Supported Synthesis of Adenosine Nucleotides and Derivatives on a Benzene-Centered Tripodal Soluble Support

Article abstract of DOI:10.1002/ejoc.202100544

The first soluble-phase synthesis of adenosine nucleotides including α,β and β,γ-methylene bisphosphonate analogues on a multi-pod support is reported. Anchoring of a 2’,3’-protected purine nucleoside to the tripodal support via the nucleobase was successfully achieved using a microwave assisted Cu(I)-catalyzed azide-alkyne cycloaddition. Then, phosphorylation was performed, followed by cleavage with aqueous ammonia to provide adenine derivatives, and finally deprotection. When using benzylamine instead of ammonia, a derivative with N⁶-benzylamine adenine as nucleobase was obtained. This methodology allows to access adenosine 5’-mono, di and triphosphates, as well as various analogues of pharmacological interest in modest to good yields.

Full text of DOI:10.1002/ejoc.202100544

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Supported synthesis of adenosine nucleotides and derivatives on
a benzene-centred tripodal soluble support
Authors: Lucie Appy, Suzanne Peyrottes, and Béatrice Roy
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202100544
Link to VoR: https://doi.org/10.1002/ejoc.202100544
01/2020

10.1002/ejoc.202100544
European Journal of Organic Chemistry
FULL PAPER
Supported synthesis of adenosine nucleotides and derivatives on
a benzene-centred tripodal soluble support
Lucie Appy,[a] Suzanne Peyrottes,[a] and Béatrice Roy*[a]
[a]
Dr. L. Appy, Dr. S. Peyrottes, Dr. B. Roy
Nucleosides & Phosphorylated Effectors Team
Institute for Biomolecules Max Mousseron (IBMM)
UMR 5247 CNRS, University of Montpellier, ENSCM
Campus Triolet, cc 1705
Place Eugène Bataillon
34095 Montpellier, France
E-mail: beatrice.roy@umontpellier.fr
Supporting information for this article is given via a link at the end of the document.
Abstract: The first soluble-phase synthesis of adenosine nucleotides
including a,b and b,g-methylene bisphosphonate analogues on a
multipod support is reported. Anchoring of a 2’,3’-protected purine
nucleoside to the tripodal support via the nucleobase was successfully
achieved using a microwave assisted CuI-catalyzed azide-alkyne
cycloaddition. Then, phosphorylation was performed, followed by
cleavage with aqueous ammonia to provide adenine derivatives, and
finally deprotection. When using benzylamine instead of ammonia, a
derivative with N6-benzylamine adenine as nucleobase was obtained.
This methodology allows to access adenosine 5’-mono, di and
triphosphates, as well as various analogues of pharmacological
interest in modest to good yields.
nucleotides remains an appealing area of research. Soluble-
supported synthesis has received continuous interest in the field
of oligonucleotides as an alternative approach to solid-phase
synthesis.[11] Several protocols reported the preparation of both
DNA and RNA oligomers of limited length in gram scale without
any special equipment. The 3’-terminal protected nucleoside is
attached through its 2’ or 3’ hydroxyl via a linker to the soluble
support and elongation is carried out in the 3'→5' direction.
Interestingly, this synthetic strategy in solution involves a smaller
excess of building blocks, the scale up procedure is
straightforward and expensive solid support materials is not
required.
To formulate our work plan, we considered (i) anchoring of the
nucleoside to the support through the nucleobase, (ii) synthesis
of the supported nucleoside and phosphorylation steps, and (iii)
suitable cleavage conditions considering the stability of the final
products. In this regard, the Huisgen 1,3-dipolar cycloaddition of
azides (1,3-dipolar partner) and alkynes (dipolarophile) to form
1,2,3-triazoles has become an in-valuable tool for biorthogonal
conjugation,[12] with many applications in the field of nucleoside,
nucleotide, and oligonucleotide chemistry.[13] In 2010, Mathew
and co-workers reported the synthesis of new C(6) 1,2,3-triazole
Introduction
Nucleotides and their conjugates represent a valuable class of
biomolecules involved in key functional and structural roles in
cells. Their analogues have also found applications as therapeutic
[1]
and diagnostic agents,[2] as well as tools for the study of
biochemical and pharmacological processes.[3,4] Several
chemical methods for their preparation have been developed,[5]
implying classical solution synthesis, solid-phase synthesis,[6] and
liquid-phase synthesis using a polymer as support[7,8] or ionic
liquids.[9] New reagents and strategies have also emerged
recently.[10] Still, solution phase dominates the field. The use of
soluble supports has been regarded as an approach that could
combine the advantageous features of both solution and solid-
phase syntheses. In this regard, we previously reported strategies
using polyethyleneglycol (PEG) support to gain access to 5’-
polyphosphorylated derivatives of cytidine and analogues. This
last was based on the anchoring of the exocyclic amino group of
the pyrimidine to basolabile succinyl or traceless carboxylethoxy
linkers, followed by regioselective 5’-phosphorylation and release
from the support.[7,8] However, its application to purine derivatives
failed.[8] Therefore, we turned our attention to an alternative
soluble-supported approach to anchor purines. So far, a single
and short report on ionic-tag-assisted synthesis of 2’-
deoxyadenosine-5’-triphosphate (dATP) has been reported.[9]
Thus, a soluble-phase methodology affording purine-containing
adenosine derivatives via
a
CuI-catalyzed azide-alkyne
cycloaddition (CuAAC).[14] Noteworthy, whatever the conditions
used to perform the 1,3-dipolar cycloaddition of 6-azido-9-(b-D-
ribofuranosyl)purine and phenylacetylene, the main product of the
reaction was adenosine, originating from the reduction of the
azide moiety. However, the “click reaction” could be
accomplished using a C(6) acetylene nucleoside as dipolarophile
in the presence of azides under microwave activation.[14] The
same year, Lakshman and co-workers reported successful
CuAAC reaction of sugar-protected C(6) azidopurine nucleosides
with alkynes affording the corresponding 1,4-disubstituted
triazolyl products.[15] Several reports from Turk’s group have also
focused on CuAAC reactions between 2,6-diazidopurine
nucleosides and terminal alkynes.[16,17] They showed that 2,6-bis-
triazolylpurine nucleosides could undergo regioselective
nucleophilic aromatic substitution with S- and N-nucleophiles at
the C(6) position.[16] Similarly, SNAr-Arbuzov reactions using
triakylphosphites
derivatives.[18]
afforded
C(6)
phosphonate
purine
1
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10.1002/ejoc.202100544
European Journal of Organic Chemistry
FULL PAPER
N
N
NHZ
N3
N
N
N
N
N
N
N
N
O
N
N
O
P
O
O
P
O
P
1. Cleavage
1. Click chemistry
O
X
P O
O
N
HO
O
X
O
N
N
O
O
O
2. Deprotection
O
O
O
O
O
m
2. Functionalization
in the 5' position
m
2
HO OH
m = 0, 1, 2
O
O
O
O
X = O, CH2
Z = H, Bn
3
1
Scheme 1. Retrosynthetic strategy for the synthesis of nucleotides and derivatives.
