Synthesis of 3′-triazoyl-dinucleotides as precursors of stable Phe-tRNAPhe and Leu-tRNALeu analogues

Article abstract of DOI:10.1016/j.bmcl.2014.06.027

We report here the synthesis of stable Phe-tRNA^Phe and Leu-tRNA^Leu analogues containing a 1,2,3-triazole ring instead of the ribose-amino acid ester bond. The 1,2,3-triazole ring is generated by dipolar cycloaddition of alkyne Phe and Leu analogues to 3′-azido-3′- deoxyadenosine via the Cu^I-catalysed Huisgen, Meldal, Sharpless 1,3-cycloaddition. The corresponding triazoyl pdCpA dinucleotides, obtained by classical phosphoramidite chemistry, were enzymatically ligated to 22-nt or 74-nt RNA generating stable Phe-tRNA^Phe analogues containing the acceptor stem or full tRNA moieties, respectively. These molecules represent useful tools to study the contribution of the RNA and amino acid moieties in stabilization of aminoacyl-tRNA/protein complexes.

Full text of DOI:10.1016/j.bmcl.2014.06.027

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3231–3233
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 30-triazoyl-dinucleotides as precursors of stable
Phe-tRNAPhe and Leu-tRNALeu analogues
Marco Santarem a, Matthieu Fonvielle b, Nicolas Sakkas c, Guillaume Laisné c, Maryline Chemama a,
Jean-Philippe Herbeuval c, Emmanuelle Braud c, Michel Arthur b, Mélanie Etheve-Quelquejeu c,
⇑
a Institut Parisien de Chimie Moléculaire, CNRS UMR 7201, Université Pierre et Marie Curie Paris 6, 4, place Jussieu, 75005 Paris, France
b Centre de Recherche des Cordeliers, LRMA, Equipe 12, INSERM UMR S 1138, Université Pierre et Marie Curie–Paris 6, UMR S 1138, Paris F-75006, France;
Université Paris Descartes, Sorbonne Paris Cité, UMR S 1138, Paris F-75006 France
C Chemistry & Biology Nucleo(s)tides & Immunology for Therapy (CBNIT), CNRS UMR8601, Université Paris Descartes, PRES Sorbonne Paris Cité, 45 rue des Saints-Pères,
75270 Paris Cedex 06, France
a r t i c l e i n f o
a b s t r a c t
We report here the synthesis of stable Phe-tRNAPhe and Leu-tRNALeu analogues containing a 1,2,3-triazole
ring instead of the ribose-amino acid ester bond. The 1,2,3-triazole ring is generated by dipolar cycload-
dition of alkyne Phe and Leu analogues to 30-azido-30-deoxyadenosine via the CuI-catalysed Huisgen, Mel-
dal, Sharpless 1,3-cycloaddition. The corresponding triazoyl pdCpA dinucleotides, obtained by classical
phosphoramidite chemistry, were enzymatically ligated to 22-nt or 74-nt RNA generating stable
Phe-tRNAPhe analogues containing the acceptor stem or full tRNA moieties, respectively. These molecules
represent useful tools to study the contribution of the RNA and amino acid moieties in stabilization of
aminoacyl-tRNA/protein complexes.
Article history:
Received 28 May 2014
Revised 9 June 2014
Accepted 10 June 2014
Available online 19 June 2014
Keywords:
Aminoacyl-tRNA
Click chemistry
Cyclodipeptides synthases
Dinucleotides
Ó 2014 Elsevier Ltd. All rights reserved.
Cycloaddition
Aminoacyl-tRNAs have important roles in a variety of biological
processes in addition to protein synthesis by ribosomes.1 Recently,
cyclodipeptide synthases (CDPSs) were shown to use aminoacyl-
tRNAs as substrates to catalyze the formation of the two peptide
bonds of various cyclodipeptides2 (Fig. 1). The cyclodipeptides
form the diketopiperazine (DKP) scaffold of a variety of natural
products with interesting biological properties, including antibac-
terial,3 antiviral,4 antitumoral,5 immunosuppressive6 and anti-
inflammatory7 activities. The first member of the CDPS family to
be identified was AlbC from Streptomyces noursei, which has been
showed to use mainly Phe-tRNAPhe and Leu-tRNALeu as substrates
to preferentially synthesize cyclo(
tures of AlbC, YvmC-Blic, and Rv2275 CDPSs revealed a compact
/b structure containing a Rossman-fold domain8 and similarities
L-Phe-L
-Leu)2. The crystal struc-
Figure 1. Synthesis of cyclodipeptides by CDPS. R = Phe and Leu for AlbC from
Streptomyces noursei; A = Adénosine.
a
with aminoacyl-tRNA synthases (aaRS). These findings suggest that
all CDPSs share a common aaRS-like architecture and catalytic
mechanism. Moreover, biochemical data indicated that YvmC-Blic
the RNA moiety of the substrates remains elusive since no crystal
structures of CDPS in complexes with aminoacyl-tRNAs have been
resolved. In such substrates, the ester bond connecting the amino
acid residue to the 20 or 30 position of the terminal nucleotide is
labile with half-lives from 10 min to 1 h at neutral pH.9 Trans-
esterification between the 20 and 30 positions occurs in the absence
of enzyme with a rate and thermodynamic equilibrium constant of
5 sꢀ1 and 1, respectively.10 We have shown that triazole rings act
binds tRNA and generates cyclo(L-Leu-L-Leu) via formation of an
aminoacyl-enzyme intermediate.8c However, recognition mode of
⇑
Corresponding author. Tel.: +33 0142864026.
E-mail address: melanie.etheve-quelquejeu@parisdescartes.fr (M. Etheve-
Quelquejeu).
http://dx.doi.org/10.1016/j.bmcl.2014.06.027
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

