol) in dry THF
(7 mL) under Ar at 0 °C, was added NaH (60% in oil, 218 mg,
5.46 mmol) and MeI (170 μL, 2.73 mmol). After stirring 6 h at
r.t., the reaction mixture was neutralised with a saturated solu-
tion of NH4Cl. After concentration, the residue was extracted
with CH2Cl2 (3 × 100 mL). The combined organic layers were
dried over MgSO4, filtered, evaporated and purified by column
chromatography over silica gel (petroleum ether/CH2Cl2: 1/4)
to afford 14 as a yellow solid (514 mg, 94%).
From 19: To a solution of 19 (77 mg, 0.21 mmol) in aceto-
nitrile (5 mL) under Ar at 0 °C, was added K2CO3 (116 mg,
0.84 mmol) and propargyl bromide (80% in toluene, 94 µL,
0.844 mmol) drop by drop. After one night stirring at r.t., the
mixture was filtrated on silica gel and the solvent evapored
under reduced pressure. The residue was then purified by
chromatography on silica gel (petroleum ether/AcOEt: 4/1) to
afford 14 (44 mg, 52%). Rf = 0.42 (petroleum ether/CH2Cl2:
1/4); mp: 155–157 °C; IR (ATR): 3234, 2976, 1685, 1602, 1509,
1
972, 843, 704 cm−1; RMN H (CDCl3, 400 MHz): δ 8.60 (s, 1H,
NH), 7.93–7.90 (m, 2Har), 7.65–7.62 (m, 2Har), 7.55–7.42 (m,
3Har), 7.27–7.22 (m, 2Har), 3.99–3.94 (m, 4H, 2CH2), 3.12 (d,
2H, J = 21.6 Hz, CH2), 1.24–1.20 (m, 6H, 2CH3); RMN 13C
(CDCl3, 100 MHz): δ 166.1 (Cq), 137.4 (Cq), 131.8, 130.4, 130.3,
128.7, 127.4 (CHar); 127.2 (d, J = 10 Hz, Cq), 120.6 (CHar), 62.3
(CH2), 33.2 (d, J = 138 Hz, CH2), 16.5 (CH3); HRMS (ESI) m/z:
Calcd for C18H22NO4P [M + H]+: 348.1365; Found: 348.1363.
4-[(1E,3E)-4-(Benzoylaminophenyl)buta-1,3-dienyl]-N-methyl-
N-(tert-butoxycarbonyl)aniline (17)
1476, 1431, 1365, 1255, 1152, 1109, 989, 850, 803 cm−1; RMN To a suspension of NaH (60% in oil, 576 mg, 14.4 mmol) in
1H (CDCl3, 400 MHz): δ 7.36 (d, 2H, J = 8.2 Hz, 2Har), 7.33 (d, dry THF (20 mL) under Ar at 0 °C, were added dropwise a solu-
2H, J = 8.2 Hz, 2Har), 7.16 (d, 2Har, J = 8.2 Hz, 2Har), 6.91–6.74 tion of 16 (1.876 g, 5.401 mmol) in dry THF (10 mL) and a
(m, 4H, 2Har, 2CH), 6.58 (d, 1H, J = 15.1 Hz, CHv), 6.53 (d, solution of 15 (942 mg, 3.60 mmol) in dry THF (20 mL). After
1H, J = 15.1 Hz, CHv), 4.02 (d, 2H, J = 2.3 Hz, CH2), 3.23 (m, stirring 6 h at r.t., the reaction mixture was neutralised with a
3H, CH3), 2.96 (s, 3H, CH3), 2.17 (t, 1H, J = 2.3 Hz, CHu), 1.45 saturated solution of NH4Cl. After concentration, the residue
(s, 9H, tBu); RMN 13C (CDCl3, 100 MHz): δ 154.7, 148.5, 142.6, was extracted with CH2Cl2 (3 × 100 mL). The combined organic
134.8 (Cq); 132.9, 130.1, 129.7 (CH); 127.5 (CHar), 127.5 (Cq), layers were dried over MgSO4, filtered, evaporated and purified
126.2 (CHar), 126.0 (CH), 125.5, 114.1 (CHar); 80.4, 79.2 (Cq); by column chromatography over silica gel (CH2Cl2/AcOEt:
72.2 (CH), 42.3 (CH2), 38.6, 37.2, 28.4 (CH3); HRMS (ESI) m/z: 1/0.05) to afford 17 as a yellow solid (914 mg, 56%). Rf = 0.61
Calcd for C26H31N2O2 [M + H]+: 403.2385; Found: 403.2380.
(CH2Cl2/AcOEt: 1/0.05); mp: 212–214 °C; IR (ATR): 3326, 2926,
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1700, 1646, 1601, 1587, 1514, 1411, 1365, 1321, 1254, 1153, 4-[(1E,3E)-4-(4-Aminophenyl)buta-1,3-dien-1-yl]-N-methyl-N-
1110, 984, 854, 804 cm−1; RMN 1H (DMSO-d6, 400 MHz): (prop-2-yn-1-yl)aniline (20)
δ 10.32 (s, 1H, NH), 7.98–7.94 (m, 2Har), 7.80 (d, 2H, J = 8.7 Hz,
Boc deprotection of compound 13 (131 mg, 0.337 mmol)
2Har), 7.62–7.46 (m, 7Har), 7.25 (d, 2H, J = 8.7 Hz, 2Har),
according to the general procedure afforded 20 (99 mg, 100%)
7.10–7.00 (m, 2H, 2CHv), 6.73–6.68 (m, 2H, 2CHv), 3.18 (s,
as a solid. mp: 188–190 °C; IR (ATR): 3394, 2967, 2920, 2878,
3H, CH3), 1.41 (s, 9H, tBu); RMN 13C (CDCl3, 100 MHz):
2584, 1596, 1508, 1465, 986, 853 cm−1; RMN 1H (CD3OD,
δ 165.7, 154.8, 143.2, 137.4, 135.1, 134.6, 133.9 (Cq); 132.1,
400 MHz): δ 7.66 (d, 2H, J = 8.7 Hz, 2Har), 7.63 (d, 2H, J =
132.0, 129.2 (CHv); 129.0, 128.8, 127.2, 127.1, 126.6, 125.6,
8.3 Hz, 2Har), 7.57 (d, 2H, J = 8.7 Hz, 2Har), 7.37 (d, 2H, J =
120.4 (CHar); 80.6 (Cq), 37.3, 28.5 (CH3); HRMS (ESI) m/z: Calcd
8.7 Hz, 2Har), 7.14–7.10 (m, 2H, 2CHv), 6.81–6.77 (m, 2H,
for C29H30N2O3 [M + H]+: 455.5680; Found: 455.5682.
2CHv), 4.49 (d, 2H, J = 2.3 Hz, CH2), 3.31 (s, 3H, CH3), 3.20
(m, 1H, CHu); RMN 13C (CD3OD, 100 MHz): δ 141.3, 140.1,
4-[(1E,3E)-4-(N-Methyl-N-benzoylaminophenyl)buta-1,3-dienyl]-
N-methyl-N-(tert-butoxycarbonyl)aniline (18)
139.8 (Cq); 133.3, 132.9, 132.2, 131.9 (CH); 130.7 (Cq), 129.0,
124.4, 122.3 (CHar); 80.6 (Cq), 74.0 (CH), 49.4 (CH2), 44.0
(CH3); HRMS (ESI) m/z: Calcd for C20H20N2O [M + H]+:
289.1705; Found: 289.1708.
