Extended targeting potential and improved synthesis of Microcin C analogs as antibacterials

Article abstract of DOI:10.1016/j.bmc.2011.07.052

Microcin C (McC) (1) is a potent antibacterial compound produced by some Escherichia coli strains. McC functions through a Trojan-Horse mechanism: it is actively taken up inside a sensitive cell through the function of the YejABEF-transporter and then pro

Full text of DOI:10.1016/j.bmc.2011.07.052

Bioorganic & Medicinal Chemistry 19 (2011) 5462–5467
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Extended targeting potential and improved synthesis of Microcin C analogs
as antibacterials
Gaston H.M. Vondenhoff a, Svetlana Dubiley c, Konstantin Severinov b,c, Eveline Lescrinier a, Jef Rozenski a,
Arthur Van Aerschot a,
⇑
a Rega Institute for Medical Research, Laboratory of Medicinal Chemistry, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
b Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
c Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Microcin C (McC) (1) is a potent antibacterial compound produced by some Escherichia coli strains. McC
functions through a Trojan-Horse mechanism: it is actively taken up inside a sensitive cell through the
function of the YejABEF-transporter and then processed by cellular aminopeptidases. Processed McC
(2) is a non-hydrolysable aspartyl-adenylate analog that inhibits aspartyl-tRNA synthetase (AspRS). A
new synthesis is described that allows for the production of a wide variety of McC analogs in acceptable
amounts. Using this synthesis a number of diverse compounds was synthesized with altered target spec-
ificity. Further characteristics of the YejABEF transporters were determined using these compounds.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 5 May 2011
Revised 20 July 2011
Accepted 23 July 2011
Available online 3 August 2011
Keywords:
Microcin C analogs
Antibiotics
Drug design
1. Introduction
inside the sensitive cell, McC is metabolized by peptide-deformyl-
ase (PDF) and next by several broad specificity aminopeptidases.8
Bacterial antibiotic resistance is a global problem and high
resistance profiles against antibiotics currently in use are ever
increasing. Hereto, different resistance mechanisms have been
developed by bacteria of which many are based on either enzy-
matic detoxification or excretion of the drug via an efflux process.1
To overcome these deficiencies in our armamentarium against
pathogens, new classes of antibiotics need to be developed.2–4
Since the discovery of antibiotics, nature has been the predominant
source of anti-microbial compounds or at least served as a source
of initial lead compounds. Even today, nature provides new antimi-
crobial compounds that can be used to tackle the shortcomings in
our antibiotics library. However, many valuable and intrinsically
interesting natural compounds often lack the necessary properties
such as bioavailability and drug uptake. Alternatively, some natu-
ral examples use a Trojan Horse strategy to smuggle active cargo
into the cell using either siderophore conjugates as iron carrying
species5 or peptidic sequences recognized by a specific reporter.6
We herein consider natural peptide-nucleotide inhibitor of enteric
bacteria Microcin C (McC, 1, Fig. 1) as a lead compound in the
development of a new class of antibiotics.
This causes the active compound (i.e., aspartyl-phosphoramidate-
adenosine, (2)) to be released. This product resembles Asp-AMP
(3), the activated aspartic-acid that normally would be esterified
to the 30-hydroxyl-group of tRNAAsp. The processed McC, however,
contains a more stable N–P bond which is not hydrolyzed inside the
active pocket of AspRS and thus functions as an inhibitor of this
enzyme.9
The production of McC is catalyzed by several enzymes encoded
in the same operon that codes for the McC heptapeptide.10 The
attachment of the heptapeptide chain to adenosine via the phos-
phoramidate linker can only be achieved if the C-terminal amino
acid is asparagine, which is indeed encoded by the last codon of
the mccA gene coding for the heptapetide. In view of the mecha-
nism of formation of the phosphoramidate bond, no other amino
acid at the seventh position of the McC-peptide would allow for
further maturation into a functional McC.11 Upon activation and
coupling of the peptide chain, the C-terminal asparagine is hereby
modified into aspartic acid.12 In analogy with the strong inhibitory
properties of 2 on AspRS, it is well known that aminoacyl-sulfa-
moyl-adenosine compounds can also target different aaRSs, but
these compounds suffer from poor bioavailability. Therefore, the
attachment of an uptake enhancer, such as the hexapeptide unit
of McC, could improve their potency and would allow targeting
virtually any aaRS.
McC functions through a Trojan-Horse mode of action, whereby
the peptide moiety is recognized by a YejABEF-peptide transporter
which transports the prodrug across the bacterial membrane.7 Once
In earlier work three McC-analogs were synthesized which
contained different amino acids at the seventh position of the
⇑
Corresponding author.
E-mail address: arthur.vanaerschot@rega.kuleuven.be (A. Van Aerschot).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.052

G. H. M. Vondenhoff et al. / Bioorg. Med. Chem. 19 (2011) 5462–5467
5463
for the synthesis of aaSAs 7a–g only protected at the amino acid
side chain.
