The design and synthesis of inhibitors of pantothenate synthetase

Article abstract of DOI:10.1039/b609482a

Pantothenate synthetase catalyses the ATP-dependent condensation of d-pantoate and β-alanine to form pantothenate. Ten analogues of the reaction intermediate pantoyl adenylate, in which the phosphodiester is replaced by either an ester or sulfamoyl group, were designed as potential inhibitors of the enzyme. The esters were all modest competitive inhibitors, the sulfamoyls were more potent, consistent with their closer structural similarity to the pantoyl adenylate intermediate. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b609482a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
The design and synthesis of inhibitors of pantothenate synthetase†
Kellie L. Tuck,a S. Adrian Saldanha,a Louise M. Birch,a Alison G. Smithb and Chris Abella
Received 4th July 2006, Accepted 8th August 2006
First published as an Advance Article on the web 30th August 2006
DOI: 10.1039/b609482a
Pantothenate synthetase catalyses the ATP-dependent condensation of D-pantoate and b-alanine to
form pantothenate. Ten analogues of the reaction intermediate pantoyl adenylate, in which the
phosphodiester is replaced by either an ester or sulfamoyl group, were designed as potential inhibitors
of the enzyme. The esters were all modest competitive inhibitors, the sulfamoyls were more potent,
consistent with their closer structural similarity to the pantoyl adenylate intermediate.
Introduction
Pantothenate 1 (vitamin B5) is a key precursor of the 4ꢀ-
phosphopantetheine moiety of coenzyme A (CoA) and the acyl
carrier protein (ACP). Both CoA and ACP are necessary cofactors
for cell growth and are involved in essential biosynthetic pathways.
Pantothenate 1 (Scheme 1) is biosynthesised in micro-organisms,
Fig. 1 Compounds reported as potential inhibitors of pantothenate
plants, and fungi, but not in animals,1 and the enzymes of the
pantothenate pathway are considered to be potential herbicide
and antimicrobial targets.
synthetase.10,11
been synthesised, however none showed significant inhibition of
pantothenate synthetase under a variety of assay conditions.10,11
A
The pathway to pantothenate 1 is best understood in Escherichia
coli, where it comprises four enzymatic reactions.2,3 The final trans-
formation, to produce pantothenate 1, is catalysed by pantothen-
ate synthetase (EC 6.3.2.1, Scheme 1), encoded by the panC gene.
Pantothenate is biosynthesised by the condensation of D-pantoate
and b-alanine. One equivalent of ATP is used, resulting in the
formation of AMP and pyrophosphate (PPi). The Mg2+-dependent
reaction consists of two sequential steps, initial pantoyl adenylate
2 formation, followed by subsequent nucleophilic attack on the
activated carbonyl by b-alanine (Scheme 1).4 The kinetic mech-
anism of Mycobacterium tuberculosis pantothenate synthetase
has been shown to be a BiUniUniBiPingPong mechanism.4,5
Pantothenate synthetase is a member of the aminoacyl-tRNA
synthetase superfamily and the mechanism for formation of the
pantoyl adenylate 2 is similar to that for formation of the acyl
adenylate intermediate.6 Several inhibitors of aminoacyl-tRNA
synthetases are known which mimic the aminoacyl adenylate
intermediate.7–9
non-hydrolysable analogue of ATP, AMPCPP, has been shown
to inhibit M. tuberculosis pantothenate synthetase, with a Ki
of 290 lM.4 No mimics of the pantoyl adenylate 2 have been
described.
The crystal structures of the apoenzyme forms of E. coli
pantothenate synthetase (PDB code: 1iho)6 and M. tuberculosis
pantothenate synthetase have been published, as well as several
very informative complexes of the M. tuberculosis enzyme includ-
ing the pantoyl adenylate 2 bound at the active site (PDB code:
1n2h).12–14 Pantothenate synthetase exists as a homodimer. Each
subunit has two well-defined domains, an N-terminal Rossmann
fold which contains the active site cavity and a C-terminal domain
which acts as a hinged lid for this cavity. ATP binds into the
N-terminal domain,6,13 and pantoate binds into a pocket on the
other domain. For E. coli pantothenate synthetase it has been
proposed that the binding of ATP is accompanied by a hinge
bending action that moves the two domains of the enzyme closer
together from an open apo-structure to a closed structure.6 The
ternary M. tuberculosis pantothenate synthetase structure adopts
a closed conformation, believed to be catalytically relevant.
In this paper we describe the preparation of a closed model
of E. coli pantothenate synthetase, the synthesis of a number of
pantoyl adenylate analogues (compounds 3–12, Fig. 2), molecular
docking studies of these analogues into the active site of E.
coli pantothenate synthetase, and their inhibition of E. coli
pantothenate synthetase.
No potent inhibitors of pantothenate synthetase have been
reported. Several potential inhibitors, pantoate analogues or
phosphonate analogues of pantoate/pantothenate (Fig. 1) have
aUniversity Chemical Laboratory, Lensfield Road, Cambridge, UK CB2
1EW. E-mail: ca26@cam.ac.uk; Fax: +44 1223 3336362; Tel: +44 1223
333405
bDepartment of Plant Sciences, Downing Street, Cambridge, UK CB2 3EA.
E-mail: as25@plant.sciences.cam.ac.uk; Fax: +44 1223 333953; Tel: +44
1223 333900
† Electronic supplementary information (ESI) available: Diagram of
binding interactions made by pantoyl adenylate in the active site of M.
tuberculosis pantothenate synthetase based on the crystal structure of
pantothenate synthetase in complex with pantoyl adenylate 2. Figure
generated using LIGPLOT15 (from PDB coordinates 1n2h). Diagram of
binding interactions made by pantoyl adenylate 2 in the model of the
close structure of the active site of E. coli pantothenate synthetase. Figure
generated using LIGPLOT.15. See DOI: 10.1039/b609482a
Results
Preparation of the closed model of E. coli pantothenate synthetase
A sequence alignment of E. coli and M. tuberculosis pantothenate
synthetase was performed with the CLUSTALW 1.82 software
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Scheme 1 Reaction catalysed by pantothenate synthetase.
Fig. 2 Analogues of pantoyl adenylate 2 described in this paper.
program (http://www2.ebi.ac.uk/clustalw).15 This revealed that
the active sites of the two enzymes possess a high degree of
sequence similarity. The residues M30, H37, N58, Q61, G149,
D152 and Q155 in E. coli are conserved as M40, H47, N69, Q72,
G158, D161 and Q164 in M. tuberculosis.
diagrams show the importance of the hydrogen bonds between the
water molecule and the hydroxyl group at the C2ꢀ, the aspartate
residue and the glycine residue.
Docking studies and design of inhibitors
The high degree of similarity in the active sites of E. coli and
M. tuberculosis pantothenate synthetase, the number of conserved
residues and the fact that the M. tuberculosis crystal structures
were in the required conformation to be catalytic, led to the
construction of a model for the closed structure of E. coli
pantothenate synthetase using the M. tuberculosis pantothenate
synthetase (PDB code: 1n2h) structure. The position of pantoyl
adenylate 2 was modelled into the E. coli active site. An overlay of
a subunit of the two structures is shown in Fig. 3A (the protein-
ligand interactions for M. tuberculosis pantothenate synthetase
(PDB code: 1n2h) and the modelled closed form of E. coli
pantothenate synthetase are shown as LIGPLOT diagrams16 in
The structure of M. tuberculosis pantothenate synthetase (PDB
code: 1n2h), was prepared using SYBYL 6.5.17 The pantoyl
adenylate 2, the glycerol molecule, the ethanol molecules and all
the water molecules (except the key structural water, see above)
were removed from the crystal structure. Hydrogens were added
to the amino acid residues and the structure was minimised
using a Tripos force field.17 The closed model structure of E.
coli pantothenate synthetase was prepared as described in the
Experimental section. GOLD (version 2.1.1)18 was used to dock
small molecules into the protein binding site. The structures of
the ligands were prepared using the program SYBYL 6.517 and
energy-minimised using the Tripos force field. A maximum of
25 independent GOLD runs were executed for each ligand.18
The dockings were allowed to terminate early if the root-mean-
squared deviation (RMSD) between the heavy atoms of the top
the Supplementary Information (ESI)†). Of note is a molecule
ꢀ
˚
of water bound 3.3 A away from the C2 ribose hydroxyl of the
pantoyl adenylate 2 which mediates hydrogen bonds from the
ribose hydroxyl(s) to the protein backbone. This key structural
water molecule is present in all pantothenate synthetase crystal
structures that include ribose-containing ligands. The LIGPLOT
˚
five ranked conformations was less than 1.5 A. The GoldScore
scoring function was used in all cases.
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˚
Fig. 3 A) Overlay of M. tuberculosis pantothenate synthetase from the crystal structure (2.0 A, PDB code: 1n2h, shown in blue) with the modelled
closed conformation of E. coli pantothenate synthetase (shown in green). The bound pantoyl adenylate 2 is also shown. The diagram was generated using
Weblab Viewer Pro (Molecular Simulations Inc) and Povray (http://www.povray.org/). B) GOLD docking results of the ligands in the active site of the
closed model of E. coli pantothenate synthetase (1) pantoyl adenylate 2, (2) inhibitor (2R)-12. These representations were produced using the UCSF
Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH
P41 RR-01081).28
Pantoyl adenylate 2 was initially docked as a control into the
active sites of E. coli and M. tuberculosis pantothenate synthetase,
and then compared to either the pantoyl adenylate–enzyme
complex (M. tuberculosis) or the pantoyl adenylate–enzyme model
(E. coli). The docked conformation corresponded to that of the
˚
crystallographically observed pantoyl adenylate 2 to within 0.6 A
RMSD.
