Microcin C and Albomycin Analogues with Aryl-tetrazole Substituents as Nucleobase Isosters Are Selective Inhibitors of Bacterial Aminoacyl tRNA Synthetases but Lack Efficient Uptake

Article abstract of DOI:10.1002/cbic.201200174

In 1998, Cubist Pharmaceuticals patented a series of aminoacyl tRNA synthetase (aaRS) inhibitors based on aminoacyl sulfamoyladenosines (aaSAs), in which the adenine was substituted by aryl-tetrazole moieties linked to the ribose fragment by a two-carbon spacer. Although potent and specific inhibitors of bacterial IleRS, these compounds did not prove successful in vivo due to low cell permeability and strong binding to serum albumin. In this work, we attempted to improve these compounds by combining them with microcin C (McC) or albomycin (i.e., siderophore-drug conjugate (SDC)) transport modules. We found that aryl-tetrazole variants of McC and albomycin still lacked antibacterial activity. However, these compounds were readily processed by E. coli aminopeptidases with the release of toxic aaRS inhibitors. Hence, the lack of activity in whole-cell assays was due to an inability of the new compounds to be taken up by the cells, thus indicating that the nucleotide moieties of McC and albomycin strongly contribute to facilitated transport of these compounds inside the cell. Synthetases' Achilles' heel? Selective inhibition of bacterial aminoacyl tRNA synthetases was previously accomplished by using aminoacyl sulfamoyladenosines and substituting aryltetrazole moieties for the adenine heterocycle. While these compounds did not prove successful in vivo, conjugation to peptidic Trojan horses or siderophore drug conjugate (SDC) transport modules envisaged improved uptake.

Full text of DOI:10.1002/cbic.201200174

DOI: 10.1002/cbic.201200174
Microcin C and Albomycin Analogues with Aryl-tetrazole
Substituents as Nucleobase Isosters Are Selective
Inhibitors of Bacterial Aminoacyl tRNA Synthetases but
Lack Efficient Uptake
Gaston H. Vondenhoff,[a] Bharat Gadakh,[a] Konstantin Severinov,[b, c] and
Arthur Van Aerschot*[a]
In 1998, Cubist Pharmaceuticals patented a series of aminoacyl
tRNA synthetase (aaRS) inhibitors based on aminoacyl sulfa-
moyladenosines (aaSAs), in which the adenine was substituted
by aryl-tetrazole moieties linked to the ribose fragment by
a two-carbon spacer. Although potent and specific inhibitors
of bacterial IleRS, these compounds did not prove successful in
vivo due to low cell permeability and strong binding to serum
albumin. In this work, we attempted to improve these com-
pounds by combining them with microcin C (McC) or albomy-
cin (i.e., siderophore–drug conjugate (SDC)) transport modules.
We found that aryl-tetrazole variants of McC and albomycin
still lacked antibacterial activity. However, these compounds
were readily processed by E. coli aminopeptidases with the
release of toxic aaRS inhibitors. Hence, the lack of activity in
whole-cell assays was due to an inability of the new com-
pounds to be taken up by the cells, thus indicating that the
nucleotide moieties of McC and albomycin strongly contribute
to facilitated transport of these compounds inside the cell.
Introduction
Increasing resistance to antibiotics is a major problem world-
wide and provides the stimulus for development of new bacte-
rial inhibitors. The process of bacterial protein synthesis is a va-
lidated target of antibiotic action. Aminoacyl-tRNA synthetases
(aaRSs) play an indispensable role in protein synthesis. These
proteins aminoacylate tRNAs with cognate amino acids in
a two-stage process whereby the amino acid is first activated
with adenosine monophosphate (AMP) and is esterified in
a second step to the 2’- or 3’-hydroxy group of the 3’-end of
respective tRNA. The best known aaRS inhibitors to date are
aminoacyl sulfamoyl adenosines that are poorly hydrolyzable
isosteres of aminoacyladenylates, which are used by aaRSs as
reactive intermediates to esterify the amino acids to their cog-
nate tRNA.[1] To date, a wealth of compounds was synthesized
that target various aaRSs.[1–5] Unfortunately, these compounds
show low selectivity towards microbial aaRSs or suffer from
other limitations such as poor cell permeability.
against the human homologue (an especially profound differ-
ence is seen with CB168 (2)). Of the numerous compounds of
this class that have been synthesized and tested, only CB432
(3) showed moderate activity against a broad range of bacteria
including Staphylococcus aureus, Staphylococcus epidermidis,
Staphylococcus saprophyticus, Streptococcus pneumoniae Strep-
tococcus pyogenes, Enterococcus faecium, Enterococcus faecalis,
Bacillus subtilis, Haemophilus influenzae, Klebsiella pneumoniae,
and Moraxella catarrhalis, with MIC values ranging between 2
and 100 mgmLÀ1 [7]
In addition, 3 showed activity during treat-
.
ment of mice infected with S. pyogenes.[7] However, this com-
pound could not be further pursued as a potential antibiotic
due to its high affinity to serum albumin.[7]
Microcin C (4a, McC) is a natural compound produced by
Enterobacteriaceae. McC consist of an adenosine to which
a heptapeptide is attached through a C-terminal phosphoa-
mide bond. The biosynthesis and mode of action of McC have
A U.S. patent by Cubist Pharmaceuticals (Lexington, USA)[6]
reported the synthesis and evaluation of a new type of aaRS
inhibitors. Although still based upon aminoacyl sulfamoylade-
nosine, these analogues contain an aryl-tetrazole in place of
the adenine moiety, connected through a two-carbon linker to
ribose. The tetrazole moiety is linked to either one or two five-
or six-membered heterocycles. The advantage of these com-
pounds compared to aaSAs is their high selectivity for bacterial
aaRSs relative to the human homologues. The two most
important compounds, along with IleSA (1), are depicted in
Scheme 1, and their inhibitory properties are listed in Table 1.
As can be seen, aryl-tetrazole-containing compounds 2 and 3
exhibit good activity against E. coli IleRS and poor activity
[a] Dr. G. H. Vondenhoff, B. Gadakh, Prof. Dr. A. Van Aerschot
Laboratory of Medicinal Chemistry, Rega Institute for Medical Research
KU Leuven, P.B. 1030, Minderbroedersstraat 10, 3000 Leuven (Belgium)
E-mail: Arthur.VanAerschot@REGA.KULeuven.be
[b] Prof. Dr. K. Severinov
Department of Molecular Biology and Biochemistry, Waksman Institute
Rutgers, The State University of New Jersey
190 Frelinghuysen Road, Piscataway, NJ 08854 (USA)
[c] Prof. Dr. K. Severinov
Institutes of Molecular Genetics and Gene Biology
Russian Academy of Sciences
Leninskiy Prospekt 14, 119991 Moscow (Russia)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200174.
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A. Van Aerschot et al.
been reviewed comprehensively
elsewhere.[8] In E. coli, the single
locus yejABEF, encoding an inner
membrane ABC transporter, is
responsible for the uptake of
McC.[6] Transport through the
outer membrane is facilitated by
OmpF. Once inside the cell, McC
is metabolized by a peptide de-
formylase and one of three ami-
nopeptidases, PepA, PepB, or
PepN, to liberate a nonhydrolyza-
ble analogue of aspartyl adeny-
late (4b). This compound is able
to selectively inhibit AspRS.[9] Be-
cause of its facilitated transport
with subsequent processing and
release of a toxic moiety, McC
belongs to the Trojan horse class
of antibacterial agents.
Albomycin (5a) is another nat-
ural aaRS inhibitor that relies on
a Trojan horse strategy to in-
crease the uptake of a toxic
moiety that is poorly permeable
on its own. Albomycin is a so-
called siderophore–drug conju-
gate (SDC), with the siderophore
consisting of a tripeptide of N-5-
acetyl-N-5-hydroxy-l-ornithines,
mimicking the siderophore ferri-
chrome. The siderophore is C-
terminally attached to a 4’-thio-
6’-amino heptonic acid through
a special linker. A modified cyto-
sine resembling a nucleobase is
attached at the 1’-position. Albo-
mycin is taken up by the FhuA–
TonB complex,
a ferrichrome
transport mechanism present in
many bacteria and capable of
transporting siderophores and,
in some cases, sideromycins,
across bacterial membranes.[10]
Once inside the cell, it is re-
leased from iron by ferric reduc-
tase and processed by PepN
with release of the active moiety
5b, which resembles seryl ade-
nylate and inhibits SerRS.[11]
We were interested to deter-
mine whether compounds de-
signed by Cubist Pharmaceuti-
cals, when attached to transport
modules of McC and albomycin,
could function as aryl-tetrazole-
containing Trojan horse inhibi-
Scheme 1. IleRS inhibitors with aryl-tetrazole substitutes for adenine, and chemical structures for microcin C and
albomycin.
