Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: New design with improved properties

Article abstract of DOI:10.1016/j.bmc.2011.11.032

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthetic pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin. The enzyme is essential for microorganisms, is absent from humans, and is not the target for any existing antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. Previously, we characterized the reaction trajectory of HPPK-catalyzed pyrophosphoryl transfer and synthesized a series of bisubstrate analog inhibitors of the enzyme by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups. Here, we report a new generation of bisubstrate analog inhibitors. To improve protein binding and linker properties of such inhibitors, we have replaced the pterin moiety with 7,7-dimethyl-7,8- dihydropterin and the phosphate bridge with a piperidine linked thioether. We have synthesized the new inhibitors, measured their K_d and IC ₅₀ values, determined their crystal structures in complex with HPPK, and established their structure-activity relationship. 6-Carboxylic acid ethyl ester-7,7-dimethyl-7,8-dihydropterin, a novel intermediate that we developed recently for easy derivatization at position 6 of 7,7-dimethyl-7,8- dihydropterin, offers a much high yield for the synthesis of bisubstrate analogs than that of previously established procedure.

Full text of DOI:10.1016/j.bmc.2011.11.032

Bioorganic & Medicinal Chemistry 20 (2012) 47–57
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin
pyrophosphokinase: New design with improved properties
Genbin Shi a, Gary Shaw a, Yu-He Liang a, Priadarsini Subburaman a, Yue Li b,
Yan Wu b, Honggao Yan b, Xinhua Ji a,
⇑
a Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702, USA
b Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthetic
pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin. The
enzyme is essential for microorganisms, is absent from humans, and is not the target for any existing
antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. Previously,
we characterized the reaction trajectory of HPPK-catalyzed pyrophosphoryl transfer and synthesized a
series of bisubstrate analog inhibitors of the enzyme by linking 6-hydroxymethylpterin to adenosine
through 2, 3, or 4 phosphate groups. Here, we report a new generation of bisubstrate analog inhibitors.
To improve protein binding and linker properties of such inhibitors, we have replaced the pterin moiety
with 7,7-dimethyl-7,8-dihydropterin and the phosphate bridge with a piperidine linked thioether. We
have synthesized the new inhibitors, measured their Kd and IC50 values, determined their crystal struc-
tures in complex with HPPK, and established their structure–activity relationship. 6-Carboxylic acid ethyl
ester-7,7-dimethyl-7,8-dihydropterin, a novel intermediate that we developed recently for easy deriva-
tization at position 6 of 7,7-dimethyl-7,8-dihydropterin, offers a much high yield for the synthesis of
bisubstrate analogs than that of previously established procedure.
Received 16 September 2011
Revised 8 November 2011
Accepted 16 November 2011
Available online 23 November 2011
Keywords:
Antibacterial
Bisubstrate
Folate
HPPK
Pterin
Published by Elsevier Ltd.
1. Introduction
HPPK catalyzes the transfer of pyrophosphate from ATP to
6-hydroxymethyl-7,8-dihydropterin (HP) (Fig. 1A).18 The reaction
Folate cofactors are essential for life.1 Mammals derive folates
from their diet, whereas most microorganisms must synthesize fo-
lates de novo.2 Therefore, the folate pathway has been a promising
target for developing antimicrobial agents.3–9 For example, inhibi-
tors of dihydropteroate synthase and dihydrofolate reductase, two
key enzymes in the pathway, are currently used in clinic as antibi-
otics.10–12 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokin-
ase (HPPK, E.C. 2.7.6.3) is another key enzyme in the pathway. It
is essential for microorganisms, is absent from mammals, and is
not the target for any existing antibiotics. Therefore, the enzyme
has been explored as an attractive target for developing novel anti-
microbial agents.13–17
follows an apparently ordered kinetic mechanism with MgATP
binding to the enzyme first followed by rapid addition of HP; the
Kd for the binding of MgATP to HPPK is 2.6–4.5
for the binding of HP to the HPPKꢀMgATP complex is in the sub-
M range.19–21 In contrast, the Kd for the binding of HP to ligand-
lM, and the Kd
l
free HPPK is in the mM range.22 The cellular ATP concentration is
estimated to be ꢁ3 mM.23 Therefore, the enzyme is essentially in
the MgATP-bound form, ready for the addition of HP. The products
of the reaction are AMP and 6-hydroxymethyl-7,8-dihydropterin
pyrophosphate (HPPP, Fig. 1A) and the release of reaction products
is rate limiting.21 In-depth structural and mechanistic studies of
the enzyme ensured that HPPK is currently the best understood
pyrophosphokinase.24,25
Two types of HPPK inhibitors have been developed previously.24
Type 1 inhibitors are HP derivatives, including 6-hydroxymethyl-
7,7-dimethyl-7,8-dihydropterin (HP-1, Fig. 1B)13,15 and 6-hydroxy-
methyl-7-methyl-7-phenethyl-7,8-dihydropterin (HP-3, Fig. 1C).16
Type 2 inhibitors are bisubstrate analogues P1-(6-hydroxymethylp-
terin)-P2-(50-adenosyl)diphosphate, P1-(6-hydroxymethylpterin)-
P3-(50-adenosyl)triphosphate, P1-(6-hydroxymethylpterin)-P4-(50-
adenosyl)tetraphosphate (HPnA; n = 2, 3, or 4, Fig. 1D).17 However,
Abbreviations: AMPCPP, a
,b-methyleneadenosine 50-triphosphate; HP, 6-hydroxy-
methyl-7,8-dihydropterin; HP-1, 6-hydroxymethyl-7,7-dimethyl-7,8-dihydropterin;
HP-3,6-hydroxymethyl-7-methyl-7-phenethyl-7,8-dihydropterin;HP2A,P1-(6-hydrox-
ymethylpterin)-P2-(50-adenosyl)diphosphate; HP3A, P1-(6-hydroxymethylpterin)-P3-
(50-adenosyl)triphosphate; HP4A, P1-(6-hydroxymethylpterin)-P4-(50-adenosyl)tetra-
phosphate; HPPK, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase; HPPP,
6-hydroxymethyl-7,8-dihydropterin pyrophosphate; PDB, Protein Data Bank.
⇑
Corresponding author. Tel.: +1 301 846 5035; fax: +1 301 846 6073.
E-mail address: jix@mail.nih.gov (X. Ji).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2011.11.032

48
G. Shi et al. / Bioorg. Med. Chem. 20 (2012) 47–57
1CBK).15 Furthermore, in the HPPKꢀHP-1 structure, the 7,7-dimethyl
group of HP-1 interacts favorably with the W89 side chain of HPPK.
As aforementioned, the phosphate bridge of HP4A carries several
negative charges that may lead to poor bioavailability. Besides, the
intact ATP moiety as part of the inhibitor may result in unwanted
interaction with other kinases. At this stage of the development,
we have modified the HP4A structure in two respects. First, we have
replaced the phosphate bridge with a piperidine linkage (Fig. 2C).
Second, we have introduced two methyl groups at position 7 of
the pterin moiety (Fig. 2D). Using the piperidine, which is seen in
other drug molecules, we can eliminate the negative charges carried
by the linkage and also diminish unwanted binding of the inhibitor
by other kinases. Introducing the 7,7-dimethyl group, we can effec-
tively prevent the hydrogen bond donor N8 from being oxidized and
thereby promote the formation of the hydrogen bond between N8
and carbonyl oxygen of L45. The resulting structures fit nicely in
the active center of HPPK (not shown).
