Bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: Transition state analogs for high affinity binding

Abstract

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate biosynthesis pathway. It catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). HPPK is essential for microorganisms but absent in mammals; therefore, it is an attractive target for developing novel antimicrobial agents. Previously, based on our studies of the structure and mechanism of HPPK, we created first-generation bisubstrate inhibitors by linking 6-hydroxymethylpterin to adenosine through phosphate groups, and developed second-generation inhibitors by replacing the phosphate bridge with a linkage that contains a piperidine moiety. Here, we report third-generation inhibitors designed based on the piperidine-containing inhibitor, mimicking the transition state. We synthesized two such inhibitors, characterized their protein-binding and enzyme inhibition properties, and determined their crystal structures in complex with HPPK, advancing the development of such bisubstrate analog inhibitors.

1. Introduction

Folates are essential cofactors for life. Mammals obtain folate cofactors from their diet with an active transport system, whereas most microorganisms must synthesize folates de novo via a biosynthesis pathway.¹ Therefore, the folate biosynthesis pathway is one of the principal targets for developing antimicrobial agents.^2,3 The folate pathway is composed of seven enzymes, among which four are absent in mammals. These four enzymes are dihydroneopterin aldolase (DHNA), 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), dihydropteroate synthase (DHPS), and dihydrofolate synthase (DHFS). DHPS is the target of sulfonamides, the first ever antimicrobial chemotherapy.⁴ HPPK catalyzes the transfer of a pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). It is not only an attractive molecular target for developing novel antimicrobial agents,^5–8 but also the best understood pyrophosphokinase.^9,10 Based on a wealth of structural and mechanistic information,^10,11 we have previously developed two generations of bisubstrate inhibitors of Escherichia coli HPPK. The first-generation inhibitors were created by linking 6-hydroxymethylpterin to adenosine through phosphate groups,¹² and the second-generation was developed by replacing the phosphate bridge with a piperidine-containing linkage.^13–15 As such, they all feature linked purine and pterin moieties.

The binding affinity of ATP to HPPK is Mg²⁺ dependent. The K_d value of ATP in the presence of Mg²⁺ (MgATP) is 2.60 μM.¹⁶ The binding affinity of HP to HPPK is MgATP dependent. In the presence of MgATP, the K_d value of HP is 0.17 μM.¹⁷ Therefore, the K_d value of MgATP can be used to indicate the potency of bisubstrate inhibitors of HPPK. The best of our first-generation inhibitors was HP₄A (Table 1).¹² Between the purine and pterin moieties of HP₄A, there are four connected phosphate groups. Although the inhibitor has a much higher affinity than MgATP, the linker of HP₄A carries many negative charges, resulting in poor bioavailability. The second-generation inhibitors are represented by HP-18 (Table 1).¹³ Unlike the phosphate bridge in HP₄A, the linkage of HP-18 has more drug-like properties. Furthermore, its affinity to the enzyme is already comparable with that of MgATP (Table 1). This was encouraging because further optimization of protein-inhibitor interactions could result in potent inhibitors.

Table 1.

K_d and IC₅₀ values of E. coli HPPK substrates and bisubstrate inhibitors

Ligand	Structure	Kd (μM)	IC50(μM)	Reference
HP		0.17 ± 0.01a	N/Ab	Previously reported17
ATP		2.60 ± 0.06c	N/A	Previously reported16
HP4A		0.47 ± 0.04	0.44 ± 0.16	Previously reported12
HP-18		2.55 ± 0.15	3.16 ± 0.34	Previously reported13
HP-73		0.19 ± 0.03	0.38 ± 0.06	This work
HP-75		0.047 ± 0.007	N/Dd	This work

^aMeasured in the presence of a non-hydrolyzable ATP analog (100 μM) and MgCl₂ (10 mM).

^bN/A, not applicable.

^cMeasured in the presence of MgCl₂ (8 mM).

^dN/D, not determined.

After we reported the second-generation bisubstrate inhibitors, HPPK became even more attractive. Chhabra and coworkers discovered that 8-mercaptoguanine (8MG) could inhibit HPPK.¹⁸ Dennis, Yun, and coworkers designed and synthesized more 8MG derivatives against Staphylococcus aureus and E. coli enzymes.^19–21 Palayam and coworkers used computational methods to design inhibitors of Salmonella enterica serovar typhi HPPK.²² Fragment-based screening on S. aureus HPPK was also performed by Chhabra and colleagues.²³ Here, we report our third-generation bisubstrate inhibitors of HPPK, HP-73 and HP-75. Developed on the basis of high-resolution crystal structures that revealed features of the transition state, these inhibitors exhibit significantly improved potency (Table 1).

2. Results

2.1. Structure-based design of HP-73

Using a non-hydrolyzable ATP analog, AMPCPP, we previously determined the crystal structure of the HPPK:MgAMPCPP:HP complex [Protein Data Bank (PDB) entry 1Q0N].²⁴ It reveals the catalytic center assembly of the enzyme, which is so far the most similar structure to the transition state. Our HPPK:HP-18 structure (PDB entry 3UDV)¹³ shows that the interactions between HPPK and HP-18’s pterin and adenosine moieties remain the same as in the catalytic center assembly, indicating no further improvement is feasible regarding these interactions. In contrast, the interactions between HPPK and HP-18’s linkage that connects the pterin and adenosine moieties are significantly different from the catalytic center assembly. As shown in Fig. 1A, the triphosphate moiety of AMPCPP interacts directly with amino acid side chains of W89, R92, H115, Y116, and R121, and indirectly with D95 and D97 via the Mg²⁺ ion in the HPPK:MgAMPCPP:HP structure.²⁴ We divided these interactions into three groups. First, the two Mg²⁺ ions coordinate with 11 atoms, 3 from the phosphate groups of AMPCPP, 1 from HP, 4 from the D95 and D97 side chains, and 3 from water molecules (Group-1); second, the γ-phosphate group of AMPCPP forms 5 hydrogen bonds with the W89, H115, Y116, and R121 side chains (Group-2); and third, the α-phosphate group of AMPCPP forms two hydrogen bonds with the guanidinium group of R92 (Group-3, Fig. 1A). In the HPPK:HP-18 structure, Group-1 interactions are replaced by the electrostatic interactions between the D97 side chain and the piperidine nitrogen, whereas Group-2 and Group-3 interactions are missing, providing room for structural modification of the bisubstrate inhibitor (Fig. 1B). Because there are more interactions in Group-2 than in Group-3, we focused on Group-2 first in designing next generation inhibitors. When the HP-18 molecule from the HPPK:HP-18 structure was superimposed on the HP and AMPCPP molecules in the HPPK:MgAMPCPP:HP structure, it became obvious that the introduction of a carboxylic group into the piperidine ring system could mimic the γ-phosphate group of ATP (Fig. 2A), resulting in the design of HP-73.