The inhibitors were designed to be analogues of the pantoyl
adenylate 2. Ten compounds were chosen (3–12), where the labile
phosphodiester is replaced with either an ester or sulfamoyl group
and the pantoyl functionality is modified. The (2S)-diastereomer
of ligands 3 and 8 and each stereoisomer of the ligands 4–7
and 10–12 were docked into the active site of M. tuberculosis
pantothenate synthetase and the active site of the closed model
of E. coli pantothenate synthetase. The docking results suggested
that the ligands would bind in the same orientation as the pantoyl
adenylate 2 in the active site. The top docking results of the pantoyl
adenylate 2 and the ligand (2S)-12 in the active site of E. coli
pantothenate synthetase are shown in Fig. 3B.
Scheme 2 Reagents and conditions: (i) DEAD, PPh3, THF, 16 h; (ii)
TFA–H2O, 3 h.
was added as a co-catalyst to improve the solubility of NaH.21
The required acids, 18 and 19, were obtained after ring opening
of the protected lactone and derivatisation to the corresponding
Synthesis of ester analogues 3–7
The amino ester analogue 3 was prepared from commercially
available N-Boc-L-tert-leucine. The N-Boc protected amino acid
was coupled with 2ꢀ,3ꢀ-isopropylideneadenosine, under Mitsunobu
conditions (Scheme 2).19 Deprotection of both the acetonide
and N-Boc protecting groups was accomplished under acidic
conditions to give the ester 3.
Ester analogue 4 was synthesised in three steps from pina-
colone (Scheme 3). The required ketoacid 14 was synthesised by
permanganate oxidation of the methyl group.20 Coupling of the
ketoacid 14 with 2ꢀ,3ꢀ-isopropylideneadenosine gave the protected
ester 15, which was deprotected under acidic conditions to
give 4.
The methoxy ether ester analogues 5 and 6 were synthe-
sised in five steps from a-hydroxy-v-butyrolactone and ( )-
pantolactone respectively (Scheme 4). In both cases the secondary
alcohol of the lactone was protected as a 2-methoxyethoxymethyl
(MEM) ether to give compounds 16 and 17, and 15-crown-5
Scheme 3 Reagents and conditions: (i) KMnO4, NaOH(aq), 0 ◦C, 1 h, 22 ◦C,
2 h; (ii) DCC, CH2Cl2, 16 h; (iii) TFA–H2O, 3 h.
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Synthesis of sulfamoyl analogues 8–12
The synthesis of N-acylsulfamoyls 8–12 was based on literature
procedures for the preparation of isoleucyl- and tyrosyl-tRNA
synthetase inhibitors.7,9,22 The required 2ꢀ,3ꢀ-protected adenosi-
nesulfamate 25 was prepared from 2ꢀ,3ꢀ-isopropylideneadenosine
and sulfamyl chloride using
a modification of literature
conditions.7,22,23 Previous work had shown that the 5ꢀ-O–SO2NH2
group was more reactive than the 6-NH2 amino group on the
adenine ring.24 Hence the adenosinesulfamate derivative 25 was
directly acylated with appropriately activated carboxylic acid
derivatives to give the N-acylsulfamates.
Sulfonamide analogues 8 and 9 were prepared by coupling the
adenosinesulfamate 25 with the hydroxysuccinimide ester of N-
Boc-L-tert-leucine and the diketone 14 respectively (Schemes 6 and
7). The hydroxysuccinimide ester was formed by reacting N-Boc-
L-tert-leucine or the diketone 14 with N-hydroxysuccinimide to
give the activated acid (26 or 28).25 The hydroxysuccinimide esters
were unstable to purification by chromatography. Deprotection of
the N-acylsulfamates (27 or 29) under acidic conditions gave the
required sulfamoyls.
Scheme 4 Reagents and conditions: (i) NaH, 15-crown-5, MEMCl, 0 ◦C,
warm to 22 ◦C, 15 h (ii) H2O–MeOH, KOH, 80 ◦C, 4 h; (iii) NaH,
15-crown-5, MeI, 0 ◦C, warm to 22 ◦C, 15 h; (iv) DEAD, PPh3, THF,
16 h; (v) TFA–H2O, 3 h.
methoxy ether. Coupling of the acids 18 and 19 with 2ꢀ,3ꢀ-
isopropylideneadenosine, under Mitsunobu conditions, gave the
protected esters (20 or 21 respectively). The final compounds,
analogues 5 and 6, were obtained after removal of the acetal
protecting group.
Ester analogue
7 was prepared from 3,3-dimethyl-1,2-
butanediol in four steps (Scheme 5). The required acid 22
was obtained after oxidation of 3,3-dimethyl-1,2-butanediol with
Pt–C in the presence of oxygen. Protection of the secondary
alcohol with a dichloroacetate group gave the dichloroacetate ester
23 in good yield. This protected 3-dimethyl-2-hydroxybutanoic
acid 23 was coupled with 2ꢀ,3ꢀ-isopropylideneadenosine under
Mitsunobu conditions to give the protected ester 24. Deprotection
under acidic conditions gave the corresponding ester 7 in good
yield.
Scheme 6 Reagents and conditions: (i) hydroxysuccinimide, DME, DCC,
0
◦C, 15 h; (ii) DBU, DCM, 16 h; (iii) TFA–H2O, 16 h.
Scheme 5 Reagents and conditions: (i) Pt–C, NaOH(aq), CH2CH2, O2(g)
,
70 ◦C, 5 h; (ii) ClCOCHCl2, 80 ◦C, 4 h; (iii) DEAD, PPh3, THF, 16 h; (iv)
TFA–H2O, 3 h.
Scheme 7 Reagents and conditions: (i) hydroxysuccinimide, DME, DCC,
◦C, 15 h; (ii) DBU, DCM, 16 h; (iii) TFA–H2O, 3 h.
0
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Scheme 8 Reagents and conditions: (i) EDC, DMAP, CH2Cl2, HOC6F5, 15 h; (ii) DBU, DMF, 16 h; (iii) TFA–H2O, 3 h.
Due to the instability of the hydroxysuccinimide esters a
different activating group was tried. Consequently, the sulfamoyl
analogues 10 and 11 were prepared by coupling 2ꢀ,3ꢀ-protected
adenosinesulfamate 25 with the pentafluorophenol esters of the
hydroxy acids 18 or 19 (Scheme 8). Purification by chromatography
resulted in an oil which was a mixture in which the desired product
(32 or 33) was identified by NMR spectroscopy and LCMS
analysis. Removal of both the acetonide and MEM protecting
groups was accomplished under acidic conditions. The vigorous
conditions required to remove the MEM protecting group led to
a mixture of products from which the sulfamoyls (10 or 11) were
purified.
Sulfonamide analogue 12 was also prepared by coupling 2ꢀ,3ꢀ-
protected adenosinesulfamate 25 with the pentafluorophenol ester
of the protected acid 23 (Scheme 9). Purification by chromatogra-
phy led to a mixture of products including some loss of the diacetyl
protecting group. Deprotection of the acetonide protecting group
was accomplished under acidic conditions which resulted in
several products, from which 12 was purified.
Assay results
E. coli pantothenate synthetase was assayed using a coupled assay
that measures the formation of AMP spectrophotometrically.26
The kinetic parameters were obtained by plotting the rate as a
function of concentration of varied substrate and curve fitting to
the Michaelis–Menten equation. Control experiments were carried
out to show that the compounds did not inhibit the coupling
enzymes.
The ester analogues, (2S)-3 and the racemic compounds 4–7,
were tested as inhibitors against E. coli pantothenate synthetase.
In all cases the inhibition studies were carried out using a
constant concentration of pantoate and b-alanine and varying
concentrations of ATP. The Ki values were obtained by plotting the
rate as a function of the concentration of ATP at different inhibitor
concentrations and curve fitting to the equation for competitive
inhibition (Table 1).27 All the compounds were found to be modest
reversible inhibitors of E. coli pantothenate synthetase, and were
competitive with ATP. The degree of inhibition is relatively
insensitive to the structure of the acyl group, with the Kis ranging
from 1.9 mM (for 4) to 18.2 mM (for 6). This suggests that most
of the binding is coming from the adenine and ribose groups in
the ATP pocket, and that the varied acyl group cannot reach far
enough into the pantoate binding pocket to pick up the specific
binding interactions associated with recognition of the pantoyl
group in the reaction intermediate 2.
Table 1 Inhibiton results for the ester analogues, varying concentrations
of ATPa
Compound
Ki/mM
Inhibition
3
4
5
6
7
9.5 1.2
1.9 0.22
3.5 0.4
18.2 4.2
2.5 0.2
Competitive
Competitive
Competitive
Competitive
Competitive
a [E. coli pantothenate synthetase] 50 nM. Km ATP 1.75 mM, ( )-pantoate
1.45 mM, b-alanine 0.31 mM, kcat = 1.4 s−1 at 25 ◦C.
Scheme 9 Reagents and conditions: (i) EDC, DMAP, CH2Cl2, HOC6F5,
15 h; (ii) DBU, DMF, 16 h (iii) TFA–H2O, 16 h.