Based on these data from the literature, we envisaged to anchor
a C-6 azidopurine nucleoside to a soluble support possessing
The synthesis of compound
2
started from inexpensive
phloroglucinol and was performed according to a procedure
adapted from the literature.[24] Reaction of phloroglucinol with an
excess of propargyl bromide and potassium carbonate in dry N,N-
dimethylformamide (DMF) for 12 h at room temperature afforded
the trialkyne 2 in 96% yield. On the other hand, 6-azido-9-b-D-
ribofuranosylpurine 3 was synthesized in two steps from 6-chloro-
9-b-D-ribofuranosylpurine (Scheme 2a) by reaction with hydrazine
hydrate, followed by treatment with sodium nitrite in aqueous
acetic acid at 0 °C (70% overall yield).[25] Then, the 2’,3’-O-
isopropylidene protecting group was introduced by treatment with
2,2-dimethoxypropane in the presence of a catalytic amount of p-
toluene sulfonic acid (PTSA) to afford 1 in 92% yield.[26] Both 6-
azidopurine and their nucleoside derivatives can exist in the
alternate tetrazolyl forms.[15,25,27] As shown in Scheme 2b, only the
tetrazole form of 1 is observed in DMF-d7 while both forms coexist
in CDCl3 (see the Supporting Information). Despite this
equilibrium, it has been shown that azide-tetrazole mixtures can
undergo successful CuAAC reactions to afford 1,4-disubstituted
triazoles.[15]
terminal alkynes via
a
copper-catalyzed azide-alkyne
cycloaddition. Cleavage from the support using an ammonia
solution would allow introduction of an amino function in place of
the triazole, and therefore release adenine as a nucleobase in the
final product (Scheme 1). In addition, substitution of the triazole
by various nucleophiles would enable access to structural
diversity. Finally, one major advantage of using a soluble support
is the opportunity to run purifications by size-exclusion
chromatography or precipitation.[11]
Herein, we report an original soluble-phase synthesis of
adenosine 5’-mono and polyphosphates using a tripodal support.
Synthetic methylene bisphosphonates of therapeutic interest
were also obtained. These include a,b-methylene ADP and its N6-
benzyl derivative, as well as b,g-methylene ATP. The advantage
of methylene bisphosphonates is the extreme stability of the P–C
bonds, which precludes any enzymatic cleavage. Hence, such
compounds can serve as chemical probes to investigate the
mechanism of phosphoryl transfer processes, as well as enzyme
inhibitors (e.g. ecto-5’-nucleotidase) with great potential as
immune-oncology agents.[19-21]
Results and Discussion
As the CuAAC is not achievable with unprotected 6-azido-9-b-D-
ribofuranosylpurine[14] but works well on the protected glycosyl
derivatives,[15] we decided to install a protecting group on the 2’,3’-
hydroxyl positions of the nucleoside, thus allowing to introduce
(poly)phosphate or bisphosphonate moieties into the 5’-position.
Our first attempts to link 2’,3’-isopropylidene 6-azido-9-b-D-
ribofuranosylpurine 1 (Scheme 1) to tetrapodal pentaerythritol-
based supports such as (2-propynyloxymethyl)methane[22] or
pentaerythrityltetrakis(3-ethynylphenyl)ether[23] via CuI-mediated
azide-alkyne ligation were unsuccessful. Indeed, we faced
several issues such as very low yields together with difficult
isolation and characterization of the supported products (data not
Scheme 2. a) Preparation of the azidonucleoside 1. Reaction conditions and
yields. (i) NH2NH2.H2O, 0 °C, 2 h; (ii) NaNO2, AcOH, 0 °C, 2 h, 70% (2 steps);
(iii) dimethoxypropane, PTSA, DMF, 40 °C, 2 h, 92%. b) Azide-tetrazole
equilibrium of 1.
shown). We then considered supports having
a
1,3,5-
trisubstituted benzene core decorated with terminal alkynes.
Gratefully, 1,3,5-tris(prop-2-yn-1-yloxy)benzene 2 allowed us to
achieve our goals. As outlined in Scheme 1, the supported
nucleoside can readily be obtained by “click chemistry” between
azidonucleoside
(dipolarophile).
1
(1,3-dipolar partner) and derivative 2
2
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10.1002/ejoc.202100544
European Journal of Organic Chemistry
FULL PAPER
At the outset, the best conditions described for the CuAAC
reaction of sugar-protected C-6 azidopurine nucleosides with
phenylacetylene were considered.[15] However, only reduction of
1 into 2’,3’-O-isopropylidene adenosine was observed in the
presence of CuSO4/Na ascorbate in a biphasic CH2Cl2/H2O
system (data not shown). Indeed, reduction of the azide moiety to
the corresponding amine is a competing process in Cu-mediated
azide–alkyne ligation.[14-15]
N
N
N
N
N
N
N
N
N
i, ii
N
iii, iv, v
N
O
P
H
O
4
O
O
O
P
O
N
N
N
O
O
O
O
O
O O
Thus, optimization was carried out in DMF in the presence of 4
equiv. of azidonucleoside per support, CuI as catalyst and N,N-
diisopropylethylamine (DIPEA) as additive.[28] While no
cycloaddition was observed at room temperature (Table 1, entry
1), heating the reaction mixture at 80 °C afforded the 1,4-triazole
4 in 30 min (Table 1, entry 2). The reaction was easily monitored
by TLC since 4 shows fluorescence extinction at 254 nm as well
as a blue spot upon irradiation at 365 nm. To our delight, the
conversion was improved when microwave (MW) activation was
applied,[28] allowing to reduce both reaction time and temperature.
The best conditions involved the use of CuI for 10 min at 40 °C
under microwave irradiations, giving rise to the supported
nucleoside 4 in 97% yield, after precipitation from iced water
(Table 1, entry 3). Therefore, in agreement with previous reports
on click reactions with C-6 azidonucleosides,[14,15] we found that
the copper-catalyzed cycloaddition of azide 1 and trialkyne 2 can
be performed in excellent yields. Before carrying on the
phosphorylation steps, we assessed the feasibility of cleavage of
the supported nucleoside. Pleasingly, treatment of 4 with
NH4OH/N-methylpyrrolidone (4:1) at 50 °C for 24 h afforded
quantitatively 2’,3’-isopropylidene adenosine (data not shown).
We thus moved forward with the next steps.
3
3
5
6
Scheme 3. Preparation of supported compound 6. Reaction conditions and
yields: (i) diphenylphosphite (20 equiv.), anhydrous pyridine, rt, 20 min; (ii)
triethylamine/water 1:1, rt, 1 h, 82%; (iii) bis(trimethylsilyl)acetamide (20 equiv.),
DMF, 50 °C, 2 h; (iv) DCSO (10 equiv.), rt, 2 h; (v) triethylamine/water 1:1, rt, 1 h,
60%.