Figure 1.

Schematic illustration of protein-ligand interactions revealed by previous crystal structures. (A) The HPPK:MgAMPCPP:HP complex (PDB entry 1Q0N). (B) The HPPK:HP-18 complex (PDB entry 3UDV). Polypeptide chains are shown as ribbon diagrams with helices (spirals) in cyan, strands (arrows) in orange, and loops (tubes) in gray. Ligands are shown as sticks in atomic color scheme (C in white, N in blue, O in red, P in orange, and S in orange). Mg²⁺ ions and water oxygen atoms are represented by spheres in green and red, respectively. Metal coordination bonds are indicated with solid lines; hydrogen bonds are indicated with dashed lines.

Figure 2.

Design, affinity, and structure of HP-73. (A) The design of HP-73 based on the HPPK:MgAMPCPP:HP (PDB entry 1Q0N) and HPPK:HP-18 (PDB entry 3UDV) structures. Superposition of HP-18 (C in magenta, N in blue, O in red, P in orange, and S in orange) with the AMPCPP and HP molecules (C in white, N in blue, O in red, P in orange, and S in orange) of the HPPK:MgAMPCPP:HP complex suggests the introduction of a carboxylic group into the piperidinyl moiety to mimic the γ-phosphate of ATP. (B) Affinity measurement of HP-73 binding to HPPK by ITC. The exothermic binding isotherm (Top) was integrated to give the enthalpy change plotted as a function of the molar ratio of HP-73/HPPK (Bottom). The integrated enthalpy change data were used to calculate the dissociation constant K_d. (C) Schematic illustration of protein-ligand interactions observed in the crystal structure of the HPPK:HP-73 complex. The HP-73 molecule (C in white, N in blue, O in red, P in orange, and S in orange) is outlined with 2F_o-F_c electron density map (in blue, contoured at 1.1 σ).

2.2. Chemical synthesis and protein-binding and enzyme inhibition properties of HP-73

As illustrated in Scheme 1, reaction of compound 1 with benzylamine in ethanol gave compound 2. When 2 was treated with glyoxylic acid in aqueous acetonitrile, compound 3 was isolated as the sole product. Hydrolysis of 3 with 1 M NaOH gave compound 4. The mixture of triphenylphosphine and diisopropyl azodicarboxylate in tetrahydrofuran was added to the mixture of 4 and thioacetic acid in tetrahydrofuran, resulting in compound 5. Sodium methoxide reacted with 5 to form the thiol, followed by the reaction with 2′,3′-O-isopropylidene-5′-O-toluene-p-sulfonyl adenosine to give compound 6. The benzyl group in 6 was removed by hydrogenation under Pd/C catalysis, yielding compound 7. The subsequent reaction of 7 with (2-bromo-ethyl)-carbamic acid tert-butyl ester provided the key intermediate, compound 8. Under the TFA/DCM conditions, cleavage of the BOC protection group yielded compound 9 that contained an amino group that was used to link 9 to 2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic acid using the coupling reagent HATU to give compound 10. Compound 10 was hydrolyzed by 1 M lithium hydroxide solution to give the final product, HP-73 (11).

Scheme 1.

Reagents and conditions: (a) benzylamine, ethanol, 75° C, 70% yield. (b) glyoxylic acid monohydrate, acetonitrile/water (1:1), rt, 70% yield. (c) 1M NaoH, rt, 80% yield. (d) triphenylphosphine, diisopropyl azodicarboxylate, thioacetic acid, THF, 0 °C to rt, 80% yield. (e) 2′,3′-O-isopropylidene-5′-O-toluene-p-sulfonyl adenosine, sodium methoxide, DMF, rt to 80 °C, 66% yield. (f) Pd/C, MeOH, rt, 95% yield. (g) 2-(Boc-amino)ethyl bromide, K₂CO₃, CH₃CN, 50 °C, 69% yield. (h) TFA/DCM, 0 °C to rt, 90% yield. (i) compound 9, HATU, DIPEA, DMF, rt, 55%. (j) LiOH/H₂O, MeOH, rt, 50% yield.

The affinity of HP-73 was determined by isothermal titration calorimetry (ITC, Fig. 2B). The K_d value of HP-73 is 0.19 ± 0.03 μM, indicating a 13-fold improvement of potency over HP-18 (Table 1). The IC₅₀ measurement was carried out as described¹² with minor adjustments, suggesting a comparable enzyme inhibition to HP₄A and an 8-fold stronger inhibition over HP-18 (Table 1).

2.3. Structure of the HPPK:HP-73 complex and the design of HP-75

The HPPK:HP-73 structure (Table 2) contains an HPPK (residues 1–83 and 87–158), an HP-73, and 171 water molecules in the asymmetric unit. Electron density for residues 84–86 of HPPK was not observed, and hence, these four residues are presumably disordered. The structure shows that the carboxylic group in the piperidine system of HP-73 indeed mimics the γ-phosphate of AMPCPP (Fig. 2C). In the HPPK:MgAMPCPP:HP structure, the γ-phosphate of AMPCPP interacts with the side chains of W89, H115, Y116, and R121 (Fig. 1A), whereas in the HPPK:HP-73 structure, the carboxylic group interacts with the side chains of H115, Y116, and R121 (Fig. 2C). We conclude that the 13-fold potency improvement of HP-73 over HP-18 is due to the interactions between the protein and the carboxylic group in the piperidine ring system of HP-73.

Table 2.

