Exploring the ribose sub-pocket of the substrate-binding site in escherichia coli IspE: Structure-based design, synthesis, and biological evaluation of cytosines and cytosine analogues

Article abstract of DOI:10.1002/ejoc.201200296

The enzymes of the non-mevalonate pathway for the isoprenoid biosynthesis are promising targets for the development of selective drugs for the treatment of important infectious diseases. This pathway is used by plants, many eubacteria, and apicomplexan pr

Full text of DOI:10.1002/ejoc.201200296

Job/Unit: O20296
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FULL PAPER
DOI: 10.1002/ejoc.201200296
Exploring the Ribose Sub-Pocket of the Substrate-Binding Site in Escherichia
coli IspE: Structure-Based Design, Synthesis, and Biological Evaluation of
Cytosines and Cytosine Analogues
Andri P. Schütz,[a] Sho Osawa,[a] Jennifer Mathis,[a] Anna K. H. Hirsch,[b] Bruno Bernet,[a]
Boris Illarionov,[c] Markus Fischer,*[c] Adelbert Bacher,*[c] and François Diederich*[a]
Keywords: Enzymes / Ligand design / Inhibition / Molecular recognition
The enzymes of the non-mevalonate pathway for the iso-
prenoid biosynthesis are promising targets for the develop-
ment of selective drugs for the treatment of important infec-
ribose sub-pocket of the CDP-ME binding site. Therefore, we
synthesized cytosine- and 2-aminopyridine-based inhibitors
with various substituents targeting this sub-pocket at the en-
tious diseases. This pathway is used by plants, many eubac- zyme active site. As cytosines display unexpectedly low solu-
teria, and apicomplexan protozoa, including major human
pathogens such as Plasmodium falciparum and Mycobacte-
rium tuberculosis, but not by humans who use the mevalon-
ate pathway. In this work, we report on the design, synthesis,
and biological evaluation of new ligands for the E. coli en-
zyme IspE. The focus of the study lies in the analysis of the
bilities in aqueous solution, special efforts were made to in-
crease the water solubility of some compounds while main-
taining the good binding affinities measured in earlier stud-
ies. In vitro studies showed IC50 values in the low micromolar
to submicromolar range against E. coli IspE.
ate pathway. In 1999, Jomaa et al. reported curing malaria
in rodents with fosmidomycin,[4] thereby validating the en-
zymes of the non-mevalonate pathway as highly attractive
drug targets. Fosmidomycin is a potent inhibitor of the sec-
ond enzyme in the pathway, 1-deoxy-d-xylulose 5-phos-
phate reductoisomerase (IspC). Clinical trials with fosmido-
mycin are ongoing[5] as is the active search for more potent
derivatives.[6]
Introduction
Protozoan parasites belonging to the genus Plasmodium
are the causative agents of malaria. Half of the world’s hu-
man population lives in malaria-endangered regions, caus-
ing each year 300–500 million infections and more than one
million deaths.[1] Although different drug combinations[2]
are used in the treatment of malaria, an increasing number
of multi-drug-resistant Plasmodium strains have been re-
ported,[1a,2a,3] clearly showing the urgency for the develop-
ment of new antimalarial drugs.
A promising strategy for developing new compounds
against parasitic diseases involves targeting enzymes that
are only present in the pathogens and not in the human
host. In plasmodial parasites, the biosynthesis of the two
essential isoprenoid precursor molecules, isopentenyl di-
phosphate (IPP) and dimethylallyl diphosphate (DMAPP),
proceeds by the non-mevalonate pathway (Scheme 1),
whereas in humans it proceeds exclusively by the mevalon-
A rapidly increasing number of X-ray crystal structures
of the seven enzymes of the non-mevalonate pathway have
recently been deposited in the PDB (RCSB Protein Data
Bank).[7] On the basis of this structural information, our
research group has designed and prepared the first families
of ligands for the kinase IspE [4-diphosphocytidyl-2-C-
methyl-d-erythritol (CDP-ME) kinase, EC 2.7.1.148], the
fourth enzyme in the pathway.[8] Kinase IspE requires ATP
and Mg2+ for the phosphorylation of the 2-hydroxy group
of CDP-ME, which affords 4-diphosphocytidyl-2-C-methyl-
d-erythritol 2-phosphate (CDP-ME2P; Scheme 1).[9]
We chose E. coli IspE as a surrogate for Plasmodium fal-
ciparum and Mycobacterium tuberculosis IspE.[8c–8e] Until
today, no IspE X-ray crystal structure of the Plasmodium
enzyme could be solved as expression and purification of
these enzymes has been rather difficult. The structural data
on M. tuberculosis IspE were published only after the com-
pletion of this work. Interestingly, its rather conserved
CDP-ME binding domain shows a high structural homol-
ogy with the CDP-ME site of E. coli IspE.[8g] Earlier re-
search yielded the 1-thiolanylated 5-ethynylated cytosine
[a] Laboratorium für Organische Chemie, ETH Zürich,
Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland
Fax: +41-44-632-11-09
E-mail: diederich@org.chem.ethz.ch
[b] Stratingh Institute for Chemistry, University of Groningen,
Nijenborgh 7, 9747 AG Groningen, The Netherlands
[c] Hamburg School of Food Science, Institut für
Lebensmittelchemie, Universität Hamburg,
Grindelallee 117, 20146 Hamburg, Germany
Fax: +49-40-428-38-43-42
E-mail: markus.fischer@hsfs.uni-hamburg.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200296.
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FULL PAPER
Scheme 1. Non-mevalonate pathway for the biosynthesis of the isoprenoid precursors IPP and DMAPP. DXS: 1-deoxy-d-xylulose 5-
phosphate synthase (EC 2.2.1.7); IspC: 1-deoxy-d-xylulose 5-phosphate reductoisomerase (EC 1.1.1.267); IspD: 4-diphosphocytidyl-2-C-
methyl-d-erythritol transferase (EC 2.7.7.60); IspF: 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (EC 4.6.1.12); IspG: 2-C-
methyl-d-erythritol 2,4-cyclodiphosphate reductase (EC 1.17.4.3); IspH: 1-hydroxy-2-methyl-(2E)-butenyl 4-diphosphate reductase (EC
1.17.1.2).