NH2
N
N
N
The hexapeptide was assembled on a super-acid-sensitive resin,
allowing the use of moderately acid-sensitive protecting groups.
The fully protected, formylated hexapeptide (fM-R(Pbf)-T(tBu)-G-
N(Trt)-A) was then coupled to the partially protected aaSAs using
standard peptide-synthesis reagents such as DIC, HOBt, and DIPEA.
The obtained products 8a–g were subsequently deprotected yield-
ing the respective McC-analogs (9a–e).
Following the here described methodology, a new set of analogs
was created, in order to further extend the synthesis and evalua-
tion of McC-analogs carrying different amino acids at the seventh
position. These compounds carried either an aspartic acid (9a),
alanine (9b), leucine (9c), glycine (9d), lysine (9e), isoleucine (9f)
or valine (9g) as the target-determining amino acid. With these
analogs also an N-terminal formyl group was included as it was
found to improve the recognition by the YejABEF-transporter.
Identity and purity of the final heptapeptidic compounds 9a–g
was established by 1D and 2D NMR and MS/MS analysis. A combi-
nation of 2D HSQC–TOCSY and HMBC experiments allowed unam-
biguous assignment of all signals and confirmed the amino acid
sequence of the peptide chain, which was further corroborated
by MS/MS analysis.
O
P
O
N
fMRTGNAD
N-
O
O
HO
OH
1
+H3N
NH2
N
N
O
P
O
O
N
N
N-
O
+H3N
-O
O
HO
OH
O
2
+H3N
NH2
N
N
N
O
O
O
N
O
P
O
+H3N
-O
O-
HO
OH
2.2. Antibacterial activity of McC analogs
O
3
The growth inhibitory properties on McC-sensitive Escherichia
coli were determined for all new compounds by measuring the
optical density reached by identical cell cultures in wells of
microtiter plates in the presence of various concentrations of the
respective inhibitors.
Figure 1. Chemical structures related to the natural product: McC (1) in unpro-
cessed form; metabolized McC (2) in its active form as bioisoster of the natural
AspRS intermediate aspartyl-adenylate (3).
As shown earlier, the intracellular target is determined by the
C-terminal amino acid, which remains attached onto the sulfa-
moyl-adenosine following intracellular metabolization.13 To facili-
tate the activity evaluation and the mechanism of action studies of
newly synthesized compounds, an E.coli tester strain Ara-Yej
(BW39758) was used, where the genomic yejABEF operon is under
control of the arabinose-inducible araBAD promoter. In the pres-
ence of L-arabinose higher amounts of Yej-transporters will be
displayed at the inner-membrane. The wild-type E. coli K-12
BW28357 and the Ara-Yej strain were either grown in LB-medium
with or without 5 mM (L)-arabinose. The antibacterial activities of
the various McC-analogs (9a–g) were determined by monitoring
the optical density of suspensions of cell-cultures. In parallel,
E. coli with disrupted yej-, or pepA,B,N-genes, grown on M63
agar-plates were used to confirm the McC mode of action.
As can be seen in Figure 2, compounds 1 (reference), 9a, 9b, and
McC-analog (i.e., XD-SA, XE-SA, XL-SA, whereby X stands for the
N-terminal hexapeptide part of McC; D, E, and L for aspartic acid,
glutamic acid and leucine respectively; and SA stands for 50-O-sul-
famoyl-adenosine).13 These compounds lacked a N-terminal for-
myl group. All three proved to be active and work via the same
mechanism as natural McC, which is through removal of the
hexapeptide part following transporter mediated uptake and con-
comitant inhibition of the respective tRNA synthetase by the
metabolite. Following these results, we now report on the develop-
ment of a new synthetic approach to improve the synthetic yield of
the synthesis and to extend the range of different McC-like com-
pounds that could be synthesized. This improved procedure is used
to generate and characterize a new series of McC analogs targeting
various aaRSs and to further characterize the requirements for effi-
cient transport by the YejABEF transporter.
9e displayed activity at concentrations ꢁ0.63
lM when challenged
2. Results
with Ara-Yej cells cultured in arabinose containing medium. In
contrast to 9b, the more hydrophobic compounds 9c, 9d, 9f, and
9g showed reduced activity. E. coli cells with a disrupted yej gene
(with concomitant loss of the active transport modality) and cells
without functional aminopeptidases A, B and N (with loss of
formation of the active principle) proved resistant to these
compounds, indicating the same mode of action as McC.
