Synthesis and biological evaluation of non-isomerizable analogues of Ala-tRNAAla

Article abstract of DOI:10.1039/c3ob41206g

Aminoacyl-tRNAs serve as amino acid donors in many reactions in addition to protein synthesis by the ribosome, including synthesis of the peptidoglycan network in the cell wall of bacterial pathogens. Synthesis of analogs of aminoacylated tRNAs is critica

Full text of DOI:10.1039/c3ob41206g

Organic &
Biomolecular Chemistry
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Synthesis and biological evaluation of non-
isomerizable analogues of Ala-tRNAAla
Cite this: Org. Biomol. Chem., 2013, 11,
6161
Denia Mellal,a,b Matthieu Fonvielle,c,d,e Marco Santarem,a,b Maryline Chemama,a,b
Yoann Schneider,a,b Laura Iannazzo,f,g Emmanuelle Braud,f,g Michel Arthur*c,d,e and
Mélanie Etheve-Quelquejeu*a,b,f,g
Aminoacyl-tRNAs serve as amino acid donors in many reactions in addition to protein synthesis by the
ribosome, including synthesis of the peptidoglycan network in the cell wall of bacterial pathogens. Syn-
thesis of analogs of aminoacylated tRNAs is critical to further improve the mechanism of these reactions.
Here we have described the synthesis of two non-isomerizable analogues of Ala-tRNAAla containing an
amide bond instead of the isomerizable ester that connects the amino acid with the terminal adenosine
in the natural substrate. The non-isomerizable 2’ and 3’ regioisomers were not used as substrates by
FemXWv, an alanyl-transferase essential for peptidoglycan synthesis, but inhibited this enzyme with IC50
of 5.8 and 5.5 µM, respectively.
Received 11th June 2013,
Accepted 22nd July 2013
DOI: 10.1039/c3ob41206g
www.rsc.org/obc
the terminal adenine. In natural aminoacyl-tRNAs, the ami-
noacyl residue is linked by an ester bond to the 2′ or 3′
Introduction
Aminoacyl-tRNAs (aa-tRNAs) participate in a vast repertoire of hydroxyls of the terminal adenosine of the tRNA (Fig. 1). An
reactions, including synthesis of peptidoglycan precursors uncatalyzed transesterification reaction leads to isomerization
and tetrapyrrole, modification of bacterial membrane lipids, between the O2′ and O3′ positions in aqueous solution with a
and N-terminal labeling of proteins targeted to proteolysis.1 thermodynamic equilibrium and a rate of 1 and 5 s−1, respecti-
Aminoacyl-tRNAs also participate in secondary metabolism vely,5 as evaluated by NMR analysis of acylated mononucleo-
including synthesis of antibiotics2 and cyclic dipeptides.3 The tides. Aminoacyl-tRNA synthetases, which charge the 20
corresponding tRNA-dependent aminoacyl-transferases have natural amino acids, fall into two classes with respect to the
only recently been investigated revealing a high diversity in the hydroxyl used for aminoacylation.6 The ribosome A site is
catalytic mechanisms and protein structures. Recognition of specific for the 3′ isomer and the position of the acyl-group is
aminoacyl-tRNAs by the transferases and by components of conserved during peptide bond formation and translocation to
the translation machinery potentially involves different mecha- the P site.7 This implies that the aminoacyl residue migrates
nisms relevant to enzyme specificity and competition between from the 2′ to the 3′ position in the case of aminoacyl-tRNA
different pathways for the same aminoacyl-tRNA pools.
This has stimulated interest in the chemical mutagenesis4 of
aminoacyl-tRNA to assess the role of specific groups in enzyme
efficacy and specificity.
One important aspect of tRNA recognition by aminoacyl-
transferases is the regiospecificity for the 2′ or 3′ position of
aInstitut Parisien de Chimie Moléculaire, CNRS UMR 7201, F-75005 Paris, France.
E-mail: melanie.etheve-quelquejeu@parisdescartes.fr
bUniversité Pierre et Marie Curie - Paris 6, F-75005 Paris, France
cCentre de Recherche des Cordeliers, LRMA, Equipe 12, University Pierre et Marie
Curie - Paris 6, UMR S 872, Paris, F-75006, France.
E-mail: michel.arthur@crc.jussieu.fr; Fax: (+33)1-4325-6812
dUniversity Paris Descartes, Sorbonne Paris Cité, UMR S 872, Paris F-75006, France
eINSERM, U872, Paris, F-75006, France
fLaboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, équipe
CBNIT, Université Paris Descartes, UMR 8601, Paris, F-75005, France
gCNRS, UMR 8601, Paris, F-75005, France
Fig. 1 Ala-tRNAALa: the natural substrate of FemXwv; 8a and 8b: Ala-tRNAAla
analogues; Pmn: Puromycin.
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synthetases that acylate the 2′ OH. The presumably uncata- the ribosome19 and in the active site of GatCAB amido
lyzed trans-esterification reaction is thought to be kinetically transferase.20
limiting for protein synthesis despite the elevated rate reported
Two strategies for synthesis of RNA adducts containing an
for model molecules.5 We have recently shown that trans-esteri- amide linkage at the 3′-terminus have been reported. The
fication is also required in the case of the aminoacyl transfer- amide bond could be introduced directly into the full RNA or
ase FemXWv, which adds an alanyl residue to the side chain into the nucleoside, which is then linked to a solid support
of peptidoglycan precursors in the Gram-positive bacterium and provides the precursor for solid phase synthesis. For the
Weissella viridescens.8 In this pathway, alanyl-tRNA synthetase first strategy, direct coupling of an activated amino acid to an
acylates the 3′ position of tRNAAla but FemXWv transfers Ala 18-nt 3′-amino-RNA has been described in a single report.11b
only from the 2′ position.8 Since FemXWv displays similar Efficient coupling was only obtained for formyl-methionine.
affinities for Ala-tRNAAla analogues in which Ala has been For the second strategy, classical peptidic21 or Staudinger
blocked at the 2′ or 3′ position by removing the adjacent coupling21c has been used to link activated amino acids to
hydroxyl, we have proposed that the enzyme binds the 3′ regio- amino- or azido-deoxyadenosine, respectively. Here we report
isomer formed by alanyl-tRNA synthetase. This implies that an alternative to solid phase synthesis that relies on enzymatic
trans-esterification occurs in the catalytic site of FemXWv to ligation. The amide linkage is introduced into the protected
produce the 2′ isomer used in the aminoacyl transfer reaction. adenosine, followed by nucleotidic coupling to obtain
This would enable FemXWv to efficiently compete with the the corresponding fully deprotected dinucleotide, which is
ribosome for the 3′ isomer formed by alanyl-tRNA synthetase.