2.2. Synthesis
Compounds 7, 8, and 16–19 are new, whereas the rest are either
known or commercially available. Using commercially available 20,
30-isopropylideneadenosine (1, Scheme 1), we followed the Sakami
method31 to synthesize 20,30-O-isopropylidene-50-O-toluene-p-sul-
fonyl adenosine (2), during which an anhydrous pyridine solution
of1wasshakenwithp-toluenesulfonylchloride. Thesyntheticinter-
mediate4-acetylsulfanyl-piperidine-1-carboxylic acid tert-butyles-
ter (4) was synthesized by using the method of Plettenburg et al.32 in
which potassium thioacetate and 4-bromo-piperidine (3) were
heated in DMF. According to a modified procedure based on an exist-
ing protocol,33 4 reacted with sodium methoxide to form the thiol,
followed by the reaction with 2 to give 4-[6-(6-amino-purin-9-yl)-
2,2-dimethyl-tetrahydro-furo[3,4-d][1,3]dioxol-4-ylmethylsulfa-
nyl]-piperidine-1-carboxylicacid tert-butylester(5). Under theTFA/
DCM condition, cleavage of the BOC protection group yielded 9-[2,2-
dimethyl-6-(piperidin-4-ylsulfanylmethyl)-tetrahydro-furo[3,4-
d][1,3]dioxol-4-yl]-9H-purin-6-ylamine (6) and the subsequent
reaction of 6 with (2-bromo-ethyl)-carbamic acid tert-butyl ester
provided the key intermediate (2-{4-[6-(6-amino-purin-9-yl)-2,
2-dimethyl-tetrahydro-furo[3,4-d][1,3]dioxol-4-ylmethylsulfa-
nyl]-piperidin-1-yl}-ethyl)-carbamic acid tert-butyl ester (7). The
deprotection of 7 yielded 2-[1-(2-amino-ethyl)-piperidin-4-ylsulfa-
nylmethyl]-5-(6-amino-purin-9-yl)-tetrahydro-furan-3,4-diol (8)
that contained an amino group that would be used to link 8 to the
pterin moiety.
Figure 1. Reaction and molecular structures of previous HPPK inhibitors. (A)
Reaction catalyzed by HPPK and chemical structures of substrate HP and product
HPPP. (B-D) HPPK inhibitors HP-1,13,15 HP-3,16 and HPnA (n = 2,3, or 4).17
HP-1 is degraded during prolonged incubation with HPPK,15 the
phenethyl groupof HP-3 disturbsthe geometry of enzyme active site
significantly,16,24 which may lead to low binding affinity, and the
phosphate bridge of HPnA carries many negative charges,17 which
may lead to poor bioavailability. Although none of these compounds
are considered useful inhibitors of HPPK,26 they provide valuable
information for further development. Here, we present improved
bisubstrate analog inhibitors of HPPK, which we have designed on
the basis of useful features of aforementioned inhibitors.
2. Results and discussion
2.1. Design
In the catalytic complex of HPPK,16,27,28 pterin is recognized by
HPPK via five hydrogen bonds (Fig. 2A); so is adenosine (Fig. 2B).
The phosphate bridge, however, is bound optimally only when the
phosphoryl transfer reaction is about to occur. One effective ap-
proach to enhance the specificity and potency in enzyme inhibition
is todesign multisubstrate analogs.29,30 Among the three bisubstrate
analogs we have previously developed, HP4A is the most potent
A total of four pterin moieties were synthesized, including
6-bromomethylpterin (11, Scheme 2), 6-bromomethyl-7,7-dime
thyl -7,8-dihydropterin (13, Scheme 3), 6-carbaldehyde-7,7-
dimethyl-7,8-dihydropterin (14, Scheme 3), and 6-carboxy-7,7-
dimethyl-7,8-dihydropterin (16, Scheme 3).
inhibitor of HPPK (Kd = 0.47 and IC50 = 0.44 l
M).17 The overall struc-
ture of the protein in the HPPKꢀMgHP4A complex [Protein Data
Bank (PDB) entry 1EX8]17 is similar to that observed in the
HPPKꢀMgAMPCPPꢀHP complex (PDB entry 1Q0N)27 and the pterin
and adenosine moieties of HP4A superimpose well with their coun-
terparts in the ternary complex.17 The pterin–protein and adeno-
sine–protein interactions are conserved in the two structures with
one exception. In HPPKꢀMgAMPCPPꢀHP, N8 of HP is hydrogen
bonded to the backbone carbonyl oxygen of L45 (Fig. 2A). In
HPPKꢀMgHP4A, however, this hydrogen bond does not exist because
the pterin moiety of HP4A is oxidized (Fig. 1D) and thus N8 cannot
serve as a hydrogen bond donor. Under normal conditions, HP is
readily oxidized at the 7 and 8 positions, which can be effectively
prevented by the introduction of two methyl groups at position 7
as those in HP-1 (Fig. 1B). As expected, the hydrogen bond between
N8 of the inhibitor and carbonyl oxygen of L45 has been observed in
the crystal structure of HPPK in complex with HP-1 (PDB entry
Compound 11 was synthesized as described.14 Briefly, 2,4-dia-
mino-6-(hydroxymethyl)pteridine hydrochloride (9, Scheme 2)
was treated with dibromotriphenylphosphorane in N,N-dimethyl-
acetamide to give 6-bromomethyl-pteridine-2,4-diamine (10). In
48% hydrobromic acid, 10 was converted through hydrolytic deam-
ination to 11 (Scheme 2).
The other three pterin moieties were derived from 2-amino-7,
8-dihydro-6,7,7-trimethylpteridin-4(3H)-one (12, Scheme 3) synthe-
sized by a procedure of Al-Hassan et al.14 Subsequently, 12 was
converted to 13 by Stuart’s method34 using bromine in acetic acid solu-
tion, oxidized by SeO2 in DMF to give 14, or converted to 16 through 2-
amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic
acid ethyl ester (15) by heating 12 with bromine in ethanol solution fol-
lowed by ester bond hydrolysis in base and methanol (Scheme 3). The
conditions for the conversion of 12 into 14, and the intermediate (15)
for the derivation of 16 from 12 are as recently described.35

G. Shi et al. / Bioorg. Med. Chem. 20 (2012) 47–57
49
Figure 2. Protein-ligand interaction and inhibitor design. (A and B) Optimized hydrogen bond interactions between HPPK and HP/ATP on the basis of the
HPPKꢀMgAMPCPPꢀHP (PDB entry 1Q0N), HPPKꢀMgATPꢀHP-3 (1DY3), and HPPKꢀMgAMPCPPꢀHP-analogs (3IP0) structures. (C and D) New design of bisubstrate analog
inhibitors of HPPK.
The final step of the synthesis is outlined in Scheme 4, resulting in
three final products: 2-amino-6-[(2-{4-[5-(6-amino-purin-9-yl)-3,
4-dihydroxy-tetrahydro-furan-2-ylmethylsulfanyl]-piperidin-1-yl}-
ethylamino)-methyl]-3H-pteridin-4-one (17), 2-amino-6-[(2-{4-
[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-yl-
meth ylsul fanyl]-piperidin-1-yl}-ethylamino)-methyl]-7,7-di-
methyl-7,8-dihydr o-3H pteridin-4-one (18), and 2-amino-7,7-
(Table 1) and the crystal structures of all three complexes
(HPPKꢀ17, HPPKꢀ18, and HPPKꢀ19) were determined (Table 2).
