Synthesis of 3′-fluoro-trna analogues for exploring nonribosomal peptide synthesis in bacteria

Article abstract of DOI:10.1002/cbic.201402523

Aminoacyl-tRNAs (aa-tRNAs) participate in a vast repertoire of metabolic pathways, including the synthesis of the peptidoglycan network in the cell walls of bacterial pathogens. Synthesis of aminoacyl-tRNA analogues is critical for further understanding the mechanisms of these reactions. Here we report the semi-synthesis of 3′-fluoro analogues of Ala-tRNA^Ala. The presence of fluorine in the 3′-position blocks Ala at the 2′-position by preventing spontaneous migration of the residue between positions 2′ and 3′. NMR analyses showed that substitution of the 3′-hydroxy group by fluorine in the ribo configuration favours the S-type conformation of the furanose ring of terminal adenosine A76. In contrast, the N-type conformation is favoured by the presence of fluorine in the xylo configuration. Thus, introduction of fluorine in the ribo and xylo configurations affects the conformation of the furanose ring in reciprocal ways. These compounds should provide insight into substrate recognition by Fem transferases and the Ala-tRNA synthetases.

Full text of DOI:10.1002/cbic.201402523

DOI: 10.1002/cbic.201402523
Full Papers
Synthesis of 3’-Fluoro-tRNA Analogues for Exploring
Non-ribosomal Peptide Synthesis in Bacteria
[a]
[a]
[b, c]
[a]
Laura Iannazzo, Guillaume Laisnꢀ, Matthieu Fonvielle,
Emmanuelle Braud, Jean-
[a]
[b, c]
[a]
Philippe Herbeuval, Michel Arthur,
and Mꢀlanie Etheve-Quelquejeu*
Aminoacyl-tRNAs (aa-tRNAs) participate in a vast repertoire of
metabolic pathways, including the synthesis of the peptidogly-
can network in the cell walls of bacterial pathogens. Synthesis
of aminoacyl-tRNA analogues is critical for further understand-
ing the mechanisms of these reactions. Here we report the
the 3’-hydroxy group by fluorine in the ribo configuration fa-
vours the S-type conformation of the furanose ring of terminal
adenosine A76. In contrast, the N-type conformation is fav-
oured by the presence of fluorine in the xylo configuration.
Thus, introduction of fluorine in the ribo and xylo configura-
tions affects the conformation of the furanose ring in recipro-
cal ways. These compounds should provide insight into sub-
strate recognition by Fem transferases and the Ala-tRNA syn-
thetases.
Ala
semi-synthesis of 3’-fluoro analogues of Ala-tRNA . The pres-
ence of fluorine in the 3’-position blocks Ala at the 2’-position
by preventing spontaneous migration of the residue between
positions 2’ and 3’. NMR analyses showed that substitution of
Introduction
Aminoacyl-tRNAs (aa-tRNAs) are a source of ester-activated
amino acids. In addition to their role in protein synthesis by
Among the tRNA-dependent aminoacyl transferases, Fem
transferases participate in peptidoglycan synthesis in Gram-
positive bacteria (Scheme 1A). Because these enzymes are
essential for resistance to b-lactam antibiotics in major patho-
gens, including Staphylococcus aureus, Streptococcus pneumo-
niae and Enterococcus faecalis, they are considered attractive
targets for the development of new antibiotics active against
[1]
the ribosome, they participate in various metabolic pathways.
The vicinal hydroxy groups in positions 2’ and 3’ of the termi-
nal nucleotide in a tRNA (A76) have pivotal roles. The tRNAs
[
2]
are esterified by aminoacyl-tRNA synthetases, which catalyse
the transfer of a specific aminoacyl residue from an adenylate
to the 2’- or the 3’-hydroxy group of A76 (Scheme 1A). Isomer-
isation of the ester between positions 2’ and 3’ occurs in the
absence of any enzyme with a rate and a thermodynamic equi-
[6]
multi-resistant bacteria. FemXWv of Weissella viridescens, the
model enzyme of the Fem family, transfers l-Ala from Ala-tRNA
to the peptidoglycan precursor (Scheme 1A). We have previ-
ꢀ
1
[3]
librium of the order of 5 s and 1, respectively. The A site of
ously determined the regiospecificity of FemX and shown
Wv
[7]
the ribosome is specific for the 3’-O-aminoacyl isomer, and the
that transfer of Ala occurs from the 2’-position. We have also
shown that the 3’-OH group is not essential for catalysis, al-
though a 240-fold reduction in the turnover number was ob-
served for the 3’-deoxy analogue. We proposed that the 3’-OH
group participates in proton shuttling in the substrate-assisted
3
’-linkage is conserved in the product of the peptidyl transfer
[
4]
reaction (Scheme 1A). The 2’-hydroxy group of peptidyl-tRNA
at the P site of the ribosome was reported to be essential, with
6
7
a 10 –10 -fold reduction in the rate of peptide bond formation
[
5]
[7,8]
being reported for a 2’-deoxy analogue. It was proposed that
the critical role of the 2’-OH group reflected substrate-assisted
catalytic mechanism (Scheme 1B).
Chemical modification of the terminal nucleotide of tRNAs
(A76) is critical for better understanding of the pivotal roles of
the vicinal 2’- or 3’-hydroxy groups in protein and peptidogly-
can synthesis. Two analogues—the deoxy or the fluoro ana-
logues, in which one of the hydroxy groups has been replaced
[5a]
catalysis in the peptidyltransferase centre of the ribosome.
[a] Dr. L. Iannazzo, G. Laisnꢀ, Dr. E. Braud, Dr. J.-P. Herbeuval,
Dr. M. Etheve-Quelquejeu
Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques
Team CBNIT, Universitꢀ Paris Descartes, CNRS UMR 8601
[5a,9]
[5a,10]
by hydrogen
or fluorine,
respectively—are mainly used
in this context. Both substitutions freeze the amino acid resi-
due at the 2’- or 3’-position. The deoxy analogues lack both
the hydrogen bond donor and acceptor characters of the hy-
droxy group. The fluoro analogues have distinct characteristics
because fluorine mimics the polarity of the hydroxy group and
7
5006 Paris (France)
E-mail: melanie.etheve-quelquejeu@parisdescartes.fr
[b] Dr. M. Fonvielle, Dr. M. Arthur
Centre de Recherche des Cordeliers, LRMA, Equipe 12
Universitꢀ Pierre et Marie Curie
Paris 6, UMR S 1138, 75006 Paris (France)
[11]
is a weak hydrogen bond acceptor. In addition, the electron-
[c] Dr. M. Fonvielle, Dr. M. Arthur
withdrawing nature of fluorine strongly affects sugar pucker-
Universitꢀ Paris Descartes, INSERM, UMR S 1138
Sorbonne Paris City, 75006 Paris (France.
[12]
ing.
Previous studies have reported on the introduction of 2’-
deoxy-2’-fluoroadenosine at the extremities of peptidyl-tRNAs
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201402523.
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Ala
Scheme 1. A) Schematic representation of Ala-tRNA regioisomers in protein and peptidoglycan synthesis. B) FemX transfers an alanyl residue from Ala-
Ala
tRNA to the side chain of l-Lys in the peptidoglycan precursor UDP-N-acetyl-muramyl-pentapeptide (UM5K). The 3’-OH group may be involved in proton
shuttling during catalysis.
[5a,10a]
and aminoacyl-tRNAs by a two-step enzymatic process.
Firstly, the terminal CA nucleotides were removed from the 3’-
Lys
end of natural tRNA by two rounds of periodate/aniline/poly-
[
13]
nucleotide kinase reactions.
Secondly, the CCA-adding
enzyme was used to restore the full tRNA sequence with cyto-
sine triphosphate (for position 75) and 2’-deoxy-2’-fluoroade-
[5a]
nosine triphosphate (for position 76) as the substrates. We
report here an alternative to this enzymatic route. Our strategy
relies on chemical synthesis of a fluorinated dinucleotide
(
pdCpA-F), followed by ligation to incomplete tRNAs lacking
[
14]
the terminal pCpA dinucleotide. The ligation reaction, cata-
lysed by T4 RNA ligase, restores the complete tRNA sequence.
We report the application of this strategy to the synthesis of
Ala
3
’-fluoro-tRNA analogues with the fluorine in the xylo and
ribo configurations and the acylated derivative of the former
analogue (Scheme 2). In addition, we have explored the con-
formational preference of the corresponding dinucleotide by
NMR spectroscopy.
Ala
Results and Discussion
Scheme 2. A) and B) Fluoro analogues of tRNA in the xylo and ribo config-
Ala
urations, respectively. C) Fluoro analogue of Ala-tRNA in the xylo configura-
Synthesis
tion.
Syntheses of 2’-deoxy-2’-fluoroadenosine have been widely de-
[
15]
scribed, whereas those of 3’-deoxy-3’-fluoroadenosine have
For compatibility with the dinucleotide synthesis, protecting
groups were changed in two steps (Scheme 4). Firstly, 4a and
4b were fully deprotected (TFA), to afford 5a and 5b in 94
and 76% yields, respectively. Secondly, silyl ether derivatives of
5a and 5b were obtained by treatment with TBDMSCl to give
6a and 6b. These were treated with benzoyl chloride to afford
7a and 7b in 90 and 100% yields, respectively. The 5’-silyl
[
16]
been less studied. Fluorinated nucleosides can be synthes-
ised either by fluorination of preformed nucleosides or by con-
densation of fluorine-substituted furanose moieties with suita-
ble heterocyclic bases. In this study we followed the first strat-
[16a,17]
egy, starting from the known tritylated compound 1
Scheme 3).
