Efficient access to peptidyl-RNA conjugates for picomolar inhibition of non-ribosomal FemXWv aminoacyl transferase

Article abstract of DOI:10.1002/chem.201201999

Peptidyl-RNA conjugates have various applications in studying the ribosome and enzymes participating in tRNA-dependent pathways such as Fem transferases in peptidoglycan synthesis. Herein a convergent synthesis of peptidyl-RNAs based on Huisgen-Sharpless

Full text of DOI:10.1002/chem.201201999

FULL PAPER
DOI: 10.1002/chem.201201999
Efficient Access to Peptidyl-RNA Conjugates for Picomolar Inhibition of
Non-ribosomal FemXWv Aminoacyl Transferase
Matthieu Fonvielle,[a] Dꢀnia Mellal,[b] Delphine Patin,[c] Maxime Lecerf,[a]
Didier Blanot,[c] Ahmed Bouhss,[c] Marco Santarem,[b] Dominique Mengin-Lecreulx,[c]
Matthieu Sollogoub,[b] Michel Arthur,*[a] and Mꢀlanie Ethꢁve-Quelquejeu*[b]
Dedicated to Professor Michel Therisod on the occasion of his 65th birthday
Abstract: Peptidyl–RNA conjugates
have various applications in studying
the ribosome and enzymes participat-
ing in tRNA-dependent pathways such
as Fem transferases in peptidoglycan
synthesis. Herein a convergent synthe-
sis of peptidyl–RNAs based on Huis-
gen–Sharpless cycloaddition for the
final ligation step is developed. Azides
and alkynes are introduced into tRNA
and UDP-MurNAc-pentapeptide, re-
spectively. Synthesis of 2’-azido RNA
helix starts from 2’-azido-2’-deoxyade-
nosine that is coupled to deoxycytidine
by phosphoramidite chemistry. The re-
sulting dinucleotide is deprotected and
ligated to a 22-nt RNA helix mimicking
the acceptor arm of Ala-tRNAAla by
T4 RNA ligase. For alkyne UDP-
MurNAc-pentapeptide, meso-cystine is
enzymatically incorporated into the
peptidoglycan precursor and reduced,
and l-Cys is converted to dehydroala-
nine with O-(mesitylenesulfonyl)hy-
droxylamine. Reaction of but-3-yne-1-
thiol with dehydroalanine affords the
alkyne-containing UDP-MurNAc-pen-
tapeptide. The CuI-catalyzed azide
alkyne cycloaddition reaction in the
presence of tris[(1-hydroxypropyl-1H-
1,2,3-triazol-4-yl)methyl]amine provid-
ed the peptidyl-RNA conjugate, which
was tested as an inhibitor of non-ribo-
somal FemXWv aminoacyl transferase.
The bi-substrate analogue was found to
inhibit FemXWv with an IC50 of (89Æ
9) pm, as both moieties of the peptidyl–
RNA conjugate contribute to high-af-
finity binding.
Keywords: click chemistry · inhibi-
tors · peptidyl–RNA conjugates ·
RNA · transferases
Introduction
ing stability and cell permeability of short interfering
RNAs.[1] Two strategies are currently explored for synthesis
of peptidyl–RNA conjugates. In the divergent method, the
peptide and oligonucleotide fragments are sequentially as-
sembled by solid-phase synthesis on the same support. In
the convergent method, the peptide and oligonucleotide
fragments are independently synthesized prior to ligation.
The first method is limited by the lack of a suitable solid
support to assemble the peptide and oligonucleotide moiet-
ies and the lack of a uniform protection strategy suitable for
any peptide sequence. A promising approach relies on poly-
merization of 3’-aminoacylamino-3’-deoxyadenosine linked
to the solid support at the 2’ position.[2] The modified adeno-
sine provides the C-terminal extremity of the peptide chain
and the 3’ terminus of the oligonucleotides. Automated syn-
thesis proceeds by Fmoc solid-phase synthesis followed by
standard automated DMT/phosphoramidite synthesis. Suc-
cessful protection of amino acid side chains is currently lim-
ited to Ser, Thr, Tyr, Asp and Glu.[3] To incorporate Arg in
peptidyl–RNA conjugates, a convergent approach was de-
veloped which relies on a final native chemical ligation
step.[4] This requires synthesis of a cysteinyl 3’-adenosine an-
alogue resulting in the obligate presence of a cysteinyl resi-
due at the C terminus of the peptide moiety of the conju-
gate. Here we develop a convergent synthetic route to pep-
tidyl–RNA conjugates based on the Huisgen–Sharpless CuI-
Peptidyl–RNA conjugates have various applications in func-
tional and structural studies of the ribosome and for improv-
[a] Dr. M. Fonvielle,+ M. Lecerf, Dr. M. Arthur
Centre de Recherche des Cordeliers, LRMA, Equipe 12
Universitꢀ Pierre et Marie Curie - Paris 6, UMR S 872
Paris 75006 (France)
Universitꢀ Paris Descartes, Sorbonne Paris Citꢀ
UMR S 872, Paris 75006 (France)
INSERM, U872, Paris, 75006 (France)
Fax : (+33)1-4325-6812
E-mail: michel.arthur@crc.jussieu.fr
[b] D. Mellal,+ Dr. M. Santarem, Dr. M. Sollogoub,
Dr. M. Ethꢁve-Quelquejeu
Institut Parisien de Chimie Molꢀculaire, CNRS UMR 7201
Universitꢀ Pierre et Marie Curie - Paris 6
4, place Jussieu, 75005 Paris (France)
Fax : (+33)1-4427-5504
E-mail: melanie.etheve-quelquejeu@parisdescartes.fr
[c] D. Patin, Dr. D. Blanot, Dr. A. Bouhss, Dr. D. Mengin-Lecreulx
Laboratoire des Enveloppes Bactꢀriennes et Antibiotiques
Institut de Biochimie et de Biophysique Molꢀculaire et
Cellulaire, UMR 8619, CNRS
Universitꢀ Paris-Sud, 91405 Orsay (France)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201201999.
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catalyzed azide–alkyne cycloaddition reaction (Cu-AAC)
and apply the method to the synthesis of a picomolar inhibi-
tor of non-ribosomal FemXWv aminoacyl transferase.
with the translation machinery for a common pool of Ala-
tRNAAla [9]
The aminoacyl-transfer reaction catalyzed by
.