3232
M. Santarem et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3231–3233
Scheme 2. Synthesis of 30-triazoyl-dinucleotides
A
and A0. (a) CuSO4, sodium
ascorbate, THF/H2O, rt, 24 h; (b) (i) Ac-dC-PCNE, tetrazole, rt, 1 h, CH3CN, (ii) I2,
H2O/pyr/THF, rt, 30 min, (iii) TCA, CH2Cl2, rt, 30 min; (c) (i) iPr2NP(OCH2CH2CN)2,
tetrazole, CH3CN, rt, 1 h, (ii) I2, H2O/pyr/THF, rt, 30 min, (iii) MeNH2, rt, 24 h; (d)
HCl/THF/MeOH (1:2:1), rt, 24 h.
Figure 2. Structure of stable Phe-tRNAPhe and Leu-tRNALeu analogues (A) and
retrosynthesis (B).
3b, the phosphoramidite approach was used to obtain the corre-
sponding dinucleotides A and A0 (Scheme 2). For the latter
transformation, commercially available deoxycytidine phospho-
ramidite is mixed with nucleosides 3a or 3b and tetrazole in ace-
tonitrile to form the corresponding phosphite triester
intermediates. The crude products were oxidized with a solution
of iodine and treated with trichloroacetic acid to afford
compounds 4a and 4b in 33% and 70% yields, respectively. A
phosphorylation step in 50-position of 4a and 4b was necessary
as surrogates of the ester bond and provide non-hydrolysable and
non-isomerisable analogues of Ala-tRNAAla 11
These analogues are
.
good candidates for crystallization of enzyme/aminoacyl-tRNA
complexes and for the investigation of catalytic mechanisms, as
recently shown for the Fem transferases involved in bacterial cell
wall synthesis.12
We describe here the synthesis of triazoyl-dinucleotides A and
A0, mimicking the terminal sequence pCpA of Phe-tRNAPhe and
Leu-tRNALeu (Fig. 2A). The general strategy for the preparation of
the target compounds A and A0 is outlined in Figure 2B. We
planned to synthesize dinucleotides by coupling monomers 3a
and 3b with Ac-dC-CE phosphoramidite. The key step to obtain
monomers 3a and 3b was the cycloaddition of alkyne amino acids
with azido-adenosine 1. Knowing that AlbC catalyzes essentially
the synthesis of cyclodipeptide Phe-Phe and Phe-Leu, we focused
our attention on the synthesis of alkynes 2a and 2b (Scheme 1)
providing mimics of Phe and Leu. Conversion of N-Boc-
aminomethylesters into the corresponding alkynes 2 was achieved
because T4 RNA ligase catalyzes the ligation of
a
50-
phosphate-dinucleotide to the 30-OH extremity of an incomplete
tRNA (see below). In order to obtain the two protected dinucleo-
tides, compounds 4a and 4b were mixed with the phosphitylation
reagent bis(2-cyanoethyl) diisopropylphosphoramidite in the
presence of tetrazole. Removal of acetyl, cyanoethyl and benzoyl
groups with a solution of methylamine afforded the desired com-
pounds 5a and 5b in 44% and 42% yields, respectively. Eventually,
the TBS and N-Boc protecting groups were removed by stirring
with a solution of HCl during 24 h, producing the deprotected
dinucleotides A and A0 in 55% and 52% yields, respectively.
The Phe-tRNAPhe analogues were obtained by enzymatic liga-
tion of dinucleotides A with 74- and 22-nt RNA molecules
(Fig. 3). Addition of pdCpA to these RNAs generate the sequence