The 3’-xylo-Fluoro analogue 4a was directly obtained by
(
ether groups were removed with the aid of a 1:1 TFA/H O mix-
2
using diethylaminosulfur trifluoride (DAST) as the fluorinating
ture to provide 8a and 8b in 90 and 81% yields, respectively.
agent (50% yield). For the synthesis of 3’-ribo-fluoro analogue
Compounds were purified by column chromatography and
1
13
19
4
b (Scheme 3), compound 1 was sequentially oxidised to
characterised by H, C and F NMR spectroscopy and mass
spectrometry analysis.
ketone 2b with Dess–Martin periodinane and reduced to 3b
in an overall yield of 47%. Fluorination of 3b was achieved
with DAST (67%).
Dinucleotides 11 a and 11 b were obtained by the phosphor-
amidite approach (Scheme 5). Phosphodiesters 9a and 9b
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Scheme 3. a) Dess–Martin periodinane, CH
EtOH/H O, 08C then RT, 2 h, 3b (50%); c) DAST, pyridine, CHCl
50%), 4b (67%).
2
Cl
2
, RT, 4 h, 2b (95%); b) NaBH
4
,
2
3
, RT, 24 h, 4a
(
Scheme 4. a) TFA, CH
2
Cl
2
, RT, 1 h, 5a (94%), 5b (76%); b) TBDMSCl, DMAP,
pyridine, RT, overnight, 6a (36%), 6b (22%), c) BzCl, pyridine, RT, overnight,
a (90%), 7b (quant.); d) TFA/H O 1:1, THF, RT, 1 h, 8a (90%), 8b (81%).
were prepared from tetrazole-activated deoxycytidine phos-
7
2
phoramidite Ac-dC-PCNE (Scheme 5) and nucleosides 8a and
8
b, respectively. After oxidation of the phosphite intermediate
and removal of the dimethoxytrityl group by treatment with
trichloroacetic acid, 9a and 9b were obtained in 72 and 70%
yields, respectively. Phosphorylation of 9a and 9b with bis-(2-
cyanoethyl)diisopropylphosphoramidite gave 10a and 10b in
to favour the N-type conformer. For the 3’-ribo configuration
(6B), the gauche effect prevails over the anomeric effect, and
[18,19]
the sugar preferentially adopts the S-type conformation
(Scheme 6).
8
0 and 87% yields, respectively. The final removal of acetyl,
Several equations have been proposed in efforts to deduce
the relative abundances of S- and N-type conformations from
benzoyl (Bz) and cyanoethyl groups was performed with meth-
ylamine, to afford 11 a and 11 b in 34 and 46% yields, respec-
tively. Dinucleotides 11 a and 11 b were purified by RP-HPLC
and fully characterised by H, C, F and P NMR spectrosco-
py and mass spectrometry analysis.
1
[12c]
H NMR spectra.
Here we have used the “10-Hertz rule-of-
thumb” (% S-type=10 J1’,2’) applied to the J1’,2’ constant in
1
13
19
31
1
H NMR spectra collected in CD OD (Table 1). The NMR spectra
3
show, as expected, that 3’-deoxy-3’-fluoroadenosine with fluo-
rine in the xylo configuration (compound 5a) has a preference
for the N type conformation, whereas its counterpart with fluo-
rine in the ribo configuration (compound 5b, Table 1) prefers
the S type. More precisely, we observed 20 and 65% of S-type
conformer for nucleosides 5a and 5b, respectively, and 22 and
Conformation study
Nucleosides and their analogues are known to exist in equilib-
rium between S- and N-type conformations, based on furanose
ring puckering. Factors that influence the preferred conforma-
tions of furanose rings in nucleosides and nucleotides are the
anomeric effect (AE, O4’-C1-N1/9 stereoelectronic effect), the
gauche effect (GE) and the steric effects of the bases. In 3’-
substituted 3’-deoxyadenosine systems, it has been shown
that the proportions of S- or N-type conformers are mostly de-
termined by the gauche effect of (X3’-C3’-C4’-04’), provided
that X3’ is an electron-withdrawing group, as is the case for
our fluorinated compounds (Scheme 6). In the 3’-xylo configu-
ration (6A), the gauche and anomeric effects act in cooperation
Table 1. J1’,2’ coupling constants and S values (in CD
fluoroadenosine derivatives.
3
OD) of 3’-deoxy-3’-
S [%]
[18]
Compounds
J1’,2’ [Hz]
xylo-F-adenosine 5a
xylo-F-pdCpA 11 a
xylo-F-Ala-pdCpA 12a
ribo-F-adenosine 5b
ribo-F-pdCpA 11 b
2.0
2.2
2.4
6.5
8.0
20
22
24
65
80
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activities, we have also evaluated
the influence of the solvent on
the conformation equilibrium.
1
H NMR spectra of dinucleo-
tide 11 a were acquired in three
solvents (Figure 1): J1’,2’ values of
2.7, 2.5 and 2.2 Hz were found in
water, DMSO and methanol, re-
spectively. Thus, a decrease in
the polarity of the solvent result-
ed in a marginal decrease in the
J1’,2’ coupling constant. This
result confirms that the gauche
effect is the main factor that
drives the equilibrium, because
this effect is not influenced by
the polarity of the solvent,
unlike the anomeric effect,
which involves dipole–dipole in-
teraction in addition to orbital
interaction. This result also sug-
gests that hydrogen bonding is
not involved in the stabilisation
[18]
of the N-type conformer.
Aminoacylation
Scheme 5. a) i: Tetrazole, CH
2
Cl
2
, RT, 1 h; ii: I
2
, H
2
O/Pyr/THF, RT, 30 min; iii: TCA, CH
Cl , RT, 1 h; ii: I
3 2 2
0a (80%), 10b (87%); c) CH NH , EtOH/H O, RT, 24 h, 11 a (34%), 11 b (46%).
2
Cl
2
, RT, 30 min, 9a (72%), 9b
(70%); b) i: bis(2-cyanoethyl)diisopropylphosphoramidite, tetrazole, CH
2
2
2
, H O/Pyr/THF, RT, 30 min,
2
Chemical aminoacylation of the
fluorinated dinucleotide 11 a
with l-Ala was performed as pre-
1
[14]
viously described.
Before the
coupling reaction, the ammonium salt 11 a was converted into
the tetrabutylammonium salt to enhance its solubility. The tet-
rabutylammonium salt of 11 a, dissolved in DMF, reacted with
the activated pentenoyl-cyanomethyl ester of l-alanine to give
12a in 10% yield (Scheme 7). The pentenoyl group is easily
cleavable under conditions that did not affect the tRNA
Scheme 6. Conformation equilibrium of A) xylo-, and B) ribo-3’-fluoroadeno-
sine. The arrows between C3’ and C4’ (A and B) show the gauche effect (GE),
which involves interaction between the best donor (sC3’–H3’) and the best
acceptor (s*C4’–O4’) orbitals. The arrow between O and C1’ (A) indicates the
interaction between the s*C1’–N9 orbital and the lone electron pair of O4’,
which contributes to the anomeric effect (AE). The N-type conformer is sta-
bilised by the GE and by the AE in (A). The S-type conformer is stabilised by
the GE in (B).
80% of S-type for the corresponding dinucleotides 11 a and
1
1 b, respectively. These values are similar to those reported in
[12c,19a,b]
the literature for 3’-fluoro analogues of adenosine.
These results show that the proportions of the conformers
were not affected by conversion of nucleosides into the corre-
sponding dinucleotides.
The first set of NMR spectra was acquired in CD OD
1
3
Figure 1. Selected areas of the H NMR spectra of compound 11 a in D O,
2
(Table 1). Because aqueous solutions are relevant to biological
3
DMSO and CD OD.
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+
4
Scheme 7. a) DOWEX NBu ; b) DMF, RT, 24 h, 12a (10% over two steps).
moiety. The fluoro-Ala-dinucleotide analogue was characterised Conclusion
1
19
31
by H, F and P NMR spectroscopy and by mass spectrome-
1
try. The coupling constant J1’,2’ determined by H NMR was
We report synthetic routes to 3’-fluorinated dinucleotides with
the xylo and ribo configurations, as well as to the acylated
form of the xylo isomer. These dinucleotides were efficiently
ligated to synthetic RNA molecules, thus providing access to
2
.4 Hz (Table 1), thus indicating that acylation at 2’-OH hardly
affected the ribose conformation.
Ala
Ala
the corresponding fluoro analogues of tRNA and Ala-tRNA .
These molecules are non-isomerisable analogues of the sub-
strates of alanyl-tRNA synthetases and FemX transferases, re-
spectively. We also analysed the conformations of the 3’-fluori-
nated sugar components and showed that the xylo and ribo
stereoisomers preferentially adopt the N and S conformations,
respectively. This was observed for the nucleosides, the dinu-
cleotides and the acylated xylo-configured dinucleotide. The
conformations were not affected by the polarity of the solvent.