Addition of water/NEt3 to the reaction mixture resulted in rapid
hydrolysis of the nucleoside phenyl H-phosphonate diester
intermediate (not isolated) to 5, which was easily purified by size-
exclusion chromatography. Silylation of
5
with N,O-
bis(trimethylsilyl)acetamide (BSA) allowed the quantitative
formation of the bis(trimethylsilyl)-phosphite, as shown by 31P
NMR (Figure 1). Then, oxidation with (−)-(8,8-dichlorocamphoryl-
sulfonyl)oxaziridine
(DCSO)
to
the
corresponding
bis(trimethylsilyl)phosphate, followed by hydrolysis gave rise to
the supported nucleotide 6 in 60% overall yield (Scheme 3). A
widely used P(V) method to prepare 5’-polyphosphorylated
derivatives involves phosphorimidazolide intermediates.[5a,c] Thus,
activation of tri- or tetra-alkylammonium salts of a 5’-nucleotide
with N,N’-carbonyldiimidazole (CDI) in dry DMF, followed by
addition of the selected phosphoryl reagent affords a wide range
of nucleotide derivatives. Accordingly, activation of supported
nucleoside monophosphate 6 by an excess of CDI allowed to
obtain quantitatively phosphorimidazolide 7 (Scheme 4), as
shown by 31P NMR (singlet at −10.6 ppm). After completion, the
reaction mixture was treated with phosphate or pyrophosphate to
First, we considered phosphorylation with POCl3 in
triethylphosphate according to a protocol optimized for PEG-
supported synthesis.[7,8] However, these conditions led to
degradation and low yields (data not shown). We then used P(III)
chemistry involving a H-phosphonate intermediate (Scheme
3).[7,29] Accordingly, the nucleoside 5’-H-phosphonate monoester
5 was prepared through transesterification of diphenylphosphite
with 4 in pyridine.
afford the supported nucleoside 5’-diphosphate
8 or 5’-
triphosphate 9, respectively (Scheme 4). Isolation was easily
performed by size-exclusion chromatography since the final
compounds elute rapidly due to their high size compared to the P-
reagents. Yields ranged from 68 to 78% over 3 steps.
Table 1. Optimization of reaction conditions for the click reaction.
Entry[a]
Conditions
4, yield (%)[b]
1
2
3
rt, 24 h
–
80 °C, 30 min
MW, 40 °C, 850 W, 10 min
71
97
Figure 1. Stepwise conversion of 5 to 6 monitored by 31P NMR. Aliquots of the
reaction medium were taken at time intervals and diluted in a deuterated solvent
for analysis. a) Before addition of BSA (D2O), b) 2 h after addition of BSA
(CDCl3), c) 2 h after addition of DCSO (CDCl3), d) 1 h after addition of NEt3/H2O
(DMSO-d6).
[a] Reaction conditions: 2 (0.2 mmol), 1 (0.8 mmol, 4.0 equiv), CuI (0.06 mmol,
0.3 equiv), DIPEA (1.2 mmol, 6.0 equiv), DMF (2.0 mL). [b] Isolated yield after
precipitation.
3
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10.1002/ejoc.202100544
European Journal of Organic Chemistry
FULL PAPER
N
N
N
N
N
NH2
N
N
N
N
N
N
N
N
N
3
iii or iv
v, vi, vii
v, vi, vii
O
P
i,ii
O
P
O
P
O
P
O
P
N
6
N
O
O
O
O
N
O
O
O
N
N
N
O
O
O
O
O
O
O
O
m
3 Na
v, vi, vii
O
O
HO OH
O
O
3
11 from 8
7
NH2
NH2
8 : m = 1
9 : m = 2
N
N
N
N
N
O
P
O
P
O
O
P
O
O
O
P
O
N
O
O
N
N
O
O
O
O
O
O
4 Na
2 Na
HO OH
HO OH
10
12 from 9
Scheme 4. Access to the supported nucleotides 7–9 and final compounds 10–12. Reaction conditions and yields: (i) CDI (6 equiv.), DMF, rt, 1 h; (ii) MeOH (20
equiv.), rt, 30 min; (iii) tri-n-butylammonium phosphate (15 equiv), rt, 20 h, 78% for 8; (iv) bis(tri-n-butylammonium) pyrophosphate (15 equiv), rt, 20 h, 68% for 9;
(v) NH4OH 28%, 50 °C, 1-2 h; (vi) Dowex H+, H2O/MeOH (v/v 1:1), 5h; (vii) Dowex Na+, 40-66% over 3 steps.
1H NMR characterization of supported nucleoside and nucleotides
was puzzling due to peak broadening that was presumably
associated with intermolecular interactions. Nevertheless, 31P
NMR and HRMS were consistent with the expected structures.
Supported nucleotides 6 and 8−9 were first treated with
concentrated aqueous ammonia (Scheme 4). After completion of
the reaction, monitored by HPLC, the adenosine derivatives were
purified by RP-18 chromatography, followed by ion exchange on
Dowex-Na+, and fully characterized (see the Supporting
Information). In the second step, removal of the acetonide group
was performed using Dowex-H+ in H2O/MeOH. The resin was
filtered off and then cation exchange was performed to provide
AMP 10, ADP 11 and ATP 12 as sodium salts. Pleasingly, we
found that the two first steps could be carried out in a single pot,
therefore increasing yields. Accordingly, compounds 10−12 were
obtained in 40–66% (Scheme 4).
methylene bisphosphonates were obtained by 5’-phosphorylation
of 4 with methylene bis(phosphonic dichloride) (Scheme 5).[30]
The reaction was carried out at 0 °C, with a 20-fold excess of the
P-reagent in trimethylphosphate, and then quenched by addition
of aqueous triethylammonium bicarbonate buffer (TEAB). The
desired compound 13a was obtained together with the
deprotected side product 13b, probably due to HCl formation
during the reaction. After
a straightforward size-exclusion
chromatography, a 1:1 mixture of the triethylammonium salts of
these two bisphosphonates was obtained in 37% yield. This
mixture could be directly used in the next steps, i.e. cleavage with
concentrated aqueous ammonia and deprotection, to give rise to
adenosine 5’-O-a,b-methylenediphosphate 14 in 31% yield
(Scheme 5). In addition, to extend the scope of our methodology,
we also performed the cleavage step with benzylamine instead of
ammonia. Accordingly, treatment of the mixture of 13a and 13b
with benzylamine in water, followed by a deprotection step and
cation exchange gave rise to compound 15 in 30% yield (Scheme
5).
