Organic Letters
Letter
Scheme 1. Synthesis of 4′-Thioacid Nucleoside Derivatives
hydrophobic reactive moieties compared to other click
reactions (e.g., tetrazine, norbornene, cyclooctyne, etc.).
Despite these interesting properties, the sulfo-click reaction
has surprisingly never been applied to nucleic acid derivatives
if we exclude the thioacid-azide ligation performed on a non-
activated 3′-azido-3′-deoxythymidine with thioacetic and
thiobenzoic acids at 60 °C for 36 h reported by Williams
1
0a
and co-workers.
Considering our interest in the development of 5′-modified
nucleoside derivatives for the formation of new internucleo-
1
5
side linkages and the potential of the sulfo-click reaction to
generate functional bioconjugates, we decided to explore its
compatibility with nucleic acids by focusing on the
introduction of the thioacid moiety at the 5′ position. We
describe herein our endeavor and demonstrate that 4′-
thioacid-modified deoxyribo- and ribonucleosides can be
readily conjugated to various small molecules in water at
very low concentrations.
The synthesis of 4′-thioacid nucleosides was developed
through an efficient three-step sequence from 3′-O-TBDMS
nucleosides (Scheme 1). 5′-Hydroxyl nucleosides 1a−d were
oxidized to carboxylic acids 2a−d, respectively, using TEMPO
16
in the presence of BAIB as a co-oxidant. A coupling reaction
with 9-fluorenylmethylthiol (Fm) generated corresponding
thioesters 3a−d in high yields. Previous works have reported
the in situ generation of thioacids in the context of the sulfo-
13b,17
click reaction from base-sensitive thioesters.
However,
Fm thioesters have been shown to be stable in aqueous media
at physiological pH. Therefore, we decided to generate
unprotected 4′-thioacid nucleoside derivatives 4a−d by
treatment with TBAF in THF, leading to the desired
compounds in the form of tetrabutylammonium (TBA)
thiocarboxylate salts. To prevent possible hydrolysis of the
thioacids under acidic or basic aqueous media, the
thiocarboxylate nucleosides were purified as such by normal
phase chromatography. It is noteworthy that all thiocarbox-
ylate nucleoside salts were completely stable at −20 °C for
months but are likely to degrade at rt.
a
b
Yield over two steps after N-Boc deprotection. Yield over two steps
after 3′-O-TBDMS deprotection.
conjugated to all thioacetates 4a−d leading to various 5′-
labeled nucleosides in excellent yields, including biotin, which
is often coupled to biomolecules as a tool for biological
1
8
19
21
applications, and the hydrosoluble fluorophore Cy 5.0, used
22
for in vivo imaging (Scheme 3). Similarly, 5′,5′-dinucleotides
bearing a non-natural N-acyl sulfonamide linkage were also
obtained in good to excellent yields. Remarkably, the sulfo-
click reaction proceeded efficiently with sulfonyl azide 6b
demonstrating the orthogonality of the reaction in the
presence of a free primary amine. Ultimately, we were able
Then, a series of sulfonyl azide partners were synthesized in
one step upon classical peptide coupling conditions by using
2
0
the 2-aminoethanesulfonyl azide as a short linker. Different
classes of labels were selected to demonstrate the versatility of
the sulfo-click reaction applied to nucleoside derivatives; these
include biotin 6a, an amino acid 6b, a modified nucleoside 6c,
a fluorophore 6d, and a peptide 6e (Scheme 2).
a
Table 1. Kinetic Studies of the Sulfo-Click Reaction
concentration (μM)
000
00
half-conversion (h)
quantitative conversion (h)
1
1
0.6
2
7
∼5
∼20
∼40
A preliminary kinetic study run with 4′-thiocarboxylate
uridine 4d and sulfonyl azide thymidine 6c showed that the
reaction reached complete conversion within 10 min at rt
under aqueous conditions [10 mM sulfonyl azide in a 25:75
1
0
a
Reactions were performed at 37 °C with 1 equiv of the Cy 5.0
3
sulfonyl azide derivative and 2 equiv of 4′-thiocarboxylate thymidine
in a mixture of aqueous 50 mM TEAA buffer (pH 7) and NMP (50%
at 1 mM, 5% at 100 μM, and 0.5% at 10 μM).
Figure S2). Under the aforementioned conditions, the sulfonyl
azides previously synthesized (Scheme 2) were successfully
B
Org. Lett. XXXX, XXX, XXX−XXX
Clavé, Guillaume