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Table 2 Inhibition results for the sulfamoyl analoguesa
were kept constant and that of the third substrate was varied.
The Ki values were obtained by plotting the rate as a function of
the concentration of the varied substrate at different inhibitor
concentrations, and curve fitting to equations for competitive,
non-competitive or un-competitive inhibition (Table 2).27 The
inhibition data for compound 12 versus ATP, pantoate and b-
alanine are shown in Fig. 4.
Compound
Varying substrate
Ki/lM
65
Inhibition
8
ATP
7
competitive
competitive
uncompetitive
competitive
competitive
uncompetitive
competitive
competitive
uncompetitive
competitive
competitive
uncompetitive
competitive
competitive
uncompetitive
pantoate
b-alanine
ATP
pantoate
b-alanine
ATP
pantoate
b-alanine
ATP
pantoate
b-alanine
ATP
160 13
250 23
9
14
15
2
2
120
9
10
11
12
0.8 0.1
ND
Conclusions
ND
30
54
4
5
The esters 3–7 and sulfamoyl compounds 8–12 were designed
as analogues of the intermediate in the pantothenate synthetase
reaction, pantoyl adenylate 2. The compounds were synthesised
and assayed against E. coli pantothenate synthetase. The sul-
famoyls 8–12 were approximately 100-fold more potent inhibitors
than their corresponding ester analogues, reflecting their closer
structural similarity with pantoyl adenylate, and suggesting that
the interactions that the phosphate group makes with the enzyme
contribute significantly to binding. The inhibition values for these
compounds ranged from 300 nM (compound 12, versus ATP) to
250 lM (compound 8, versus b-alanine).
144 17
0.3 0.05
0.8 0.05
1.1 0.08
pantoate
b-alanine
a [E. coli pantothenate synthetase] 50 nM, ND—not determined, insuffi-
cient compound, Km (ATP) 1.75 mM, ( )-pantoate 1.45 mM, b-alanine
0.31 mM, kcat = 1.4 s−1 at 25 ◦C.
Inhibition studies with the sulfamoyl analogues, (2S)-8 and
the racemic compounds 10–12, were carried out against E. coli
pantothenate synthetase. The inhibition by these compounds was
measured against varying concentrations of ATP, pantoate and
b-alanine. In all assays the concentration of two of the substrates
The sulfamoyl groups 8–12 were competitive inhibitors against
ATP and pantoate and uncompetitive inhibitors against b-
alanine. This was somewhat unexpected as it has previously been
reported that the kinetic catalytic mechanism of M. tuberculosis
Fig. 4 Steady state inhibition studies of E. coli pantothenate synthetase with inhibitor (2SR)-12, A) versus ATP, data were fitted to the equation for
competitive inhibition, B) versus pantoate, data were fitted to the equation for competitive inhibition, C) versus b-alanine, data were fitted to the equation
for uncompetitive inhibition. GraFit software (version 5.0.6, Erithacus Software Limited, http://www.erithacus.com/grafit/) was used to construct
Michaelis–Menten plots of the kinetic data and to calculate Ki values for competitive inhibition.
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pantothenate synthetase involves ordered binding of ATP followed
by pantoate.4,5 It is interesting however that pantoate can be
soaked into a crystal of apoenzyme, suggesting that binding of
ATP is not absolutely required for pantoate binding.13 Sulfamoyl
analogue 12, the most potent inhibitor, is structurally most similar
to the pantoyl adenylate 2. The pantoate fragment differs only
by replacement of the primary hydroxyl group with a proton.
The inclusion of a primary methyoxy ether in 11 results in
a hundred-fold decrease in potency. This result was consistent
with docking studies which revealed that 11 was too large to
bind in the same orientation as the pantoyl adenylate 2, due
to the combined presence of the methoxy ether and the gem-
dimethyl groups. Docking studies of compound 10, which had the
gem-dimethyl groups removed, showed that this compound could
bind in the same orientation as the pantoyl adenylate 2 and
this was mirrored by an increase in potency. Replacement of
the secondary hydroxyl in compound 12 with a ketone (com-
pound 9) or amino group (compound 8) resulted in less potent
molecule, indicating that the secondary hydroxyl group is required
for activity. All compounds were found to be poor inhibitors
of pantothenate kinase and ketopantoate reductase (data not
showm).
GOLD docking
All ligands and the receptor were prepared using SYBYL 6.5 and
used as MOL2 files. The structures of the ligands were energy-
minimised using the Tripos force-field prior to docking. Each
ligand was docked into the active site of pantothenate synthetase
(M. tuberculosis. and E. coli) using GOLD 2.1.1 in 25 independent
genetic algorithm (GA) runs, and for each of these a maximum
of 100 000 GA operations were performed on a single population
of 50 individuals. Operator weights for crossover, mutation and
migration in the entry box were used as default parameters (95, 95
and 10, respectively), as well as the initial hydrogen bonding (4.0
˚
˚
A) and external van der Waals (2.5 A) parameters. The position of
˚
the active site was introduced and the radius was set to 10 A, with
the automatic active site detection on. The “flip ring corners”,
“flip amide bonds” and “flip planer N” flags were switched on,
while “internal H-bonds” was switched off.
Protein expression
The panC gene was subcloned into SmaI–EcoRI-digested pUC19,
such that the panC gene was under the control of the lacZ
promoter. For overexpression of pantothenate synthetase, E. coli
cells were grown overnight at 37 ◦C on LB medium containing
ampicillin (100 mg ml−1). The lacZ gene was induced by the
addition of IPTG (70 mg ml−1) at the start of the culture.
Pantothenate synthetase was purified as described previously,6 and
stored in aliquots as concentrated solutions in glycerol–Tris buffer
at −20 ◦C.
This paper reports the first synthesis of a submicromolar in-
hibitor of pantothenate synthetase. The structure of the inhibitor is
consistent with the mechanism of the enzyme proceeding through
a pantoyl adenylate intermediate. The kinetics of the inhibition
suggest that a random order binding of the two substrates, ATP
and pantoate, is possible for the E. coli enzyme.
Experimental
Pantothenate synthetase assay
Design of the closed model of E. coli pantothenate synthetase
Pantothenate synthetase activity was assayed by coupling the
formation of AMP to the reactions of myokinase, pyruvate kinase,
and lactate dehydrogenase as described previously26 except that
Tris buffer was used. The decrease in absorbance of NADH
at 340 nm (e340 = 6220 M−1 cm−1) was measured in a 96-well
plate using a Biotek Powerwave XS plate reader running KC4
v3.2 Biotek instrument software. Conversion of ( )-pantolactone
to ( )-sodium pantoate was achieved by treatment of ( )-
pantolactone with 1 molar equivalent of sodium hydroxide.
The standard assay contained 100 mM Tris, pH 7.5, 10 mM
MgCl2, 5 mM ATP, 3 mM b-alanine, 1.5 mM potassium phos-
phoenolpyruvate, 150 lM NADH, 5 units of myokinase, 10 units
of pyruvate kinase, and 10 units of lactate dehydrogenase and
E. coli pantothenate synthetase (50 nM) in a total volume of
160 lL at 25 C. After incubation for 5 min at 25 C, reactions
were initiated by the addition of sodium pantoate (4 mM, 40 lL,
preincubated to 25 ◦C). The rate of pantothenate formation is
proportional to the rate of oxidation of NADH, two molecules of
NADH are oxidized for each molecule of pantothenate formed.
The apparent KM values for ( )-pantoate, ATP and b-alanine
The N-terminal domains of E. coli pantothenate synthetase
(PDB code: 1iho) and M. tuberculosis pantothenate synthetase
complexed with the pantoyl adenylate 2 (PDB code: 1n2h) were
superimposed using Swiss-PDBViewer. The backbone RMSD was
˚
found to be 1.0 A for this region. This domain corresponds to the
Rossmann fold and is assumed to stay rigid. Particular attention
was then paid to the hinge region between the domains.
The program SYBYL was used to modify the torsion angles
along the backbone around the central proline (P176 in E. coli
and P180 in M. tuberculosis) so that the C-terminal domains
overlapped. The proline ring conformation was changed in order
to accommodate the backbone torsion angle changes. The back-
bone torsion angles were modified near residues 175–177, with
no amide torsion (x) being moved by more than 4 degrees. The
region between residues 174–180 was then minimised in SYBYL
to allow for reorientation of the sidechains. The active site of the
protein was then modelled by extracting the position observed
in the (superimposed) M. tuberculosis pantothenate synthetase
structure, merging the pantoyl adenylate into the new active site
and then conducting a minimisation of the intermediate and
the surrounding sidechains using a substructure minimisation
in SYBYL (hydrogens were added to both the protein and the
intermediate). The key water molecule from the M. tuberculosis
pantothenate synthetase structure was retained in the active
site.
◦
◦
were 1.45
0.38 mM, 1.75
0.05 mM and 0.31
0.04 mM,
respectively, and kcat was 1.4 s−1. GraFit software (version 5.0.6,
Erithacus Software Limited, http://www.erithacus.com/grafit/)
was used to construct Michaelis–Menten plots of the kinetic data.
All experiments were performed in duplicate and the mean values
are reported.
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Inhibition studies
Tetrahydrofuran (THF) and diethyl ether (Et2O) were distilled
over a mixture of lithium aluminium hydride and calcium hydride.