To a solution of 17 (48 mg, 0.11 mmol) in dry THF (2 mL)
under Ar at 0 °C, was added NaH (60% in oil, 13 mg,
0.33 mmol) and MeI (14 μL, 0.22 mmol). After stirring one
night at r.t., the reaction mixture was neutralised with a satu-
rated solution of NH4Cl. After concentration, the residue was
extracted with CH2Cl2 (3 × 30 mL). The combined organic
layers were dried over MgSO4, filtered, evaporated and purified
by column chromatography over silica gel (petroleum ether/
AcOEt: 4/1) to afford 18 as a yellow solid (48 mg, 90%). Rf =
0.25 (petroleum ether/AcOEt: 4/1); mp: 74–76 °C; IR (ATR):
1694, 1643, 1600, 1512, 1364, 1283, 1152, 1106, 972, 840,
O-Alkylation of 5′-azido-5′-deoxy-adenosine (21)
To a suspension of NaH (60% in oil, 159 mg, 3.98 mmol) in
dry DMF (18 mL) at 0 °C, was added in portion compound 21
(1.054 g, 3.607 mmol) under Ar. The reaction mixture was
stirred at 0 °C for 2 h, then cooled to −45 °C, to which tert-
butyl bromoacetate (587 μL, 3.98 mmol) was added slowly.
After one night reaction at r.t., the reaction mixture was neu-
tralised with a saturated solution of NH4Cl. After solvent evap-
oration, the residue was extracted with AcOEt (3 × 100 mL).
The combined organic layers were washed with saturated
NH4Cl (3 × 200 mL), dried over MgSO4, filtered, evaporated
and purified by column chromatography over silica gel
(CH2Cl2/EtOH: 10/0.5) to afford to 5′-azido-5′-deoxy-2′,3′-di-O-
(tert-butoxycarbonylmethyl)-adenosine (22) (360 mg, 18%),
5′-azido-5′-deoxy-2′-O-(tert-butoxycarbonylmethyl)-adenosine (23)
(269 mg, 18%) and 5′-azido-5′-deoxy-3′-O-(tert-butoxycarbonyl-
methyl)-adenosine (24) (94 mg, 6%) as white solids. By using
NaH (60% in oil, 117 mg, 2.92 mmol, 2 equiv.) in DMF (8 mL),
21 (427 mg, 1.46 mmol), and tert-butyl bromoacetate (431 μL,
1
700 cm−1; RMN H (CDCl3, 400 MHz): δ 7.38–7.18 (m, 11Har),
6.98 (d, 2H, J = 8.2 Hz, 2Har), 6.88–6.84 (m, 2H, 2CHv),
6.64–6.53 (m, 2H, 2CHv), 3.50 (s, 3H, CH3), 3.26 (s, 3H, CH3),
1.46 (s, 9H, tBu); RMN 13C (CDCl3, 100 MHz): δ 170.5, 154.7,
144.0, 143.2, 136.0, 135.6, 134.2 (Cq); 132.7, 131.4, 129.9, 129.8
(CHv); 128.8, 128.7, 127.9, 127.0, 126.6, 125.5 (CHar); 80.6
(Cq), 38.4, 37.3, 28.4 (CH3). HRMS (ESI) m/z: Calcd for
C30H33N2O3 [M + H]+: 469.2486; Found: 469.2488.
4-[(1E,3E)-4-(N-Methylaminophenyl)buta-1,3-dienyl]-N-methyl-
N-(tert-butoxycarbonyl)aniline (19)
To a solution of 18 (1.356 g; 2.89 mmol) in EtOH (40 mL) at 2.92 mmol, 2 equiv.), compounds 22 (265 mg, 35%), 25
r.t., was added NaOH (4 M, 72 mL, 289 mmol). The reaction (5′-azido-5′-deoxy-2′,3′-di-O-(tert-butoxycarbonylmethyl)-N6,N6-
mixture was stirred at reflux during 8 h and 48h at r.t. After di-(tert-butoxycarbonylmethyl)-adenosine) (142 mg, 13%) and
solvent evaporation, the residue was extracted with CH2Cl2 (3 × 26 (5′-azido-5′-deoxy-2′,3′-di-O-(tert-butoxycarbonylmethyl)-N6-
80 mL). The combined organic layers were dried over MgSO4, (tert-butoxycarbonylmethyl)-adenosine) (74 mg, 8%) were
filtered, evaporated and purified by column chromatography obtained as white solids.
over silica gel (petroleum ether/AcOEt: 4/1) to afford 19 as a
22: Rf = 0.24 (CH2Cl2/EtOH: 10/0.5); mp: 48–50 °C; [α]D
red solid (766 mg, 73%). Rf = 0.33 (petroleum ether/AcOEt: +53.4 (c 1.0, MeOH); IR (ATR): 3331, 3176, 2979, 2935, 2103,
4/1); mp: 182–184 °C; IR (ATR): 3398, 2974, 2929, 1691, 1603, 1742, 1641, 1368, 1235, 1145, 848 cm−1; RMN 1H (CD3OD,
1512, 1365, 1258, 1153, 1109, 987, 851, 804 cm−1; RMN 400 MHz): δ 8.30 (s, 1H, H2), 8.20 (s, 1H, H8), 6.19 (d, 1H, J =
1H (CDCl3, 400 MHz): δ 7.34 (d, 2H, J = 8.7 Hz, 2Har), 7.30 (d, 4.6 Hz, H1′), 4.97 (dd, 1H, J = 4.6, 5.0 Hz, H2′), 4.47 (dd, 1H, J =
2Har, J = 8.3 Hz, 2Har), 7.18 (d, 2Har, J = 8.3 Hz, 2Har), 6.90 (dd, 5.0, 5.5 Hz, H3′), 4.34–4.32 (m, 1H, H4′), 4.29–4.27 (m, 2H,
1H, J = 10.5, 15.1 Hz, CHv), 6.77 (dd, 1H, J = 10.5, 15.1 Hz, CH2), 4.24–4.22 (m, 2H, CH2), 3.72–3.70 (m, 2H, H5′), 1.49 (s,
CHv), 6.62–6.52 (m, 4H, 2CHar, 2CHv), 3.87 (s, 1H, NH), 3.30 9H, tBu), 1.37 (s, 9H, tBu); RMN 13C (CD3OD, 100 MHz):
(s, 3H, CH3), 2.85 (s, 3H, CH3), 1.47 (s, 9H, tBu); RMN 13C δ 171.3, 170.9 (Cq); 157.3 (C6), 153.9 (C2), 150.5 (C4), 141.7 (C8),
(CDCl3, 100 MHz): δ 154.8, 149.1, 142.6, 135.0 (Cq); 133.4, 120.7 (C5), 89.0 (C1′), 82.9 (Cq), 82.8 (C4′), 81.4 (C2′), 79.2 (C3′),
129.9, 129.7 (CHv); 127.8 (CHar), 126.8 (Cq), 126.2, 125.5 69.0 (CH2), 52.8 (C5′), 28.4, 28.3 (CH3); HRMS (ESI) m/z: Calcd
(CHar); 125.2 (CHv), 112.5 (CHar), 80.4 (Cq), 37.3, 30.7, 28.5 for C22H33N8O7 [M + H]+: 521.2472; Found: 521.2463.
(CH3). HRMS (ESI) m/z: Calcd for C23H29N2O [M + H]+:
365.2224; Found: 365.2224.