Finally, we expanded the scope of our strategy to relevant
bisphosphonates such as a,b-methylene ADP and its N6-benzyl
derivative which are a potent inhibitors of ecto-5’-nucleotidase
(CD73) and exhibit pharmacological interest.[19-21] Supported a,b-
4
i, ii
N
N
N
HN
N
NH2
N
N
N
N
N
N
N
N
O
P
O
P
O
P
O
P
O
P
O
P
vi,iv,v
iii,iv,v
O
CH2
O
O
CH2
O
O
CH2
O
N
N
N
O
O
O
O
O
O
O
O
O
3 Na
3 Na
RO OR
HO OH
HO OH
15
14
3
13a: R = C(CH3)2 (2',3'-isopropylidene)
13b: R = H
Scheme 5. Phosphorylation, cleavage and deprotection steps to give a,b-bisphosphonates analogues 14 and 15. Reaction conditions: (i) CH2(POCl2)2 (20 equiv.),
trimethyl phosphate, 0 °C, 8 h; (ii) TEAB buffer, 0.5 M pH 7.6, rt, 15 min; (iii) NH4OH 28%, 50 °C, 1-2 h; (iv) Dowex H+, H2O/MeOH (v/v 1:1), 5h; (v) Dowex Na+; (vi)
Benzylamine, H2O, 50 °C, 1 h. Yields: 30−31% for the last 3 steps.
4
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10.1002/ejoc.202100544
European Journal of Organic Chemistry
FULL PAPER
were distilled from CaH2, trimethyl phosphate over BaO under reduced
pressure. Solids were dried over KOH pellets or P2O5 under reduced
pressure at rt. Moisture sensitive reactions were performed under argon
atmosphere using oven-dried glassware. Tri-n-butylammonium
phosphate,[32] bis(tri-n-butylammonium) pyrophosphate[33] and bis(tri-n-
butylammonium) methylenebisphosphonate[34] were obtained following
previously published procedures. Reactions that required heating were
performed in a hot plate/heating block apparatus with internal temperature
control. Microwave irradiation experiments were carried out in an Anton
Paar microwave apparatus with continuous irradiation power of 850 W.
Sealed reaction vessels were used and the reaction temperature was
monitored with an internal probe. Analytical thin-layer chromatography
was performed with commercial plates of silica gel 60, visualized with
short-wavelength UV light (254 nm), and stained with a 5% ethanolic
sulphuric acid, or using KMnO4 dip with sub-sequent heating. Preparative
flash chromatography was performed on silica gel 60 (particle size: 0.040–
0.063 mm). Size exclusion chromatography was performed using Bio Gel
P–2 Gel fine and H2O as eluant. Ion exchange chromatography was
carried out by percolation on a Dowex 50W-X8 (Na+ form) resin and H2O
as eluant followed by lyophilization. HPLC-UV analysis was performed
using C18 3.5 µm 4.6 ´ 100 mm column. The mobile phase consisted of:
(A) Trifluoroacetic acid 0.1% in water and (B) ACN applied at a flow rate
of 0.3 mL.min-1 in a gradient mode. A linear 15 min gradient from 0% to
100% B, followed by 5 min isocratic 100% B, and then a linear 1 min
N
N
N
N
N
N
3
O
O
P
O
P
i
7
O
P CH2
O
O
O
N
O
O
O
O
O
16
ii,iii, iv
NH2
N
N
O
O
O
O
P
CH2
P
O
P
O
N
N
O
O
O
O
4 Na
HO OH
17
Scheme 6. Preparation of b,g-methylene triphosphate 17. Reaction conditions.
(i) bis(tri-n-butylammonium) methylene bisphosphonate (15 equiv), rt, 20 h,
53%; (ii) NH4OH 28%, 50 °C, 1-2 h; (iii) Dowex H+, H2O/MeOH (v/v 1:1), 5h; (iv)
Dowex Na+, 58% over 3 steps.
gradient from 100% to 0 % B was used. 1H NMR (300 or 400 MHz),13
C
NMR (101, 126 or 151 MHz), and 31P NMR (162 MHz) spectra were
obtained in CDCl3 (dH 7.26 ppm, dC 77.16 ppm) or DMSO-d6 (dH 2.50 ppm,
dC 39.52 ppm) or D2O (dH 4.79 ppm). Data are reported as follow: chemical
shift (d, ppm), multiplicity (s = singlet; d = doublet; dd = double doublet; t =
triplet; q, quartet; bs = broad signal; and m = multiplet or not resolved
signal). Coupling constants (J) are quoted in Hertz. High-resolution mass
spectra (HRMS) were recorded on a Q-TOF Synapt-G2-S instrument using
electrospray ionization (ESI) in positive or negative ion polarity mode.
Finally, supported adenosine b,g-methylene triphosphate 16 was
obtained by conversion of 6 to 7 and then substitution of the
imidazole with methylene bis(phosphonate) as shown in Scheme
6. Compound 16 was subjected to cleavage with NH4OH, removal
of the protecting group and ion exchange to afford adenosine b,g-
methylene triphosphate 17 in 38% yield.
6-Azido-9-(2’,3’-O-isopropylidene-
b
-D-ribofuranosyl)purine
(1).
Compound 3 (2 g, 6.83 mmol, 1 equiv) was dissolved in dry DMF (46 mL),
then 2,2-dimethoxypropane (4.2 mL, 34.12 mmol, 5 equiv) and p-
toluenesulfonic acid (300 mg, 1.74 mmol, 0.25 equiv) were added. The
reaction mixture was stirred at 40 °C for 2 h, under an argon atmosphere.
The volatiles were evaporated under reduced pressure and the residue
was dissolved in water (100 mL). The aqueous layer was extracted with
ethyl acetate (4 ´ 50 mL) and the combined organic layers were washed
with water (3 ´ 30 mL), dried on Na2SO4 and concentrated under reduced
pressure. The desired compound was isolated as a white solid (2.108 g,
92% yield). Rf = 0.40 (CH2Cl2/MeOH 95:5). In CDCl3, this compound is
present as a mixture of tetrazole and azido forms. Signals of each form are
detailed in the following NMR description (A for azide and T for tetrazole,
T/A 25/75 in CDCl3). 1H NMR (400 MHz, CDCl3): d = 9.55 (s, 1H, T), 8.63
(s, 1H x 3, A), 8.57 (s, 1H, T), 8.10 (s, 1H x 3, A), 6.28 (d, J = 3.5 Hz, 1H,
T), 5.94 (d, J = 4.7 Hz, 1H x 3, A), 5.21 (dd, J = 6.1, 3.5 Hz, 1H, T), 5.20–
5.16 (m, 1H x 3, A), 5.14–5.06 (m, 1H + 1H x 3, A+T), 4.59–4.57 (m, 1H,
T), 4.54–4.52 (m, 1H x 3, A), 4.05–3.78 (m, 2H + 2H x 3, A+T), 1.67 (s, 3H,
T), 1.64 (s, 3H x 3, A), 1.40 (s, 3H, T), 1.37 (s, 3H x 3, A); 13C NMR (101
MHz, CDCl3): d = 154.4, 152.1, 151.0, 143.6, 143.0, 134.2, 125.6, 114.4,
94.4, 93.3, 86.8, 86.4, 84.7, 83.3, 81.7, 81.5, 63.4, 63.1, 29.8, 27.7, 27.4,
Conclusion
We have developed a new synthetic approach of adenine
nucleoside phosphoesters and methylenebisphosphonate
derivatives using a tripodal soluble support. Immobilization of the
C(6) azidopurine nucleoside to the alkyne trisubstituted benzene
core decorated with terminal alkynes was performed efficiently by
using the CuAAC reaction. Phosphorylation followed by cleavage
from the support and removal of the protecting groups gave the
desired products in modest to good yields. Using benzylamine
instead of ammonia in the cleavage step allowed formation of a
nucleotide with N6-benzylamine adenine as nucleobase. Thus,
the use of other nucleophiles could gain access to relevant N6-
substituted ATP, such as analogues described for the “bump and
hole” approach proposed by the Shokat research group.[31] The
opportunity to follow the reactions by HPLC or 31P NMR
spectroscopy is one of the attractive features of this soluble-
supported approach. Interestingly, most of the isolation
procedures are based on straightforward precipitation or size-
chromatography, which led to minimize the overall process time
compared to solution synthesis.