enzymatically ligated to unprotected RNA. Two analogues of
Two types of chemical modifications have been introduced Ala-tRNAAla 8 containing a 2′- or 3′-amide linkage were gener-
into aa-tRNAs to block trans-esterification between the 2′ and ated by this approach and evaluated as inhibitors or substrates
3′ positions. In a first approach, the adjacent hydroxyl is of FemXWv, a model aminoacyl transferase that catalyzes an
removed or replaced by a methoxy group.9 This leads to the essential step of peptidoglycan synthesis (Fig. 1).
desired stable isomers but also introduces potentially con-
founding factors, due to modification of the furanose confor-
mation of the terminal nucleotide. In addition, the vicinal
Results and discussion
hydroxyl may have a crucial role in substrate-assisted catalysis
Synthesis of nucleosides and dinucleotides
by participating in proton shuttle as proposed for peptide
bond formation by the ribosome and by FemXWv.8 In a second
The synthesis (Scheme 1) started with the hydrogenolysis of
6-N-benzoyl-3′-azido-2′,5′-bis-O-(tert-butyldimethylsilyl)-3′-deoxy-
adenosine 1a8 and 6-N-benzoyl-2′-azido-3′,5′-bis-O-(tert-butyldi-
methylsilyl)-2′-deoxyadenosine 1b8 to afford compounds 2a
and 2b in quantitative yield. N-Boc-L-alanine was used as this
protecting group is stable during phosphoramidite coupling
and easily removed in the last step of dinucleotide synthesis.
Coupling of N-Boc-L-Ala to protected amino-nucleosides 2a
approach, the ester bond connecting the amino acid residue
with the tRNA is substituted by a stable bond that prevents iso-
merization. Phosphate, phosphoramidate, triazole, and oxadia-
zole provide such stable analogues that retain an adjacent
hydroxyl.9,10 Phosphate and phosphoramidate have the poten-
tial advantage to mimic the tetrahedral transition state of
aminoacyl-transfer reactions. Triazole or oxadiazole rings act as
stable isosters of esters, both with respect to geometries and
stereoelectronic properties.10 However, these five-membered
rings increase the distance between the ribose and the amino
acid since they harbor an additional carbon in comparison to
the ester of natural aa-tRNAs. Aminoacyl-tRNA analogues were
also generated by replacement of the ester bond by an amide
linkage11 (Fig. 1). Puromycin has been the first analogue of aa-
tRNA shown to act as a translation inhibitor by entering the
ribosomal A site and participating in peptide bond formation
with the nascent peptidyl chain, thereby blocking further poly-
peptide elongation.12 Puromycin analogues have been deve-
loped in which the methyl groups of the nucleobase have
been removed and the amino acid moiety has been modified.13
Puromycin and its analogues have been broadly used to
solve the X-ray structure of nucleoprotein complexes. This
structural approach has been instrumental to mechanistic
studies of the peptidyl transfer center of the ribosome,14,9c
the editing domain of aa-tRNA synthetases (ThrRS,15 LeuRS,16
and IleRS),17 and the catalytic cavity of L/F transferase.18
Puromycin analogues have also been used to assess stereo-
Scheme 1 (a) H2, Lindlar’s cat., MeOH, RT, 12 h; (b) L-AlaNHBoc, EDCI, DMAP,
and regio-chemical constraints on peptide bond formation in CH2Cl2, 0 °C to RT, 18 h; (c) TFA, THF–H2O, 0 °C, 12 h.
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Fig. 2 (A) Magnification of JRES-NMR spectra of 7a and 7b. (B) Coupling con-
stants for adenosine H1’ and S% values in D2O of 7a and 7b.
Conformational study
Nucleosides and their analogues are known to exist in equili-
Scheme 2 (a) (i) Tetrazole, CH2Cl2, RT, 1 h; (ii) I2, H2O/Pyr/THF, RT, 30 min; brium between S- and N-type conformations based on their
(iii) TCA, CH2Cl2, RT, 30 min; (b) (i) bis(2-cyanoethyl)diisopropylphosphoramidite,
furanose-ring puckering. Conformational modifications of the
furanose ring produce major structural changes in natural
tetrazole, CH2Cl2, RT, 1 h; (ii) I2, H2O/Pyr/THF, RT, 30 min; (iii) CH3CN, EtOH/H2O,
RT, 24 h; (c) TBAF, THF then TFA/H2O, RT, 15 min.
nucleic acids and have important consequences for their bio-
logical functions. Altona and co-workers reported that 1H–1H
coupling constants J1′2′, J2′3′and J3′4′ vary with ribose confor-
and 2b was done by EDCI/DMAP-mediated amide bond for-
mation in dichloromethane overnight at room temperature.
The 3′-L-Ala-amido-3′-deoxyadenosine 3a and the 2′-L-Ala-
amido-2′-deoxyadenosine 3b were obtained in 62% and 74%
yields, respectively. Selective deprotection of the tert-butyldi-
methylsilyl group at position 5′ was performed in the presence
of TFA to afford 4a and 4b in 64% and 78% yields, respectively.
Dinucleotides 7a and 7b were obtained by the phosphor-
amidite approach (Scheme 2). Nucleosides 4a and 4b were
coupled to commercial deoxycytidine phosphoramidite
(Ac-dC-CE-phosphoramidite) in the presence of tetrazole. The
crude products were oxidized with iodine and treated with tri-
chloroacetic acid to afford compounds 5a and 5b in 54% and
72% yields, respectively. Phosphorylation of 5a and 5b with
bis(2-cyanoethyl)diisopropylphosphoramidite in tetrazole, fol-
lowed by deprotection of cyanoethyl, acetyl, and benzoyl groups
with methylamine afforded 6a and 6b, respectively. The N-Boc
and TBS protecting groups were removed in 6a and 6b by treat-
ing with a solution of TBAF and then with a solution of TFA/H2O
for 15 min, producing 7a and 7b. The two deprotection steps
were performed without purification of compounds 6a and 6b.
Compounds 7a and 7b were obtained from 5a and 5b in 40%
and 42% yields, respectively. COSY NMR experiments were
performed to assign all protons of dinucleotides 7a and 7b.
Carbons were assigned by HSQC and HMBC experiments.
mation and several equations have been proposed to predict
the relative abundance of S- and N-type conformations.22 Here,
we used the “10-Hertz rule-of-thumb” (% S-type = 10J1′–2′) to
1
determine the pucker preference of compounds 7a and 7b. H
NMR was not sufficient to determine J1′–2′ since the signal of
the anomeric proton of adenosine (H1′) overlapped with other
signals (H5 of the cytidine base for 7a and H1′ of the cytosine
for 7b). The J1′–2′ constant was successfully determined by 2D
1H J-resolved ( JRES) NMR spectroscopy, which provided well-
resolved signals for adenosine H1′ (Fig. 2). The deduced S%
values in D2O were 25 and 78 for 7a and 7b, respectively. The
N-type conformation is also preponderant for puromycin,
which is 3′-substituted (Fig. 3).23
Enzymatic ligation
Ala-tRNAAla analogues 8a and 8b were obtained by ligation of
dinucleotides 7a and 7b to a 22-nt RNA helix, which mimics
the acceptor arm of tRNAAla (helixAla: 5′-GGG GCC UUA GCU
CAG GCU CCA C-3′) (Fig. 4). The ligation reaction was per-
formed with T4 RNA ligase, as previously described.24 Com-
pounds 8a and 8b were purified by anion exchange
chromatography and analyzed by denaturing PAGE.
