Exploring structural motifs necessary for substrate binding in the active site of Escherichia coli pantothenate kinase

Article abstract of DOI:10.1016/j.bmc.2014.04.030

The coenzyme A (CoA) biosynthetic enzymes have been used to produce various CoA analogues, including mechanistic probes of CoA-dependent enzymes such as those involved in fatty acid biosynthesis. These enzymes are also important for the activation of the pantothenamide class of antibacterial agents, and of a recently reported family of antibiotic resistance inhibitors. Herein we report a study on the selectivity of pantothenate kinase, the first and rate limiting step of CoA biosynthesis. A robust synthetic route was developed to allow rapid access to a small library of pantothenate analogs diversified at the β-alanine moiety, the carboxylate or the geminal dimethyl group. All derivatives were tested as substrates of Escherichia coli pantothenate kinase (EcPanK). Four derivatives, all N-aromatic pantothenamides, proved to be equivalent to the benchmark N-pentylpantothenamide (N5-pan) as substrates of EcPanK, while two others, also with N-aromatic groups, were some of the best substrates reported for this enzyme. This collection of data provides insight for the future design of PanK substrates in the production of useful CoA analogues.

Full text of DOI:10.1016/j.bmc.2014.04.030

Bioorganic & Medicinal Chemistry 22 (2014) 3083–3090
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Exploring structural motifs necessary for substrate binding in the
active site of Escherichia coli pantothenate kinase
⇑
Emelia Awuah, Eric Ma, Annabelle Hoegl, Kenward Vong, Eric Habib, Karine Auclair
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montréal, Québec H3A 0B8, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 February 2014
Revised 8 April 2014
Accepted 16 April 2014
Available online 24 April 2014
The coenzyme A (CoA) biosynthetic enzymes have been used to produce various CoA analogues, including
mechanistic probes of CoA-dependent enzymes such as those involved in fatty acid biosynthesis. These
enzymes are also important for the activation of the pantothenamide class of antibacterial agents, and
of a recently reported family of antibiotic resistance inhibitors. Herein we report a study on the selectivity
of pantothenate kinase, the first and rate limiting step of CoA biosynthesis. A robust synthetic route was
developed to allow rapid access to a small library of pantothenate analogs diversified at the b-alanine
moiety, the carboxylate or the geminal dimethyl group. All derivatives were tested as substrates of Esch-
erichia coli pantothenate kinase (EcPanK). Four derivatives, all N-aromatic pantothenamides, proved to be
equivalent to the benchmark N-pentylpantothenamide (N5-pan) as substrates of EcPanK, while two oth-
ers, also with N-aromatic groups, were some of the best substrates reported for this enzyme. This collec-
tion of data provides insight for the future design of PanK substrates in the production of useful CoA
analogues.
Keywords:
Enzyme ligand
Kinase
Coenzyme A
Structure–activity relationship
Inhibitor
Substrate selectivity
Ó 2014 Published by Elsevier Ltd.
1. Introduction
coli.5–10 Although the bacteriostatic effect of pantothenamides has
been recognized since the 1950s, their exact mechanism of action
The biosynthesis of coenzyme A (CoA), an essential cofactor, is a
process that is ubiquitously present in all living organisms. It is
most commonly a five-step process from pantothenate (vitamin
B5). Pantothenate kinase (PanK, EC 2.7.1.33) is the first and rate-
limiting step of this process and is characterized by the ATP-depen-
dent transformation of pantothenate to phosphopantothenate,1 as
shown in Figure 1. Three main types of PanK have been identified,
namely Types I, II and III, based on differences in structural fold, reg-
ulation, substrate affinity, and specificity.2 Type I PanK enzymes,
typically associated with bacteria, have gained significant interest
due to the low sequence similarity to their human counterparts.3
Their structural and amino acid sequence similarity is considered
high (52–91% similarity), with many predicted active site residues
conserved among all isoforms (Fig. S1). Numerous studies have
focused on developing PanK inhibitors and substrates.4 Of these,
pantothenamides are particularly interesting due to their antibac-
terial activity against Staphylococcus aureus and Escherichia
in bacteria has only been clarified in the last 15 years.5–15 Interest-
ingly, pantothenamides are substrates of E. coli PanK (EcPanK).