(Ϯ)-1 as the most active inhibitor of E. coli IspE (median and thietanedimethanol (Ϯ)-6. Earlier work on inhibitors
inhibitory concentration IC50
=
1.7Ϯ0.2 μm, Ki
=
of IspF[10] showed that 2-aminopyridines are a reasonable
0.29Ϯ0.10 μm; Figure 1). The corresponding 1-cyclopent- substitute for cytosines (for cytosine substitutes, see ref.[11]).
ylated cytosine 2 is also a good inhibitor of E. coli IspE Therefore, we also prepared the 2-aminopyridines 7a–i
(IC50 = 16Ϯ0.5 μm, Ki = 1.5Ϯ0.2 μm).[8d] These inhibitors bearing aryl moieties to fill the ribose sub-pocket.
bind to the cytidine pocket and the ME/phosphate binding
The biological studies performed with these ligands have
site (see below), leaving the adenosine binding site open for further enhanced the understanding of the molecular re-
its natural substrate.
cognition properties of the IspE enzyme and cytosine-bind-
ing proteins in general. Cytosine replacements[11] are far less
abundant and documented than adenine replacements,[12]
for example, in kinase inhibitors,[13] or thymine surrogates,
for example, in antitumor drugs.[14] Understanding cytosine
recognition will become of growing importance in view of
the interest in cytosine methylation/demethylation at the 5-
position,[15] which is increasingly recognized as a major epi-
genetic mechanism in switching on (demethylated) or off
(methylated) genes.[16]
Results and Discussion
Figure 1. Structures and inhibition data of thiolane (Ϯ)-1 and cy-
clopentane 2.
Design
Molecular modeling (MOLOC[17]) of the co-crystal
Herein we report the design, synthesis, and biological ac- structure of E. coli IspE with bound CDP-ME and the ATP
tivity of more polar and more water-soluble O-containing analogue ADPNP [adenosine-5Ј-(γ-imino)triphosphate]
analogues of (Ϯ)-1, namely 2Ј-deoxycytidine 3 with a very (2.0 Å resolution, PDB code 1OJ4)[8a] suggested that the
similar structure, oxolane (Ϯ)-4, 4-ethoxybutanol (Ϯ)-5, central cytosine scaffold of the E. coli IspE inhibitor is
2
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Cytosines and Cytosine Analogues
Figure 2. Complexation of inhibitor (S)-1 in the cytidine binding pocket of the active site of E. coli IspE (PDB code: 1OJ4) modeled
with MOLOC.[17] Both enantiomers of (Ϯ)-1 are expected to bind with similar strength. Color code: carbon: green; nitrogen: blue;
oxygen: red; sulfur: yellow. Heteroatom distances are given in Å. This color code is maintained throughout the article, unless otherwise
stated.
sandwiched by Phe185 and partially by Tyr25 and forms
In further attempts to obtain the degree of water solubil-
three hydrogen bonds with the backbone and the side-chain ity required for co-crystallization, we targeted the 2Ј-deoxy-
of His26.[8d] This binding mode is depicted in Figure 2 for ribose derivative 3, for which modeling with MOLOC pre-
ligand (Ϯ)-1 and is strongly supported by the X-ray struc- dicted a hydrogen bond from 3Ј-OH to the side-chain of
ture of Aquifex aeolicus IspE co-crystallized with a closely Lys186 (Figure S1). The 2Ј-deoxyribose ring was expected
related inhibitor.[8d] The small and rather lipophilic cy- to adopt a similar geometry to the one seen for the ribose
clopropylsulfonamido moiety appears to contribute signifi- of CDP-ME in the co-crystal structure (1OJ4). A further
cantly to the good binding affinity of (Ϯ)-1. The sulfon- increase in water solubility was expected for 4-ethoxybut-
amido moiety, in the favorable staggered conformation with anol (Ϯ)-5, the ethoxy group of which, according to the
the N lone pair bisecting the O=S=O angle,[8d] can form molecular modeling, should engage in similar interactions
hydrogen bonds with the catalytically important residues with the amino acids of the ribose sub-pocket to those ob-
Lys10 and Asp141. The cyclopropyl ring fills a small, highly served for the cyclic analogues 3 and (Ϯ)-4 (Figure S1).
preorganized hydrophobic pocket lined by the side-chains With thiethane (Ϯ)-6 (Figure S2) we hoped to gain further
of Leu15, Leu28, and Phe185, which is not occupied by the insight into the postulated S···π interaction of (Ϯ)-1 while
substrate CDP-ME.
at the same time enhancing water solubility. One of the hy-
Keeping the C-5 substituent of (Ϯ)-1 unchanged, we droxymethyl groups of (Ϯ)-6 was expected to form a hydro-
wished to investigate the interactions of different N-1 sub- gen bond with Lys186 similar to the one displayed by the
stituents with the amino acids Tyr25, His26, and Lys186 in bound natural substrate CDP-ME, whereas the second hy-
the ribose sub-pocket of the CDP-ME binding site. Given droxymethyl group has no partner to form an additional
that the cyclopentane 2 shows a 10-fold lower binding affin- hydrogen bond and may just point into bulk water. Accord-
ity than thiolane (Ϯ)-1, an S···π interaction[18] with the ing to the modeling, both enantiomers of (Ϯ)-4–6 should
phenol ring of Tyr25 had previously been postulated to ex- fit well into the active site of E. coli IspE without any pref-
plain the better binding of the latter (Figure 2).[8d] We erence for one over the other.
hoped to test this hypothesis by evaluating the affinity of
In the series of 2-amino-5-arylpyridine-derived ligands,
the tetrahydrofuranyl derivative (Ϯ)-4, the O atom of which compounds 7a and 7g–7i feature a planar or nearly planar
is not expected to be directed into the π cloud of the phenol biaryl system, whereas in the ortho-substituted 7b–f the two
ring.[19] A co-crystal structure of bound (Ϯ)-1 could not be aromatic rings should have an increased torsional angle.[20]
obtained to prove the S···π interaction due to the low solu-
Modeling suggested that the ribose binding site in be-
bility of the ligand in pure aqueous buffer and because the tween the side-chains of Pro182 and Tyr25 is actually too
co-crystallization protocol is intolerant to even small large to enable close van der Waals interactions with an aryl
amounts of organic co-solvents.
ring in the plane of the aminopyridine ring. According to
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FULL PAPER
the molecular modeling (Figure S3), an aromatic ring ylated cytosine with (Ϯ)-14 gave 35% of the thietanylated
twisted out of the plane of the aminopyridine ring could iodocytosine (Ϯ)-15 (Scheme 3). Interestingly, a Pummerer-
possibly establish better intermolecular contacts, and we in- type reaction with the corresponding thietane 1-oxide gave
tended to verify this hypothesis in our studies.
only traces of the desired product, whereas a similar reac-
tion with uracil provided acceptable yields of the prod-
uct.[25] Debenzoylation of (Ϯ)-15 and subsequent Sonoga-
shira coupling with sulfonamide 10 afforded the desired thi-
etanylated and ethynylated cytosine (Ϯ)-6, albeit in a low
Synthesis
Iodination of 2Ј-deoxycytidine (8) according to the pro- yield (14%). The inverse sequence of reactions, coupling be-
cedure of Kumar et al.,[21] but without the use of ionic li- fore debenzoylation, failed as the coupling product decom-
quids, gave 9[22] in 57% yield (Scheme 2). Sonogashira cou- posed under the conditions of debenzoylation.