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Fig. 3 Conformers of 7a and 7b.
Fig. 6 (A) Inhibition of FemXWv by 8a and 8b. The inhibition of the transfer of
[
14C]L-Ala from 14C]L-Ala-tRNAAla to UDP-MurNAc-pentapeptide was deter-
[
mined as previously described.26 (B) Assay of 8a and 8b as substrates of FemXwv
.
The transfer of L-Ala from Ala-tRNAAla (1), 8a (2), and 8b (3) to [14C]UDP-Mur-
NAc-pentapeptide (UM5 K) was determined by rp-HPLC as previously
described.27
Fig. 4 (A) Ligation of dinucleotides to the RNA helix and (B) PAGE analysis.
Lanes 1 and 3, helixAla; lane 2, 8a; lane 4, 8b.
compounds (up to 20 µM) were not used as substrates of
FemXWv (10 µM) (Fig. 6B). Under the same conditions, transfer
of L-Ala from L-Ala-tRNAAla to UDP-MurNAc-pentapeptide
was observed at lower substrate and enzyme concentrations
(10 and 0.001 µM, respectively).
It has been previously shown that FemXWv transfers L-Ala
from the 2′-position of tRNAAla.8 In this study, FemXWv did not
catalyze the transfer of L-Ala from 2′-modified 8b, despite the
relatively high affinity of the enzyme for this compound (IC50
of 5.5 0.6 µM). This result is consistent with the fact that pur-
omycin and its analogues, which also contain an amide
instead of an ester bond, block polypeptide elongation by the
ribosome. Although amide bonds are considered chemically
stable, several peptide-bond forming enzymes catalyze amino-
acyl transfer both from amide- and ester-containing substrates.
In peptidoglycan synthesis, this is the case of D,D-transpepti-
dases that cross-link peptidoglycan precursors ending in D-Ala
and D-lactate in vancomycin-resistant enterococci.26
Fig. 5 Synthesis of peptidoglycan precursors in W. viridescens. GlcNAc:
N-acetylglucosamine; MurNAc: N-acetylmuramic acid. The black box represents
the undecaprenyl lipid carrier linked to the peptidoglycan precursor by a
pyrophosphate.
Biological tests
Compounds 8a and 8b were evaluated as inhibitors and
substrates of transferase FemXWv from W. viridescens, which
has been broadly used as a model enzyme for kinetic and
structural analyses of transferases of the Fem family.25 The
enzyme catalyzes the transfer of L-Ala from Ala-tRNAAla to
UDP-MurNAc-pentapeptide, thereby introducing the first
residue into the L-Ala-L-Ser-L-Ala side-chain of peptidoglycan
precursors (Fig. 5).
Both the 2′-modified 8b and the 3′-modified 8a inhibited
FemXWv with IC50 that were similar to each other (5.5 0.6
and 5.8 0.4 µM, respectively) and also similar to the IC50 pre-
viously reported for 3′- and 2′-triazole-substituted RNA ana-
0.1 µM, respectively).8 These
Compounds 8a and 8b inhibited FemXWv with IC50 of
5.8 0.4 and 5.5 0.6 µM, respectively (Fig. 6A), but these
logues (2.4
0.4 and 2.3
observations confirmed that FemXWv does not interact with
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the Ala moiety of the Ala-tRNAAla substrate allowing trans-acy- palladium catalyst (10 mg) at room temperature for 12 hours.
lation within the enzyme active site, as previously discussed.27 The reaction mixture was filtered through celite and the
The amide bond and the triazole linker had similar positive solvent was removed under vacuum.
effects on recognition by FemXWv in comparison to free
3′-Amino-N6-benzoyl-2′,5′-bis-O-(tert-butyl-dimethylsilyl)-3′-
deoxyadenosine (2a). Compound 1a (50 mg, 80 µmol) and
Lindlar palladium catalyst (10 mg) were reacted under a hydro-
gen atmosphere according to the general procedure to give
crude product 2a (48 mg, quantitative yield) as a white foam
which was used in the next step without further purification.
Physical data were in agreement with published data.29
tRNAAla (IC50 of 89 4 µM8).
Conclusions
We have described the synthesis of two Ala-tRNAAla analogues
which contained a stable amide bond instead of the hydroly-
sable and isomerizable ester linkage present in the natural
substrate. The compounds were inhibitors but not substrates
of aminoacyl-transferase FemXWv. H1 NMR spectroscopy, per-
formed on the dinucleotides 7a and 7b, indicated that the 3′-
and 2′-substituted analogues show a preference for N-type and
S-type conformations, respectively. The 3′-substituted ribose of
7a and puromycin display similar preference for N-type confor-
mation.24 The semi-synthetic route reported here is expected
to be applicable to various combinations of amino acid and
RNA due to the promiscuous activity of T4 RNA ligase.28
2′-Amino-N6-benzoyl-3′,5′-bis-O-(tert-butyl-dimethylsilyl)-2′-
deoxyadenosine (2b). Compound 1b (50 mg, 80 µmol) and
Lindlar palladium catalyst (10 mg) were reacted under a hydro-
gen atmosphere according to the general procedure to give
crude product 2b (46 mg, 96%) as a white foam which was
used in the next step without further purification. Rf = 0.36
(AcOEt–Et3N, 99 : 1); [α]2D5 = −54 (c = 1 in CH2Cl2); 1H NMR
(250 MHz, CDCl3) δ = 8.73 (s, 1H, H2 or H8), 8.26 (s, 1H, H2 or
3
H8), 7.99 (d, J(H–H) = 5 Hz, 2H, H–Bz), 7.44 (m, 3H, H–Bz),
3
3
5.93 (d, J(H–H) = 7.5 Hz, 1H, H1′), 4.30 (d, J(H–H) = 7.5 Hz,
1H, H3′), 4.13 (m, 1H, H4′), 3.94 (m, 1H, H2′), 3.81 (dq,
3J(H–H) = 2.5 Hz–15 Hz, 2H, H5′), 0.94 (s, 9H, tBuTBS), 0.89 (s,
9H, tBuTBS), 0.12 (2 s, 6H, 2 × MeTBS), 0.01 (2 s, 6H, 2 × MeTBS).
13C NMR (63 MHz, CDCl3) δ = 165.8 (2 × CvO–Bz), 152.74
(C2 or C8), 152.37 (Cq-Ad), 150.29 (Cq-Ad), 134.18 (Cq-Bz),
124.03 (Cq-Bz), 132.80, 128.39, 128.02 (C–Bz), 89.93 (C1′),
87.19 (C4′), 74.12 (C3′), 63.46 (C5′), 59.45 (C2′), 26.30
(C-tBuTBS), 18.53 (Cq-tBuTBS), −4.17, −4.37, −5.05, −5.15
(4 × MeTBS).