Their lethal effect on bacteria stems, at least in part, from their bio-
transformation by the CoA biosynthetic pathway into the corre-
sponding CoA analogues. The resulting antimetabolite proceeds to
Abbreviations: CoA, coenzyme A; PanK, pantothenate kinase; EcPanK, PanK
isoform from Escherichia coli; MtPanK, PanK isoform from Mycobacterium tubercu-
losis; CbPanK, PanK isoform from Coxiella burnetii; KpPanK, PanK isoform from
Klebsiella pneumonia.
⇑
Corresponding author. Tel.: +1 514 398 2822; fax: +1 514 398 3797.
E-mail address: karine.auclair@mcgill.ca (K. Auclair).
Figure 1. Reaction scheme for the PanK-catalyzed phosphorylation of pantothenate
to phosphopantothenate.
http://dx.doi.org/10.1016/j.bmc.2014.04.030
0968-0896/Ó 2014 Published by Elsevier Ltd.

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E. Awuah et al. / Bioorg. Med. Chem. 22 (2014) 3083–3090
Figure 2. Pantothenate analogues described in this work.
inhibit downstream pathways, such as fatty acid biosynthesis.8,16
Other studies have suggested that pantothenamides may, in some
strains, act as inhibitors of the CoA biosynthetic pathway.5,16
The importance of CoA analogues not only stems from their
antibacterial activity, but also from their demonstrated use as
chemical biology tools and as mechanistic probes of CoA-depen-

E. Awuah et al. / Bioorg. Med. Chem. 22 (2014) 3083–3090
3085
dent enzymes. These applications include both in vitro and in-cell
labelling of carrier proteins in polyketide and nonribosomal pep-
tide synthesis,17–19 as well as aminoglycoside resistance inhibitors
activated by the CoA biosynthetic pathway.20
studies with CoA analogues used for carrier protein labelling stud-
ies, where C-7 carbonyl group substitutions often showed detri-
mental effects.18 Synthesis of 4 and 5 (Scheme 1A) began with
first obtaining the amine required for D-pantolactone ring opening.
Although a wide range of pantothenamide derivatives have
been reported over the years, most of these derivatives are modi-
fied at the amine moiety and less is known about the effects of
modifications at other positions. Given the current and potential
utility of pantothenate derivatives, a greater understanding of their
effect on PanK is beneficial to future chemical biology and medic-
inal chemistry research. We report here on the synthesis of a
library of pantothenate analogues modified at the b-alanine, the
carboxylate or the geminal dimethyl group (Fig. 2), and on their
structure–activity relationships as EcPanK substrates.
Coupling of 1-pentylamine (13) with Boc-glycine yielded 14, which
was coupled with Boc-b-alanine to form the Boc protected amine
15. For the preparation of 5, the reaction of 13 with chloroacetyl
chloride first produced 16, to which was added tert-butyl-3-
aminopropylcarbamate to yield the Boc-protected amine 17. With
15 and 17 in hand, subsequent Boc-deprotection followed by D-
pantolactone addition afforded the desired compounds 4 and 5.
In the synthesis of guanidine-containing 6 (Scheme 1B), N,N0-di-
Boc-thiourea (18) was first reacted with ethylenediamine to pro-
duce 19, followed by coupling to 20 as previously reported21 to
yield 21. Final Boc and PMP deprotection afforded 6. For the p-chlo-
roaniline N-linked guanidine-containing 7 (Scheme 1C), imidazole
was first reacted with cyanogen bromide to form 22.22 Reaction of
22 with p-chloroaniline generated the guanidine derivative 23,
which upon addition of Boc-protected propane-1,3-diamine
2. Results
2.1. Design and synthesis
yielded intermediate 24. Boc-deprotection of 24 and
tone addition finally afforded 7. The assembly of triazole-contain-
ing derivatives and (Scheme 1D) involved 1,3-dipolar
cycloadditions with in situ azide formation to form intermediates
D-pantolac-
2.1.1. Compounds 4–12
Compounds 4–12 are designed to probe the hydrogen bonds
bridging residues Asn282 and Tyr240 to the C-7 carbonyl group
of pantothenate in EcPanK. Their importance was suggested by
8
9