2.1. Design and synthesis of McC analogs
aaSAs were created in analogy to previously published method-
ology13 with the modification that orthogonal protecting groups
were used for the side chains with respect to the alpha-amine of
the respective amino acids (see Scheme 1). Amino acylated SAs
3. Discussion
were prepared by condensation of either L-N-benzyloxycarbonyl-
aminoacyl(O-tert-butyl or tert-butylox ycarbonyl)]-O-succinimide
analogs with the adenosine derivative 4 using DBU in DMF. The
Z-group of the resulting products (5a and 5e) was next removed
by hydrogenation. The aaSAs containing an amino acid not requir-
ing protection of its side chain (5b–d and 5f–g) were prepared by
We previously suggested that deviations from the native
McC-structure could be quite extensive.13 This is further exempli-
fied here with McC analogs affording different intracellular active
compounds upon metabolization. Even if more extensive altera-
tions were incorporated, such as a lysine as the target determining
amino acid, activities were in the same range as observed for
native McC. However, the potency of the compounds 9c, 9d, 9f,
and 9g was lower in comparison to 9a, 9b, and 9e. The reduced
condensation of the respective L-N-tert-butyloxycarbonyl-amino-
acyl]-O-succinimide precursors with 4. The Boc-group was
removed by acidolysis and the TBDMS-protecting groups of all
compounds were removed using Et3Nꢀ3HF. This strategy allowed

5464
G. H. M. Vondenhoff et al. / Bioorg. Med. Chem. 19 (2011) 5462–5467
NH2
NH2
N
N
N
N
N
N
O
O
H2N
O
S O
N
O
i
O
O
N
O
ii or iii
H
N
S O
H
N
R2
TBDMSO
OTBDMS
TBDMSO
OTBDMS
R1
4
5a: R1= CH2COOtBu,
5b: R1= CH3,
R2= Z
R2= Boc
R2= Boc
R2= Boc
R2= Z
5c: R1= CH2CH(CH3)2,
5d: R1= H
5e R1=(CH2)4NHBoc,
5f: R1= CH(CH3)CH2CH3, R2= Boc
5g: R1= CH(CH3)2,
R2= Boc
NH2
N
NH2
N
N
N
N
N
iv
O
N
O
O
O
O
O
O
N
O
H
N
H
S O
TBDMSO
6a-g: R1= as in 5a-g
N
O
S O
H2N
H2N
OTBDMS
HO
OH
R1
R1
7a-g: R1= as in 5a-g
NH2
NH2
N
N
N
N
N
N
N
O
O
O
S
N
O
O
O
O
S
O
H
N
vi
H
O
v
H
N
H
N
R4
R3
N
HO
OH
R1
HO
OH
R1
8a-g: R3= f-M-R(Pbf)-T(tBu)-G-N(Trt)-A
9a-e: R4= f-M-R-T-G-N-A
9a: R1= CH2COOH
9b: R1= CH3,
9c: R1= CH2CH(CH3)2,
9d: R1= H
9e: R1= (CH2)4NH2,
9f: R1= CH(CH3)CH2CH3
9g: R1= CH(CH3)2,
Scheme 1. Reagents and conditions: General scheme affording the various McC analogs with R4 being the formylated hexapeptide. (i) N-a-CBZ-L-aminoacyl-(tBu or Boc)-
succinimide, DBU in DMF, 6 h, rt. (ii) For R2 = Z-group, H2, Pd/C in MeOH, 3 h, rt. (iii) For R2 = Boc-group of TFA/H2O (5:2), 4 h, 0 °C to rt. (iv) Et3Nꢀ3HF in THF, 16 h, rt. (v)
Protected peptide (1 equiv), HOBt (4 equiv), DIC(4 equiv) and DIEA(2 equiv) in DMF, 16 h, rt. R3 may be either Boc-, or formyl-group. (vi) TFA/thioanisole/H2O (90/2.5/7.5), 2 h,
rt.
activity for compounds with a more hydrophobic amino acid at the
C-terminal end is remarkable, as M Brown et al. found a Ki of
0.01 nM of I-SA for IleRS.14 Therefore the relatively low activity
of the compound fXI-SA (9f) must be attributed to either uptake
or metabolization issues. As shown in Figure 2 (graph 9f), the
strain with upregulated Yej-transporters is more sensitive to this
compound. Therefore it may be concluded that the Yej-transporter
discriminates between the constituencies of the peptide chains.
The polarity may be a decisive factor since compounds containing
amino acids at the seventh position with more aliphatic side chains
display lower activities in comparison to compounds containing
aspartic acid (9a) or lysine (9e), with the alanine derivative 9b as
a notable exception. However, compound 9c containing leucine
also showed a five-fold higher potency compared to 9f. Therefore
it may be concluded that not only the polarity, but also more
specific recognition by the transporter is of importance for uptake
of these compounds, as processing of the different analogs to the
respective active metabolites is straightforward. This was shown
before for the glycine analog 9d as well as for several shortened
compounds which all proved active in an in vitro assay, where in
presence of cellular extracts tRNA aminoacylation was inhibited.