Our results indicate that the populations of the N and S con-
Enzymatic ligation
Ala
The fluoro-tRNA analogues were obtained by enzymatic liga-
tion of dinucleotides 11 a, 11 b and 12a with a 22-nt RNA mol-
ecule that mimics the acceptor arm of the tRNA (Figure 2).
Ala
The ligation was performed in HEPES buffer containing the 22-
nt RNA, compound 11 a, 11 b or 12a, T4 RNA ligase, ATP and
Ala
MgCl . After ligation, the Ala-tRNA analogues were purified
2
Ala
by anion-exchange chromatography and analysed by denatur-
ing PAGE (Figure 2). The ligation reaction was efficiently cata-
lysed by T4 RNA ligase, thus indicating that this enzyme toler-
ates fluorination of the terminal adenosine moiety.
formations are likely to be similar for 3’-fluorinated tRNA and
Ala
Ala-tRNA analogues. These compounds should provide new
insight into substrate recognition by Fem transferases and Ala-
tRNA synthetases.
Figure 2. A) Ligation of 11 a, 11 b and 12a to the 22-nt RNA by T4RNA ligase. B) PAGE analysis.
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chromatography with CH Cl /MeOH (9:1) to afford 5a as a white
Experimental Section
2
2
1
foam (165 mg, 94%). H NMR (500 MHz, CD OD): d=3.90–3.99 (m,
3
General reagents and materials: Solvents were dried by standard
methods and distilled before use. Unless otherwise specified, mate-
rials were purchased from commercial suppliers and used without
further purification. TLC: precoated silica gel thin-layer sheets
2H; H5’), 4.47 (m, 1H; H4’), 4.71 (dt, J=14.0, 3.5 Hz, 1H; H2’), 5.10
(ddd, J=52.0, 3.6, 3.0 Hz, 1H; H3’), 6.09 (d, J=2.0 Hz, 1H; H1’),
8.15 (s, 1H; H2 or H8), 8.22 ppm (s, 1H; H2 or H8); C NMR
1
3
(125 MHz, CD OD): d=60.0 (d, J=10.7 Hz; C5’), 79.7 (d, J=27.1 Hz;
3
(
(
1
60 F254, Merck) and detection by charring with H SO in ethanol
10%) followed by heating. Flash chromatography: silica gel 60 ꢂ,
80–240 mesh from Merck. Spectra were recording with Bruker
C2’), 83.9 (d, J=19.8 Hz; C4’), 91.4 (C1’), 96.7 (d, J=183.2 Hz; C3’),
2
4
Ad
Ad
140.2 (C2 or C8), 140.3 (C ), 150.2 (C ), 153.8 (C2 or C8),
Ad
157.3 ppm (C ); HRMS: m/z calcd for C H FN O : 270.1002
10
13
5
3
+
spectrometers (AM 250 or Advance II 500). Chemical shifts (d) are
expressed in ppm relative to residual CHCl3 (d=7.26 ppm) or
CD OD (d=3.31 ppm) for H or relative to CDCl (d=77.16 ppm) or
[M+H] ; found: 270.0992.
1
Compound 6a: DMAP (40 mg, 0.33 mmol) and TBDMSCl (273 mg,
1.8 mmol) were successively added at 08C to a solution of 5a
(444 mg, 1.65 mmol) in pyridine (10 mL). The reaction mixture was
stirred at room temperature overnight and coevaporated with tolu-
ene in vacuo. EtOAc was added, and the aqueous layer was ex-
tracted. The organic extract was washed with brine, dried over
Na SO and concentrated in vacuo. The crude residue was purified
3
3
13
CD OD (d=049.00 ppm) for C as internal references. Signals were
3
13
assigned by COSY and HSQC ( C). HRMS was carried out with a
Bruker micrOTOF spectrometer. HPLC was performed with a HPLC
system including a reversed-phase C-18 column (250ꢃ21.2 mm)
and use of a solvent system consisting of 50 mm aqueous CH CN/
NH OAc (linear gradient from 0:100 to 63:37 in 40 min) at a flow
3
2
4
4
ꢀ1
by chromatography with CH Cl /MeOH (96:4) as the eluent, yield-
2 2
rate of 15 mLmin and UV detection at 254 nm.
1
ing 6a as a white foam (228 mg, 36%). H NMR (250 MHz, CD OD):
3
TBS
TBS
Compound 1: A mixture of adenosine (2.67 g, 10.0 mmol), DMAP
(
was heated at 808C for three days. The reaction mixture was
quenched with EtOH, concentrated in vacuo and coevaporated
with toluene. CH Cl was added to the resulting orange foam, the
mixture was washed with H O, and the combined aqueous layers
were then re-extracted with CH Cl . The combined organic layers
were dried over Na SO and concentrated in vacuo, and the crude
d=0.10 (s, 6H; Me ), 0.90 (s, 9H; tBu ), 4.00–4.36 (m, 2H; H5’),
4.45 (m, 1H; H4’), 4.66 (d, J=13.5 Hz, 1H; H2’), 5.05 (dq, J=51.2,
1.2 Hz, 1H; H3’), 6.11 (d, J=1.2 Hz, 1H; H1’), 8.11 (s, 1H; H2 or H8),
1.00 g, 8.0 mmol) and TrCl (13.9 g, 50.0 mmol) in pyridine (125 mL)
13
8.21 ppm (s, 1H; H2 or H8); C NMR (63 MHz, CD OD): d=ꢀ5.4
3
TBS
TBS
TBS
TBS
2
2
(Me ), ꢀ5.2 (Me ), 19.1 (Cq ), 26.3 (tBu ), 61.1 (d, J=10.1 Hz;
C5’), 79.8 (d, J=27.0 Hz; C2’), 84.0 (d, J=18.7 Hz; C4’), 91.5 (C1’),
2
Ad
Ad
2
2
96.5 (d, J=184.2 Hz; C3’), 120.0 (C ), 140.2 (C2 or C8), 150.2 (C ),
154.0 (C2 or C8), 157.2 ppm (C ); F NMR (471 MHz, CD OD): d=
Ad
19
2
4
3
residue was purified by chromatography with cyclohexane/EtOAc
9:1 then 8:2) as the eluent to yield 1 as a white foam (4.63 g,
ꢀ203.3 ppm (s, 1F; F3’); HRMS: m/z calcd for C H FN O Si:
16
27
5
3
+
(
384.1867 [M+H] ; found: 384.1852.
1
4
3
6%). H NMR (250 MHz, CDCl ): d=2.86 (d, J=4.3 Hz, 1H; H3’),
.00 (dd, J=10.5, 3.2 Hz, 1H; H5’a), 3.26 (dd, J=10.5, 3.5 Hz, 1H;
3
Compound 7a: BzCl (392 mL, 3.3 mmol) was added at 08C to a solu-
tion of 6a (185 mg, 0.48 mmol) in pyridine (5 mL), and the reaction
mixture was stirred at room temperature overnight. The resulting
solution was coevaporated with toluene in vacuo. The residue was
suspended in EtOAc, washed with brine and dried over Na SO .
H5’b), 4.03 (brs, 1H; H4’), 5.16 (dd, J=7.5, 4.7 Hz, 1H; H2’), 6.32 (d,
Trt
J=7.5 Hz, 1H; H1’), 7.00–7.42 (m, 45H; H ), 7.86 (s, 1H; H2 or H8),
1
3
7
7
(
1
.89 ppm (s, 1H; H2 or H8); C NMR (63 MHz, CDCl ): d=63.9 (C5’),
3
Trt Trt
0.7 (C ), 71.4 (C3’), 76.9 (C2’), 84.3 (C4’), 86.5 (C ), 87.1 (C1’), 87.8
2
4
Trt
Ad
Trt
Trt
Trt
Trt
The solvent was removed under reduced pressure, and the prod-
uct was purified by chromatography with cyclohexane/EtOAc (8:2)
C ), 121.3 (C ), 126.9 (C ), 127.1 (C ), 127.6 (C ), 127.8 (C ),
Trt
Trt
Trt
Trt
Trt
27.9 (C ), 128.1 (C ), 128.3 (C ), 128.7 (C ), 129.0 (C ), 139.5 (C2
1
Trt
Trt
Trt
Ad
Ad
as the eluent to give 7a as a white foam (300 mg, 90%). H NMR
or C8), 143.2 (C ), 143.5 (C ), 145.1 (C ), 149.3 (C ), 152.3 (C ),
54.1 ppm (C2 or C8); HRMS: m/z calcd for C H N O : 994.4332
TBS
TBS
(
250 MHz, CDCl ): d=0.11 (s, 6H; Me ), 0.91 (s, 9H; tBu ), 4.04
1
3
67
56
5
4
+
(d, J=6.7 Hz, 2H; H5’), 4.53 (ddt, J=29.2, 6.5, 2.5 Hz, 1H; H4’), 5.32
dd, J=50.0, 2.0 Hz, 1H; H3’), 5.87 (d, J=12.7 Hz, 1H; H2’), 6.56 (s,
[M+H] ; found: 994.4306.