All other reagents were purchased from Aldrich, Acros, or Fisher
Scientific, and were used without further purification. Moisture
sensitive reactions were carried out under a nitrogen atmosphere
in pre-dried glassware.
The pantothenate synthetase assay was repeated in the presence
of inhibitor. When ATP was the varied substrate the enzymatic
reaction was initiated by the addition of sodium pantoate (4 mM,
40 lL, preincubated to 25 ◦C). When sodium pantoate was
the substrate varied the enzymatic reaction was initiated by
the addition of ATP (5 mM, 40 lL, preincubated to 25 ◦C).
When b-alanine was the substrate varied the enzymatic reaction
was initiated by the addition of ( )-pantoate (3 mM, 40 lL,
preincubated to 25 ◦C).
GraFit software (version 5.0.6, Erithacus Software Lim-
ited, http://www.erithacus.com/grafit/) was used to generate
Michaelis–Menten plots of the kinetic data and calculate Ki
values for competitive inhibition, non-competitive inhibition,
uncompetitive inhibition and mixed inhibition.27 The type of
inhibition was determined by statistical analysis (F test) of the
Ki. The results are shown in Tables 1 and 2. All experiments were
performed in duplicate, unless otherwise stated, and the mean
values are reported.
(2S)-Adenosyl-2-L-amino-3,3-dimethylbutanoate (3). To a so-
lution of Boc-L-tert-leucine (347 mg, 1.5 mmol) and 2ꢀ,3ꢀ-
isopropylideneadenosine (384 mg, 1.25 mmol) in THF (20 mL)
was added DEAD (236 lL, 1.5 mmol) and triphenylphosphine
(332 mg, 1.3 mmol). After stirring at 22 ◦C for 16 h the solution was
concentrated and then diluted with EtOAc (30 mL), washed with
water (20 mL) and dried (MgSO4), and the solvent was removed
in vacuo. Purification by column chromatography (methanol–
DCM gradient 2–5% methanol) gave the protected ester 13 as
a white solid (600 mg, 92%). dH (250 MHz, CDCl3): 0.89 (s, 9H,
CH3), 1.39 (s, 3H, CH3), 1.41 (m, 9H, CH3, Boc), 1.61 (s, 3H,
CH3), 4.21 (m, 4H, H4ꢀ, H5ꢀ and H2), 5.06 (m, 1H, H3ꢀ), 5.58 (m,
1H, H2ꢀ), 6.06 (m, 1H, H1ꢀ), 7.25 (s, 2H, NH2), 7.88 (s, 1H, H2ꢀ’),
8.37 (s, 1H, H8ꢀꢀ).
The protected ester 13 (600 mg, 1.15 mmol) was dissolved
in water–TFA (2 : 4 v/v) and stirred at 22 ◦C for 3 h. The
solvent was removed in vacuo by azeotroping with ethanol
and toluene. Purification by column chromatography (methanol–
DCM gradient 2–15% methanol) gave the ester as a white solid
(244 mg, 55%). dH (250 MHz, DMSO): 0.90 (s, 9H, CH3), 4.15 (m,
1H, H3ꢀ), 4.24 (m, 1H, H4ꢀ), 4.38 (m, 2H, H5ꢀ), 4.78 (m, 1H, H2ꢀ),
5.52 (br s, 2H, NH2), 5.90 (d, 1H, J = 5.5 Hz, H1ꢀ), 7.25 (br s, 2H,
NH2), 8.12 (s, 1H, H2ꢀꢀ), 8.31 (s, 1H, H8ꢀꢀ). dC (62.5 MHz, DMSO):
26.2, 33.3, 61.0, 65.7, 70.6, 72.7, 81.5, 87.8, 119.4, 140.2, 149.5,
152.7, 156.2, 169.5. HRMS (ES) for C16H24N6O5: (MH+) calcd:
381.1808, found: 381.1805.
Chemistry—General Methods
1H NMR spectra were recorded on either a Bruker Avance DPX-
250 MHz spectrometer, a Bruker DPX-400 MHz spectrometer, or
a Bruker DPX-400 MHz spectrometer in deuterated solvents, as
specified. Splitting patterns are abbreviated as follows: s, singlet,
d, doublet, t, triplet, q, quartet and m, multiplet. 13C NMR
spectra were recorded on a Bruker DPX-400 MHz spectrometer
operating at 100 MHz or Bruker DPX-500 spectrometer operating
at 125 MHz. Samples were referenced with respect to the residual
solvent signal.
Infrared spectra were recorded on an ATI Mattson Genesis
FTIR infrared spectrometer using NaCl plates or on a Perkin
Elmer Spectrum One FTIR spectrometer using attenuated trans-
mittance reflectance (ATR).
3,3-Dimethyl-2-oxobutyric acid (14).20
To a suspension of
sodium hydroxide (2.0 g, 51.5 mmol), KMnO4 (6.1 g, 38.6 mmol) in
water (48 mL) cooled to 0 ◦C, was added a solution of pinacolone
(2.5 mL, 19.9 mmol) in water (40 mL). The solution was stirred at
0 ◦C for 1 h and then 22 ◦C for 2 h. The mixture was filtered through
a pad of celite, acidified to pH 2, extracted with Et2O (3 × 40 mL)
and dried (Na2SO4), and the solution was concentrated in vacuo.
Distillation (81 ◦C, 20 mm) gave the acid as a clear oil (2.23 g, 87%).
dH (400 MHz, CDCl3): 1.32 (s, 9H, CH3). dC (100 MHz, CDCl3):
26.1, 160.9, 201.3. mmax (NaCl): 3100 (br s), 1731 (s), 1715 (s),
1428 (s) cm−1. HRMS (ES) for C6H12O3: (MNa+) calcd: 153.0528,
found: 153.0527.
Mass spectrometry was carried out using a Micromass
Quadrapole-time of flight (Q-Tof) spectrometer. Liquid chro-
matography mass spectrometry (LCMS) was carried out using
an Alliance HT Waters 2795 Separations Module coupled to a
Waters Micromass ZQ Quadrapole Mass Analyzer. Samples were
detected using a photomultiplier detection system. Samples were
run on a gradient from 10 mM ammonium acetate containing
0.1% formic acid to 95% acetonitrile over a period of 8 minutes.
Column chromatography was performed with Kieselgel 60
(230–400 mesh). Where indicated, compounds were purified with
diaion HP-20SS resin (Supelco). TLC was performed using
commercially prepared silica gel (Kieselgel 60 0.25 mm) plates
which were visualized either with UV light (254 nm) or by
immersion in acidic ammonium molybdate solution.
HPLC purification was carried out on a Gilson HPLC system
fitted with a Gilson 118 UV–vis detector, with detection at 254 nm.
Where noted, compounds were purified using a Hichrom KR-100
C18 5 lm 250 × 4.6 mm column. Columns were eluted with a
gradient of methanol–water.
Organic solutions were dried over Na2SO4 or MgSO4, as
indicated. All organic solvents were freshly distilled prior to
use. Dichloromethane (DCM), ethyl acetate (EtOAc), methanol,
hexane and acetonitrile were distilled over calcium hydride.
3,3-Dimethyl-2-oxobutyric acid 5-(6-aminopurin-9-yl)-3,4-
dihydroxytetrahydrofuran-2-ylmethyl ester (4). To a solution of
3,3-dimethyl-2-oxobutyric acid 14 (198 mg, 1.52 mmol), 2ꢀ,3ꢀ-
isopropylideneadenosine (200 mg, 0.65 mmol) in DCM (2 mL)
was added DCC (206 mg, 0.87 mmol) portionwise. After stirring
◦
at 22 C for 16 h the solution was filtered, the solid was washed
with DCM (20 mL) and the solution was concentrated in vacuo.
Purification by column chromatography (methanol–EtOAc, 1 :
20 v/v) gave the protected ester 15 as an oily residue (184 mg,
42%). dH (500 MHz, CDCl3): 1.19 (s, 9H, t-Bu), 1.38 (s, 3H, CH3),
1.60 (s, 3H, CH3), 4.46 (m, 2H, H5ꢀ), 4.52 (m, 1H, H4ꢀ), 5.08
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(dd, 1H, J = 3.3 and 6.3 Hz, H3ꢀ), 5.44 (dd, 1H, J = 2.2 and
6.3 Hz, H2ꢀ), 6.13 (d, 1H, J = 2.2 Hz, H1ꢀ), 7.93 (s, 1H, H2ꢀꢀ),
8.32 (s, 1H, H8ꢀꢀ). dC (125 MHz, DMSO): 25.3, 25.6, 27.1, 33.9,
42.6, 64.7, 81.6, 84.2, 84.4, 90.6, 114.7, 120.0, 139.7, 149.2, 153.1,
155.7, 162.6. mmax (NaCl): 3322 (m), 2929 (m), 1740 (m), 1625 (s),
1575 (s), 1242 (s), 1007 (m) cm−1. LCMS: RT 3.7 min, (MH+)
420.