23: Rf = 0.07 (CH2Cl2/EtOH: 10/0.5); mp: 65–67 °C; [α]D
+51.9 (c 1.0, MeOH); IR (ATR): 3342, 3178, 2977, 2103, 1737,
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1641, 1599, 1368, 1243, 1139, 798 cm−1; RMN 1H (CD3OD, 1-[5′-Deoxy-2′,3′-di-O-(tert-butoxycarbonylmethyl)-adenosin-5′-
400 MHz): δ 8.29 (s, 1H, H2), 8.20 (s, 1H, H8), 6.05 (d, 1H, J = yl]-4-[(N-methyl-N-((4-(4-(4-(N-methyl-N-tert-butoxycarbonyl)
5.0 Hz, H1′), 4.91 (dd, 1H, J = 5.0, 4.6 Hz, H2′), 4.34–4.33 (m, amino)phenyl)buta-1,3-dienyl)phenyl))aminomethyl]-1H-1,2,3-
1H, H4′), 4.30 (t, 1H, J = 5.0 Hz, H3′), 4.26–4.25 (m, 2H, CH2), triazole (27)
3.70–3.69 (m, 2H, H5′), 1.47 (s, 9H, tBu); RMN 13C (CD3OD,
From alkyne 14 (212 mg, 0.523 mmol) and azido adenosine 22
100 MHz): δ 171.9 (Cq), 157.3 (C6), 153.9 (C2), 150.6 (C4), 141.3
(272 mg, 0.523 mmol), compound 27 was obtained after purifi-
(C8), 120.5 (C5), 90.2 (C1′), 83.3 (Cq), 82.9 (C4′), 81.0 (C3′), 74.2
cation by column chromatography over silica gel (CH2Cl2/
(C2′), 69.5 (CH2), 53.0 (C5′), 28.3 (CH3); HRMS (ESI) m/z: Calcd
EtOH: 10/0.4) as a yellow solid (434 mg, 90%). Rf = 0.34
(CH2Cl2/EtOH: 10/0.4); mp: 112–114 °C; [α]D +10.5 (c 1.0,
for C16H23N8O5 [M + H]+: 407.1791; Found: 407.1786.
24: Rf = 0.13 (CH2Cl2/EtOH: 10/0.5); mp: 64–66 °C; [α]D
CHCl3); IR (ATR): 2978, 1741, 1692, 1640, 1632, 1510, 1367,
+40.1 (c 1.0, MeOH); IR (ATR): 3438, 3334, 3180, 2980, 2930,
1238, 1147, 984, 845, 732 cm−1; RMN 1H (CDCl3, 400 MHz):
2102, 1737, 1641, 1247, 1140, 1082, 723 cm−1; RMN 1H
δ 8.19 (s, 1H, H2), 7.72 (s, 1H, H8), 7.35 (d, 2H, J = 8.8 Hz,
(CD3OD, 400 MHz): δ 8.30 (s, 1H, H2), 8.22 (s, 1H, H8), 6.19 (d,
2Har), 7.24 (d, 2H, J = 8.4 Hz, 2Har), 7.20 (s, 1H, CHv), 7.16 (d,
1H, J = 5.0 Hz, H1′), 4.80 (t, 1H, J = 5.0 Hz, H2′), 4.50 (dd, 1H,
2H, J = 8.4 Hz, 2Har), 6.86 (dd, 1H, J = 10.4, 15.2 Hz, CHv),
J = 5.0, 4.9 Hz, H3′), 4.22–4.21 (m, 2H, CH2), 4.20–4.17 (m, 1H,
6.72 (dd, 1H, J = 10.4, 15.2 Hz, CHv), 6.57 (d, 2H, J = 8.8 Hz,
H4′), 3.71–3.57 (m, 2H, H5′), 1.37 (s, 9H, tBu); RMN 13C
2Har), 6.53 (d, 2H, J = 15.2 Hz, 2CHv), 6.10 (d, 1H, J = 2.8 Hz,
(CD3OD, 100 MHz): δ 171.6 (Cq), 157.3 (C6), 153.9 (C2), 150.6
H1′), 5.87 (br s, 2H, NH2), 4.75–4.74 (m, 2H, CH2), 4.71 (dd,
(C4), 141.6 (C8), 120.6 (Cq), 88.7 (C1′), 84.5 (C4′), 83.3 (C2′), 83.2
1H, J = 2.8, 4.4 Hz, H2′), 4.55–4.42 (m, 4H, H3′, H4′, 2H5′),
4.34–4.17 (m, 2H, CH2), 3.25 (s, 3H, CH3), 2.90 (s, 3H, CH3),
(Cq), 71.5 (C3′), 69.6 (CH2), 52.9 (C5′), 28.2 (CH3); HRMS (ESI)
m/z: Calcd for C16H23N8O5 [M + H]+: 407.1791; Found:
1.48 (s, 9H, tBu), 1.45 (s, 9H, tBu), 1.38 (s, 9H, tBu); RMN 13C
407.1783.
(CDCl3, 100 MHz): δ 169.6, 169.5, 155.6, 154.8 (Cq); 153.0 (C2),
149.3, 148.5, 145.2, 142.7 (Cq); 140.4 (C8), 135.0 (Cq), 133.0,
129.9 (CHv); 127.7 (CHar), 126.4 (Cq), 126.3, 125.6 (CHar);
123.3 (CH), 120.5 (C5), 112.8 (CHar), 89.0 (C1′), 82.2, 80.5 (Cq);
25: Rf = 0.64 (CH2Cl2/AcOEt/EtOH: 2/1/0.1); mp: 48–50 °C;
[α]D +50.9 (c 1.0, CHCl3); IR (ATR): 2979, 2932, 2103, 1741,
1621, 1585, 1369, 1230, 1148, 1090, 848 cm−1; RMN 1H (CDCl3,
400 MHz): δ 8.29 (s, 1H, H2), 8.02 (s, 1H, H8), 6.51 (br s, 1H,
NH), 6.12 (d, 1H, J = 3.2 Hz, H1′), 4.77–4.76 (m, 1H, H2′),
4.37–4.15 (m, 8H, H3′, H4′, 3CH2), 3.78 (dd, 1H, J = 3.0, 13.3
Hz, H5′), 3.70 (dd, 1H, J = 4.6, 13.3 Hz, H5′), 1.44 (s, 27H, tBu);
RMN 13C (CDCl3, 100 MHz): δ 169.4, 169.2 (Cq); 154.5 (C6),
152.8 (C2), 148.8 (C4), 139.2 (C8), 120.7 (Cq), 88.3 (C1′), 82.1,
82.0 (Cq); 80.7 (C4′), 80.2 (C2′), 77.6 (C3′), 68.0 (CH2), 51.7 (C5′),
28.2 (CH3), 28.1 (CH3).
80.3 (C4′), 80.1 (C2′), 78.4 (C3′), 68.7, 68.6, 50.6 (CH2); 48.4 (C5′),
38.6, 37.3, 28.5, 28.3, 28.2 (CH3); HRMS (ESI) m/z: Calcd for
C48H63N10O9 [M + H]+: 923.4779; Found: 923.4774.
1-[5′-Deoxy-2′,3′-di-O-(tert-butoxycarbonylmethyl)-adenosin-5′-yl]-
4-[(N-methyl-N-((4-(4-(4-(N-tert-butoxycarbonyl)amino) phenyl)
buta-1,3-dienyl)phenyl))aminomethyl]-1H-1,2,3-triazole (28)
26: Rf = 0.94 (CH2Cl2/AcOEt/EtOH: 2/1/0.1); mp: 29–31 °C; From alkyne 13 (198 mg, 0.510 mmol) and azido adenosine 22
[α]D +26.1 (c 1.0, CHCl3); IR (ATR): 2976, 2925, 2103, 1741, (265 mg, 0.510 mmol), compound 28 was obtained after puri-
1590, 1368, 1226, 1149, 994, 848 cm−1; RMN 1H (CDCl3, fication by column chromatography over silica gel (CH2Cl2/
400 MHz): δ 8.27 (s, 1H, H2), 8.00 (s, 1H, H8), 6.12 (d, 1H, J = EtOH: 10/0.4) as a yellow solid (397 mg, 86%). Rf = 0.33
2.3 Hz, H1′), 5.00–4.89 (m, 2H, CH2), 4.68–4.67 (m, 1H, H2′), (CH2Cl2/EtOH: 10/0.4); mp: 123–125 °C; [α]D +8.1 (c 1.0,
4.57–4.07 (m, 8H, H3′, H4′, 3CH2), 3.83 (dd, 1H, J = 2.5, 13.0 CHCl3); IR (ATR): 2975, 1737, 1726, 1603, 1514, 1368, 1234,
1
Hz, H5′), 3.73 (dd, 1H, J = 3.9, 13.0 Hz, H5′), 1.46–1.41 (m, 36H, 1154, 1049, 985, 848, 800 cm−1; RMN H (CDCl3, 400 MHz): δ
tBu); RMN 13C (CDCl3, 100 MHz): δ 169.5, 169.4, 169.3, 169.2 8.19 (s, 1H, H2), 7.72 (s, 1H, H8), 7.34–7.29 (m, 4Har), 7.21 (d,
(Cq); 154.5 (C6), 152.1 (C2), 150.2 (C4), 137.8 (C8), 121.1 (Cq), 2H, J = 8.8 Hz, 2Har), 7.20 (s, 1H, CH), 6.82 (dd, 1H, J = 10.4,
88.4 (C1′), 82.0 (Cq), 81.8 (Cq), 80.5 (C4′), 80.2 (C2′), 77.4 (C3′), 15.2 Hz, CHv), 6.71 (dd, 1H, J = 10.4, 15.2 Hz, CHv), 6.62 (br
67.9 (CH2), 67.8 (CH2), 53.1 (CH2), 51.7 (C5′), 51.3 (CH2), 28.2 s, 1H, NH), 6.56 (d, 2H, J = 8.8 Hz, 2Har), 6.50 (d, 2H, J = 15.2
(CH3).