+
25.3; HRMS (ESI) m/z: [M+H]+ calcd. for C13H16N7O4 334.1264, found
334.1262.
1,3,5-Tris(prop-2-yn-1-yloxy)benzene (2).[24] Phloroglucinol (1 g, 7.9
mmol, 1 equiv) was dissolved in dry DMF (15 mL), under an inert
atmosphere, and K2CO3 (5.47 g, 40 mmol, 5 equiv) was added. After 30
min at rt, propargyl bromide (3.54 mL of an 80% w/w solution in toluene,
40 mmol, 5 equiv) was added. The reaction mixture was stirred at rt for 12
h and the solution turned dark. The crude mixture was concentrated in
vacuo. The residue was dissolved in CH2Cl2 and the organic layer was
washed with brine (3 ´ 60 mL). The aqueous layer was extracted three
times with CH2Cl2 (3 ´ 50 mL). The organic layers were combined, dried
over Na2SO4 and evaporated under reduced pressure. The product was
isolated by flash chromatography on silica gel (CH2Cl2/Cyclohexane
Experimental Section
General: All the chemicals and solvents were purchased from commercial
suppliers and used without further purification. Anhydrous DMF and
pyridine, and Et2O were commercially available, whereas MeOH and DCM
5
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202100544
European Journal of Organic Chemistry
FULL PAPER
1:1).1.83
g
(7.61 mmol, 96% yield), white solid. Rf
=
0.56
addition of (−)-(8,8-dichlorocamphorylsulfonyl)oxaziridine (491 mg, 1.7
mmol, 10 equiv). After 2 h at rt, the conversion was checked by 31P NMR
in CDCl3 and then the reaction was treated by an excess of
triethylamine/H2O 1:1, for 1 h at rt. The volatiles were evaporated under
reduced pressure and the residue was dissolved in water. The aqueous
layer was extracted three times with CH2Cl2 (3 ´ 40 mL). The aqueous
layers were combined, lyophilized. The crude product was purified by Bio
Gel P–2 Gel, fine.[35] 152 mg (0.10 mmol, 60% yield), brown solid.1H NMR
(400 MHz, D2O) d = 9.02 (bs, 1H), 8.97 (bs, 1H), 8.90 (bs, 1H), 6.44 (bs,
2H), 5.43 (bs, 2H), 5.19–5.18 (bs, 2H), 4.68 (bs, 1H), 3.95 (bs, 2H), 3.13
(CH2Cl2/cyclohexane 5:5); 1H NMR (400 MHz, CDCl3) d = 6.27 (s, 3H),
4.65 (d, J = 2.4 Hz, 6H), 2.53 (t, J = 2.4 Hz, 3H); 13C NMR (151 MHz,
CDCl3): d = 159.5, 95.5, 75.9, 56.1; HRMS (ESI) m/z: [M+H]+ calcd. for
C15H13O3+ 241.0859, found 241.0867.
6-Azido-9-(
b
-D-ribofuranosyl)purine (3).[25] 6-Chloro-(b-D-ribofuranosyl)-
purine (6 g, 21 mmol, 1 equiv) was added portionwise to hydrazin hydrate
solution (57 mL, 50-60% in water) at 0 °C. The reaction mixture was stirred
for 2 h at 0 °C, concentrated under vacuum and then co-evaporated with
isopropanol (4 ´ 40 mL). The crude residue (5.92 g) was directly used in
the next step and dissolved in a mixture of AcOH (6 mL, 105 mmol, 5 equiv)
and water (90 mL). A solution of NaNO2 (1.74 g, 25 mmol, 1.2 equiv) in
water (60 mL) was slowly added to the previous acidic solution and the
resulting mixture was stirred at 0 °C for 2 h. The precipitate was filtered-
off, washed with cold water, and then recrystallized from water (340 mL).
The desired product was isolated as a white powder (4.32 g, 70% yield).
Rf = 0.55 (CH2Cl2/MeOH 8:2); 1H NMR (300 MHz, DMSO-d6): d = 10.16 (s,
1H), 8.94 (s, 1H), 6.16 (d, J = 5.2 Hz, 1H), 5.64 (d, J = 5.9 Hz, 1H), 5.31
(d, J = 5.3 Hz, 1H), 5.12 (t, J = 5.4 Hz, 1H), 4.58 (dd, J = 10.8, 5.2 Hz, 1H),
4.22 (dd, J = 9.5, 4.9 Hz, 1H), 4.05–4.01 (m, 1H), 3.79–3.54 (m, 2H); 13C
NMR (101 MHz, DMSO-d6): d = 145.4, 142.6, 141.9, 136.1, 120.2, 88.4,
(q, J = 7.3 Hz, 2H), 1.64 (s, 3H) , 1.41 (s, 3H), 1.21 (t, J = 7.3 Hz , 3H); 31
P
NMR (162 MHz, D2O) d = 2.9; HRMS (ESI) m/z: [M+H]+ calcd. for
C54H55N21O24P3+ 1474.2591, found 1474.2849. HPLC: rt = 4.44 min (lmax
= 285 nm).
General procedure for the synthesis of 8-9 via phosphorimidazolide
7 (Scheme 4). Compound 6 (1 equiv) was dried by co-evaporation with
anhydrous pyridine and the residue was dissolved in anhydrous DMF (6
mL/0.1 mmol). Then CDI (6 equiv) was added and the reaction mixture
was stirred at rt for 1 h. The conversion to the intermediate 7 was
monitored by 31P NMR. After completion, the reaction mixture was treated
by an excess of dry MeOH (20 equiv) and stirring was pursued for 30 min.