Experimental part
General reagents and materials
Solvents were dried using standard methods and distilled
before use. Unless otherwise specified, materials were pur-
chased from commercial suppliers and used without further
purification. TLC: precoated silica gel thin layer sheets 60 F254
(Merck) and detection by charring with 10% H2SO4 in ethanol
followed by heating. Flash chromatography: silica gel 60 Å,
180–240 mesh from Merck. Spectra were recorded using
Bruker spectrometers ARX 250 for 1H (250.13 MHz) and 13C
(62.89 MHz), AC 400 for 31P (161.97 MHz), Bruker Avance II
600 MHz for 1H and 13C in D2O as indicated below. Chemicals
shifts (δ) are expressed in ppm relative to residual CHCl3
(δ 7.26) or CHD2OD (δ 3.31) or HDO (δ 4.79) for 1H, CDCl3
(δ 77.16) or CD3OD (δ 49.00) for 13C as internal references and
H3PO4 (δ 0) for 31P as external references. Signals were
assigned based on COSY and DEPT 135 (13C). High-resolution
mass spectroscopy (HRMS) was recorded using a Bruker
micrOTOF spectrometer. High-performance liquid chromato-
graphy (HPLC) was performed on a HPLC system with a
reverse phase C-18 column (250 mm × 21.2 mm; HYPERSIL
HSC18; Thermoelectron Corporation) using a solvent system
consisting of 50 mM aqueous NH4OAc–CH3CN (linear gradient
from 100 : 0 to 50 : 50 in 50 min) at a flow rate of 17 mL min−1
and UV detection at 260 nm. Fast protein liquid chromato-
graphy (FPLC) was performed using an AKTA purifier
(Amersham Pharmacia Biotech). Optical rotations were carried
out using a PerkinElmer Model 341 Polarimeter.
General procedure for the coupling step
4-Dimethylaminopyridine (1.2 eq., 0.260 mmol) and 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide (1.2 eq., 0.260 mmol)
were added to a mixture of Boc-L-Ala (1.2 eq., 0.260 mmol) in
anhydrous dichloromethane (1.0 mL) at 0 °C. After being
stirred at 0 °C for 15 min., a solution of 2a–b diluted in anhy-
drous dichloromethane (1.5 mL) was added to the mixture.
After being stirred at room temperature for 18 hours, the
solvent was removed under vacuum. The crude product was
subjected to column chromatography and eluted with EtOAc–
cyclohexane (5 : 5).
N6-Benzoyl-2′,5′-bis-O-(tert-butyl-dimethylsilyl)-3′-(S)-methyl-
carbamate 3′-deoxyadenosine (3a). Compound 2a (0.13 g,
0.217 mmol) and Boc-L-Ala (49 mg, 0.260 mmol) were reacted
according to the general procedure to give compound 3a
(104 mg, 62%) as a yellow oil. Rf = 0.39 (cyclohexane–EtOAc:
1
5/5); [α]2D0 = −35.5 (c = 1 in CHCl3); H NMR (400 MHz, CDCl3)
δ = 9.33 (bs, 1H, NH-Ad), 8.77 (s, 1H, H2 or H8), 8.55 (s, 1H,
3
H2 or H8), 8.01 (d, J(H–H) = 9.0 Hz, 2H, H–Bz), 7.45–7.56 (m,
3H, H–Bz), 6.55–6.58 (bs, 1H, NH-amide), 6.12 (d, 3J(H–H) =
4.0 Hz, 1H, H1′), 4.88 (d, 3J(H–H) = 12 Hz, 1H, NH-Boc),
4.58–4.64 (m, 2H, H2′ et H4′), 3.84–4.15 (m, 4H, H3′, H5′ and
CHα), 1.43 (s, 9H, tBu-Boc), 1.34–1.37 (d, 3J(H–H) = 9.0 Hz, 3H,
CH3α), 0.89–0.94 (m, 18H, H-tBuTBS), 0.03–0.13 (m, 12H,
General procedure for reduction of azido-nucleosides
A mixture of 1 (1.0 eq., 80 µmol) in methanol (4.2 mL) was sub- H-MeTBS); 13C NMR (100 MHz, CDCl3) δ = 172.8 (CvO–Bz),
jected to hydrogenation (1 atm) in the presence of Lindlar 164.9 (CvO-amide), 155.2 (Cq-Ad), 152.8 (C2 or C8), 151.3
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(CvO-Boc), 149.6 (Cq-Ad), 141.2 (C2 or C8), 133.9 (Cq-Bz), 91.2 (C1′), 85.4 (C4′), 80.5 (Cq-tBuBoc), 74.9 (C2′), 61.9 (C5′),
132.7 (C–Bz), 128.9 (C–Bz), 128.0 (C–Bz), 123.0 (Cq), 89.6 (C1′), 51.0 (C3′ and CHα), 28.4 (C-tBuBoc), 25.8 (C-tBuTBS), 18.3
84.5 (C3′), 80.5 (Cq-tBuBoc), 76.5 (C3′), 62.5 (C5′), 50.3 (C2′), (CH3Ala), 18.0 (Cq-tBuTBS), −4.8 (C-MeTBS), −5.0 (C-MeTBS);
28.3 (C-tBuBoc), 26.1 (C-tBuTBS), 25.8 (C-tBuTBS), 18.7 (CH3), HRMS (ESI): m/z: calcd for C31H45N7O7SiNa1: 678.3042, found:
18.6 (Cq-tBuTBS), 18.0 (Cq-tBuTBS), −4.7 (C-MeTBS), −5.0 678.3100 [M + Na+].
(C-MeTBS), −5.3 (C-MeTBS), −5.5 (C-MeTBS); HRMS (ESI): m/z:
N6-Benzoyl-3′-O-(tert-butyl-dimethylsilyl)-2′-(N-tert-butyloxy-
calcd for C37H59N7O7Si2Na1: 792.3907, found: 792.3908 carbonyl-L-alanine)amido-3′-deoxyadenosine (4b). Compound
[M + Na+].