The HPPKꢀ17 structure (Fig. 3A) contains one HPPK (residues
1–158), one compound 17, one ethylene glycol, and 106 water
molecules. The HPPKꢀ18 structure (Fig. 3B) contains one HPPK
(residues 1–82, 87–158), one compound 18, one acetate ion,
and 88 water molecules. The HPPKꢀ19 structure (Fig. 3C) con-
tains one HPPK (residues 1–82, 87–158), one compound 19,
one acetate ion, and 91 water molecules. Residues 83–86 in
HPPKꢀ18 and HPPKꢀ19 were not observed and presumably
disordered.
dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic
acid
(2-{4-[5-(6-amino-purin-9-yl)-3 4-dihydroxy-tetrahydro-furan-2-
ylmethylsulfanyl]-piperidin-1-yl}-e thyl)-amide (19). Compound
17 was synthesized by allowing 8 and 11 to react. Compound 18
was derived from 8 and 13 in DMF solution with potassium carbon-
ate. Compound 19 was obtained by two procedures: the procedure
developed by Yoo and Li36 using copper–silver catalysis and aqueous
tert-butyl hydroperoxide (Method A) and the procedure developed
by us35 using the new intermediate 15 (Method B) with a signifi-
cantly improved yield.
2.5. Structure–activity relationship
2.5.1. Protein conformation and inhibitor binding
HPPK has three flexible loops (Loop 1, residues 8–15; Loop 2, resi-
dues 43–53; Loop 3, residues 82–92), among which Loop 3 undergoes
dramatic conformational changes during catalysis.22,38 The catalytic
trajectory of HPPK can be described by six consecutive states: ligand-
free HPPK, HPPKꢀMgATP, HPPKꢀMgATPꢀHP, HPPKꢀMgAMP-PP-HP (the
transition state), HPPKꢀAMPꢀHPPP, and HPPKꢀHPPP.21,39 In the HPPKꢀ
MgATP state, Loop 3 exhibits open conformation as seen in the
HPPKꢀMgADP (PDB entry 1EQM) and HPPKꢀMgAMPPCP (PDB entry
1EQ0) structures.38 Loop 3 is also open in the HPPKꢀAMPꢀHPPP state
(PDB entry 1RAO).39 In the HPPKꢀMgATPꢀHP state, in contrast, Loop 3
is closed as seen in the HPPKꢀMgAMPCPPꢀHP structure (PDB entry
1Q0N).27 In the HPPKꢀMgATP state, however, Loop 3 has been seen
to have a closed conformation in the HPPKꢀMgAMPCPP complex
(PDB entry 2F65),40 suggesting that Loop 3 in the HPPKꢀMgATP state
is dynamic.25
2.3. HPPK binding and inhibition
The Kd and IC50 measurements were carried out as de-
scribed.37,17 The Kd value is >150
for 19, and the IC50 value is >100
l
l
M for 17 and 2.55 0.15
M for 17 and 3.16 0.34
l
l
M
M
for 19. Compound 18 was not stable, and therefore, its Kd and
IC50 were not determined. These data show that 19 has a 60-fold
increased binding affinity and 30-fold increased inhibition ability
over 17.
2.4. Crystal structures of compounds 17–19 each in complex
with HPPK
The polypeptide chains in the HPPKꢀ17, HPPKꢀ18, and HPPKꢀ19
structures superimpose well except for Loop 3 (Fig. 4A). The aden-
osine moieties in the three inhibitors superimpose well; so do the
Although 18 was not stable, it was stabilized when bound to
HPPK. HPPK in complex with 17, 18, or 19 was crystallized

50
G. Shi et al. / Bioorg. Med. Chem. 20 (2012) 47–57
NH2
N
NH2
N
N
N
N
N
N
N
HO
TsO
O
O
TsCl
Py
H
H
O
H
H
O
H
H
H
H
O
O
NH
1
2
2
N
N
N
N
O
N
S
O
O
H
H
O
O
N
O
N
Br
O
S
H
H
O
O
CH3COSK
O
CH3ONa
4
5
3
NH
NH2
N
2
N
N
N
N
N
H
N
O
H
N
N
N
O
N
HN
S
S
O
Br
O
CF3COOH
DCM, 2 hr
O
O
H
H
O
H
H
H
H
H
H
O
O
O
7
6
NH2
N
N
N
H2N
N
N
S
CF3COOH
DCM, O/N
O
H
H
H
H
OH OH
8
Scheme 1.
O
NH2
NH2
N
N
N
N
N
HN
Br
N
OH
Ph3P/Br2
N
Br
40% HBr
N
N
H2N
H2N
N
N
H2N
10
11
9
Scheme 2.
O
O
N
N
N
HN
HN
H2N
Br
HBr
HOAc
N
N
H
N
H
H2N
13
12
SeO2 DMF
O
N
O
O
N
CHO
COOEt
COOH
N
N
HN
HN
HN
H2N
NaOH
N
N
H
N
N
H
N
H
H2N
H2N
CH3OH/H2O
14
15
16
Scheme 3.

G. Shi et al. / Bioorg. Med. Chem. 20 (2012) 47–57
51
NH2
N
N
N
O
N
14
K2CO3
N
N
13
S
S
HN
N
6
O
11
+
8
H
8
N
H
H
N
H2N
H
H
OH OH
17
NH2
N
N
O
N
N
O
N
K2CO3
N
N
HN
H2N
N
H
13
14
+
+
8
8
H
H
N
H
H
H
OH OH
18
NH2
N
CuI2, AgIO3
N
CaCO3, T-HYDRO
O
O
N
N
O
N
N
N
S
HN
H2N
N
H
OR
H
H
N
H
HATU, DIPEA
DMF
H
H
OH OH
19
16
+
8
Scheme 4.
Table 1
Crystallization conditions
2.5.2. Protein conformation and inhibitor structure
To form the catalytic assembly of HPPKꢀMgATPꢀHP in the closed
conformation, conserved residues N10 (of Loop 1) and Q50 (of Loop
2) function as the center of a hydrogen bond network with the
backbone carbonyl groups of P47 and P51 of Loop 2 and with the
backbone amide and/or carbonyl groups of W89, P91, and R92 of
Loop 3.27 This hydrogen bond network couples all three loops
and helps stabilize the complex and seal the active center. The
structures show that such a hydrogen bond network exists in
HPPKꢀ18 and HPPKꢀ19, but not in HPPKꢀ17 (Fig. 4B).
HPPKꢀ17
HPPKꢀ18
3UDE
HPPKꢀ19
PDB entry code
3UD5
3UDV
Protein solution
HPPK (mg/mL)
Compound 17
Compound 18
Compound 19
Tris–HCl [mM (pH)]
10
Saturated
10
10
Saturated
20 (8.0)
Saturated
20 (8.0)
20 (8.0)
Reservoir solution
PEG 3350 [%(w/v)]
CH3ꢀCOONH4 (mM)
Bis–Tris [mM (pH)]
HEPES [mM (pH)]
25
25
200
100 (8.5)
20
200
The only differences between 18 and 17 are in positions 7 and 8
of the pterin moiety (Scheme 4). In 18, the 7,7-dimethyl group
keeps the NH group at position 8 in its reduced form, allowing
the formation of the hydrogen bond between N8 and the carbonyl
oxygen of L45 (N8Pterinꢀ ꢀ ꢀOL45). In contrast, the dimethyl group is
absent from 17 and the pterin moiety is oxidized with an N instead
of an NH group at position 8, rendering the formation of the
N8Pterinꢀ ꢀ ꢀOL45 hydrogen bond impossible.