FemXWv may proceed by a substrate-assisted catalytic mech-
anism involving direct attack of the amino group of the pep-
tidoglycan precursor on the carbonyl group of Ala attached
to the 2’ position of the terminal ribose.[9] Our target (Fig-
ure 1B) is a bi-substrate composed of a peptidoglycan pre-
cursor analogue covalently linked to an RNA helix mimick-
ing the tRNAAla acceptor arm. The convergent synthesis of
the bi-substrate (Scheme 1) is based on a Cu-AAC reaction
between alkyne UDP-MurNAc-pentapeptide 9 and 2’-azido
RNA helix 11.
Aminoacyl–tRNAs participate in various biosynthetic
pathways as donors of activated aminoacyl residues in addi-
tion to their role in protein synthesis by the ribosome.[5]
Among non-ribosomal aminoacyl transferases, FemXWv
from Weissella viridescens[6] catalyzes transfer of Ala from
Ala-tRNAAla to the amino group of l-Lys at the 3-position
of peptidoglycan precursors[7] (Figure 1A). Enzymes of the
Fem family are attractive targets for the development of an-
tibiotics active against resistant bacteria, since the residues
incorporated by the enzymes are essential for the last cross-
linking step of peptidoglycan polymerization in several im-
portant b-lactam-resistant pathogens such as staphylococci,
pneumococci and streptococci.[8] Blocking Fem activity
would therefore result in production of incomplete precur-
sors acting as chain terminators that block formation of the
essential stress-bearing peptidoglycan layer of the bacterial
cell wall.
The alanyl-tRNA substrate of FemXWv is produced by the
alanyl-tRNA synthetase that aminoacylates the 3’-position
of the terminal adenosine residue (A76) of tRNAAla (Fig-
ure 1A).[7] The resulting 3’-acylated tRNA binds to the ac-
ceptor site and delivers Ala to the ribosome. The same 3’
Ala-tRNAAla regioisomer binds to FemXWv with high affini-
ty, although the enzyme catalyzes aminoacyl transfer only
from the 2’-position following trans-esterification within the
active site.[9] This enables FemXWv to efficiently compete
Results and Discussion
For synthesis of 2’-azido RNA helix 11, we started from the
2’-azido-2’-deoxyadenosine 1 (Scheme 1).[9,10] Coupling of 1
with commercially available Ac-dC-PCNE afforded dinu-
cleotide 2, which was phosphorylated and deprotected to
obtain 3. An additional deprotection step afforded dinucleo-
tide 4, which was ligated to 22-nt RNA helix 10 by T4 RNA
ligase to obtain 24-nt azido RNA helix 11.
Alkyne UDP-MurNAc-pentapeptides 9a and 9b were ob-
tained by semisynthesis (Scheme 1). Briefly, UDP-MurNAc-
pentapeptide analogue 6, which contained meso-cystine in-
stead of l-Lys, was obtained enzymatically by sequential ad-
dition of l-Ala, d-Glu, meso-cystine and d-Ala-d-Ala to
UDP-MurNAc by Mur synthetases MurC, D, E and F from
Escherichia coli, respective-
ly.[11] The natural substrate of
E. coli MurE, meso-diaminopi-
melic acid (meso-DAP), differs
from meso-cystine only by re-
placement of a methylene by a
disulfide group. This account
for successful incorporation of
meso-cystine instead of meso-
DAP by MurE, albeit at the
cost of 28-fold reduction in
enzyme catalytic efficiency
(data not shown). The meso-
cystine-containing
MurNAc-pentapeptide
UDP-
6 was
reduced by dithiothreitol to
afford l-cysteine-containing
UDP-MurNAc-pentapeptide 7.
Transformation of l-Cys of 7
into dehydroalanine of 8 was
achieved with O-(mesitylene-
sulfonyl)hydroxylamine
(MSH) by using procedures
developed
for
proteins,[12]
which proved compatible with
the peptidoglycan precursor.
In the last step, reaction of
but-3-yne-1-thiol with dehy-
Figure 1. Participation of Ala-tRNAAla in protein and peptidoglycan synthesis (A). Target peptidyl-RNA con-
jugate (B). The RNA helix mimics the acceptor arm (red) and ACCA extremity (blue) or tRNAAla
.
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Efficient Access to Peptidyl–RNA Conjugates
FULL PAPER
Scheme 1. Convergent synthesis of peptidyl–RNA conjugates 13a and 13b. a) 1. Tetrazole, CH2Cl2, RT, 1 h; 2. I2, 30 min, RT; 3. TCA, CH2Cl2, RT,
30 min; b) 1. bis(2-cyanoethyl)diisopropylphosphoramidite, tetrazole, CH2Cl2, RT, 1 h; 2. I2, RT, 30 min; 3. CH3NH2, RT, 24 h; c) HCl (6m)/THF/CH3OH,
24 h; d) 22 nt RNA helixAla, T4 RNA ligase; e) 1. l-Ala, MurC; d-Glu, MurD; 2. meso-Cystine, MurE; 3. d-Ala-d-Ala, MurF; f) DTT, Tris-HCl buffer,
pH 8.0, 2 h, RT; g) MSH, DMF, 2 h, RT; h) (S)-But-3-ynyl ethanethioate, DMF, 2 h, 378C; i) CuSO4, THPTA, Na ascorbate, 48C, 24 h.
droalanine afforded UDP-MurNAc-pentapeptide analogues
9a and 9b, which contained the desired alkyne. Diaster-
eoisomers 9a and 9b, which resulted from non-stereoselec-
tive thiol conjugate addition[13] to dehydroalanine 8, were
purified by HPLC.
and 13b were purified by denaturing polyacrylamide gel
electrophoresis (Figure 2A). These initial conditions were
optimized by increasing the concentrations of UDP-
MurNAc-pentapeptide 9a (20 mm) and RNA helix 11
(1 mm), which increased the yield from 4 to 36%.