pling of 9 with the known sulfonamide 10[8c] gave the 5-
The 2-fluorothietane (Ϯ)-14 shows characteristic J(H,F)
1
ethynylated 2Ј-deoxycytidine 3 in 60% yield. The syntheses couplings in the H NMR spectrum. The F atom couples
of (Ϯ)-4 and (Ϯ)-5 started from 5-iodocytosine (11).[23] For with the geminal 2-H [2J(H,F) = 63.3 Hz], the cis-oriented
the acetalization of 11 at N-1, the intermediate formation 4-H [4J(H,F) = 9.0 Hz], and the cis-oriented methylene
of 2-O-silylated 5-iodocytosine[24] is crucial for acceptable group [4J(H,F) = 3.0 Hz and Ͻ 1.5 Hz (line-broadening)].
yields. The TsOH-catalyzed addition of silylated 11 to 2,3- Nishizono et al.[25] only reported the geminal J(H,F) cou-
2
dihydrofuran yielded (Ϯ)-12 (19% yield), whereas the reac- pling. The coupling of the pseudoequatorial F atom of (Ϯ)-
tion with 2-ethoxyoxolane and TMSOTf led to the butanol 14 with 4-Hcis is a W coupling transmitted through S and
(Ϯ)-13 (18% yield). Sonogashira coupling of (Ϯ)-12 and C-2 of the slightly puckered thietane ring, whereas a close
(Ϯ)-13 with sulfonamide 10 gave the desired target com- contact is responsible for the couplings with the H atoms
pounds (Ϯ)-4 and (Ϯ)-5 in yields of 58 and 59%, respec- of the cis-methylene group. A W coupling of 6 Hz between
tively (Scheme 2).
F and 4-Hcis of a more strongly puckered thietane S-oxide
The procedure of Nishizono et al.[25] was applied to the has previously been observed.[26] Calculations with Spar-
synthesis of the 2-fluorothietane (Ϯ)-14 by starting from tan 10[27] (B3LYP/6-31* level of theory) correlated with the
2,2-bis(hydroxymethyl)-1,3-propanediol (five steps). Sil- weak puckering of (Ϯ)-14 and the strong puckering of thiet-
ylation under Vorbrüggen conditions[24] of iodocytosine 11 ane S-oxide, as indicated by the S–C1–C2–C3 torsion
and AgClO4-promoted condensation of the resulting sil- angles of 8 and 28°, respectively (Scheme 3).
Scheme 2. Synthesis of inhibitors 3, (Ϯ)-4, and (Ϯ)-5. Reagents and conditions: (a) N-iodosuccinimide (NIS), CH2Cl2, 22 °C, 57%; (b) 10,
NEt3, [Pd(PPh3)2Cl2], CuI, DMF, 22 °C, 60% of 3, 58% of (Ϯ)-4, and 59% of (Ϯ)-5; (c) hexamethyldisilazane (HMDS), 75 °C, 2,3-
dihydrofuran, TsOH·H2O (cat.), CH2Cl2, 22 °C, 19%; (d) HMDS, 60 °C, 2-ethoxyoxolane, TMSOTf, TsOH·H2O (cat.), CH2Cl2, 22 °C,
18%.
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Cytosines and Cytosine Analogues
Scheme 3. Synthesis of inhibitor (Ϯ)-6. Reagents and conditions: (a) HMDS, reflux; (Ϯ)-14, AgClO4, SnCl2, CH2Cl2, 22 °C, 35%;
(b) NaOMe, MeOH/CH2Cl2, 22 °C; 10, NEt3, [Pd(PPh3)2Cl2], CuI, DMF, 22 °C, 14%.
Scheme 4. Synthesis of inhibitors 7a–7i. Reagents and conditions: (a) 10, [Pd(PPh3)2Cl2], CuI, NEt3, THF, 25 °C, 87%; (b) 18a–i,
[Pd(dppf)Cl2]·CH2Cl2, 2 m aq. Na2CO3, 1,2-dimethoxyethane (DME), reflux; 59% of 7a, 54% of 7b, 43% of 7c, 58% of 7d, 56% of 7e,
40% of 7f, 60% of 7g, 69% of 7h, and 25% of 7i.
Compounds 7a–i were synthesized by two consecutive drogenase reduces pyruvate to lactate with 1 equiv. of
cross-coupling reactions. Sonogashira coupling of 2-amino- NADH. The NADH consumption was monitored at a
5-bromo-3-iodopyridine (16), which has previously been wavelength of 340 nm as NAD+ does not absorb light at
prepared by Beard et al.,[28] with sulfonamide 10 yielded the this wavelength. The photometric assay has previously been
ethynylated 5-bromopyridine 17 (Scheme 4), the common proven to work effectively for similar compounds by using
precursor for the desired biaryls. Suzuki coupling reactions a direct NMR spectroscopic assay.[31]
of 17 with the commercially available boronic acids 18a–g,
According to the biological data in Table 1, the pre-
the known boronic acid 18h,[29] and the pinacol boronate viously reported ligand (Ϯ)-1 remains the best inhibitor of
18i[30] led to the targeted compounds 7a–i in moderate to E. coli IspE. Besides providing an optimal volume occu-
good yields (7a–h: 40–69%; 7i: 25%), (Scheme 4).
pancy for the ribose binding site, inhibitor (Ϯ)-1 has also
been proposed to benefit from an S···π interaction with
Tyr25. The results presented herein apparently disprove this
proposal. Oxolane (Ϯ)-4 shows nearly the same binding af-
Biological Results
The IC50 values for ligands 3, (Ϯ)-4–6, and 7a–i were finity as thiolane (Ϯ)-1, which would not have been ex-
determined in an enzyme-coupled photometric assay[31] by pected if the latter can undergo a particular interaction that
using E. coli IspE, pyruvate kinase, and lactate dehydro- is absent in the complex of (Ϯ)-4. According to the molecu-
genase. Per catalytic turnover, 1 equiv. of ATP was used for lar modeling, both five-membered rings can adopt favorable
the phosphorylation of CDP-ME to CDP-ME2P, which conformations with their heteroatom either approaching
was restored by the pyruvate kinase under the conversion the tyrosine ring or turning away from it. We propose that
of phospoenol pyruvate (PEP) to pyruvate. Lactate dehy- the oxolanyl ring of (Ϯ)-4 adopts a conformation similar to
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Table 1. Inhibitory activities of inhibitors (Ϯ)-1, 2, 3, (Ϯ)-4–6, and 7a–i for E. coli IspE and calculated physicochemical properties.
