Synthesis of stable aminoacyl-tRNA analogues containing triazole as a bioisoster of esters

Article abstract of DOI:10.1002/chem.200801563

Aminoacyl-tRNAs have important roles in a variety of biological processes, including protein synthesis by ribosomes, targeting of proteins for degradation by the proteasome, and bacterial cell wall synthesis. Here we describe the synthesis of stable amino

Full text of DOI:10.1002/chem.200801563

FULL PAPER
DOI: 10.1002/chem.200801563
Synthesis of Stable Aminoacyl-tRNA Analogues Containing Triazole as a
Bioisoster of Esters
Maryline Chemama,[a] Matthieu Fonvielle,[b, c, d] Michel Arthur,[b, c, d]
Jean-Marc Valꢀry,[a] and Mꢀlanie Etheve-Quelquejeu*[a]
Abstract: Aminoacyl-tRNAs have im-
portant roles in a variety of biological
processes, including protein synthesis
by ribosomes, targeting of proteins for
degradation by the proteasome, and
bacterial cell wall synthesis. Here we
describe the synthesis of stable amino-
acyl-tRNA analogues containing 1,4-
and 1,5-substituted 1,2,3-triazole rings.
The procedure involves i) Cu- and Ru-
catalysed cycloadditions of 3’-azidoade-
nosine and alkynes, which produced
the 1,4 and 1,5 regioisomers of the tri-
azoles, respectively, ii) coupling be-
tween the resulting triazole-deoxyade-
nosine derivatives and a deoxycytidine
phosphoramidite, and iii) the enzymatic
ligation of the substituted dinucleotides
were characterized by MS spectrome-
1
try and H, 31P and 13C NMR spectros-
copy and were assayed for inhibition of
FemXWv, an alanyltransferase essential
for the formation of the peptidoglycan
network of Gram-positive bacterial
pathogens. The low IC50 values ob-
tained (2 to 4 mm) indicate that the
five-membered triazole rings acted as
bioisosters of esters and can be used
for the design of stable aminoacyl-
tRNA analogues.
with
a 22 nt RNA microhelix that
mimics the acceptor arm of tRNA. Nu-
cleoside and nucleotide compounds
Keywords:
click chemistry
inhibitors · triazoles
aminoacyl-tRNA
·
·
·
dinucleotides
Introduction
stituted with a conserved stem pentapeptide.[1] In most
Gram-positive bacteria the subunit contains an additional
side chain linked to the third position of the stem pentapep-
tide. The side chains display considerable interspecies se-
quence diversity, consisting, for example, of five glycines in
Staphylococcus aureus and of the sequence l-Ser-l-Ala or l-
Ala-l-Ala in Streptococcus pneumoniae.[2] The side chains
are assembled by transferases of the Fem family that have
the particularity of using aminoacyl-tRNAs as substrates.[3]
These enzymes have a pivotal role in peptidoglycan synthe-
sis because the side chains supply the branching points to
cross-link peptides from adjacent glycan chains, an essential
reaction catalysed by the d,d-transpeptidase catalytic
domain of penicillin-binding proteins (PBPs).[4] For this
reason, Fem transferases are considered attractive targets
for the development of novel antibiotics active against mul-
tiply resistant bacteria.[5]
Peptidoglycan is an essential component of the bacterial cell
envelope because it provides mechanical protection against
the osmotic pressure of the cytoplasm. The peptidoglycan
subunit (Figure 1) consists of b-1,4-linked N-acetylglucosa-
mine (GlcNAc) and N-acetylmuramic acid (MurNAc) sub-
[a] M. Chemama, Prof. J.-M. Valꢀry, Dr. M. Etheve-Quelquejeu
UMR 7613
Synthꢁse, Structure et Fonction de Molꢀcules Bioactives
Universitꢀ Pierre et Marie Curie
4 place Jussieu, case 179
75252 Paris Cedex (France)
Fax : (+33)0144275513
E-mail: quelque@ccr.jussieu.fr
[b] Dr. M. Fonvielle, Dr. M. Arthur
Centre de Recherche des Cordeliers, LRMA
Equipe 12, INSERM, U872
Recently, we have developed the synthesis of novel ami-
[6]
75006 Paris (France)
noacyl-tRNA analogues for inhibition of FemXWv
,
the pro-
[c] Dr. M. Fonvielle, Dr. M. Arthur
Universitꢀ Pierre et Marie Curie, Paris 6
UMR S 872
totypic enzyme of the Fem family. The inhibitor
A
(Scheme 1) contains a 1,2,4-oxadiazole ring as a mimic of
the natural 3’-aminoacyl ester B (Figure 2). Oxadiazoles, in-
cluding 1,2,4-oxadiazoles, are stable analogues of esters,[7]
due to their geometries and electronic properties.[8] The oxa-
diazole-containing FemXWv inhibitor differs from previously
75006 Paris (France)
[d] Dr. M. Fonvielle, Dr. M. Arthur
Universitꢀ Paris Descartes, UMR S 872
75006 Paris (France)
Chem. Eur. J. 2009, 15, 1929 – 1938
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1929

Figure 2. Ala-tRNAAla substrate of FemXWv (B). Stable analogues (C, C’)
of tRNAAla containing triazole as ester isoster.
The synthesis of the FemXWv inhibitor[6] (A in Scheme 1)
starts from the 3’-cyanodeoxyadenosine, which is converted
into the oxadiazole derivative by treatment with hydroxyl-
amine in methanol and condensation with activated Boc-l-
alanine. The oxadiazole nucleoside is then coupled with the
deoxycytidine in the classical phosphoramidite approach.
The resulting dinucleotide is phosphorylated and ligated
with an RNA microhelix by T4 RNA ligase to afford com-
pound A. This oxadiazole-tRNA was shown to inhibit
FemXWv with an IC50 value of 1.4 mm, indicating that the
five-membered heterocycle ring was acting as an ester surro-
gate.
For this report we have developed novel aminoacyl-tRNA
analogues each containing a triazole ring instead of an oxa-
diazole ring (C and C’ in Figure 2). These units are hetero-
cyclic structural motifs with considerable medicinal poten-
tial, because they are more than just passive linkers, due to
their high potential for association with biological targets
through hydrogen bonding and dipole interactions.[11] They
can be obtained by azide–alkyne cycloaddition (Huisgenꢃs
1,3-dipolar cycloaddition).[12] A remarkable development of
this approach, based on the CuI-catalysed reaction, afforded
regioselective formation of the 1,4-substituted 1,2,3-triazole,
reported as the first “click chemistry” reaction.[13] This has
led to growing use of triazoles for drug discovery. In the
field of nucleoside and nucleotide chemistry, Huisgen–
Sharpless cycloaddition has been used as powerful linking
reaction to obtain a variety of bioconjugates,[14] modified
bases,[15] sugar residues[16] and altered phosphodiester back-
bones.[17] Here we have applied
Figure 1. Synthesis of bacterial cell wall peptidoglycan. Arrows show pep-
tide bond orientation CO!NH. GlcNAc: N-acetylglucosamine.
MurNAc: N-acetylmuramic acid.
reported aminoacyl-tRNA analogues used in the study of
aminoacyl-tRNA synthetases and of the peptidyl-transferase
centre of the ribosome.[9] In those studies, the ester linkages
at the 3’-end of the aminoacyl- or peptidyl-tRNAs have
been typically replaced by amide linkages (puromycin ana-
logues) or by phosphate or phosphoramidate groups that
mimic the tetrahedral transition state formed during the
aminoacyl transfer reactions. A deoxyadenosine 3’-phospho-
nate analogue has also been reported to inhibit MurM, a
transferase of the Fem family (IC50 =100 mm).[10]
this approach to the synthesis
of aminoacyl-tRNA analogues
C and C’ and have shown that
these compounds inhibit the
FemXWv cell wall target.
Results and Discussion
Synthesis: The general strategy
(Scheme 2) for the preparation
of the target compounds C and
Scheme 1. Stable analogue of tRNAAla (A).
1930
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Aminoacyl-tRNA Analogues
FULL PAPER
Scheme 2. General strategy for the preparation of Ala-tRNAAla analogues C and C’.
C’ was based on enzymatic ligation of 13 or 16 with a 22 nt
RNA microhelix that mimics the acceptor arm of tRNA.
This approach, initially developed for the introduction of
non-natural amino acids in proteins,[18] involves the chemical
synthesis of an aminoacylated dinucleotide and its enzymatic
coupling to an incomplete RNA lacking the terminal dinu-
cleotide unit. We planned to develop the synthesis of dinu-
cleotides 13 or 16 by coupling between Ac-dC-CE phosphor-
amidite and nucleosides 6 or 9a. The key step to obtaining 6
and 9a was to be a cycloaddition between a 3’-azido-adeno-
sine derivatives and alkynes.