100 (6.5)
100 (7.5)
Crystals
Appear (days)
Reach final size (days)
Shape
7
14
7
14
14
21
Thin plate
0.15 ꢂ 0.10
ꢂ 0.005
Thin plate
0.10 ꢂ 0.05
ꢂ 0.005
Thin plate
0.15 ꢂ 0.10
ꢂ 0.005
Dimension (mm)
The impact of the formation of N8Pterinꢀ ꢀ ꢀOL45 is twofold. First, it is
one of the five hydrogen bonds for the recognition of HP by HPPK
(Fig. 2A) and therefore contributes to protein-ligand affinity. Second,
by anchoringL45 it optimizes the conformationof Loop 2 and Loop-2
residues, especially Q50, favoring the formation of the hydrogen
bond network that couples and stabilizes the three loops (Fig. 4B).
Taken together, these structures show that the N8Pterinꢀ ꢀ ꢀOL45 hydro-
gen bond facilitates the formation of the hydrogen bond network
that couples the three loops as seen in the closed HPPKꢀMgATPꢀHP
state. A good inhibitor, such as 19, should not impair the coupling
of these loops.
pterin moieties. The linkers in 18 and 19 superimpose well, but the
linker in 17 displays a different conformation. In HPPKꢀ17, Loop 3
displays the open conformation (Fig. 5A). During HPPK catalysis,
the open Loop 3 facilitates HP binding and product release. For en-
zyme inhibition, however, the open conformation of Loop 3 has
negative impact on binding, which is in agreement with the Kd va-
lue for the binding of 17 to HPPK (>150
lM). As aforementioned,
the Kd for the binding of MgATP to HPPK is 2.6–4.5
l
M. Therefore,
As aforementioned, the 7,7-dimethyl group in HPPKꢀHP-1 inter-
acts with the side chain of W89.15 This hydrophobic interaction is
also observed in HPPKꢀ19, whereas it is not possible in HPPKꢀ17
(Fig. 4B). In addition to the 7,7-dimetyl group, 19 has a new car-
bonyl group next to the pterin moiety (Scheme 4). It does not, how-
ever, affect the conformation of the protein.
17 is not a good inhibitor. In contrast, Loop 3 assumes the closed
conformation in the HPPKꢀ18 and HPPKꢀ19 structures although
residues 83–86 in the loop are disordered (Figs. 4A and 5B). During
HPPK catalysis, a closed Loop 3 seals the active center for the
reaction to occur. For enzyme inhibition, a closed Loop 3 enhances
the affinity for the enzyme and consequently the potency of an
inhibitor. Consistently, compound 19 shows a much higher binding
2.5.3. Inhibitor structure and stability
affinity (Kd = 2.55
of MgATP, suggesting the compound is a good inhibitor of HPPK.
l
M). The Kd value of 19 is comparable with that
Compound 17 is stable, but 18 tends to degrade when not in
complex with HPPK. The only difference between 17 and 18 is that

52
G. Shi et al. / Bioorg. Med. Chem. 20 (2012) 47–57
Table 2
Crystal data, X-ray diffraction, and structures
HPPKꢀ17
HPPKꢀ18
HPPKꢀ19
PDB entry code
3UD5
3UDE
3UDV
Crystal
Space group
Unit cell parameters: a (Å)
b (Å)
c (Å)
b(o)
C2
P21212
52.91
70.98
36.38
90
P21212
53.00
70.64
36.25
90
79.98
52.77
36.69
102.70
Matthews coefficient (Å3/Da)
2.1
1.9
1.9
Data
Overall (last shell)
30.00–1.90 (1.97–1.90)
10236 (717)
6.5 (5.3)
87.0 (61.7)
Overall (last shell)
30.00–1.82 (1.89–1.82)
11525 (650)
6.1 (2.5)
89.7 (51.9)
Overall (last shell)
30.00–1.79 (1.85–1.79)
11716 (658)
6.0 (2.8)
87.6 (50.4)
Resolution (Å)
Unique reflections
Redundancy
Completeness (%)
a
Rmerge
0.089 (0.320)
16.2 (3.8)
0.084 (0.561)
16.7 (1.3)
0.074 (0.322)
19.1 (2.3)
I/
r
Refinement
Overall (last shell)
29.84–2.00 (2.13–2.00)
9252 (1203)
90.7 (72.0)
824 (107)
Overall (last shell)
29.48–1.88 (1.98–1.88)
10902 (1180)
93.5 (73.0)
949 (104)
0.178 (0.243)
0.237 (0.309)
Overall (last shell)
29.92–1.88 (1.98–1.88)
10754 (1150)
92.8 (72.0)
920 (98)
0.210 (0.253)
0.275 (0.288)
Resolution (Å)
Unique reflections
Completeness (%)
Data in the test set
R-work
0.158 (0.164)
0.205 (0.262)
R-free
Structure
Protein non-H atoms/B (Å2)
Ligand atoms/B (Å2)
Water oxygen atoms/B (Å2)
Rmsd
1448/30.0
49/44.4
106/38.3
1389/28.1
47/31.3
88/36.1
1399/26.4
48/40.7
91/32.8
Bond lengths (Å)
Bond angles (°)
Coordinate error (Å)
Ramachandran plotb
Favored regions (%)
Disallowed regions (%)
0.012
1.302
0.54
0.012
1.382
0.50
0.013
1.376
0.48
98.7
0.0
97.3
0.0
97.3
0.0
a
Rmerge
=
R
|(I ꢃ <I>)|/
R(I), where I is the observed intensity.
b
Obtained using Ramachandran data by Lovell and coworkers.52
Figure 3. Schematic illustration of crystal structures. (A) The HPPKꢀ17 complex. (B) The HPPKꢀ18 complex. (C) The HPPKꢀ19 complex. Polypeptide chains are shown as a
ribbon diagrams with helices (spirals) in cyan, strands (arrows) in orange, and loops (tubes) in grey. Ligands are shown as sticks in atomic color scheme (C in grey, N in blue, O
in red, P in orange, and S in orange).
the pterin moiety in 17 is oxidized, but it is reduced in 18. Com-
pound 19 is stable. The only difference between 18 and 19 is that
the methylene group (–CH2–) at position 13 in 18 becomes a car-
bonyl group (–CO–) in 19 (Scheme 4).
disorder and differences indicate that the protein–linker interac-
tions are not optimized in HPPKꢀ19 although the three loops of
the enzyme are coupled and the complex assumes the overall
closed conformation. We need to further modify the linker
between the pterin and adenosine moieties of 19, which we
believe will lead to higher binding affinity. In the
HPPKꢀMgATPꢀHP state, the phosphate bridge is recognized by
conserved HPPK side chains R82, R92, H115, and R121
(Fig. 2B). Therefore, hydrogen bond acceptors should be intro-
duced into the linker.