(
Bz
Bz
Compound 4a: Compound 1 (2.00 g, 2.0 mmol) was dissolved in
CHCl3 (100 mL), and pyridine (4 mL) was added to the solution.
DAST (1.32 mL, 10.0 mmol) was then added, and the solution was
stirred at room temperature for 24 h. The reaction mixture was
1H; H1’), 7.33–7.64 (m, 9H; H ), 7.86 (d, J=7.2 Hz, 4H; H ), 8.05 (t,
J=6.7 Hz, 2H; H ), 8.38 (s, 1H; H2 or H8), 8.65 ppm (s, 1H; H2 or
Bz
1
3
TBS
TBS
H8); C NMR (63 MHz, CDCl ): d=ꢀ5.4 (Me ), ꢀ5.3 (Me ), 18.4
3
TBS
TBS
(Cq ), 25.9 (tBu ), 59.6 (d, J=9.1 Hz; C5’), 79.8 (d, J=31.6 Hz;
C2’), 83.3 (d, J=19.7 Hz; C4’), 87.5 (C1’), 92.7 (d, J=185.4 Hz; C3’),
quenched with aqueous NaHCO , and the two layers were separat-
3
Ar
Ar
Ar
Ar
Ar
Ar
ed. The organic layer was then washed with water, and the com-
bined aqueous layers were re-extracted with CH Cl . The combined
127.1 (C ), 128.2 (C ), 128.5 (C ), 128.8 (C ), 129.6 (C ), 130.1 (C ),
130.2 (C ), 133.1 (C ), 133.6 (C ), 134.1 (C ), 134.3 (C ), 143.0 (C2
or C8), 152.0 (C ), 152.6 (C2 or C8), 152.7 (C ), 164.7 (C ), 170.9
(C=O), 172.3 ppm (C=O);
Ar
Ar
Ar
Ar
Ar
2
2
Ar
Ar
Ar
organic layers were dried over Na SO , concentrated in vacuo and
2
4
19
purified by chromatography with cyclohexane/EtOAc (9:1) as the
F NMR (471 MHz, CDCl3): d=
1
eluent to afford 4a as a white foam (1.00 g, 50%). H NMR
ꢀ202.65 ppm (s, 1F; F3’); HRMS: m/z calcd for C H FN O Si:
37
39
5
6
+
(
250 MHz, CDCl ): d=3.24–3.31 (m, 1H; H5’a), 3.43–3.50 (m, 1H;
696.2653 [M+H] ; found: 696.2630.
3
H5’b), 3.77 (d, J=51.7 Hz, 1H; H3’), 4.20 (dt, J=29.7, 6.25 Hz, 1H;
H4’), 4.38 (d, J=14.5 Hz, 1H; H2’), 6.45 (s, 1H; H1’), 6.90 (s, 1H;
Compound 8a: TFA/H O (1:1, 1.32 mL, 17.5 mmol) was added at
2
Trt
08C to a solution of 7a (240 mg, 0.35 mmol) in THF (5 mL). The re-
action mixture was stirred at RT for 1 h and quenched with aque-
ous NaHCO EtOAc was added, and the two layers were separated.
NH), 7.15–7.35 (m, 45H; H ), 7.62 (s, 1H; H2 or H8), 8.10 ppm (s,
1
9
1
H; H2 or H8); F NMR (471 MHz, CDCl ): d=ꢀ199.60 ppm (s, 1F;
3
+
3.
F3’); HRMS: m/z calcd for C H FN O : 996.4278 [M+H] ; found:
67
55
5
3
The organic layer was then washed with brine, and the combined
aqueous layers were re-extracted with EtOAc. The combined or-
ganic layers were dried over Na SO , and concentrated in vacuo.
9
96.4258.
Compound 5a: Trifluoroacetic acid (645 mL, 8.45 mmol) was added
at 08C to a solution of 4a (64 mg, 0.65 mmol) in CH Cl (6 mL). The
2
4
Purification by chromatography with cyclohexane/EtOAc (4:6) as
2
2
1
reaction mixture was allowed to warm to RT and further stirred for
h. Water and CH Cl were then added to the resulting solution.
the eluent afforded 8a as a white foam (180 mg, 90%). H NMR
1
(250 MHz, CDCl ): d=3.98 (d, J=5.5 Hz, 2H; H5’), 4.49 (dq, J=27.5,
2
2
3
After filtration, the aqueous layer was lyophilised and purified by
4.4 Hz, 1H; H4’), 5.32 (d, J=52.0 Hz, 1H; H3’), 5.90 (d, J=13.5 Hz,
&
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Full Papers
Bz
Ar
Ar
Ad
Ad
1
7
H; H2’), 6.46 (d, J=1.7 Hz, 1H; H1’), 7.30–7.36 (m, 4H; H ), 7.42–
2.0 Hz; H ), 8.03–8.06 (m, 2H; H ), 8.44 (s, 1H; H2 or H8 ), 8.64
Bz
Bz
Ad
Ad
19
.49 (m, 4H; H ), 7.58–7.63 (m, 1H; H ), 7.85 (d, J=7.2 Hz, 4H;
H ), 8.02 (d, J=7.5 Hz, 2H; H ), 8.36 (s, 1H; H2 or H8), 8.63 ppm
s, 1H; H2 or H8); C NMR (63 MHz, CDCl ): d=59.3 (d, J=9.1 Hz;
(s, 1H; H2 or H8 , diast), 8.78 ppm (s, 1H; NH); F NMR
Bz
Bz
31
(471 MHz, CDCl ): d=ꢀ201.54 ppm (s, 1F; F3’); P NMR (101 MHz,
3
1
3
(
CDCl ): d=ꢀ3.06 (s, 1P), ꢀ2.54 ppm (s, 1P); HRMS: m/z calcd for
3
3
+
C5’), 78.5 (d, J=30.6 Hz; C2’), 82.6 (d, J=19.7 Hz; C4’), 87.1 (C1’),
C H FN O P : 1152.2818 [M+H] ; found: 1152.2823.
51
49
11 16 2
Ar
Ar
Ar
9
(
(
1
2.9 (d, J=187.4 Hz; C3’), 127.2 (C ), 128.0 (C ), 128.7 (C ), 128.8
Ar Ar Ar Ar Ar Ar
Compound 11a: Dinucleotide 10a (166 mg, 0.14 mmol) was dis-
solved in a solution of MeNH (5m, EtOH/H O 1:1; 14 mL), and the
reaction mixture was stirred at RT for 24 h. After concentration
under reduced pressure, the residue was purified by HPLC. The
appropriate fractions were collected and lyophilised, to give the
C ), 129.4 (C ), 130.0 (C ), 133.0 (C ), 133.9 (C ), 134.1 (C ), 143.2
Ar
Ar
Ar
2
2
C2 or C8), 151.8 (C ), 152.4 (C2 or C8), 152.6 (C ), 164.6 (C ),
1
9
72.3 ppm (C=O); F NMR (471 MHz, CDCl ): d=ꢀ202.46 ppm (s,
3
+
1
F; F3’); HRMS: m/z calcd for C H FN O : 582.1789 [M+H] ;
31
25
5
6
found: 582.1771.
phosphorylated ammonium salt 11 a as a white foam (34 mg,
1
3
4%). t =15 min; H NMR (500 MHz, CD OD): d=2.17–2.23 (m,
R
3
Compound 9a: A tetrazole solution in CH CN (0.45m, 1.3 mmol,
.8 mL) and adenosine derivative 8a (0.13 mmol, 75 mg) in CH Cl2
2
350 mL) were added to a solution of phosphoramidite Ac-dC-PCNE
0.32 mmol, 250 mg) in CH Cl (350 mL). After this system had been
stirred at RT for 1 h, an iodine solution (0.1m) in THF/H O/pyridine
3
Cyt
Cyt
Ad
1
(
H; H2’ ), 2.51–2.54 (m, 1H; H2’ ), 4.03 (m, 1H; H5’ ), 4.09–4.13
2
(
(
Ad
Cyt
Cyt
m, 1H; H5’ ), 4.24–4.29 (m, 2H; H5’ ), 4.34 (s, 1H; H4’ ), 4.66
Ad Ad Cyt
(
m, 1H; H2’ ), 4.74 (d, J=13.7 Hz, 1H; H4’ ), H3’ masked in
2
2
Ad
residual H O peak of CD OD, 5.20 (dt, J=51.5, 1.3 Hz, 1H; H3’ ),
2
3
2
Cyt
Ad
5
.95 (d, J=7.9 Hz, 1H; H5 ), 6.12 (s, 1H; H1’ ), 6.35–6.38 (m, 1H;
Cyt Cyt Ad Ad
(
75:2:20, 3.2 mL) was added. After 30 min, the reaction mixture
H1’ ), 8.05 (d, J=7.8 Hz, 1H; H6 ), 8.21 (s, 1H; H2 or H8 ),
was diluted with EtOAc, washed with water, saturated Na S O so-
2
2
3
Ad
Ad
13
8
4
2
9
(
1
.24 ppm (s, 1H; H2 or H8 ); C NMR (125 MHz, CD OD): d=
3
lution and brine, dried over anhydrous Na SO and concentrated
under reduced pressure. The residue was then stirred with a TCA
solution (3%) in CH Cl (7.1 mL) at RT for 30 min. The reaction mix-
ture was diluted with CH Cl , and the organic phase was washed
with a saturated aqueous NaHCO solution and brine, dried over
Na SO and concentrated. The crude product was purified by flash
column chromatography with CH Cl /MeOH (96:4) as the eluent to
yield 9a as a white foam (90 mg, 72%). H NMR (250 MHz, CDCl ):
d=2.13–2.21 (m, 3H; Me ), 2.26–2.35 (m, 1H; H2’ ), 2.16 (s, 3H;
2
4
Cyt
Cyt
Cyt
Cyt
0.7 (C2’ ), 63.4 (C5’ ), 66.0 (C5’ ), 77.6 (C3’ ), 79.7 (d, J=
Ad
Ad
Cyt
Cyt
7.1 Hz; C4’ ), 82.4 (d, J=30.3 Hz; C2’ ), 86.7 (C4’ ), 87.3 (C1’ ),
2
2
Ad
Cyt
Ad
1.2 (C1’ ), 96.5 (C5 ), 96.6 (d, J=182.2 Hz; C3’ ), 120.0, 140.2
2
2
Ad
Cyt
Ad
C2 or C8 ), 142.9 (C6 ), 150.4, 153.9 (C2 or C8 ), 157.3, 158.2,
3
Cyt
19
67.6, 178.9 ppm (C=O );
F NMR (471 MHz, CD OD): d=
3
2
4
31
ꢀ
203.16 ppm (s, 1F; F3’); P NMR (203 MHz, CD OD): d=ꢀ0.94 (s,
3
2
2
1
1P), 1.28 ppm (s, 1P); HRMS: m/z calcd for C H FN O P :
19
26
8
12 2
3
+
Ac
Cyt
639.1151 [M+H] ; found: 639.1125.