Scheme 1. Reagents and conditions: (a) Boc-glycine, EDC, HOBt, DIPEA, THF, rt, 16 h, 94%; (b) TFA, DCM, rt, 1 h; (c) Boc-b-alanine, EDC, HOBt, DIPEA, THF, rt, 16 h, 60% over 2
steps; (d) -pantolactone, EtOH, TEA, 80 °C, 16 h, 40% over 2 steps; (e) chloroacetyl chloride, THF, rt, 16 h, 89%; (f) tert-butyl 3-aminopropylcarbamate, K2CO3, DMF, 50 °C,
16 h, 60%; (g) ethylenediamine, DMF, rt, 30 min, 82%; (h) EDC, DIPEA, HOBt, THF, rt, 16 h, 79%; (i) TFA, DCM, rt, 3 h; (j) 80% AcOH, rt, 16 h, 66% over 2 steps; (k) p-chloroaniline,
THF, rt, 12 h, 88%; (l) Boc-propane-1,3-diamine, THF, 65 °C, 24 h, 86%; (m) TFA, DCM, rt, 1 h; (n) -pantolactone, EtOH, TEA, 75 °C, 24 h, 59% over 2 steps; (o) 1-bromopentane,
NaN3, CuSO4ꢀ5H2O, sodium ascorbate, DMF, H2O, 100 °C, 16 h, 81%; (p) 1-pentyne, NaN3, CuSO4ꢀ5H2O, sodium ascorbate, DMF, H2O, 100 °C, 16 h, 26%; (q) TFA, DCM, rt, 1 h; (r)
-pantolactone, EtOH, NEt3, 75 °C, 16 h, 14–46% over 2 steps; (s) pentanal, 1% AcOH/MeOH, rt, 5 h; (t) NaBH4, MeOH, 0 °C–rt, 1 h; (u) TFA, DCM, rt, 1 h; (v) -pantolactone,
EtOH, NEt3, 75 °C, 24 h, 52% over 4 steps; (w) butyric acid, EDC, HOBt, DIPEA, THF, rt, 16 h, 53%; (x) TFA, DCM, rt, 1 h; (y) Boc-b-alanine, EDC, DMAP, DIPEA, THF, rt, 16 h, 64%
over 2 steps; (z) -pantolactone, EtOH, NEt3, 75 °C, 16 h, 11% over 2 steps.
D
D
D
D
D

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E. Awuah et al. / Bioorg. Med. Chem. 22 (2014) 3083–3090
Scheme 2. Reagents and conditions: (a) PMPCH(OMe)2, CSA, DMF, rt, 16 h, 75%; (b) NHS, DCC, THF, rt, 6–24 h, 90%; (c) R-NH2, DCM, rt, 16 h, 69–94%; (d) 80% AcOH, rt, 16 h,
71–94%; (e) microwave, HATU, DIPEA, DMF, 60 °C, 30 min; (f) 80% AcOH, rt, 16 h, 82–89% over 2 steps; (g) R-NH2, EDC, HOBt, DIPEA, THF, rt, 16 h, 60–98%; (h) TFA, DCM, rt,
1 h; (i) D-pantolactone, EtOH, NEt3, 75 °C, 16 h, 10–53% over 2 steps.
Scheme 3. Reagents and conditions: (a) Compound 53, HATU, DMF, DIPEA, 80 °C, 16 h, 72%; (b) 80% AcOH, rt, 16 h, 90%; (c) triclosan, Ph3P, DIAD, THF, rt, 24 h; (d) 80% AcOH,
rt, 16 h, 50% over 2 steps.
26 and 28, respectively. Deprotection and
D
-pantolactone addition
atives 35–52 were synthesized and used in this study to probe the
potential of pi–pi interactions with PanK. Np-Pan (34) was also re-
synthesized because no kinetic data were reported.24 For 34, 35–
39, 41–42, 44–46, and 51–52 (Scheme 2A), synthesis started with
the 1,3-diol protection of commercially available pantothenate as
a p-methoxyphenyldimethylacetal to yield 53, followed by activa-
tion to a N-succinamyl pantothenic ester 54.25 The desired com-
mercially available amines were then reacted separately with 54,
followed by dilute acidic removal of the acetal protecting group
to afford the N-linked derivatives 34, 35–37, 39, 41–42, 45, and
51–52 in good yields. Deactivated amines such as aniline, 3-meth-
ylpyridin-2-amine, and 4-aminopyrimidine under standard reac-
tion conditions resulted in lower yields (10–20%) of 38, 44, and
46, respectively. However, under microwave irradiation at 60 °C
for 30 min and using HATU as the coupling agent, higher yields
(82–89%) of 38, 44, and 46 were achieved. In our hands, previous
synthetic protocols for pantothenamide analogues using diph-
enylphosphorylazide to activate pantothenate were found to gen-
then afforded 8 and 9. Carboxylate-containing pantothenamide 10
(Scheme 1E) was assembled by first reductive amination of 3-N-
Boc protected 2,3-diaminopropanoic acid 29 with pentanal to
afford 30, which was needed for nucleophilic ring opening of D-
pantolactone following Boc deprotection. Synthesis of the known
sulfonate-containing 11 used commercially available taurine (after
NaOH treatment and precipitation) in the nucleophilic ring open-
ing of D
-pantolactone to yield 11, as previously reported.23 Finally,
compound 12 (Scheme 1F) was prepared from two subsequent
amide couplings of Boc-protected ethylenediamine (31) with buty-
ric acid and with Boc-b-alanine to afford 33. Deprotection of the
Boc group followed by
D-pantolactone addition yielded 12.