Crystal data, X-ray diffraction, and structures

PDB Entry Code	pending	pending
Crystal	EcHPPK:HP-73	EcHPPK:HP-75
Space group	P21212	C2
Unit cell parameters:
a, b, c (Å)	53.01, 70.09, 36.27	61.45, 42.80, 56.28
a, β, γ(°)	90, 90, 90	90, 101.54, 90
Data	Overall (last shell)	Overall (last shell)
Resolution (Å)	30.00–1.60 (1.66–1.60)	30.00–1.48 (1.53–1.48)
Unique reflections	17716 (1399)	22571 (1503)
Redundancy	5.0 (3.1)	5.4 (2.6)
Completeness (%)	95.7 (77.1)	94.6 (64.3)
Rmergea	0.067 (0.581)	0.123 (0.325)
Rpimb	0.030 (0.349)	0.053 (0.201)
I/σ	18.8 (1.6)	9.3 (2.4)
Refinement	Overall (last shell)	Overall (last shell)
Resolution (Å)	29.94–1.60 (1.68–1.60)	28.97–1.49 (1.57–1.49)
Unique reflections	17689 (2053)	22492 (2526)
Completeness (%)	95.7 (80.0)	95.1 (79.0)
Data in the test set	1000 (117)	1000 (118)
R-work	0.184 (0.282)	0.164 (0.230)
R-free	0.224 (0.322)	0.201 (0.266)
Structure
Protein non-H atoms / B (Å2)	1400 / 24.9	1803 / 24.7
Ligand atoms / B (Å2)	62 / 26.2	64 / 21.5
Water oxygen atoms / B (Å2)	171 / 33.5	268 / 40.6
RMSD
Bond lengths (Å)	0.009	0.005
Bond angles (°)	1.033	0.926
Coordinate error (A)	0.19	0.11
Ramachandran plot
Favored regions (%)	98.0	98.0
Disallowed regions (%)	0.0	0.0

^aR_merge = Σ_h Σ_i|(I_h − <I_h,i>)| / Σ_h Σ_i (I_h,i), where I is the observed intensity.

^bR_pim = Σ_h [1/ (/n_h − 1)]^1/2 Σ_i|<I_h> − I_h,i|/Σ_h Σ_i I_h,i, where I is the observed intensity.

Previously, we showed that residues R82 and R92 play not only essential but also dynamic roles in the catalytic cycle of the enzyme.^25,26 Our structures indicate that R92 first binds to the α-phosphate group of ATP and then shifts to interact with the β-phosphate when the pyrophosphoryl transfer is about to occur.²⁵ As shown in Fig. 1A, the guanidinium group of R92 forms two hydrogen bonds with the α-phosphate of AMPCPP in the HPPK:MgAMPCPP:HP structure (PDB entry 1Q0N).²⁴ Comparative structural analysis suggests that these Group-3 interactions could be recovered by replacing the -S- bridge in HP-73 with a sulfonyl group (Fig. 3A), leading to the design of HP-75.

Figure 3.

Design, affinity, and structure of HP-75. (A) Design of HP-75 based on the HPPK:MgAMPCPP:HP (PDB entry 1Q0N) and HPPK:HP-73 (this work) structures. Superposition of HP-73 (C in magenta, N in blue, O in red, P in orange, and S in orange) with the AMPCPP and HP molecules (C in white, N in blue, O in red, P in orange, and S in orange) of the HPPK:MgAMPCPP:HP complex suggests replacement of the sulfur bridge with a sulfonyl group to mimic the α-phosphate of ATP. (B) Affinity measurement of HP-75 binding to HPPK by ITC. The exothermic binding isotherm (Top) was integrated to give the enthalpy change plotted as a function of the molar ratio of HP-75/HPPK (Bottom). The integrated enthalpy change data were used to calculate the dissociation constant K_d. (C) Schematic illustration of protein-ligand interactions observed in the crystal structure of the HPPK:HP-75 complex. The HP-75 molecule (C in white, N in blue, O in red, P in orange, and S in orange) is outlined with 2F_o-F_c electron density map (in blue, contoured at 1.1 σ).

2.4. Chemical synthesis and protein-binding affinity of HP-75

Our first attempt at HP-75 synthesis was not successful because the ((5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)sulfonyl moiety was not stable. Its methyl group could not be removed by lithium hydroxide as achieved in the last step of HP-73 synthesis (Scheme 1). Therefore, a second attempt was made to replace the methyl group with a benzyl group in two steps. First, the methyl group was removed from 8 in 1 M lithium hydroxide methanol solution to get compound 12, and second, 12 reacted with benzyl bromide to give compound 13 (Scheme 2). Compound 13 was oxidized by Oxone to give compound 14. Under the TFA/DCM conditions, cleavage of the BOC protection group from 14 yielded compound 15. To avoid possible cyclization byproduct of 15, it was not purified. Instead, the deprotected amino group of 15 was used immediately to link the compound to 2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic acid in the presence of coupling reagent HATU to produce compound 16. Compound 16 was deprotected by Pd/C Et₃SiH to generate the final product, HP-75 (17).

Scheme 2.

Reagents and conditions: (a) LiOH/H₂O, MeOH, rt, 70% yield. (b) benzyl bromide, K₂CO₃, dimethylacetamide, rt, 60% yield. (c) Oxone, pH 5, MeOH/H₂O, rt, 35% yield. (d) TFA/DCM, H₂O, 0 °C to rt. (e) compound 9, HATU, DIPEA, DMF, rt, 25% yield. (f) Pd/C, triethylsilane, THF, rt, 27% yield.

The affinity of HP-75 was determined by ITC (Fig. 3B). The K_d value of HP-75 is 0.047 ± 0.007 μM, indicating a 54- and 4-fold improvement of potency over HP-18 and HP-73, respectively (Table 1). The IC₅₀ measurement was not successful because HP-75 was not sufficiently stable under the experimental conditions.

2.5. Crystal structure of the HPPK:HP-75 complex

The HPPK:HP-75 structure (Table 2) contains an HPPK (residues 1–158), an HP-75, and 268 water molecules in the asymmetric unit. As expected, the introduced sulfonyl group of HP-75 mimics the α-phosphate of AMPCPP (Fig. 3C). In the HPPK:MgAMPCPP:HP structure, the α-phosphate of AMPCPP interacts with the guanidinium group of R92 via two hydrogen bonds (Fig. 1A), whereas in the HPPK:HP-75 structure, the sulfonyl group contacts the side chain of R92 with a single hydrogen bond (Fig. 3C). We conclude that the 4-fold potency improvement of HP-75 over HP-73 is due to the recovery of a Group-3 interaction between HPPK and the ligand molecule.

Our HPPK:HP-75 structure shows that the recovered Group-3 interaction stabilizes Loop-3, a flexible loop between R82 and R92, which is required for the assembly of the active center and therefore is essential for catalysis.^27,28 Previously, we showed that Loop-3 settles down over and seals the active center when the reaction is about to occur.^24,25 However, residues 83–86 in Loop-3 of the HPPK:HP-18 structure are disordered (PDB entry 3UDV)¹³ and residues 84–86 in the HPPK:HP-73 structure are disordered (Fig. 4A). In contrast, Loop-3 is complete and ordered in the HPPK:HP-75 structure (Fig. 4B). Our structures show that HP-75 also has an impact on the conformation of Loop-1. In the HPPK:HP-75 complex, Loop-1 assumes the same conformation as seen in the HPPK:MgAMPCPP:HP complex (Fig. 4B); whereas in the HPPK:HP-73 complex, it exhibits a significantly different conformation (Fig. 4A). We conclude that the HPPK:HP-75 structure, among all structures of HPPK in complex with bisubstrate inhibitors, represents the best mimic of the transition state assembly of the enzyme.