The protected ester 15 (184 mg, 0.43 mmol) was dissolved in
water (0.4 mL) and cooled to 0 ◦C. TFA (1 mL) was added
dropwise and the reaction mixture was stirred and allowed to
22 ◦C over 3 h. The solvent was removed in vacuo by azeotroping
with ethanol and toluene. Purification by column chromatography
(methanol–EtOAc gradient 1 : 20 v/v) gave the ester as a white
solid (30 mg, 19%).dH (400 MHz, MeOD): 1.20 (s, 9H, t-Bu), 4.34
(m, 2H, H5ꢀ), 4.41 (t, 1H, J = 5.0 Hz, H4ꢀ), 4.57 (m, 1H, H3ꢀ),
4.73 (t, 1H, J = 4.9 Hz, H2ꢀ), 6.07 (d, 1H, J = 4.7 Hz, H1),
8.23 (s, 1H, H2ꢀꢀ), 8.33 (s, 1H, H8ꢀꢀ). dC (100 MHz, MeOD): 26.0,
43.6, 65.6, 71.9, 75.4, 83.3, 90.2, 120.5, 141.6, 150.6, 152.6, 156.5,
164.3. mmax (ATR): 3449 (m), 1677 (s), 1615 (s), 1454 (m), 1203 (s),
1088 (s) cm−1. HRMS (ES) calcd for C16H21N5O6: (MNa+) calcd:
380.1570, found: 380.1583.
4-Methoxy-2-(2-methoxyethoxymethoxy)-butyric acid (18).
To a solution of 2-methoxyethoxymethoxy-v-butyrolactone 16
(3.0 g, 16 mmol) in water (30 mL) and methanol (20 mL) was
added potassium hydroxide (750 mg, 19 mmol). After heating
at 70 ◦C for 4 h the solvent was removed in vacuo to give an
oil. This oil was dissolved in THF (40 mL) and 15-crown-5 (150
lL), cooled to 0 ◦C and sodium hydride (1.28 mg, 22 mmol) was
added carefully. After 10 min iodomethane (2.49 mL, 40 mmol)
was added and the solution was stirred at 22 ◦C for 15 h. A
solution of NaOH(aq) (10 mL of 1 M) was added carefully and the
◦
solution was stirred at 60 C for 2 h. The solution was acidified
to pH 3, extracted with EtOAc (3 × 25 mL), successively washed
with water (25 mL) and brine (25 mL), dried (MgSO4) and
concentrated in vacuo. The acid was obtained as an oil (1.5 g,
43%). dH (250 MHz, CDCl3): 2.00 (m, 2H, H2), 3.31 (s, 3H,
OCH3), 3.36 (s, 3H, OCH3), 3.51 (m, 4H, H7 and H4), 3.74 (m,
2H, CH2O), 4.31 (s, 1H, H2), 4.78 (m, 2H, H6). dC (100 MHz,
CDCl3): 30.9, 56.7, 57.3, 66.3, 68.3, 70.0, 71.1, 94.0, 174.6. mmax
(NaCl): 1745 (s) cm−1. HRMS (ES) for C9H18O6: (MNa+) calcd:
223.1182, found: 223.1168.
4-Methoxy-2-(2-methoxyethoxymethoxy)-3,3-dimethylbutyric
acid (19). To a solution of 4,4-dimethyl-2-methoxyethoxy-
methoxy-v-butyrolactone 17 (1.0 g, 4.6 mmol) in water (20 mL)
and methanol (5 mL) was added potassium hydroxide (310 mg,
5.5 mmol). After heating at 80 ◦C for 4 h the solvent was removed
in vacuo to give an oil. This oil was dissolved in THF (30 mL)
and 15-crown-5 (100 lL) and cooled to 0 ◦C, and sodium hydride
(480 mg, 12 mmol), was added carefully. After 10 min iodomethane
(1.24 mL, 20 mmol) was added and the solution was stirred
at 22 ◦C for 15 h. A solution of NaOH(aq) (10 mL of 1 M)
was added carefully and the solution was stirred at 60 ◦C for
5 h. The solution was acidified to pH 3, extracted with EtOAc
(3 × 25 mL), successively washed with water (25 mL) and brine
(25 mL), dried (MgSO4) and concentrated in vacuo. Purification
by column chromatography (EtOAc–hexane 1 : 1 v/v) gave the
acid as an oil (410 mg, 39%). dH (250 MHz, CDCl3): 1.06 (s,
3H, CH3), 1.11 (s, 3H, CH3), 3.20 (AB quartet, 2H, J = 8.9 Hz,
CH2O), 3.33 (s, 3H, OCH3), 3.39 (s, 3H, OCH3), 3.51 (t, 2H,
J = 4.7 Hz, OCH2), 3.74 (m, 2H, CH2O), 4.06 (s, 1H, H2), 4.80
(AB quartet, 2H, J = 6.9 Hz, H8). dC (62.5 MHz, CDCl3): 20.5,
21.2, 58.6, 70.5, 73.1, 74.2, 78.0, 81.7, 87.5, 172.8. mmax (NaCl):
1739 (s) cm−1. HRMS (ES) for C11H22O6: (MNa+) calcd 273.1314,
found 273.1314.
2-Methoxyethoxymethoxy-v-butyrolactone (16). To a suspen-
sion of sodium hydride (60% oil dispersion, 3.5 g, 88 mmol) and
15-crown-5 (0.65 g, 8 mmol) in THF (100 mL), cooled to 0 ◦C, was
adde◦d a-hydroxy-v-butyrolactone (5.0 g, 49 mmol). After stirring
at 0 C for 10 min MEMCl (6.7 g, 55 mmol) and THF (50 mL)
were added dropwise and the solution was stirred at 22 ◦C for
15 h. The reaction was quenched carefully with water (30 mL)
and the THF was removed in vacuo. The residue was diluted
with EtOAc (3 × 50 mL), dried (MgSO4) and solvent removed
in vacuo to yield the lactone as an oil (9.3 g, 100%) which was
used without further purification. dH (250 MHz, CDCl3): 2.31
(m, 1H, H3), 2.56 (m, 1H, H3), 3.38 (s, 1H, OCH3), 3.54 (m,
2H, OCH2), 3.74 (m, 2H, OCH2), 4.31 (m, 3H, H2 and H4),
4.90 (AB quartet, 2H, J = 6.9 Hz, H5, OCH2O). dC (62.5 MHz,
CDCl3): 29.8, 59.0, 65.8, 67.5, 70.3, 71.6, 94.9, 174.8. mmax: 1786
(s) cm−1. HRMS (ES) for C8H14O5: (MNa+) calcd: 213.0739,
found: 213.0738.
4,4-Dimethyl-2-methoxyethoxymethoxy-v-butyrolactone (17).
To a suspension of sodium hydride (60% oil dispersion, 6.0 g,
150 mmol), 15-crown-5 (1.3 g, 16 mmol) in THF (100 mL) cooled
to 0 ◦C, was added 4,4-dimethyl-a-hydroxy-c-butyrolactone
(100 mmol, 13.2 g). After stirring at 0 ◦C for 10 min MEMCl
(14.7 g, 120 mmol) was added and the solution was stirred at 22 ◦C
for 15 h. The reaction was quenched carefully with water (50 mL)
and the THF was removed in vacuo. The residue was diluted with
EtOAc (3 × 100 mL) and dried (MgSO4), and the solvent was
removed in vacuo to yield the lactone as an oil (22 g, 100%) which
was used without further purification. dH (250 MHz, MeOD):
1.09 (s, 3H, CH3), 1.19 (s, 3H, CH3), 3.38 (s, 3H, OCH3), 3.55
(m, 2H, methylene protons), 3.79 (m, 2H, methylene protons),
3.89 (AB quartet, 2H, J = 8.8 Hz, H4), 4.11 (s, 1H, H2), 4.82 (d,
1H, J = 6.8 Hz, OCH2O), 5.07 (d, 1H, J = 6.8 Hz, OCH2O). dC
(62.5 MHz, MeOD): 19.4, 23.1, 40.2, 59.0, 67.5, 70.6, 71.6, 78.4,
95.1, 175.0. mmax (NaCl): 1739 (s) cm−1. HRMS (ES) for C10H18O5:
(MNa+) calcd: 241.1052, found: 241.1054.
(2RS)-Adensoyl-2-hydroxy-4-methoxybutyrate
(205 lL, 1.3 mmol) and triphenylphosphine (332 mg,
1.3 mmol) were added to solution of 4-methoxy-2-(2-
(5). DEAD
a
methoxyethoxymethoxy)-butyric acid 18 (332 mg, 1.5 mmol)
and 2ꢀ,3ꢀ-isopropylideneadenosine (306 mg, 1.0 mmol) in THF
(30 mL). After stirring at 22 ◦C for 16 h the solution was
concentrated and then diluted with EtOAc (30 mL), washed with
water (20 mL) and dried (MgSO4), and the solvent was removed in
vacuo. Purification by column chromatography (methanol–DCM
gradient 2–15% methanol) gave the protected ester 20 as a white
solid (600 mg, 80%). dH (250 MHz, CDCl3): 1.36 (s, 3H, CH3),
1.58 (s, 3H, CH3), 1.94 (m, 2H, H3), 3.25 (s, 2H, H5), 3.33 (s,
3H, MEM group), 3.35 (m, 4H, MEM group and H4), 3.66
(m, 2H, MEM group), 4.22 (m, 3H, H2, H4ꢀ and H5ꢀ), 4.71 (m,
2H, MEM group), 5.04 (m, 1H, H3ꢀ), 5.42 (m, 1H, H2ꢀ), 6.08
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(m, 1H, H1ꢀ), 6.30 (m, 2H, NH2), 7.91 (s, 1H, H2ꢀꢀ), 8.32 (s, 1H,
H8ꢀꢀ).