2.1.2. Compounds 35–52
The recently reported crystal structure of KpPanK bound to Np-
Pan (34) suggests that PanK substrates may interact with the
enzyme via
p–p interactions with nearby Tyr180 and Phe259,
and potentially with Tyr240, Tyr258, and Tyr262.24 As such, deriv-
erate diphenyl hydrogen phosphate as
a byproduct which

E. Awuah et al. / Bioorg. Med. Chem. 22 (2014) 3083–3090
3087
contaminated the final product.7 For the synthesis of 40, 43, and
49–50 (Scheme 2B), Boc-b-alanine (56) was first coupled to the
desired commercially available amine to produce 57a–d. Subse-
ble and must be used right away. Subsequent amide coupling fol-
lowed by deprotection afforded the desired pantothenamides 62-
67.
quent deprotection followed by
D-pantolactone addition then
afforded the desired compounds 40, 43, and 49–50.
2.2. Biological studies
Two approaches were envisaged to assemble the triclosan
derivatives 47 and 48 (Scheme 3). Synthesis of the amide-linked
47 was achieved from amine 58 which was synthesized according
to a literature protocol.26 Subsequent coupling of 58 to 53 afforded
59, followed by deprotection to yield 47. Synthesis of the ether-
linked triclosan derivative 48 on the other hand began with
Following the synthesis of 4–12, 35–52, and 62–67, their sub-
strate activity with EcPanK was measured and the results are com-
piled in Table 1.
While transformation of compounds 6–7, 11, 47–48, 51–52, and
66–67 by EcPanK is below our detection limit, complementary
studies show that they did not inhibit the enzyme either (data
not shown), suggesting that the compounds are likely not binding
EcPanK. All other derivatives are substrates of EcPanK, yet their
overall activity (kcat/Km) vary greatly from one to the other. A
few compounds (3, 38, 39, 41, 42, 44 and 45) show comparable
activity to that of pantetheine, while 34 and 40 are some of the rare
compounds to rival the preferred substrate pantothenate.
PMP-protected D
-pantothenol 60,25 which underwent a Mitsunobu
reaction with triclosan to afford 61. PMP deprotection yielded the
desired product 48.
2.1.3. Compounds 62–67
Finally, a previous study with C-2 geminal dialkylpantothena-
mides suggests that they might be good PanK substrates.11 Crystal
structure analysis points to possible interactions of the alkyl group
with a hydrophobic pocket consisting of Val97, Leu201, and Ile290.