3b (0.20 g, 0.260 mmol) was reacted according to the general
N6-Benzoyl-3′,5′-bis-O-(tert-butyl-dimethylsilyl)-2′-(S)-methyl- procedure to give compound 4b (133 mg, 78%) as a white
carbamate 2′-deoxyadenosine (3b). Compound 2b (0.13 g, foam. Rf = 0.22 (EtOAc); [α]2D0 = −83 (c = 1 in CHCl3); H NMR
1
0.217 mmol) and Boc-L-Ala (49 mg, 0.260 mmol) were reacted (250 MHz, CDCl3) δ = 9.33 (ls, 1H, NHBz), 8.74 (s, 1H, H2 or
according to the general procedure to give compound 3b H8), 8.01 (H2 or H8), 7.97 (d, 3J(H–H) = 7.5 Hz, 2H, H–Bz), 7.49
(144 mg, 74%) as a white powder. Rf = 0.32 (cyclohexane–EtOAc: (m, 3H, H–Bz), 6.78 (m, 1H, NHCO), 6.05 (m, 1H, OH), 5.84
1
3
7/3) [α]2D0 = −69 (c = 1 in CH2Cl2); H NMR (250 MHz, CDCl3) (d, J(H–H) = 7.5 Hz, 1H, H1′), 5.20 (m, 1H, H2′), 5.01 (m, 1H,
δ = 9.12 (ls, 1H, NHBz), 8.79 (s, 1H, H2 or H8), 8.02 (d, 3J(H–H) = NHBoc), 4.58 (d, J(H–H) = 5 Hz, 1H, H3′), 4.13 (m, 1H, H4′),
3
7.5 Hz, 2H, H–Bz), 7.51 (m, 3H, H–Bz), 6.99 (m, 1H, NHCO), 4.05 (m, 1H, HAla), 3.85 (m, 2H, H5′), 1.42 (s, 9H, tBu-Boc),
6.14 (d, J(H–H) = 7.5 Hz, 1H, H1′), 5.02 (m, 1H, H2′), 4.75 (m, 1.15 (d, 3J(H–H) = 7.5 Hz, 3H, CH3Ala), 0.96 (s, 9H, tBuTBS),
3
1H, NHBoc), 4.49 (d, J(H–H) = 2.5 Hz, 1H, H3′), 4.18 (m, 1H, 0.15, 0.14 (2 s, 6H, 2 × MeTBS); 13C NMR (63 MHz, CDCl3) δ =
3
3
H4′), 4.10 (m, 1H, HAla), 3.85 (dq, J(H–H) = 5 Hz–7.5 Hz, 2H, 165.8 (2 × CvO–Bz), 152.7 (C2 or C8), 152.3, 150.2 (2 × CqAd),
H5′), 1.42 (s, 9H, tBuBoc), 1.15 (d, 3J(H–H) = 7.5 Hz, 3H, 134.1 (CqBz), 132.8 (CBz), 128.3, 128.0 (CBz), 124.0 (CqBz), 89.9
CH3Ala), 0.99, 0.92, (2 s, 18H, 2 × tBuTBS), 0.17 (2 s, 6H, (C1′), 87.1 (C4′), 74.1 (C3′), 63.4 (C5′), 59.4 (C2′), 26.3
2 × MeTBS), 0.10 (2 s, 6H, 2 × MeTBS). 13C NMR (63 MHz, (tBuTBS),18.8 (Cq tBuTBS), 18.45 (CH3Ala), −4.17, −4,37
CDCl3) δ = 173.0 (CvO–Bz), 153.0 (C2 or C8), 141.86 (C2 or C8) (2 × MeTBS). HRMS (ESI+): m/z: calcd for C31H45N7O7SiNa:
151.5, 149.9 (2 × Cq-Ad), 134.1 (Cq-Bz), 133.0 (C–Bz), 128.8 678.3042; found 678.3041 [M + Na+].
(C–Bz), 128.2 (C–Bz), 123.2 (CqAd), 87.6 (C4′), 87.0 (C1′),
General procedure for dinucleotides synthesis
73.2 (C3′), 63.5 (C5′), 56.4 (C2′), 28.6 (tBuBoc), 26.3, 26.2
(2 × C-tBuTBS), 18.8, 18.4 (Cq-tBuTBS), 14.26 (C-CH3Ala) −4.1, Coupling reaction was directly carried out in a commercial
−4.2, −5.0, −5.1 (C-MeTBS); HRMS (ESI): m/z: calcd for Ac-dC-CE phosphoramidite vial (250 mg, 324 µmol) equipped
C37H59N7O7Si2Na: 792.3907; found: 792.7258 [M + Na+].
with a flat magnetic stirrer under an argon atmosphere. The
adenosine derivative (130 µmol) was added first followed by
anhydrous CH2Cl2 (350 µL). A 0.45 M solution of tetrazole in
General procedure for selective desilylation
To a stirred solution of 3 (1.0 eq., 0.126 mmol) in THF (2 mL) CH3CN (2.9 mL) was then added slowly to start the reaction.
was added aqueous TFA (2 mL, TFA–H2O: 1/1) at 0 °C. After The mixture was stirred at room temperature for 1 hour (TLC
stirring for 12 hours at 0 °C, the reaction mixture was neutral- monitoring) and a 0.1 M solution of I2 (3.3 mL, H2O/Pyr/THF
ized with saturated aqueous NaHCO3 and diluted with ethyl 2 : 20 : 75) was added. The reaction was stirred at room temp-
acetate (10 mL). After separation, the organic phase was erature for 30 minutes, diluted with EtOAc and washed succes-
washed with H2O (10 mL) and brine (10 mL), dried over anhy- sively with aqueous saturated Na2S2O3 solution and brine. The
drous Na2SO4 and evaporated at reduced pressure. The crude organic layer was dried over anhydrous Na2SO4, concentrated
product was subjected to column chromatography and eluted to dryness. A 0.18 M solution of trichloroacetic acid (7.2 mL)
with EtOAc–cyclohexane (5 : 5).
was finally added to the resulting residue and the mixture was
N6-Benzoyl-2′-O-(tert-butyl-dimethylsilyl)-3′-(N-tert-butyloxy- stirred at room temperature for 30 minutes, diluted with
carbonyl-L-alanine)amido-3′-deoxyadenosine (4a). Compound CH2Cl2 and washed successively with aqueous ice-saturated
3a (0.10 g, 0.129 mmol) was reacted according to the general NaHCO3 solution and brine. The organic layer was dried over
procedure to give compound 4a (54 mg, 64%) as a white foam. anhydrous Na2SO4 and evaporated under vacuum. The crude
Rf = 0.20 (EtOAc); [α]2D5 = −40.2 (c = 1 in CHCl3); 1H NMR product was purified by preparative TLC (CH2Cl2–MeOH 9 : 1)
(400 MHz, CDCl3) δ = 9.31 (bs, 1H, NH-Ad), 8.78 (s, 1H, H2 affording the desired compound as two diastereomers.