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Aryl-tetrazole Substituents in McC and Albomycin Analogues
Table 1. Respective inhibition constants of compounds 1–3 on IleRS iso-
lated from different organisms [mgmLÀ1].
S. aureus
E. faecalis
E. coli
Human
IleSA (1)
CB168 (2)
CB432 (3)
2.9
5
0.5
2
20
9
2
1.3
3.2
20
3000
450
tors. We concentrated on two different lead compounds for
this analysis, CB168, which was chosen for its great potential
as a selective inhibitor of bacterial IleRS, and CB432, because
of its broad-range antibacterial activity, low level inhibition of
human IleRS, and moderate efficacy in an animal model. Our
expectation was that Trojan horse inhibitors based on aryl-tet-
razole-containing aaRS inhibitors, would be superior to natural
compounds for further development.
Scheme 2. Synthesis of 5-aryl tetrazoles. A) Synthesis of 5-phenyl tetrazole:
a) benzonitrile, NaN3, HOAc in nBuOH, 96 h, 1258C. B) Synthesis of 5-(4-phe-
noxy)phenyltetrazole tetrazole: b) 4-phenoxybenzonitrile, dibutyltinoxide, tri-
methylsilylazide in toluene, 5 h, 958C; c) work-up with NaOH (1.6 n).
pounds were selectively deprotected to liberate the 5’-hydroxy
group, affording 17 and 18. Sulfamoylation of the latter com-
pounds according to previous protocols[16] gave 19 and 20.
Previously, we described a convenient method for the coupling
of peptides to sulfamoylated adenosine (Scheme 4).[17,18] Analo-
gous procedures were followed here to obtain compounds
32–36 as described in Scheme 3. SDCs 38 and 39 were synthe-
sized by coupling of compound 2 or 3 to [N-2-(benzyloxycar-
bonyl)-N-5-acetyl-N-5-O-acetyl-l-ornithinyl]-[N-5-acetyl-N-5-O-
acetyl-l-ornithinyl]-N-5-acetyl-N-5-O-acetyl-l-ornithine (37) by
using HBTU. The synthesis of this hydroxamate-based sidero-
phore 37 was described earlier by Miller et al.[19] Subsequent
removal of the three O-acetyl groups with DIPEA and removal
of the Cbz group by hydrogenation yielded compounds 38
and 39 (Scheme 5).
Results
Design and synthesis of McC and albomycin analogues
carrying a 2-carbon- linked aryl-tetrazole as an adenine
substitute
As mentioned above, two of the compounds developed by
Cubist Pharmaceuticals (2 and 3) were used as lead com-
pounds for our study. Compound 2 was further modified by
several strategies: by converting it into an McC analogue
(yielding compound 32), and by replacing the isoleucine
moiety of compound 32 with leucine (33) or aspartic acid as
found in McC (34). In addition, compound 1 was also convert-
ed into a SDC by coupling it to a hydroxamate-based sidero-
phore (37), affording compound 38. Lead compound 3 was
likewise converted into a McC analogue by coupling it to the
McC peptide, affording compound 35. In related compound
36, the N-terminal formyl group of the McC peptide was omit-
ted, and the alanine at position 6 was replaced by a serine.
The expectation was that this compound would be processed
more efficiently once inside the cell, due to the expected lack
of requirement for peptide deformylase caused by the pres-
ence of a possible endopeptidase cleavage site. Finally, com-
pound 3 was coupled with the hydroxamate-based sidero-
phore (37), affording compound 39.
The 5-phenyltetrazole (7) was synthesized by following a
method described by Herbst and Wilson.[12] The 5-(4-phenoxy)-
phenyltetrazole (9) was synthesized as described by Hill et al.[6]
(Scheme 2). Following the example of Cubist Pharmaceuticals,
the design of starting materials 2 and 3 was retained. However,
the synthetic procedure was modified, largely based on work
by Lee et al.,[13] Mandal et al.[14] and most importantly, Ohrui
et al.[15] (Scheme 3). Starting from ribose (10), the 2’- and 3’-hy-
droxy groups were protected with an isopropylidene moiety
and the 5’-hydroxy with a p-anisyl-diphenylmethyl (MMTr) pro-
tecting group to afford 12. Following the method described
by Ohrui et al.,[15] a Wittig–Moffat reaction was carried out, pro-
viding compound 13. Reduction of the ester, followed by a Mit-
sunobu reaction with tetrazole derivative 7 or 9, yielded com-
pounds 15 and 16, respectively. Subsequently, these com-
Antibacterial activity of McC and albomycin analogues
The ability of new McC-like compounds to inhibit growth of
E. coli was determined. In addition to wild-type E. coli K-12
BW28357, a derivative strain Ara-Yej (BW39758) with the ge-
nomic yejABEF operon under control of the arabinose-inducible
araBAD promoter was tested. In the absence of arabinose, the
Ara-Yej strain is virtually resistant to McC. However, when in-
duced, the Ara-Yej strain is more sensitive to McC and its ana-
logues than the wild-type strain due to higher levels of the
YejABEF transporter. Both strains were grown in LB medium
with or without 5 mm (l)-arabinose in the presence of various
concentrations of compounds 32–36. Unfortunately, with the
exception of the McC control, none of the compounds showed
activity.
The antibacterial activities of all compounds against
S. aureus (ATCC 6538), S. epidermidis RP62A (ATCC 35984), Pseu-
domonas aeruginosa PAO1, Sarcina lutea (ATCC 9341), and Can-
dida albicans CO11 were also tested. The positive control, com-
pound 2, was active against S. aureus, in agreement with the
original findings of Cubist Pharmaceuticals and also showed
activity against C. albicans at 50 mm. Among the newly synthe-
sized compounds, only 39 showed statistically significant, al-
though very weak, activity against S. aureus and C. albicans
after 11 h incubation as shown in a Student’s T-test (p=0.01)
(Figure 1). To a lesser extent, C. albicans was also inhibited after
11 h (p=0.1).
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Scheme 3. Synthesis of aryl tetrazole derivatives. a) d(À)-ribose, H2SO4, acetone, 30 min, RT; b) 11, monomethoxytrityl-Cl, pyridine, 16 h, RT; c) 12, carbethoxy-
methylenetriphenylphosphorane, CH3CN, 8 h, 958C; d) 13, LiAlH4, THF, 4 h, RT; e) 14, Ph3P, DEAD, 5-phenyltetrazole or 5-(4-phenoxy)phenyltetrazole, THF, 08C;
f) 15 or 16, ether/HOAc, 10 h, 558C; g) 17 or 18, sulfamoylchloride, DMAc, 08C; h) 19 or 20, N-a-Cbz or Boc-l-aminoacyl-(tBu)-succinimide, DBU in DMF, 6 h,
RT.
To understand the reason(s) for the disappointing lack of an-
tibacterial activity of new compounds, in vitro analyses were
performed, and the ability of all compounds targeting IleRS
(compounds 2, 3, 32, 35, 36, 38, and 39) to inhibit the tRNAIle
aminoacylation reaction in E. coli extracts was determined
(Figure 2). All compounds showed good activity against IleRS
in wild-type cell extracts and, with the exception of com-
pounds 2 and 3, were devoid of activity in extracts prepared
from McC- and albomycin-resistant cells lacking peptidases
PepA, PepB, and PepN. The results thus indicate that new com-
pounds are processed in an expected way, and the released
active moiety is able to inhibit the IleRS target (compounds 2
and 3 do not require processing for target inhibition).