Cyt
Ad or Cyt
CH CN, H2’ ), 3.75 (brs, 2H; H5’
H4’ ), 4.50–4.54 (m, 2H; H5’
5
(
), 4.19–4.29 (m, 3H; CH O,
2
2
Compound 12a: Dowex ion-exchange beads in tetrabutylammoni-
um form were added to a solution of pdCpdA·3NH4 salt 11 a in
Cyt
Ad or Cyt
Ad
), 4.63–4.83 (m, 1H; H4’ ),
+
Cyt
Ad
.21–5.25 (m, 1H; H3’ ), 5.43 (dd, J=49.9, 3.4 Hz, 1H; H3’ ), 5.94
Ad Cyt
Millipore water (4 mL), and the resulting mixture was stirred at RT
for 30 min. The mixture was filtered, and the aqueous solution was
lyophilised to afford pdCpdA as a tetrabutylammonium salt. The
residue was added to a solution of N-pent-4-enoyl-l-alanine cyano-
methyl ester (24 mg, 0.11 mmol) in DMF (250 mL). The reaction mix-
ture was stirred for 24 h at RT, diluted to a total volume of 900 mL
with a 1:2 CH CN/NH OAc (50 mm) solution and purified on a semi-
d, J=14.0 Hz, 1H; H2’ ), 6.13–6.19 (m, 1H; H1’ ), 6.54 (d, J=
Ad
Bz
Cyt
1
.7 Hz, 1H; H1’ ), 7.29–7.39 (m, 5H; H , H5 ), 7.42–7.48 (m, 4H;
Bz Bz Bz
H ), 7.61–7.71 (m, 1H; H ), 7.82 (d, J=7.6 Hz, 4H; H ), 8.00 (d, J=
Bz
Cyt
Ad
Ad
8
8
.1 Hz, 2H; H ), 8.18–8.22 (m, 1H; H6 ), 8.39 (s, 1H; H2 or H8 ),
Ad
Ad
Ac
13
.61 (s, 1H; H2 or H8 ), 9.84 ppm (brs, 1H; NH ); C NMR
Ac
Cyt
(
(
63 MHz, CDCl ): d=19.7 (CH CN), 24.8 (Me ), 39.9 (C2’ ), 61.4
3 2
Ad or Cyt Ad or Cyt Cyt
3
4
C5’
), 62.7 (CH O), 65.5 (C5’
), 79.2 (C3’ ), 79.6 (d, J=
Ad Cyt
2
preparative C-18 reversed-phase column. After lyophilisation of the
appropriate fractions, dinucleotide 12a was obtained as a white
foam (4 mg, 10%). t =18 min (CH CN/NH OAc 0:100!63:37 over
Ad
1
9.3 Hz; C4’ ), 80.16 (d, J=30.7 Hz; C2’ ), 86.5 (C4’ ), 87.3
Ad or Cyt
Ad or Cyt
Ad
(
C1’
), 87.4 (C1’
), 93.1 (d, J=187.3 Hz; C3’ ), 97.0, 127.1,
27.9, 128.7, 128.8, 129.4, 130.0, 133.2, 133.9, 134.2, 143.1 (C2 or
C8 ), 145.2 (C6 ), 151.8 (C2 or C8 ), 152.6, 152.7, 152.8, 155.5
C=O), 162.7 (C=O), 164.7 (C=N), 171.6 (C=O), 172.4 ppm (C=O);
HRMS: m/z calcd for C H FN O P: 966.2623 [M+H] ; found:
R
3
4
1
1
4
2
0 min); H NMR (250 MHz, CD OD): d=1.42 (d, J=7.4 Hz, 3H; Me),
3
Ad
Cyt
Ad
Ad
Cyt
.15–2.24 (m, 1H; H2’ ), 2.31 (s, 4H; 2ꢃCH ), 2.47–2.59 (m, 1H;
H2’ ), 4.06–4.12 (m, 2H; H5’
.34 (s, 1H; H4’ ), 4.43–4.69 (m, 3H; H2’ , H4’ , CH ), H3’ and
2
(
Cyt
Ad or Cyt
Ad or Cyt
), 4.24–4.29 (m, 2H; H5’
),
+
Cyt
Ad
Ad
Cyt
45
42
9
13
4
a
9
66.2596.
CH= masked in residual H O peak of CD OD, 5.40 (d, J=50.5 Hz,
2
3
Ad
Cyt
1
6
7
H; H3’ ), 5.77–5.85 (m, 2H; CH=), 5.96 (d, J=7.6 Hz, 1H; H5 ),
Ad Cyt
Compound 10a:
(
(
Bis(2-cyanoethyl)diisopropylphosphoramidite
124 mg, 0.45 mmol) was added to a solution of dinucleotide 9a
176 mg, 0.18 mmol) in CH Cl (3 mL), followed by a solution of tet-
razole in CH CN (0.45m, 4 mL, 1.8 mmol). The reaction mixture was
stirred at RT for 3 h, and an iodine solution (0.1m, 4.5 mL,
.45 mmol) was added. After having been stirred at RT for 1 h, the
mixture was diluted in EtOAc and washed successively with
Na S O and brine. The organic layer was dried over anhydrous
.27 (d, J=2.4 Hz, 1H; H1’ ), 6.36–6.41 (m, 1H; H1’ ), 8.09 (d, J=
Ad
Cyt
Ad
Ad
.5 Hz, 1H; H6 ), 8.19 (s, 1H; H2 or H8 ), 8.30 ppm (s, 1H; H2
Ad
19
2
2
or H8 ); F NMR (471 MHz, CD OD): d=ꢀ203.18 ppm (s, 1F; F3’);
3
31
3
P NMR (203 MHz, CD OD): d=ꢀ0.93 (s, 1P), 0.62 ppm (s, 1P);
3
+
HRMS: m/z calcd for C H FN O P : 792.1941 [M+H] ; found:
27
37
9
14 2
0
7
92.2001.