Hz, 2CHv), 6.10 (d, 1H, J = 2.8 Hz, H1′), 5.89 (s, 2H, NH2),
4.74–4.73 (m, 2H, CH2), 4.70 (dd, 1H, J = 2.8, 4.0 Hz, H2′),
4.55–4.42 (m, 4H, H3′ + H4′ + 2H5′), 4.32 (d, 1H, J = 16.8 Hz,
CHCO), 4.24 (d, 1H, J = 16.8 Hz, CHCO), 4.21 (d, 1H, J = 16.8
General procedure for CuAAC
To a solution of alkyne (1 equiv.) and azide (1 equiv.) in a Hz, CHCO), 4.19 (d, 1H, J = 16.8 Hz, CHCO), 2.89 (s, 3H, CH3),
mixture of DMF/H2O (1/0.1, 2.2 mL mmol−1 alkyne) at r.t. 1.51 (s, 9H, tBu), 1.48 (s, 9H, tBu), 1.38 (s, 9H, tBu); RMN 13C
under Ar, were added CuSO4·5H2O (0.4 equiv.) and Na ascor- (CDCl3, 100 MHz): δ 169.6, 169.5, 155.6 (Cq); 153.0 (C2), 152.8,
bate (0.4 equiv.). After completion of reaction (4 h at r.t.), the 149.3, 148.4, 145.2 (Cq); 140.4 (C8), 137.4, 132.9 (Cq); 132.5,
solvent was removed in vacuo and the residue diluted in AcOEt 130.1, 128.8 (CHv); 127.6, 126.9 (CHar); 126.6 (Cq), 125.8
and brine. The aqueous layer was extracted with AcOEt (2×). (CHv), 123.3 (CH), 120.5 (C5), 118.7, 112.8 (CHar); 89.0 (C1′),
The combined organic layers were washed with brine (3×), 82.2, 80.7 (Cq); 80.3 (C4′), 80.1 (C2′), 78.4 (C3′), 68.7, 68.6, 50.6
dried over MgSO4, filtered, evaporated and purified by column (CH2); 48.4 (C5′), 38.6, 28.5, 28.3, 28.2 (CH3); HRMS (ESI) m/z:
chromatography over silica gel to afford the triazole.
Calcd for C47H61N10O9 [M + H]+: 909.4623; Found: 909.4618.
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Organic & Biomolecular Chemistry
1-[5′-Deoxy-3′-O-(tert-butoxycarbonylmethyl)-adenosin-5′-yl]-4-
[(N-methyl-N-((4-(4-(4-(N-methyl-N-tert-butoxycarbonyl)amino)
phenyl)buta-1,3-dienyl)phenyl))aminomethyl]-1H-1,2,3-
triazole (29)
1-[5′-Deoxy-2′-O-(tert-butoxycarbonylmethyl)-adenosin-5′-yl]-4-
[(N-methyl-N-((4-(4-(4-(N-methyl-N-tert-butoxycarbonyl)amino)
phenyl)buta-1,3-dienyl)phenyl))aminomethyl]-1H-1,2,3-
triazole (31)
From alkyne 14 (44 mg, 0.109 mmol) and azido adenosine 24 From alkyne 14 (64 mg, 0.159 mmol) and azido adenosine 23
(44 mg, 0.109 mmol), compound 29 was obtained after purifi- (65 mg, 0.159 mmol), compound 31 was obtained after purifi-
cation by column chromatography over silica gel (CH2Cl2/ cation by column chromatography over silica gel (CH2Cl2/
EtOH: 10/0.6) as a yellow solid (73 mg, 83%). Rf = 0.31 EtOH: 10/0.6) as a yellow solid (112 mg, 87%). Rf = 0.12
(CH2Cl2/EtOH: 10/0.5); mp: 139–141 °C; [α]D −7.2 (c 1.0, (CH2Cl2/EtOH: 10/0.6); mp: 109–111 °C; [α]D +15.1 (c 1.0,
CHCl3); IR (ATR): 2972, 2921, 1692, 1640, 1602, 1511, 1367, CHCl3); IR (ATR): 3321, 3171, 2974, 2930, 1734, 1691, 1641,
1
1249, 1146, 1107, 985, 846 cm−1; RMN H (CDCl3, 400 MHz): 1602, 1511, 1367, 1247, 1145, 985, 910, 843 cm−1; RMN 1H
δ 8.18 (s, 1H, H2), 7.67 (s, 1H, H8), 7.33 (d, 2H, J = 8.8 Hz, (CDCl3, 400 MHz): δ 8.27 (s, 1H, H2), 7.70 (s, 1H, H8), 7.35 (d,
2Har), 7.21 (d, 2H, J = 8.8 Hz, 2Har), 7.16 (d, 2H, J = 8.4 Hz, 2H, J = 8.4 Hz, 2Har), 7.26 (s, 1H, CH), 7.23 (d, 2H, J = 8.4 Hz,
2Har), 7.07 (s, 1H, CHv), 6.87 (dd, 1H, J = 10.4, 15.2 Hz, 2Har), 7.17 (d, 2H, J = 8.4 Hz, 2Har), 6.87 (dd, 1H, J = 10.4,
CHv), 6.71 (dd, 1H, J = 10.4, 15.2 Hz, CHv), 6.61–6.49 (m, 15.2 Hz, CHv), 6.73 (dd, 1H, J = 10.4, 15.2 Hz, CHv), 6.63
4H, 2CHv, 2Har), 6.13 (s, 2H, NH2), 5.89 (d, 1H, J = 2.0 Hz, (d, 2H, J = 8.8 Hz, 2Har), 6.54 (d, 1H, J = 15.2 Hz, CHv), 6.53
H1′), 4.73 (dd, 1H, J = 4.4, 14.8 Hz), 4.62–4.58 (m, 2H), (d, 1H, J = 15.2 Hz, CHv), 6.02 (d, 1H, J = 4.8 Hz, H1′), 5.87
4.52–4.47 (m, 2H), 4.43–4.34 (m, 2H), 4.36 (d, 1H, J = 17.2 Hz, (s, 2H, NH2), 4.77 (dd, 1H, J = 4.2, 14.6 Hz), 4.72 (dd, 1H, J =
CHCO), 4.11 (d, 1H, J = 17.2 Hz, CHCO), 3.64 (s, 1H, OH), 6.2, 14.6 Hz), 4.61 (t, 1H, J = 4.8 Hz), 4.57 (br s, 2H),
3.24 (s, 3H, CH3), 2.85 (s, 3H, CH3), 1.46 (s, 9H, tBu), 1.45 (s, 4.45–4.37 (m, 2H), 4.24 (d, 1H, J = 17.2 Hz, CHCO), 3.99 (d,
9H, tBu); RMN 13C (CDCl3, 100 MHz): δ 171.4, 155.7, 154.8 1H, J = 17.2 Hz, CHCO), 3.25 (s, 3H, CH3), 2.95 (s, 3H, CH3),
(Cq); 153.1 (C2), 149.2, 148.4, 145.2, 142.7 (Cq); 140.2 (C8), 1.45 (s, 18H, tBu); RMN 13C (CDCl3, 100 MHz): δ 170.5, 155.9,
134.9 (Cq), 133.0, 129.9, 129.8 (CHv); 127.6 (CHar), 126.4 (Cq), 154.7 (Cq); 153.1 (C2), 149.3, 148.4, 145.1, 142.6 (Cq); 140.0
126.3 (CHar), 125.6 (CHv), 125.5 (CHar), 123.3 (CH), 120.3 (C8), 134.9 (Cq), 132.9, 129.8 (CHv); 127.6 (CHar), 126.3 (Cq),
(C5), 112.7 (CHar), 90.3 (C1′), 83.4 (Cq), 81.5 (C4′), 80.5 (Cq), 126.2, 125.4 (CHar); 123.3 (CH), 120.3 (C5), 112.8 (CHar), 88.1
79.8 (C3′), 72.9 (C2′), 69.9, 50.3 (CH2); 48.3 (C5′), 38.5, 37.3, (C1′), 83.2 (Cq), 83.0 (C4′), 82.2 (C2′), 80.4 (Cq), 70.4 (C3′), 69.5,
28.4, 28.1 (CH3); HRMS (ESI) m/z: Calcd for C42H52N10O7 [M + 51.1 (CH2); 48.2 (C5′), 38.5, 37.2, 28.4, 28.0 (CH3); HRMS (ESI)
H]+: 809.4098; Found: 809.4093.