FULL PAPER
clogP[a]
clogD[a] at pH 8.0
IC50 [μm]
Ki[b] (calcd.) [μm]
Ki[c] (exptl.) [μm]
(Ϯ)-1
2
1.46Ϯ0.76
2.02Ϯ0.63
–2.41Ϯ0.81
–0.27Ϯ0.73
0.17Ϯ0.73
–0.19Ϯ0.86
2.02Ϯ1.06
2.57Ϯ1.06
3.08Ϯ1.06
1.47Ϯ1.09
1.72Ϯ1.09
2.18Ϯ1.08
1.83Ϯ1.07
1.73Ϯ1.07
2.61Ϯ1.07
1.32
1.89
–2.27
–0.41
0.04
–0.33
2.01
2.56
3.07
1.46
1.71
2.17
1.82
1.72
2.60
1.7Ϯ0.2
16Ϯ0.5
36Ϯ2
2.0Ϯ0.2
152Ϯ0.9
103Ϯ7
Ն 500
480Ϯ44
Ͼ 500
242Ϯ24
341Ϯ24
273Ϯ35
193Ϯ9
Ͼ 500
0.6
5.1
13.6
0.7
57
38.6
–
180
–
91
128
102
138
–
0.29Ϯ0.1
1.5Ϯ0.2
3
–
–
–
–
(Ϯ)-4
(Ϯ)-5
(Ϯ)-6
7a
128Ϯ12
7b
–
–
–
–
–
–
–
–
7c
7d
7e
7f
7g
7h
7i
Ͼ 500
–
[a] clogP and clogD values were calculated by using Advanced Chemistry Development (ACD/Labs) Software V12.5 (© 1994–2012 ACD/
Labs). [b] Ki values were calculated from IC50 values and Km = 150 μm (E. coli IspE) from the Cheng–Prusoff equation.[33] [c] Ki values
were measured in a spectrophotometric assay by varying the substrate concentration from 0 to 400 μm.
the furanosyl ring of the natural substrate in which the O
Conclusions
atom does not point into the π cloud of the phenol side-
We have described herein the design, synthesis, and in
vitro evaluation of a series of inhibitors of the kinase IspE
(E. coli). Valuable insights into the molecular recognition
properties of the substrate binding site of E. coli IspE have
chain. The cyclopentyl derivative 2[8d] binds more weakly
than (Ϯ)-1 and (Ϯ)-4 by approximately a factor of eight.
The heteroatoms of the alicyclic heterocycles induce local
dipoles, which could provide a better interaction with the
been obtained. (1) Low-molecular-weight inhibitors with
protein than the cyclopentyl substituent.[32]
IC50 values of ca. 1 μm are obtained when appropriately
functionalized cytosines direct a five-membered alicyclic
heterocycle into the ribose sub-pocket. (2) Direct interac-
Polar, water-solubility-enhancing substituents are not
well tolerated near the ribose binding site (Table 1). 2Ј-De-
oxycytidine 3, the only enantiomerically pure compound of
tions of heterocyclic S atoms with the phenolic ring of
this series, shows an approximately 20-fold larger IC50 value
Tyr25 are not effective; however, the local dipoles induced
by the O or S atoms in the alicyclic heterocycles enhance
their binding relative to cyclopentyl-substituted ligands by
than (Ϯ)-1 or (Ϯ)-4. The 4-ethoxybutanol derivative (Ϯ)-5
binds more weakly than (Ϯ)-1 and (Ϯ)-4 by almost a factor
of 100. Inhibitors 3, (Ϯ)-5, and (Ϯ)-6 are moderately water-
nearly an order of magnitude. (3) Substituted aminopyr-
soluble, and co-crystallization experiments are ongoing to
idines are potential cytosine substitutes, although the aryl-
provide a deeper insight into the molecular recognition
substituted derivatives prepared in this study are rather
modes in the cytidine binding pocket.
modest binders; the ribose sub-pocket does not seem to be
an effective aryl-binding site. (4) Although flexible polar
functional groups do not enhance but rather reduce binding
affinity, water-soluble ligands are obtained that should now
The planar biaryl ligand 7g exhibits a weak binding af-
finity (IC50 = 193 μm), whereas the other planar derivatives
(7a, 7h, and 7i) show no inhibition within the measurement
limits of the biological assay (IC50 Ն 500 μm, Table 1). The
enable successful co-crystallization with IspE.
twisted biaryls 7b–7f, with the exception of 7c, show mea-
surable inhibition of E. coli IspE. Methyl ether 7d, 3-meth-
ylated thiophene 7f, and thioether 7e display similar com-
plexation strengths, which rules out any contributions of an
Experimental Section
S···π interaction to the binding of 7e and 7f. Overall, there Materials and General Methods: Reagents were purchased reagent-
grade or, if necessary, purified by distillation. Dry solvents (DMF,
CH2Cl2, MeOH, and THF) were purified by a solvent-drying sys-
tem from LC Technology Solutions Inc. S-105 under nitrogen (H2O
content Ͻ 10 ppm as determined by Karl-Fischer titration). All re-
actions were performed in oven-dried glassware under Ar or N2.
TLC: Glass sheets coated with SiO2 60 F254 from Merck, visualiza-
tion by UV light at 254 nm, and staining with a solution of KMnO4
(1.5 g) and K2CO3 (10 g) in H2O (150 mL). Column chromatog-
raphy: SiO2 (60) from Fluka. Melting points were determined in
an open capillary by using a Büchi-510 apparatus. IR spectra were
recorded with a Perkin–Elmer BX FT-IR spectrophotometer (ATR
is no substantial difference in the binding affinities of
planar and twisted biaryls. A direct comparison between
the binding data for ligands with cycloalkyl or alicyclic het-
erocycles filling the ribose sub-pocket and those with aro-
matic rings is not possible as the change from cytosine to its
substitute aminopyridine lowers the overall binding affinity.
Nevertheless, the data seem to suggest that the ribose sub-
pocket in the substrate binding site of E. coli IspE is better
filled by cycloalkyl and alicyclic heterocyclic residues than
by aromatic rings.