Hence, metabolization is a prerequisite, but not an issue for the
activity of the different congeners.15 Neither is amino acid usage
per se a decisive factor in the activity profile, as many McC variants
obtained by mutagenesis proved active.11 The latter methodology
however, did not allow for analogs with a different amino acid at
the seventh position, targeting different tRNA synthetases.
The synthetic scheme as presented here is
a significant
improvement upon the earlier reported synthesis with up to
15-fold improved overall yields. The new methodology allows for
the preparation of sufficient amounts of different McC analogs,
allowing further studies on the biology of this fascinating class of
compounds and enabling the design of further improved analogs.
Following this method, new compounds could be created that
may not only inhibit other aaRSs, but even other targets. Further-
more, compounds that suffer from low bioavailability could be
improved following the proposed synthetic scheme in order to turn
these potential drugs in Trojan Horse-like compounds.
4. Materials and methods
4.1. Chemistry
General: Reagents and solvents were purchased from commer-
cial suppliers (Acros, Sigma–Aldrich, Bachem, Novabiochem) and
used as provided, unless indicated otherwise. DMF and THF were
analytical grade and were stored over 4 Å molecular sieves. For
reactions involving Fmoc-protected amino acids and peptides,
DMF for peptide synthesis (low amine content) was used. All other
solvents used for reactions were analytical grade and used as

G. H. M. Vondenhoff et al. / Bioorg. Med. Chem. 19 (2011) 5462–5467
5465
Figure 2. In vitro screening of McC analogs. Cell growth of the wild-type E. coli and of the Ara-Yej strain is shown in presence of different concentrations of 9a–g and the
reference compound 1 with or without arabinose induction.
0
provided. Reactions were carried out in oven-dried glassware un-
der a nitrogen atmosphere and stirred at room temperature, unless
indicated otherwise.
detector. Final products were purified using a PLRP-S 100 ÅA column
connected to a Merck-Hitachi L6200A Intelligent pump. Eluent
compositions are expressed as v/v.
Standard 1H and 13C NMR spectra of the compounds were re-
corded on a Bruker UltraShield Avance 300 MHz or 500 MHz spec-
trometer. Spectra were recorded in DMSO-d6 or D2O. The chemical
shifts are expressed as d values in parts per million (ppm), using
the residual solvent peaks (DMSO: 1H, 2.50 ppm; 13C, 39.60 ppm;
HOD: 1H, 4.79 ppm with dioxane added for 13C, 67.19 ppm) as a
reference. Coupling constants are given in Hertz (Hz). The peak
patterns are indicated by the following abbreviations: bs = broad
singlet, d = doublet, m = multiple, q = quadruplet, s = singlet and
t = triplet. High resolution mass spectra were recorded on a quad-
rupole time-of-flight mass spectrometer (Q-Tof-2, Micromass,
Manchester, UK) equipped with a standard ESI interface; samples
4.1.1. Synthesis of 50-O-[N-[
L-Aspartyl(O-tert-butyl)]-sulfamoyl]
adenosine (7a)
A solution of Z-Aspartyl(O-tert-butyl)-O-Su (1 g, 2.38 mmol,
1.0 equiv),
50-O-sulfamoyl-20,30-di-O-(tert-butyldimethylsilyl)-
adenosine (4) (1.23 g, 2.14 mmol, 0.9 equiv) and DBU (356 L,
l
2.38 mmol, 1.0 equiv) in DMF (7 mL) was stirred at rt for 8 h under
nitrogen atmosphere. DMF was evaporated under reduced
pressure. Next, the reaction mixture was purified by flash chroma-
tography (CH2Cl2, 1% Et3N, 2.5–10% MeOH). Fractions containing
the desired product were evaporated giving a yellow oil (5a). Yield:
1.7 g (90%). ESI-MS [M+H+]: C38H62N7O11SSi2 Calculated: 880.4
Found: 880.1
were infused in 2-propanol/H2O (1:1) at 3 l
L minꢂ1. High resolu-
tion NMR spectra of the final compounds 9a–g were recorded on
a Bruker Avance II 600 with a gradient cryoprobe. Methodology
and 1D and 2D NMR spectra for analysis of the different analogs
9a–g are included in the Supplementary data.
For TLC, precoated aluminium sheets were used (Merck, Silica
gel 60 F254). The spots were visualized by UV light. Column chro-
matography was performed on ICN silica gel 60A 60–200. For size
exclusion chromatography, a 2 ꢃ 30 cm column of Sephadex LH-20
was used as the solid phase and MeOH/H20 (7:3 v/v) as the eluent.