Figure 4.

Structural comparison. (A) Superimposed HPPK:MgAMPCPP:HP (PDB entry 1Q0N, in white) and HPPK:HP-73 (this work, in pink and magenta) structures. The three flexible loops of HPPK are indicated and the disordered fragment of Loop-3 between residues 83–87 is indicated. (B) Superimposed HPPK:HP:MgAMPCPP (in white) and HPPK:HP-76 (this work, in palecyan and cyan) structures.

3. Discussion

The HPPK:MgAMPCPP:HP structure (PDB entry 1Q0N) provided the basis for structure-based development of bisubstrate analog inhibitors (Fig. 1A). Our approach involved dividing the HPPK-ligand interactions into Mg²⁺-mediated Group-1, γ-phosphate-mediated Group-2, and α-phosphate-mediated Group-3. Our strategy of first recovering Group-2 interactions by mimicking the γ-phosphate of ATP by the carboxylic group is practical and beneficial, resulting in HP-73 with a greatly improved binding affinity (Fig. 2). Further improvement was achieved by recovering Group-3 interactions by mimicking the α-phosphate of ATP by a sulfonyl group, leading to HP-75, which exhibits the highest binding affinity to date (Fig. 3). Together, these two modifications, starting from HP-18, are responsible for the 54-fold potency improvement (Table 1), representing a unique example for structure-based design.

Among the amino acid side chains that are involved in protein-ligand interactions, W89 is located near the tip of Loop-3. This residue plays an important role when the catalytic center of the enzyme needs to be sealed by contacting the γ-phosphate of ATP as seen in the HPPK:MgAMPCPP:HP structure.²⁴ Although the HPPK:HP-75 structure represents the best mimic of the transition state assembly among complexes of HPPK with bisubstrate inhibitors, the conformation of Loop-3 and the positioning of W89 are different from that in the HPPK:MgAMPCPP:HP complex (Fig. 4B). Instead of a sealed catalytic center (Fig. 5A), the catalytic center in the HPPK:HP-75 complex is only partially closed (Fig. 5B). Regardless of this, however, HP-75 exhibits the highest binding affinity in vitro.

Figure 5.

Schematic illustration of (A) the sealed active center upon pyrophosphoryl transfer as observed for the HPPK:MgAMPCPP:HP complex (PDB entry 1Q0N) and (B) the open active center as observed for the HPPK:HP-75 complex (this work). HPPK is shown as a molecular surface in white or palecyan. Ligands and ions are shown as spheres in atomic color scheme (N in blue, O in red, P in orange, S in orange, and C in white or cyan).

Both HP-73 and HP-75 are excellent inhibitors of HPPK because their binding affinities are substantially better than that of MgATP (Table 1). As bisubstrate analog inhibitors, they inhibit not only the binding of MgATP, but also the subsequent binding of HP. Nonetheless, these compounds do not exhibit antibacterial activity in vivo. They cannot kill bacteria, nor do they inhibit bacterial growth, for which the most likely reason is their lack of cell permeability. Smart strategies have recently been developed to circumvent cell permeability problem, among which the employment of either a siderophore molecule or a cell-penetrating peptide to bring potential antibiotics into the cell is most suitable for further development of our bisubstrate inhibitors of HPPK. Siderophores are small, high-affinity iron-chelating molecules; secreted by microorganisms, they primarily transport iron across cell membranes.²⁹ A series of siderophore-antibiotic conjugates has been synthesized and, as expected, studies have shown that such conjugates make it possible to design antibiotics with improved cell permeability.³⁰ Cell-penetrating peptides are positively charged peptides of 5–30 residues in length; they are able to penetrate into cell membranes and thereby deliver therapeutics into cells.³¹ It has been shown that conjugation of cell-penetrating peptide to antimicrobial peptides enhances antibacterial activity.³² We will be using these two strategies to further advance the development of bisubstrate analog inhibitors of HPPK.

4. Experimental methods

4.1. Chemistry

4.1.1. General methods

All chemicals were purchased from Sigma-Aldrich. Starting materials and solvents were used without further purification. Anhydrous reactions were conducted under a positive pressure of dry Argon. Reactions were monitored by TLC on Baker-flex Silica Gel IB-F (J. T. Baker). All compounds and intermediates were purified by flash chromatography performed on a Teledyne ISCO Combiflash Rf system, using RediSep Rf columns, and a Biotage Isolera One system. Ion exchange chromatography was performed using strata Scx (50 μm particle size, 70 Å pore) resin cartridges. Preparative high-pressure liquid chromatography (HPLC) was conducted with a Waters 2545 system using a Waters 2998 photodiode array detector and Phenomenex C₁₈ columns (250 mm × 21.2 mm, 5 μm particle size, 110 Å pore) at a flow rate of 15 mL/min. A binary solvent system consisting of A = 0.1% aqueous TFA and B = 0.1% TFA in acetonitrile was employed with the gradients as indicated. The ¹H and ¹³C NMR data were obtained on a Bruker 500 MHz spectrometer and reported in ppm relative to TMS (tetramethylsilane). Mass spectra were measured with an Agilent 1100 series LC/Mass Selective Detector, an 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 column (Narrow Bore RR 2.1 mm × 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.

4.1.2. Methyl 4-(acetylthio)-1-benzylpiperidine-2-carboxylate (5)

Triphenylphosphine (7.55g, 28.8 mmol) and diisopropyl azodicarboxylate (5.59 ml, 28.4 mmol) were mixed in dry tetrahydrofuran (200 ml) at 0 °C. Then, methyl 1-benzyl-4-hydroxypiperidine-2-carboxylate (3.6g, 14.5 mmol) and thioacetic acid (2.07 ml, 29.0 mmol) was added to the mixture and stirred at room temperature overnight. The solid was filtered, the solvent was removed under vacuum, and the residue was purified by silica gel chromatography, eluting with hexane/ethyl acetate, to afford 3.53 g (80%) of compound 5 as colorless oil. NMR δH (500 MHz; CD₃SOCD₃), 1.70 (2H, m), 2.00 (2H, m), 2.21 (2H, m), 2.33 (3H,s), 2.81 (1H, m), 3.17 (1H, m), 3.98 (1H, s), 4.11 (1H, s), 3.75 (3H, s), 7.39 (5H, s); δ¹³C (176 MHz; CD₃SOCD₃), 194.67 (1C), 170.77 (1C), 130.31 (1C), 129.96 (1C), 128.81 (4C), 60.45 (1C), 58.52 (1C), 52.72 (1C), 47.56 (1C), 36.60 (1C), 33.13 (1C), 33.05 (1C), 29.38 (1C); MS (ESI-MS) calculated for C₁₆H₂₁NO₃S (MH⁺): 308.12; found: 308.30.