Finally, the phosphorylated reagent (15 equiv) was added and the reaction
mixture stirred at rt for 20 h. The solvents were removed under reduced
pressure and the residue was dissolved in water. The product was isolated
by chromatography on Bio Gel P–2 Gel, fine, followed by freeze-drying.[36]
85.7, 74.5, 70.0, 60.9; HRMS (ESI) m/z: [M+H]+ calcd. for C10H12N7O4
294.0951, found 294.0952.
+
Supported nucleoside (4). Compound 2 (50 mg, 0.2 mmol, 1 equiv) was
dissolved in dry DMF (2 mL) in a 10 mL glass vial equipped with magnetic
stirring bar. Then compound 1 (277 mg, 0.83 mmol, 4 equiv), CuI (13 mg,
0.066 mmol, 0.3 equiv) and dry DIPEA (0.209 mL, 1.2 mmol, 6 equiv) were
added to the solution. The vial was sealed and heated under microwave
irradiation at 40 °C for 10 min. The conversion was monitored by TLC
(AcOEt/Cyclohexane 6:4, KMnO4 dip) until starting material disappeared.
The formation of the desired product was observed at l = 365 nm
(CH2Cl2/MeOH 9:1). The volatiles were removed in vacuo, then the crude
product was dissolved in 30 mL CH2Cl2 and 30 mL water was added. The
aqueous layer was extracted three times with CH2Cl2 (3 ´ 15 mL). The
combined organic layers were dried over Na2SO4 and concentrated. The
crude product was purified by precipitation in iced water.[35] 240 mg (0.19
mmol, 97% yield), brown solid. 1H NMR (400 MHz, CDCl3): d = 9.14 (bs,
1H), 8.91 (bs, 1H), 8.44 (bs, 1H), 6.38 (bs, 1H), 6.11 (d, J = 4 Hz, 1H),
5.34–5.10 (m, 3H), 4.58 (bs, 2H), 4.03–3.80 (m, 2H), 1.67 (s, 3H), 1.40 (s,
3H); HRMS (ESI) m/z: [M+H]+ calcd. for C54H58N21O15+ 1240.4416, found
1240.4423. HPLC: rt = 5.05 min (lmax = 286 nm).
Supported phosphorimidazolide intermediate (7). 31P NMR (162 MHz,
D2O) d = -10.6 (s).
Supported 2’,3’-O-isopropylidene adenosine 5’-diphosphate (8).
Compound 8 was prepared from 6 (140 mg, 0.067 mmol) according to the
general procedure. 90 mg (0.052 mmol, 78% yield), brown solid. 1H NMR
(400 MHz, D2O) d = 8.67 (bs, 2H), 7.44 (bs, 1H), 6.30 (bs, 2H), 5.43–5.21
(m, 4H), 4.22–4.11 (m, 3H), 1.72 (s, 3H), 1.49 (s, 3H); 31P NMR (162 MHz,
D2O) d = - 10.8 (d, J = 20.8 Hz), –11.7 (d, J = 20.8 Hz). HPLC: rt = 4.16
min (lmax = 284 nm).
Supported 2’,3’-O-isopropylidene adenosine 5’-triphosphate (9).
Compound 9 was prepared from 6 (140 mg, 0.067 mmol) according to the
general procedure. 50 mg (0.025 mmol, 68% yield), brown solid. 1H NMR
(400 MHz, D2O) d = 8.71 (br, 2H), 7.43 (s, 1H), 6.28 (s, 2H), 5.50–5.00 (m,
4H), 4.26 (bs, 3H), 1.66 (s, 3H), 1.45 (s, 3H); 31P NMR (162 MHz, D2O) d
= –10.5 (bs, Pg), –11.7 (bs, Pa), –22.9 (bs, Pb); HPLC: rt = 4.24 min (lmax
= 284 nm).
Supported 2’,3’-O-isopropylidene adenosine H-phosphonate (5).
Diphenyl phosphite (0.305 mL, 1.6 mmol, 20 equiv) was added to a
solution of compound 4 (100 mg, 0.08 mmol, 1 equiv) in anhydrous
pyridine (3.75 mL). The reaction mixture was stirred at rt during 20 min.
The conversion was checked by TLC (CH2Cl2/MeOH 9:1). The reaction
mixture was treated with a solution of triethylamine and water (1.25 mL,
1:1) and stirred for 1 h at rt, then the volatiles were removed under reduced
pressure. The residue was dissolved in water and the aqueous layer was
extracted three times with CH2Cl2 (3 ´ 20 mL). The aqueous layer was
freeze-dried. The crude product was purified by Bio Gel P–2 Gel fine.[35]
93 mg (0.06 mmol, 82% yield), brown solid.1H NMR (400 MHz, CH3OD) d
= 8.75 (bs, 1H), 8.61 (bs, 1H), 7.42 (bs, 1H), 6.62 (d, JPH = 644 Hz, 1H),
6.27 (bs, 1H), 6.07 (bs, 1H), 5.43-5.42 (bs, 2H), 5.19-5.18 (bs, 2H),
4.68-4.65 (bs, 1H), 4.09 (bs, 2H), 3.27 (q, J = 7.3 Hz, 2H), 1.67 (s, 3H),
1.46 (s, 3H), 1.25 (t, J = 7.3 Hz, 3H); 31P NMR (162 MHz, CH3OD) d = 5.9
(s); HRMS (ESI) m/z: [M+H]+ calcd. for C54H61N21O21P3+ 1432.3558, found
1432.3556. HPLC: rt = 3.51 min (lmax = 285 nm).
General procedure for the one-pot cleavage and removal of the
protecting group (Scheme 4): The supported 2’,3’-O-isopropylidene
adenine derivative (1 equiv) was dissolved in 1 mL of concentrated
aqueous ammonia solution. The reaction mixture was stirred at 50 °C in a
sealed tube for 1–2 h. After evaporation under reduced pressure, the crude
product was dissolved in H2O/MeOH (1:1, 2 mL) and Dowex resin (H+ form,
50 mg) was added. The mixture was heated at 50 °C for 5 h. The
conversion was checked by HPLC. The resin was removed by filtration and
the filtrate was percolated through a column of Dowex resin 50WX8 (Na+
form) followed by freeze-drying. The last purification step was performed
with either method A or B. Method A: The crude product was applied to a
plug of C18-AQ-RP-silica, eluted with a gradient of water to methanol, and
the product-containing fractions were combined and concentrated under
reduced pressure. Method B: The crude product was purified by DEAE-
Sephadex using a linear gradient from 10-3 M to 0.6 M TEAB pH 7.5. Pure
fractions were combined and concentrated under high vacuum.
Supported 2’,3’-O-isopropylidene AMP (6). Compound 5 (294 mg, 0.17
mmol, 1 equiv) was dissolved in 10 mL of anhydrous DMF, and
bis(trimethylsilyl)acetamide (0.83 mL, 3.3 mmoles, 20 equiv) was added.