2.5.4. Partial disorder of Loop 3 and protein–linker interaction
Although 19 is a good inhibitor of HPPK, Loop 3 residues 83–
86 in HPPKꢀ19 are disordered (Fig. 4A). Furthermore, all three
loops in HPPKꢀ19 display noticeable differences when compared
with those in the HPPKꢀMgAMPCPPꢀHP structure (Fig. 5B). Such

G. Shi et al. / Bioorg. Med. Chem. 20 (2012) 47–57
53
Figure 4. Structural comparison in stereo. (A) The HPPKꢀ17 (in orange), HPPKꢀ18 (in magenta), and HPPKꢀ19 (in cyan) structures are superimposed. Proteins are shown as C
a
traces and ligands as sticks. (B) Coupled loops in HPPKꢀ19 (in cyan, those in HPPKꢀ18 are not shown for clarity) versus uncoupled loops in HPPKꢀ17 (in orange). Hydrogen
bonds are indicated by dashed lines in black, but the N8Pterinꢀ ꢀ ꢀOL45 interaction is highlighted in magenta. Labels are color coded: blue for HPPKꢀ19, red for HPPKꢀ17, and black
for the common features in both. For clarity, most side chains that are not involved in loop coupling are not shown.
R121 (Fig. 2B). None of these side chains interacts favorably with the
linker of any of the three compounds (17–19). Further development
of bisubstrate analog inhibitors should allow favorable interactions
between the linker of inhibitor and the four positively charged side
chains of HPPK. Residues R82 and R92 define the boundary of Loop 3.
Favored interactions between these two side chains and inhibitor
should lead to a completely ordered and closed Loop 3 in the
protein–inhibitor complex.
3. Conclusions
We report a new generation of bisubstrate analog inhibitors of
HPPK, compounds 17, 18, and 19 (Scheme 4). In complex with
HPPK, these compounds share a common adenosine-protein inter-
action pattern, but their pterin moieties interact with the protein
differently. In HPPKꢀ18 and HPPKꢀ19, the N8Pterinꢀ ꢀ ꢀOL45 hydrogen
bond is formed between pterin atom N8 (donor) of the inhibitor
and carbonyl oxygen (acceptor) of L45, whereas in HPPKꢀ17, the
formation of this hydrogen bond is impossible. The structures
show that the formation of N8Pterinꢀ ꢀ ꢀOL45 is sufficient to facilitate
the coupling of the three loops of HPPK in the manner as seen in
the HPPKꢀMgAMPCPPꢀHP complex.27 Without the N8Pterinꢀ ꢀ ꢀOL45
interaction, the HPPKꢀ17 complex exhibits the open conformation
without the coupling of the three loops and the binding affinity
4. Experimental methods
4.1. Modeling
Design and docking of new inhibitors into the active site of
HPPK were carried out with program packages CNS and O.41,42
The model complex was subject to geometry optimization using
the conjugate gradient method developed by Powell;43 Engh
and Huber geometric parameters were used as the basis of the
force field.44
of 17 is low (Kd > 150
l
M). With the formation of N8Pterinꢀ ꢀ ꢀOL45
,
in contrast, the HPPKꢀ19 complex displays the closed conformation
due to the coupling of the three loops and the affinity of 19 is ꢁ60-
fold higher. Introducing the 7,7-dimethyl groups into 17 results in
18, which triggers the transition of the open conformation (in
HPPKꢀ17) into the closed form (in HPPKꢀ18). Nevertheless, 18 is
not stable when it is not in complex with the enzyme. It is the
replacement of the methylene group in N@CH–CH2–NH of 18 with
the carbonyl group in N@CH–CO–NH of 19 that improves the
chemical stability (Scheme 4).
4.2. Chemistry
4.2.1. General methods
All chemicals were purchased from Sigma–Aldrich except that
compound 1 was purchased from TCI America. Starting materials
and solvents were used without further purification. Anhydrous
reactions were conducted under a positive pressure of dry N2. Reac-
tions were monitored by TLC, on Baker-flex Silica Gel IB-F (J. T.
Baker). All compounds and intermediates were purified by flash
With and without the N8Pterinꢀ ꢀ ꢀOL45 interaction, the binding
mode of the linker is also different (Fig. 4A), underscoring the impor-
tance of an ordered and closed Loop 3 for optimized protein–inhib-
itor interaction. For pyrophosphoryl transfer, the phosphate groups
of ATP interact with four conserved side chains, R82, R92, H115, and

54
G. Shi et al. / Bioorg. Med. Chem. 20 (2012) 47–57
Figure 5. Comparison of new with related structures in stereo. (A) The HPPKꢀ17 (in orange, this work) and HPPKꢀAMPꢀHPPP (in blue, PDB entry 1RAO) structures are
superimposed. (B) The HPPKꢀ19 (in cyan, this work) and HPPKꢀMgAMPCPPꢀHP (in purple, PDB entry 1Q0N) structures are superimposed. Proteins are shown as C
a traces and
ligands as sticks.
chromatography performed on Teledyne ISCO Combiflash Rf system
using RediSep Rf columns. Ion exchange chromatography was per-
formed using strata Scx (50 lm particle size, 70 Å pore) resin car-
tridges. Preparative high pressure liquid chromatography (HPLC)
was conducted using a Waters 600E system using a Waters 2487
5 mmol, 0.5 equiv) was added and then stirred at 50 °C for a few hours
before another dose of 2-(Boc-amino)ethyl bromide (1.12 g, 5 mmol,
0.5 equiv) was added. After the reaction was finished, the mixture
was evaporated, diluted with EtOAc (100 mL), and washed with satu-
rated aqueous NaHCO3 (2 ꢂ 50 mL) and brine (50 mL). The residue
from concentrating the organic layer was purified by silica gel chroma-
tography, eluting with 10% DCM/methanol, to afford 5.43 g (99%) of the
title compound as white foam. NMR d H (400 MHz; CD3OD), 1.39 (3H,
s), 1.42–1.48 (13H, m), 1.58 (3H, s), 1.72–1.99 (4H, m), 2.37 (2H, t),
2.53–2.58 (1H, m), 2.75–2.82 (2H, m), 3.17 (2H, t), 4.33 (1H, m), 5.07
(1H, m), 5.58 (1H, m), 6.18 (1H, d), 8.23 (1H, s), 8.28 (1H, s); d 13C
(100 MHz; CD3OD), 158.32 (1C), 157.46 (1C), 154.03 (1C), 150.25
(1C), 142.05 (1C), 120.65 (1C), 115.33 (1C), 91.88 (1C), 88.87 (1C),
85.25 (1C), 85.07 (1C), 80.05 (1C), 58.75 (1C), 54.18 (2C), 42.29 (1C),
38.41 (1C), 33.56 (1C), 33.44 (1C), 33.28 (1C), 28.75 (3C), 27.35 (1C),
25.48 (1C); MS (ESI) calculated for C25H39N7O5S [M+H]+ 550.27, found
550.1.
dual
k
absorbance detector and Phenomenex C18 columns
m particle size, 110 Å pore) at a flow rate
(250 mm ꢂ 21.2 mm, 5
l
of 10 mL/min. A binary solvent systems consisting of A = 0.1% aque-
ous TFA and B = 0.1% TFA in acetonitrile was employed with the gra-
dients as indicated. 1H and 13C NMR data were obtained on a Varian
400 MHz spectrometer and are reported in ppm relative to TMS (tet-
ramethylsilane). Mass spectra were measured with Agilent 1100
series LC/Mass Selective Detector, Agilent 1200 LC/MSD-SL system
and Thermoquest Surveyor Finnigan LCQ deca. Chemical purity
was determined by HPLC analysis with a Zorbax Eclipse plus C18 col-
umn (Narrow Bore RR 2.1 mm ꢂ 50 mm, 3.5 micron; flow rate of
0.3 mL/min; solvent, methanol:H2O gradient, 0.1% acetic acid;
detection at 260 nm), confirming >95% purity.