4.1.3. Methyl-4-((((3aS,4S,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)thio)-1-benzylpiperidine-2-carboxylate (6)

Methyl 4-(acetylthio)-1-benzylpiperidine-2-carboxylate (3.08 g, 10 mmol) reacted with sodium methoxide (2.67 ml, 4.5 M, 12mmol) in DMF (20 ml) under argon. Ten minutes later, 2′,3′-O-isopropylidene-5′-O-toluene-p-sulfonyl adenosine (4.62g, 10mmol) was added the mixture and stirred at 80 °C overnight. The solvent was removed under vacuum, and the residue was extracted with ethyl acetate and purified by silica gel chromatography, eluting with hexane/ethyl acetate, to afford 3.66 g (66%) of compound 6 as white crystal powder. NMR δH (500 MHz; CD₃SOCD₃), 1.34 (3H, s), 1.55 (3H, s), 1.67 (1H, m), 1.96–2.08 (2H, m), 3.01 (2H, m), 3.11 (2H, m), 4.04 (1H, m), 4.15 (1H, m), 3.76 (3H, s), 4.27 (1H, s), 4.99 (1H, s), 5.48 (1H, m), 6.22 (1H, m), 7.43 (7H,s), 8.39 (1H, s), 8.53(1H, s); δ¹³C (176 MHz; CD₃SOCD₃), 169.73 (1C), 153.70 (1C), 149.36 (1C), 148.77 (1C), 141.90 (1C), 130.98 (1C), 129.19 (7C), 119.56 (1C), 117.72 (1C), 89.93 (1C), 86.60 (1C), 83.71 (1C), 83.58 (1C), 59.94 (1C), 58.24 (1C), 53.27 (1C), 47.27 (1C), 36.92 (1C), 32.58(1C), 27.25 (1C), 27.58 (1C), 25.60 (1C); MS (ESI-MS) calculated for C₂₇H₃₄N₆O₅S (MH⁺): 555.23; found: 555.30.

4.1.4. Methyl 4-((((3aS,4S,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)thio)piperidine-2-carboxylate (7)

Compound 6 (0.76 g, 1.37 mmol) was stirred over Pd/C (100 mg, 10% by wt.) in MeOH under 1 atm H₂. After 16 h, the Pd/C was removed by filtration, the solvent was removed under vacuum, and the residue was purified by silica gel chromatography, eluting with 15% DCM/methanol, to afford 0.61g (95%) of compound 7 as a white powder. NMR δH (500 MHz; CD₃SOCD₃), 1.34 (3H, s), 1.54 (3H, s), 1.67 (1H, m), 1.96–2.08 (2H, m), 2.86 (2H, m), 3.09 (2H, m), 3.25 (1H, m), 3.75 (3H, s), 4.29 (1H, s), 5.0 (1H, s), 5.48 (1H, m), 6.21 (1H, m), 8.36 (1H, s), 8.51(1H, s); δ13C (176 MHz; CD₃SOCD₃), 169.21 (1C), 154.10 (1C), 149.87 (1C), 148.74 (1C), 141.79 (1C), 119.59 (1C), 117.84 (1C), 89.95 (1C), 86.59 (1C), 83.73 (1C), 83.61 (1C), 53.59 (1C), 52.87 (1C), 37.06 (1C), 32.80(1C), 31.03(1C), 30.59 (1C), 27.52 (1C), 27.33 (1C), 25.60 (1C); MS (ESI-MS) calculated for C₂₀H₂₈N₆O₅S (MH+): 465.18; found: 465.30.

4.1.5. Methyl4-((((3aS,4S,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)thio)-1-(2-((tert-butoxycarbonyl)amino)ethyl)piperidine-2-carboxylate (8)

To a solution of compound 7 (4.65 g, 10 mmol) and potassium carbonate (2.76 g, 20 mmol) in 100 mL acetonitrile, 2-(Boc-amino)ethyl bromide (1.12g, 5 mmol) was added and then stirred at 50 °C for a few hours before another dose of 2-(Boc-amino)ethyl bromide (1.12g, 5 mmol) was added. After the reaction was finished, the mixture was evaporated, diluted with EtOAc (100 mL), and washed with saturated aqueous NaHCO₃ (2 × 50 mL) and brine (50 mL). The residue from concentrating the organic layer was purified by silica gel chromatography, eluting with 10% DCM/methanol, to afford 4.19 g (69%) of compound 8 as white foam. NMR δH (500 MHz; CD₃SOCD₃), 1.31 (3H, s), 1.35 (9H, m), ), 1.52 (3H, s), 1.74 (1H, m), 2.04 (1H, m), 2.06 (1H, m), 2.21 (1H, m), 2.36 (2H, m), 2.84 (2H, m), 3.21 (2H, s), 3.28 (1H, s), 3.57 (1H, m), 3.71 (3H, s), 3.74 (3H, s), 4.28 (1H, m), 4.96 (1H, m), 5.41 (1 H, m), 6.19 (1 H, m), 6.95 (NH, s), 8.40 (1 H, s), 8.55(1 H, s); δ¹³C (176 MHz; CD₃SOCD₃), 168.00 (1C), 156.80 (1C), 151.78 (1C), 148.38 (1C), 147.05 (1C), 142.59 (1C), 119.34 (1C), 117.81 (1C), 114.28 (1C), 89.97 (1C), 86.39 (1C), 83.84 (1C), 84.45 (1C), 79.56 (1C), 59.87 (1C), 53.82 (1C), 48.39(1C), 36.25(1C), 35.37 (1C), 32.67 (1C), 28.56 (1C), 28.42 (1C), 27.14 (1C), 25.42 (1C); MS (ESI-MS) calculated for C₂₇H₄₁N₇O₇S (MH⁺): 608.28; found: 608.30.