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Cytosines and Cytosine Analogues
unit, Golden Gate) neat or as a film. NMR spectra were recorded
at 22 °C with a Varian Gemini 300 or Bruker DRX 400 spectrome-
ter with an automated sample changer by using the solvent peak
as internal reference. Coupling constants (J) are given in Hz. Reso-
nance multiplicities are described as s (singlet), br. s (broad singlet),
d (doublet), t (triplet), q (quartet), quint (quintet), and m (mul-
tiplet). High-resolution mass spectra (HRMS) were recorded with a
Bruker maXis-ESI-Q-TOF (ESI) or Micromass (Waters) AutoSpec
Ultima-EI-EBE-MS (EI) spectrometer. The masses are given as m/z
with [M]+ representing the molecular ion. Elemental analyses were
performed by the Mikrolabor at the Laboratorium für Organische
Chemie, ETH Zürich. Nomenclature is given in accord with nucleic
acid nomenclature or proposals of the computer program ACD/
Name. Experimental procedures for the synthesis of compounds
7b–i are available in the Supporting Information.
4.08 (s, 2 H, CH2NH), 4.20 (td, J = 8.2, 4.2 Hz, 1 H, 5ЈЈ-H), 5.86
(dd, J = 6.2, 2.6 Hz, 1 H, 2ЈЈ-H), 7.61 (s, 1 H, 6Ј-H) ppm. 13C
NMR (75 MHz, CDCl3/CD3OD, 6:1): δ = 5.36 (2 C), 23.33, 24.60,
30.12, 33.10, 63.63, 70.22, 74.81, 88.29, 91.39, 142.79, 154.62,
164.61 ppm. IR (ATR): ν = 3426 (w), 3324 (w), 2961 (w), 2874 (w),
˜
2235 (w), 1631 (s), 1611 (s), 1572 (s), 1471 (s), 1276 (m), 1249 (m),
1180 (m), 1121 (w), 1066 (s), 1032 (m), 950 (m), 922 (m), 897 (m),
779 (s), 736 (m), 619 (s) cm–1. HRMS (ESI): m/z (%) = 339.1121
(100) [M + H]+, 269.0703 (99) [M – C4H6O + H]+. HRMS (ESI):
calcd. for C14H19N4O4S+ [M + H]+ 339.1122; found 339.1121;
calcd. for C10H13N4O3S+ [M – C4H6O + H]+ 269.0703; found
269.0703.
(؎)-N-{3-[1-(1-Ethoxy-4-hydroxybutyl)cytosin-5-yl]prop-2-yn-1-yl}-
cyclopropanesulfonamide [(؎)-5]: A solution of (Ϯ)-13 (100 mg,
0.28 mmol), 10 (100 mg, 0.62 mmol), and NEt3 (0.2 mL,
1.44 mmol) in DMF (4 mL) was degassed by freeze–pump–thaw
cycles performed under Ar. [Pd(PPh3)2Cl2] (52 mg, 0.04 mmol) and
CuI (32 mg, 0.07 mmol) were added, and the mixture was stirred at
25 °C for 6 h. Concentration and chromatography (SiO2; CH2Cl2/
MeOH, 97:3) yielded (Ϯ)-5 (64 mg, 59%) as an orange, viscous oil.
Rf = 0.38 (SiO2; CH2Cl2/MeOH, 95:5). 1H NMR (300 MHz,
CDCl3/CD3OD, 6:1): δ = 0.90–0.99 (m, 2 H, 2,3-H), 1.06–1.18 (m,
2 H, 2,3-H), 1.11 (t, J = 7.1 Hz, 3 H, OCH2CH3), 1.44–1.60 (m, 2
H, 2ЈЈ-H), 1.61–1.72 (m, 2 H, 3ЈЈ-H), 3.39 (q, J = 7.0 Hz, 2 H,
OCH2CH3), 3.50–3.56 (m, 2 H, 4ЈЈ-H), 4.08 (s, 2 H, CH2NH), 5.63
(t, J = 6.1 Hz, 1 H, 1ЈЈ-H), 7.59 (s, 1 H, 6Ј-H) ppm. 13C NMR
(75 MHz, CDCl3/CD3OD, 6:1): δ = 5.50 (2 C), 8.62, 27.35, 30.24,
32.29, 33.27, 61.11, 65.05, 68.55, 74.55, 86.37, 92.04, 142.68,
Enzyme-Coupled Photometric Assay for IC50 Determination: Assay
mixtures were prepared as previously described[31] with some minor
modifications: A solution (60 μL) containing 100 mm Tris-HCl (pH
= 8.0), 10 mm MgCl2, 2 mm dithiothreitol, 2.5 mm phosphoenolpyr-
uvate potassium salt, 2 mm ATP, 0.46 mm NADH, 1 U of lactate
dehydrogenase, 1 U of pyruvate kinase, and 1 U of IspE protein
were added to the inhibitor solution (60 μL, final concentration of
8–1000 μm). The reaction was started by addition of CDP-ME
(60 μL, final concentration 250 μm) and monitored at a wavelength
of 340 nm at 27 °C.
5-[3-(Cyclopropylsulfonylamino)prop-1-yn-1-yl]-2Ј-deoxycytidine (3):
A solution of 9 (100 mg, 0.29 mmol), 10 (100 mg, 0.62 mmol), and
NEt3 (0.2 mL, 1.4 mmol) in DMF (5 mL) was degassed by freeze–
pump–thaw cycles performed under Ar. [Pd(PPh3)2Cl2] (42 mg,
0.06 mmol) and CuI (27 mg, 0.14 mmol) were added, and the mix-
ture was stirred at 25 °C for 4 h. Concentration and chromatog-
raphy (SiO2; CH2Cl2/MeOH, 93:7) yielded 3 (64 mg, 60%) as a
brown solid (containing triethylammonium salt). Rf = 0.32 (SiO2;
CH2Cl2/MeOH, 92:8). [α]2D2 = +15.0 (c = 0.07, CH2Cl2/MeOH, 1:1).
M.p. 154–157 °C. 1H NMR (300 MHz, CDCl3/CD3OD, 5:1): δ =
0.75–0.86 (m, 2 H, 2ЈЈ,3ЈЈ-H), 0.89–0.97 (m, 2 H, 2ЈЈ,3ЈЈ-H), 1.88–
1.99 (m, 1 H, 2Ј-H), 2.15–2.25 (m, 1 H, 2Ј-H), 2.31 (tt, J ≈ 8.0,
3.9 Hz, 1 H, 1ЈЈ-H), 3.54 (br. d, J = 12.7 Hz, 1 H, 5Ј-H), 3.64 (br.
d, J = 11.9 Hz, 1 H, 5Ј-H), 3.75 (br. s, 1 H, 4Ј-H), 3.88 (s, 2 H,
NCH2), 4.15 (br. s, 1 H, 3Ј-H), 5.96 (br. s, 1 H, 1Ј-H), 8.07 (s, 1 H,
6-H) ppm. 13C NMR (100 MHz, CDCl3/CD3OD, 5:1): δ = 5.01 (2
C), 29.86, 33.11, 41.04, 60.97, 69.82, 74.41, 86.60, 87.31, 90.72,
155.05, 164.07 ppm. IR (ATR): ν = 3411 (w), 3244 (w), 3049 (w),
˜
2974 (w), 2914 (w), 2277 (w), 1661 (m), 1631 (s), 1614 (s), 1536
(m), 1481 (s), 1444 (m), 1387 (m), 1367 (m), 1303 (m), 1275 (m),
1244 (m), 1201 (m), 1150 (m), 1061 (s), 989 (m), 912 (m), 838 (m),
803 (m) 779 (s), 661 (m), 640 (s) cm–1. HRMS (ESI): m/z (%) =
407.1360 (30) [M + Na]+, 269.0692 (100) [M – C6H12O2 + 2 H]+.