Preparative HPLC of peptides was done using a Waters Xbridge
preparative C18 (19 ꢃ 150 mm) column connected to a Waters
1525 binary HPLC pump and a Waters 2487 dual absorbance
The obtained product 5a (1.7 g) was dissolved in MeOH at 0 °C
and Pd/C (0.17 g) was added. The solution was stirred under hydro-
gen atmosphere for 3 h. The mixture was filtered and evaporated
yielding a white–yellow foam (6a). Yield: 1.1 g (76%). The product
6a (1.1 g) was carefully dried and dissolved in THF (15 mL) and
Et3Nꢀ3HF (1 mL). After 3 h, another 0.8 mL of Et3Nꢀ3HF was added
and the reaction mixture was stirred for another 22 h. The reaction
mixture was evaporated and the reaction mixture was purified by
flash chromatography (CH2Cl2, 5–20% MeOH). Fractions containing
the desired product were evaporated, yielding 7a: 670 mg (88%)1H
NMR (D2O): 1.04–1.09 (t, 9H, Et3N, J = 7.16), 1.44 (s, 9H, tBu),
2.67–2.75 (q, 6H,Et3N CH2, J = 7.12), 3.68–3.72 (t, 2H, Asp H,
a

5466
G. H. M. Vondenhoff et al. / Bioorg. Med. Chem. 19 (2011) 5462–5467
J = 5.70), 4.12–4.19 (m, 4H, 30, 40, 50), 4.63–4.67 (t, 1H, 20H, J = 5.36),
5.94–5.96 (d, 1H, 10, J = 5.82), 7.31 (s, 1H, NH2), 8.18 (s, 1H, 2H),
8.41 (s, 1H, 8H).
1H NMR (D2O): 1.26–1.44 (m, Lys-
c
e
and d), 1.38 (s, 9H, tBu),
), 3.75–3.79 (t, 2H, Lys-a,
1.77–1.84 (m, Lys-b), 2.87 (m, 2H, Lys-
J = 5.77), 4.41–4.46 (m, 4H, 30,40,50), 4.50–4.53 (t, 1H, 20, J = 4.26),
6.12–6.14 (d, 1H, J = 5.7), 8.26 (s, 1H, 2H), 8.43 (s, 1H, 8H)
13C NMR (D2O): 27.02 (tBu-CH3), 35.71 (Asp-b-C), 51.67 (Asp-
a-
C), 68.14 (50-C), 70.08 (30-C), 74.14 (20-C), 82.05 (40-C), 83.88
(tBu-C), 87.37 (10-C), 118.06 (5-C), 139.49 (8-C), 148.31(4-C),
152.19 (2-C), 154.74 (6-C), 170.50 and 173.81 (Asp- C@O).
ESI-MS calcd for C18H26N7O9S [MꢂH]ꢂ: 516.2; found: 515.8.
13C NMR (D2O): 21.89 (Lys-
c
), 28.26 (tBu-CH3), 29.12 (Lys-d),
31.14 (Lys-b), 40.15 (Lys-
e
), 56.03 (Lys-
a
), 68.97 (50-C), 70.76
(30-C), 74.73 (20-C), 82.87 (40-C), 87.69 (10-C), 119.57 (5-C), 140.39
(8-C), 149.68 (4-C), 153.57 (2-C), 156.23 (6-C), 158.77
(Boc –C–O), 176.31 (Lys –C@O).
4.1.2. Synthesis of 50-O-[N-[
A solution of Boc-Alanyl-O-Su (1 g, 3.5 mmol, 1.0 equiv), com-
pound 4 (1.8 g, 3.2 mmol, 0.9 equiv) and DBU (450 L, 3 mmol,
L
-alanyl]-sulfamoyl] adenosine (7b)
ESI-MS calcd for C21H35N8O9S [M+H]+: 575.22; found: 575.00.
l
4.1.6. Synthesis of 50-O-[N-[
L-isoleucyl]-sulfamoyl]adenosine
0.9 equiv) in DMF (7 mL) was stirred at rt for 8 h under nitrogen
atmosphere. DMF was evaporated under reduced pressure. Next,
the reaction mixture was purified by flash chromatography
(CH2Cl2, 1% Et3N, 2.5–10% MeOH). Fractions containing the desired
product were evaporated giving a yellow oil (5b). Yield: 2.09 g
(90%). Compound 5b was next treated with TFA/H2O (5/2 v/v) for
5 h at rt, after which the volatiles were evaporated to yield the
product 6b. Compound 6b was carefully dried and dissolved in
THF (15 mL) and Et3Nꢀ3HF (1 mL). After 3 h, another 0.8 mL of
Et3Nꢀ3HF was added and the reaction mixture was stirred further
for 22 h. The reaction mixture was evaporated and the residue
was purified by flash chromatography (CH2Cl2, 5–20% MeOH).
Fractions containing the desired product 7b were evaporated.
Yield: 710 mg (63%)
(7f)
This compound was synthesized analogously to compound 7b.