4.1.6. Methyl1-(2-(l2-azanyl)ethyl)-4-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)thio)piperidine-2-carboxylate (9)

To a solution of compound 8 (3.04 g, 5 mmol) in 50 mL DCM, 10 ml TFA was added dropwise at −20 °C and then stirred at room temperature overnight. After the reaction was finished, the solvent was removed under vacuum, and the residue was purified by silica gel chromatography, eluting with 15% DCM/methanol, to afford 2.1 g (90%) of compound 9 as white foam. NMR δH (500 MHz; CD₃SOCD₃, 1.45 (2H, m), 1.75 (2H, m), 2.12 (2H, m), 2.64 (2H, m), 2.73 (2H, m), 2.86 (4H, m), 3.57 (3H, s), 4.18 (1H, m), 4.93 (1H, m), 5.46 (1H, m), 6.18 (1H, m), 7.53 (NH, s), 8.31 (1H, s), 8.47(1H, s); δ¹³C (176 MHz; CD₃SOCD₃), 172.32 (1C), 152.35 (1C), 149.15 (1C), 148.72 (1C), 142.38 (1C), 119.38 (1C), 117.48 (1C), 88.14 (1C), 73.49 (1C), 72.91 (1C), 60.81 (1C), 52.16 (1C), 51.87(1C), 46.83(1C), 38.14 (1C), 36.64 (1C), 34.55 (1C), 32.37 (1C), 32.17 (1C); MS (ESI-MS) calculated for C₁₉H₂₉N₇O₅S (MH⁺): 468.20; found: 468.30.

4.1.7. Methyl1-(2-(2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydropteridine-6-carboxamido)ethyl)-4-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)thio)piperidine-2-carboxylate (10)

To a solution of 6-carboxy-7,7-dimethyl-7,8-dihydropterin (94.8 mg, 0.4 mmol), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (167.3, 0.44 mmol), and compound 9 (186.8mg, 0.4 mmol) in anhydrous DMF (40 mL) was added DIPEA (209 uL, 1.2 mmol). After 18 h, the solvent was evaporated under high vacuum, the reaction residue was purified by HPLC to provide compound 10 (150.9 mg, 55%) as a pale yellow solid. NMR δH (500 MHz; CD₃SOCD₃), 1.46 (6H, s), 1.74 (1H, m), 2.09 (1H, m), 2.22 (1H, m), 2.69(1H, m), 2.73 (1H, m), 2.89 (1H, m), 2.94 (1H, m), 2.98 (2H, m), 3.23(2H, m), 3.47 (2H, m), 3.71 (3H, s), 4.03 (1H, m), 4.16 (1H, m), 4.41 (1H, m), 4.7 (1H, m), 5.93 (1H, m), 6.75 (1H, s), 7.39 (1H, s), 8.39 (1H, s), 8.57(1H, s); δ¹³C (176 MHz; CD₃SOCD₃), 164.62 (1C), 158.30 (1C), 155.54 (1C), 155.41 (1C), 153.44 (1C), 149.26 (1C), 148.97 (1C), 143.31 (1C), 141.90 (1C), 119.44 (1C), 101.01 (1C), 88.23 (1C), 84.83 (1C), 84.59 (1C), 73.42 (1C), 72.99 (1C), 53.71 (1C), 48.23 (1C), 38.71 (1C), 36.63 (1C), 36.20 (1C), 34.56 (1C), 32.80 (1C), 31.16 (1C), 28.31 (2C); MS (ESI-MS) calculated for C₂₈H₃₈N₁₂O₇S (MH⁺): 687.27; found: 687.30.

4.1.8. (2R,4R)-1-(2-(2-Amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydropteridine-6-carboxamido)ethyl)-4-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)thio)piperidine-2-carboxylic acid (11, HP-73)

To a solution of lithium hydroxide monohydrate (22.0 mg, 0.53 mmol) in water (0.2 mL) at 0 °C was added dropwise a solution of compound 10 (54.9 mg, 0.08 mmol) in methanol (0.8 mL). The resulting mixtures were stirred at room temperature overnight. The reaction mixtures were evaporated to dryness, and the reaction residue was purified by HPLC to provide compound 11 (26.9 mg, 50%) as a yellow solid. NMR δH (500 MHz; CD₃SOCD₃), 1.32 (3H, S), 1.48 (3H, s), 1.57 (1H,m), 1.80 (1H, m), 2.23 (1H, m), 2.30 (1H, m), 2.69(1H, m), 2.71 (1H, m), 2.77 (1H, m), 2.91 (1H, m), 3.00 (1H, m), 3.16 (1H, m), 4.03 (1H, m), 4.15 (1H, m), 4.25 (1H, m), 4.71 (1H, m), 5.89 (1H, m), 6.21 (1H, s), 7.28 (1H, s), 8.15 (1H, s), 8.37(1H, s); δ¹³C (176 MHz; CD₃SOCD₃), 176.78 (1C), 164.60 (1C), 156.52 (2C), 155.29 (1C), 153.13 (2C), 149.94 (1C), 140.18 (1C),119.52 (1C), 104.57 (1C), 87.86 (1C), 84.46 (1C), 83.92 (1C), 73.22 (1C), 72.99 (1C), 72.55 (1C), 64.68 (1C), 53.85 (1C), 53.36 (1C), 47.12 (1C), 38.70 (1C), 33.58 (1C), 30.45 (1C), 27.75 (2C); MS (ESI-MS) calculated for C₂₇H₃₆N₁₂O₇S (MH⁺): 673.26; found: 673.30.

4.1.9. 4-((((3aS,4S,6R,6aR)6-(6-Amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)thio)-1-(2-((tert-butoxycarbonyl)amino)ethyl)piperidine-2-carboxylic acid (12)

To a solution of lithium hydroxide monohydrate (222.0 mg, 5.3 mmol) in water (2.0 mL) at 0 °C was added dropwise a solution of compound 8 (485.6 mg, 0.8 mmol) in methanol (8.7 mL). The resulting mixtures were stirred at room temperature overnight. The reaction mixtures were evaporated to dryness, the reaction residue was purified by HPLC to provide compound 12 (332.1 mg, 70%) as a white powder. NMR δH (500 MHz; CD₃SOCD₃), 1.33 (3H, s), 1.38 (9H, m), 1.54 (3H, s), 2.01 (2H, m), 2.86 (4H, m), 2.96 (2H, m), 3.18 (2H, m), 3.26 (2H, m), 3.34 (2H, m), 4.25 (1H, m), 5.01 (1H, m), 5.50 (1H, m), 6.19 (1H, m), 8.22 (1H, s), 8.38(1H, s); δ¹³C (176 MHz; CD₃SOCD₃), 162.22 (1C), 158.50 (1C), 158.24 (1C), 155.22 (1C), 148.16 (1C), 141.28 (1C), 118.99 (1C), 113.40 (1C), 113.36 (1C), 89.35 (1C), 85.84 (1C), 83.23 (1C), 83.09 (1C), 78.43 (1C), 60.03 (1C), 42.63 (1C), 35.35 (1C), 28.70 (2C), 28.58 (3C), 27.38 (1C), 27.36 (1C), 25.63 (1C), 25.59 (1C); MS (ESI-MS) calculated for C₂₆H₃₉N₇O₇S (MH⁺): 594.26; found: 594.30.