78.4, 178.3. HRMS (ES) for C6H12O3: (MNa+) calcd: 155.0684,
found: 155.0689.
The protected ester 20 (600 mg, 1.2 mmol) was stirred for 3 h at
22 ◦C in water–TFA (2 : 4 v/v). The solvent was removed in vacuo
by azeotroping with ethanol and toluene. Purification by column
chromatography (methanol–DCM gradient 2–30% methanol)
gave the ester as a white solid (125 mg, 27%). dH (250 MHz, CDCl3):
1.74 (m, 2H, H3), 3.15 (s, 2H, H5), 3.25 (m, 2H, H4), 4.25 (m, 3H,
H2, H4ꢀand H5ꢀ), 4.60 (m, 1H, H3ꢀ), 5.51 (m, 2H, OH, H2ꢀ), 5.90
(d, 1H, J = 5.5 Hz, H1ꢀ), 7.00 (m, 2H, NH2), 8.13 (s, 1H, H2ꢀꢀ),
8.32 (s, 1H, H8ꢀꢀ). dC (125 MHz, DMSO): 34.0, 58.6, 61.8, 64.3,
67.7, 70.5, 73.5, 81.7, 88.0, 119.4, 140.0, 149.5, 152.7, 156.2, 174.0.
mmax (ATR): 3321 (w), 1643 (s), 1603 (s), 1478 (m), 1203 (m), 1075
(s) cm−1. HRMS (ES) for C15H19N5O7: (MH+) calcd: 384.1519,
found: 384.1508.
2-Dichloroacetoxy-3,3-dimethylbutanoic
acid
(23). 3,3-
Dimethyl-2-hydroxybutanoic acid 22 (500 mg, 3.8 mmol) was
added to dichloroacetyl chloride (2 mL, 20 mmol) and heated at
80 ◦C for 4 h. After removal of the excess dichloroacetyl chloride
in vacuo the acid was obtained as an oil (680 mg, 2.8 mol, 73%).
dH (250 MHz, CDCl3): 1.12 (s, 9H, CH3, H4), 4.78 (s, 1H, H2),
6.06 (s, 1H, H6). dC (62.5 MHz, CDCl3): 26.0, 34.2, 63.9, 81.8,
167.0. mmax (NaCl): 1735 (s) cm−1.
(2RS)-Adenosyl-2-hydroxy-3,3-dimethylbuyrate (7). To a so-
lution of 2-dichloroacetoxy-3,3-dimethylbutyric acid 23 (194 mg,
0.8 mmol) and 2ꢀ,3ꢀ-isopropylideneadenosine (270 mg, 0.88 mmol)
in THF (20 mL) were added DEAD (158 lL, 1.0 mmol) and
triphenylphosphine (256 mg, 1.0 mmol). After stirring at 22 ◦C
for 16 h the solution was concentrated in vacuo to remove the
THF. The residue was taken up in EtOAc (30 mL), washed with
water (20 mL) and dried (MgSO4), and the solvent was removed
in vacuo. Purification by column chromatography (methanol–
DCM gradient 2–10% methanol) gave the protected ester 24
as an oil (150 mg, 35%). dH (250 MHz, CDCl3): 0.89 (s, 9H,
CH3), 1.38 (s, 3H, CH3), 1.58 (s, 3H, CH3), 3.72 (m, 1H, H2ꢀ),
4.37 (m, 3H, H4ꢀ and H5ꢀ), 5.08 (m, 1H, H3ꢀ), 5.30 (m, 1H,
H2ꢀ), 6.09 (m, 1H, H1ꢀ), 6.21 (s, 1H, CH), 8.21 (s, 1H, H2ꢀꢀ),
8.27 (s, 1H, H8ꢀꢀ).
The protected ester 24 (150 mg, 0.28 mmol) was dissolved in
pyridine–water (1 : 1 v/v, 2 mL, pH 7.5) and stirred for 16 h
at 22 ◦C. The solvent was removed in vacuo and the residue
was dissolved in water–TFA (2 : 4 v/v) and stirred at 22 ◦C
for 3 h. The solvent was removed in vacuo by azeotroping with
ethanol and toluene. Purification by column chromatography
(methanol–DCM gradient 2–20% methanol) gave the ester 7
as a white solid (90 mg, 82%). dH (250 MHz, MeOD): 0.91
(s, 9H, CH3), 3.82 (s, 1H, H2), 4.39 (m, 4H, H3ꢀ, H4ꢀ and
H5ꢀ), 4.74 (m, 1H, H2ꢀ), 6.03 (d, 1H, J = 5.7 Hz, H1ꢀ), 8.21
(s, 1H, H2ꢀꢀ), 8.34 (s, 1H, H8ꢀꢀ). dC (62.5 MHz, DMSO): 25.0,
34.5, 63.6, 70.6, 73.9, 78.6, 82.2, 88.9, 122.2, 140.7, 150.6, 154.7,
157.1, 173.1. HRMS (ES) for C16H24N6O5: (MH+) calcd: 382.1726,
found: 382.1737.
(2RS)-Adensoyl-3,3-dimethyl-2-hydroxy-4-methoxybutyrate (6).
DEAD (221 lL, 1.4 mmol) and triphenylphosphine (358 mg,
1.4 mmol) were added to
a solution of 4-methoxy-2-(2-
methoxyethoxymethoxy)-3,3-dimethylbutyric acid 19 (236 mg,
1.0 mmol) and isopropylideneadenosine (368 mg, 1.2 mmol) in
◦
THF (30 mL). After stirring at 22 C for 16 h the solution was
concentrated and then diluted with EtOAc (30 mL), washed with
water (20 mL) and dried (MgSO4), and the solvent was removed in
vacuo. Purification by column chromatography (methanol–DCM
gradient 2–15% methanol) gave the protected ester 21 as a white
solid (385 mg, 73%). dH (250 MHz, CDCl3): 0.95 (s, 6H, H4), 1.36
(s, 3H, CH3), 1.58 (s, 3H, CH3), 3.16 (s, 2H, H5), 3.31 (m, 3H,
MEM group), 3.32 (s, 3H, H6), 3.46 (m, 2H, MEM group), 3.65
(m, 2H, MEM group), 4.01 (m, 1H, H2), 4.38 (m, 2H, H5ꢀ), 4.49
(m, 1H, H4ꢀ), 4.69 (m, 1H, MEM group), 5.08 (m, 1H, H3ꢀ), 5.45
(m, 1H, H2ꢀ), 5.65 (br s, 2H, NH2), 6.12 (m, 1H, H1ꢀ), 7.96 (s, 1H,
H2ꢀꢀ), 8.36 (s, 1H, H8ꢀꢀ).
The protected ester 21 (385 mg, 0.73 mmol) was stirred for 3 h at
22 ◦C in water–TFA (2 : 4 v/v). The solvent was removed in vacuo
by azeotroping with ethanol and toluene. Purification by column
chromatography (methanol–DCM gradient 2–30% methanol)
gave the ester as a white solid (110 mg, 37%). dH (250 MHz,
DMSO): 0.83 (s, 6H, H4), 3.16 (s, 2H, H5), 3.17 (s, 3H, H6), 3.90 (s,
1H, H2), 4.19 (m, 4H, H3ꢀ, H4ꢀ and H5ꢀ), 4.67 (m, 1H, H2ꢀ), 5.93 (d,
1H, J = 5.2 Hz, H1ꢀ), 7.42 (br s, 2H, NH2), 8.17 (s, 1H, H2ꢀꢀ), 8.39 (s,
1H, H8ꢀꢀ). dC (62.5 MHz, DMSO): 20.5, 21.2, 55.0, 58.6, 64.1, 70.5,
73.1, 74.2, 78.0, 81.8, 87.5, 119.1, 139.9, 149.4, 152.2, 155.7, 172.8.
mmax (ATR): 3434 (m), 1732 (m), 1644 (s), 1601 (m), 1477 (w), 1150
(s) cm−1. HRMS (ES) for C17H25N5O7: (MH+) calcd: 412.1824,
found: 412.1828.
2ꢀ,3ꢀ-Isopropylidene-5ꢀ-O-sulfamoyladenosine (25). To an ice-
cooled solution of chlorosulfonylisocyanate (1 mL, 1.6 g,
11.3 mmol) in acetonitrile (10 ml), was added water (0.2 ml),
dropwise. The mixture was warmed to 22 ◦C and stirred for
15 min. The solution was concentrated in vacuo by azeotroping
with toluene (10 mL) to give chlorosulfonylamine as a white
powder (1.4 g, 100% crude yield).