of the full tRNAPhe or its acceptor arm, respectively. The ligation
reaction is catalyzed by T4 RNA ligase, which tolerates a wide
range of modifications of the adenosine, including a triazole ring
in 20 or 30 position.11a The ligation was performed in HEPES buffer
containing the 74- and 22-nt RNAs, compound A, T4 RNA ligase,
ATP and MgCl2. After two hours, the Phe-tRNAPhe analogues were
purified by anion-exchange chromatography and analyzed by
denaturating polyacrylamide gel electrophoresis (Fig. 3).
by a Seyferth–Gilbert homologation of the
a-amino aldehydes
using the Bestmann-Ohira reagent.13 Alkynes 2a14 and 2b14b were
obtained in 73% and 45% yields, respectively, (Scheme 1).
Cycloaddition of organic azides and alkynes is the most direct
route to 1,2,3-triazoles.15 The use of CuI as a catalyst has the
advantage of providing exclusively the 1,4-disubstitued 1,2,3-
triazole regioisomers. Alkynes 2a and 2b reacted with the known
30-azido-20-O-(tert-butyldimethylsilyl)-30-deoxyadenosine 111a in
presence of copper(II) sulfate and sodium ascorbate. The 30-
triazoyl deoxyadenosine 3a and 3b were obtained in 82% and
61% yields, respectively, (Scheme 2). From nucleosides 3a and
In summary, we synthesised two dinucleotides pdCpA,
containing
a
30-terminal triazoyl-deoxyadenosine mimicking
the 30-terminal sequence of Phe-tRNAPhe and Leu-tRNALeu
.
These molecules are stable analogues of aminoacyl-tRNAs because
the triazole rings act as bio-isoster of the ester bond and provide
non-hydrolysable and non-isomerisable analogues of Phe-tRNAPhe
and Leu-tRNALeu. Dinucleotide A underwent enzymatic ligation to
a 22nt-microhelix and to a 74nt-RNA to afford two Phe-tRNAPhe
analogues. These compounds are currently investigated as ligands
for co-crystallization with cyclodipeptide synthetase AlbC.
Scheme 1. Synthesis of alkynes 2a and 2b. (a) DIBAL-H, CH2Cl2, ꢀ78 °C, 1 h; (b)
K2CO3, MeOH, 0 °C 1 h, then rt, 24 h.

M. Santarem et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3231–3233
3233
Figure 3. Ligation of A to 22-nt by T4 RNA ligase (A); Ligation of A to 74-nt by T4 RNA ligase (B).
6. Waring, P.; Beaver, J. Gen. Pharmacol. 1996, 27, 1311.
Acknowledgments
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The ANR CyDiPepS and MimictRNA supported this work.
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Masson, C.; Fonvielle, M.; Pernodet, J. L.; Charbonnier, J. B.; Gondry, M. Nucleic
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Supplementary data
Supplementary data (experimental details for the preparation
of nucleosides and nucleotides) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2014.06.027.
12. (a) Fonvielle, M.; de La Sierra-Gallay, I. L.; El-Sagheer, A. H.; Lecerf, M.; Patin, D.;
Mellal, D.; Mayer, C.; Blanot, D.; Gale, N.; Brown, T.; van Tilbeurgh, H.; Etheve-
Quelquejeu, M.; Arthur, M. Angew. Chem., Int. Ed. 2013, 52, 7278; (b) Fonvielle,
M.; Mellal, D.; Patin, D.; Lecerf, M.; Blanot, D.; Bouhss, A.; Santarem, M.;
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