4.1.10. Benzyl-4-((((3aS,4S,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)thio)-1-(2-((tert-butoxycarbonyl)amino)ethyl)piperidine-2-carboxylate (13)

To a solution of compound 12 (118.6 mg, 0.2 mmol) and potassium carbonate (33.8 mg, 0.24 mmol) in 20 mL dimethylacetamide, benzyl bromide (41.0 mg, 0.24 mmol) was added and stirred at room temperature for 24 hours. It was evaporated under high vacuum and the residue was dissolved in a water methanol mixture and purified by HPLC to give compound 13 (82.0mg, 60%). NMR δH (500 MHz; CD₃SOCD₃), 1.32 (3H, s), 1.37 (9H, m), ), 1.38 (3H, s), 1.52 (2H, d), 1.71 (2H, m), 2.17 (2H, m), 2.36 (2H, m), 2.63 (2H, m), 2.73 (1H, m), 2.83 (2H, m), 2.87 (2H, m), 2.97 (2H, m), 3.25 (2 H, m), 4.25 (1H, m), 4.98 (1H, s), 5.21 (1H, m), 5.50 (1H, m), 6.19 (1H, m), 7.35–7.42 (5H, m), 8.23 (1H, s), 8.40(1H, s); δ¹³C (176 MHz; CD₃SOCD₃), 157.71 (1C), 157.47 (1C), 155.67 (1C), 152.75 (1C), 148.55 (1C), 140.50 (1C), 135.03 (1C), 128.52 −128.48 (5C), 128.39 (1C), 128.10 (1C), 119.18 (1C), 113.34 (1C), 113.27 (1C), 89.37 (1C), 89.25 (1C), 85.79 (1C), 83.32 (1C), 83.28 (1C), 83.23 (1C), 83.11 (1C), 71.13 (1C), 59.95 (1C), 54.13 (1C), 37.81 (1C), 28.17 (3C), 26.86 (2C), 25.15 (1C), 25.08 (1C); MS (ESI-MS) calculated for C₃₃H₄₅N₇O₇S (MH⁺): 684.31; found: 684.30

4.1.11. Benzyl-4-((((3aS,4S,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)sulfonyl)-1-(2-((tert-butoxycarbonyl)amino)ethyl)piperidine-2-carboxylate (14)

To a solution of potassium hydrogen persulfate (0.18 g, 0.3 mmol), water (5 mL), 0.5 M potassium carbonate was carefully added to until the final pH of the aqueous solution was 5. The Oxone solution was added dropwise to a solution of compound 13 (68.3mg 0.1mmol) in methanol (5 mL), which was cooled to 0 °C while stirring, and the reaction mixture was stirred at room temperature. The solution was extracted three times between ethyl acetate and water, and the organic layer was dried over MgSO₄. The organic layer was concentrated and compound 14 was obtained after purification by flash chromatography (25mg, 35% ) NMR δH (500 MHz; CD₃SOCD₃), 1.33 (3H, s), 1.35 (9H, m), 1.55 (3H, s), 1.71 (2H, m), 2.17 (2H, m), 2.46 (4H, m), 2.96 (2H, m), 3.01 (2H, m), 3.26 (2H, m), 3.72 (2H, m), 4.54 (1H, m), 5.03 (2 H, s), 5.17 (1H, m), 5.50 (1H, m), 6.27 (1H, m), 7.32–7.38 (5H, m), 8.19 (1H, s), 8.35(1H, s); δ¹³C (176 MHz; CD₃SOCD₃), 171.03 (1C), 156.25 (1C), 155.64 (1C), 152.75 (1C), 148.47 (1C), 140.44 (1C), 136.01 (1C), 128.42 (2C), 128.02 (2C), 127.93 (1C), 119.32 (1C), 113.51 (1C), 113.32 (1C), 89.96 (1C), 83.92 (1C), 83.08 (1C), 81.80 (1C), 80.68 (1C), 77.53 (1C), 65.49 (1C), 58.34 (1C), 55.51 (1C), 54.11 (1C), 52.01 (1C), 44.77 (1C), 37.81 (1C), 28.24 (3C), 26.83 (1C), 25.92 (1C), 25.09 (1C), 24.20 (1C); MS (ESI-MS) calculated for C₃₃H₄₅N₇O₉S (MH⁺): 716.29; found: 716.30.

4.1.12. Benzyl-1-(2-(2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydropteridine-6-carboxamido)ethyl)-4-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)sulfonyl)piperidine-2-carboxylate (16)

To a solution of compound 14 (25mg, 0.035 mmol) in 5 mL DCM, 1 mL TFA was added dropwise at 0 °C and then stirred at room temperature overnight. After the reaction was finished, the solvent was removed under vacuum. The product (15) was added to a solution of 6-carboxy-7,7-dimethyl-7,8-dihydropterin (9.5mg, 0.04 mmol). After adding O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (19 mg, 0.05 mmol) in anhydrous DMF (5 mL) and DIPEA (17.5 μL, 0.1 mmol), the reaction mixture was stirred at room temperature overnight, the solvent was evaporated under high vacuum, and the reaction residue was purified by HPLC to give compound 16 (6.95mg, 25%). NMR δH (500 MHz; CD₃SOCD₃), 1.43 (6H, S), 1.90 (1H, m), 2.11(1H,m), 2.21 (1H, m), 2.35 (1H, m), 2.63 (1H, m), 2.73(1H, m), 2.88 (1H, m), 3.23 (1H, m), 3.54 (1H, m), 3.63 (1H, m), 3.72 (1H, m), 3.89 (1H, m), 4.25 (1H, m), 4.33 (1H, m), 4.52 (1H, m), 4.63 (1H, m), 5.04 (1H, m), 5.36 (1H, m), 5.96 (1H, m), 6.25 (1H, s), 7.28–7.33 (5H, m), 8.25 (1H, s), 8.45(1H, s); δ¹³C (176 MHz; CD₃SOCD₃), 164.19 (1C), 158.22 (1C), 157.06 (1C),155.42 (2C), 149.36 (1C), 148.95 (1C), 141.18 (1C), 128.89 (2C), 128.55 (1C), 128.47 (1C), 119.49 (1C), 118.18 (1C), 115.82 (1C), 101.02 (1C), 90.41 (1C), 88.80 (1C), 82.48 (1C), 81.95 (1C), 81.22 (1C), 78.53 (1C), 73.50 (1C), 53.66 (1C), 36.24 (1C), 31.22 (1C), 28.26 (2C); MS (ESI-MS) calculated for C₃₄H₄₂N₁₂O₉S (MH⁺): 795.29; found: 795.30.