Optimization of the cycloaddition reaction[14] was per-
formed with 24-nt azido RNA helix 11 and commercially
available 1,8-nonadiyne, which afforded compound 12 (Fig-
ure 2A). The reaction conditions for this critical step were
azido-RNA helix 11 (0.1 mm), nonadiyne (5 mm), copper sul-
fate (0.5 mm), sodium ascorbate (5 mm) and the tris[(1-hy-
droxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine (THPTA)
ligand (3.5 mm) in water for 24 h at 78C. The THPTA
ligand[15] was used to stabilize CuI in aqueous buffer and
avoid RNA degradation. These conditions provided the ex-
pected triazole analogue 12 in 70% yield, as detected by gel
electrophoresis (Figure 2A) and mass spectrometry (calcu-
lated and observed masses of 7771.8 and 7772.1, respective-
ly). The same approach was used to connect alkyne UDP-
MurNAc-pentapeptides 9a and 9b (100 mm) to azido RNA
helix 11 (50 mm; Scheme 1). The resulting bi-substrates 13a
Interaction of 13a with FemXWv was analyzed by size-ex-
clusion chromatography (Figure 3A). In the presence of a
threefold molar excess of FemXWv, 13a was quantitatively
eluted as a complex. Highly purified bi-substrate 13a was
recovered from the corresponding fractions following
phenol extraction to remove the protein (Figure 3B) and an-
alyzed by mass spectrometry (calculated and observed
masses of 8828.6 and 8828.4, respectively).
The RNA helices were tested as inhibitors of FemXWv by
using a coupled assay[16] that relies on aminoacylation of
tRNAAla by aminoacyl-tRNA synthetase AlaRS and transfer
of Ala from the resulting Ala-tRNAAla to UDP-MurNAc-
pentapeptide by FemXWv (Figure 2B). RNA helix 10 inhibit-
ed FemXWv with an IC50 of (89Æ4) mm.[9] Low affinity is ac-
counted for by the presence of vicinal hydroxyl groups in
the terminal adenosine, which are also present in the
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M. Arthur, M. Ethꢁve-Quelquejeu et al.
IC50 of 89Æ9 pm, whereas dia-
stereoisomer 13b inhibited
FemXWv with an IC50 of 2.3Æ
0.2 nm. The l and d configura-
tions of Cys were assigned to
diastereoisomers 13a and 13b,
respectively, based on the
higher affinity of FemXWv for
13a and the presence of l-Lys
at the analogous position of
the natural substrate. Together,
these results indicate that the
RNA
helix
and
UDP-
MurNAc-peptide moieties of
bi-substrate 13a both contrib-
uted to high-affinity binding to
FemXWv
.
Conclusion
We have developed a conver-
gent method to synthesize pep-
tidyl–RNA conjugates based
on Huisgen–Sharpless cycload-
dition for the final ligation
step. The reaction conditions
reported here are fully compat-
ible with two types of biomole-
cules, RNA and the peptido-
glycan precursor. We also
show that introduction of de-
hydroalanine as electrophilic
site by using the MSH reagent
is compatible with the peptido-
glycan precursor containing
Figure 2. Inhibition of FemXWv. Structure of tRNA analogues and analysis by denaturing PAGE (A). IC50 of
the tRNA analogues (B).
nucleotide
(UDP),
sugar
(MurNAc) and peptide (l-Ala-
g-d-Glu-l-Cys-d-Ala-d-Ala)
moieties. Finally, association of
both partners of the enzymatic
acyl-transfer reaction in pep-
tidyl-RNA 13a induces an
18000-fold increase in FemXWv
inhibition efficiency and paves
the way for the design of anti-
bacterial agents acting on this
new target.
Figure 3. Isolation of the complex containing 13a and FemXWv. Size-exclusion chromatography of the complex
(A) and denaturing PAGE analysis of purified 13a (B).
tRNAAla product of the FemXWv-catalyzed reaction.[9] In
agreement, substitution of 2’-hydroxyl by an azido group
(helix 11) led to a 56-fold decrease in the IC50 ((1.6Æ
0.3) mm). Addition of nonadiyne to 11 by the Cu-AAC reac-
tion (compound 12) led to a further eightfold decrease in
the IC50 ((0.20Æ0.02) mm). Thus, the nonadiyne carbon
chain may mimic the l-Lys side chain of UDP-MurNAc-
pentapeptide. Bi-substrate 13a inhibited FemXWv with an
Experimental Section
General reagents and materials: Solvents were dried by standard meth-
ods and distilled before use. Unless otherwise specified, materials were
purchased from commercial suppliers and used without further purifica-
tion. TLC: precoated silica gel thin layer sheets 60 F254 (Merck). Flash
chromatography: silica gel, 180–240 mesh (Merck). Optical rotations
were measured on a PerkinElmer 341 digital polarimeter. Spectra were
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Efficient Access to Peptidyl–RNA Conjugates
FULL PAPER
recorded on Bruker AVANCE 400 spectrometer for 1H (400 MHz), 13C
(101 MHz) and 31P (122 or 162 MHz) in CDCl3, D2O or DMSO. Chemi-
cals shifts are expressed in parts per million (ppm) relative to residual
(Cq), 96.8 (C5Cyt), 87.8 (C1’Ad), 87.3 (C1’Cyt), 77.5 (C3’Cyt), 71.9 (C3’Ad),
62.6 (C2’Ad), 61.5 (C5’Cyt), 61.3 (C5’Ad), 39.7 (C2’Cyt), 25.8 (tBuTBS), 25.0
(CH3Ac), 18.5 (CH2CN), 18.1 (CqTBS), À4.5, À4.8 ppm (2 ꢃ MeTBS);
31P NMR (1H-decoupled, 122 MHz, DMSO): d=À2.60, À2.66 (2s); ESI+
MS: m/z calcd for C37H47N12O11PNaSi+ [M+Na]+: 917.2886; found:
917.3253.