HRMS (ESI): calcd. for C16H24N4NaO5S+ [M + Na]+ 407.1352;
found 407.1360; calcd. for C10H13N4O3S+ [M – C6H12O2 + 2H]+
269.0703; found 269.0692.
(؎)-N-(3-{1-[3,3-Bis(hydroxymethyl)thietan-2-yl]cytosin-5-yl}prop-
2-yn-1-yl)cyclopropanesulfonamide [(؎)-6]: A solution of (Ϯ)-15
(200 mg, 0.34 mmol) in 1 m NaOMe in MeOH (7.5 mL) and
CH2Cl2 (7.5 mL) was stirred at 22 °C for 3 h. After the addition of
a satd. aq. solution of NaHCO3 (10 mL), the mixture was diluted
with CH2Cl2/iPrOH (3:1) (3ϫ 20 mL). The organic phase was sep-
arated, washed with brine, dried with Na2SO4, filtered, and concen-
trated under reduced pressure. A solution of the residue, 10 (90 mg,
0.6 mmol), and NEt3 (0.25 mL, 1.8 mmol) in DMF (8 mL) in a
Schlenk flask was degassed by freeze–pump–thaw cycles performed
under Ar and then treated with [Pd(PPh3)2Cl2] (42 mg, 0.06 mmol)
and CuI (28 mg, 0.07 mmol). The mixture was stirred at 25 °C for
18 h, diluted with CH2Cl2/iPrOH (3:1), and washed with a satd.
aq. solution of NaHCO3 and brine. The organic layer was dried
91.77, 144.41, 154.97, 164.67 ppm. IR (ATR): ν = 3332 (w, br.),
˜
3208 (w, br.), 2923 (w), 2233 (w), 1642 (s), 1601 (m), 1533 (m), 1497
(m), 1436 (m), 1323 (s), 1299 (m), 1260 (m), 1190 (m), 1141 (s),
1089 (s), 1055 (s), 950 (m), 889 (m), 836 (m), 782 (m), 726 (s), 646
(m) cm–1. HRMS (ESI): m/z (%) = 385.1177 (100) [M + H]+,
269.0699 (52) [M
–
C5H9O3]+. HRMS (ESI): calcd. for
C15H21N4O6S+ [M + H]+ 385.1176; found 385.1177.
(؎)-N-{3-[1-(Oxolan-2-yl)cytosin-5-yl]prop-2-yn-1-yl}cyclopropane-
sulfonamide [(؎)-4]: A solution of (Ϯ)-12 (100 mg, 0.32 mmol), 10 with Na2SO4, filtered, and concentrated under reduced pressure.
(100 mg, 0.62 mmol), and NEt3 (0.2 mL, 1.44 mmol) in DMF
(4 mL) was degassed by freeze–pump–thaw cycles performed under
Ar. [Pd(PPh3)2Cl2 ] (52 mg, 0.04 mmol) and CuI (32 mg,
0.07 mmol) were added, and the mixture was stirred at 25 °C for
6 h. Concentration and chromatography (SiO2; CH2Cl2/MeOH,
97:3) yielded (Ϯ)-4 (64 mg, 58%) as an off-white solid. Rf = 0.42
Chromatography (SiO2; CH2Cl2/MeOH, 93:7) afforded (Ϯ)-6
(18 mg, 14 %) as an off-white solid. Rf = 0.38 (SiO2; CH2Cl2/
MeOH, 93:7). M.p. 131–134 °C. 1H NMR (400 MHz, CDCl3/
CD3OD, 6:1): δ = 0.89–0.97 (m, 2 H, 2,3-H), 1.05–1.12 (m, 2 H,
2,3-H), 2.42 (tt, J = 8.0, 4.9 Hz, 1 H, 1-H), 2.72 and 2.76 (2 d, J =
9.3 Hz, 2 H, 4ЈЈ-H), 3.39 and 3.45 (2 d, J = 11.4 Hz, 2 H, CH2OH),
(SiO2; CH2Cl2/MeOH, 95:5). M.p. 118–122 °C. 1H NMR 3.52 and 3.79 (2 d, J = 11.6 Hz, 2 H, CH2OH), 4.02 (s, 2 H, CH2N),
(300 MHz, CDCl3/CD3OD, 6:1): δ = 0.90–0.99 (m, 2 H, 2,3-H),
1.11 (td, J = 6.8, 4.8 Hz, 2 H, 2,3-H), 1.70–1.88 (m, 1 H, 4ЈЈ-H),
5.78 (s, 1 H, 2ЈЈ-H), 8.36 (s, 1 H, 6Ј-H) ppm. 13C NMR (75 MHz,
CDCl3/CD3OD, 6:1): δ = 5.27 (2 C), 23.83, 30.04, 32.99, 55.49,
1.90–2.05 (m, 2 H, 3ЈЈ,4ЈЈ-H), 2.26–2.42 (m, 1 H, 3ЈЈ-H), 2.49 (tt, 59.52, 60.70, 65.37, 74.42, 90.69, 91.69, 146.02, 156.09, 164.53 ppm.
J = 8.0, 4.9 Hz, 1 H, 1-H), 3.92 (td, J = 8.5, 6.7 Hz, 1 H, 5ЈЈ-H), IR (ATR): ν = 3329 (w), 3200 (w), 2926 (w), 2868 (w), 2235 (w),
˜
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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/KAP1
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Pages: 11
M. Fischer, A. Bacher, F. Diederich et al.
FULL PAPER
1637 (s), 1595 (m), 1496 (s), 1404 (m), 1322 (s), 1297 (s), 1239 (m), 60.97, 65.13, 69.00, 90.30, 150.19, 160.03, 167.53 ppm. IR (ATR):
1190 (w), 1140 (s), 1040 (s), 888 (s), 827 (m), 784 (s), 704 (s) cm–1.