3
or H8), 8.38 (s, 1H, H2 or H8), 8.03 (d, J(H–H) = 9.0 Hz, 2H,
tert-Butyl (2R)-1-((2S,3R,4R,5R)-2-((((2R,3S,5R)-5-(4-acetamido-
H–Bz), 7.49–7.52 (m, 3H, H–Bz), 6.80–6.81 (bs, 1H, NH-amide), 2-oxopyrimidin-1(2H)-yl)-2-(hydroxymethyl)tetrahydrofuran-
3
5.95 (d, J(H–H) = 4.0 Hz, 1H, H1′), 4.84–4.87 (m, 3H, NH-Boc, 3-yloxy)(2-cyanoethoxy)phosphoryloxy)methyl)-5-(6-benzamido-
H2′ and OH), 4.50 (m, 1H, H3′), 4.16–4.26 (m, 2H, H4′ 9H-purin-9-yl)-4-(tert-butyldimethylsilyloxy)tetrahydrofuran-
and CHα), 3.85–4.03 (m, 2H, H5′), 1.48 (s, 9H, tBu-Boc), 1.36 3-ylamino)-1-oxopropan-2-ylcarbamate (5a). According to the
(d, 3J(H–H) = 8.0 Hz, 3H, CH3α), 0.88 (m, 9H, H-tBuTBS), general procedure, adenosine derivative 4a (84 mg, 129 µmol)
0.01–0.04 (m, 6H, H-MeTBS); 13C NMR (100 MHz, CDCl3) δ = was coupled to Ac-dC-PCNE-phosphoramide to give dinucleo-
173.9 (CvO–Bz), 164.8 (CvO-amide), 155.4 (Cq-Ad), 152.7 (C2 tide 5a (74 mg, 54%) as two diastereomers. 1H NMR (400 MHz,
or C8), 150.8 (CvO-Boc), 150.1 (Cq-Ad), 142.2 (C2 or C8), 133.7 CDCl3) δ = 9.63 (bs, 1H, NHBz), 8.74 (s, 1H, H2 or H8), 8.46 (s,
(Cq-Bz), 132.9 (C–Bz), 128.9 (C–Bz), 128.0 (C–Bz), 123.9 (Cq), 1H, H2 or H8), 8.26 (m, 1H, H6Cyt), 8.08, 7.46 (m, 5H, H–Bz)
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7.31 (m, 1H, H5Cyt), 6.17 (m, 1H, H1′Cyt), 6.07 (m, 1H, H1′Ad
)
in a 5 M solution of MeNH2 (large excess EtOH/H2O 1 : 1). The
5.20 (m, 1H, NHBoc), 4.83 (m, 2H, H2′Ad/H4′Ad), 4.63 (m, 1H, reaction was stirred for 12 hours at room temperature and
H3′Ad), 4.16–4.37 (m, 6H, H4′Cyt/HαAla/CH2O/H5′Cyt), 3.63–3.83 concentrated under reduced pressure. The residue was purified
(m, 3H, H5′Ad/H3′Cyt), 2.69–2.78 (m, 3H, CH2CN/H2′Cyt), 2.41 by HPLC. After the appropriate fractions were collected and
(m, 1H, H2′Cyt), 2.10 (m, 3H, CH3NHAc), 1.33 (s, 9H, tBuBoc), lyophilized, the phosphorylated product was obtained as a
1.24 (m, 3H, CH3Ala), 0.87 (s, 9H, tBuTBS) 0.06–0.20 (2 s, 6H, NH4+ salt.
2 × MeTBS); 13C NMR (100 MHz, CDCl3) δ = 173.9, 173.7 (CvOBoc),
tert-Butyl (2S)-1-((2S,3R,4R,5R)-2-((((2R,3S,5R)-5-(4-amino-2-
171.3, 171.0 (CvOAc), 162.7 (CvOCyt), 155.7 (CvOBz), 152.8 oxopyrimidin-1(2H)-yl)-2-(phosphonooxymethyl)tetrahydrofuran-
(C2 or C8), 151.3 (Cq), 149.9 (Cq), 145.2 (C2 or C8), 133.1 3-yloxy)(hydroxy)phosphoryloxy)methyl)-5-(6-amino-9H-purin-
(CqBz), 132.9, 129.0, 128.9, 128.4, 128.3 (CHBz), 123.7 (CqAd), 9-yl)-4-(tert-butyldimethylsilyloxy)tetrahydrofuran-3-ylamino)-
116.9 (CN), 96.9 (C5Cyt), 87.7 (C1′Ad), 86.6 (C1′Cyt), 80.6 (C4′Ad), 1-oxopropan-2-ylcarbamate (6a). According to the general pro-
77.4 (C4′Cyt), 75.5 (C3′Ad), 66.3 (C5′Cyt), 62.8 (CH2O), 61.4 cedure, dinucleotide 5a (72 mg, 69.2 µmol) was reacted with
(C5′Ad), 50.8 (C2′Ad), 49.9 (CHα), 40.0 (C2′Cyt), 28.5 (tBuBoc), bis(2-cyanoethyl)diisopropylphosphoramidite (94 mg, 346
25.9 (tBuTBS), 25.0 (CH3Ac), 18.9 (CH2CH2CN), 18.5 (Cq tBuTBS), µmol) in the presence of tetrazole (3.08 mL, 1.38 mmol) and
18.3 (CH3 Ala), −4.5, −4.8 (2 × MeTBS); 31P NMR (162 MHz, then oxidized with I2 (3.46 mL, 346 µmol) and finally a 5 M
CDCl3) −2.9; HRMS (ESI): m/z: calcd for C45H62N11NaO14PSi: solution of MeNH2 (20 mL) to give after lyophilization the
1062.3883, found: 1062.3516 [M + Na+].
NH4 salt phosphorylated product 6a (64 mg) as a yellow
+
tert-Butyl (2R)-1-((2R,3R,4S,5R)-5-((((2R,3S,5R)-5-(4-acetamido- solid. 1H NMR (250 MHz, MeOD) δ = 8.53 (s, 1H, H2Ad or H8Ad),
2-oxopyrimidin-1(2H)-yl)-2-(hydroxymethyl)tetrahydrofuran-3- 8.21 (s, 1H, H2Ad or H8Ad), 7.90 (d, 3J(H–H) = 7.5 Hz, 1H, H6Cyt),
yloxy)(2-cyanoethoxy)phosphoryloxy)methyl)-2-(6-benzamido- 6.22 (m, 1H, H1′Cyt), 6.07 (m, 1H, H1′Ad), 5.90 (d, 3J(H–H) = 7.5 Hz,
9H-purin-9-yl)-4-(tert-butyldimethylsilyloxy)tetrahydrofuran-3- 1H, H5Cyt), 4.81 (m, 1H, H2′Ad), 4.65 (m, 1H, H3′Ad), 4.11–4.26 (m,
ylamino)-1-oxopropan-2-ylcarbamate (5b). According to the 6H, H3′Cyt/H4′Cyt/CHα/H4′Cyt/H5′Ad), 3.72 (m, 2H, H5′Cyt), 2.50 (m,
general procedure, adenosine derivative 4b (84 mg, 129 µmol) 1H, H2′Cyt), 2.16 (m, 1H, H2′Cyt), 1.44 (s, 9H, H-tBuBoc), 1.33 (d,
was coupled to Ac-dC-PCNE-phosphoramide to give dinucleo- 3J(H–H) = 7.5 Hz, CH3), −0.04 (s, 3H, H-MeTBS), −0.03 (s, 3H,
tide 5b (92 mg, 72%) as two diastereomers. 1H NMR (250 MHz, H-MeTBS); HPLC retention time: 7.87 min.