To a solution of 2ꢀ,3ꢀ-isopropylideneadenosine (1.0 g, 3.3 mmol),
DCM (10 ml) and DBU (1 mL) was added a solution of
chlorosulfonylamine (1.4 g) in DCM (5 mL) over 5 min. The
solution was stirred for 15 h, and then the solvent was removed
in vacuo. Column chromatography (methanol–ethyl acetate 10 :
90 v/v) gave the sulfamoyl as a white solid (751 mg, 58%). dH
(400 MHz, MeOD): 1.32 (s, 3H, CH3, H6), 1.53 (s, 3H, CH3, H7),
4.26–4.39 (AB of ABX, 2H, H5), 4.54 (X of ABX, 1H, H4), 5.12
(dd, J = 2.8 and 6.0 Hz, 1H, H3), 5.41 (dd, 1H, J = 2.4 and
3,3-Dimethyl-2-hydroxybutanoic acid (22). A solution of 3,3-
dimethyl-1,2-butanediol (2.8 g, 23.7 mmol), Pt–C (10%, 800 mg)
and NaOH (3.7 g, 93 mmol) in water (60 ml) under a constant
gentle flow of oxygen, was stirred at 70 ◦C for 5 h. The
mixture was filtered through a pad of celite, acidified to pH 3
and the solution was concentrated in vacuo. The solution was
diluted with EtOAc (50 mL), filtered to remove NaCl(s) and
dried (MgSO4), and the solvent was removed in vacuo to yield
the acid as an oil (1.5 g, 46%) which was >95% pure by 1H
NMR spectroscopy. dH (250 MHz, MeOD): 0.99 (s, 9H, CH3,
H4), 3.74 (s, 1H, H2). dC (62.5 MHz, MeOD): 25.6, 62.1,
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6.0 Hz, H2), 6.24 (d, 1H, J = 2.4 Hz, H1), 8.21 (s, 1H, H2ꢀ), 8.27
(s, 1H, H8ꢀ). dC (100 MHz, MeOD): 26.0, 28.0, 70.5, 83.5, 86.0,
86.3, 92.3, 116.2, 121.0, 142.0, 150.8, 154.6, 157.9. mmax (ATR):
3122 (m), 2100 (w), 1686 (s), 1650 (s), 1374 (s), 1105 (s) cm−1.
HRMS (ES) for C13H18N6O6SNa: (MNa+): calcd 409.0906, found
409.0927.
To a solution of 2ꢀ,3ꢀ-isopropylidene-5ꢀ-O-sulfamoyladenosine
25 (598 mg, 1.5 mmol) and DBU (500 lL) in DCM (10 mL)
was added 3,3-dimethyl-2-oxobutyric acid 2,5-dioxopyrrolidin-1-
yl ester 28 (820 mg, 3.6 mmol) portionwise. After the reaction
◦
was stirred at 22 C for 16 h, the solvent was removed in vacuo.
The residue was purified by column chromatography (EtOAc–
MeOH 90 : 10 v/v) and 29 was obtained as a white solid
(524 mg, 70%). dH (400 MHz, MeOH): 1.22 (s, 9H, t-Bu), 1.39
(s, 3H, CH3), 1.62 (s, 3H, CH3), 4.31 (t, 2H, J = 4.3 Hz, H5ꢀ),
4.56 (m, 1H, H4ꢀ), 5.16 (dd, 1H, J = 2.2 and 6.1 Hz, H3ꢀ),
5.39 (dd, 1H, J = 3.0 and 6.1 Hz, H2ꢀ), 6.25 (d, 1H, J =
2.9 Hz, H1ꢀ), 8.23 (s, 1H, H2ꢀꢀ), 8.42 (s, 1H, H8ꢀꢀ). dC (125 MHz,
MeOD): 25.6, 27.1, 27.5, 30.0, 33.8, 50.0, 70.2, 83.3, 85.7, 115.4,
120.2, 141.4, 150.5, 154.1, 157.4, 167.5, 174.6. LCMS: Rt 4.1,
(MH+) 499.
The protected compound 29 (50.2 mg, 0.10 mmol) was stirred
for 3 h at 22 ◦C in water–TFA (2 : 4 v/v). The solvent was
removed in vacuo by azeotroping with ethanol and toluene to
give a white solid (40 mg, 87%). Purification by diaion HP-
20SS chromatography (THF–water gradient 0 to 5%) gave the
sulfamoyl as a white solid (25 mg, 54%). dH (400 MHz, D2O):
1.10 (s, 9H, t-Bu), 4.36 (m, 2H, H5ꢀ), 4.77 (m, 3H, H4ꢀ, H3ꢀ, H2ꢀ),
4.98 (m, 1H, H2ꢀ), 6.10 (d, 1H, J = 5.1 Hz, H1ꢀ), 8.35 (s, 1H,
H2ꢀꢀ), 8.50 (s, 1H, H8ꢀꢀ). dC (125 MHz, D2O): 24.6, 30.6, 57.9,
69.6, 74.7, 82.3, 92.6, 130.6, 139.4, 144.1, 148.8, 152.6, 175.9,
198.8. mmax (ATR): 3324 (w), 2928 (m), 1684 (s), 1436 (m), 1134
(s) cm−1. HRMS (ES) for C16H23N6O8S: (MH+) calcd: 459.1298,
found: 459.1284.
(2RS)-5ꢀ-O-(3,3-Dimethyl-2-aminobutyrylsulfamoyl) adenosine
(8). To Boc-L-tert-leucine (388 mg, 1.55 mmol) and hydroxy-
succinimide (355 mg, 3.08 mmol) in DME (10 mL), cooled to
0
0
◦C, was added DCC (438 mg, 2.12 mmol). After sitting at
◦C for 15 h the solution was filtered and the solvent was
removed in vacuo. The residue was dissolved in Et2O (20 mL)
and filtered, and the solvent was removed in vacuo to give the
hydroxysuccinimide ester 26 as a white solid (318 mg, 62%).
The product was unstable to purification by chromatography. dH
(400 MHz, CDCl3): 1.12 (s, 9H, CH3), 1.45 (s, 9H, CH3), 2.82 (s,
4H, CH2), 4.42 (d, 1H, J = 7.2 Hz, H-a), 5.08 (d, 1H, J = 7.2 Hz,
NH). HRMS (ES) for C15H24N2O6Na: (MNa+) calcd 351.1532,
found 351.1540.
To a solution of isopropylidene sulfamoyl adenoside 25
(350 mg, 0.90 mmol) and DBU (500 lL) in DCM (5 mL) was
added 2-tert-butoxycarbonylamino-3,3-dimethylbutyric acid 2,5-
dioxopyrrolidin-1-yl ester 26 (330 mg, 1.00 mmol) portionwise.
The reaction was stirred at 22 ◦C for 16 h, then the solvent
was removed in vacuo and the residue was purified by column
chromatography (EtOAc). Compound 27 was obtained as a white
solid (263 mg) which included DBU impurities (5% by 1H NMR
spectroscopy). dH (400 MHz, MeOH): 1.28 (s, 9H, t-Bu), 1.37 (s,
3H, CH3), 1.51 (s, 9H, t-Bu), 1.59 (s, 3H, CH3), 4.21 (br s, 1H,
CH), 4.53 (br s, 2H, H5ꢀ), 4.59 (m, 1H, H4ꢀ), 5.16 (m, 1H, H3ꢀ),
5.33 (dd, 1H, J = 3.2, 5.8 Hz, H2ꢀ), 6.21 (d, 1H, J = 3.1 Hz, H1ꢀ),
8.36 (s, 1H, H2ꢀꢀ), 8.55 (s, 1H, H8ꢀꢀ). mmax (ATR): 3118 (m), 2101
(m), 1686 (s), 1647 (s), 1604 (s), 1374 (s), 1178 (s), 1079 (s) cm−1.
LCMS: Rt 4.0, (MH+) 600.
The protected compound 27 (82 mg, 0.14 mmol) was stirred for
16 h at 22 ◦C in water–TFA (2 : 4 v/v). The solvent was removed
in vacuo by azeotroping with ethanol and toluene. Purification
by diaion HP-20SS chromatography (THF–water gradient 0 to
5%) gave the sulfamoyl as a white solid (48 mg, 76% yield). dH
(250 MHz, D2O): 1.07 (s, 9H, H4), 4.38 (m, 4H, H2, H4ꢀ and
H5ꢀ), 4.63 (m, 1H, H3ꢀ), 4.79 (m, 1H, H2ꢀ), 6.09 (m, 1H, H1ꢀ), 8.18
(s, 1H, H2ꢀꢀ), 8.51 (s, 1H, H8ꢀꢀ). dC (125 MHz, D2O): 25.7, 32.8,
64.4, 67.5, 70.6, 74.6, 82.8, 88.1, 118.7, 139.8, 149.3, 152.5, 155.8,
174.1. mmax (ATR): 2990 (m), 1634 (s), 1600 (s), 1335 (m), 1150 (s),
1044 (s) cm−1. HRMS (ES) for C16H25N7O7SNa: (MNa+) calcd:
482.1434, found: 482.1428.
(2RS)-5ꢀ-O-(2-Hydroxy-4-methoxybutyrylsulfamoyl) adenosine
(10). To a solution of 4-methoxy-2-(2-methoxyethoxymethoxy)-
butyric acid 18 (500 mg, 2.3 mmol) and EDC (600 mg, 4 mmol) in
DCM (30 mL) were added pentafluorophenol (752 mg, 4 mmol)
and DMAP (50 mg, 0.4 mmol). After stirring at 22 ◦C for 15 h the
solution was washed with water (2 × 20 mL), dried (Na2SO4) and
concentrated in vacuo. Purification by column chromatography
(EtOAc–toluene 1 : 1 v/v) gave the pentafluorophenol ester 30
(529 mg, 60%). dH (250 MHz, MeOD): 2.19 (m, 2H, OCH2), 3.35
(s, 3H, OCH3), 3.39 (s, 3H, OCH3), 3.58 (s, 4H, H7 and H4),
3.77 (m, 2H, CH2O), 4.69 (m, 1H, H2), 4.84 (AB quartet, 2H,
J = 6.8 Hz, OCH2O). dF (376 MHz, MeOD): −152.1, −156.9,
−163.4.