m/z: Calcd for C42H53N10O7 [M + H]+: 809.4098; Found:
809.4093.
1-[5′-Deoxy-3′-O-(tert-butoxycarbonylmethyl)-adenosin-5′-yl]-4-
[(N-methyl-N-((4-(4-(4-(N-tert-butoxycarbonyl)amino)phenyl)
buta-1,3-dienyl)phenyl))aminomethyl]-1H-1,2,3-triazole (30)
1-[5′-Deoxy-2′-O-(tert-butoxycarbonylmethyl)-adenosin-5′-yl]-4-
[(N-methyl-N-((4-(4-(4-(N-tert-butoxycarbonyl)amino)phenyl)
buta-1,3-dienyl)phenyl))aminomethyl]-1H-1,2,3-triazole (32)
From alkyne 13 (41 mg, 0.106 mmol) and azido adenosine 23
(43 mg, 0.106 mmol), compound 30 was obtained after purifi- From alkyne 13 (56 mg, 0.144 mmol) and azido adenosine 23
cation by column chromatography over silica gel (CH2Cl2/ (59 mg, 0.144 mmol), compound 32 was obtained after purifi-
EtOH: 10/0.6) as a yellow solid (71 mg, 84%). Rf = 0.31 (CH2Cl2/ cation by column chromatography over silica gel (CH2Cl2/
EtOH: 10/0.5); mp: 123–125 °C; [α]D −5.8 (c 1.0, CHCl3); EtOH: 10/0.6) as a yellow solid (100 mg, 87%). Rf = 0.12
IR (ATR): 3680 (br), 3303 (br), 2987, 2900, 1726, 1605, 1514, (CH2Cl2/EtOH: 10/0.6); mp: 132–134 °C; [α]D +13.5 (c 1.0,
1369, 1241, 1159, 1050 cm−1; RMN 1H (CDCl3, 400 MHz): δ CHCl3); IR (ATR): 1725, 1632, 1601, 1514, 1368, 1239, 1156,
1
8.17 (s, 1H, H2), 7.67 (s, 1H, H8), 7.29 (m, 4H, Har), 7.19 (d, 2H, 1052, 985, 843 cm−1; RMN H (CDCl3, 400 MHz): δ 8.27 (s, 1H,
J = 8.8 Hz, 2Har), 7.07 (s, 1H, CHv), 6.79 (dd, 1H, J = 10.4, 15.2 H2), 7.69 (s, 1H, H8), 7.34–7.29 (m, 4Har), 7.26 (s, 1H, CHv),
Hz, CHv), 6.69 (dd, 1H, J = 10.4, 15.2 Hz, CHv), 6.51 (d, 2H, 7.22 (d, 2H, J = 8.8 Hz, 2Har), 6.84 (dd, 1H, J = 10.6, 15.2 Hz,
J = 9.2 Hz, 2Har), 6.48 (d, 1H, J = 15.2 Hz, CHv), 6.46 (d, 1H, J CHv), 6.72 (dd, 1H, J = 10.6, 15.2 Hz, CHv), 6.62 (d, 2H, J =
= 15.2 Hz, CHv), 6.30 (br s, 2H, NH2), 5.89 (d, 1H, J = 2.4 Hz, 8.8 Hz, 2Har), 6.60 (br s, 1H, NH), 6.51 (d, 1H, J = 15.2 Hz,
H1′), 4.72 (dd, 1H, J = 4.4, 14.6 Hz), 4.63 (dd, 1H, J = 2.4, 4.8 CHv), 6.50 (d, 1H, J = 14.8 Hz, CHv), 6.02 (d, 1H, J = 5.2 Hz,
Hz, H2′), 4.59 (dd, 1H, J = 3.2, 14.4 Hz), 4.51–4.33 (m, 4H), 4.35 H1′), 5.92 (br s, 2H, NH2), 4.77 (dd, 1H, J = 4.4, 14.6 Hz), 4.71
(d, 1H, J = 17.2 Hz, CHCO), 4.11 (d, 1H, J = 17.2 Hz, CHCO), (dd, 1H, J = 6.4, 14.6 Hz), 4.60 (t, 1H, J = 4.8 Hz), 4.57 (br s,
3.63 (br s, 1H, OH), 2.83 (s, 3H, CH3), 1.50 (s, 9H, tBu), 1.45 (s, 2H), 4.54–4.53 (m, 1H), 4.44–4.41 (m, 1H), 4.39–4.37 (m, 1H),
9H, tBu); RMN 13C (CDCl3, 100 MHz): δ 171.3, 155.8 (Cq); 153.0 4.23 (d, 1H, J = 17.2 Hz, CHCO), 3.99 (d, 1H, J = 17.2 Hz,
(C2), 152.8, 149.1, 148.3, 145.1 (Cq); 140.2 (C8), 137.4, 132.8 CHCO), 2.94 (s, 3H, CH3), 1.52 (s, 9H, tBu), 1.44 (s, 9H, tBu);
(Cq); 132.4, 130.1, 128.7 (CHv); 127.6, 126.8 (CHar); 126.5 (Cq), RMN 13C (CDCl3, 100 MHz): δ 170.6, 155.9 (Cq); 153.1 (C2),
125.7 (CHv), 123.3 (CH), 120.2 (C5), 118.7, 112.7 (CHar); 90.2 152.8, 149.4, 148.3, 145.2 (Cq); 140.1 (C8), 137.4, 132.8 (Cq);
(C1′), 83.3 (Cq), 81.5 (C4′), 80.6 (Cq), 79.8 (C3′), 72.8 (C2′), 69.9, 132.4, 130.1, 128.7 (CHv); 127.5, 126.8 (CHar); 126.5 (Cq),
50.3 (CH2); 48.2 (C5′), 38.4, 28.4, 28.1 (CH3); HRMS (ESI) m/z: 125.7 (CHv), 123.3 (CH), 120.4 (C5), 118.7, 112.9 (CHar); 88.1
Calcd for C41H51N10O7 [M + H]+: 795.3942; Found: 795.3937.