4.2.3. 2-[1-(2-Amino-ethyl)-piperidin-4-ylsulfanylmethyl]-5-(6-
amino-purin-9-yl)-tetrahydro-furan-3,4-diol (8)
4.2.2. (2-{4-[6-(6-Amino-purin-9-yl)-2,2-dimethyl-tetrahydro-
furo[3,4-d][1,3]dioxol-4-ylmethylsulfanyl]-piperidin-1-yl}-eth
yl)-carbamic acid tert-butyl ester (7)
To a solution of (2-{4-[6-(6-amino-purin-9-yl)-2,2-dimethyl-
tetrahydro-furo[3,4-d][1,3]dioxol-4-ylmethylsulfanyl]-piperidin-
1-yl}-ethyl)-carbamic acid tert-butyl ester (7) (2.75 g, 5 mmol,
1 equiv) in 50 mL DCM, 10 mL TFA was added dropwise at
ꢃ20 °C and then stirred at room temperature overnight. After the
reaction was finished, the solvent was removed under vacuum,
To a solution of 9-[2,2-dimethyl-6-(piperidin-4-ylsulfanylmethyl)-
tetrahydro-furo[3,4-d][1,3]dioxol-4-yl]-9H-purin-6-ylamine
(6)
(4.06 g, 10 mmol, 1 equiv) and potassium carbonate (2.76 g, 20 mmol,
2 equiv) in 100 mL acetonitrile, 2-(Boc-amino)ethyl bromide (1.12 g,

G. Shi et al. / Bioorg. Med. Chem. 20 (2012) 47–57
55
and the residue was purified by silica gel chromatography, eluting
with 15% DCM/methanol, to afford 1.84 g (90%) of the title com-
pound as white foam. NMR d H (400 MHz; CD3OD), 1.46–1.54
(4H, m), 1.84–1.98 (4H, m), 2.41 (2H, t), 2.65–2.82 (3H, m), 2.89–
3.02 (2H, m), 4.20 (1H, m), 4.37 (1H, m), 4.80 (1H, m), 6.0 (1H,
d), 8.21 (1H, s), 8.31 (1H, s); d 13C (100 MHz; CD3OD), 157.40
(1C), 153.97 (1C), 150.73 (1C), 141.55 (1C), 120.61 (1C), 90.19
(1C), 86.00 (1C), 74.72 (1C), 73.97 (1C), 59.76 (1C), 54.32 (2C),
42.64 (1C), 38.66 (1C), 33.67 (2C), 33.13 (1C); MS (ESI) calculated
for C17H27N7O3S [M+H]+ 410.27, found 410.1.
perature. The reaction was allowed to stir overnight at 40 °C. The
crude reaction was purified by HPLC (H2O:Methanol = 2:3) to pro-
vide 19 (3.77 mg, 0.006 mmol, 30%) as a pale yellow solid. NMR d
H (400 MHz; CD3OD), 1.58 (6H, s), 1.76–2.28 (8H, m), 2.85–3.06
(3H, m), 3.25 (2H, m) 3.61 (2H, m), 4.22 (1H, m), 4.33 (1H, m), 4.74
(1H, m), 6.05 (1H, d, J = 4.8), 8.37 (1H, s), 8.46 (1H, s); d 13C
(100 MHz; DMSO-d6), 164.22 (1C), 157.79 (1C), 155.09 (1C),
155.00 (1C), 148.84 (1C), 143.01 (1C), 141.49 (1C), 118.96 (1C),
117.62 (1C), 114.69 (1C), 100.66 (1C), 87.64 (1C), 84.18 (1C), 72.96
(1C), 72.53 (1C), 55.31 (1C), 53.27 (2C), 51.90 (1C), 37.83 (1C),
33.44 (2C), 31.97 (1C), 29.85 (1C), 27.71 (2C); HRMS (ESI-MS) calcu-
lated for C26H36N12O5S (MH+): 629.2725; found: 629.2708.
4.2.4. 2-Amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-
6-carboxylic acid (16)
The intermediate 15 was synthesized as described.35 To a solu-
tion of 15 (265 mg, 1 mmol, 1 equiv) in methanol (5 mL) was added
a solution of sodium hydroxide (2 M, 2 mmol, 2 equiv). After stir-
ring for two hours, the reaction mixture was acidified to pH 2 with
1 M HCl, the product was precipitated, and the precipitate washed
once with water and then dried to obtain 16 (213 mg, 0.9 mmol,
90%). NMR d H (400 MHz; CD3OD), 1.59 (H, s); d 13C (100 MHz;
CD3OD), 166.22 (1C), 161.58 (1C), 157.23 (1C), 156.67 (1C),
142.07 (1C), 101.93 (1C), 36.50 (1C), 29.12 (2C); MS (ESI) calcu-
lated for C9H11N5O3 ([M+H]+) 238.09, found 238.10.
4.2.8. Compound 19 (Method B)
To a solution of 16 (190 mg, 0.8 mmol, 1 equiv), O-(7-aza-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate (HATU) (334.6, 0.88 mmol, 1.1 equiv), and compound 8
(327.2 mg, 0.8 mmol, 1 equiv) in anhydrous DMF (100 mL) was
added DIPEA (4.18 lL, 2.4 mmol, 3 equiv). After 18 h, the solvent
was evaporated under high vacuum, the reaction residue was puri-
fied by HPLC (H2O:Methanol = 2:3) to provide 19 (377 mg,
0.6 mmol, 75%) as a pale yellow solid. NMR and HRMS (ESI-MS) data
are found in Section 4.2.7.
4.2.5. 2-Amino-6-[(2-{4-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-
tetrahydro-furan-2-ylmethylsulfanyl]-piperidin-1-yl}-ethylam
ino)-methyl]-3H-pteridin-4-one (17)
4.3. Fluorometric titration
The dissociation constants of the inhibitors were measured as
described37 for the fluorometric measurement of the dissociation
constant of Ant-ATP with modifications. Briefly, the inhibitors were
dissolved in dimethylsulfoxide. Dimethylsulfoxide concentrations
were kept within 1.7% during the titration experiments and control
experiments showed that dimethylsulfoxide at these concentra-
tions had no effects on activity (substrate binding and catalysis)
of the enzyme. The excitation and emission wavelengths were
420 and 450 nm, respectively, for 17, but 450 and 480, respec-
tively, for 19. The excitation and emission slits were 1 and 4 nm,
respectively. The titration was performed by adding aliquots of a
To a solution of 8 (100.0 mg, 0.244 mmol, 1 equiv) and potas-
sium carbonate (337.9 mg, 2.44 mmol, 10 equiv) in 20 mL dimeth-
ylacetamide, 11 (81.7 mg, 0.244 mmol, 1 equiv) was added and
stirred at room temperature for 24 h. It was evaporated under high
vacuum and the residue was dissolved in water methanol mixture
and purified by HPLC to give 17 (71.0 mg, 0.122 mmol, 50%) as a
yellowish powder. NMR d H (400 MHz; CD3OD), 1.89 (4H, m),
2.28–2.32 (4H, m), 3.05–3.3 (3H, m), 3.53 (2H, m), 3.63 (2H, m),
4.22 (1H, m), 4.35 (1H, t, J = 5.2), 4.41 (2H, s), 4.74 (1H, t, J = 5.2),
6.05 (1H, d, J = 4.8), 8.37 (1H, s), 8.47 (1H, s), 8.76 (1H, s); d 13C
(100 MHz; DMSO-d6), 160.62 (1C), 158.77 (1C), 158.44 (1C),
158.09 (1C), 154.34 (1C), 149.85 (1C), 148.99 (1C), 141.52 (1C),
141.09 (1C), 127.94 (1C), 119.02 (1C), 87.64 (1C), 84.07 (1C),
72.84 (1C), 72.58 (1C), 51.37 (2C), 48.33 (1C), 41.16 (1C), 40.36
(1C), 37.52 (1C), 32.26 (2C), 31.97 (1C); HRMS (ESI-MS) calculated
for C24H32N12O4S (MH+): 585.2463; found: 585.2460.