1H NMR (D2O): 0.84–0.89 (t, 3H, Ile-d, J = 7.38), 0.97–0.99 (d, 3H,
Ile-c0-H, J = 7.02), 1.1–1.6 (m, 2H, Ile-
cA/B-H), 1.9–2,1 (m, 1H, Ile-
b-H), 3.75–3.76(d, 1H, Ile-a
-H), 4.41–4.50 (m, 4H, 30,40,50),
4.54–4.57 (t, 1H, 20-H, J = 4.6), 6.12–6.14 (d, 1H, 10-H, J = 5.31),
8.24 (s, 1H, 2H), 8.41 (s, 1H, 8H).
13C NMR (D2O): 11.5 (, Ile-d-CH3), 15.1 (Ile-c0-CH3), 24.7 (Ile-
c-
CH2), 36.9 (Ile-b-CH), 60.7 (Ile-a
-CH), 68.9 (50-C), 70.79 (30-C),
74.69 (20-C), 82.94 (40-C), 87.93 (10-C), 119.13 (5-C), 140.43 (8-C),
149.47 (4-C), 153.05 (2-C), 155.79 (6-C), 175.61 (Ile- C@O).
HR-MS calcd for C16H24N7O9S1 [MꢂH]ꢂ: 458.1548; found:
458.1450.
1H NMR (D2O): 1.22–1.27 (t, 9H, Et3N-CH3, J = 7.34), 1.43–1.45
(d, 3H, Ala-b-H, J = 7.2), 2.61–2.68 (q, 6H, Et3N–CH2, J = 7.35),
4.1.7. Synthesis of 50-O-[N-[
L-valyl]-sulfamoyl] adenosine (7g)
This compound was synthesized analogously to compound 7b.
3.45–3.52 (q, 1H, Ala-a
-H, J = 7.19), 4.35–4.41 (m, 4H, 30, 40, 50,),
1H NMR (D2O):0.96–0.98 (0.84-0.89 (t, 3H, Ile-d, J = 7.38),
4.45–4.48 (t, 1H, 20-H, J = 4.7), 6.08–6.10 (d, 1H, 10-H, J = 6.0), 7.28
(bs, 2H, NH2, exchangable with D2O), 8.22 (s, 1H, 2H), 8.36 (s, 1H,
8H).
0.97–0.99 (d, 3H, Ile-c0-H, J = 7.02), 1.1–1.6 (m, 2H, Ile-
c
A/B-H),
1.9–2,1 (m, 1H, Ile-b-H), 3.75–3.76(d, 1H, Ile-
a-H), 4.41–4.50 (m,
4H, 30,40,50), 4.54–4.57 (t, 1H, 20-H, J = 4.6), 6.12–6.14 (d, 1H, 10-H,
J = 5.31), 8.24 (s, 1H, 2H), 8.41 (s, 1H, 8H).
13C NMR (D2O): 17.07 (Ala-b-C), 52.01 (Ala- -C), 68.89 (50-C),
a
70.75 (30-C), 74.71 (20-C), 82.88 (40-C), 88.03 (10-C), 119.17 (5-C),
140.36 (8-C), 149.45 (4-C), 153.11 (2-C), 156.00(6-C), 177.12
(Asp- C@O).
13C NMR (D2O): 16.94(Val-
c
B-CH3), 18.56 (Ile-
cA-CH3), 30.45
(Val-b-CH), 61.45 (Val-
a
-CH), 68.91 (50-C), 70.78 (30-C), 74.65
(20-C), 82.95 (40-C), 87.90 (10-C), 119.20 (5-C), 140.31 (8-C),
149.54 (4-C), 153.47 (2-C), 156.15 (6-C), 176.38 (Val- C@O).
ESI-MS calcd for C15H22N7O7S [MꢂH]ꢂ: 444.45; found: 444.30.
ESI-MS calcd for C13H18N7O7S [MꢂH]ꢂ: 416.4; found: 416.2.
4.1.3. Synthesis 50-O-[N-[
L-leucyl]-sulfamoyl] adenosine (7c)
This compound was synthesized analogously to compound 7b.
4.1.8. Synthesis of fMRTGNAD-SA (9a)
1H NMR (D2O): 0.87–0.89 (d, 3H, Leu-d, J = 6), 0.89–0.90 (d, 3H,
The peptide Formyl-methionyl-arginyl(2,2,4,6,7-pentamethyl
dihydrobenzofuran-5-sulfonyl)-threonyl(tBu)-glycyl-asparaginyl
(trityl)-alanyl-OH was synthesized on a 2-chlorotrityl chloride
resin using standard Fmoc-based solid phase peptide chemistry.
The protected peptide was cleaved from the resin using a mixture
of HOAc/Trifluoroethanol/DCM (1/1/8, v/v) in 30 min. Following
Leu-d, J = 6), 1.64–1.73 (m, 3H, Leu-c, b-H), 3.75–3.76(m, 1H,
Leu-a
-H), 4.42–4.45 (m, 3H, 30,,50), 4.50–4.52 (t. 1H, 40-H, J = 4.8)
4.74-4.76 (t, 1H, 20-H, J = 4.8), 6.15–6.16 (d, 1H, 10-H, J = 5.31),
8.42 (s, 1H, 2H), 8.51 (s, 1H, 8H).