Compound 2b: A solution of Dess–Martin periodinane (4.86 g,
11.4 mmol) in CH Cl (40 mL) was added at 08C to a solution of
2
2
3
Na SO and concentrated to dryness. Purification by flash column
2
4
2
2
chromatography with CH Cl /MeOH (96:4) as the eluent afforded
1 (3.77 g, 3.8 mmol) in CH Cl2 (40 mL), and the reaction mixture
2
2
2
the desired phosphorylated dinucleotide 10a as a white foam
was stirred at room temperature for 4 h. The reaction mixture was
then cooled to 08C and quenched with an aqueous mixture of
NaHCO3 and Na S O . The two layers were separated, and the
1
(
2
(
4
166 mg, 80%). H NMR (500 MHz, CDCl ): d=2.20 (s, 3H; CH ),
3 3
Cyt
.30–2.34 (m, 1H; H2’ ), 2.73–2.78 (m, 6H; 3ꢃCH CN), 2.83–2.88
2
Cyt
2
2
3
Cyt
Cyt
Ad
m, 1H; H2’ ), 4.26–4.58 (m, 11H; 3ꢃCH ꢀO, H4’ , H5’ , H5’ ),
organic layer was washed with aqueous NaHCO and brine, dried
2
3
Ad
Cyt
.74–4.81 (m, 1H; H4’ ), 5.16–5.17 (m, 1H; H3’ ), 5.46 (d, J=
over MgSO4 and concentrated in vacuo. Purification by flash
Ad
Ad
5
1.0 Hz, 1H; H3’ ), 5.95–6.01 (m, 1H; H2’ ), 6.15–6.19 (m, 1H;
column chromatography with cyclohexane/EtOAc (8:2) as the
Cyt
Ad
Ar
1
H1’ ), 6.57 (s, 1H; H1’ ),7.34–7.41 (m, 5H; H ), 7.45–7.50 (m, 4H;
eluent afforded 2b as
a
white foam (3.60 g, 95%). H NMR
Ar
Ar
Ar
H ), 7.61–7.64 (m, 1H; H ), 7.83–7.84 (m, 4H; H ), 7.95 (d, J=7.5,
(250 MHz, CDCl ): d=3.49–3.66 (m, 2H; H5’), 4.66 (brs, 1H; H4’),
3
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7
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&
These are not the final page numbers! ÞÞ

Full Papers
5
.71 (d, J=6.1 Hz, 1H; H2’), 6.12 (d, J=6.1 Hz, 1H; H1’), 7.31–7.34
CD OD): d=63.0 (d, J=11.8 Hz; C5’), 74.5 (d, J=15.8 Hz; C4’), 86.3
3
Trt
Trt
Trt
(
m, 8H; H ), 7.40–7.44 (m, 8H; H ), 7.49–7.57 (m, 15H; H ), 7.61–
(d, J=21.7 Hz; C2’), 90.0 (C1’), 94.5 (d, J=182.9 Hz; C3’), 142.1 (C2
Trt
13
Ad
Ad
Ad
7
.65 (m, 15H; H ), 7.99 ppm (s, 1H; H2 or H8); C NMR (63 MHz,
or C8), 150.0 (C ), 153.4 (C2 or C8), 157.6 (C ), 163.4 ppm (C );
Trt
19
CDCl ): d=64.01 (C5’), 71.4 (C2’), 80.8 (C4’), 85.6 (C1’), 87.2 (C ),
F NMR (471 MHz, CD OD): d=ꢀ199.90 ppm (s, 1F; F3’); HRMS:
3
3
Trt
+
8
1
2
9.4 (C ), 121.1, 127.0, 127.2, 127.3, 127.5, 127.9, 128.0, 128.6,
28.7, 128.8, 129.1, 138.9, 143.3, 145.1, 148.6, 152.0, 153.8,
08.1 ppm (C=O); HRMS: m/z calcd for C H N O : 992.4176
m/z calcd for C H FN O : 270.1002 [M+H] ; found: 270.0992.
1
0
13
5
3
Compound 6b: Compound 5b (227 mg, 0.84 mmol) in pyridine
(5 mL) was cooled to 08C, and DMAP (20 mg, 0.17 mmol) and
TBDMSCl (140 mg, 0.92 mmol) were successively added. The reac-
tion mixture was stirred overnight at RT and then concentrated in
vacuo and coevaporated with toluene. EtOAc was added, and the
aqueous layer was extracted. The combined organic extracts were
washed with brine, dried over Na SO and concentrated in vacuo.
67
54
5
4
+
[M+H] ; found: 992.4145.
Compound 3b: Compound 2b (1.68 g, 1.69 mmol) was suspended
in EtOH/H O (15:1, 70 mL), and the suspension was cooled to 08C.
NaBH4 (73.4 mg, 1.94 mmol) was added, and the mixture was
allowed to warm to room temperature and stirred for 1 h 30.
NaHCO3 was then added, and the mixture was extracted with
CHCl . The organic layer was washed with water, dried over Na SO
4
and concentrated in vacuo. Purification by flash column chroma-
tography with cyclohexane/EtOAc (8:2) as the eluent gave 3b as
a white foam (837 mg, 50%). H NMR (500 MHz, CDCl ): d=3.44–
2
2
4
The crude residue was purified by chromatography with CH Cl /
2
2
MeOH (96:4) as the eluent, yielding 6b as a white foam (71 mg,
3
2
1
TBS
22%). H NMR (250 MHz, CD OD): d=0.12 (s, 6H; Me ), 0.93 (s,
3
TBS
9H; tBu ), 3.92 (d, J=3.4 Hz, 2H; H5’), 4.41 (dq, J=25.2 Hz,
1
2.1 Hz, 1H; H4’), 4.91–4.95 (m, 1H; H2’), 5.10 (dd, J=54.3 Hz,
4.3 Hz, 1H; H3’), 6.10 (d, J=7.2 Hz, 1H; H1’), 8.24 (s, 1H; H2 or H8),
3
3
3
5
.47 (m, 1H; H5’a), 3.53–3.57 (m, 1H; H5’b), 3.97 (dd, J=11.0,
1
3
.4 Hz, 1H; H3’), 4.31 (quint, J=3.5 Hz, 1H; H4’), 4.57 (s, 1H; H2’),
8.31 ppm (s, 1H; H2 or H8); C NMR (63 MHz, CD OD): d=ꢀ5.4
3
Trt
TBS
TBS
TBS
.45 (s, 1H; H1’), 6.97 (d, J=13.6 Hz, 2H; H ), 7.12–7.14 (m, 4H;
(Me ), 19.2 (Cq ), 26.3 (tBu ), 64.0 (d, J=11.7 Hz; C5’), 75.3 (d,
J=19.5 Hz; C2’), 85.2 (d, J=23.3 Hz; C4’), 88.3 (C1’), 93.6 (d, J=
181.8 Hz; C3’), 120.3 (Cq), 141.0 (C2 or C8), 150.9 (Cq), 152.5 (C2 or
C8), 156.4 ppm (Cq); HRMS: m/z calcd for C H FN O Si: 384.1867
Trt
Trt
Trt
H ), 7.16–7.17 (m, 4H; H ), 7.20–7.24 (m, 13H; H ), 7.26–7.27 (m,
5
Trt
Trt
Trt
H; H ), 7.31–7.33 (m, 6H; H ), 7.35–7.37 (m, 6H; H ), 7.39–7.41
Trt
13
(
m, 6H; H ), 7.76 ppm (s, 1H; H2 or H8); C NMR (125 MHz,
16
27
5
3
Trt
+
CDCl ): d=62.6 (C5’), 77.0 (C3’), 82.6 (C2’), 84.6 (C4’), 87.1 (C ), 88.7
[M+H] ; found: 384.1848.
3
Trt
(
C ), 92.39 (C1’), 121.5, 126.9, 127.1, 127.7, 127.8, 128.0, 128.2,
Compound 7b: BzCl (150 mL, 1.29 mmol) was added at 08C to a so-
lution of 6b (71 mg, 0.18 mmol) in pyridine (2 mL), and the reac-
tion mixture was stirred at room temperature overnight. The result-
ing solution was coevaporated with toluene in vacuo. The residue
was suspended in EtOAc, washed with brine and dried over
Na SO . The solvent was removed under reduced pressure, and the
1
1
28.8, 128.9, 129.0, 140.7, 143.8, 143.9, 144.8, 146.4, 151.0,
+
54.3 ppm; HRMS: m/z calcd for C H N O : 994.4332 [M+H] ;
67
56
5
4
found: 994.4306.
Compound 4b: Compound 3b (1.37 g, 1.37 mmol) was dissolved
in CHCl (20 mL), and pyridine (33 mmol, 2.7 mL) was added to the
3
2
4
solution. DAST (837 mL, 6.3 mmol) was then added at 08C, and the
solution was stirred at room temperature for 24 h. The reaction
product was purified by chromatography with cyclohexane/EtOAc
1
(8:2) as the eluent to give 7b (129 mg, quant.). H NMR (500 MHz,
TBS
TBS
mixture was quenched with aqueous NaHCO , and the two layers
CDCl ): d=0.15 (s, 3H; Me ), 0.16 (s, 3H; Me ), 0.95 (s, 9H;
3
3
TBS
were separated. The organic layer was then washed with water,
and the combined aqueous layers were re-extracted with CH Cl .
tBu ), 3.92–4.01 (m, 2H; H5’), 4.57 (d, J=25.7 Hz, 1H; H4’), 5.50
(dd, J=54.1, 4.8 Hz, 1H; H3’), 5.90 (dq, J=21.0, 4.1 Hz, 1H; H2’),
2
2
Bz
The combined organic layers were dried over Na SO , concentrated
6.66 (d, J=7.7 Hz, 1H; H1’), 7.26–7.34 (m, 4H; H ), 7.40–7.47 (m,
2
4
Bz
Bz
Bz
in vacuo and purified by chromatography with cyclohexane/EtOAc
8:2) as the eluent to afford 4b as a white foam (927 mg, 67%).