1
CHCl3 (d=7.26) or HDO (d=4.79) for H and CDCl3 (d=77.16) for 13C
as internal references. Signals were assigned on the basis of DEPT 135,
COSY, HSQC and HMBC. High-resolution mass spectra were carried re-
corded on a Bruker micrOTOF spectrometer. Unless otherwise specified,
HPLC was performed on a HPLC system with C18 reverse-phase col-
umns (analytical column, 250ꢃ4.6 mm, HYPERSIL-100 C18; semipre-
parative column, 250ꢃ21.2 mm, HYPERSIL HS C18; Thermoelectron
Corporation). The solvent system consisted of 50 mm aqueous
NH4OAc:CH3CN with a linear gradient (100:0 to 67:33) applied from 5
to 50 min at a flow rate of 1 (analytical) or 10 mLminÀ1 (semiprepara-
tive) and UV detection at 260 nm. For separation of UDP-MurNAc-pep-
tides, fast protein liquid chromatography (FPLC) was performed on an
ꢄkta purifier system (Amersham Pharmacia Biotech) with an analytical
C18 reverse-phase column (Nucleosil 100-3 C18, 250ꢃ4.6 mm, Macherey-
Nagel). The solvent system consisted of 50 mm aqueous NH4OAc/CH3CN
with a linear gradient (100:0 to 95:5) applied from 7 to 37 min at a flow
rate of 1 mLminÀ1 and UV detection at 260 nm. Analytical denaturing
polyacrylamide gel electrophoresis (PAGE) was performed in gels (20ꢃ
20ꢃ0.1 cm) containing acrylamide (13%, w/v), bis-acrylamide (4.4%, w/
v), urea (8m), tetramethylethylenediamine (TEMED, 0.04% v/v), ammo-
nium persulfate (0.08% w/v) and 1ꢃ TBE buffer (pH 8.2) [Tris (89 mm),
borate (89 mm), EDTA (2 mm)]. Gels were loaded (lane width 6 mm)
with RNA helices (100 pmol) in a volume of 10 mL containing bromophe-
nol blue (0.01% w/v) and glycerol (25% v/v). Electrophoresis was per-
formed for 120 min at 600 V, and gels stained with ethidium bromide
(0.5 mgmLÀ1) were imaged with a Herolab E.A.S.Y 429K camera. Prepa-
rative denaturing PAGE was performed under the same conditions,
except that 1 nmol of RNA helices was loaded per lane and the concen-
tration of ethidium bromide used for staining was reduced to
0.25 mgmLÀ1. RNA bands were cut off from gels with a sterile scalpel
and electroeluted for 2 h at 100 V in dialysis bags (cut-off 1 kDa; Spec-
tra/Por 7 Dialysis Membrane, Spectrum Labs). RNA helices were con-
centrated to a final volume of 250 mL by ultrafiltration (Microcon, cut-off
3 kDa, Millipore), purified by gel filtration (Sephadex G-75 column 10/
300 GL, GE Healthcare) equilibrated in Tris-HCl buffer (25 mm, pH 7.5)
containing NaCl (100 mm) and MgCl2 (5 mm).
ACHUTNGRENUN[G (2R,3S,5R)-5-[4-Amino-2-oxopyrimidin-1ACHTUNGTERN(NUGN 2H)-yl]-3-({[(2R,3S,4R,5R)-5-
(6-amino-9H-purin-9-yl)-4-azido-3-(tert-butyldimethylsilyloxy)tetrahydro-
furan-2-yl]methoxy}oxidophosphoryloxy)tetrahydrofuran-2-yl]methyl hy-
drogenphosphate
(3):
Bis(2-cyanoethyl)diisopropylphosphoramidite
(88 mg, 320 mmol, 2.5 equiv) was added to phosphotriester 2 (115 mg,
130 mmol). Ultradry CH2Cl2 (400 mL) was added, followed by tetrazole in
CH3CN (2.8 mL, 1.3 mmol). The mixture was stirred at room tempera-
ture for 1 h, and I2 in THF:H2O:pyridine, (75:2:20, 3.2 mL, 320 mmol)
was added. After being stirred at room temperature for 30 min, the mix-
ture was diluted with EtOAc (5 mL) and washed successively with satu-
rated aqueous Na2S2O3 solution (5 mL) and brine (5 mL). The organic
layer was dried over anhydrous MgSO4 and concentrated to dryness. The
crude intermediate was dissolved in 15 mL of aqueous 5m MeNH2. The
reaction was stirred for 12 h at room temperature and concentrated
under reduced pressure. The ammonium salt of dinucleotide 3 was puri-
fied by HPLC, lyophilized and recovered as a white solid (35 mg, 33%).
Rf =0.2 (CH2Cl2:MeOH 94:6); 1H NMR (400 MHz, D2O): d=8.47 (s,
1H, H2Ad or H8Ad), 8.30 (s, 1H, H2Ad or H8Ad), 7.86 (d, J=7.7 Hz, 2H,
H6Cyt), 6.19 (d, J=4.8 Hz, 1H, H5Cyt), 6.13–6.06 (m, 2H, H1’Ad, H1’Cyt),
4.97 (t, J=5.5 Hz, 1H, H3’Ad), 4.89–4.84 (m, 1H, H3’Cyt), 4.34–4.28 (m,
2H, H4’Ad, H4’Cyt), 4.24–4.10 (m, 2H, H5’Ad), 4.03 (s, 2H, H5’Cyt), 2.42
(ddd, J=7.9, 5.4, 2.7 Hz, 1H, H2’Cyt), 1.93–1.85 (m, 1H, H2’Cyt), 1.00 (s,
9H, tBuTBS), 0.28 (s, 3H, MeTBS), 0.27 ppm (s, 3H, MeTBS); HPLC reten-
tion time: 24.7 min (MeCN:NH4OAc 0:100 to 50:50); ESIÀ MS: m/z
calcd for C25H37N11O12P2SiÀ [MÀH]À: 774.1951; found: 774.1959.