Cycloaddition: Cycloaddition of organic azides and al-
kynes is the most direct route to 1,2,3-triazoles. In this study,
we used two different catalysts to achieve this reaction: the
CuI catalyst, which has the advantage of exclusively provid-
ing the 1,4-disubstituted 1,2,3-triazole regioisomers,[13] and
ride (BzCl) to afford 2 in 89% yield. The selective removal
of the silyl group by treatment with trifluoroacetic acid gave
3 in 84% yield. Nucleoside 3 was converted into the 1,4-dis-
ubstituted 1,2,3-triazoles 5a–c through Huisgen–Sharpless
1,3-dipolar cycloadditions with alkynes 4a–c,[21] in the pres-
ence of copper(II) sulfate and sodium ascorbate.
The use of alkynes 4a–c provides mimics of Ala, Gly and
Phe and shows the potential for application to various
amino acid residues in the field of modified nucleoside syn-
thesis. The 1,4-disubstituted 1,2,3-triazoles 5a–c were ob-
tained in 99, 89, and 81% yields respectively, from 3. Very
high yields for the formation of triazole-nucleosides were
also obtained under the same conditions from nucleosides 1
or 8, which differ from 3 in their protecting groups (results
not shown).
To obtain the regioisomer 1,5-disubstituted 1,2,3-triazoles,
we started from the dibenzoyl-nucleoside 3 in the presence
the [Cp*RuClACHTUNGTRENNUNG(PPh3)2] catalyst, which has recently been de-
scribed for regioselective synthesis of 1,5-disubstituted 1,2,3-
triazole systems.[19] The synthesis (Scheme 3) started from
the known 3’-azido-2’,5’-bis-O-(tert-butyldimethylsilyl)-3’-de-
oxyadenosine (1),[20] which was protected with benzoyl chlo-
of alkynes 4a–c and [Cp*RuClACHTUNGETRNNU(G PPh3)2]. From these starting
materials we variously observed a low yield (9% from 4a)
or no pure products from the two other derivatives 4b and
4c. These poor results were partly due to the removal of
Scheme 3. Synthesis of 3’-triazole-nucleosides: a) BzCl, pyridine, then H2O; b) TFA/H2O, 6 h, 0 8C; c) CuSO4, sodium ascorbate, THF, H2O, RT, 24 h ;
d) NH4OH; e) BzCl, pyridine, then H2O and NH4OH; f) [Cp*RuCl(PPh3)2], C6H6, reflux, 2 h.
AHCTUNGTRENNUNG
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1931

M. Etheve-Quelquejeu et al.
one of the benzoyl groups in
compound 3. To bypass this
problem, we synthesized
8
(Scheme 3) from 1 through a
protection step with benzoyl
chloride and a selective depro-
tection of the 5’-hydroxy group
with trifluoroacetic acid (45%
yield over the two steps) and
used it under the same condi-
tions to afford the 1,5-disubsti-
tuted triazoles 9a and 9c in 37
and 39% yields. These results
show that the efficiencies of
the cycloadditions in providing
triazole-nucleosides is depen-
Figure 3. Comparison of the downfield regions of the 1H NMR spectra of 9a (top) and 6 (bottom) in CDCl3.
dent on the natures of alkynes and azido derivatives in the
ruthenium-catalysed reactions. In contrast, the copper-cata-
lysed reaction (a “click reaction”) provides high yields with
various compounds.
Experimental Section). The 1H NMR signals for H3’, H1’
and Htriazole for compound 6 appeared at 5.18, 6.15 and
7.51 ppm, respectively, whereas in compound 9a the signals
were shifted downfield (5.62, 6.42 and 7.64 ppm, respective-
ly). In their 13C NMR spectra, CHtriazole and C3’ of the nu-
cleoside were observed at 122.8 and 63.1 ppm, respectively,
for compound 6 and at 129.5 and 60.4 ppm, respectively, for
9a.
Structural assignment: The use of the ruthenium catalyst
[Cp*RuClACHTUNGTRENNUNG(PPh3)2] has been reported mainly to provide 1,5-
disubstituted 1,2,3-triazoles, although the nature of the azide
component can also affect the regioselectivity, leading to
mixtures of 1,5- and 1,4-disubstituted triazoles.[19b] The struc-
ture of triazole 9a was thus confirmed by comparison with
Phosphoramidite coupling: The dinucleotides 13 and 16
were obtained by the phosphoramidite approach
(Scheme 4). Nucleosides 6 and 9a were coupled in the pres-
ence of tetrazole with the commercially available deoxycyti-
dine phosphoramidite (Ac-dC-CE-phosphoramidite). Re-
1
compound 6. Their H and 13C NMR spectra were very simi-
lar, except for H3’, H1’ and Htriazole in the 1H NMR
(Figure 3) and for the C3’ and CHtriazole in the 13C NMR (see
Scheme 4. a) Ac-dC-CE phosphoramidite, tetrazole, CH2Cl2, RT, 1 h; b) I2, 30 min, RT; c) TCA, CH2Cl2, 30 min, RT; d) bis(2-cyanoethyl)diisopropyl-
phosphoramidite, tetrazole, CH2Cl2, 1 h, RT; e) CH3NH2, 24 h, RT; f) HCl (6n)/THF/CH3OH, 24 h.
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Aminoacyl-tRNA Analogues
FULL PAPER
placement of cytidine with deoxycytidine simplified the syn-
thesis because it eliminated the 2’-OH reactive group with-
out affecting biological activity.[22] After the coupling step,
the crude products were oxidized with iodine and treated
with trichloroacetic acid to afford compounds 11 and 14 in
69% and 70% yields, respectively. Phosphorylation of 11
and 14 with bis(2-cyanoethyl)diisopropylphosphoramidite in
tetrazole, followed by removal of cyanoethyl, acetyl and
benzoyl groups with methylamine, afforded 12 and 15 in 54
and 64% yields. The N-Boc and TBS protecting groups
were removed by stirring with HCl for 24 h, producing 13
and 16 in 44 and 58% yields.
Figure 4. Analysis of C and C’ by polyacrylamide gel electrophoresis. C=
5’-GGGGCCUUAGCUCAGGCUCCACdCA-(1,4-disubstituted 1,2,3-tri-
azole)-ethanamine-3’;
C’=5’-GGGGCCUUAGCUCAGGCUCCACd-
CA-(1,5-disubstituted 1,2,3-triazole)ethanamine-3’; 22 nt=microhelix 5’-
GGGGCCUUAGCUCAGGCUCCAC-3’;
GGGGCCUUAGCUCAGGCUCCACCA-3’.
24 nt=microhelix
5’-
Enzymatic ligation: Aminoacyl-tRNAs are obtained through
the coupling of protected aminoacylated dinucleotides with
tRNAs lacking the 3’ terminal pdCpA moiety by use of
T4 RNA ligase.[18] Stable aminoacyl-pdCpA analogues are
also substrates of T4 RNA ligase,[6] and 13 and 16 were ligat-
ed to the 3’-end of an RNA microhelix with this enzyme
(Scheme 5). In this study we chose a 22 nt RNA microhelix
tidoglycan precursor UDP-MurNAc-pentapeptide in order
to introduce the first residue of an l-Ala-l-Ser-l-Ala side
chain. Inhibition of FemXWv was tested in a radioactive cou-
pled assay as previously described.[6] Inhibition of the trans-
fer of l-Ala by FemXWv re-
vealed IC50 values of 2.4ꢀ
0.4 mm for
C
and of 4.1ꢀ
0.4 mm for C’ (Figure 5). The
low IC50 values indicate that
the triazole unit can be used as
an ester bioisoster.
Compound C’ was only
slightly less active than C (4.1
versus 2.4 mm). This result sug-
gests that the ethylamine sub-
stituent of the triazole, which
has different orientations in
the two compounds, has little
effect on the interaction of the
inhibitors with the enzyme.