ν = 3411 (w), 3244 (w, br.), 3049 (w), 2974 (w), 2914 (w), 2878 (w),
˜
HRMS (ESI): m/z (%) = 401.0945 (100) [M + H]+. HRMS (ESI):
2227 (w), 1661 (m), 1614 (s), 1536 (m), 1481 (s), 1444 (m), 1387
(m), 1367 (m), 1303 (m), 1275 (m), 1244 (m), 1201 (m), 1150 (w),
1061 (s), 989 (w), 966 (m), 912 (w), 838 (m), 803 (m) 779 (s) cm–1.
HRMS (ESI): m/z (%) = 376.0127 (31) [M + Na]+, 237.9471 (100)
[C4H5IN3O]+. HRMS (ESI): calcd. for C10H16IN3NaO3+ [M + Na]
+
calcd. for C15H21N4O5S2 [M + H]+ 401.0948; found 401.0945.
Typical Example of the Suzuki Coupling of Pyridyl Bromide 17.
Synthesis of N-[3-(2-Amino-5-phenyl-3-pyridyl)prop-2-yn-1-yl]cyclo-
propanesulfonamide (7a): Pyridyl bromide 17 (170 mg, 0.50 mmol)
and phenylboronic acid (18a; 120 mg, 1.0 mmol) were added to a
+
376.0129; found 376.0127.
mixture of DME (18 mL) and 2 m aq. Na2CO3 (0.5 mL) in a (؎)-[2-(5-Iodocytosin-1-yl)thietane-3,3-diyl]dimethanediyl Dibenz-
Schlenk flask. After degassing and addition of [Pd(dppf)Cl2]
·CH2Cl2 (41 mg, 0.05 mmol), the mixture was heated at reflux for
18 h and concentrated under reduced pressure. Chromatography
(SiO2; hexane/EtOAc, 1:1) afforded 7a (100 mg, 59%) as an orange
oil. 1H NMR (400 MHz, CDCl3): δ = 1.01–1.08 (m, 2 H, 2,3-
oate [(؎)-15]: A suspension of 11 (130 mg, 0.55 mmol) in HMDS
(4.0 mL) was heated at reflux for 50 min. Excess HMDS was evapo-
rated under reduced pressure. A solution of the residue in CH2Cl2
(4.0 mL) was cooled to 0 °C, treated with a solution of (Ϯ)-14[25]
(200 mg, 0.55 mmol) in CH2Cl2 (3.0 mL), AgClO4 (114 mg,
0.55 mmol), and SnCl2 (104 mg, 0.55 mmol), and stirred at 22 °C
Htrans), 1.22–1.28 (m, 2 H, 2,3-Hcis), 2.56 (tt, J = 8.0, 4.8 Hz, 1 H,
1-H), 4.24 (d, J = 5.5 Hz, 2 H, NCH2), 4.81 (s, 1 H, NH), 5.26 (br. for 70 h. The suspension was diluted with EtOAc and CH2Cl2 and
s, 2 H, NH2), 7.28–7.45 (m, 5 H, arom. H), 7.69 (d, J = 2.3 Hz, 1 washed with a satd. solution of NaHCO3, water, and brine. The
H, 4Ј-H), 8.27 (d, J = 2.3 Hz, 1 H, 6Ј-H) ppm. 13C NMR (75 MHz, combined aqueous phases were extracted with CH2Cl2/iPrOH
CDCl3): δ = 5.94 (2 C), 30.7, 33.7, 79.6, 91.4, 102.1, 125.9 (2 C),
126.5, 127.0, 128.8 (2 C), 137.0, 138.5, 146.0, 158.1 ppm. IR (ATR):
(3:1). The combined organic phases were dried with Na2SO4, fil-
tered, and the solvents evaporated. Chromatography (SiO2; CH2Cl2
Ǟ CH2Cl2/MeOH, 95:5) yielded (Ϯ)-15 (106 mg, 35%) as a yellow
ν = 3460 (w), 3360 (w), 3061 (w), 2228 (w), 1618 (m), 1460 (s),
˜
1402 (m), 1326 (s), 1303 (s), 1242(m), 1142 (s), 1068 (s), 985 (m), solid. Rf = 0.24 (SiO2; CH2Cl2/MeOH, 95:5). 1H NMR (400 MHz,
891 (s), 828 (m), 758 (s), 690 (s), 668 (m) cm–1. HRMS (ESI): m/z
(%) = 328.1115 (11) [M + H]+ . HRMS (ESI): calcd. for
C17H17N3O2S+ [M + H]+ 328.1114; found 328.1115. C17H17N3O2S
CDCl3/CD3OD, 6:1): δ = 2.90 and 3.18 (2 d, J = 12.0 Hz, 2 H, 4-
H), 4.27 and 4.39 (2 d, J = 12.3 Hz, 2 H, CH2O), 4.52 and 4.72 (2
d, J = 12.3 Hz, 2 H, CH2O), 6.18 (s, 1 H, 2-H), 7.32–7.37 (m, 4 H,
(327.40): C 62.37, H 5.23, N 12.83; found C 61.87, H 5.35, N 12.83. arom. H), 7.44–7.50 (m, 2 H, arom. H), 7.83–7.85 (m, 2 H, arom.
H), 7.96–7.99 (m, 2 H, arom. H), 8.40 (s, 1 H, 6-H) ppm. 13C NMR
(؎)-5-Iodo-1-(oxolan-2-yl)cytosine [(؎)-12]: A solution of 11[23]
(100 MHz, CDCl3/CD3OD, 6:1): δ = 24.80, 53.49, 56.78, 58.01,
(500 mg, 2.11 mmol) in hexamethyldisilazane (HMDS, 4 mL) was
63.19, 66.75, 128.54 (2 C), 128.63 (2 C), 128.86, 129.55, 129.76 (2
stirred at 75 °C for 30 min, and then the solution was concentrated
under reduced pressure. A solution of the residue in CH2Cl2
C), 129.81 (2 C), 133.35, 133.49, 148.27, 155.29, 163.56, 165.79,
166.00 ppm. IR (ATR): ν = 3300 (w), 3063 (w), 2947 (w), 2696 (w),
˜
(10 mL) was treated with 2,3-dihydrofuran (160 mg, 2.2 mmol) and
TsOH·H2O (4 mg), stirred at 22 °C for 48 h, and the solvents were
evaporated. Chromatography (SiO2; CH2Cl2/MeOH, 97:3) yielded
(Ϯ)-12 (120 mg, 19%) as a white solid. Rf = 0.37 (SiO2; CH2Cl2/
MeOH, 96:4). M.p. Ͼ 180 °C (decomp.). 1H NMR (300 MHz,
CDCl3/CD3OD, 6:1, assignment based on the HSQC spectrum): δ
= 1.68–1.85 (m, 1 H, 3Ј-H), 1.88–2.05 (m, 2 H, 3Ј,4Ј-H), 2.24–2.42
(m, 1 H, 4Ј-H), 3.91 (td, J = 8.5, 6.5 Hz, 1 H, 5Ј-H), 4.15 (td, J =
1718 (s), 1693 (m), 1631 (m), 1601 (m), 1584 (w), 1491 (w), 1450
(m), 1428 (w), 1398 (w), 1369 (w), 1337 (w), 1314 (m), 1262 (s),
1176 (m), 1094 (s), 1069 (m), 1051 (w), 1025 (m), 978 (w), 917 (w),
846 (w), 803 (w), 763 (w), 705 (s), 685 (m), 675 (m), 660 (w) cm–1.