A similar result was obtained for compounds 27 and 33
(Figure 3): these LeuRS inhibitors showed activity against
LeuRS in wild-type cell extracts. In extracts prepared from cells
lacking aminopeptidase activity, compound 33 was inactive
while compound 27 showed activity. Finally, compound 34, ex-
pected to target AspRS, was devoid of inhibitory activity even
in wild-type cell extracts.
which are well-known nonhydrolyzable isosteres of aminoacyl
adenylates. It was found that selectivity of aminoacyl sulfamoyl
adenosines can be increased by replacing adenine with a two-
carbon-linked aryl-tetrazole. Unfortunately, these compounds
were shown to exhibit low whole-cell activity and to bind
avidly to serum albumin. Previously, we have shown that McC
and its synthetic analogues show great potential as antibacteri-
al compounds, and this concept could be further exploited by
endowing different antibacterial compounds with the McC
peptide to enhance their uptake.[17,18] A related Trojan horse
approach was investigated by several groups using sidero-
phore-conjugated antibiotics.[20] In order to improve the anti-
bacterial potency of the Cubist compounds, we successfully
converted them into McC or SDC analogues. The whole-cell
activity as well as the inhibitory action of these compounds
against their respective aaRSs in in vitro aminoacylation experi-
ments was determined.
Three important conclusions can be drawn from these re-
sults. First, synthetic SDC-prodrugs have often been found
unable to release the active moiety, which therefore cannot
reach its target,[21] making this approach ineffective. Our results
clearly show that, although no antibacterial activity was ob-
served with new McC or SDC analogues, these compounds did
show strong activity in cell extracts, and the activity was de-
pendent on processing by cellular peptidases. Hence, it must
be concluded that all compounds were efficiently metabolized,
Discussion
In search of new antibacterial compounds targeting aaRSs, re-
searchers from Cubist Pharmaceuticals identified a number of
new entities that are based on aminoacyl sulfamoyladenosines,
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Aryl-tetrazole Substituents in McC and Albomycin Analogues
presented in this study could
surpass issues related to the re-
lease of the active moiety ob-
served in previous work. Second-
ly, it must be concluded that, in
the case of McC analogues, the
absence of antibacterial activity
against E. coli is attributable to
failure of uptake. In our previous
work,[17,18] it was clearly shown
that the YejABEF transporter, re-
sponsible for McC uptake, toler-
ates modifications to the McC
peptide moiety relatively well. In
this paper, the only modification
compared to natural McC or its
active chemically synthesized an-
alogues targeting LeuRS or IleRS
is the replacement of the ade-
nine base by two-carbon-linked
aryl-tetrazoles. Therefore, the
YejABEF transporter is able to
recognize the nucleotide moiety
of McC and may be a selective
transporter of peptidyl adeny-
lates or derivatives with very
close resemblance. The same
may be true for SDCs that rely
on FhuA and the TonB complex
for efficient uptake. Further re-
search is required to resolve this
issue. However, it is clearly
shown that substitution of the
base results in absence of the
ability to pass the bacterial
membrane of the tested E. coli
strains. Therefore, it would be
highly desirable to investigate
whether efficient uptake by one
or both transport systems can
be obtained by modifying the
two-carbon-linked aryl-tetrazole
moiety. Attempts to synthesize
SDCs by conjugation of the side-
rophore presented here with
simple aminoacyl-sulfamoyl-ade-
nosines proved ineffective due
to the instability of these com-
pounds.
In spite of all our attempts to
promote uptake of well-known
inhibitors, none of the newly
synthesized congeners showed
antibacterial activity, while in
vitro, all compounds (excluding
Scheme 4. General procedure for the preparation of McC analogues 32–36: a) for R3 =Z-group, H2, Pd/C in MeOH,
3 h, RT; b) for R3 =Boc group, TFA/H2O (5:2), 4 h, 08C to RT; c) protected peptide (f-MRTGNA for 28–30 or Boc-
MRTGNS for 31), HOBt, DIC, DIPEA, DMF, 16 h, RT; d) TFA/thioanisole/H2O (90:2.5:7.5), 2 h, RT.
liberating the active moiety which inhibited the aminoacyla-
tion reaction of IleRS and LeuRS. This shows that the approach
compound 34) proved potent inhibitors of either LeuRS or
IleRS. However, since compound 34 did not inhibit AspRS, it
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A. Van Aerschot et al.
metabolized,[17] it is unlikely that this observation re-
sults from problems during metabolism. This sug-
gests that LeuRS and IleRS can accommodate and ef-
fectively bind the aryl-tetrazole moiety substitution
for adenine, while AspRS does not allow for such
modification. An explanation for this observation
may be given by the fact that class II aaRSs, in con-
trast to class I aaRSs, have a well conserved ATP bind-
ing site in which ATP is stacked between a phenylala-
nine of motif 2 and an arginine in motif 3. In addi-
tion, Brick et al. and Nakama et al. showed that there
is no initial hydrogen bonding of the adenine N-6
group with the KMSKS loop for isoleucyl and tyrosyl-
tRNA synthetases.[22,23] Therefore, adenine mimics
such as the aryl-tetrazoles presented in this manu-
script may be accepted as good isosteres of adenine.
Experimental Section
Scheme 5. General procedure for the preparation of hydroxamate-based siderophore–
IleRS conjugated inhibitors, carrying either a phenyl tetrazole (2) or a phenoxyphenyl
tetrazole (3) moiety. a) Protected peptide, HBTU, Et3N in DMF, 16 h, RT; b) DIPEA (6%)
in MeOH, 5 h, RT; c) H2, Pd/C in aqueous DMF with FeCl3, 3 h, RT.
Reagents and solvents were purchased from commercial
suppliers (Acros, Sigma–Aldrich, Bachem, Novabiochem)
and used as provided, unless otherwise indicated. DMF
and THF were of analytical grade and were stored over
4 ꢁ molecular sieves. For reactions involving Fmoc-pro-
tected 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 provided. Reactions were carried
out in oven-dried glassware under a nitrogen atmosphere with stir-
ring at room temperature, unless otherwise indicated.
can be concluded that not all modifications of the amino acid
constituency are allowed. Since we have shown before that all
McC analogues comprising l-amino acids can be efficiently
Figure 2. Inhibition of the aminoacylation reaction. A) IleRS inhibition in S30
extracts of McC-sensitive E. coli cells. B) IleRS in S30 extracts of McC-resistant
E. coli cells lacking peptidases A, B, and N. In all tests, the different extracts
were incubated with the respective IleRS inhibitors (25 mm), with samples
taken after 15 min for evaluation of the aminoacylation reaction. The
amounts of aminoacylated tRNAIle were measured through liquid scintillation
counting and normalized versus the respective control.
Figure 1. Inhibitory activity of compounds 3 and 39 against S. aureus and
C. albicans at 50 mm concentrations. OD600 refers to the optical density of the
cell culture measured at 600 nm. Marginal but statistically significant inhibi-
tion of S. aureus by compound 39 was found.
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Aryl-tetrazole Substituents in McC and Albomycin Analogues
3.16 mmol, 3.0 equiv), and trimethylsilylazide (679 mL, 5.17 mmol,
5.0 equiv) were dissolved in toluene (5 mL) and stirred at reflux for
5 h, after which the reaction mixture was cooled to room tempera-
ture. Additional toluene (100 mL) was added to this mixture, and
the organic layer was extracted twice with 50 mL NaOH (1.6 n).
The aqueous layer was subsequently washed twice with 50 mL di-
ethyl ether, after which the aqueous layer was acidified to pH 6
with concentrated HCl. The aqueous layer was then extracted with
EtOAc. The organic layer was dried over Na2SO4, filtered, and
evaporated, yielding 215 mg (87%) of the desired compound:
13C NMR (75 MHz, [D6]DMSO): d=119.49, 120.24, 120.52, 125.36,
129.90, 131.23, 156.41, 160.16 ppm; HR-MS: calcd for C13H10N4O1:
239.0933 [M+H]+; found: 239.0918.