The 1-aminoethyl group was
Scheme 5. Reaction was performed at 378C for 120 min in HEPES buffer (50 mm, 500 mL) containing the 22 nt
RNA microhelix (20 nmol), compound 13 or 16 (200 nmol), T4 RNA ligase (3.1 mg), DMSO (10%), ATP
(1 mm), MgCl2 (15 mm).
introduced in
C and C’ in
order to mimic the amine and
to mimic the acceptor arm of tRNA, because oxadiazole-
containing analogues with full-length tRNA (76 nt) or mi-
crohelix RNA (24 nt) had previously been shown to inhibit
FemXWv with similar efficiencies (IC50 =0.17 and 1.4 mm, re-
spectively).[6] The ligation was performed in HEPES buffer
containing the RNA microhelix (22 nt), compound 13 or 16,
T4 RNA ligase, ATP and MgCl2. After two hours, com-
pounds C and C’ had been obtained and were purified by
anion-exchange chromatography. Fractions containing the li-
gation product were analysed by denaturing polyacrylamide
gel electrophoresis (Figure 4), which revealed that the mi-
crohelix (22 nt) had been quantitatively converted into the
24 nt triazole-containing oligonucleotides.
the methyl side chain of l-Ala. FemXWv had previously been
shown to catalyse aminoacyl transfer from Gly-tRNAGly, in-
dicating that the methyl group of l-Ala is not essential for
activity.[24] This observation could account for the similar be-
haviour of C and C’ as inhibitors.
Conclusions
We have described an efficient method for the synthesis of a
novel class of stable analogues of aminoacyl-tRNAs. In con-
trast with native aminoacyl-tRNAs, which bear readily hy-
drolysable ester linkages, the analogues each contained a
stable five-membered triazole ring. The use of “click
chemistry” techniques afforded 3’deoxy-3’-triazole-nucleo-
sides in very high yields, and cycloadditions catalysed by
copper or ruthenium allowed the 1,4- and 1,5-regioisomers
of the triazoles to be obtain from the same starting materi-
In vitro inhibition assays: FemXWv from Weissella viridescens
has been used as a model enzyme for kinetics and structural
analyses of transferases of the Fem family.[23] The enzyme
catalyses the transfer of l-Ala from Ala-tRNAAla to the pep-
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1933

M. Etheve-Quelquejeu et al.
17 mLminꢁ1 and UV detection at 260 nm. Fast protein liquid chromatog-
raphy (FPLC) was performed with an AKTA purifier (Amersham Phar-
macia Biotech). Optical rotations were carried out on a Perkin–Elmer
Model 341 polarimeter.
3’-Azido-6-N,N-dibenzoyl-2’,5’-bis-O-(tert-butyldimethylsilyl)-3’-deoxy-
adenosine (2): Benzoyl chloride (975 mL, 8.40 mmol) was added dropwise
at 08C to a solution of 1 (875 mg, 1.68 mmol) in anhydrous pyridine
(50 mL). The mixture was stirred at room temperature for 4 h, and the
solution was concentrated to dryness. The residue was dissolved in
CH2Cl2 and washed with H2O, saturated NaHCO3 and brine. The com-
bined organic layers were dried over anhydrous Na2SO4. After removal
of the solvents, the crude product was purified on a silica gel column
with elution with EtOAc/cyclohexane 2:8 to give 2 (1.09 g, 89%) as a
white foam. Rf =0.6 (EtOAc/cyclohexane 2:8); [a]2D5 =ꢁ20.2 (c=0.5 in
1
CHCl3); H NMR (250 MHz, CDCl3): d=8.65 (s, 1H; H2 or H8), 8.37 (s,
1H; H2 or H8), 7.93–7.78 (m, 4H; H-Bz), 7.53–7.29 (m, 6H; H-Bz), 6.08
(d, 3JH,H =4.5 Hz, 1H; H1’), 4.88 (t, 3JH,H =4.7 Hz, 1H; H2’), 4.22 (dd,
3JH,H =2.7, 5.2 Hz, 1H; H4’), 4.10–3.99 (m, 2H; H3’/H5’a), 3.83 (dd,
2
3JH,H =2.8, JH,H =11.7 Hz, 1H; H5’b), 0.93 (s, 9H; H-tBuTBS), 0.83 (s, 9H;
H-tBuTBS), 0.12 (s, 6H; H-MeTBS), 0.04 (s, 3H; H-MeTBS), ꢁ0.02 ppm (s,
13
3H; H-MeTBS); C NMR (63 MHz, CDCl3): d=172.3 (C=O Bz), 152.8
ꢁ
(Cq-Ad), 152.4 (C2 or C8), 151.9 (Cq-Ad), 143.4 (C2 or C8), 134.2 (Cq-
Bz), 133.0 (C-Bz), 129.6 (C-Bz), 128.8 (C-Bz), 88.9 (C1’), 82.6 (C4’), 77.1
(C2’), 62.7 (C5’), 61.3 (C3’), 26.1 (C-tBuTBS), 25.6 (C-tBuTBS), 18.6 (Cq-
tBuTBS), 18.0 (Cq-tBuTBS), ꢁ4.9 (C-MeTBS), ꢁ5.1 (C-MeTBS), ꢁ5.2 (C-
MeTBS), ꢁ5.3 ppm (C-MeTBS); HRMS (ESI): m/z: calcd for
C36H49N8O5Si2: 729.3359 [M+H]+; found: 729.3359.
Figure 5. Inhibition of FemXWv by compounds C and C’. Curves represent
nonlinear regressions (four-parameter logistic curve, sigma Plot 9.0) of
3’-Azido-6-N,N-dibenzoyl-2’-O-(tert-butyldimethylsilyl)-3’-deoxyadeno-
sine (3): Aqueous TFA (5 mL, 1:1) was added at 08C to a stirred solution
of 2 (459 mg, 0.630 mmol) in THF (7 mL). After having been stirred for
6 h at 08C, the reaction mixture was neutralized with saturated aqueous
NaHCO3 and diluted with EtOAc. After separation, the organic phase
was washed with H2O and brine, dried over anhydrous Na2SO4 and con-
centrated at reduced pressure. The residue was subjected to flash chro-
*
experimentally determined values. ) IC50 =2.4ꢀ0.4 mm for C Hillslope
*
1.03ꢀ0.05; R=0.9998, and ) IC50 =4.1ꢀ0.4 mm for C’ Hillslope 1.0ꢀ
0.1; R=0.9986.
als. The IC50 values of C and C’ for FemXWv indicate that
their triazole rings are good ester bioisosters. The strategy is
of broad interest for the design of inhibitors of various types
of enzymes that use aminoacyl-tRNA as substrates.
matography with elution with EtOAc/cyclohexane (:7 to provide
3
(324 mg, 84%) as a white foam. Rf =0.24 (EtOAc/cyclohexane 3:7);
[a]2D5 =ꢁ36.1 (c=1 in CHCl3); 1H NMR (250 MHz, CDCl3): d=8.65 (s,
1H; H2 or H8), 8.09 (s, 1H; H2 or H8), 7.92–7.80 (m, 4H; H-Bz), 7.50
(m, 2H; H-Bz), 7.35 (m, 4H; H-Bz), 5.82 (d, 3JH,H =7.5 Hz, 1H; H1’),
5.70 (d, 3JH,H =10.2 Hz, 1H; OH), 5.28 (dd, 3JH,H =5.5, 7.5 Hz, 1H; H2’),
4.25 (d, 3JH,H =5.4 Hz, 1H; H3’), 4.16 (s, 1H; H4’), 3.83 (dd, 3JH,H =2.8,
2JH,H =11.6 Hz, 2H; H5’a/H5’b), 0.78 (s, 9H; H-tBuTBS), ꢁ0.12 (s, 3H; H-
MeTBS), ꢁ0.53 ppm (s, 3H; H-MeTBS); 13C NMR (63 MHz, CDCl3): d=
Experimental Section
ꢁ
172.1 (C=O Bz), 153.1 (Cq-Ad), 151.8 (C2 or C8), 145.0 (C2 or C8),
General reagents and materials: Solvents were dried by standard meth-
ods and were distilled before use. Unless otherwise specified, materials
were purchased from commercial suppliers and were used without fur-
133.9 (Cq-Bz), 133.3 (C-Bz), 129.6 (C-Bz), 128.9 (C-Bz), 90.8 (C1’), 85.4
(C4’), 74.9 (C2’), 63.6 (C3’), 63.4 (C5’), 25.7 (C-tBuTBS), 17.9 (Cq-tBuTBS),
ꢁ5.1 (C-MeTBS), ꢁ5.8 ppm (C-MeTBS); HRMS (ESI): m/z: calcd for
C30H34N8O5SiNa: 637.2308 [M+Na]+; found: 637.2309.