ACHUTNGRENUN[G (2R,3S,5R)-5-[4-Amino-2-oxopyrimidin-1ACHTUNGTERN(NUGN 2H)-yl]-3-({[(2R,3S,4R,5R)-5-
(6-amino-9H-purin-9-yl)-4-azido-3-hydroxytetrahydrofuran-2-yl]methox-
y}oxidophosphoryloxy)tetrahydrofuran-2-yl]methyl hydrogenphosphate
(4): Partially protected dinucleotide 3 (34 mg, 44 mmol) was treated with
aqueous 6m HCl:THF:MeOH (1:2:1, 2.8 mL) at room temperature for
24 h. The reaction mixture was concentrated in vacuo, diluted with water
(3.5 mL) and washed with CH2Cl2 (3.5 mL). The aqueous layer was
evaporated under reduced pressure, and the ammonium salt of dinucleo-
tide 4 was purified by HPLC, lyophilized and recovered as a white solid
(13.9 mg, 44%). 1H NMR (400 MHz, D2O): d=8.49 (s, 1H, H2Ad or
H8Ad), 8.25 (s, 1H, H2Ad or H8Ad), 7.81 (d, J=7.7 Hz, 1H, H6Ad), 6.13–
6.10 (m, 2H, H5Cyt, H1’Ad), 6.08 (d, J=8.0 Hz, 1H, H1’Cyt), 4.87–4.84 (m,
2H, H2’Ad, H3’Cyt), 4.35 (br, 1H, H3’Ad), 4.29 (br, 2H, H4’Cyt, H4’Ad),
4.22–4.12 (m, 2H, H5’Ad), 4.02 (dd, J=4.6, 2.6 Hz, 2H, H5’Cyt), 2.40 (ddd,
J=7.7, 5.7, 1.9 Hz, 1H, H2’aCyt), 1.92 À1.85 ppm (m, 1H, H2’bCyt);
13C NMR (101 MHz, D2O): d=155.0 (C2Ad or C8Ad), 152.5 (C2Ad or
C8Ad), 141.8 (C6Cyt), 139.7(Cq), 96.0 (C1’Cyt), 85.7 (C1’Ad), 85.5 (C5Cyt),
84.7 (C4’Cyt, C4’Ad), 83.5 (C3’Ad), 70.4 (C2’Ad), 64.9 (C3’Cyt), 64.5 (C5’Cyt),
64.2 (C5’Ad), 38.1 ppm (C2’Cyt); 31P NMR (1H-decoupled, 162 MHz, D2O):
d=0.19, À1.12 (2s); HPLC retention time: 19.2 min (MeCN:NH4OAc
ACHTUNGTRENNUNG(2R,3S,5R)-5-[4-Acetamido-2-oxopyrimidin-1(2H)-yl]-2-(hydroxyme-
thyl)tetrahydrofuran-3-yl[(2R,3S,4R,5R)-4-azido-5-(6-benzamido-9H-
purin-9-yl)-3-(tert-butyldimethylsilyloxy)tetrahydrofuran-2-yl]methyl 2-
cyanoethyl phosphate (2):
A 0.45m solution of tetrazole in MeCN
(2.9 mL, 1.29 mmol) and Ac-dC-PCNE (250 mg, 0.324 mmol) were added
to a solution of 1[9,10] (66 mg, 0.130 mmol) in CH2Cl2 (700 mL) at room
temperature under argon atmosphere. After stirring for 1 h at room tem-
perature, 0.1m iodine in THF:H2O:pyridine (75:2:20, 3.3 mL) was added.
After 30 min, the reaction mixture was diluted with EtOAc (10 mL),
washed with water (10 mL), aqueous saturated Na2S2O3 solution (10 mL)
and brine (10 mL), dried over anhydrous MgSO4, filtered and concentrat-
ed at reduced pressure. The residue was then stirred with 0.18m trichloro-
acetic acid solution in dichloromethane (7.2 mL) at room temperature
for 30 min. The reaction mixture was diluted with CH2Cl2 (5 mL) and the
organic phase was washed with water (5 mL), aqueous saturated
NaHCO3 solution at 08C (5 mL) and brine (5 mL), dried over anhydrous
MgSO4, filtered and concentrated. The crude product was purified by
preparative TLC with CH2Cl2:MeOH (90:10) as eluent to give 2 (two di-
astereoisomers, ratio 1:1, 95 mg, 82%). 1H NMR (400 MHz, CDCl3): d=
9.44 (br, 1H, NHAc), 8.75 (s, 1H, H2Ad or H8Ad), 8.31 (s, 1H, H2Ad or
H8Ad), 8.16 (dd, J=7.5, 1.9 Hz, 1H, H6Cyt), 8.02 (d, J=1.3 Hz, 2H, Bz),
7.58 (ddd, J=7.2, 5.1, 1.6 Hz, 1H, Bz), 7.49 (ddd, J=8.3, 6.9, 2.5 Hz, 2H,
Bz), 7.31 (dd, J=7.5, 2.8 Hz, 1H, H5Cyt), 6.15–6.05 (m, 2H, H1’Ad, H1’Cyt),
5.07 (ddd, J=8.6, 5.9, 2.6 Hz, 1H, H3’Cyt), 4.97–4.93 (m, 1H, H3’Ad), 4.80–
4.75 (m, 1H, H2’Ad), 4.27–4.15 (m, 5H, OCH2, H4’Ad, H5’Cyt), 3.77 (dd,
J=8.7, 2.7 Hz, 2H, H5’Ad), 3.47 (s, 3H, MeAc), 2.76–2.71 (m, 2H,
CH2CN), 2.68–2.57 (m, 1H, H2’aCyt), 2.36–2.29 (m, 1H, H2’bCyt), 0.97 (s,
9H, tBuTBS), 0.21 (s, 3H, MeTBS), 0.20 ppm (s, 3H, MeTBS); 13C NMR
(101 MHz, CDCl3): d=170.7 (C=OBz), 162.6 (C=OBz), 155.5 (C2Ad or
C8Ad), 145.3 (C6Cyt), 142.5, (C2Ad or C8Ad), 133.0, 128.9, 128.3 (Bz), 116.5
0:100 to 25:75 from
7
to 37 min); ESIÀ MS: m/z calcd for
C19H24N11O12P2À [MÀH]À: 660.1087; found: 660.1116.
UDP-MurNAc-l-Ala-g-d-Glu-meso-cystine (5): The reaction mixture
(100 mL) contained Tris-HCl (100 mm, pH 8.6), MgCl2 (40 mm), potassi-
um phosphate (20 mm), ATP (5 mm), UDP-MurNAc-l-Ala-d-Glu
(0.1 mm),[11] meso-cystine (0.5 mm) and His-tagged MurE from E. coli
(8.7 mg).[17] After 18 h at 378C under gentle stirring, the magnesium
phosphate precipitate was removed by centrifugation and the supernatant
was lyophilized. Product 5 was purified by gel filtration on a Sephadex
G-25 column (115ꢃ2 cm) in water.[18] Yield, 8.4 mmol (80%). The reac-
tion product was used without further purification, although analytical
HPLC revealed the presence of UDP-MurNAc-l-Ala-d-Glu (ca. 10%)
in addition to 5.
UDP-MurNAc-l-Ala-g-d-Glu-meso-cystine-d-Ala-d-Ala (6): The reac-
tion mixture (8 mL) contained Tris-HCl (100 mm, pH 8.6), MgCl2
(30 mm), ATP (5 mm), UDP-MurNAc-l-Ala-g-d-Glu-meso-cystine
5
(1 mm), d-Ala-d-Ala (3 mm) and His-tagged MurF from E. coli
Chem. Eur. J. 2012, 00, 0 – 0
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M. Arthur, M. Ethꢁve-Quelquejeu et al.