4H; H ), 7.53–7.57 (m, 1H; H ), 7.85–7.87 (m, 4H; H ), 8.02–8.04
Bz
(
(m, 2H; H ), 8.48 (s, 1H; H2 or H8), 8.67 ppm (s, 1H; H2 or H8);
1
13
TBS
TBS
H NMR (500 MHz, CDCl ): d=3.01–3.03 (m, 1H; H5’a), 3.29–3.32
C NMR (125 MHz, CDCl ): d=ꢀ5.4 (Me ), ꢀ5.3 (Me ), 18.4
3
3
TBS
TBS
(
m, 1H; H5’b), 3.58 (dd, J=52.7, 3.7 Hz, 1H; H3’), 4.96 (dt, J=27.4,
(Cq ), 26.0 (tBu ), 62.9 (d, J=10.8 Hz; C5’), 75.2 (d, J=16.0 Hz;
C2’), 84.7 (d, J=23.5 Hz; C4’), 85.0 (C1’), 90.53 (d, J=188.8 Hz; C3’),
128.4, 128.5, 128.57, 129.5, 130.1, 133.0, 133.8, 134.1, 143.0, 151.9,
152.5, 152.2, 165.3, 170.8, 172.2 ppm; HRMS: m/z calcd for
4
8
7
.0 Hz, 1H; H4’), 5.25 (dq, J=22.1, 4.0 Hz, 1H; H2’), 6.22 (d, J=
Trt Trt
.1 Hz, 1H; H1’), 6.98–7.03 (m, 7H; H ), 7.07–7.12 (m, 12H; H ),
Trt
Trt
.15–7.18 (m, 4H; H ), 7.21–7.24 (m, 6H; H ), 7.27–7.31 (m, 10H;
Trt
Trt
+
H ), 7.40–7.41 (m, 6H; H ), 7.76 (s, 1H; H2 or H8), 7.81 ppm (s,
1
C H FN O Si: 696.2653 [M+H] ; found: 696.2633.
37
39
5
6
1
3
H; H2 or H8); C NMR (125 MHz, CDCl ): d=62.9 (d, J=10.8 Hz;
3
Compound 8b: Compound 7b (129 mg, 0.18 mmol) in THF (5 mL)
C5’), 74.7 (d, J=16.2 Hz; C2’), 82.6 (d, J=23.5 Hz; C4’), 86.7 (C1’),
Trt
Trt
was cooled to 08C, and TFA/H O (1:1, 709 mL, 9.25 mmol) was
2
8
1
1
7.1 (C ), 87.5 (C ), 90.6 (d, J=184.1 Hz; C3’), 121.6, 126.9, 127.1,
added. The reaction mixture was stirred at RT for 1 h, and then the
27.3, 127.7, 127.8, 128.6, 129.0, 139.7, 143.3, 145.0, 149.0, 152.1,
1
9
reaction was quenched with aqueous NaHCO . EtOAc was added,
3
53.9 ppm; F NMR (471 MHz, CDCl ): d=ꢀ192.34 (s, 1F; F3’);
3
+
and the two layers were separated. The organic layer was then
washed with brine, and the combined aqueous layers were re-ex-
tracted with EtOAc. The combined organic layers were dried over
Na SO and concentrated in vacuo. Purification by chromatography
HRMS: m/z calcd for C H FN O : 996.4289 [M+H] ; found:
67
55
5
3
9
96.4262.
Compound 5b: Trifluoroacetic acid (1.15 mL, 14.9 mmol) was
2
4
added at 08C to a solution of 4b (1.15 g, 1.15 mmol) in CH Cl2
with cyclohexane/EtOAc (4:6) as the eluent afforded 8b as a white
2
1
(
10 mL). The reaction mixture was allowed to warm to RT and
foam (86 mg, 81%). H NMR (250 MHz, CDCl ): d=3.80–3.89 (m,
3
stirred for 1 h. Water and CH Cl were then added to the resulting
1H; H5’a), 3.98–4.03 (m, 1H; H5’b), 4.62 (d, J=30.5 Hz, 1H; H4’),
5.57 (dd, J=54.4, 4.5 Hz, 1H; H3’), 5.68 (brs, 1H; OH), 6.08 (dq, J=
23.1, 4.3 Hz, 1H; H2’), 6.33 (d, J=7.8 Hz, 1H; H1’), 7.32–7.40 (m,
2
2
solution, the mixture was filtered, and the combined aqueous
layers were lyophilised and purified by chromatography with
CH Cl /MeOH (9:1) to afford 5b as a white foam (235 mg, 76%).
Bz
Bz
Bz
4H; H ), 7.43–7.51 (m, 4H; H ), 7.53–7.62 (m, 1H; H ), 7.83–7.86
2
2
1
Bz
Bz
H NMR (500 MHz, CD OD): d=3.87–3.78 (m, 2H; H5’), 4.43 (d, J=
(m, 4H; H ), 7.96–8.00 (m, 2H; H ), 8.20 (s, 1H; H2 or H8),
3
13
2
.81 Hz, 1H; H4’), H2’ masked in residual H O peak of MeOD, 5.13
8.65 ppm (s, 1H; H2 or H8); C NMR (63 MHz, CDCl ): d=62.3 (d,
2
3
(
1
dd, J=55.7, 3.5 Hz, 1H; H3’), 6.01 (d, J=6.5 Hz, 1H; H1’), 8.17 (s,
H; H2 or H8), 8.28 ppm (s, 1H; H2 or H8); C NMR (125 MHz,
J=11.2 Hz; C5’), 74.1 (d, J=14.9 Hz; C2’), 86.5 (d, J=21.9 Hz; C4’),
89.9 (C1’), 91.4 (d, J=187.9 Hz; C3’), 128.3, 128.6, 128.8, 128.9,
13
&
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ÝÝ These are not the final page numbers!

Full Papers
Cyt
Cyt
Ad
1
1
29.5, 129.9, 133.2, 133.8, 133.9, 144.4 (C2 or C8), 151.8, 152.0,
52.9 (C2 or C8), 165.0 (C=O ), 172.1 ppm (C=O ); F NMR
6.35 (m, 1H; H1’ ), 7.97 (d, J=7.8 Hz, 1H; H6 ), 8.20 (s, 1H; H2
Ad Ad Ad 13
Bz
Bz
19
or H8 ), 8.61 ppm (s, 1H; H2 or H8 ); C NMR (63 MHz, CD OD):
3
Cyt
Cyt
Ad
Cyt
Ad
(
471 MHz, CDCl ): d=ꢀ197.60 ppm (s, 1F; F3’); HRMS: m/z calcd
d=40.5 (C2’ ), 66.0 (C5’ or C5’ ), 66.1 (C5’ or C5’ ), 74.9 (d,
Ad Cyt Ad
3
+
for C H FN O : 582.1789 [M+H] ; found: 582.1770.
J=15.7 Hz; C4’ ), 77.8 (C3’ ), 84.2 (d, J=21.8 Hz; C2’ ), 86.8
3
1
25
5
6
Cyt
Cyt
Ad
Ad
(
(
1
C4’ ), 87.2 (C1’ ), 87.4 (C1’ ), 94.3 (d, J=176.6 Hz; C3’ ), 96.6
Cyt Ad Ad Cyt Ad Ad
Compound 9b: A tetrazole solution in CH CN (0.45m, 1 mmol,
2
3
C5 ), 119.8, 140.8 (C2 or C8 ), 142.8 (C6 ), 151.3 (C2 or C8 ),
.3 mL) and adenosine derivative 8b (0.1 mmol, 60 mg) in CH Cl2
19
2
54.1, 158.1, 167.4, 178.2 ppm (C=O); F NMR (471 MHz, CD OD):
3
(
(
350 mL) were added to a solution of phosphoramidite Ac-dC-PCNE
0.32 mmol, 250 mg) in CH Cl (350 mL). After the system had been
31
d=ꢀ199.30 ppm (s, 1F; F3’); P NMR (101 MHz, CD OD): d=ꢀ1.13
3
2
2
(
s, 1P), 0.94 ppm (s, 1P); HRMS: m/z calcd for C H FN O P :
19
24
8
12 2
stirred at RT for 1 h, an iodine solution (0.1m) in THF/H O/pyridine
+
2
6
37.0973 [MꢀH] ; found: 637.0833.