Synthesis of 5-O-p-anisyldiphenylmethyl-2,3-O-isopropylidene-b-
Figure 3. Inhibition of LeuRS by compounds 27 and 33. In all tests, the dif-
ferent extracts were incubated with the respective IleRS inhibitors (25 mm),
with samples taken after 15 min for evaluation of the aminoacylation reac-
tion. The amounts of aminoacylated tRNAIle were measured through liquid
scintillation counting and normalized versus the respective control.
d-ribofuranose (12):
A
solution of d(À)-ribose (10) (6.50 g,
43.16 mmol, 1 equiv), acetone (80 mL), and H2SO4 (0.2 mL) was
stirred for 30 min at room temperature under argon. NaHCO3
(0.43 g, 5.14 mmol, 0.12 equiv) was added, and the mixture was
stirred for 30 min, filtered, and the volatiles evaporated, yielding
2’,3’-O-isopropylidene-b-d-ribofuranose (11). The crude product
was dissolved in pyridine (30 mL). Next, a solution of monometh-
oxytrityl-chloride (MMTr-Cl) (10 g, 32 mmol, 0.67 equiv) in pyridine
was added. This mixture was stirred overnight at room tempera-
ture under argon. The volatiles were evaporated, and the residue
was purified by flash chromatography (20% EtOAc in hexane with
0.1% Et3N). Fractions containing the desired product, as deter-
mined by TLC analysis, were evaporated to yield product 12 (2.8 g,
6.054 mmol, 43% calculated over two steps): 13C NMR (75 MHz,
CDCl3): d=24.98, 25.31, 26.36, and 26.75 (2ꢂisopropylidene CH3),
55.39 (O-CH3), 65.19 and 65.56 (C5’), 79.68 and 82.27(C1’), 80.32
and 86.23 (C3’), 82.41 and 87.20 (C2’), 87.44 and 88.04 (C4’), 98.22,
103.66, 112.41, 113.33, 113.48 (trityl), 113.56 (C-isopropylidene),
127.51, 128.26, 128.63, 130.63, 134.79, 135.33, 143.73, 144.40,
159.04 ppm (aromatic signals); ESI-MS calcd for C28H30O6Na: 485.19
[M+Na]+; found: 484.80.
1H and 13C NMR spectra of the compounds dissolved in [D6]DMSO
or D2O were recorded on a Bruker UltraShield Avance 300 MHz or
500 MHz spectrometer. Chemical shifts are expressed as d values in
parts per million (ppm) using residual solvent peaks (DMSO: 1H,
d=2.50 ppm; 13C, d=39.52 ppm; HOD: 1H, d=4.79 ppm) as a refer-
ence. Coupling constants are given in Hertz (Hz). The peak patterns
are indicated by the following abbreviations: brs=broad singlet,
d=doublet, m=multiplet, q=quadruplet, s=singlet, and t=trip-
let. High resolution mass spectra were recorded on a quadrupole
time-of-flight mass spectrometer (Q-Tof-2, Micromass, Manchester,
UK) equipped with a standard ESI interface; samples were infused
in propan-2-ol/H2O (1:1) at 3 mLminÀ1
.
Precoated aluminium sheets were used for TLC (Merck, Silica gel
60 F254). Spots were visualized by UV light at 254 nm. Column chro-
matography was performed on ICN silica gel 60A 60–200. Prepara-
tive HPLC of peptides was done using a Waters Xbridge prepara-
tive C18 (19ꢂ150 mm) column connected to a Waters 1525 binary
HPLC pump and a Waters 2487 dual absorbance detector. Final
products were purified using a PLRP-S 100ꢁ column connected to
a Merck-Hitachi L6200A Intelligent pump. Eluent compositions are
expressed as v/v. Purity was checked by analytical HPLC on a Inertsil
ODS-3 (C-18; 4.6ꢂ100 mm) column, connected to a Shimadzu LC-
20AT pump using a Shimadzu SPD-20A UV detector. Recordings
were performed at 254 and 214 nm.
Synthesis of ethyl-3,6-anhydro-7-O-p-anisyldiphenylmethyl-2-
deoxy-4,5-O-isopropylidene-d-allo-heptanoate (13): Product 12
(11.0 g, 23.8 mmol, 1 equiv) and carbethoxymethylene-triphenyl-
phosphorane (12.45 g, 35.7 mmol, 1.5 equiv) were dissolved in
CH3CN (100 mL). The solution was stirred at reflux for 8 h, then was
evaporated to yield a pale yellow oil. The product was purified by
flash chromatography (22% EtOAc in hexane). Fractions containing
the desired product were evaporated, affording product 13
(12.50 g, 23.485 mmol, 99%): 13C NMR (75 MHz, CDCl3): d=14.44
(CH3 ethyl), 25.89 and 27.77 (2ꢂCH3 isopropylidene), 39.00 (C2),
55.38 (C1), 60.85 (CH2 ethyl), 64.44 (C7), 81.17 (C3), 82.57 (C5),
83.77 (C4), 84.54 (C6), 86.61 (MMTr-C(Ph3)), 113.34 (trityl), 114.39 (C-
isopropylidene), 127.10, 128.03, 128.70, 130.65, 135.71, 144.57,
144.62, and 158.81 (aromatic signals), 170.75 ppm (C=O); ESI-MS
calcd for C32H36O7Na: 555.24 [M+Na]+; found: 554.83.
Synthesis of 5-phenyltetrazole (7): Benzonitrile (6) (15.5 mL),
sodium azide (13 g, 15.5 mL) and acetic acid (11.4 mL) were dis-
solved in n-BuOH (60 mL) and stirred at reflux. After 3 d, another
portion of sodium azide (2.60 g), acetic acid (3.8 mL), and nBuOH
(20 mL) were added, and the mixture was stirred for another 24 h.
The mixture was cooled to 08C, and a white precipitate formed.
Water (250 mL) was added, and the mixture was concentrated to
100 mL. The resulting solution was neutralized with 1n NaOH and
was then washed with toluene (2ꢂ100 mL). The water layer was
acidified with 1 n HCl until a white precipitate formed. This pre-
cipitate was washed with cold water (08C) and recrystallized from
20% isopropanol in water. The white crystals were filtered, yielding
11.31 g (77.4 mmol, 39%): 13C NMR (75 MHz, [D6]DMSO): d=124.23
(i-aryl), 127.08 (o-aryl), 129.52 (m-aryl), 131.36 (p-aryl), 155.38 ppm
(C-tetrazole); ESI-MS calcd for C7H7N4: 147.07 [M+H]+; found:
146.90.
Synthesis of 3,6-anhydro-7-O-p-anisyldiphenylmethyl-2-deoxy-
4,5-O-isopropylidene-d-allo-heptitol (14): Compound 13 (12.50 g,
23.48 mmol, 1 equiv) was dried overnight in vacuo. THF (10 mL)
was added, then LiAlH4 (1.34 g, 35.23 mmol, 1.5 equiv) was careful-
ly added to the solution. After 30 min, the reaction was quenched
by adding propan-2-ol (50 mL). The volatiles were evaporated, and
the product was purified by flash chromatography (40% EtOAc in
hexane). Fractions containing the desired product 14 were collect-
ed and evaporated to yield 9.5 g (19.38 mmol, 83%): 13C NMR
(125 MHz, [D6]DMSO): d=25.95 (CH3 isopropylidene), 27.88 (CH3
isopropylidene), 36.09 (C2), 55.55 (CH3 para-anisyl), 61.21 (C1),
64.40 (C7), 82.48 (C3), 83.65 (C5), 84.35 (C4), 85.10 (C6), 86.71 (C-
Synthesis of 5-(4-phenoxy)phenyltetrazole (9): 4-Phenoxybenzo-
nitrile (8) (200 mg, 1.02 mmol, 1.0 equiv), dibutyltinoxide (79 mg,
ChemBioChem 0000, 00, 1 – 12
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A. Van Aerschot et al.
trityl), 113.48 (C-isopropylidene), 114.82, 127.25,128.17, 128.76,
130.72, 144.60, 144.66, and 158.94 ppm (aromatic signals); ESI-MS
calcd for C30H34O6: 513.22 [M+H]+; found: 513.00.
1H NMR (500 MHz, [D6]DMSO): d=1.32 (s, 3H; CH3), 1.48 (s, 3H;
CH3), 2.31–2.48 (m, 2H; C1), 3.21–3.33 (m, 2H; C7), 3.99–4.02 (m,
2H; C6, C3), 4.48–4.52 (dd, J=4.6, 6.5 Hz, 1H; C4), 4.71–4.74 (dd,
J=2.8/6.5 Hz, 1H; C5), 4.79–4.84 (m, 2H; C2), 7.51–7.53 (m, 3H;
m/p-aryl), 8.09–8.13 ppm (m, 2H; o-aryl, J=1.5 Hz); 13C NMR
(125 MHz, [D6]DMSO): 26.44 and 28.46 (2CH3 isopropylidene), 34.99
(C2), 52.03 (C1), 71.08 (C7), 83.89 (C3), 84.03 (C5), 84.29 (C4), 86.68
(C6), 116.52 (C-isopropylidene), 128.59 (C-aryl), 129.43 (i-aryl),
130.93 (C-aryl), 132.41 (p- C-aryl), 167.05 (tetrazole); ESI-MS calcd
for C17H26N5O6S1: 426.14 [M+H]+; found: 426.15.