ther purification. TLC: precoated thin-layer silica gel sheets (60 F254
,
Merck) and detection by charring with H2SO4 in ethanol (10%), followed
by heating. Flash chromatography: silica gel (60 ꢄ, 180–240 mesh,
Merck). Spectra were recorded on Bruker spectrometers: ARX 250 for
1H NMR (250.13 MHz) and 13C NMR (62.89 MHz), AC 400 for 31P NMR
(161.97 MHz), Avance III 500 for 1H NMR (500.11 MHz) and 13C NMR
(125.75 MHz), and DRX 500 for 31P NMR (202.31 MHz), in CDCl3,
[D6]DMSO, CD3OD or D2O as indicated below. Chemical shifts (d) are
expressed in ppm relative to residual CHCl3 (d=7.26 ppm), CHD2OD
(d=3.31 ppm), CHD2SOCD3 (d=2.50 ppm) or HDO (d=4.79 ppm) for
1H, and to CDCl3 (d=77.16 ppm), CD3OD (d=49.00 ppm) or
CD3SOCD3 (d=39.52 ppm) for 13C as internal references, and to H3PO4
(d=0 ppm) for 31P as external reference. Signals were attributed on the
basis of COSY and DEPT 135 (13C). High-resolution mass spectrometry
General procedure for Cu-catalysed cycloadditions: A solution of aq
sodium ascorbate (1m, 0.2 equiv, 16 mL, 16 mmol) and aq CuSO4 (7.5%,
0.1 equiv, 81 mL, 8 mmol) was added at 08C to a mixture of 3 (1.0 equiv,
81 mmol) and one of the compounds 4a–c (1.1 equiv, 89 mmol) in H2O/
THF (3 mL, 1:1). The heterogeneous mixture was stirred vigorously at
room temperature until complete consumption of the reactants was indi-
cated by TLC analyses. After removal of THF under reduced pressure,
water (3 mL) was added, and the product was extracted with EtOAc (3ꢅ
10 mL). The combined organic layers were washed with brine, dried over
anhydrous Na2SO4 and concentrated in vacuo. The crude product was
subjected to column chromatography with elution with EtOAc/cyclohex-
ane 5:5.
6-N,N-Dibenzoyl-2’-O-(tert-butyldimethylsilyl)-3’-(S)-tert-butyl-1-(1H-
1,2,3-triazol-4-yl)ethylcarbamate-3’-deoxyadenosine (5a): Compound
(HRMS) was carried out with
a LTQ Orbitrap mass spectrometer
(Thermo Fisher Scientific, Inc.) by electrospray ionization (ESI+ or
ESIꢁ) at the Mass Spectrometry Centre of the University Pierre & Marie
Curie (Paris). High-performance liquid chromatography (HPLC) was
3
(50 mg, 81 mmol) and alkyne 4a (15 mg, 89 mmol) were treated as de-
scribed in the general procedure to give compound 5a (55 mg, 99%) as a
white solid. Rf =0.71 (EtOAc/cyclohexane 8:2); [a]2D5 =ꢁ67.2 (c=1 in
performed on
a HPLC system with a reversed-phase C-18 column
1
CHCl3); H NMR (250 MHz, CDCl3): d=8.65 (s, 1H; H2 or H8), 8.16 (s,
(250 mmꢅ21.2 mm, HYPERSIL HSC18, Thermoelectron Corporation)
with a solvent system consisting of aqueous NH4OAc (50 mm)/CH3CN
(linear gradient from 100:0 to 50:50 over 50 min) at a flow rate of
1H; H2 or H8), 7.82 (d, 3JH,H =7.4 Hz, 4H; H-Bz), 7.51 (s, 1H; Htriazole),
7.51–7.41 (m, 2H; H-Bz), 7.32 (t, 3JH,H =7.6 Hz, 4H; H-Bz), 6.14 (d,
1934
www.chemeurj.org
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 1929 – 1938

Aminoacyl-tRNA Analogues
FULL PAPER
3JH,H =6.9 Hz, 1H; H1’), 5.77 (d, 3JH,H =10.3 Hz, 1H; OH), 5.30–5.10 (m,
3H,H2’/H4’/NH), 4.93 (m, 2H; H3’/CH), 4.12–3.96 (m, 1H; H5’a), 3.82–
3.66 (m, 1H; H5’b), 1.52 (d, 3JH,H =6.9 Hz, 1H; CH3), 1.41 (s, 9H; tBu-
(CH3), 17.5(Cq-tBuTBS), ꢁ5.00 (C-MeTBS), ꢁ5.72 ppm (C-MeTBS); HRMS
(ESI): m/z: calcd for C32H45N9O6SiNa: 702.3160 [M+Na]+; found:
702.3154.
Boc), 0.55 (s, 9H; H-tBuTBS), ꢁ0.23 (s, 3H; H-MeTBS), ꢁ0.61 ppm (s, 3H;
3’-Azido-6-N-benzoyl-2’,5’-bis-O-(tert-butyldimethylsilyl)-3’-deoxyadeno-
sine (7): Benzoyl chloride (953 mL, 8.2 mmol) was added dropwise at 08C
to a solution of 1 (0.855 g, 1.64 mmol) in anhydrous pyridine (12 mL).
The mixture was stirred for 2 h at room temperature. Ice-water (10 mL)
was added, and the mixture was allowed to stir for 30 min at 08C. Aque-
ous NH4OH solution (30% 10 mL) was then added, and the reaction was
stirred for 1 hour at 08C and for another 3 h at room temperature. The
reaction mixture was washed with aqueous saturated NH4Cl solution (2ꢅ
50 mL), water (2ꢅ50 mL) and brine (2ꢅ50 mL). The organic layers were
dried over Na2SO4. After removal of the solvents, the residue was puri-
fied on a flash column of silica gel with elution with EtOAc/cyclohexane
2:8 to give 7 (629 mg, 61%) as a white solid. Rf =0.70 (EtOAc/cyclohex-
13
H-MeTBS); C NMR (63 MHz, CDCl3): d=172.0 (C=O Bz), 155.2 (Cq-
ꢁ
ꢁ
Ad), 153.0 (C2 or C8), 151.8 (C=O Boc), 149.59 (Cq-Ad), 145.0 (C2 or
C8), 133.8 (Cq-Bz), 133.2 (C-Bz), 129.5 (C-Bz), 128.8 (C-Bz), 122.7 (C-
triazole), 91.3 (C1’), 84.7 (C4’), 79.6 (Cq-tBuBoc), 73.6 (C2’), 63.3 (C5’),
62.8 (C3’), 42.6 (CH), 28.4 (C-tBuBoc), 25.2 (C-tBuTBS), 21.2 (CH3), 17.4
(Cq-tBuTBS), ꢁ5.1 (C-MeTBS), ꢁ5.9 ppm (C-MeTBS); HRMS (ESI): m/z:
calcd for C39H49N9O7SiNa: 806.3422 [M+Na]+; found: 806.3416.
6-N,N-Dibenzoyl-2’-O-(tert-butyldimethylsilyl)-3’-(S)-tert-butyl-1-(1H-
1,2,3-triazol-4-yl)methylcarbamate-3’-deoxyadenosine (5b): Compound 3
(50 mg, 81 mmol) and alkyne 4b (14 mg, 89 mmol) were treated as de-
scribed in the General Procedure to give compound 5b (56 mg, 89%) as
a white solid. Rf =0.59 (EtOAc/cyclohexane 8:2); [a]2D5 =ꢁ75.7 (c=1 in
CHCl3); 1H NMR (250 MHz, CD3OD): d=8.84 (s, 1H; H2 or H8), 8.67
(s, 1H; H2 or H8), 7.97 (s, 1H; Htriazole), 7.84 (d, 3JH,H =7.2 Hz, 4H; H-
Bz), 7.54 (t, 3JH,H =7.4 Hz, 2H; H-Bz), 7.39 (t, 3JH,H =7.6 Hz, 4H; H-Bz),
1
ane 5:5); [a]2D5 =ꢁ14.7 (c=1 in CHCl3); H NMR (250 MHz, CDCl3): d=
9.39 (brs, 1H; NH), 8.78 (s, 1H; H2 or H8), 8.40 (s, 1H; H2 or H8), 8.01
3
(d, JH,H =9.0 Hz, 2H; H-Bz), 7.50 (m, 3H; H-Bz), 6.11 (d, 3JH,H =5.0 Hz,
1H; H1’), 4.86 (t, 3JH,H =5.0 Hz, 1H; H2’), 4.26 (m, 1H; H4’), 4.07 (m,
2H,H3’/H5’a), 3.84 (dd, 3JH,H =2.5, 2JH,H =12.5 Hz, 1H; H5’b), 0.94 (s,
9H; H-tBuTBS), 0.86 (s, 9H; H-tBuTBS), 0.15 (s, 6H; H-MeTBS), 0.06 (s,
3H; H-MeTBS), ꢁ0.06 ppm (s, 3H; H-MeTBS); 13C NMR (63 MHz,
3
3
6.38 (d, JH,H =5.5 Hz, 1H; H1’), 5.49 (m, 1H; H3’), 5.19 (t, JH,H =6.0 Hz,
1H; H2’), 4.92 (m, 1H; H4’), 4.33 (s, 2H; CH2), 3.91 (dd, 2JH,H =10.0 Hz,
2H; H5’a/H5’b), 1.44 (s, 9H; H-tBuBoc), 0.60 (s, 9H; H-tBuTBS), ꢁ0.19 (s,
3H; H-MeTBS), ꢁ0.39 ppm (s, 3H; H-MeTBS); 13C NMR (63 MHz,
ꢁ
CDCl3): d=165.0 (C=O Bz), 152.4 (C2 or C8), 141.3 (C2 or C8), 151.5,
ꢁ
149.7 (Cq-Ad), 133.3 (Cq-Bz), 133.8 (C-Bz), 128.8 (C-Bz), 128.1 (C-Bz),
89.1 (C1’), 82.3 (C4’), 77.2 (C2’), 62.5 (C5’), 60.9 (C3’), 26.1 (C-tBuTBS),
25.7 (C-tBuTBS), 18.6 (Cq-tBuTBS), 18.0 (Cq-tBuTBS), ꢁ4.9 (C-MeTBS), ꢁ5.0
(C-MeTBS), ꢁ5.2 (C-MeTBS), ꢁ5.4 ppm (C-MeTBS); HRMS (ESI): m/z:
calcd for C29H44N8O4Si2Na: 647.2922 [M+Na]+; found: 647.2916.