(
75:2:20, 2.6 mL) was added. After 30 min, the reaction mixture
Ligation of fluorinated dinucleotides to RNA helices: Dinucleoti-
was diluted with EtOAc, washed with water, saturated Na S O so-
2
2
3
helix
des 11 a, 11 b and 12a were ligated to RNA
(5’-GGGGC CUUAG
lution and brine, dried over anhydrous Na SO and concentrated
2
4
CUCAG GCUCC AC-3’), thus mimicking the acceptor arm of the
under reduced pressure. The residue was then stirred with a solu-
tion of TCA (3%) in CH Cl (5.7 mL) at RT for 30 min. The reaction
Ala
[7]
helix
tRNA with purified T4 RNA ligase. RNA
was synthesised by
2
2
the phosphoramidite method and purified by polyacrylamide gel
electrophoresis (Eurogentec). The ligation reaction was performed
mixture was diluted with CH Cl , and the organic phase was
2
2
washed with saturated aqueous NaHCO solution and brine, dried
3
at 168C for 12 h in HEPES buffer (50 mm, pH 7.5) containing
over Na SO and concentrated. The crude product was purified by
2
4
helix
RNA
(40 mm), dinucleotide (400 mm), T4 RNA ligase
flash column chromatography with CH Cl /MeOH (96:4) as the
2
2
ꢀ
1
1
(0.6 mgmL ), DMSO (10%), ATP (1 mm) and MgCl (15 mm). Com-
eluent to yield 9b as a white foam (70 mg, 70%). H NMR
2
Cyt
pounds A, B and C were purified by anion-exchange chromatogra-
phy (DEAE column, DNAPac-100, Dionex) with a linear gradient of
ammonium acetate (pH 8.0, 25–2500 mm) containing acetonitrile
(
250 MHz, CDCl ): d=2.16 (s, 3H; CH ), 2.29–2.41 (m, 1H; H2’ ),
3 3
Cyt Cyt
2
3
5
.69–2.76 (m, 3H; CH CN, H2’ ), 3.75 (s, 2H; H5’ ), 4.19–4.27 (m,
2
Cyt Ad Ad
H; CH O, H4’ ), 4.48 (brs, 2H; H5’ ), 4.68 (m, 1H; H4’ ), 5.15–
2
Cyt
Ad
Cyt
(0.5%). Fractions containing the ligation product were identified
.19 (m, 2H; H3’ ), 5.67 (m, 1H; H3’ ), 6.01–6.21 (m, 2H; H1’
H2’ ), 6.53, 6.56 (d, J=7.2 Hz, 1H; H1’ , diast), 7.28–7.43 (m, 9H;
H ), 7.52–7.58 (m, 1H; H ), 7.79 (d, J=7.4 Hz, 4H; H ), 7.94–7.97
m, 2H; H ), 8.17–8.22 (m, 1H; H ), 8.52, 8.53 (s, 1H; H2 or H8 ,
,
Ad
Ad
by denaturing polyacrylamide gel electrophoresis, lyophilised, re-
suspended in RNAse-free water (Sigma) and stored at ꢀ208C.
Ar
Ar
Ar
Ar
Ar
Ad
Ad
(
Ad
Ad
diast), 8.64, 8.65 (s, 1H; H2 or H8 , diast), 8.82 ppm (s, 1H; NH);
1
9
31
F NMR (471 MHz, CDCl ): d=ꢀ200.69 ppm (s, 1F; F3’); P NMR Acknowledgements
3
(
101 MHz, CDCl ): d=ꢀ2.94 ppm (s, 1P); HRMS: m/z calcd for
3
+
C H FN O P: 966.2623 [M+H] ; found: 966.2615.
4
5
42
9
13
The ANR MimictRNA supported this work.
Compound 10b: Bis(2-cyanoethyl)diisopropylphosphoramidite
(
(
50 mg, 0.18 mmol) was added to a solution of dinucleotide 9b
Keywords: aminoacyl-tRNAs
nucleosides · peptidoglycan · transferases · tRNA
·
dinucleotides
·
fluoro-
70 mg, 0.07 mmol) in CH Cl (2 mL), followed by a solution of tet-
2
2
razole in CH CN (0.45m, 1.6 mL, 0.70 mmol). The reaction mixture
3
was stirred at RT for 3 h, and an iodine solution (0.1m) in THF/H O/
2
[
1] a) J. Shepherd, M. Ibba, FEBS Lett. 2013, 587, 2895–2904; b) H. von Dçh-
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pyridine (75:2:20, 1.8 mL) was added. After having been stirred at
RT for 1 h, the mixture was diluted with EtOAc and washed succes-
sively with Na S O and brine. The organic layer was dried over an-
[
[
2
2
3
hydrous Na SO and concentrated to dryness. Purification by flash
2
4
[4] K. S. Huang, J. S. Weinger, E. B. Butler, S. A. Strobel, J. Am. Chem. Soc.
2006, 128, 3108–3109.
column chromatography with CH Cl /MeOH (96:4) as the eluent
2
2
afforded the desired phosphorylated dinucleotide 10b as a white
[5] a) J. S. Weinger, K. M. Parnell, S. Dorner, R. Green, S. A. Strobel, Nat.
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[6] U. Kopp, M. Roos, J. Wecke, H. Labischinski, Microb. Drug Resist. 1996, 2,
29–41.
1
foam (72 mg, 87%). H NMR (250 MHz, CDCl ): d=2.17 (s, 3H; CH ),
3
3
Cyt
Cyt
2
4
.27–2.39 (m, 1H; H2’ ), 2.71–2.75 (m, 7H; 3ꢃCH CN, H2’ ),
2
Cyt Cyt Ad
.25–4.50 (m, 11H; 3ꢃCH O, H4’ , H5’ , H5’ ), 4.70 (m, 1H;
2
Ad
Cyt
Ad
H4’ ), 5.16 (brs, 1H; H3’ ), 5.70 (m, 1H; H3’ ), 6.03–6.21 (m, 2H;
H1’ , H2’ ), 6.52–6.57 (m, 1H; H1’ ), 7.29–7.48 (m, 9H; H ),
.54–7.59 (m, 1H; H ), 7.80 (d, J=7.3 Hz, 4H; H ), 7.93–8.00 (m,
H; H ), 8.51, 8.53 (s, 1H; H2 or H8 , diast), 8.65 (s, 1H; NH or
Cyt
Ad
Ad
Ar
[
7] M. Fonvielle, M. Chemama, M. Lecerf, R. Villet, P. Busca, A. Bouhss, M.
Ar
Ar
7
3
Etheve-Quelquejeu, M. Arthur, Angew. Chem. Int. Ed. 2010, 49, 5115–
Ar
Ad
Ad
5119; Angew. Chem. 2010, 122, 5241–5245.
Ad
Ad
Ad
Ad
31
H2 or H8 ), 8.83 ppm (s, 1H; NH or H2 or H8 ); P NMR
[
8] M. Fonvielle, I. L. de La Sierra-Gallay, A. H. El-Sagheer, M. Lecerf, D. Patin,
D. Mellal, C. Mayer, D. Blanot, N. Gale, T. Brown, H. van Tilbeurgh, M.
Etheve-Quelquejeu, M. Arthur, Angew. Chem. Int. Ed. 2013, 52, 7278–
(
101 MHz, CDCl ): d=ꢀ2.91 (s, 1P), ꢀ2.51 ppm (s, 1P); HRMS: m/z
3
+
calcd for C H FN O P : 1152.2818 [M+H] ; found: 1152.2889.
5
1
49
11 16 2
7281; Angew. Chem. 2013, 125, 7419–7422.
Compound 11b: Dinucleotide 10b was dissolved in a solution
5m) of MeNH (EtOH/H O 1:1; 3 mL), and the reaction mixture was
[9] a) Y. W. Huang, M. Sprinzl, Angew. Chem. Int. Ed. 2011, 50, 7287–7289;
Angew. Chem. 2011, 123, 7425–7427; b) M. M. Changalov, G. D. Ivanova,
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D. D. Petkov, ChemBioChem 2005, 6, 992–996.
(
2
2
stirred at RT for 24 h. After concentration under reduced pressure,
the residue was purified by HPLC. The appropriate fractions were
collected and lyophilised, to give the phosphorylated ammonium
salt 11 b as a white foam (20 mg, 46%). t =15 min (CH CN/
[
10] a) J. L. Brunelle, J. J. Shaw, E. M. Youngman, R. Green, RNA 2008, 14,
R
3
1526–1531; b) A. Charafeddine, W. Dayoub, H. Chapuis, P. Strazewski,
1
NH OAc 0:100!63:37 over 40 min); H NMR (250 MHz, CD OD):
4
3
Chem. Eur. J. 2007, 13, 5566–5584.
Cyt
Cyt
d=2.04–2.08 (m, 1H; H2’ ), 2.33–2.41 (m, 1H; H2’ ), 4.06–4.12
[
[
11] P. Liu, A. Sharon, C. K. Chu, J. Fluorine Chem. 2008, 129, 743–766.
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Kulkarni, N. Martin-Pintado, P. Lindovska, M. Thomson, C. Gonzalez, M.
Ad
Cyt
Cyt
Ad
(
m, 4H; H5’ , H5’ ), 4.29 (s, 1H; H4’ ), 4.52 (m, 1H; H2’ ),
Ad Cyt Ad
H4’ , H3’ and H3’ masked in residual H O peak of CD OD,
2
3
Cyt
Ad
5
.94 (d, J=7.5 Hz, 1H; H5 ), 6.15 (d, J=8.0 Hz, 1H; H1’ ), 6.29–
ChemBioChem 0000, 00, 0 – 0
www.chembiochem.org
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ꢁ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

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Received: September 10, 2014
2
Published online on && &&, 0000
&
ChemBioChem 0000, 00, 0 – 0
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ꢁ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

FULL PAPERS
Fluorinated aminoacyl-tRNA ana-
L. Iannazzo, G. Laisnꢀ, M. Fonvielle,
E. Braud, J.-P. Herbeuval, M. Arthur,
M. Etheve-Quelquejeu*
logues: Semisynthetic routes to fluoro
Ala
Ala
analogues of tRNA and Ala-tRNA
have been developed. These molecules
are non-isomerisable analogues of the
substrates of alanyl-tRNA synthetases
and FemX transferases and should pro-
vide new insight into substrate recogni-
tion by these enzymes.
&
& – &&
Synthesis of 3’-Fluoro-tRNA Analogues
for Exploring Non-ribosomal Peptide
Synthesis in Bacteria
ChemBioChem 0000, 00, 0 – 0
www.chembiochem.org
11
ꢁ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