500
lM 19 stock solution to an HPPK solution. The initial HPPK
concentration and volume were 10
l
M and 2 mL, respectively.
The Kd values were obtained by nonlinear least-squares regression
of the data to Eq. 1 as described37
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
D
FmolðKd þ Et þ Lt ꢃ ðKd þ Et þ LtÞ ꢃ 4EtLtÞ
D
Fobs
¼
ð1Þ
2
4.2.6. 2-Amino-6-[(2-{4-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-
tetrahydro-furan-2-ylmethylsulfanyl]-piperidin-1-yl}-ethylam
ino)-methyl]-7,7-dimethyl-7,8-dihydro-3H pteridin-4-one (18)
To a solution of 8 (100.0 mg, 0.244 mmol, 1 equiv) and potas-
sium carbonate (337.9 mg, 2.44 mmol, 10 equiv) in 20 mL dimeth-
ylacetamide, 13 (89.0 mg, 0.244 mmol, 1 equiv) was added and
stirred at room temperature for 24 h. It was evaporated under high
vacuum and the residue was extracted by methanol. It was evapo-
rated again and the residue was used for direct analysis without
where
D
Fobs and DFmol are observed and molar fluorescence
changes caused by binding, Et is the total concentration of HPPK,
and Lt is the total concentration of the inhibitor. As an example,
the fluorometric titration of HPPK with compound 19 is shown in
Figure 6A.
4.4. Enzyme inhibition assay
IC50 measurements were carried out as described17 except for
the concentrations of reaction mixtures, which contained 1 nM
Escherichia coli HPPK, 2
further purification. MS (ESI) calculated for
C26H38N12O4S
([M+H]+) 615.29, found 615.10.
lM ATP, 1 lM HP, 5 mM MgCl2, 25 mM
DTT, and a trace amount of [
a
-
32P]-ATP (ꢁ1
lCi) in 100 mM Tris,
4.2.7. 2-Amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridi
ne-6-carboxylic acid (2-{4-[5-(6-amino-purin-9-yl)-3,4-dihydro
xy-tetrahydro-furan-2-ylmethylsulfanyl]-piperidin-1-yl}-ethyl)
-amide (19, Method A)36
pH 8.3. IC50 values were obtained by fitting the data to a logistic
equation by nonlinear least-squares regression of the data to Eq.
2 as described45
Compound 8 (12.3 mg, 0.03 mmol, 1.5 equiv) was mixed withCuI
þ vmax
ꢃ
vmin
v
¼
vmin
ð2Þ
½Iꢄ
(0.0388 mg,
0.0002 mmol,
1.0 mol %),
AgIO3
(0.057 mg,
1 þ IC
50
0.0002 mmol, 1.0 mol %), and CaCO3 (2.2 mg, 0.022 mmol,
1.1 equiv) in DMF (0.2 mL). Compound 14 (4.5 mg, 0.020 mmol,
1.0 equiv) and T-HYDROÒ (70 wt % in H2O, 0.00315 mL, 0.022 mmol,
1.1 equiv) were added under an inert atmosphere (N2) at room tem-
where
v is the reaction rate, vmin the minimum reaction rate, vmax
the maximum reaction rate, and [I] the concentration of the inhib-
itor. The inhibition of HPPK by compound 19 is shown in Figure 6B.

56
G. Shi et al. / Bioorg. Med. Chem. 20 (2012) 47–57
Resource in the Structural Biophysics Laboratory, an Agilent 1200
LC/MSD-SL system in the Chemical Biology Laboratory, and a Ther-
moquest Surveyor Finnigan LCQ deca maintained by the Compara-
tive Carcinogenesis Laboratory of National Cancer Institute at
Frederick. X-ray diffraction data were collected at the Southeast
Regional Collaborative Access Team (SER-CAT) 22-ID and 22-BM
beamlines at the Advanced Photon Source (APS), Argonne National
Laboratory (ANL).
The coordinates and structure factors have been deposited in
the PDB under entry codes 3UD5 (HPPKꢀ17), 3UDE (HPPKꢀ18),
and 3UDV (HPPKꢀ19).
Supplementary data
Supplementary data (the 1H and 13C spectra of compounds 7, 8,
16, 17, and 19) associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmc.2011.11.032.
References and notes
1. Blakley, R. L.; Benkovic, S. J. In Folates and Pterins; Blakley, R. L., Benkovic, S. J.,
Eds.; John Wiley & Sons, Inc.: New York, 1984.
2. Hitchings, G. H.; Burchall, J. J. Adv. Enzymol. Relat. Areas Mol. Biol. 1965, 27, 417.
3. Cohen, M. L. Science 1992, 257, 1050.
4. Neu, H. C. Science 1992, 257, 1064.
5. Kunin, C. M. Ann. Intern. Med. 1993, 118, 557.
6. Levy, S. B. Adv. Exp. Med. Biol. 1995, 390, 1.
7. Murray, B. E. Adv. Intern. Med. 1997, 42, 339.
8. Bermingham, A.; Derrick, J. P. Bioessays 2002, 24, 637.
9. Walsh, C. Nat. Rev. Microbiol. 2003, 1, 65.
10. Hughes, D. T. D. Sulphonamides. Antibiotic and Chemotherapy, 7th ed.; Churchill
Livingstone: New York, 1997.
11. Hughes, D. T. D. In Antibiotics and Chemotherapy; O’Grady, F., Lambert, H. P.,
Finch, R. G., Greenwood, D., Eds.; Churchill Livingstone: New York, 1997; pp
346–356.
12. Zinner, S. H.; Mayer, K. H. In Principles and Practice of Infectious Diseases;
Mandell, G. L., Bennett, J. E., Dolin, R., Eds.; Churchill Livingstone: Philadelphia,
2005; pp 440–451.
Figure 6. Kd and IC50 measurements. (A) Fluorometric titration of HPPK with
compound 19. (B) Inhibition of HPPK by 19.
13. Wood, H. C. S. In Chemistry and Biology of Pteridines; Pfleiderer, W., Ed.; Walter
de Gruyter: Berlin-New York, 1975.
14. Al-Hassan, S. S.; Cameron, R. J.; Curran, A. W. C.; Lyall, W. J. S.; Nicholson, S. H.;
Robinson, D. R.; Stuart, A.; Suckling, C. J.; Stirling, I.; Wood, H. C. S. J. Chem. Soc.,
Perkin Trans. 1 1985, 1645.