(C1′), 83.3 (Cq), 83.1 (C4′), 82.3 (C2′), 80.6 (Cq), 70.4 (C3′), 69.6,
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51.1 (CH2); 48.3 (C5′), 38.6, 28.4, 28.0 (CH3); HRMS (ESI) m/z: (CD3OD, 400 MHz): δ 8.35(s, 1H, H2), 8.33 (s, 1H, H8), 7.94 (s,
Calcd for C41H51N10O7 [M + H]+: 795.3942; Found: 795.3937.
1H, CHv), 7.65 (d, 2H, J = 8.8 Hz, 2CHar), 7.52 (d, 2H, J = 8.8
Hz, 2CHar), 7.45 (d, 2H, J = 8.8 Hz, 2CHar), 7.35 (d, 2H, J = 8.8
Hz, 2CHar), 7.11–7.00 (m, 2H, 2CHv), 6.75 (d, 1H, J = 14.8 Hz,
CHv), 6.70 (d, 1H, J = 14.8 Hz, CHv), 6.20 (d, 1H, J = 4.4 Hz,
H1′), 4.81–4.76 (m, 5H), 4.48–4.31 (m, 6H), 3.05 (s, 3H, CH3);
1-[5′-Deoxy-adenosin-5′-yl]-4-[(N-methyl-N-((4-(4-(4-(N-tert-
butoxycarbonyl)amino)phenyl)buta-1,3-dienyl)phenyl))
aminomethyl]-1H-1,2,3-triazole (33)
From alkyne 13 (57 mg, 0.15 mmol) and azido adenosine 21 RMN 13C (CD3OD, 100 MHz): δ 172.4, 172.1, 151.6, 149.7 (Cq);
(43 mg, 0.15 mmol), compound 33 was obtained after purifi- 145.4 (C2), 145.2 (Cq), 144.5 (C8), 140.5, 140.2, 139.5 (Cq);
cation by column chromatography over silica gel (CH2Cl2/ 133.5, 132.7, 132.6, 131.7 (CHv); 130.8 (Cq), 129.8 (CHv),
EtOH: 9/1) as a yellow solid (80 mg, 80%). Rf = 0.49 (CH2Cl2/ 129.0, 124.5, 123.2 (CHar); 121.2 (C5), 89.7 (C1′), 82.5 (C4′), 81.6
EtOH: 9/1); mp: 162–164 °C; IR (ATR): 3347 (br s), 1709, 1641, (C2′), 79.2 (C3′), 68.6, 68.5, 55.1 (CH2); 52.9 (C5′), 45.1 (CH3);
1
1603, 1514, 1369, 1314, 1237, 1159, 1053, 980 cm−1; RMN H HRMS (ESI) m/z: Calcd for C34H37N10O7 [M + H]+: 697.2846;
(DMSO-d6, 400 MHz): δ 9.40 (s, 1H, NH), 8.21 (s, 1H, H2), 8.14 Found: 697.2841.
(s, 1H, H8), 7.75 (s, 1H, CH), 7.42–7.34 (m, 6H, NH2 + 4Har),
7.23 (d, 2H, J = 8.7 Hz, 2Har), 6.89 (dd, 1H, J = 10.6, 15.6 Hz,
CHv), 6.77 (dd, 1H, J = 10.5, 15.1 Hz, CHv), 6.69 (d, 2H, J =
9.2 Hz, 2Har), 6.53 (d, 1H, J = 15.1 Hz, CHv), 6.52 (d, 1H, J =
15.1 Hz, CHv), 5.88 (d, 1H, J = 5.0 Hz, H1′), 5.61 (d, 1H, J = 6.0
Hz, H2′), 5.47 (d, 1H, J = 4.6 Hz, H3′), 4.71–4.66 (m, 2H, CH2),
4.65–4.62 (m, 1H, H4′), 4.51 (s, 2H, 2OH), 4.23–4.20 (m, 2H,
2H5′), 2.91 (s, 3H, CH3), 1.47 (s, 9H, tBu); RMN 13C (DMSO-d6,
100 MHz): δ 156.1 (Cq), 152.7 (C2), 149.3, 148.3, 143.7 (Cq);
139.9 (C8), 138.5, 132.2 (Cq); 131.5, 129.8, 128.3 (CHv); 127.3,
126.4 (CHar); 125.4, 125.2 (Cq); 123.6 (CH), 119.2 (C5), 118.2,
112.6 (CHar); 87.8 (C1′), 82.2 (C2′), 79.1 (Cq), 72.6 (C4′) 70.9 (C3′),
51.2 (CH2), 46.8 (C5′), 38.1, 28.1 (CH3); HRMS (ESI) m/z: Calcd
for C35H40N10O5 [M + H]+: 681.3261; Found: 681.3163.
1-[5′-Deoxy-3′-O-(carboxymethyl)-adenosin-5′-yl]-4-[(N-methyl-
N-((4-(4-(4-(N-methyl)amino)phenyl)buta-1,3-dienyl)phenyl))
aminomethyl]-1H-1,2,3-triazole (3)
Deprotection of compound 29 (35 mg, 0.043 mmol) according
to the general procedure afforded 3 (30 mg, 100%) as a red
solid. mp: 172–174 °C; IR (ATR): 3327 (br), 2925, 1737, 1687,
1604, 1509, 1460, 1427, 1242, 1142, 1085, 1000, 824 cm−1
;
RMN 1H (CD3OD, 400 MHz): δ 8.43 (s, 1H, H2), 8.36 (s, 1H,
H8), 8.03 (s, 1H, CHv), 7.70 (d, 2H, J = 8.8 Hz, 2Har), 7.59 (d,
2H, J = 8.4 Hz, 2Har), 7.50 (d, 2H, J = 8.8 Hz, 2Har), 7.48 (d, 2H,
J = 8.4 Hz, 2Har), 7.16–7.09 (m, 2H, 2CHv), 6.85–6.72 (m, 2H,
2CHv), 6.05 (d, 1H, J = 5.2 Hz, H1′), 4.89–4.80 (m, 4H), 4.73 (t,
1H, J = 5.0 Hz), 4.53–4.49 (m, 1H), 4.42 (d, 1H, J = 17.0 Hz,
CHCO), 4.35 (d, 1H, J = 17.0 Hz, CHCO), 4.28 (t, 1H, J = 5.0
Hz), 3.35 (s, 3H, CH3), 3.09 (s, 3H, CH3); RMN 13C (CD3OD,
100 MHz): δ 173.0, 151.6, 149.7 (Cq); 145.4 (C2), 144.3 (C8),
140.6, 140.3, 138.2, 137.1 (Cq); 133.4, 132.9, 132.6, 132.2
(CHv); 131.7 (Cq), 129.4 (CH), 129.2, 129.1, 123.7, 123.3
(CHar); 120.9 (C5), 90.8 (C1′), 82.6 (C4′), 80.9 (C3′), 74.3 (C2′),
68.9, 55.0 (CH2); 52.9 (C5′), 45.0, 37.9 (CH3); HRMS (ESI) m/z:
Calcd for C33H36N10O5 [M + H]+: 653.2948; Found: 653.2943.