Synthesis of 3,6-anhydro-7-O-p-anisyldiphenylmethyl-1,2-di-
deoxy-4,5-O-isopropylidene-1-[5-phenyltetrazole]-d-allo-heptitol
(15): Product 14 (9.35 g, 19.1 mmol, 1.0 equiv) and triphenylphos-
phine (Ph3P, 10.0 g, 38.2 mmol, 1.0 equiv) were combined and
dried in vacuo. THF (140 mL) was added, and the mixture was
cooled to 08C. Diethyl 1,2-diazenedicarboxylate (DEAD) was slowly
added to the cooled mixture over 1 h, after which 7 (3.6 g,
24.8 mmol, 0.33 equiv) was added. The mixture was stirred for 3 h
at room temperature under argon, after which the volatiles were
evaporated and the product was purified using flash chromatogra-
phy (10% MeOH in chloroform). All fractions containing the de-
sired product were collected and evaporated, yielding 15 (9.0 g,
14.6 mmol, 76%): 13C NMR (125 MHz, CDCl3): d=25.91 and 27.81
(2CH3 isopropylidene), 31.91 (C2), 50.46 (C1), 55.54 (O-CH3), 64.56
(C7), 81.66 (C3), 82.71 (C5), 83.82 (C4), 85.01 (C6), 114.78 (C-isopro-
pylidene), 113.49, 127.17, 127.29, 127.81, 128.19, 128.76, 129.18,
130.56, 130.70, 135.75, 144.59, 158.95, and 165.45 ppm (aromatic
signals); ESI-MS calcd for C37H39N4O5: 641.27 [M+H]+; found:
640.79.
Synthesis of 3,6-anhydro-2-deoxy-4,5-O-isopropylidene-1-[5-
phenyltetrazole]-d-allo-heptitol-7-[N-(Boc-Ile)-sulfamate]
Product 19 (95 mg, 0.22 mmol, 1.0 equiv) was dried with N-tert-
butoxycarbonyl-isoleucine-O-succinimide (110 mg, 0.33 mmol,
(21):
1.5 equiv) in vacuo. Diazobicyclo[5,4,0]undecene, (DBU, 50 mL,
1.5 equiv) was added and this mixture was stirred for 8 h. The vola-
tiles were evaporated, and the product was purified using flash
chromatography (40% hexane in EtOAc). Fractions containing the
product were collected and evaporated, yielding product 21
(20 mg, 0.03 mmol, 14%): 13C NMR (75 MHz, CDCl3): d=11.58 (Ile-d-
CH3), 16.04 (Ile-g-CH3), 25.21 (Ile-g’-CH3), 25.73 (isopropylidene CH3),
27.59 (isopropylidene CH3), 28.71 (tBu-Boc), 33.33 (C2), 37.45 (Ile-b-
CH2), 50.17 (C1), 63.16 (Ile-a-CH), 69.11 (C7), 80.32 (C3), 81.80 (C5),
82.33 (C4), 84.82 (C6), 115.17 (C-isopropylidene), 127.18 (C-aryl),
127.67 (i-C-aryl), 129.19 (C-aryl), 130.63 (p-C-aryl), 157.23 (C=O Boc),
165.44 (tetrazole), 182.3 ppm (C=O Ile); ESI-MS calcd for
C28H43N6O9S: 637.27 [MÀH]À; found: 637.11.
Synthesis of 3,6-anhydro-2-deoxy-7-hydroxy-4,5-O-isopropyli-
dene-1-[5-phenyltetrazole]-d-allo-heptitol (17): Product 15 (5.6 g,
9.1 mmol, 1.0 equiv) was dissolved in diethyl ether/HOAc (20 mL,
1 v/v) and refluxed for 10 h. The mixture was evaporated and neu-
tralized with NaHCO3(sat.). The mixture was then extracted with
ether (2ꢂ20 mL) and washed with brine (20 mL). The organic layer
was dried (Na2SO4), filtered, and evaporated. The product was
easily purified by flash chromatography (60% EtOAc in hexane) to
Synthesis of 3,6-anhydro-2-deoxy-1-[5-phenyltetrazole]-d-allo-
heptitol-7-[N-(Ile)-sulfamate] (2): A mixture of TFA/water (5:2) was
added to 21 (20 mg, 0.03 mmol, 1 equiv), and the mixture was
allowed to react for 2 h. The volatiles were evaporated, then the
residue was coevaporated with toluene. The desired product was
purified using HPLC (10% MeOH in water) to yield 1 (4.5 mg,
0.01 mmol, 30%): 1H NMR (500 MHz, MeOD): d=0.95–0.98 (t, J=
7.4 Hz, 3H; Ile-d-CH3), 1.05–1.06 (d, J=7.0 Hz, 3H; Ile-g’-CH3), 1.23–
1.31 (m, 1H; Ile-gB-CH2), 1.58–1.66 (m, 1H; Ile-gA-CH2), 1.99 (m, 1H;
Ile-b-CH), 2.19–2.26 and 2.41–2.47 (m, 2H; C2), 3.57–3.57 (d, J=
4.0 Hz, 1H; Ile-a-CH), 3.78–3.81 (m, 1H; C4), 3.83–3.85 (t, J=5.5 Hz,
1H; C5), 4.00–4.03 (dd, J=3.7/7.7 Hz, 1H; C6), 4.11–4.13 (dd, J=
4.5, 9.6 Hz, 1H; C3), 4.17–4.24 (dAB, JA,4’ =3.63, JA,B =10.81 Hz, 1H;
C7), 4.90 (m, 2H; C1), 7.50–7.51 (m, 3H; m/p-phenyl), 8.09–
8.11 ppm (m, 2H; o-phenyl); 13C NMR (125 MHz, MeOD): d=12.18
(Ile-d-CH3), 15.56 (Ile-g-CH3), 25.60 (Ile-g’-CH2), 34.19 (C2), 38.15 (Ile-
b-CH), 51.40 (C1), 61.22 (Ile-a-CH2), 70.25 (C7), 73.00 (C3), 76.06
(C5), 80.70 (C6), 83.43 (C4), 127.77 (i-C-aryl), 128.72 (o-C-aryl),
130.13 (m-C-aryl), 131.58 (p-C-aryl), 166.22 (tetrazole), 173.99 ppm
(C=O Ile); HR-MS calcd for C20H29N6O7S: 497.1819 [MÀH]À; found:
497.1822.
1
yield 1.2 g (3.4 mmol, 38%): H NMR (500 MHz, [D6]DMSO): d=1.25
(s, 3H; CH3), 1.40 (s, 3H; CH3), 2.17–2.33 (m, 2H; C1), 3.44–3.46
(tAB, JAB =11.60 Hz, JA,OH ꢀJA,6 ꢀ5.4 Hz, JB,OH ꢀJB,6 ꢀ5.3 Hz, 2H; C7),
3.83–3.87 (m, 2H; C6, C3), 4.45–4.47 (dd, J=4.2/6.5, 1H; C4), 4.59–
4.61 (dd, J=3.3, 6.5 Hz, 1H; C5), 4.79–4.84 (m, 2H; C2), 4.87–4.88
(t, J=5.5 Hz, 1H; OH), 7.54–7.58 (m, 1H; m/p-aryl), 8.05 (d, J=
1.5 Hz, 1H; o-aryl), 8.07 ppm (d, J=1.8 Hz, 1H; o-aryl); 13C NMR
(125 MHz, [D6]DMSO): d=25.34 and 27.23 (2CH3 isopropylidene),
32.88 (C2), 49.95 (C1), 61.88 (C7), 80.94 (C3), 81.90 (C5), 83.99 (C4),
84.84 (C6), 113.00 (C-isopropylidene), 126.39 (C-aryl), 127.05 (i-aryl),
129.33 (C-aryl), 130.59 (p-aryl), 164.11 ppm (tetrazole); ESI-MS calcd
for C17H23N4O4: 347.17 [M+H]+; found: 346.74.
Synthesis of 3,6-anhydro-2-deoxy-4,5-O-isopropylidene-1-[5-
phenyltetrazole]-d-allo-heptitol-7-sulfamate (19): Chlorosulfonyl
isocyanate (2.3 mmol, 198 mL) and formic acid (2.2 mmol, 85.2 mL)
were combined at 08C. A white foam formed, and the mixture was
allowed to come to room temperature. After 15 min, the mixture
was cooled to 08C again, and CH3CN (1 mL) was added. This mix-
ture was stirred for 15 min. The resulting product was used as such
for the next step.