(90 mg).[19] After 3 h of incubation at 378C, the reaction mixture was
lyophilized and product 6 was purified on a Sephadex G-25 column
(115ꢃ2 cm) in water. Yield, 7 mmol (90%). ESI MS: m/z calcd for
rically (e260 nm =2.26 105 mÀ1 cmÀ1; 1.4 nmol, 70%). ESI MS: m/z calcd for
C236H296N93O164P23 [M]: 7771.8; found: 7772.1.
RNA-helix-UDP-MurNAc-pentapeptide bi-substrate (13a and 13b): 2’-
Azido-RNA-helix 11 was coupled to 9a or 9b by Cu-AAC reaction. The
mixture (200 mL in phosphate buffer, 25 mm, pH 8.0), which contained 2’-
azido-RNA-helix 11 (5 nmol, 25 mm), 9a or 9b (20 nmol, 50 mm), CuSO4
(0.5 mm), Na ascorbate (5 mm) and THPTA (3.5 mm), was incubated at
48C for 24 h. Compounds 13a and 13b were purified by denaturing
PAGE and gel filtration as described above for compound 12 (e=2.36ꢃ
105 mÀ1 cmÀ1 at 260 nm, 200 pmol; 4%). The procedure was optimized by
increasing the concentration of 9a (20 mm, 200 nmol) and 11 (1 mm,
10 nmol) in the ligation step and by purifying 13a by anion-exchange
chromatography (DNAPac PA-100, Dionex) with a solvent system con-
sisting of a 30 min linear gradient from 25 to 2500 mm of NH4OAc
(pH 8.0) containing 0.5% CH3CN at a flow rate of 1 mLminÀ1. Com-
pound 13a was identified by denaturing PAGE and its concentration was
determined spectrophotometrically (e260 nm =2.36ꢃ105 mÀ1 cmÀ1; 3.6 nmol,
36%). ESI MS: m/z calcd for C268H346N101O190P25S [M]: 8828.6; found:
8828.4.
À
C40H62N9O28P2S2 1242.2626 [MÀH]À; found 1242.1827. The reaction
product was used without further purification although analytical HPLC
revealed the presence of UDP-MurNAc-l-Ala-d-Glu (ca. 10%) in addi-
tion to 6.
UDP-MurNAc-l-Ala-g-d-Glu-l-Cys-d-Ala-d-Ala (7): Dithiothreitol
(15.43 mg, 100 mmol) was added to 6 (3.3 mm, 3 mmol) in Tris-HCl buffer
(50 mm, pH 8.0). After 2 h of incubation at room temperature, the prod-
uct was purified by size-exclusion chromatography on a Sephadex G-25
column (30ꢃ1 cm) in water, lyophilized, dissolved in water and UV-
quantified (e260 nm =10000mÀ1 cmÀ1, 2.7 mmol, 90% yield). Purity was as-
sessed by analytical FPLC on a C18 column and product 7 was used with-
out further purification. Retention time: 22.0 min; ESI MS: m/z calcd for
C37H57N8O26P2SÀ [MÀH]À: 1123.2585; found 1123.1970.
UDP-MurNAc-l-Ala-g-d-Glu-dehydroalanine-d-Ala-d-Ala (8): O-Mesi-
tylenesulfonylhydroxylamine (MSH)[12] in DMF (7 mm, 7.04 mmol) was
added to a solution of 7 (1.4 mm, 704 nmol) in phosphate buffer (50 mm,
pH 8.0) at room temperature. Total conversion of Cys to dehydroalanine
was typically obtained after 2 h of incubation at room temperature, as as-
sessed by determining the sulfhydryl concentration with Ellmanꢅs re-
agent. Product 8 was purified by size-exclusion chromatography on a Se-
phadex G-25 column (30ꢃ1 cm) in water followed by FPLC on a C18
column. Product 8 was lyophilized, dissolved in water and UV-quantified
(e260 nm =10000mÀ1 cmÀ1, 200 nmol, 28% yield). Retention time: 20.2 min;
Analysis of the interaction of 13a with FemXWv by size-exclusion chro-
matography: Bi-substrate 13a (200 pmol) was incubated with FemXWv
(1200 pmol) for 10 min at 48C in 500 mL of buffer A (25 mm Tris-HCl,
pH 7.5; 100 mm NaCl; 5 mm MgCl2). The FemXWv-13a complex was
loaded onto a Sephadex G-75 column (10/300 GL, GE Healthcare) equi-
librated with buffer A and elution was performed at a flow rate of
0.8 mLminÀ1 at room temperature. Relevant fractions (5 mL) were
pooled, concentrated to
a final volume of 500 mL by ultrafiltration
À
ESI MS: m/z calcd for C37H55N8O26P2 [MÀH]À: 1089.2708; found:
(3 kDa, Microcon, Millipore) and incubated for 10 min at 658C followed
by 5 min at 08C. Compound 13a was extracted twice with phenol
(500 mL; phenol, water-saturated, stabilized, pH 4.1; Eurobio) and once
with a phenol:chloroform solution (5:1 v:v, 500 mL, Sigma). The product
was purified on a Sephadex 75 10/300 GL column in buffer A. Com-
pound 13a was concentrated to a final volume of 250 mL by ultrafiltra-
tion. The overall yield of the size-exclusion chromatography purification
1089.2710.
UDP-MurNAc-l-Ala-g-d-Glu-l-Cys(S-but-3-ynyl)-d-Ala-d-Ala (9a) and
UDP-MurNAc-l-Ala-g-d-Glu-d-Cys(S-but-3-ynyl)-d-Ala-d-Ala
(9b):
NaOH (1m, 10 mmol) was added at room temperature to (S)-but-3-ynyl
ethanethioate in DMF (1m, 10 mmol). After 10 min phosphate buffer
(80 mL, 250 mm, pH 8.0) and compound 8 in water (1 mm, 100 nmol) were
sequentially added and the mixture was incubated for 2 h at 378C. The
mixture of 9a and 9b was purified by size-exclusion chromatography on
a Sephadex G-25 column (30ꢃ1 cm), lyophilized and dissolved in water.