CD3OD): d=173.6 (C=O Bz), 158.2 (Cq-Ad), 154.02 (Cq-Ad), 153.2 (C2
or C8), 147.1 (Cq-Ad), 146.5 (C2 or C8), 135.30 (Cq-Bz), 134.4 (C-Bz),
130.5 (C-Bz), 129.9 (C-Bz), 129.4 (Cq-triazole), 125.4 (C-triazole), 91.2
(C1’), 84.7 (C4’), 80.4 (Cq-tBuBoc), 76.6 (C2’), 63.2 (C3’), 62.7 (C5’), 36.5
(CH2), 28.8 (C-tBuBoc), 25.8 (C-tBuTBS), 18.4 (Cq-tBuTBS), ꢁ5.01 (C-
MeTBS), ꢁ5.31 ppm (C-MeTBS); HRMS (ESI): m/z: calcd for
C38H47N9O7SiNa: 792.3265 [M+Na]+; found: 792.3260.
3’-Azido-6-N-benzoyl-2’-O-(tert-butyldimethylsilyl)-3’-deoxyadenosine
(8): Aqueous TFA (6 mL, 1:1) was added at 08C to a stirred solution of 7
(594 mg, 0.95 mmol) in THF (8 mL). After having been stirred for 8 h at
08C, the reaction mixture was neutralized with aqueous saturated
NaHCO3 solution and diluted with EtOAc. After separation, the organic
phase was washed with water and brine, dried over anhydrous Na2SO4
and concentrated under reduced pressure. The residue was subjected to
flash chromatography with elution with EtOAc/cyclohexane 2:8 to pro-
vide 8 (359 mg, 74%) as a white foam. Rf =0.62 (EtOAc); [a]2D5 =+7.8
(c=1 in CHCl3); 1H NMR (250 MHz, CDCl3): d=9.39 (brs, 1H; NH),
8.75 (s, 1H; H2 or H8), 8.05 (s, 1H; H2 or H8), 8.01 (d, 3JH,H =9.0 Hz,
2H; H-Bz), 7.51 (m, 3H; H-Bz), 6.05 (brs, 1H; OH), 5.81 (d, J=7.5 Hz,
1H; H1’), 5.29 (dd, 3JH,H =5.0, 7.5 Hz, 1H; H2’), 4.24 (d, 3JH,H =7.5 Hz,
1H; H3’), 4.13 (s, 1H,H4’), 3.91 (m, 1H; H5’a), 3.70 (m,1H; H5’b), 0.75
6-N,N-Dibenzoyl-2’-O-(tert-butyldimethylsilyl)-3’-(S)-tert-butyl-1-(1H-
1,2,3-triazol-4-yl)-2-phenylethylcarbamate-3’-deoxyadenosine (5c): Com-
pound 3 (50 mg, 81 mmol) and alkyne 4c (22 mg, 89 mmol) were treated
as described in the General Procedure to give compound 5c (57 mg,
81%) as a white solid. Rf =0.71 (EtOAc/cyclohexane 8:2); [a]2D5 =ꢁ72.1
(c=1 in CHCl3); 1H NMR (250 MHz, CD3OD): d=8.84 (s, 1H; H2 or
H8), 8.67 (s, 1H; H2 or H8), 7.91 (s, 1H; Htriazole), 7.89–7.79 (m, 4H; H-
Bz), 7.54 (m, 2H; H-Bz), 7.39 (m, 4H; H-Bz), 7.24 (m, 5H; H-Bz), 6.36
(d, 3JH,H =5.3 Hz, 1H; H1’), 5.49 (m, 1H; H3’), 5.19 (m, 1H; H2’), 5.08
(m, 1H; CH), 4.88 (m, 1H; H4’), 4.03–3.74 (m, 2H; H5’a/H5’b), 3.29–
2.94 (m, 2H; CH2), 1.34 (s, 9H; H-tBuBoc), 0.59 (s, 9H; H-tBuTBS), ꢁ0.21
(s, 3H; H-MeTBS), ꢁ0.39 ppm (s, 3H; H-MeTBS); 13C NMR (63 MHz,
(s, 9H; H-tBuTBS), ꢁ0.12 (s, 3H; H-MeTBS), ꢁ0.49 ppm (s, 3H; H-MeTBS);
ꢁ
CD3OD): d=173.6 (C=O Bz), 157.4 (Cq), 154.0 (Cq), 153.2 (C2 or C8),
13
ꢁ
C NMR (63 MHz, CDCl3): d=164.7 (C=O Bz), 152.4 (C2 or C8), 150.7
150.4 (Cq), 146.5 (C2 or C8), 139.2 (Cq), 135.3 (Cq-Ar), 134.3 (C-Ar),
130.5 (C-Ar), 129.9 (C-Ar), 129.4 (C-Ar), 127.5 (Cq-triazole), 124.6 (C-
triazole), 91.1 (C1’), 84.7 (C4’), 80.2 (Cq-tBuBoc), 76.6 (C2’), 63.2 (C3’),
62.6 (C5’), 50.0 (CH), 42.5 (CH2), 28.7 (C-tBuBoc), 25.9 (C-tBuTBS),
18.5(Cq-tBuTBS), ꢁ5.00 (C-MeTBS), ꢁ5.28 ppm (C-MeTBS); HRMS (ESI):
m/z: calcd for C45H53N9O7SiNa: 882.3735 [M+Na+]; found: 882.3729.
(Cq-Ad), 143.1 (C2 or C8), 133.5 (Cq-Bz), 133.0 (C-Bz), 128.9 (C-Bz),
128.0 (C-Bz), 91.0 (C1’), 85.4 (C4’), 74.9 (C2’), 63.3 (C3’), 60.9 (C5’), 25.2
(C-tBuTBS), 15.2 (Cq-tBuTBS), ꢁ5.0 (C-MeTBS), ꢁ5.8 ppm (C-MeTBS);
HRMS (ESI): m/z: calcd for C23H31N8O4SiNa: 533.2057 [M+Na]+;
found: 533.2052.
General procedure for Ru-catalysed cycloadditions: A mixture of azide 8
(1.0 equiv, 0.2 mmol), alkyne 4a or 4c (2.5 equiv) and [Cp*RuClACHTUNGTRENNUNG(PPh3)2]
6-N-Benzoyl-2’-O-(tert-butyldimethylsilyl)-3’-(S)-tert-butyl-1-(1H-1,2,3-
triazol-4-yl)ethylcarbamate-3’-deoxyadenosine (6): Compound 5a (97 mg,
12.4 mmol) was stirred in CH2Cl2/NH4OH (8 mL, 1:1) for 4 h at room
temperature. The reaction mixture was washed with aqueous satured
NH4Cl solution, water and brine. The organic phase was dried over anhy-
drous Na2SO4. The solvent was removed under reduced pressure, and the
crude residue was purified by flash chromatography (EtOAc/cyclohexane
(0.1 equiv, 0.02 mmol) in anhydrous benzene (1 mL) was heated at reflux
at 8088C for 24 h. The progress of the reaction was monitored by TLC.
The mixture was then cooled and evaporated under reduced pressure.
The product was purified by flash chromatography with elution with
EtOAc/cyclohexane 6:4.