Synthesis of peptide coupled 3,6-anhydro-2-deoxy-1-[5-phenyl-
tetrazole]-d-allo-heptitol-7-[N-(Ile)-sulfamate] (32): f-MR(Pbf)T-
(tBu)GN(Trt)A-OH (7 mg, 0.006 mmol, 2 equiv) was combined with
HOBt (1.5 mg, 0.01 mmol, 3 equiv) and dried in vacuo. DMF
(0.5 mL) and DIC (1.6 mL, 0.01 mmol, 3 equiv) were added, and the
solution was stirred for 1 h. Previously obtained product 1 (1.5 mg,
0.003 mmol, 1 equiv) and DIPEA (1 mL, 0.006 mmol, 2 equiv) were
added. This solution was stirred overnight, then the evaporated
desired product was purified on a Porapak-column (25% to 100%
CH3CN in water). The protecting groups were removed by addition
of TFA/water/thioanisol (92.5:7.5:2.5, v/v/v). This solution was
stirred for 2 h, then the mixture was coevaporated with toluene.
Next, the mixture was purified using HPLC (2% CH3CN in water).
Compound 17 (450 mg, 1.3 mmol) was dissolved in dimethylaceta-
mide (DMAc, 2.3 mL) at room temperature. This solution was
cooled to 08C, and cooled chlorosulfonamide was added. This mix-
ture was stirred for 50 min from 08C to room temperature. After
50 min, the mixture was cooled to 08C again, and Et3N (0.46 mL)
was added. The mixture was stirred for 15 min, then MeOH
(2.3 mL) was added, and the mixture was stirred for another
15 min. The solvents were evaporated, and the desired product
was redissolved in EtOAc and washed twice with NaHCO3 (50 mL)
and once with brine (50 mL). The organic layer was dried (Na2SO4),
filtered, and evaporated to yield 19 (410 mg, 0.96 mmol, 74%):
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Aryl-tetrazole Substituents in McC and Albomycin Analogues
HR-MS calcd for C45H71N16O16S2: 1155.4676 [MÀH]À; found:
nals); 13C NMR (125 MHz, MeOD): d=13.03 (Ile-d-CH3), 16.39 (Ile-g-
CH3), 26.56 (Ile-g’-CH3), 34.99 (C2), 30.06 (Ile-b-CH2), 52.18 (C1),
62.27 (Ile-a-CH2), 70.65 (C7), 73.86 (C3), 76.86 (C5), 81.59 (C4), 84.34
(C6), 107.38–161.84 ppm (aromatic signals); HR-MS calcd for
C26H33N6O8S: 589.2081 [MÀH]À; found: 589.2090.
1155.4664.
Synthesis of 3,6-anhydro-2-deoxy-1-[5-phenyltetrazole]-d-allo-
heptitol-7-[N-[[N-5-acetyl-N-5-O-hydroxyl-l-ornithinyl]-[N-5-
acetyl-N-5-O-hydroxyl-l-ornithinyl]-[N-5-acetyl-N-5-O-hydroxyl-l-
ornithinyl]-l-Ile)-sulfamate] (38): Et3N (13 mL, 0.1 mmol, 2 equiv)
was added to compound 1 (25 mg, 0.05 mmol) dissolved in DMF
(0.5 mL). This mixture was next combined with a mixture of 37
(32.4 mg, 0.05 mmol, 1 equiv) and HBTU (23 mg, 0.06 mmol,
1.2 equiv), which were dissolved in DMF (0.5 mL). The reaction mix-
ture was stirred overnight at room temperature under argon. The
following day, the DMF was evaporated, and the residue was redis-
solved in EtOAc. The organic layer was washed twice with water.
Subsequently, the EtOAc was evaporated, and the product was dis-
solved in water/CH3CN, during which the volume of CH3CN was
kept to a minimum. This mixture was then purified over a Porapak
column (0 to 100% CH3CN in water). Fractions containing the
product were dried, affording the desired product, still containing
side products (yield: 21 mg, 34%): ESI-MS calcd for C55H77N12O21S:
1273.5 [MÀH]À; found: 1273.1.
Synthesis of peptide coupled 3,6-anhydro-2-deoxy-1-[5-(4-phe-
noxy)phenyltetrazole]-d-allo-heptitol-7-[N-(l-Ile)-sulfamate] (35):
Synthesis was performed, analogous to the preparation of 32, to
afford 1.0 mg (18% total yield) of the compound 35: HR-MS calcd
for C51H75N16O17S2: 1247.4938 [MÀH]À; found: 1247.4900.
Synthesis of peptide coupled 3,6-anhydro-2-deoxy-1-[5-(4-phe-
noxy)phenyltetrazole]-d-allo-heptitol-7-[N-(l-Ile)-sulfamate] (36):
The required hexapeptide Boc-MR(Pbf)T(tBu)GN(Trt)S(tBu)-OH was
prepared in an analogous manner to the formylated derivative
f-MR(Pbf)T(tBu)GN(Trt)A-OH:[18]
HR-MS
calcd
for
[MÀH]À:
C69H97N10O15S2 1369.6581; found: 1369.6570. Synthesis analogous
to the preparation of 32 was performed to afford 1.0 mg (18%
total yield) of compound 36: HR-MS calcd for C51H76N16O18S2:
1263.4887 [MÀH]À; found: 1263.4774.
The crude product (10.5 mg, 8.2 mmol) was dissolved in MeOH con-
taining 6% DIPEA and was stirred for 24 h. The product was dried
and dissolved in H2O/CH3CN. The product was next purified by RP-
HPLC (5 to 80% CH3CN in H2O), yielding 1.8 mg (5%) of the desired
compound: 13C NMR (125 MHz, MeOD): d=12.08 (CH3 DIPEA),
13.20 (Ile-d-CH3), 16.37 (Ile-g-CH3), 17.38 (DIPEA), 19.41 (Orn- g-CH2),
20.27 (N-acetyl-CH3), 25.79 (Ile-g’-CH2), 30.29 (Orn-b-CH2), 34.15
(C2), 38.59 (Ile-b-CH), 43.79 (DIPEA-CH2), 51.43 (C1), 54.21 (Orn-a-
CH), 55.83 (DIPEA-CH), 61.62 (Ile-a-CH2), 67.76 (Cbz-CH2), 69.81 (C7),
73.08 (C3), 76.10 (C5), 80.45 (C6), 83.73 (C4), 127.77–138.46 (aro-
matic signals), 158.77 (C=O Cbz), 166.15 (tetrazole), 173.84 ppm
(C=O Ile); HR-MS calcd for C49H71N12O18S: 1147.4730 [MÀH]À;
found: 1147.4774.
Synthesis of 3,6-anhydro-2-deoxy-1-[5-phenoxyphenyltetrazole]-
d-allo-heptitol-7-[N-[[N-5-acetyl-N-5-O-hydroxyl-l-ornithinyl]-[N-
5-acetyl-N-5-O-hydroxyl-l-ornithinyl]-[N-5-acetyl-N-5-O-hydroxyl-
l-ornithinyl]-l-Ile)-sulfamate] (39): Synthesis analogous to the
preparation of 38 was performed to afford 1.3 mg (20% total yield)
of compound 39: HR-MS calcd for C55H75N12O19S: 1239.4992
[MÀH]À; found: 1239.4967.
Synthesis of 3,6-anhydro-2-deoxy-4,5-O-isopropylidene-1-[5-
phenyltetrazole]-d-allo-heptitol-7-[N-(Boc-Leu)]-sulfamate (23):
Synthesis was performed, analogous to the preparation of 21, to
afford 550 mg (82% total yield) of compound 23: 13C NMR
(75 MHz, CDCl3): d=22.18 (Leu-dB-CH3), 23.41 (Leu-dA-CH3), 25.13
(Leu-g-CH) 25.74 (CH3 isopropylidene), 27.60 (CH3 isopropylidene),
28.68 (tBu Boc), 33.35 (C2), 41.27 (Leu-b-CH3), 50.18 (C1), 56.51
(Leu-a), 69.71 (C7), 80.74 (C3), 81.88 (C5), 82.28 (C4), 84.83 (C6),
115.26 (C-isopropylidene), 127.21 (C-aryl), 127.66 (i C-aryl), 129.22
(C-aryl), 130.67 (p-C-aryl),156.69 (Boc C=O), 165.48 ppm (tetrazole);
ESI-MS calcd for C28H43N6O9S: 639.28 [M+H]+; found: 639.20.