Diastereoisomers 9a and 9b were purified by FPLC on a C18 column,
lyophilized and dissolved in RNAse-free water (Sigma) (e260 nm =10
000mÀ1 cmÀ1; 9a: 25 nmol, 25%; 9b:25 nmol, 25%). Retention times:
36.5 and 33.9 min for 9a and 9b, respectively; ESI MS: m/z calcd for
C41H61N8O26P2SÀ 1175.2898 [MÀH]À; found: 1175.2820 and 1175.2893 for
9a and 9b, respectively.
step was 43% (85 pmol).
[16]
IC50 determination: W. viridescens FemXWv
,
E. faecalis alanyl-tRNA
synthetase (AlaRS)[7a] and T7 RNA polymerase[6c] were purified as previ-
ously described. Full-length tRNAAla (5’-GGGGCCUUAGCUCAG-
CUGGGAGAGCGCCUGCUUUGCACGCAGGAGGUCAGCGGU-
UCGAUCCCGCUAGGCUCCACCA-3’) was obtained by in vitro tran-
scription using T7 RNA polymerase.[6c] Inhibition of FemXWv was tested
in a radioactive coupled assay[8a] involving acylation of tRNAAla by
[14C]Ala and transfer of [14C]Ala from the resulting [14C]Ala-tRNAAla to
UDP-MurNAc-l-Ala-g-d-Glu-l-Lys-d-Ala-d-Ala (UDP-MurNAc-penta-
2’-Azido-RNA helix (11): 2’-Azido-dinucleotide 4 was ligated to RNA-
helix 10 (5’-GGGGCCUUAGCUCAGGCUCCAC-3’) mimicking the ac-
ceptor arm of the tRNAAla[6c] with purified T4 RNA ligase.[6c] RNA-helix
10 was synthesized by the phosphoramidite method and purified by poly-
acrylamide gel electrophoresis (Eurogentec). The ligation reaction was
performed at 378C for 120 min in 500 mL of HEPES buffer (50 mm,
peptide) to form UDP-MurNAc-l-Ala-g-d-Glu-l-Lys(l-ACTHNUTRGNEUNG
[14C]Ala)-d-Ala-
d-Ala ([14C]UDP-MurNAc-hexapeptide). The standard assay mixture
(10 mL) contained Tris-HCl (50 mm, pH 7.5), alanyl-tRNA synthetase
(800 nm), ATP (7.5 mm), b-mercaptoethanol (2 mm), MgCl2 (12.5 mm), l-
[14C]Ala (50 mm, 3700 BqnmolÀ1, ICN, Orsay, France), tRNAAla (0.4 mm),
UDP-MurNAc-pentapeptide (50 mm), FemXWv (5 nm) and various con-
centrations of compounds 10, 11 and 12. Reactions were performed at
308C for 10 min and stopped at 958C for 10 min. Inhibition of FemXWv
by 13a and 13b was tested under the same conditions, except for the
volume of the mixture (100 mL and 10 mL, respectively), FemXWv concen-
tration (0.05 mm and 0.5 mm, respectively) and incubation time (100 min
for both). The reaction mixture was lyophilized and dissolved in water
(10 mL). l-[14C]Ala and [14C]UDP-MurNAc-hexapeptide were separated
by descending paper chromatography (Whatman 4MM, Elancourt,
France) with isobutyric acid:1m ammonium hydroxide (5:3). Radioactive
spots were identified by autoradiography, cut out and counted by liquid
scintillation. IC50 were determined by plotting residual activity versus in-
hibitor concentration. Curves represent fits of a four-parameter logistic
function of experimental values (Sigma Plot 9.0).
pH 7.5) containing RNA helix 10 (40 nmol), 2’-azido-dinucleotide
4
(400 nmol), T4 RNA ligase (0.6 mg), DMSO (10%), ATP (1 mm) and
MgCl2 (15 mm). The product of the ligation reaction, 11, was purified by
anion-exchange chromatography (DNAPac PA100, 250ꢃ4 mm; Dionex)
with a linear ammonium acetate gradient (25 to 2500 mm, pH 8.0) con-
taining 0.5% acetonitrile, which was applied for 30 min at a flow rate of
[6b]
1 mLminÀ1
.
Fractions containing the ligation product were identified
by PAGE, lyophilized, dissolved in RNAse-free water and stored at
À208C (e260 nm =2.26 105 mÀ1 cmÀ1; 15.4 nmol, 46%); ESI MS: m/z calcd
for C227H284N93O164P23 [M]: 7651.6; found: 7651.5.
Nonadiyne derivative of 2’-azido-RNA-helix (12): 2’-Azido-RNA-helix 11
was coupled to 1,8-nonadiyne by CuI-catalyzed azido–alkyne cycloaddi-
tion (Cu-AAC) reaction. The mixture (50 mL in water) contained 2’-
azido-RNA-helix 11 (2 nmol, 100 mm), 1,8-nonadiyne (5 mm), CuSO4
(0.5 mm), Na ascorbate (5 mm) and the CuI-stabilizing ligand THPTA
(3.5 mm).[15a] After 24 h of incubation at 48C, Cu–THPTA complex and
Na ascorbate were removed by gel filtration on a Sephadex G-25 column
(30ꢃ1 cm) in water. Product 12 was purified by denaturing PAGE fol-
lowed by gel filtration. Concentration was determined spectrophotomet-
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ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
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Efficient Access to Peptidyl–RNA Conjugates
FULL PAPER
Bioorg. Med. Chem. Lett. 2007, 17, 4654; e) J. L. Mainardi, R. Villet,
T. D. Bugg, C. Mayer, M. Arthur, FEMS Microbiol. Rev. 2008, 32,
386.
Acknowledgements
This work was supported by the European Community (EUR-INTA-
FAR, Project N8 LSHM-CT-2004-512138, 6th PCRD). D.M. was support-
ed by a research fellowship from the Rꢀgion Ile-de-France.
[9] M. Fonvielle, M. Chemama, M. Lecerf, R. Villet, P. Busca, A.
Bouhss, M. Etheve-Quelquejeu, M. Arthur, Angew. Chem. 2010,
122, 5241; Angew. Chem. Int. Ed. 2010, 49, 5115.
[10] M. Chemama, M. Fonvielle, M. Lecerf, D. Mellal, H. Fief, M.
Arthur, M. Etheve-Quelquejeu, Curr Protoc Nucleic Acid Chem.
2011, 4, 44.
[1] N. Venkatesan, B. H. Kim, Chem. Rev. 2006, 106, 3712.