2ꢀ,3ꢀ-Isopropylidene-5ꢀ-O-sulfamoyladenosine 25 (386 mg,
1 mmol), DMF (10 mL), and DBU (230 lL, 1.5 mmol) were
combined and stirred at 22 ◦C for 10 min. 4-Methoxy-2-(2-
methoxyethoxymethoxy)-butyric acid pentafluorophenol ester 30
(529 mg, 1.36 mmol) was added and the reaction was stirred
at 30 ◦C for 16 h. The residue was diluted with ethyl acetate
(30 mL), washed with water (20 mL), dried (Na2SO4) and
concentrated in vacuo. Purification by column chromatography
(methanol–DCM 2–30% methanol) gave an oil (100 mg), which
was a mixture of products including the desired product 32 by
LCMS.
The protected compound 32 (50 mg by LCMS analysis, max.
0.08 mmol) was stirred for 40 h at 22 ◦C in water–TFA (2 :
4 v/v). The solvent was removed in vacuo by azeotroping with
ethanol and toluene. Purification by HPLC gave the sulfamoyl
10 as a white solid (10 mg, 2% yield from 2ꢀ,3ꢀ-isopropylidene-
5ꢀ-O-sulfamoyladenosine 25). dH (400 MHz, MeOD): 2.01 (m,
(2RS)-5ꢀ-O-(3,3-Dimethyl-2-oxobutyrylsulfamoyl)
adenosine
(9). To 3,3-dimethyl-2-oxobutyric acid 14 (504 mg, 3.9 mmol)
and hydroxysuccinimide (646 mg, 5.6 mmol) in DME (10 mL),
cooled to 0 ◦C, was added DCC (939 mg, 4.5 mmol). After sitting
at 0 ◦C for 15 h the solution was filtered and the solvent removed
in vacuo. To further precipitate remaining DCU the residue was
resuspended in Et2O (20 mL) and filtered, and the solvent was
removed in vacuo to give the activated compound 32 as a white
solid (444 mg, 50%). The product was unstable to purification by
chromatography. dH (400 MHz, CDCl3): 1.34 (s, 9H, CH3), 2.89
(s, 4H, CH2).
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2H, H3), 3.22 (t, 2H, J = 6.9 Hz, H4), 3.30 (s, 3H, H5),
4.35 (m, 1H, H2), 4.43 (m, 4H, H3ꢀ, H4ꢀ and H5ꢀ), 4.65 (m,
1H, H2ꢀ), 6.14 (d, 1H, J = 4.8 Hz, H1ꢀ), 8.40 (s, 1H, H2ꢀꢀ),
8.55 (s, 1H, H8ꢀꢀ). dC (125 MHz, D2O): 35.8, 37.5, 52.6, 58.9,
69.9, 71.9, 76.2, 84.3, 90.4, 120.4, 143.6, 145.7, 150.1, 152.0,
174.4. HRMS (ES) for C15H23N6O9S: (MH+) calcd: 463.1247,
found: 463.1263.
Purification by column chromatography (methanol–DCM 2–
30% methanol) gave a white solid (368 mg). This contained
several products in which the desired product 35 was detected
by LCMS:
The protected compound 35 (386 mg, max. 0.77 mmol) was
stirred for 16 h at 22 ◦C in water–TFA (2 : 4 v/v). The solvent
was removed in vacuo by azeotroping with ethanol and toluene.
Purification by diaion HP-20SS chromatography (THF–water
gradient 0 to 5%) gave the sulfamoyl 12 as a white solid (33 mg, 7%
yield from isopropylidene sulfamoyl adenoside 25). dH (250 MHz,
D2O): 0.79 (s, 9H, H4), 3.58 (m, 1H, H2), 4.30 (m, 4H, H3ꢀ, H4ꢀ
and H5ꢀ), 4.65 (m, 1H, H2ꢀ), 6.05 (m, 1H, H1ꢀ), 8.25 (s, 1H, H2ꢀꢀ),
8.29 (s, 1H, H8ꢀꢀ). dC (125 MHz, D2O): 25.8, 36.8, 70.7, 72.8,
76.5, 83.5, 85.1, 89.7, 121.2, 142.3, 151.6, 155.4, 158.1, 183.3.
mmax (ATR): 3110 (w), 1633 (s), 1599 (s), 1376 (m), 1150 (s), 1040
(s) cm−1. HRMS (ES) for C16H25N6O8S: (MH+) calcd: 461.1454,
found: 461.1460.
(2RS)-5ꢀ -O-(3,3-Dimethyl-2-hydroxy-4-methoxybutyrylsulfa-
moyl) adenosine (11). To
a solution of 4-methoxy-2-(2-
methoxyethoxymethoxy)-3,3-dimethylbutyric acid 19 (500 mg,
2.1 mmol) and EDC (600 mg, 4 mmol) in DCM (30 mL) was
added pentafluorophenol (752 mg, 4 mmol) and DMAP (25 mg,
0.2 mmol). After stirring for 15 h at 22 ◦C, the solution was washed
with water (2 × 20 mL), dried (Na2SO4) and concentrated in vacuo.
Purification by column chromatography (EtOAc–toluene 1 : 1 v/v)
gave the pentafluorophenol ester 31 (255 mg, 30%). d H (250 MHz,
MeOD): 1.00 (s, 3H, CH3), 1.02 (s, 3H, CH3), 3.20 (AB quartet,
2H, J = 8.9 Hz, CH2O), 3.34 (s, 3H, OCH3), 3.38 (s, 3H, OCH3),
3.53 (m, 2H, OCH2), 3.76 (m, 2H, CH2O), 4.06 (s, 1H, H2), 4.75
(AB quartet, 2H, J = 6.9 Hz, H8). dF (376 MHz, MeOD): −151.5,
−158.2, −163.1. mmax: 1790 (s) cm−1.
Acknowledgements
We gratefully acknowledge financial support from the BBSRC and
Christ’s College, Cambridge.
2ꢀ,3ꢀ-Isopropylidene-5ꢀ-O-sulfamoyladenosine 25 (232 mg,
0.6 mmol), DMF (10 mL), and DBU (179 lL, 1.2 mmol)
were combined and stirred at 22 ◦C for 10 min. 4-Methoxy-
2-(2-methoxyethoxymethoxy)-3,3-dimethylbutyric acid pentaflu-
orophenol ester 31 (180 mg, 0.45 mmol) was added and the
reaction mixture was stirred at 30 ◦C for 16 h. The solvent
was removed in vacuo and the residue was dissolved with ethyl
acetate (30 mL), washed with water (20 mL), dried (Na2SO4) and
concentrated in vacuo. Purification by column chromatography
(methanol–DCM 2–30% methanol) gave an oily residue (77 mg).
This was a mixture in which the desired product 33 was detected by
LCMS.
References
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The protected compound 33 (max. 77 mg, 0.12 mmol) was
stirred for 16 h at 22 ◦C in water–TFA (2 : 4 v/v). The solvent
was removed in vacuo by azeotroping with ethanol and toluene.
Purification by HPLC gave the sulfamoyl 11 as a white solid (8 mg,
4% yield from 2ꢀ,3ꢀ-isopropylidene-5ꢀ-O-sulfamoyladenosine 25).
dH (250 MHz, MeOD): 0.94 (m, 3H, H4), 0.97 (m, 3H, H5), 3.15
(m, 2H, H6), 3.29 (s, 3H, H7), 3.89 (s, 1H, H2), 4.38 (m, 4H, H3ꢀ,
H4ꢀ and H5ꢀ), 4.63 (m, 1H, H2ꢀ), 6.05 (m, 1H, H1ꢀ), 8.25 (s, 1H,
H2ꢀꢀ), 8.29 (s, 1H, H8ꢀꢀ). dC (125 MHz, D2O): 20.8, 21.0, 38.2, 58.6,
68.1, 70.2, 73.9, 75.6, 77.4, 82.4, 87.1, 118.7, 139.7, 149.1, 152.8,
155.7, 180.3. mmax (ATR): 3257 (w), 1672 (s), 1648 (s), 1439 (w),
1325 (w), 1180 (s) cm−1. HRMS (ES) for C17H27N6O9S: (MH+)
calcd: 491.1156, found: 491.1577
(2RS)-5ꢀ-O-(3,3-Dimethyl-2-hydroxybutyrylsulfamoyl) adeno-
sine (12). To a solution of 2-dichloroacetoxy-3,3-dimethylbutyric
acid 23 (209 mg, 0.88 mmol) and EDC (600 mg, 1 mmol) in DCM
(30 mL) was added pentafluorophenol (752 mg, 2.2 mmol) and
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◦
17 Tripos SYBYL v6.5, Tripos, Inc, South Hanley Road, St Louis,
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16 h. Then the solution was washed with water (2 × 20 mL), dried
(Na2SO4) and concentrated in vacuo to give the pentafluorophenol
34 (500 mg, max. 0.88 mmol). This was directly reacted with 2ꢀ,3ꢀ-
isopropylidene-5ꢀ-O-sulfamoyladenosine 25 (386 mg, 1 mmol),
DMF (10 mL), and DBU (225 lL, 1.5 mmol). After stirring
at 30 ◦C for 16 h, the solution was concentrated in vacuo.
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3610 | Org. Biomol. Chem., 2006, 4, 3598–3610
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©