8:2) to give 6 (77 mg, 92%) as a white solid. Rf =0.49 (EtOAc); [a]D25
=
6-N-Benzoyl-2’-O-(tert-butyldimethylsilyl)-3’-(S)-tert-butyl-1-(1H-1,2,3-
1
ꢁ51.0 (c=0.8 in MeOH); H NMR (250 MHz, CDCl3): d=9.30 (brs, 1H;
triazol-5-yl)ethylcarbamate-3’-deoxyadenosine (9a): Azide
0.2 mmol), alkyne 4a (83 mg, 0.5 mmol) and [Cp*RuCl(PPh3)2] (16 mg,
0.02 mmol) were treated as described in the General Procedure. The
product 9a (49 mg, 37%) was obtained as yellow oil. Rf =0.68
8 (100 mg,
NH), 8.86 (s, 1H; H2 or H8), 8.09 (s, 1H; H2 or H8), 8.03–8.00 (m, 2H;
AHCTUNGTRENNUNG
H-Bz), 7.60–7.50 (m, 3H; H-Bz), 7.48 (s, 1H; Htriazole), 6.14 (d, JH,H
=
3
7.5 Hz, 1H; H1’), 5.32 (m, 1H; H2’), 5.18 (m, 2H; H3’/NH), 4.96 (m, 2H;
a
3
CH/H4’), 4.11–3.75 (dd, 2JH,H =12.5 Hz, 2H; H5’a/H5’b), 1.56 (d, JH,H
=
(EtOAc); [a]2D5 =ꢁ37.8 (c=1 in MeOH); 1H NMR (250 MHz, CDCl3):
d=9.32 (brs, 1H; NH), 8.85 (s, 1H; H2 or H8), 8.08 (s, 1H; H2 or H8),
8.03 (m, 2H; H-Bz), 7.83 (s, 1H; Htriazole), 7.56 (m, 3H; H-Bz), 6.41 (d,
3JH,H =7.2 Hz, 1H; H1’), 6.14 (brs, 1H; NH), 5.60 (m, 1H; H3’), 5.41 (t,
3JH,H =7.1 Hz, 1H; H2’), 4.96 (m, 1H; CH), 4.81 (s, 1H; H4’), 4.20–3.96
(m, 2H; H5’a/H5’b), 1.61 (d, 3JH,H =5.0 Hz, 3H; CH3), 1.45 (s, 9H; H-
tBuBoc), 0.54 (s, 9H; H-tBuTBS), ꢁ0.12 (s, 3H; H-MeTBS), ꢁ0.72 ppm (s,
7.5 Hz, 3H; CH3), 1.42 (s, 9H; H-tBuBoc), 0.55 (s, 9H; H-tBuTBS), ꢁ0.20
(s, 3H; H-MeTBS), ꢁ0.56 ppm (s, 3H; H-MeTBS); 13C NMR (63 MHz,
ꢁ
CDCl3): d=164.8 (C=O Bz), 155.3 (Cq), 152.6 (C2 or C8), 150.5 (Cq),
143.3 (C2 or C8), 133.2 (Cq-Bz), 129.0 (C-Bz), 128.0 (C-Bz), 124.2 (Cq-
Bz), 122.8 (C-triazole), 91.6 (C1’), 84.9 (C4’), 79.7 (Cq-tBuBoc), 73.7 (C2’),
63.5 (C5’), 63.1 (C3’), 42.7 (CH), 28.5 (C-tBuBoc), 25.2 (C-tBuTBS), 21.3
Chem. Eur. J. 2009, 15, 1929 – 1938
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1935

M. Etheve-Quelquejeu et al.
13
3H; H-MeTBS); C NMR (63 MHz, CDCl3): d=172.1 (C=O Bz), 152.6
(C2 or C8), 143.4 (C2 or C8), 132.1 (C-Bz), 129.5 (C-triazole), 129.0 (C-
Bz), 128.7 (C-Bz), 92.0 (C1’), 86.1 (C4’), 73.5 (C2’), 63.4 (C5’), 60.4 (C3’),
39.8 (CH), 28.4 (C-tBuBoc), 25.1 (C-tBuTBS), 20.2 (CH3), ꢁ4.9 (C-MeTBS),
ꢁ5.9 ppm (C-MeTBS); HRMS (ESI): m/z: calcd for C32H45N9O6SiNa:
702.3160 [M+Na]+; found: 702.3154.
to give dinucleotide 14 (97 mg, 70%) as two diastereomers. 1H NMR
ꢁ
(250 MHz, CDCl3): d=9.60 (m, 2H; NHAc/NHBz), 8.73 (s, 1H; H2Ad or
3
H8Ad), 8.41 (d, J=7.0, 1H; H6Cyt), 7.83 (d, JH,H =7.3, 3H; H2Ad or H8Ad
/
H-Bz), 7.41 (m, 5H; H5Cyt/H-Bz/Htriazole), 6.34 (s, 1H; H1’Ad), 6.09 (m,
2H; H1’Cyt/NH), 5.51 (m, 1H; H3’Ad), 5.03 (m, 3H; H2’Ad/H4’Ad/H3’Cyt),
4.50 (m, 1H; CH), 4.22 (m, 5H; H4’Cyt/H5’Cyt/CH2O), 3.72 (m, 2H;
H5’Ad), 2.68 (m, 4H; H2’Ad/H2’aCyt/CH2CN), 2.34 (m, 1H; H2’bCyt), 2.20 (s,
3H; H-MeAc), 1.60 (m, 3H; CH3), 1.40 (s, 9H; H-tBuBoc), 0.07 (2 s, 9H;
H-tBuTBS), ꢁ0.20 (2ꢅs, 3H; H-MeTBS), ꢁ0.43 ppm (2ꢅs, 3H; H-MeTBS);
31P NMR (1H decoupled, 162 MHz, CDCl3): d=ꢁ0.15, 1.98 ppm (2ꢅs,
diast). HRMS (ESI): m/z: calcd for C46H63N13O13P1SiNa: 1086.3995
[M+Na]+; found: 1086.3989.
6-N,N-Dibenzoyl-2’-O-(tert-butyldimethylsilyl)-3’-(S)-tert-butyl-1-(1H-
1,2,3-triazol-5-yl)-2-phenylethylcarbamate-3’-deoxyadenosine (9c): Azide
8 (100 mg, 0.2 mmol), alkyne 4c (120 mg, 0.5 mmol) and [Cp*RuCl-
ACHTUNGTRENNUNG(PPh3)2] (16 mg, 0.02 mmol) were treated as described in the General
Procedure. The product 9c (57 mg, 39%) was obtained as a yellow oil.
Rf =0.72 (EtOAc); [a]2D5 =ꢁ42.0 (c=1 in CHCl3); 1H NMR (250 MHz,
CDCl3): d=9.36 (brs, 1H; NH), 8.82 (s, 1H; H2 or H8), 8.10 (s, 1H; H2
or H8), 8.03 (m, 2H; H-Bz), 7.61 (s, 1H; Htriazole), 7.53–7.47 (m, 8H; H-
Bz/H-Ph), 6.49 (d, 3JH,H =7.5 Hz, 1H; H1’), 6.09 (brs, 1H; NH), 5.39 (t,
3JH,H =7.5 Hz, 1H; H2’), 5.01 (m, 1H; H4’), 4.24–3.87 (m, 4H; CH/H3’/
H5’a/H5’b), 3.53–3.05 (m, 2H; CH2 Ph), 1.21 (s, 9H; H-tBuBoc), 0.55 (s,
General procedure for dinucleotide phosphorylation: Bis(2-cyanoethyl)-
diisopropylphosphoramidite (5 equiv) was added neat to the flask con-
taining the dinucleotide (1 equiv). Anhydrous CH2Cl2 (3.5 mLmmolꢁ1
)
was then added, followed by a solution of tetrazole in CH3CN (0.45m,
20 equiv). The mixture was stirred at room temperature for 1 hour, and a
solution of I2 (0.1m, 5 equiv) was added. After having been stirred at
room temperature for 30 min, the mixture was diluted with EtOAc and
washed successively with aqueous saturated Na2S2O3 solution and brine.