Compounds 62–67 were synthesized to probe this pocket and
study consequent effects on substrate activity. Synthesis of C-2
geminal dialkylpantothenamide derivatives 62–67 was achieved
3. Discussion
Crystal structures are reported for Type I PanK isoforms from
four species: Escherichia coli (EcPanK),14,28 Mycobacterium tuber-
culosis (MtPanK),29–31 Coxiella burnetii (CbPanK; PDB 3TQC), and
Klebsiella pneumoniae (KpPanK).24 The reported crystal structure
by adapting
a protocol previously reported by our group
(Scheme 4).11 The known diethyl ester 68,11 was first reacted in
two successive alkylations using the method developed by Seebach
and Frater27 to generate derivatives 70a–f via 69a–f. Reduction to
the triol followed by selective 1,3-diol protection, left the terminal
alcohol of 71a–f available for oxidation to the corresponding acids
72a–f, via Dess–Martin Periodinane oxidation of the alcohol to the
aldehyde followed by Pinnick oxidation. The acids 72a–f are unsta-
Table 1
EcPanK kinetic parameters
Km
(
lM)
kcat (sꢁ1
)
kcat/Km (mMꢁ1 sꢁ1
)
Pantothenate (1)
exp
21
34
3
6
1.07 0.05
0.76 0.03
50
21
7
2
lit.6
Pantetheine (2)
exp
lit.17
N5-Pan (3)
4
5
6
60 10
91 10
1.03 0.06
0.32 0.02
0.93 0.04
1.12 0.05
0.13 0.03
17
3.5 0.4
26
10
0.1 0.1
3
36
3
2
1
120 10
1200 400
NAa
7
NA
8
9
190 20
1000 200
700 300
NA
0.96 0.04
0.49 0.06
0.23 0.05
5.1 0.6
0.5 0.1
0.3 0.2
10
11
12
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
62
63
64
65
66
67
240 30
1.09 0.07
1.24 0.06
1.19 0.11
0.75 0.18
1.03 0.05
1.02 0.05
1.15 0.05
1.13 0.05
0.46 0.03
1.02 0.05
0.94 0.08
0.78 0.04
1.15 0.06
1.44 0.09
4.5 0.6
33
4
38
5
>1000
>1000
350 20
0.5 0.1
0.3 0.1
2.9 0.3
43
67
32
22
50
4
6
3
7
7
24
17
35
21
20
7
3
2
4
6
3
2
3
1
130 30
39
5
20
11
110 10
450 50
NA
3.2 0.4
NA
200 100
400 200
NA
0.45 0.09
0.18 0.05
1.9 0.9
0.4 0.2
NA
230 20
1200 400
590 60
110 10
NA
1.17 0.06
0.28 0.06
0.89 0.06
1.04 0.06
5.1 0.5
0.2 0.1
1.5 0.2
Scheme 4. Reagents and conditions: (a) LDA, THF, ꢁ78 °C, 15 min; (b) R1-X,
ꢁ78 °C–rt, 16 h, 67–75%; (c) R2-X, 78 °C-rt, 16 h, 29–58%; (d) LAH, THF, 70 °C, 16 h;
(e) PMPCH(OMe)2, CSA, DCM, rt, 4 h, 50–86% over 2 steps; (f) DMP, DCM, rt, 2 h; (g)
NaClO2, NaHPO4, acetone, DCM, H2O, rt, 15 min; (h) piperidine, DMF, rt, 30 min; (i)
EDC, DIPEA, HOBt, THF, rt, 16 h, 42–51% over 4 steps; (j) 80% AcOH, rt, 16 h, 64–81%.
9
1
NA
a
NA—no observable activity up to 1 lM.

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E. Awuah et al. / Bioorg. Med. Chem. 22 (2014) 3083–3090
Figure 3. Highlight of the conserved active site residues involved in substrate binding; (A) EcPanK (PDB 1SQ5) in complex with pantothenate (1); (B) KpPanK (PDB 4F7W) in
complex with 3; (C) KpPanK (PDB 4GI7) in complex with 34 and in a conformation where sandwich stacking interactions form with Tyr180, or (D) KpPanK (PDB 4GI7) in
interactions form with Phe259 and water-mediated hydrogen bonding between the pyridyl nitrogen and Arg243,
p–p
complex with 34 in a conformation where T-shaped p–p
Phe244, and Tyr240.
of EcPanK in complex with pantothenate predicts several amino
acids involved in stabilizing pantothenate binding.28 These include
Asp127 and His177 forming hydrogen bonds with the C1 and C3
hydroxyl groups of pantothenate, respectively; Tyr175 hydrogen
bonding with the C-4 carbonyl oxygen via H2O; and Asn282 and
Tyr240 hydrogen bonding with the terminal C-7 carboxylate of
pantothenate (Fig. 3A). Recently, KpPanK was also crystallized with
3 and 34,24 where analysis confirms similar roles of Asp127,
His177, Tyr175, Asn282, and Tyr240 for substrate binding, whereas
Asn282 and Tyr240 may hydrogen bond not only with the C-7 car-
boxylate of pantothenate but also with the C-7 amides of 3 and 34
(Fig. 3B–D). It is also interesting to note that 34 is able to adopt two
different substrate conformations in the enzyme. One conforma-
tion has the pyridine moiety either positioned parallel to Tyr180
The next series of molecules probed whether the hydrogen
bonding acceptor and donor nature of the C-7 carboxylate/amide
could be mimicked with triazoles,32,33 guanidines, or sulfonate.