13C NMR (D2O): 20.3 (Leu-dB-CH3), 21.45 (Leu-dA-CH3), 23.51
(Leu-
c
-CH), 39.46 (Leu-b-CH2), 53.25 (Leu-
a
-CH), 68.67 (50-C),
RP-HPLC purification, the peptide (20 mg, 16.13
and HOBt (9 mg, 64.52 mol, 4.0 equiv) were dissolved in DMF
(500 L) and DIC (10 L, 64.52 mol, 4.0 equiv) was added. This
mixture was stirred for 1 h at rt under argon atmosphere. DIPEA
(7.5 L, 40.33 mol, 2.5 equiv) was added and the mixture was
added to the adenosine analog 7a (16.68 mg, 32.26 mol,
lmol, 1.0 equiv)
69.49 (30-C), 73.76 (20-C), 81.88 (40-C), 88.02 (10-C), 118.29 (5-C),
142.06 (8-C), 144.17 (4-C), 147.91 (2-C), 149.50 (6-C), 174.14
(Leu- C@O).
l
l
l
l
ESI-MS calcd for C16H26N7O7S [M+H]+: 460.48; found: 460.16.
l
l
l
4.1.4. Synthesis of 50-O-[N-[
This compound was synthesized analogously to compound 7b.
L
-glycyl]-sulfamoyl] adenosine (7d)
2.0 equiv) and stirred for 16 h at rt under argon. Next, the volatiles
were evaporated and the residue was taken up in a mixture of
CH3CN/water. This was purified on a PoraPak RxnÒ column (CH3CN
25–100% in water). The fractions containing the product were
evaporated yielding 8a. The product 8a was subsequently depro-
tected using a mixture of 90% TFA, 7.5% H2O and 2.5% thioanisole.
The volatiles were evaporated and co-evaporated 3 times with tol-
uene (10 mL). The remaining title product (9a, fMRTGNAD-SA) was
redissolved in H2O, filtered and purified by RP-HPLC (solvent A:
25 mM TEAB in H2O; solvent B: 25 mM TEAB in CH3CN; see Supple-
mentary data for HPLC analysis of all final compounds).
1H NMR (D2O): 3.70 (s, 2H,
a
-H), 4.41–4.50 (m, 4H, 30,40,50),
4.69–4.73 (t, 1H, 20-H, J = 4.6), 6.12–6.14 (d, 1H, 10-H, J = 5.31),
8.15 (s, 1H, 2H), 8.34 (s, 1H, 8H).
13C NMR (D2O): 42.72 (Gly-
a
-C), 68.11 (50-C), 70.06 (30-C),
74.06 (20-C), 82.15 (40-C), 87.35 (10-C), 118.43 (5-C), 139.54 (8-C),
148.73 (4-C), 152.37 (2-C), 156.15 (6-C), 172.69 (Gly –C@O).
ESI-MS calcd for
402.31.
C
12H16N7O7S [MꢂH]ꢂ: 402.37; found:
4.1.5. Synthesis of 50-O-[N-[
L
-lysyl(N-tert-butyloxycarbonyl)]-
Yield (as calculated over coupling and deprotection): 1.398 mg
(13.9%). HR-MS calcd for C39H60N17O18S2 [MꢂH]ꢂ: 1118.3822;
found: 1118.3789.
sulfamoyl]adenosine (7e)
This compound was synthesized analogously to compound 7a.

G. H. M. Vondenhoff et al. / Bioorg. Med. Chem. 19 (2011) 5462–5467
5467
4.1.9. Synthesis of fMRTGNAA-SA (9b)
Acknowledgments
Further synthesis to obtain 9b (yield: 2.3 mg (11%)) was per-
formed analoguously to the synthesis of 9a. HR-MS calcd for
C
G.H.M.V. is recipient of a Belgian Agency for Innovation by Sci-
ence and Technology (IWT) fellowship (SB 81116). We are grateful
for practical assistance with the synthetic procedures by MSc stu-
dent Bart Blanchart during his lab rotation and we thank Chantal
Biernaux for very helpful assistance in editing the manuscript.
38H62N17O16S2 [M+H]+: 1076.4002; found: 1076.3849.
4.1.10. Synthesis of fMRTGNAL-SA (9c)
Further synthesis to obtain 9c (yield: 0.7 mg (7%)) was per-
formed analoguously to the synthesis of 9a. HR-MS calcd for
C
41H68N17O16S2 [M+H]+: 1118.4471; found: 1118.4482.