4.5. Crystallization, X-ray diffraction, structure solution, and
refinement
15. Hennig, M.; Dale, G. E.; D’Arcy, A.; Danel, F.; Fischer, S.; Gray, C. P.; Jolidon, S.;
Muller, F.; Page, M. G.; Pattison, P.; Oefner, C. J. Mol. Biol. 1999, 287, 211.
16. Stammers, D. K.; Achari, A.; Somers, D. O.; Bryant, P. K.; Rosemond, J.; Scott, D.
L.; Champness, J. N. FEBS Lett. 1999, 456, 49.
17. Shi, G.; Blaszczyk, J.; Ji, X.; Yan, H. J. Med. Chem. 2001, 44, 1364.
18. Shiota, T. In Chemistry and Biochemistry of Folates; Blakley, R. T., Benkovic, S. J.,
Eds.; John Wiley & Sons: New York, 1984; pp 121–134.
19. Shi, G.; Gong, Y.; Savchenko, A.; Zeikus, J. G.; Xiao, B.; Ji, X.; Yan, H. Biochim.
Biophys. Acta 2000, 1478, 289.
Crystals were grown in sitting drops at 19 1 °C. Crystallization
conditions are summarized in Table 1. A Hydra II Plus crystalliza-
tion robot (Matrix Technologies, Hudson, New Hampshire, USA)
and Crystal Screen kits from Hampton Research (Laguna Niguel,
California, USA) were used. X-ray diffraction data were collected
at 100 K with an MARCCD detector mounted at the synchrotron
Beamline 22 at the Advanced Photon Source, Argonne National
Laboratory. Data processing was carried out with the HKL2000 pro-
gram suite.46 The structure was solved by Fourier synthesis start-
ing with a homologous structure: PDB entry 1EQM for HPPKꢀ17,
3ILJ for HPPKꢀ18, and 3UDE for HPPKꢀ19. Multiple conformations
of amino acid residues, ligands, and solvent molecules were re-
moved from the starting models. Structure solution and refinement
were done with PHENIX.47 All graphics work, including model
building and rebuilding, was performed with COOT.48 The struc-
tures were verified with annealed omit maps and the geometry
was assessed using PROCHECK49 and WHAT IF.50 The statistics of
X-ray diffraction data and structures are summarized in Table 2.
Illustrations were prepared with PyMOL.51
20. Bermingham, A.; Bottomley, J. R.; Primrose, W. U.; Derrick, J. P. J. Biol. Chem.
2000, 275, 17962.
21. Li, Y.; Gong, Y.; Shi, G.; Blaszczyk, J.; Ji, X.; Yan, H. Biochemistry 2002, 41, 8777.
22. Xiao, B.; Shi, G.; Chen, X.; Yan, H.; Ji, X. Structure 1999, 7, 489.
23. Neuhard, J.; Nygaard, P. In Escherichia coli and Salmonella typhimurium: Cellular
and Molecular Biology; Neidhardt, F. C., Ingraham, J. L., Low, K. B., Magasanik, B.,
Schaechter, M., Umbarger, H. E., Eds.; ASM Press: Washington, DC, 1987; pp
445–473.
24. Derrick, J. P. In Folic Acid and Folates; Litwack, G., Ed.; Academic Press: Oxford,
UK, 2008; pp 411–433.
25. Yan, H.; Ji, X. Protein Pept. Lett. 2011, 18, 328.
26. Kompis, I. M.; Islam, K.; Then, R. L. Chem. Rev. 2005, 105, 593.
27. Blaszczyk, J.; Shi, G.; Yan, H.; Ji, X. Structure 2000, 8, 1049.
28. Blaszczyk, J.; Li, Y.; Shi, G.; Yan, H.; Ji, X. Biochemistry 2003, 42, 1573.
29. Broom, A. D. J. Med. Chem. 1989, 32, 2.
30. Radzicka, A.; Wolfenden, R. Methods Enzymol. 1995, 249, 284.
31. Sakami, W. Biochem. Prep. 1961, 8, 5.
32. Plettenburg, O.; Hofmeister, A.; Goerlitzer, J.; LÖHn, M.; WO Patent WO/2008/
077,552, 2008.
33. Isakovic, L.; Saavedra, O. M.; Llewellyn, D. B.; Claridge, S.; Zhan, L.; Bernstein,
N.; Vaisburg, A.; Elowe, N.; Petschner, A. J.; Rahil, J. Bioorg. Med. Chem. Lett.
2009, 19, 2742.
34. Stuart, A. U.S. Patent 4,036,961, 1977.
35. Shi, G.; Ji, X. Tetrahedron Lett. 2011, 52, 6174.
36. Yoo, W. J.; Li, C. J. J. Am. Chem. Soc 2006, 128, 13064.
37. Li, Y.; Wu, Y.; Blaszczyk, J.; Ji, X.; Yan, H. Biochemistry 2003, 42, 1581.
38. Xiao, B.; Shi, G.; Gao, J.; Blaszczyk, J.; Liu, Q.; Ji, X.; Yan, H. J. Biol. Chem. 2001,
276, 40274.
Acknowledgements
This research was supported by NIH Grant GM51901 (H.Y.),
NIAID Trans NIH/FDA Intramural Biodefense Program
Y3-RC-8007-01 (X.J.), and the Intramural Research Program of the
NIH, National Cancer Institute, Center for Cancer Research. Mass
spectrometry experiments were conducted on an Agilent 1100
series LC/Mass Selective Detector maintained by the Biophysics

G. Shi et al. / Bioorg. Med. Chem. 20 (2012) 47–57
57
39. Blaszczyk, J.; Shi, G.; Li, Y.; Yan, H.; Ji, X. Structure (Camb) 2004, 12, 467.
40. Li, G.; Felczak, K.; Shi, G.; Yan, H. Biochemistry 2006, 45, 12573.
41. Brünger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros, P.; Grosse-
Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.; Pannu, N. S.; Read, R. J.;
Rice, L. M.; Simonson, T.; Warren, G. L. Acta Crystallogr. D 1998, 54, 905.
42. Jones, T. A.; Kjeldgaard, M. Methods Enzymol. 1997, 277, 173.
43. Powell, M. J. D. Math. Prog. 1977, 12, 241.
47. Adams, P. D.; Grosse-Kunstleve, R. W.; Hung, L. W.; Ioerger, T. R.; McCoy, A. J.;
Moriarty, N. W.; Read, R. J.; Sacchettini, J. C.; Sauter, N. K.; Terwilliger, T. C. Acta
Crystallogr. D 2002, 58, 1948.
48. Emsley, P.; Cowtan, K. Acta Crystallogr. D 2004, 60, 2126.
49. Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M. J. Appl.
Crystallogr. 1993, 26, 283.
50. Vriend, G. J. Mol. Graph. 1990, 8(52–56), 29.
44. Engh, R. A.; Huber, R. Acta Crystallogr. A 1991, 47, 392.
45. Graeser, D.; Neubig, R. R. In Signal Transduction; Milligan, G., Ed.; IRL Press:
Oxford, 1992; pp 1–30.
51. DeLano, W. L.; Delano Scientific: San Carlos, CA, 2002.
52. Lovell, S. C.; Davis, I. W.; Arendall, W. B., 3rd; de Bakker, P. I.; Word, J. M.;
Prisant, M. G.; Richardson, J. S.; Richardson, D. C. Proteins: Struct. Funct. Genet.
2003, 50, 437.
46. Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307.