4.1.13. (2R,4R)-1-(2-(2-Amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydropteridine-6-carboxamido)ethyl)-4-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)sulfonyl)piperidine-2-carboxylic acid (17, HP-75)

To a solution of compound 16 (10 mg, 0.013 mmol) in 5 mL THF, 5 mg Pd/C (10%) was added and 55 μL triethylsilane was added dropwise. The reaction mixture was stirred overnight at room temperature, filtered, and washed with methanol, and the solvent was evaporated under high vacuum to yield compound 17 (2.5 mg, 27%). MS (ESI-MS) calculated for C₂₇H₃₆N₁₂O₉S (MH⁺): 705.24; found: 705.30. The purity of the product was about 90%, suitable for binding studies by ITC and co-crystallization with HPPK. However, compound 17 was not stable during further purification with HPLC and the amount of resulting material was not adequate for NMR studies. Co-crystallization with HPPK stabilized the compound and the crystal structure of the HPPK:HP-75 complex at 1.48-Å resolution confirms the molecular structure of the compound.

4.2. Isothermal Titration Calorimetry

E. coli HPPK was prepared as described.¹⁶ ITC experiments were performed at 25 °C on a MicroCal iTC200 system (Malvern, PA, USA). HPPK were dialyzed extensively against an ITC buffer (50 mM sodium phosphate, pH 8.0) while HP-73 (or HP-75) was dissolved in the ITC buffer. One aliquot of 0.5 μL followed by 18 aliquots of 2.1 μL of 40 μM HP-73 (or HP-75) were injected at 750 r.p.m. into the calorimeter cell (volume 200.7 μL) that contained 5 μM HPPK. Blank experiments were performed by replacing the protein sample with buffer and the resulting data subtracted from the experimental data during analyses. The integrated interaction heat values were normalized as a function of protein concentration, and the data were fit with MicroCal Origin 7.0 software. Binding was assumed to be at one site to yield the binding affinity K_a (1/K_d), stoichiometry, and other thermodynamic parameters.

4.3. Enzyme inhibition assay

IC₅₀ measurements were carried out as described¹² except for the concentrations of the reaction mixture, which contained 1 nM E. coli HPPK, 2 μM ATP, 1 μM HP, 5 mM MgCl₂, 25 mM DTT, and a trace amount of [α−³²P]-ATP (~1 μCi) in 100 mM Tris, pH 8.0. Briefly, the total volume was 50 μl and the experiments were conducted at 30 °C. The reaction was initiated by the addition of the enzyme and stopped 30 min later by the addition of 6 μL of 0.5 M EDTA. The radioactive reactant and product were separated by thin-layer chromatography, using a PEI-cellulose plastic plate (EMD) with 0.3 M KH₂PO₄ as the mobile phase, and quantified by a Phosphor-Imager system (Amersham Typhoon TRIO). The IC₅₀ value was obtained by fitting the data to a logistic equation by nonlinear least-squares regression.

4.4. Crystallization, X-ray diffraction, structure solution, and refinement

Crystal Screen kit Index from Hampton Research (Laguna Niguel, California, USA) was used with a Gryphon crystallization robot (Art Robbins Instrument, Sunnyvale, California, USA) for crystallization of the HPPK:HP-73 complex. Crystal Screen kit Wizard from Emerald BioSystem (Bainbridge Is, Washington, USA) was used with a Mosquito crystallization robot (SPT Labtech Ltd., Hertfordshire, UK) for crystallization of the HPPK:HP-75 complex. Crystals of HPPK:HP-73 were grown at 19±1 °C in sitting drops containing 0.6 μL protein solution (10 mg/mL HPPK with saturated HP-73 in 20 mM Tris-HCl, pH 7.0) and 0.2 μL well solution (28% PEG2000, in 0.1 M Bis-Tris, pH 6.5). Crystals of HPPK:HP-75 were grown at 19 ± 1 °C in sitting drops containing 0.1 μl protein solution (10 mg/mL HPPK with saturated HP-75 in 20 mM Tris-HCl, pH 7.0) and 0.05 μL well solution (30% PEG8000 in 0.1 M Imidazole, pH 8.0). A crystal of HPPK:HP-73 was flash-frozen in liquid nitrogen. A crystal of HPPK:HP-75 was soaked in a cryoprotectant solution containing 75% (v/v) well solution and 25% (v/v) ethylene glycol, and flash-frozen in liquid nitrogen. X-ray diffraction data were collected at 100K at the synchrotron Beamline 22 of the Advanced Photon Source, Argonne National Laboratory. Data processing was carried out for HPPK:HP-73 and HPPK:HP-75 with the HKL2000 and HKL3000 program suite, respectively.³³ The HPPK:HP-73 structure was solved by Fourier Synthesis with our 1.88-Å structure of HPPK:HP-18 (PDB entry 3UDV)¹³ as the starting model after multiple conformations of amino acid residues, ligands, and solvent molecules were removed. The HPPK:HP-75 structure was solved by Molecular Replacement with the HPPK:HP73 structure (this study) as the search model after multiple conformations of amino acid residues, ligands, and solvent molecules were removed. Structure solution and refinement were carried out with PHENIX.³⁴ All graphics work, including model building and rebuilding, was performed with COOT.³⁵ The structures were verified with annealed omit maps and the geometry was assessed following the PDB structure validation procedure. The statistics of X-ray diffraction data, refinement, and final structures are summarized in Table 2. Illustrations were prepared with PyMOL (Schrödinger, LLC.).

Abbreviations:

HPPK

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase

HP

6-hydroxymethyl-7,8-dihydropterin

8MG

8-mercaptoguanine

PDB

Protein Data Bank