The results of the kinetic studies imply that guanidine groups (as
in 6 and 7) are inadequate mimics in this context. Docking 6 into
EcPanK suggest that the guanidine group might be better posi-
tioned near the hydroxyl group of Tyr240 (Fig. 4B). The aromatic
group of 7 may however compete for Tyr240 thus preventing inter-
action between the guanine group of 7 and Tyr240. Triazole 8 on
the other hand was a better mimic but still suffered a loss of activ-
ity (kcat/Km = 5.1 mMꢁ1 sꢁ1) compared to that of 3 (kcat/Km = 26 -
mMꢁ1 sꢁ1). Docking studies predict that hydrogen bonding with
Asn282 and Tyr240 should be preserved (Fig. 4C), and hence steric
effects might explain the observed loss. This hypothesis was veri-
fied with compound 9, which has similar steric bulk as that of 8
but with a triazole moiety not positioned to hydrogen bond with
Asn282 and Tyr240 (Fig. 4C). As expected 9 showed vastly reduced
activity (kcat/Km = 0.5 mMꢁ1 sꢁ1) compared to 8.
forming
mation has the pyridine moiety positioned perpendicular to
Phe259 forming T-shaped interactions (Fig. 3D).
p–p stacking interactions (Fig. 3C), and the second confor-
p–p
Based on the kinetic data obtained with EcPanK and crystal
structure analysis, we were able to identify and verify several
aspects of the enzyme binding pocket that are crucial for influenc-
ing the binding affinity and activity of PanK substrates. As men-
tioned above, the 3D structures of PanK suggest that the
hydrogen bonds between the C-7 carboxylate of pantothenate
and both Asn282 and Tyr240 can be sustained by an amide group
(C-7 amide of N5-Pan). This is supported by very similar Km value
Mimicking the carboxylate group was attempted with a sulfo-
nate, however the sulfonate-containing 11 was not a substrate of
PanK. A possible explanation for this observation is that the
enzyme binding pocket may not tolerate the bulky tetrahedral
geometry of the sulfonate group. This is further supported by the
loss of activity for 10 (kcat/Km = 0.3 mMꢁ1 sꢁ1), which is a derivative
of pantothenate with a side chain at C-6.
(21 vs 36
ing EcPanK was confirmed with the 10 times difference in binding
affinity between the amide-containing 4 (kcat/Km = 10 mMꢁ1 sꢁ1
compared to its amide-deficient counterpart, (kcat/Km
0.1 mMꢁ1 sꢁ1). This is also supported by modeling (Fig. 4A).
l
M). The importance of the C-7 carbonyl group for bind-
Compounds 4, 5, and 12 were synthesized with either C-9 or C-10
amide groups. All of these compounds (kcat/Km = 0.1–1 mMꢁ1 sꢁ1
)
)
displayed reduced activity compared to 3 (kcat/Km = 26 mMꢁ1 sꢁ1).
This is consistent with the lack of hydrogen bond acceptors/donors
in the pantothenate binding pocket besides those aligned with
5
=

E. Awuah et al. / Bioorg. Med. Chem. 22 (2014) 3083–3090
3089
Figure 5. Highlight of the hydrophobic pocket consisting of residues Val97, Leu201,
and Ile290 from EcPanK (green) in complex with (yellow), and modeled
3
a
conformation of 65 (turquoise) and its potential reach into the hydrophobic binding
pocket of EcPanK.
Another interesting observation with 34 was the position of the
pyridyl nitrogen and its water-mediated hydrogen bonding with
Tyr240, Phe244, and Arg243 of KpPanK.24 Our studies show that
the pyridyl nitrogen brings
a sizable contribution (e.g. 39,
kcat/Km = 17 mMꢁ1 sꢁ1), and its position matters (e.g. 42,
kcat/Km = 20 mMꢁ1 sꢁ1; 43, kcat/Km = 7 mMꢁ1 sꢁ1). The importance
of the aromatic ring was further confirmed using the amine-contain-
ing, six membered ring substituted 35, 36, and 37 which all showed a
loss of activity with EcPanK (kcat/Km = 0.3–2.9 mMꢁ1 sꢁ1) compared
to 38–45. As an extra control for the increase bulk of sp3 over sp2
hybridized carbons, the less bulky linear polyamine-containing 51
and 52 were also tested, and, as predicted they were not significantly
transformed by EcPanK.