CDCl3) δ = 8.62 (1 s, 1H, H2 or H8), 8.27 (1 s, 1H, H2 or H8),
General procedure for N-Boc and O-TBS deprotection
8.15 (m, 1H, H6Cyt), 7.91, 7.41 (m, 5H, H–Bz) 7.29 (m, 1H,
H5Cyt), 6.09 (m, 1H, H1′Cyt), 6.03 (m, 1H, H1′Ad) 5.86 (bs, 1H, The partially protected dinucleotides 6a–b were treated first
OH), 4.94 (m, NHBoc), 4.83 (m, 1H, H2′Ad), 4.63–4.61 (m, 1H, with a 1 M solution of TBAF in THF (0.45 eq., 0.152 mmol)
H3′Ad), 4.28–4.26 (m, 2H, H4′Ad/HαAla) 4.16, 4.14 (m, 3H, and then with a solution of TFA (50% aq.) at room temperature
CH2O/H3′Cyt), 4.01 (m, 2H, H5′Cyt), 3.63 (m, 1H, H4′Cyt), 3.51, for 15 min. The reaction mixture was then concentrated under
3.22 (m, 2H, H5′Ad), 2.68 (m, 2H, CH2CN), 2.66 (m, 1H, H2′Cyt), vacuum, diluted with water and washed with CH2Cl2. The
2.15 (m, 1H, H2′Cyt), 2.03 (s, 3H, CH3NHAc), 1.26 (s, 9H, tBuBoc), aqueous layer was evaporated under reduced pressure and the
1.04 (d, 3J(H–H) = 7.5 Hz, CH3Ala), 0.84 (s, 9H, tBuTBS) 0.05, residue was purified by HPLC. After the appropriate fractions
0.03, (2 s, 6H, 2 × MeTBS). 13C NMR (63 MHz, CDCl3) δ = 173.7 were collected and lyophilized, the final dinucleotide was
(CvOBoc), 171.2 (CvOAc), 162.5 (CvOCyt), 155.9 (CvOBz)152.2 obtained as a NH4+ salt.
(C2 or C8), 151.4 (Cq), 149.8 (Cq), 144.7 (C2 or C8) 132.7
((2R,3S,5R)-5-(4-Amino-2-oxopyrimidin-1(2H)-yl)-3-((((2S,3S,4R,5R)-
(CqBz), 132.6 (CBz), 128.5 (CBz), 128.0 (CBz), 122.8 (CqAd), 116.3 5-(6-amino-9H-purin-9-yl)-3-((R)-2-aminopropanamido)-4-hydroxy-
(CN), 97.0 (C5Cyt), 87.1 (C1′Ad), 86.8 (C1′Cyt), 80.0 (C4′Ad), 79.0 tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryloxy)tetra-
(C4′Cyt), 71.2 (C3′Ad), 67.3 (C5′Cyt), 62.7 (CH2O), 60.8 (C5′Ad), hydrofuran-2-yl)methyl dihydrogen phosphate (7a). According
49.7 (C2′Ad), 39.8 (C2′Cyt), 27.9 (tBuBoc), 24.2 (tBuTBS), 24.0 to the general procedure, dinucleotide 6a (64 mg, 9 µmol) was
(CH3Ac), 24.2 (CHα), 19.2 (CH2CH2CN), 17.7 (CH3Ala), −5.0 first reacted with a 1 M solution of TBAF in THF (152 μL) and
(2 × MeTBS). HRMS (ESI): m/z: calcd for C45H62N11NaO14PSi: then with a solution of TFA (50% aq., 3 mL) at room tempera-
1062.3883, found: 1062.3646 [M + Na+].
ture for 15 minutes. After purification by HPLC, the final di-
+
nucleotide 7a was obtained as a NH4 salt (20 mg, 40% yield)
General procedure for dinucleotides phosphorylation
as a white solid. 1H NMR (600 MHz, D2O) δ = 8.52 (s, 1H, H2Ad
Bis(2-cyanoethyl)diisopropylphosphoramidite (5.0 eq.) was or H8Ad), 8.33 (s, 1H, H2Ad or H8Ad), 8.00 (d, J(H–H) = 7.8 Hz,
3
added neat into the flask containing the dinucleotide (1.0 eq.). H6Cyt), 6.13 (dd, J(H1′–H2′) = 2.4 Hz, J(H5–H6) = 7.8 Hz, 2H,
3
3
Anhydrous CH2Cl2 (3.5 µL µmol−1) was then added, followed H1′Ad/H5Cyt), 6.08 (m, 1H, H1′Cyt), 4.70–4.80 (m, 3H, H2′Ad
/
by the 0.45 M solution of tetrazole in CH3CN (20 eq.). The H3′Ad/H3′Cyt), 4.48 (m, 1H, H4′Ad), 4.30 (m, 1H, H4′Cyt), 4.22
mixture was stirred at room temperature for 1 hour and a 0.1 M (m, 2H, H5′Ad/Hα), 3.98 (m, 3H, H5′Cyt/H5′Ad), 2.45 (m, 1H,
solution of I2 (5.0 eq.) was added. After being stirred at H2′Cyt), 2.06 (m, 1H, H2′Cyt), 1.55 (d, 3J(H–H) = 7.2 Hz, 3H,
room temperature for 30 minutes, the mixture was diluted CH3Ala); 13C NMR (151 MHz, CDCl3) δ = 171.2 (CvO), 163.3
with EtOAc and washed successively with aqueous saturated (CvOCyt), 160.1, 152.9, 152.7, 149.4, 149.1, (Cq), 148.4 (C2 or
Na2S2O3 solution and brine. The organic layer was dried over C8), 143.6 (C6Cyt), 141.1 (C2 or C8), 95.3 (C5Cyt), 89.4 (C1′Ad),
anhydrous Na2SO4, concentrated to dryness and then dissolved 85.2 (C1′Cyt), 85.1 (C4′Cyt), 80.1 (C4′Ad), 76.1 (C3′Cyt), 73.3
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(C3′Ad), 64.6 (C5′Ad), 64.1 (C5′Cyt), 50.8 (C2′Ad), 49.1 (CHα), 38.6 (Whatman 4MM, Elancourt) with isobutyric acid–ammonia,
(C2′Cyt), 16.7 (CH3Ala); 31P NMR (1H decoupled, 242 MHz, D2O) 1 M (5 : 3 per vol). Radioactive spots were identified by auto-
−
−0.57, −0.51; HRMS (ESI): m/z: calcd for C22H31N10O13P2
:
radiography, cut out, and counted by a liquid scintillation.
705.1553, found: 705.1111 [M − H+]; HPLC retention time:
9.26 min.