The reaction mixture was stirred at 50 °C for 2 h affording the silylated
phosphite intermediate. The conversion was checked by 31P NMR in
CDCl3. The mixture was cooled to rt and then oxidation was carried out by
Adenosine 5’-monophosphate disodium salt (10).[37] The general
procedure was carried out starting from 6 (236 mg, 0.11 mmol). The crude
product was purified according to method A. 17 mg (0.043 mmol, 40%
yield), white solid. 1H NMR (400 MHz, D2O): d = 8.55 (s, 1H), 8.17 (s, 1H),
6.07 (d, J = 6.0 Hz, 1H), 4.77–4.73 (m, 1H), 4.45 (dd, J = 5.0, 3.4 Hz, 1H),
6
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202100544
European Journal of Organic Chemistry
FULL PAPER
4.31–4.30 (m, 1H), 3.94 (t, J = 3.7 Hz, 2H); 13C NMR (126 MHz, D2O): d =
155.5, 152.7, 149.0, 140.1, 118.5, 86.6, 84.8 (d, J = 8.8 Hz), 74.4, 70.7,
63.3; 31P NMR (162 MHz, D2O) d = 3.6 (s); HRMS (ESI) m/z: calcd for
C10H13N5O7P– [M-H]– 346.0558, found 346.0561. HPLC: rt = 3.21 min
(lmax = 258 nm).
Supported 2’,3’-O-isopropylidene adenosine 5’- O- , -methylene-
b g
triphosphate (16). Compound 6 (1 equiv) was dried by co-evaporation
with anhydrous pyridine and the residue was dissolved in anhydrous DMF
(6 mL/0.1 mmol). Then CDI (6 equiv) was added and the reaction mixture
was stirred at rt for 1 h. The conversion to the intermediate 7 was
monitored by 31P NMR. After completion, the reaction mixture was treated
by an excess of dry MeOH (20 equiv) and stirring was pursued for 30 min.
Finally, bis(tri-n-butylammonium) methylene bisphosphonate (15 equiv)
was added and the reaction mixture stirred at rt for 20 h. The solvents were
removed under reduced pressure and the residue was dissolved in water.
The product was isolated by chromatography on Bio Gel P–2 Gel, fine,
followed by freeze-drying.[36] The reaction was performed starting from 6
(140 mg, 0.067 mmol). 114 mg (0.036 mmol, 53% yield), brown solid. 1H
NMR (400 MHz, D2O) d = 8.69 (bs, 2H), 7.43 (bs, 1H), 6.34 (bs, 2H), 5.43–
5.28 (m, 4H), 4.23-4.00 (m, 3H), 3.08 (bs, 4H), 2.35 (bs, 2H, PCH2P), 1.62
(bs, 3H), 1.33 (bs, 3H), 0.88 (bs, 6H); 31P NMR (162 MHz, D2O) d = 15.3
(s, Pg), 8.0 (s, Pb), –11.7 (s, Pa). HPLC: rt = 4.25 min (lmax = 284 nm).
Adenosine 5’-diphosphate trisodium salt (11).[38] The general
procedure was carried out starting from 8 (190 mg, 0.07 mmol). The crude
product was purified according to method A. 23 mg (0.047 mmol, 66%
yield), white solid. 1H NMR (400 MHz, D2O) d = 8.45 (s, 1H), 8.15 (s, 1H),
6.06 (d, J = 5.6 Hz, 1H), 4.70 (m, 1H), 4.52 (m, 1H), 4.33 (bs, 1H), 4.17 (m,
2H); 13C NMR (126 MHz, D2O): d = 155.4, 152.2, 148.9, 139.7, 118.4, 86.8,
83.8 (d, J = 9.0 Hz), 74.2, 70.1, 64.7 (d, J = 4.9 Hz); 31P NMR (202 MHz,
D2O): d = - 9.2 (d, J = 20.7 Hz), -11.1 (d, J = 20.7 Hz); HRMS (ESI) m/z:
+
[M-H]- calcd. for C10H16N5O10P2 428.0367, found 428.0369. HPLC: rt =
3.08 min (lmax = 258 nm).
Adenosine 5’-triphosphate tetrasodium salt (12).[39] The general
procedure was carried out starting from 9 (190 mg, 0.09 mmol). The crude
product was purified according to method A. 31 mg (0.053 mmol, 58%
yield), white solid. 1H NMR (400 MHz, D2O) d = 8.43 (s, 1H), 8.14 (s, 1H),
6.05 (d, J = 6 Hz, 1H), 4.70-4.49 (m, 2H), 4.32-4.31 (m, 2H), 4.15-4.14
(m, 1H); 13C NMR (126 MHz, D2O): d = 155.5, 152.6, 149.1, 139.8, 118.5,
86.8 (d, J = 31.2 Hz), 83.9 (dd, J= 17.6 Hz, 9.0 Hz), 74.2 (d, J = 7.9 Hz),
70.3, 65.1 (d, J = 5.2 Hz); 31P NMR (162 MHz, D2O) d = -9.2 (bs, Pg), -11.0
Adenosine 5’-O- ,
b g
-methylenetriphosphate tetrasodium salt (17).[41]
The supported compound 16 (232 mg, 0.073 mmol, 1 equiv) was dissolved
in 1 mL of concentrated aqueous ammonia solution. The reaction mixture
was stirred at 50 °C in a sealed tube for 1–2 h. After evaporation under
reduced pressure, the crude product was dissolved in H2O/MeOH (1:1, 2
mL) and Dowex resin (H+ form, 50 mg) was added. The mixture was
heated at 50 °C for 5 h. The conversion was checked by HPLC. The resin
was removed by filtration and the filtrate was percolated through a column
of Dowex resin 50WX8 (Na+ form). The crude product was purified
according to method A. 16 mg (0.027 mmol, 38% yield), white solid. 1H
NMR (400 MHz, D2O) d = 8.47 (s, 1H), 8.20 (s, 1H), 6.08 (d, J = 5.8 Hz,
1H), 4.73 (m, 1H), 4.52 (m, 1H), 4.34 (m, 1H), 4.19–4.15 (m, 2H), 2.21 (t,
JPH = 20.2 Hz, 2H); 13C NMR (126 MHz, D2O): d = 155.6, 152.8, 149.0,
139.7, 118.5, 86.7, 83.9, 74.2, 70.3, 64.8, 29.7 (dd, J = 129.9 Hz, J = 121.4
Hz); 31P NMR (162 MHz, D2O) d = 13.3 (bs, Pg), 10.9 (m, Pb), –10.9 (d, J
–
bs, Pa), -22.6 (bs, Pb); HRMS (ESI) m/z: [M-H]– calcd. for C10H17N5O13P3
508.0030, found 508.0036. HPLC: rt = 3 min (lmax = 258 nm).