The organic layer was dried over anhydrous Na2SO4, concentrated to dry-
ness and then dissolved in a solution of MeNH2 (5m, large excess). The
reaction mixture was stirred for 12 h at room temperature and concen-
trated under reduced pressure. The residue was purified by HPLC. After
the appropriate fractions had been collected and lyophilized, the phos-
9H; H-tBuTBS), ꢁ0.25 (s, 3H; H-MeTBS), ꢁ0.67 ppm (s, 3H; H-MeTBS);
13
ꢁ
C NMR (63 MHz, CDCl3): d=164.6 (C=O Bz), 152.6 (C2 or C8), 150.6
(Cq), 143.4 (C2 or C8), 132.0 (C-Bz or C-Ph), 132.8 (C-Bz or C-Ph),
132.2 (C-Bz or C-Ph), 131.0 (C-triazole), 127.3 (C-Bz or C-Ph), 92.1
(C1’), 86.2 (C4’), 73.5 (C2’), 63.2 (C5’), 60.3 (C3’), 43.5 (CH), 29.8 (C-
CH2Ph), 28.1 (C-tBuBoc), 25.4 (C-tBuTBS), ꢁ4.8 (C-MeTBS), ꢁ5.7 ppm (C-
MeTBS); HRMS (ESI): m/z: calcd for C38H49N9O6SiNa: 778.3473; found:
778.3467 [M+Na]+.
+
phorylated product was obtained as an NH4 salt.
General procedure for dinucleotide synthesis: Coupling was directly car-
ried out under argon in a commercial Ac-dC-PCNE phosphoramidite
vial (250 mg, 324 mmol) fitted with a flat magnetic stirrer. The adenosine
derivative (130 mmol) was added first, followed by anhydrous CH2Cl2
(350 mL). A solution of tetrazole in CH3CN (0.45m, 2.9 mL) was then
added slowly to start the reaction. The mixture was stirred at room tem-
perature for 1 hour (TLC monitoring), and a solution of I2 (0.1m, 3.3 mL)
was added. The reaction mixture was stirred at room temperature for
30 min, diluted with EtOAc and washed successively with aqueous satu-
rated Na2S2O3 solution and brine. The organic layer was dried over anhy-
drous Na2SO4 and concentrated to dryness. A solution of trichloroacetic
acid (0.18m, 7.2 mL) was finally added to the resulting residue, and the
mixture was stirred at room temperature for 30 min, diluted with CH2Cl2
and washed successively with aqueous ice-saturated NaHCO3 solution
and brine. The organic layer was dried over anhydrous Na2SO4 and con-
centrated under vacuum. The crude product was purified by preparative
TLC (CH2Cl2/MeOH 9:1) to afford the desired compound as two diaste-
reomers.
Compound 12: Dinucleotide 11 (56 mg, 52.6 mmol) was treated with
bis(2-cyanoethyl)diisopropylphosphoramidite (71 mg, 263 mmol) in the
presence of tetrazole (2.76 mL, 1.05 mmol) as described in the General
Procedure and was then oxidized with I2 (3 mL, 263 mmol) to give the
+
NH4 salt of the phosphorylated product 12 (27 mg, 54%) as a white
solid. HPLC retention time: 37 min; 1H NMR (250 MHz, CD3OD): d=
3
8.69 (s, 1H; H2Ad or H8Ad), 8.21 (s, 1H; H2Ad or H8Ad), 8.09 (d, JH,H
=
7.6, 1H; H6Cyt), 8.00 (s, 1H; Htriazole), 6.78 (d, 3JH,H =5.7 Hz, 1H; H1’Ad),
6.25 (m, 1H; H1’Cyt), 5.95 (d, 3JH,H =7.6 Hz, 1H; H5Cyt), 5.57 (m, 1H;
H3’Ad), 5.21 (m, 1H; H2’Ad), 4.92 (m, 3H; H3’Cyt/H4’Ad/CH), 4.34 (s, 1H;
H4’Cyt), 4.30–4.03 (m, 4H; H5’Ad/H5’Cyt), 2.50 (m, 1H; H2’aCyt), 2.16 (m,
1H; H2’bCyt), 1.50 (d, JH,H =7.0, 3H; CH3), 1.44 (s, 9H; H-tBuBoc), 0.59 (s,
3
9H; H-tBuTBS), ꢁ0.12 (s, 3H; H-MeTBS), ꢁ0.29 ppm (s, 3H; H-MeTBS);
13C NMR (63 MHz, CD3OD): d=175.5 (C=O Boc), 165.7 (C=OCyt),
ꢁ
157.2 (Cq), 154.0 (Cq), 151.7 (C2Ad or C8Ad), 151.0 (Cq), 143.6 (C2Ad or
C8Ad), 141.2 (C6Cyt), 130.4 (Cq), 124.4 (CHtriazole), 119.9 (Cq), 116.2 (Cq),
96.2 (C5Cyt), 89.7 (C1’Ad), 87.6 (C1’Cyt), 87.1 (C4’Cyt), 82.8 (C4’Ad), 80.3
(Cq-tBuBoc), 77.7 (C3’Cyt), 76.9 (C2’Ad), 66.3 (C5’Cyt), 66.0 (C5’Ad), 63.4
(C3’Ad), 44.2 (CH), 40.8 (C2’Cyt), 28.8 (C-tBuBoc), 25.9 (C-tBuTBS), 20.9
(CH3), 18.4 (Cq-tBuTBS), ꢁ3.7 (C-MeTBS), ꢁ4.1 ppm (C-MeTBS); 31P NMR
(1H decoupled, 162 MHz, CDCl3): d=1.98, 0.17 ppm (2ꢀs).
Compound 11: Adenosine derivative 6 (88 mg, 129 mmol) was coupled to
Ac-dC-PCNE-phosphoramide as described in the General Procedure to
give dinucleotide 11 (95 mg, 69%) as two diastereomers. 1H NMR
(250 MHz, CDCl3): d=9.73 (s, 2H; NHAc/NHBz), 8.76 (s, 1H; H2Ad or
3
H8Ad), 8.39 (s, 1H; H2Ad or H8Ad), 8.16 (brs, 1H; H6Cyt), 8.02 (d, JH,H
=
Compound 15: Dinucleotide 14 (7 mg, 6.6 mmol) was treated with bis(2-
cyanoethyl)diisopropylphosphoramidite (9 mg, 33 mmol) in the presence
of tetrazole (0.345 mL, 132 mmol) as described in the General Procedure
3
7.8 Hz, 2H; H-Bz), 7.50–7.30 (m, 6H; H-Bz/Htriazole), 7.31 (d, JH,H
=
7.2 Hz, 1H; H5Cyt), 6.17 (s, 1H; H1’Ad), 6.07 (s, 1H; H1’Cyt), 5.68 (m, 1H;
H3’Ad), 5.27 (m, 1H; H2’Ad), 5.12 (m, 2H; H3’Cyt/H4’Ad), 4.96 (m, 1H;
CH), 4.55 (m, 2H; H5’Cyt), 4.19 (m, 5H; H4’Cyt/H5’Cyt/CH2O), 3.75 (m,
2H; H5’Ad), 2.73 (m, 4H; H2’Ad/H2’aCyt/CH2CN), 2.16 (s, 3H; H-MeAc),
1.52 (d, 3JH,H =6.8 Hz, 3H; CH3), 1.41 (s, 9H; H-tBuBoc), 0.68 (s, 9H; H-
tBuTBS), ꢁ0.15 (s, 3H; H-MeTBS), ꢁ0.22 ppm (s, 3H; H-MeTBS); 13C NMR
+
and was then oxidized with I2 (0.75 mL, 33 mmol) to give the NH4 salt
of the phosphorylated product 15 (4 mg, 64%) as a white solid. HPLC re-
tention time: 35 min; 31P NMR (1H decoupled, 162 MHz, CDCl3): d=
1.52, 0.55 ppm (2ꢅs).
General procedure for N-Boc and O-TBS removal: The partially protect-
ed dinucleotide 12 or 15 was treated with a mixture of aqueous HCl
(6n)/THF/MeOH 1:2:1 at room temperature for 24 h. The reaction mix-
ture was then concentrated under vacuum, diluted with water and
washed with CH2Cl2. The aqueous layer was evaporated under reduced
pressure, and the residue was purified by HPLC. After the appropriate
fractions had been collected and lyophilized, the final dinucleotide was
(63 MHz, CDCl3): d=184.7 (C=OBoc), 171.2 (C=O Ac), 162.8 (C=OCyt),
ꢁ
Ad
155.4 (C=O Bz), 152.7 (C2 or C8Ad), 151.4 (Cq), 150.1 (Cq), 145.2
ꢁ
(C6Cyt), 142.1 (C2Ad or C8Ad), 132.9 (C-Bz), 128.8 (C-Bz), 128.2 (C-Bz),
124.0 (Cq-triazole), 122.0 (CHtriazole), 96.8 (C5Cyt), 91.0 (C1’Ad), 87.6
(C1’Cyt), 86.4 (C4’Cyt), 79.4 (Cq-tBuBoc), 79.2 (C4’Ad/C3’Cyt), 75.0 (C2’Ad),
66.3 (C5’Cyt), 62.8 (CH2O), 61.2 (C5’Ad), 60.4, 55.9 (CH), 42.9 (C3’Ad), 39.7
(C2’Cyt), 29.4 (Cq-tBuTBS), 28.4 (C-tBuBoc), 25.4 (C-tBuTBS), 24.8 (C-MeAc),
+
obtained as an NH4 salt.