1-[5′-Deoxy-2′,3′-di-O-(carboxymethyl)-adenosin-5′-yl]-4-[(N-
methyl-N-((4-(4-(4-(N-methyl)amino)phenyl)buta-1,3-dienyl)
phenyl))aminomethyl]-1H-1,2,3-triazole (1)
Deprotection of compound 27 (118 mg, 0.128 mmol) according
to the general procedure afforded 1 (91 mg, 100%) as a red
solid. mp: 171–173 °C; IR (ATR): 2915 (br), 1737, 1688, 1597,
1509, 1462, 1420, 1230, 1151, 994 cm−1; RMN 1H (CD3OD,
400 MHz): δ 8.40 (s, 1H, H2), 8.39 (s, 1H, H8), 8.02 (s, 1H, CH),
7.68 (d, 2H, J = 8.4 Hz, 2Har), 7.54 (d, 2H, J = 8.8 Hz, 2Har), 7.51
(d, 2H, J = 8.4 Hz, 2Har), 7.42 (d, 2H, J = 8.8 Hz, 2CHar),
7.13–7.02 (m, 2H, 2CHv), 6.79 (d, 1H, J = 14.8 Hz, CHv), 6.72
(d, 1H, J = 14.4 Hz, CHv), 6.23 (d, 1H, J = 4.4 Hz, H1′),
1-[5′-Deoxy-3′-O-(carboxymethyl)-adenosin-5′-yl]-4-[(N-methyl-
N-((4-(4-(4-amino)phenyl)buta-1,3-dienyl)phenyl))
aminomethyl]-1H-1,2,3-triazole (4)
4.90–4.81 (m, 5H, CH2 + H2′ + 2H5′), 4.50–4.35 (m, 6H, H3′
+
Deprotection of compound 30 (31 mg, 0.039 mmol) according
H4′ + 2CH2), 3.32 (s, 3H, CH3), 3.09 (s, 3H, CH3); RMN 13C to the general procedure afforded 4 (25 mg, 100%) as a red
(CD3OD, 100 MHz): δ 172.5, 172.3, 151.7, 149.6 (Cq); 145.3 (C2), solid. mp: 182–184 °C; IR (ATR): 3320 (br), 3080 (br), 2930,
144.5 (C8), 141.2, 140.3, 139.4, 139.0, 137.1 (Cq); 133.2, 133.0, 2586, 1738, 1688, 1601, 1510, 1427, 1239, 1143, 1084, 993,
132.3, 132.0 (CHv); 129.3, 129.0, 123.6, 122.4 (CHar); 121.0 860 cm−1; RMN H (CD3OD, 400 MHz): δ 8.43 (s, 1H, H2), 8.38
1
(C5), 89.8 (C1′), 82.6 (C4′), 81.6 (C2′), 79.1 (C3′), 68.4, 54.4 (CH2); (s, 1H, H8), 8.09 (s, 1H, CHv), 7.65 (d, 2H, J = 8.4 Hz, 2Har),
52.6 (C5′), 44.3, 37.8 (CH3); HRMS (ESI) m/z: Calcd for 7.57–7.52 (m, 4Har), 7.42 (d, 2H, J = 8.4 Hz, 2Har), 7.10–7.04
C35H38N10O7 [M + H]+: 711.3003; Found: 711.2998.
(m, 2H, 2CHv), 6.78 (d, 1H, J = 14.0 Hz, CHv), 6.70 (d, 1H,
J = 14.0 Hz, CHv), 6.04 (d, 1H, J = 4.4 Hz, H1′), 4.95–4.80 (m,
4H), 4.74–4.73 (m, 1H), 4.51–4.49 (m, 1H), 4.43 (d, 1H, J = 17.0
Hz, CHCO), 4.36 (d, 1H, J = 17.0 Hz, CHCO), 4.30–4.29 (m,
1H), 3.35 (s, 3H, CH3); RMN 13C (CD3OD, 100 MHz): δ 172.9,
1-[5′-Deoxy-2′,3′-di-O-(carboxymethyl)-adenosin-5′-yl]-4-[(N-
methyl-N-((4-(4-(4-amino)phenyl)buta-1,3-dienyl)phenyl))
aminomethyl]-1H-1,2,3-triazole (2)
Deprotection of compound 28 (102 mg, 0.112 mmol) according 151.6, 149.7 (Cq); 145.4 (C2), 144.3 (C8), 140.7, 140.2, 139.6,
to the general procedure afforded 2 (99 mg, 100%) as a red 138.2 (Cq); 133.6, 132.7, 131.8 (CHv); 130.9 (Cq), 129.4 (CHar),
solid. mp: 171–173 °C; IR (ATR): 2923 (br), 2851, 1741, 1688, 129.1, 124.6, 123.3 (CHar); 120.9 (C5), 90.8 (C1′), 82.6 (C4′), 80.9
1605, 1509, 1426, 1232, 1153, 1086, 993, 850 cm−1; RMN 1H (C3′), 74.3 (C2′), 68.9, 55.0 (CH2); 52.9 (C5′), 45.1 (CH3); HRMS
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(ESI) m/z: Calcd for C32H35N10O5 [M + H]+: 639.2792; Found: 2CHar), 7.08–7.05 (m, 2H, 2CHv), 6.79–6.68 (m, 2H, 2CHv),
639.2786.
6.00 (d, 1H, J = 4.6 Hz, H1′), 4.85–4.79 (m, 2H, H5′ + H4′), 4.64
(t, 1H, J = 4,6 Hz, H2′), 4.32–4.29 (dd, 2H, J = 5.0 Hz, 8.7 Hz, H3′
+ H5′), 3.61 (s, 2H, CH2), 3.29–3.28 (m, 3H, CH3); RMN 13C
(CD3OD, 100 MHz): δ 151.8, 149.7 (Cq); 145.4 (C2), 144.5 (C8),
140.7, 140.2, 139.7, 138.5 (Cq); 133.6, 132.9, 132.5, 131.9
1-[5′-Deoxy-2′-di-O-(carboxymethyl)-adenosin-5′-yl]-4-[(N-
methyl-N-((4-(4-(4-(N-methyl)amino)phenyl)buta-1,3-dienyl)
phenyl))aminomethyl]-1H-1,2,3-triazole (5)
Deprotection of compound 31 (42 mg, 0.052 mmol) according (CHv); 130.9 (Cq), 129.1, 129.0, 124.6 (CHar); 124.5 (CHv),
to the general procedure afforded 5 (34 mg, 100%) as a red 122.8 (CHar), 121.0 (C5), 91.2 (C1′), 84.3 (C2′), 75.0 (C4′), 72.5
solid. mp: 116–118 °C; IR (ATR): 3060 (br), 2924, 1736, 1689, (C3′), 54.7 (CH2), 53.0 (C5′), 44.7 (CH3); HRMS (ESI) m/z: Calcd
1680, 1600, 1510, 1423, 1241, 1141, 857 cm−1; RMN 1H for C30H32N10O3 [M + H]+: 581.2737; Found: 581.2735.
(CD3OD, 400 MHz): δ 8.42 (s, 1H, H2), 8.41 (s, 1H, H8), 7.98 (d,
Spectroscopic characterization of NT compounds
1H, J = 2.4 Hz, CH), 7.70 (d, 2H, J = 8.8 Hz, 2Har), 7.57 (d, 2H,
J = 8.8 Hz, 2Har), 7.50 (d, 2H, J = 8.4 Hz, 2Har), 7.45–7.43 (m, Absorption spectra of NTs in DMSO or Tris buffer (20 mM
2Har), 7.16–7.06 (m, 2H, 2CHv), 6.85–6.71 (m, 2H, 2CHv), Tris-HCl, pH 7.4) were carried out at 22 °C with a Uvikon
6.23 (d, 1H, J = 4.4 Hz, H1′), 4.83–4.80 (m, 4H), 4.67 (t, 1H, J = spectrophotometer. Fluorescence excitation and emission
4.6 Hz), 4.45 (t, 1H, J = 5.2 Hz), 4.38 (d, 1H, J = 17.0 Hz, spectra under one-photon excitation condition (1-PE) were
CHCO), 4.36–4.30 (m, 1H), 4.30 (d, 1H, J = 17.0 Hz, CHCO), recorded at 22 °C on an Eclipse (Varian) fluorimeter.