Next, the compound was dissolved in 5% aqueous DMF (100 mL),
and a solution of FeCl3 (10 mL of a 0.5m solution) was added. Next,
10 mg of 10% Pd-C were added, and the reaction mixture was
stirred for 5 h under hydrogen atmosphere. Subsequently, the re-
action mixture was filtered and evaporated, yielding 1.2 mg of the
desired compound: HR-MS calcd for C41H65N12O16S: 1013.4360
[MÀH]À; found: 1013.4360.
Synthesis of 2-deoxy-4,5-dihydroxy-1-[5-phenyltetrazole]-d-allo-
heptitol-7-N-(Leu)-sulfamate (27): Synthesis analogous to the
preparation of 1 was performed to afford 460 mg (89% total yield)
Synthesis of 3,6-anhydro-2-deoxy-4,5-isopropylidene-1-[5-(4-
phenoxy)phenyltetrazole]-d-allo-heptitol-7-[N-(Boc-l-Ile)-sulfa-
mate] (22): Synthesis analogous to the preparation of 21 was per-
formed to affording 260 mg (88% total yield) of compound 22:
13C NMR (125 MHz, CDCl3): d=11.85 (Ile-d-CH3), 15.90 (Ile-g-CH3),
25.22 (Ile-g’-CH3), 25.69 (isopropylidene CH3), 27.56 (isopropylidene
CH3), 28.67 (tBu-Boc), 33.33 (C2), 37.47 (Ile-b-CH2), 50.13 (C1), 69.00
(C7), 79.95 (C3), 81.78 (C5), 82.38 (C4), 84.78 (C6), 115.01 (C-isopro-
pylidene), 118.85, 119.84, 122.41, 124.27, 128.81, 130.21, 156.61,
156.88, 159.64 (aromatic signals), 156.51 (C=O Boc), 164.98 ppm
(tetrazole); ESI-MS calcd for C34H45N6O10S: 729.29 [MÀH]À; found:
729.18.
1
of compound 27: H NMR (300 MHz, MeOD): d=0.96–0.97 (d, J=
5.9 Hz, 3H; Leu-dB-CH3), 0.98–0.99 (d, J=6.1 Hz, 3H; Leu-dA-CH3)
1.56–1.62 and 1.76–1.80 (m, 2H; C1), 1.76–1.80 (m, 1H; Leu-g-CH),
2.20–2.26 (m, 1H; Leu-bA-CH2), 2.41–2.43 (m, 1H; Leu-bB-CH2), 3.57–
3.60 (dd, J=5.2, 8.5 Hz, 1H; Leu-a), 3.77–3.80 (m, 1H; C4), 3.83–
3.85 (t, J=4.8 Hz, 1H; C5), 4.00–4.02 (dd, J=3.7/7.5 Hz, 1H; C6),
4.11–4.19 (m, 1H; C3 and C7), 4.90 (m, 2H; C2), 7.50–7.51 (m, 1H;
m/p-aryl), 8.10–8.11 ppm (dd, J=1.5, 7.26 Hz, 1H; o-aryl); 13C NMR
(75 MHz, MeOD): d=22.13 (Leu-dB-CH3), 23.23 (Leu-dA-CH3), 25.86
(Leu-g-CH), 34.14 (C2), 42.33 (Leu-b-CH2), 51.36 (C1), 55.55 (Leu-a),
69.75 (C7), 73.01 (C3), 76.02 (C5), 80.59 (C4), 83.53 (C6), 127.74 (C-
aryl), 128.71 (i-C-aryl), 130.09 (C-aryl), 131.54 (p-C-aryl), 166.20 (tet-
razole), 176.47 ppm (C=O Leu); HR-MS calcd for C20H29N6O7S:
497.1819 [MÀH]À; found: 497.1822.
Synthesis of 3,6-anhydro-2-deoxy-1-[5-(4-phenoxy)phenyltetra-
zole]-d-allo-heptitol-7-[N-(l-Ile)-sulfamate] (3): Synthesis was per-
formed, analogous to the preparation of 1, to afford 260 mg (88%
total yield) of the compound 3: 1H NMR (500 MHz, MeOD): d=
0.88–0.93 (t, J=7.4 Hz, 3H; Ile-d-CH3), 0.98–0.99 (d, J=7.0 Hz, 3H;
Ile-g’-CH3), 1.23–1.27 (m, 1H; Ile-gB-CH2), 1.52–1.57 (m, 1H; Ile-gA-
CH2), 1.93 (m, 1H; Ile-b-CH), 2.15–2.36 (m, 2H; C2), 3.48–3.49 (d,
J=4.0 Hz, 1H; Ile-a-CH), 3.73–3.81 (m, 1H; C4), 3.95–3.96 (m, 1H;
C5), 4.05–4.11 (m, 4H; C3, C1, C6, C7), 6.99–7.37 (m, aromatic sig-
Synthesis of peptide-coupled 2-deoxy-4,5-dihydroxy-1-[5-phe-
nyltetrazole]-d-allo-heptitol-7-N-(Leu)-sulfamate (33): Synthesis
was performed, analogous to the preparation of 32, to afford
1.8 mg (4% total yield) of the compound 33: HR-MS calcd for
C45H71N16O16S2: 1155.4676 [MÀH]À; found: 1155.4683.
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A. Van Aerschot et al.
Synthesis of 3,6-anhydro-2-deoxy-4,5-O-isopropylidene-1-[5-
phenyltetrazole]-d-allo-heptitol-7-[N-[N-benzyloxycarbonyl-l-as-
partyl-(O-tBu)]-sulfamate] (24): A solution of (Z)-aspartyl-(O-tert-
butyl)-O-Su (460 mg, 0.15 mmol, 2.0 equiv), 3,6-anhydro-2-deoxy-
4,5-O-isopropylidene-1-[5-phenyltetrazole]-d-allo-heptitol-7-sulfa-
mate (19) (320 mg, 0.75 mmol, 1 equiv), and DBU (171 mL,
0.9 mmol, 1.2 equiv) in DMF (5 mL) was stirred at room tempera-
ture for 8 h. DMF was evaporated under reduced pressure, then
the residue was purified by flash chromatography (10% MeOH in
CH2Cl2). Fractions containing the desired product were evaporated
to afford 24 as colorless oil (420 mg, 81%). 13C NMR (75 MHz,
CDCl3): d=25.57 (isopropylidene CH3), 27.47 (isopropylidene CH3),
28.35 (tBu), 29.84 (C2), 38.45 (Asp-CH2) 50.04 (C1), 53.59 (Ca), 67.46
(CH2-Cbz), 69.83 (C7), 81.78 (C3), 81.96 (C-tBu), 82.09 (C5), 82.46
(C4), 84.76 (C6), 115.47 (C-isopropylidene), 127.18 (C-aryl), 127.67 (i
C-aryl), 128.51 (C-aryl), 129.21 (p C-aryl), 130.64 (C-aryl), 136.44 (i C-
aryl), 157.44 (C=O Asp), 165.49 (tetrazole), 172.42 ppm (C=O Asp);
ESI-MS calcd for C38H62N7O11SSi2: 880.4 [M+H]+; found: 880.1.
nals), 165.03 ppm (tetrazole); ESI-MS calcd for C23H26N4O5Na: 461.18
[M+Na]+; found: 461.25.
Synthesis of 3,6-anhydro-2-deoxy-4,5-O-isopropylidene-1-[5-(4-
phenoxy)phenyltetrazole]-d-allo-heptitol-7-sulfamate (20): Syn-
thesis was performed, analogous to the preparation of 19, to
afford 260 mg (88% total yield) of compound 20: 13C NMR
(75 MHz, CDCl3): d=25.69 and 27.59 (2ꢂCH3 isopropylidene), 33.74
(C2), 50.28 (C1), 70.57 (C7), 81.59 (C3), 82.35 (C5), 82.74 (C4), 85.05
(C6), 118.93, 120.01, 124.43, 128.89, 130.31 ppm (aromatic signals);
HR-MS calcd for C23H28N5O7S ([M+H]+): 518.1709; found: 518.1708.