21.5 (CH3), 19.7 (CH2CN), ꢁ5.0
ACHTUNGTRENNUNG
Compound 13: The protected dinucleotide 12 (23 mg, 24.3 mmol) was
treated with a mixture of aqueous HCl (6n)/THF/MeOH (2 mL) as de-
scribed in the General Procedure to give the NH4 salt of the deprotect-
31P NMR (1H decoupled, 162 MHz, CDCl3): d=ꢁ0.68, 1.45 ppm (2ꢅs,
diast); HRMS (ESI): m/z: calcd for C46H63N13O13PSi: 1064.4175 [M+H]+;
found: 1064.4170.
+
ed dinucleotide 13 (8 mg, 44%) as a white solid. HPLC retention time:
1
Compound 14: Adenosine derivative 9a (88 mg, 129 mmol) was coupled
to Ac-dC-PCNE-phosphoramide as described in the General Procedure
13 min; [a]2D5 =ꢁ8.0 (c=0.27 in H2O); H NMR (500 MHz, D2O): d=8.57
(s, 1H; H2Ad or H8Ad), 8.32 (s, 1H; Htriazole), 8.18 (s, 1H; H2Ad or H8Ad),
1936
www.chemeurj.org
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 1929 – 1938

Aminoacyl-tRNA Analogues
FULL PAPER
7.74 (d, JH,H =7.6 Hz, 1H; H6Cyt), 6.25 (d, JH,H =6.4 Hz, 1H; H1’Ad), 6.11
(t, J=6.5 Hz, 1H; H1’Cyt), 5.81 (d, 3JH,H =7.6 Hz, 1H; H5Cyt), 5.75–5.61
(m, 1H; H3’Ad/H4’Cyt), 5.15 (m, 2H; H2’Ad/H4’Ad), 4.91–4.69 (m, 2H; CH/
H3’Cyt), 4.23 (m, 2H; H5’bAd), 4.17 (d, 2JH,H =15.0 Hz, 1H; H5’bAd), 4.01
(sl, 2H; H5’Cyt), 2.40 (m, 1H; H2’aCyt), 1.97 (m, 1H; H2’bCyt), 1.72 ppm (d,
3JH,H =7.0 Hz, 3H; H-CH3); 13C NMR (126 MHz, D2O): d=168.0 (C=
OCyt), 159.6 (Cq), 157.9 (Cq), 155.6 (C2Ad or C8Ad), 151.8 (Cq), 147.5
(Cq), 143.8 (C6Cyt), 142.0 (C2Ad or C8Ad), 128.5 (CHtriazole), 121.0 (Cq),
98.8 (C5Cyt), 89.9 (C1’Ad), 88.2 (C1’Cyt), 86.7 (C4’Cyt), 83.8 (C4’Ad), 78.5
(C3’Cyt), 77.5 (C2’Ad), 66.7 (C5’Cyt), 67.7 (C5’Ad), 64.8 (C3’Ad), 45.8 (CH),
41.2 (C2’Cyt), 20.4 ppm (CH3); 31P NMR (1H decoupled, 202 MHz, D2O):
d=1.98, 0.30 ppm (2ꢅs); HRMS (ESI): m/z: calcd for C23H31O12N12P2:
729.1665 [MꢁH]ꢁ; found: 729.1665.
lard, J. Delettrꢀ, W. Sougakoff, M. Arthur, C. Mayer, Structure 2004,
12, 257–267; c) T. E. Benson, D. B. Prince, V. T. Mutchler, K. A.
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3
3
[4] C. Goffin, J.-M. Ghuysen, Microbiol. Mol. Biol. Rev. 2002, 66, 702–
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Compound 16: The protected dinucleotide 15 (4 mg, 4.23 mmol) was
treated with a mixture of aqueous HCl (6n)/THF/MeOH (large excess)
+
as described in the General Procedure to give the NH4 salt of the de-
protected dinucleotide 16 (1.8 mg, 58%) as a white solid. [a]2D5 =ꢁ7.0
(c=0.03 in H2O); 1H NMR (500 MHz, D2O): d=8.56 (s, 1H; H2Ad or
H8Ad), 8.20 (s, 1H; H2Ad or H8Ad), 8.09 (s, 1H; Htriazole), 7.73 (d, JH,H
=
3
7.6 Hz, 1H; H6Cyt), 6.14 (m, 2H; H1’Ad/H1’Cyt), 5.91 (d, 3JH,H =7.8 Hz,
1H; H5’Cyt), 5.66 (dd, 3JH,H =2.8, 6.7 Hz, 1H; H3’Ad), 5.34 (s, 1H; H4’Ad),
5.30 (m, 1H; H2’Ad), 4.99 (dd, J=6.7, 13.5 Hz, 1H; CH), 4.79 (m, 1H;
H3’Cyt), 4.23 (m, 3H; H4’Cyt/H5’Ad), 4.00 (dd, 3JH,H =4.5, 7.5 Hz, 2H;
H5’Cyt), 2.40 (m, 1H; H2’aCyt), 1.90 (m, 1H; H2’bCyt), 1.72 ppm (d, JH,H
=
3
6.9, 3H; CH3); 13C NMR (126 MHz, D2O): d=155.4 (C2 or C8Ad), 143.4
(C6Cyt), 142.1 (C2 or C8Ad), 135.1 (CHtriazole), 98.8 (C5Cyt), 88.3 (C1’Cyt
/
C1’Ad), 86.7 (C4’Cyt), 83.1 (C4’Ad), 77.9 (C3’Cyt), 75.3 (C2’Ad), 67.6 (C5’Ad),
66.4 (C5’Cyt), 62.8 (C3’Ad), 42.5 (CH), 40.3 (C2’Cyt), 21.5 ppm (CH3);
31P NMR (1H decoupled, 202 MHz, D2O): d=2.73, 1.61 ppm (2ꢅs);
HRMS (ESI): m/z: calcd for C23H31O12N12P2: 729.1665; found: 729.1647
[MꢁH]ꢁ; HPLC retention time: 12 min.
Ligation of dinucleotides 13 or 16 with the microhelix and purification of
product C or C’: The ligation reaction was performed at 378C over
120 min in Hepes buffer (500 mL, 50 mm) containing the RNA microhelix
(22 nt, 20 nmol), compound 13 or 16 (200 nmol), T4 RNA ligase (3.1 mg),
DMSO (10%), ATP (1 mm) and MgCl2 (15 mm). Compound C was puri-
fied by FPLC (Superdex 75 HR 10/30 column, Amersham Pharmacia
Biotech) in Tris-HCl (pH 7.5, 25 mm), NaCl (100 mm), MgCl2 (5 mm) (re-
tention volume=12.40 mL, corresponding to a 21 kDa protein). After de-
salting, the product was lyophilised, dissolved in H2O (100 mL, RNAse-
free, Sigma) and quantified by UV absorption at lmax(e)=260 nm (2.3ꢅ
105). Analysis of C or C’ by denaturing polyacrylamide gel electrophore-
sis was carried out in an acrylamide gel (20 cm, 13%) containing urea
(8m) over 120 min at 600 V/300 mA/50 W.
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Radioactive coupled FemXWv assay: The standard assay used Tris-HCl
(50 mm, pH 7.5), alanyl-tRNA synthetase of E. faecalis (800 nm), ATP
(7.5 mm), MgCl2 (12.5 mm), [14C]Ala (50 mm, 3700 Bqnmolꢁ1, ICN, Orsay,
France), FemXWv (2 nm), UDP-MurNAc-pentapeptide (50 mm), tRNAAla
(0.4 mm) and inhibitor C or C’ (0 to 500 mm). The reaction was performed
at 308C for 10 min with a preincubation time of 10 min in the absence of
FemXWv for synthesis of Ala-tRNAAla by the auxiliary system. The reac-
tion was stopped at 958C for 10 min, and analysis was performed by de-
scending paper chromatography (Whatman 4MM, Elancourt, France)
with isobutyric acid/1m ammonia 5:3 v/v. Radioactive spots were identi-
fied by autoradiography, cut out and counted by liquid scintillation.
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Acknowledgements
We thank Dr. Isabelle Correia and Prof. Olivier Lequin for NMR spectra
of compounds 13 and 16.
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Received: July 31, 2008
Published online: November 26, 2008
1938
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Chem. Eur. J. 2009, 15, 1929 – 1938