[2] a) H. Moroder, J. Steger, D. Graber, K. Fauster, K. Trappl, V. Mar-
quez, N. Polacek, D. N. Wilson, R. Micura, Angew. Chem. 2009, 121,
4116; Angew. Chem. Int. Ed. 2009, 48, 4056; b) S. Terenzi, E. Biala,
N. Q. Nguyen-Trung, P. Strazewski, Angew. Chem. 2003, 115, 3015;
Angew. Chem. Int. Ed. 2003, 42, 2909.
[3] J. Steger, R. Micura, Bioorg. Med. Chem. 2011, 19, 5167.
[4] A. S. Geiermann, N. Polacek, R. Micura, J. Am. Chem. Soc. 2011,
133, 19068.
ˇ
[11] A. Babic, D. Patin, A. Boniface, M. Hervꢀ, D. Mengin-Lecreulx, S.
Pecar, S. Gobec, D. Blanot in 5th Joint Meeting on Medicinal Chemis-
ˇ
ˇ
try (Ed.: D. Kikelj), Medimond Srl, Bologna, Portoroz, Slovenia
2007, pp. 1.
[12] J. M. Chalker, S. B. Gunnoo, O. Boutureira, S. C. Gerstberger, M.
Fernandez-Gonzalez, G. J. L. Bernardes, L. Griffin, H. Hailu, C. J.
Schofield, B. G. Davis, Chem. Sci. 2011, 2, 1666.
[13] D. P. Galonic, W. A. van der Donk, D. Y. Gin, Chem. Eur. J. 2003, 9,
5997.
[5] H. von Dçhren, Nat. Chem. Biol. 2009, 5, 374.
[6] a) S. Biarrotte-Sorin, A. P. Maillard, J. Delettre, W. Sougakoff, M.
Arthur, C. Mayer, Structure 2004, 12, 257; b) M. Fonvielle, M.
Chemama, R. Villet, M. Lecerf, A. Bouhss, J. M. Valery, M. Etheve-
Quelquejeu, M. Arthur, Nucleic Acids Res. 2009, 37, 1589; c) R.
Villet, M. Fonvielle, P. Busca, M. Chemama, A. P. Maillard, J. E. Hu-
gonnet, L. Dubost, A. Marie, N. Josseaume, S. Mesnage, C. Mayer,
J. M. Valery, M. Etheve-Quelquejeu, M. Arthur, Nucleic Acids Res.
2007, 35, 6870.
[14] a) A. H. El-Sagheer, T. Brown, Chem. Soc. Rev. 2010, 39, 1388; b) E.
Paredes, S. R. Das, Chembiochem 2011, 12, 125.
[15] a) T. R. Chan, R. Hilgraf, K. B. Sharpless, V. V. Fokin, Org. Lett.
2004, 6, 2853; b) C. Besanceney-Webler, H. Jiang, T. Q. Zheng, L.
Feng, D. S. del Amo, W. Wang, L. M. Klivansky, F. L. Marlow, Y.
Liu, P. Wu, Angew. Chem. 2011, 123, 8201; Angew. Chem. Int. Ed.
2011, 50, 8051.
[16] A. P. Maillard, S. Biarrotte-Sorin, R. Villet, S. Mesnage, A. Bouhss,
W. Sougakoff, C. Mayer, M. Arthur, J. Bacteriol. 2005, 187, 3833.
[17] E. Gordon, B. Flouret, L. Chantalat, J. van Heijenoort, D. Mengin-
Lecreulx, O. Dideberg, J. Biol. Chem. 2001, 276, 10999.
[18] D. Mengin-Lecreulx, B. Flouret, J. van Heijenoort, J. Bact. 1982,
151, 1109.
[19] D. Dementin, A. Bouhss, G. Auger, C. Parquet, D. Mengin-Lecreulx,
O. Dideberg, J. van Heijenoort, D. Blanot, Eur. J. Biochem. 2001,
268, 5800.
[7] a) A. Bouhss, N. Josseaume, D. Allanic, M. Crouvoisier, L. Gut-
mann, J. L. Mainardi, D. Mengin-Lecreulx, J. van Heijenoort, M.
Arthur, J. Bacteriol. 2001, 183, 5122; b) S. S. Hegde, T. E. Shrader, J.
Biol. Chem. 2001, 276, 6998; c) R. Plapp, J. Stroming, J. Biol. Chem.
1970, 245, 3667.
[8] a) M. Chemama, M. Fonvielle, M. Arthur, J. M. Valery, M. Etheve-
Quelquejeu, Chem. Eur. J. 2009, 15, 1929; b) M. Chemama, M. Fon-
vielle, R. Villet, M. Arthur, J. M. Valery, M. Etheve-Quelquejeu, J.
Am. Chem. Soc. 2007, 129, 12642; c) E. Cressina, A. J. Lloyd, G. De
Pascale, B. J. Mok, S. Caddick, D. I. Roper, C. G. Dowson, T. D. H.
Bugg, Bioorg. Med. Chem. 2009, 17, 3443; d) E. Cressina, A. J.
Lloyd, G. De Pascale, D. I. Roper, C. G. Dowson, T. D. H. Bugg,
Received: June 6, 2012
Revised: October 11, 2012
Published online: && &&, 0000
Chem. Eur. J. 2012, 00, 0 – 0
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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These are not the final page numbers!

M. Arthur, M. Ethꢁve-Quelquejeu et al.
Peptidyl–RNA Conjugates
M. Fonvielle, D. Mellal, D. Patin,
M. Lecerf, D. Blanot, A. Bouhss,
M. Santarem, D. Mengin-Lecreulx,
M. Sollogoub, M. Arthur,*
M. Ethꢀve-Quelquejeu* ..... &&&& — &&&&
Efficient Access to Peptidyl-RNA
Conjugates for Picomolar Inhibition of
Non-ribosomal FemXWv Aminoacyl
Transferase
Conjugation of complex biomolecules:
Peptidyl–RNA conjugates have various
applications to study the ribosome and
enzymes participating in tRNA-
dependent pathways such as Fem
transferases in peptidoglycan synthesis.
Here, a convergent route for synthesis
of peptidyl–RNAs based on Huisgen–
Sharpless cycloaddition for the final
ligation step is developed (see figure).
Both moieties of the peptidyl–RNA
conjugate contribute to high-affinity
binding to FemX, providing a picomo-
lar inhibitor.
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ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
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These are not the final page numbers!