3.35 (s, 3H, CH3), 3.09 (s, 3H, CH3); RMN 13C (CD3OD, Fluorescence quantum yields (ϕ) were measured using
100 MHz): δ 172.6, 151.6, 149.7 (Cq); 145.4 (C2), 144.7 (C8), Coumarin-1 (Sigma) in ethanol as a reference (λmax,exc
=
140.6, 140.3, 140.2, 138.4, 137.0 (Cq); 133.4, 133.0, 132.6, 132.2 373 nm; λem = 447 nm). Two-photon excitation (2-PE) and
(CHv); 129.5 (CHv), 129.3, 129.1, 123.7, 123.3 (CHar); 121.1 emission spectra were recorded using a home-built set-up.10,21
(C5), 89.7 (C1′), 84.1 (C4′), 83.3 (C2′), 71.4 (C3′), 69.0, 55.1 (CH2); Briefly, a 80 MHz mode-locked Mai-Tai® Ti:Sapphire tunable
52.9 (C5′), 45.0, 37.9 (CH3); HRMS (ESI) m/z: Calcd for laser (690–1040 nm, 100 fs laser pulse; Spectra Physics) was
C33H37N10O5 [M + H]+: 653.2952; Found: 653.2943.
focused onto the sample (80 µL) placed in a quartz micro cell.
The two-photon fluorescence signal was collected at 90°, fil-
tered by a Semrock FF01-842/SP filter – to reject the residual
excitation light – and focused into an optical fiber connected
1-[5′-Deoxy-2′-O-(carboxymethyl)-adenosin-5′-yl]-4-[(N-methyl-
N-((4-(4-(4-amino)phenyl)buta-1,3-dienyl)phenyl))
aminomethyl]-1H-1,2,3-triazole (6)
to
a QE65000 spectrometer (Ocean Optics). Two-photon
Deprotection of compound 32 (49 mg, 0.062 mmol) according absorption cross-sections (σ2) of NTs were determined as pre-
to the general procedure afforded 6 (40 mg, 100%) as a red viously described.10
solid, mp: 181–183 °C; IR (ATR): 3345 (br), 1736, 1688, 1610,
1509, 1457, 1423, 1232, 1141, 995, 848, 732 cm−1; RMN 1H
Binding affinity evaluation
(CD3OD, 400 MHz): δ 8.41 (s, 1H, H2), 8.39 (s, 1H, H8), 7.99 (s, The apparent binding constants of the NTs to the neuronal
1H, CH), 7.66 (d, 2H, J = 8.8 Hz, 2Har), 7.65 (d, 2H, J = 8.8 Hz, (nNOS) and endothelial (eNOS) isoforms of nitric oxide
2Har), 7.42 (d, 2H, J = 8.8 Hz, 2Har), 7.40 (d, 2H, J = 8.8 Hz, synthase (NOS) were assessed by changes in NT intrinsic fluo-
2Har), 7.12–7.10 (m, 2H, 2CHv), 6.84–6.69 (m, 2H, 2CHv), rescence. NOS proteins were recombinantly expressed and pur-
6.21 (d, 1H, J = 4.4 Hz, H1′), 4.90–4.80 (m, 4H), 4.68 (t, 1H, J = ified as described.22 NTs were dissolved in DMSO to make
4.8 Hz), 4.43 (t, 1H, J = 5.2 Hz), 4.37–4.26 (m, 3H), 3.32 (s, 3H, ∼10 mM stock solutions, which were diluted to 300 μM in
CH3); RMN 13C (CD3OD, 100 MHz): δ 173.9, 151.7, 149.7 (Cq); buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl). Aliquots
145.3 (C2), 144.6 (C8), 141.0, 139.8, 138.8 (Cq); 133.3, 132.9, (1 μL) were added to 100 μL buffer containing NOS protein
132.2, 131.9 (CHv); 130.9 (Cq), 129.1, 129.0 (CHar); 128.9 (5 μM), and fluorescence emission spectra were recorded from
(CHv), 124.6, 122.5 (CHar); 121.1 (C5), 89.8 (C1′), 84.2 (C4′), 430–700 nm (excitation = 420 nm; slit widths = 5 nm) on a
83.3 (C2′), 71.4 (C3′), 68.9, 54.5, 52.8 (CH2); 44.4 (CH3); HRMS Shimadzu RF-5301PC fluorometer. Spectra of intrinsic protein
(ESI) m/z: Calcd for C32H35N10O5 [M + H]+: 639.2792; Found: fluorescence and unbound NT were subtracted from protein/
639.2786.
NT spectra and the fluorescence at 460 nm was plotted against
NT concentration. Resultant curves were fit to a rectangular
hyperbola y = m1 × x/(x + m2), where m1 is maximum fluo-
rescence and m2 is the apparent binding constant, and appar-
ent binding constant was determined.
1-[5′-Deoxy-adenosin-5′-yl]-4-[(N-methyl-N-((4-(4-(4-amino)
phenyl)buta-1,3-dienyl)phenyl))aminomethyl]-1H-1,2,3-
triazole (7)
Deprotection of compound 33 (38 mg, 0.056 mmol) according
to the general procedure afforded 7 (34 mg, 100%) as a red
solid; mp: 174–176 °C; IR (ATR): 2957 (br s), 2597, 1686, 1600,
Cell culture and fluorescence imaging of NTs in HUVEC and
HeLa cells
;
RMN 1H (CD3OD, Endothelial cells (HUVEC; Sigma) were cultured in Petri dishes
1506, 1413, 1225, 1130, 1064 cm−1
400 MHz): δ 8.39 (s, 1H, H2), 8.35 (s, 1H, H8), 7.98 (s, 1H, coated with 0.2% gelatin in Endothelial Cell Growth Medium
CHv), 7.64 (d, 2H, J = 8.7 Hz, 2CHar), 7.55 (d, 2H, J = 8.7 Hz, (Cell Application).23 HeLa cells (ATCC) were cultured in
2CHar), 7.43 (d, 2H, J = 9.2 Hz, 2CHar), 7.37 (d, 2H, J = 8.7 Hz, Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco®) sup-
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plemented with 10% Fetal Bovin Serum (FBS; Gibco®) and 1%
penicillin–streptomycin (Gibco®). HUVEC and HeLa cells were
cultured at 37 °C (5% CO2) to ≈95% confluence. In all experi-
ments with HUVECs, the cells were used within passage five.
Two-photon fluorescence imaging of NT compounds in living
HUVEC and HeLa cells was performed using a multiphoton
imaging set-up previously described.10,19 Briefly, HUVEC and
HeLa cells were plated in glass bottom dishes (WillCo-dish;
WillCo Wells) and treated for varying times with NT com-
pounds at a final concentration of 5 μM. Two-photon fluo-
H. J. Gukasyan, J. A. Lafontaine, S. Mehta, B. D. Patterson,
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Bioorg. Med. Chem., 2005, 13, 47–57; (b) H. J. Montgomery,
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9 (a) E. Beaumont, J. C. Lambry, C. Gautier, A. C. Robin,
S. Gmouh, V. Berka, A. L. Tsai, M. Blanchard-Desce and
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rescence images were obtained using
a SP2 confocal
microscope (Leica MicroSystems) equipped with an oil immer-
sion ×63 objective (numerical aperture, 1.32) and an incu-
bation chamber (37 °C, CO2 5%). The excitation source was a
80 MHz mode-locked Mai-Tai® Ti:Sapphire tunable laser
(720–920 nm, 100 fs laser pulse; Spectra Physics) tuned to
760 nm (emission slit setting: 450—550 nm).
Acknowledgements
This work is supported by the Agence Nationale de la
Recherche (ANR-14-CE06-0031 to A. S. S., E. D. and J. X.), by
the National Science Foundation (NSF-CHE 1415895 to
L. J. R.), by Institut d’Alembert (FR3242, to E. D. and J. X.) and
by the government of Vietnam (to N.-H. N.). We thank
Bertrand Cinquin for technical assistance.
10 Y. Li, H. Wang, B. Tarus, M. R. Perez, L. Morellato,
E. Henry, V. Berka, A.-L. Tsai, B. Ramassamy, H. Dhimane,
C. Dessy, P. Tauc, J.-L. Boucher, E. Deprez and A. Slama-
Schwok, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 12526–
12531.
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