HRMS (ESI): calcd. for C23H20IN3NaO5S+ [M + Na]+ 600.0061;
found 600.0059.
N-[3-(2-Amino-5-bromo-3-pyridyl)prop-2-yn-1-yl]cyclopropanesulf-
8.1, 4.2 Hz, 1 H, 5Ј-H), 5.84 (dd, J = 6.2, 2.8 Hz, 1 H, 2Ј-H), 7.66 onamide (17): A solution of 16 (300 mg, 1 mmol), 10 (190 mg,
(s, 1 H, 6-H) ppm. 13C NMR (100 MHz, CDCl3/CD3OD, 6:1): δ = 1.2 mmol), and NEt3 (0.42 mL, 3 mmol) in THF (23 mL) in a
27.39, 37.12, 59.39, 74.22, 92.41, 150.11, 159.30, 167.79 ppm. IR
(ATR): ν = 3426 (w), 3324 (w), 2961 (w), 2874 (w), 1631 (s), 1611
Schlenk flask was degassed and treated with [Pd(PPh3)2Cl2] (70 mg,
0.1 mmol) and CuI (38 mg, 0.1 mmol). The mixture was stirred at
˜
(s), 1572 (s), 1471 (s), 1276 (m), 1249 (m), 1180 (m), 1121 (w), 1066 25 °C for 18 h, diluted with CH2Cl2, and washed with a satd. aq.
(s), 1032 (m), 950 (m), 922 (m), 897 (m), 779 (s), 736 (s) cm–1. solution of NaHCO3 and brine. Concentration under reduced pres-
HRMS (ESI): m/z (%) = 307.9886 (100) [M + H]+, 237.9466 (86)
sure and chromatography (SiO2; hexane/EtOAc, 1:1) afforded 17
(290 mg, 87%) as a brown solid. M.p. 141–144 °C. 1H NMR
(400 MHz, CDCl3): δ = 1.04–1.07 (m, 2 H, 2,3-Htrans), 1.23–1.25
(m, 2 H, 2,3-Hcis), 2.53 (tt, J = 8.0, 4.8 Hz, 1 H, 1-H), 4.21 (d, J =
3.5 Hz, 2 H, NCH2), 4.84 (br. s, 1 H, NH), 5.15 (br. s, 2 H, NH2),
7.95 (d, J = 2.3 Hz, 1 H, 4Ј-H), 8.05 (d, J = 2.3 Hz, 1 H, 6Ј-H)
ppm. 13C NMR (100 MHz, CDCl3): δ = 5.91 (2 C), 30.85, 33.85,
79.06, 92.20, 103.76, 141.93, 149.24, 158.09 ppm.
+
[M – C4H6O + H]+. HRMS (ESI): calcd. for C8H11IN3O2 [M +
H]+ 307.9890; found 307.9886; calcd. for C4H5IN3O+ [M – C4H6O
+ H]+ 237.9477; found 237.9466.
(؎)-1-(1-Ethoxy-4-hydroxybutyl)-5-iodocytosine [(؎)-13]: A solu-
tion of 11 (1.0 g, 4.2 mmol) in HMDS (4 mL) was stirred at 60 °C
for 30 min. Excess HMDS was evaporated under reduced pressure.
A solution of the residue in CH2Cl2 (25 mL) was treated with
TMSOTf (1.86 g, 8.4 mmol), 2-ethoxyoxolane (320 mg, 4.4 mmol),
and TsOH·H2O (20 mg), stirred at 22 °C for 48 h, and the solvents
were evaporated. Chromatography (SiO2; CH2Cl2/MeOH, 97:3)
yielded (Ϯ)-13 (260 mg, 18%) as a light-orange solid. Rf = 0.52
Supporting Information (see footnote on the first page of this arti-
cle): Modeling figures referred to in the article, synthesis of amino-
pyridine ligands with aromatic rings, NMR spectra of new com-
pounds.
1
(SiO2; CH2Cl2/MeOH, 96:4). M.p. Ͼ 200 °C (decomp.). H NMR
(300 MHz, CDCl3/CD3OD, 6:1): δ = 0.97 (t, J = 7.0 Hz, 3 H,
OCH2CH3), 1.29–1.46 (m, 2 H, 3Ј-H), 1.46–1.61 (m, 2 H, 2Ј-H),
3.19–3.33 (m, 2 H, OCH2CH3), 3.36 (t, J = 6.2 Hz, 2 H, 4Ј-H),
5.47 (dd, J = 7.2, 5.1 Hz, 1 H, 1Ј-H), 7.58 (s, 1 H, 6-H) ppm. 13C
Acknowledgments
This work was supported by the Swiss National Science Founda-
NMR (100 MHz, CDCl3/CD3OD, 6:1): δ = 18.57, 31.31, 36.30, tion. The authors are indebted to Dr. Xiangyang Zhang, Louis
8
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20296
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Date: 03-05-12 16:04:20
Pages: 11
Cytosines and Cytosine Analogues
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Received: March 11, 2012
Published Online: ᭿
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Job/Unit: O20296
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Date: 03-05-12 16:04:20
Pages: 11
Cytosines and Cytosine Analogues
Enzyme Molecular Recognition
A. P. Schütz, S. Osawa, J. Mathis,
A. K. H. Hirsch, B. Bernet, B. Illarionov,
M. Fischer,* A. Bacher,*
F. Diederich* ................................... 1–11
Exploring the Ribose Sub-Pocket of the
Substrate-Binding Site in Escherichia coli
IspE: Structure-Based Design, Synthesis,
and Biological Evaluation of Cytosines and
Cytosine Analogues
We report the design, synthesis, and bio-
logical evaluation of new cytosine- and 2-
aminopyridine-based ligands to explore the
molecular recognition properties of the
ribose sub-pocket of the substrate-binding
site in the IspE protein of Escherichia coli,
a model enzyme for P. falciparum IspE.
Keywords: Enzymes / Ligand design / Inhi-
bition / Molecular recognition
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
11