Analysis of the various PanK crystal structures14,24,28–31 reveals
a cluster of residues (Val97, Leu201, and Ile290) forming a small
hydrophobic pocket in the enzyme active site (Fig. 5). To explore
whether substrates can interact with this pocket via van der Waals
forces, a series of derivatives of 3 modified at the C-2 geminal dial-
kyl position were prepared. Emphasis was placed on modifying the
C-2 alkyl substituent anti to the C-3 hydroxyl group, which is the
position on the backbone of 3 that is best suited to reach into the
pocket. Of these, the propynyl derivative 65 proved to be the most
promising, consistent with a previous study reporting the allyl
group as the best among a variety of short alkyl groups.11 A com-
bination of reduced bulk of the propynyl substituent and stronger
van der Waals forces that alkynes experience due to the increased
Figure 4. Highlight of the predicted hydrogen bonding interactions of Tyr240 and
Asn282 from EcPanK (green) with (A) 4 (yellow) and 5 (turquoise), (B) 6 (turquoise)
and 1 (orange), and (C) 8 (yellow) and 9 (grey).
polarity of its
p-elections as a result of its linearity, and might
explain this result.
C-7. Indeed the majority of residues in this area are hydrophobic,
aromatic residues.
4. Conclusion
A recent study shows that 34 may adopt two different substrate
conformations in KpPanK,24 hinting that aromatic rings might fit
We have developed the synthetic route necessary to access pan-
tothenate analogs diversified not only at the amine moiety but also
at the b-alanine moiety, at the carboxylate, or at the geminal
dimethyl group. Kinetic studies of these analogs with EcPanK
revealed a number of structure–activity relationships. While some
of our compounds proved to be equivalent to the benchmark com-
pound 3, others were some of the best substrates reported for
EcPanK. We believe that these results will be useful to guide the
future design of PanK substrates for the biosynthetic generation
of CoA analogues as probes or drug candidates.
better in EcPanK. In fact, turnover rates were better for 34 (kcat
Km = 38 mMꢁ1 sꢁ1) than for 3 (kcat/Km = 26 mMꢁ1 sꢁ1). Given these
results, potential interactions between the enzyme and sub-
/
p–p
strates were probed with additional pantothenamides containing
various aromatic substituents. There was little change among bind-
ing affinities for substrates with differing chain lengths (0–3 car-
bons) linking the aromatic substituents to the C-7 amide, with a
slight preference for 3 carbons (see 38–40). A possible explanation
for this result is that the high proportion of aromatic residues in the
substrate binding pocket (e.g. Tyr180, Phe259, Tyr240, Tyr258, and
Tyr262) allows a high degree of flexibility for
p–p interactions to
5. Experimental procedures
5.1. Docking studies
form. Overall, imidazole (e.g. 41), pyridine (e.g. 34, 42–45), and phe-
nyl groups (e.g. 38–40) were all effective for this purpose. Multi-
ring aromatics (e.g. 47–48) and bulky aromatic substituents (e.g.
49–50), however had a detrimental effect. This suggests that the
binding pocket can only accommodate one aromatic ring.
Docking was performed using FITTED via the web-based plat-
form FORECASTER.34

3090
E. Awuah et al. / Bioorg. Med. Chem. 22 (2014) 3083–3090
5.2. Expression and purification of EcPanK
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5.3. Kinetic assay of EcPanK
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Detailed synthetic protocols and compound characterizations
are provided as Supporting information.
Acknowledgments
This work was supported by Research Grants from the Canadian
Institute of Health Research (CIHR) and the National Science and
Engineering Research Council of Canada (NSERC) to K.A. CIHR pro-
vided funding as part of the Strategic Training Initiative in Chemi-
cal Biology (to K.V.), the Drug Development Training Program (to
E.A. and A.H.), and a graduate scholarship (to A.H.). The Fonds
québécois de la recherche sur la nature et les technologies (FQRNT,
to K.V.) also provided a scholarship. The authors are grateful to G.D.
Wright at McMaster University for sharing his PanK expression
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.04.030.