Supported bisphosphonates 13a and 13b. A solution of methylene
bis(phosphonic dichloride) (804 mg, 3.22 mmol, 30 equiv) in trimethyl
phosphate (1.6 mL) was added to a suspension of 4 (200 mg, 0.16 mmol,
1 equiv) in trimethylphosphate (1.6 mL) at 0 °C. The reaction mixture was
stirred at 0 °C for 8 h and conversion was monitored by HPLC. The mixture
was quenched with 30 mL of a cold 0.5 M aqueous TEAB solution (pH 7.5).
Trimethyl phosphate was extracted using tert-butyl methyl ether (3 ´ 100
mL) and the aqueous layer was lyophilized. The crude product was
dissolved in water and purified by Bio Gel P–2 Gel, fine. A mixture of
compounds 13a and 13b (1:1) was isolated in 37% yield.[36] 1H NMR (400
MHz, D2O) d = 8.74 (bs, 2H), 7.43 (bs, 1H), 6.33 (bs, 2H), 5.36–5.00 (m,
4H), 4.45–3.60 (m, 3H), 2.23 (bs, 2H, PCH2P), 1.62 (bs, 3H), 1.32 (bs, 3H);
31P NMR (162 MHz, D2O) d = 7.8 (s, Pa), 16.5 (s, Pb).
+
= 26.5 Hz, Pa); HRMS (ESI) m/z: [M+H]+ calcd. for C11H19N5O12P3
506.0238, found 506.0236. HPLC: rt = 1.86 min (lmax = 258 nm).
Acknowledgements
We thank Anaïs Depaix and Georg Rolshoven for conducting
preliminary experiments.
Adenosine 5’-O- ,
a b
-methylenediphosphate trisodium salt (14).[30,40]
The general procedure was carried out starting from a 1:1 mixture of
13a/13b (215 mg, 0.081 mmol). The crude product was purified according
to method B. 12 mg (0.024 mmol, 31% yield), white solid. 1H NMR (400
MHz, D2O) d = 8.54 (s, 1H), 8.24 (s, 1H), 6.13 (d, J = 5.3 Hz, 1H), 4.76 (m,
1H), 4.58 (t, J = 4.8 Hz, 1H), 4.37 (d, J = 3.1 Hz, 1H), 4.21–4.17 (m, 2H),
2.12 (t, JPH = 19.7 Hz, 2H); 13C NMR (101 MHz, D2O): d = 155.6, 152.9,
149.0, 140.0, 118.7, 87.0, 83.0 (d, J = 7.8 Hz), 74.2, 70.1, 63.3, 28.1 (t, J
= 123.3 Hz); 31P NMR (162 MHz, D2O) d = 20.58 (d, J = 9.2 Hz), 12.76 (d,
Conflict of interest
The authors declare no conflict of interest.
+
J = 9.2 Hz); HRMS (ESI) m/z: calcd for C11H18N5O9P2 [M+H]+ 426.0574;
Keywords: bisphosphonates • click reaction • nucleotides •
found 426.0581. HPLC: rt = 2.75 min (lmax = 258 nm).
phosphorylation • soluble-supported synthesis •
N6-benzyladenosine 5’-O-
, -methylenediphosphate trisodium salt
a b
[1] E. Groaz, S. De Jonghe, Front. Chem. 2020, 8, 616863.
[2] S. Goodwin, J. D. McPherson, W. R. McCombie, Nat. Rev Genet. 2016, 17,
333–351.
(15).[19] The general procedure was carried out starting from a 1:1 mixture
of 13a/13b (158 mg, 0.06 mmol) except that a solution of benzylamine (200
µL, 1.80 mmol, 30 equiv) in water (2 mL) was used instead of a
concentrated aqueous ammonia solution. The crude product was purified
according to method B. 10 mg (0.016 mmol, 30% yield), white solid. 1H
NMR (400 MHz, D2O) d = 8.48 (s, 1H), 8.17 (s, 1H), 7.38-7.27 (m, 5H),
6.10 (d, J = 5.2 Hz, 1H), 4.71 (d, J = 5.2 Hz, 2H), 4.55 (t, J = 4.6 Hz, 1H),
4.31 (d, J = 3.6 Hz, 1H), 4.10-4.10 (m, 2H), 2.02 (t, JPH = 19.2 Hz, 2H);
13C NMR (151 MHz, D2O): d = 154.5, 152.8, 139.5, 138.4, 128.7, 127.3,
126.8, 118.9, 86.9, 83.7 (d, J = 8.0 Hz), 74.1, 69.8, 63.0 (d, J = 4.2 Hz),
58.9, 28.5 (t, J = 124.7 Hz), 19.2; 31P NMR (162 MHz, D2O) d = 21.8 (s),
11.7 (s); HRMS (ESI) m/z: [M+H]+ calcd. for C18H24N5O9P2+ 516.104, found
516.1036. HPLC: rt = 3.28 min (lmax = 266 nm).
[3] a) S. R. Malwal, B. O'Dowd, X. Feng, P. Turhanen, C. Shin, J. Yao, B. K.
Kim, N. Baig, T. Zhou, S. Bansal, R. L. Khade, Y. Zhang, E. Oldfield, J. Am.
Chem. Soc. 2018, 140, 7568–7578; b) Y. Saito, R. H. E. Hudson, J.
Photochem. Photobiol. C 2018, 36, 48–73.
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Schakel, H. Al-Hroub, Y. Wang, F. Garofano, I. G. H. Schmidt-Wolf, H.
Zimmermann, G. G. Yegutkin, C. E. Muller, ACS Med. Chem. Lett. 2020,
11, 2253–2260.
[5] For reviews on nucleotide synthesis, see: a) B. Roy, A. Depaix, C. Périgaud,
S. Peyrottes, Chem. Rev. 2016, 116, 7854–7897; b) D. R. W. Hodgson,
7
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10.1002/ejoc.202100544
European Journal of Organic Chemistry
FULL PAPER
Adv. Phys. Org. Chem. 2017, 51, 187–219; c) H. J. Jessen, Synlett 2018,
29, 699–715.
[20] S. Bhattarai, J. Pippel, E. Scaletti, R. Idris, M. Freundlieb, G. Rolshoven, C.
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8
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10.1002/ejoc.202100544
European Journal of Organic Chemistry
FULL PAPER
Entry for the Table of Contents
N3
Adenosine derivatives
NHZ
2. Phosphorylation
3. Cleavage
4. Deprotection
1. Click
Chemistry
N
N
N
N
N
O
O P
O
O
HO
N
X
P O
O
N
N
O
O
m
O O
m = 0, 1, 2
X = O, CH2
Z = H, Bn
HO OH
A new strategy based on anchoring C(6) azidopurine nucleoside derivative to a tripodal soluble support allows to access the
corresponding adenosine nucleotides. We extended the scope of the method to methylene bisphosphonate analogues and N6-
benzylamine derivative.
Institute and/or researcher Twitter usernames: Nucleosides & Phosphorylated Effectors Team: @Nuep Team; Dr. Béatrice Roy:
@BatriceRoy1
9
This article is protected by copyright. All rights reserved.