Biological activity
Whole-cell activity determinations: The respective bacteria were
grown overnight in LB medium and cultured again the following
day in fresh LB medium or LB medium containing l-arabinose
(5 mm). Compounds were titrated in a 96-well plate using either LB
medium Æ l-arabinose (5 mm) to dilute the compounds. To each
well, 85 mL LB medium Æ l-arabinose (5 mm) was added to a total
volume of 90 mL. Next, 10 mL of bacterial cell culture, grown to
a OD600 of 0.1, was added. The cultures were next placed into
a Tecan Infinite M200 incubator and shaken at 378C; subsequently
the OD600 was determined after 8 h. All experiments have been
performed in triplicate.
Synthesis of 3,6-anhydro-2-deoxy-4,5-O-isopropylidene-1-[5-
phenyltetrazole]-d-allo-heptitol-7-[N-[l-aspartyl-(O-tert-butyl)]-
sulfamate] (25): Compound 24 (420 mg) was dissolved in a mixture
of MeOH with 4.4% HCOOH (15 mL), and Pd/C (100 mg) was
added. The mixture was stirred for 0.5 h, after which it was filtered
and evaporated to yield 25 as a white foam: (250 mg, 76%):
13C NMR (75 MHz, CDCl3): d=25.79 (isopropylidene CH3), 27.63 (iso-
propylidene CH3), 28.39 (tBu-Boc), 33.42 (C2), 35.63 (Asp-CH2) 50.27
(C1), 53.05 (Asp-Ca), 69.53 (C7), 81.91 (C3), 82.02 (C-tBu), 82.44
(C5), 83.11 (C4), 84.84 (C6), 115.02 (C-isopropylidene), 127.18 (C-
aryl), 127.75 (i C-aryl), 129.22 (C-aryl), 130.61 (p C-aryl), 165.39 (tet-
razole), 171.49 and 174.10 ppm (C=O Asp); ESI-MS calcd for
C32H31N6O9S: 555.2 [MÀH]À; found: 554.9.
Bacterial strains used for the evaluations: E. coli Ara-Yej (BW39758),
expressing the yejABEF transporter upon (l)-arabinose induction;
E. coli K-12 (BW28357) was used as the wild-type control. The anti-
bacterial activities of all compounds were determined by monitor-
ing the optical density of suspensions of cell cultures of the follow-
ing strains: Staphylococcus aureus ATCC 65388, Staphylococcus
epidermidis RP62A (ATCC 35984), Pseudomonas aeruginosa PAO1,
Sarcina lutea ATCC 9341, Candida albicans CO11.
Synthesis of peptide-coupled 3,6-anhydro-2-deoxy-4,5-O-isopro-
pylidene-1-[5-phenyltetrazole]-d-allo-heptitol-7-[N-[l-aspartyl-(O-
tert-butyl)]-sulfamate] (34): Synthesis was performed, analogous
to the preparation of 32, to afford 1.7 mg (15% total yield) of
compound 34: HR-MS calcd for C42H67N16O17S2: 1131.4311 [M+H]+;
found: 1131.4850.
Aminoacylation experiments: To assess the degree of inhibition of
the aminoacylation reaction, in vitro tests were performed using
the relevant S30 cell extracts.
Preparation of S30 cell extracts: Cells were grown in 50 mL LB
medium. After centrifution at 3000g for 10 min, the supernatant
was discarded, and the pellet was resuspended in 40 mL buffer
containing Tris·HCl or HEPES·KOH (pH 8.0, 20 mm), MgCl2 (10 mm),
KCl (100 mm). The cell suspension was centrifuged again at 3000g.
This procedure was repeated twice, then the pellet was resuspend-
ed in 1 mL of the following buffer: Tris·HCl or HEPES·KOH (pH 8.0,
20 mm), MgCl2 (10 mm), KCl (100 mm),and DTT (1 mm) and was
kept at 08C. Subsequently, the cells were sonicated for 10 s and
left at 08C for 10 min. This procedure was repeated 5–8 times,
then the lysate was centrifuged at 15000g for 30 min at 48C.
Synthesis of 3,6-anhydro-7-O-p-anisyldiphenylmethyl-1,2-di-
deoxy-4,5-O-isopropylidene-1-[5-(4-phenoxy)phenyltetrazole]-d-
allo-heptitol (16): Synthesis was performed, analogously to the
preparation of 15, to afford 410 mg (71% total yield) of compound
16: 13C NMR (75 MHz, CDCl3): d=25.93 and 27.83 (2ꢂCH3 isopropy-
lidene), 33.96 (C2), 50.45 (C1), 55.56 (O-CH3), 64.57 (C7), 81.68 (C3),
82.72 (C5), 83.83 (C4), 85.02 (C6), 86.79 (C-trityl), 114.79 (C-isopro-
pylidene), 113.49, 118.93, 119.89, 122.63, 124.28, 127.29, 128.19,
128.77, 128.85, 130.26, 130.72, 135.76, 144.61, 156.72, 158.96 (aro-
matic signals), 159.64 ppm (tetrazole); ESI-MS calcd for
C43H42N4O6Na: 733.30 [M+Na]+; found: 733.24.
tRNA aminoacylation reaction: E. coli S30 extracts (3 mL) were added
to 1 mL of solution containing inhibitor. Next, 16 mL of the follow-
ing aminoacylation mixture were added: Tris·HCl (30 mm, pH 8.0),
DTT (1 mm), bulk of E. coli tRNA (5 gLÀ1), ATP (3 mm), KCl (30 mm),
MgCl2 (8 mm), and the specified 14C-radiolabeled amino acid
(40 mm, 200 mCimmolÀ1). The reaction products were precipitated
in cold 10% TCA on Whatman 3 MM papers 5 min. after the ami-
noacylation mixture was added. The aminoacylation reaction was
carried out at room temperature. Depending on whether or not
processing was needed, variable time intervals were included be-
tween the addition of the cell extract and the addition of the ami-
noacylation mixture. After thorough washing with cold 10% TCA,
the papers were washed twice with acetone and dried on a heating
plate. Following the addition of scintillation liquid (12 mL), the
amount of radioactivity was determined in a Tri-card 2300 TR
liquid scintillation counter. Reported inhibitory values are based on
Synthesis of 3,6-anhydro-2-deoxy-7-hydroxy-4,5-O-isopropyli-
dene-1-[5-(4-phenoxy)phenyltetrazole]-d-allo-heptitol (18): Syn-
thesis was performed, analogous to the preparation of 17, to
afford 248 mg (98% total yield) of compound 18: 1H NMR
(500 MHz, CDCl3): d=1.34 (s, 3H; CH3), 1.53 (s, 3H; CH3), 2.26–2.56
(m, 2H; C1) 3.67–3.46 (ABX, JAB =12.60 Hz/JAX ꢀ3.0/JBX ꢀ3.75 Hz,
2H; C7), 3.95–4.05 (m, 2H; C6, C3), 4.34–4.38 (m, 1H; C4), 4.64–
4.89 (m, 3H; C5, C2), 7.07–7.19 and 7.36–7.41 and 8.09–8.12 ppm
(aromatic signals); 13C NMR (125 MHz, CDCl3): d=25.71 and 27.65
(2CH3 isopropylidene), 33.18 (C2), 50.23 (C1), 62.90 (C7), 81.75 (C3),
81.86 (C5), 84.79 (C4), 84.83 (C6), 115.17 (C-isopropylidene), 118.87,
119.91, 122.35, 124.30, 128.79, 130.23, 156.59, 159.72 (aromatic sig-
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Aryl-tetrazole Substituents in McC and Albomycin Analogues
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Acknowledgements
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for their help with some of the NMR spectra and to Prof. Jef Ro-
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for help with the preparation of some of the compounds. G.H.V.
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Keywords: aaRS · aminoacyl tRNA synthetases · inhibitors ·
antibiotics · microcin C · siderophores
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Received: March 12, 2012
Published online on && &&, 0000
ChemBioChem 0000, 00, 1 – 12
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FULL PAPERS
G. H. Vondenhoff, B. Gadakh, K. Severinov,
A. Van Aerschot*
Synthetases’ Achilles’ heel? Selective
inhibition of bacterial aminoacyl tRNA
synthetases was previously accom-
plished by using aminoacyl sulfamoyla-
denosines and substituting aryltetrazole
moieties for the adenine heterocycle.
While these compounds did not prove
successful in vivo, conjugation to pepti-
dic Trojan horses or siderophore drug
conjugate (SDC) transport modules en-
visaged improved uptake.
&& – &&
Microcin C and Albomycin Analogues
with Aryl-tetrazole Substituents as
Nucleobase Isosters Are Selective
Inhibitors of Bacterial Aminoacyl tRNA
Synthetases but Lack Efficient Uptake
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!