Supplementary data
4.1.11. Synthesis of fMRTGNAG-SA (9d)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.07.052.
Further synthesis to obtain 9d (yield: 1.8 mg (8%)) was per-
formed analoguously to the synthesis of 9a. HR-MS calcd for
C
37H58N17O16S2 [MꢂH]ꢂ: 1060.3689; found: 1060.3702.
References and notes
4.1.12. Synthesis of fMRTGNAK-SA (9e)
1. Bockstael, K.; Van Aerschot, A. Centr. Eur. J. Med. 2009, 4, 141.
2. Spizek, J.; Novotna, J.; Rezanka, T.; Demain, A. L. J. Ind. Microbiol. Biotechnol.
2010, 37, 1241.
3. Hogberg, L. D.; Heddini, A.; Cars, O. Trends Pharmacol. Sci. 2010, 31, 509.
4. Barker, J. J. Drug Discov. Today 2006, 11, 391.
Further synthesis to obtain 9e (yield: 1.2 mg (2%)) was per-
formed analoguously to the synthesis of 9a. HR-MS calcd for
C41H67N18O16S2 [MꢂH]ꢂ: 1131.4400; found: 1132.3250.
5. Mollmann, U.; Heinisch, L.; Bauernfeind, A.; Kohler, T.; Ankel-Fuchs, D.
Biometals 2009, 22, 615.
6. Duquesne, S.; Petit, V.; Peduzzi, J.; Rebuffat, S. J. Mol. Microbiol. Biotechnol. 2007,
13, 200.
7. Novikova, M.; Metlitskaya, A.; Datsenko, K.; Kazakov, T.; Kazakov, A.; Wanner,
B.; Severinov, K. J. Bacteriol. 2007, 189, 8361.
8. Kazakov, T.; Vondenhoff, G. H.; Datsenko, K. A.; Novikova, M.; Metlitskaya, A.;
Wanner, B. L.; Severinov, K. J. Bacteriol. 2008, 190, 2607.
9. Metlitskaya, A.; Kazakov, T.; Kommer, A.; Pavlova, O.; Praetorius-Ibba, M.; Ibba,
M.; Krasheninnikov, I.; Kolb, V.; Khmel, I.; Severinov, K. J. Biol. Chem. 2006, 281,
18033.
10. Metlitskaya, A.; Kazakov, T.; Vondenhoff, G. H.; Novikova, M.; Shashkov, A.;
Zatsepin, T.; Semenova, E.; Zaitseva, N.; Ramensky, V.; Van Aerschot, A.;
Severinov, K. J. Bacteriol. 2009, 191, 2380.
4.1.13. Synthesis of fMRTGNAI-SA (9f)
Further synthesis to obtain 9f (yield: 1.2 mg (5.6%)) was per-
formed analoguously to the synthesis of 9a. HR-MS calcd for
C
41H66N17O16S2 [MꢂH]ꢂ: 1116.4314; found: 1116.4326.
4.1.14. Synthesis of fMRTGNAV-SA (9g)
Further synthesis to obtain 9g (yield: 3.8 mg (19%)) was per-
formed analoguously to the synthesis of 9a. HR-MS calcd for
C
40H64N17O16S2 [MꢂH]ꢂ: 1102.4158; found: 1102.4165.
4.1.15. Biology
11. Kazakov, T.; Metlitskaya, A.; Severinov, K. J. Bacteriol. 2007, 189, 2114.
12. Roush, R. F.; Nolan, E. M.; Lohr, F.; Walsh, C. T. J. Am. Chem. Soc. 2008, 130, 3603.
13. Van de Vijver, P.; Vondenhoff, G. H.; Kazakov, T. S.; Semenova, E.; Kuznedelov,
K.; Metlitskaya, A.; Van Aerschot, A.; Severinov, K. J. Bacteriol. 2009, 191,
6273.
14. Brown, M. J.; Mensah, L. M.; Doyle, M. L.; Broom, N. J.; Osbourne, N.; Forrest, A.
K.; Richardson, C. M.; O’Hanlon, P. J.; Pope, A. J. Biochemistry (Mosc.) 2000, 39,
6003.
Bacteria were grown overnight in LB medium and cultured
again the following day in fresh LB medium or LB-medium contain-
ing 5 mM (
trated in a 96 well-plate using either LB-medium with or without
5 mM ( )-arabinose. To each well 85 L LB-medium with or with-
out 5 mM ( )-arabinose was added to a total volume of 90 L fol-
lowed by 10 L of bacterial cell culture. The cultures were next
incubated at 37 °C and the OD600 was determined after 8 h.
L)-arabinose to an OD600 of 0.1. Compounds were ti-
L
l
L
l
15. Vondenhoff, G. H. M.; Blanchaert, B.; Geboers, S.; Kazakov, T.; Datsenko, K. A.;
Wanner, B. L.; Rozenski, J.; Severinov, K.; Van Aerschot, A. J. Bacteriol. 2011, 193,
3618.
l