((2R,3S,5R)-5-(4-Amino-2-oxopyrimidin-1(2H)-yl)-3-((((2R,3S,4R,5R)-
5-(6-amino-9H-purin-9-yl)-4-((R)-2-aminopropanamido)-3-hydroxy-
Acknowledgements
tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryloxy)tetra- D. Mellal was supported by a research fellowship from the
hydrofuran-2-yl)methyldihydrogen phosphate (7b). According Région Ile-de-France.
to the general procedure, dinucleotide 6b (82 mg, 108.1 µmol)
was first reacted with a 1 M solution of TBAF in THF (152 μL,
0.152 mmol) and then with a solution of TFA–H2O (50% aq.,
3 mL) at room temperature for 15 minutes. After purification
Notes and references
by HPLC, the final dinucleotide 7b was obtained as a NH4+ salt
(34.4 mg, 42% yield) as a white solid. HPLC retention time:
15 min. 1H NMR (600 MHz, D2O) δ = 8.51 (s, 1H, H2Ad or
H8Ad), 8.20 (s, 1H, H2Ad or H8Ad), 8.00 (s, 1H, NH), 7.74 (d,
3J(H–H) = 3.3 Hz, 1H, H6Cyt), 6.15 (dd, 3J(H1′cyt–H2′cyt a) =
1 (a) C. S. Francklyn and A. Minajigi, FEBS Lett., 2010, 584,
366–375; (b) A. Schon, G. Krupp, S. Gough, S. Berry-Lowe,
C. G. Kannangara and D. Söll, Nature, 1986, 322, 281–
284.
2 (a) R. P. Garg, X. L. Qian, L. B. Alemany, S. Moran and
R. J. Parry, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 6543–
6547; (b) W. Zhang, I. Ntai, N. L. Kelleherc and C. T. Walsh,
Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 12249–12253.
3 (a) M. W. Vetting, S. S. Hegde and J. S. Blanchard, Nat.
Chem. Biol., 2010, 6, 797–799; (b) L. Bonnefond, T. Araib,
Y. Sakaguchib, T. Suzukib, R. Ishitania and O. Nureki,
Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 3912–3917;
(c) M. Gondry, L. Sauguet, P. Belin, R. Thai, R. Amouroux,
C. Tellier, K. Tuphile, M. Jacquet, S. Braud and
M. Courçon, Nat. Chem. Biol., 2009, 5, 414–420.
4 K. Lang, M. Erlacher, D. N. Wilson, R. Micura and
N. Polacek, Chem. Biol., 2008, 15, 485–492.
5 M. Taiji, S. Yokoyama and T. Miyazawa, Biochemistry, 1983,
22, 3220–3225.
6 J. Ling, N. Reynolds and M. Ibba, Annu. Rev. Microbiol.,
2009, 63, 61–78.
7 K. S. Huang, J. S. Weinger, E. B. Butler and S. A. Strobel,
J. Am. Chem. Soc., 2006, 128, 3108–3109.
3
3
8.8 Hz, J(H1′cyt–H2′cyt b) = 5.7 Hz, 1H, H1′Cyt), 6.13 (d, J(H1′Ad
–
H2′Ad) = 7,8 Hz, 1H, H1′Ad), 6.02 (d, 3J(H–H) = 7.2 Hz, 1H,
H5Cyt), 5.11 (m, 1H, H2′Ad), 4.77–4.75 (m, 1H, H3′Cyt), 4.65 (m,
1H, H3′Ad), 4.41–4.38 (s, 1H, H4′Ad), 4.28 (s, 1H, H4′Cyt),
4.15–4.11 (m, 2H, H5′Ad), 4.07–3.98 (m, 2H, Hα/H5′Cyt), 2.36
(m, 1H, H2′Cyt), 1.82 (m, 1H, H2′Cyt), 1.53 (d, J(H–H) = 6.6 Hz,
3
3H, CH3Ala). 13C NMR (600 MHz, CDCl3) δ = 180.4 (CvO), δ =
171 (CvO), 164.8 (CvOcyt), 155.3 (C2 or C8), 152.8 (C2 or C8),
141.3 (C6Cyt), 96.4 (C5Cyt), 85.6 (C1′Cyt), 85.1 (C1′Ad), 84.8
(C4′Ad), 84.7 (C4′Cyt), 76.4 (C3′cyt), 69.7 (C3′Ad), 64.9 (C5′Ad), 64.6
(C5′Cyt), 55.5 (C2′Ad), 48.9 (Cα), 38.0 (C2′Cyt), 16.5 (CH3Ala). 31P
NMR (1H decoupled, 162 MHz, CDCl3): 1.26, 0.2 (2 s). 31P
NMR (1H decoupled, 162 MHz, D2O) 1.24, 0.17 (2 s). HRMS
(ESI): m/z: calcd for C22H33N10O13P2: 706.1626, found:
705.1510 [M − H+].
Ligation of modified dinucleotides to RNA helices
Modified dinucleotides 7a and 7b were ligated to RNA helices
with purified T4 RNA ligase.10a Compounds 8a and 8b were
purified by anion exchange chromatography (DEAE column,
DNAPac-100, Dionex) with a linear gradient of ammonium
acetate pH 8.0 (25–2500 mM) containing 0.5% acetonitrile.
Fractions containing the ligation product were identified by
denaturing polyacrylamide gel electrophoresis,10a lyophilized,
resuspended in RNAse free water (Sigma), and stored at
−20 °C. The concentration of the inhibitors was determined
spectrophotometrically (ε = 2.26 × 105 M−1 cm−1 at 260 nm).
8 M. Fonvielle, M. Chemama, M. Lecerf, R. Villet, P. Busca,
A. Bouhss, M. Ethève-Quelquejeu and M. Arthur, Angew.
Chem., Int. Ed., 2010, 49, 5115–5119.
9 (a) J. S. Weinger, K. M. Parnell, S. Dorner, R. Green and
S. A. Strobel, Nat. Struct. Mol. Biol., 2004, 11, 1101–1106;
(b) J. S. Weinger, D. Kitchen, S. A. Scaringe, S. A. Strobel
and G. W. Muth, Nucleic Acids Res., 2004, 32, 1502–1511;
(c) T. M. Schmeing, K. S. Huang, D. E. Kitchen, S. A. Strobel
and T. A. Steitz, Mol. Cell, 2005, 20, 437–448; (d) M. Koch,
Y. Huang and M. Sprinzl, Angew. Chem., Int. Ed., 2008, 47,
7242–7245.
Inhibition of FemXWv by 8a and 8b
The assay contained Tris–HCl (50 mM, pH 7.5), alanyl-tRNA 10 (a) M. Chemama, M. Fonvielle, R. Villet, M. Arthur,
synthetase of E. faecalis (800 nM), ATP (7.5 mM), MgCl2
(12.5 mM), L-[14C]Ala (50 µM, 3700 Bq nmol−1; ICN, Orsay,
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