PGM from Pseudomonas aeruginosa

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Cite This: J. Org. Chem. XXXX, XXX, XXX−XXX

Inhibitory Evaluation of αPMM/PGM from Pseudomonas aeruginosa: Chemical Synthesis, Enzyme Kinetics, and Protein Crystallographic Study Jian-She Zhu,† Kyle M. Stiers,‡ Ebrahim Soleimani,†,§ Brandon R. Groves,† Lesa J. Beamer,*,‡ and David L. Jakeman*,†,∥ †

College of Pharmacy, Dalhousie University, 5968 College Street, Halifax, Nova Scotia B3H 4R2, Canada Biochemistry Department, University of Missouri, 117 Schweitzer Hall, Columbia, Missouri 65211, United States § Department of Chemistry, Razi University, Kermanshah 67149-67346, Iran ∥ Department of Chemistry, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada

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ABSTRACT: α-Phosphomannomutase/phosphoglucomutase (αPMM/PGM) from P. aeruginosa is involved in bacterial cell wall assembly and is implicated in P. aeruginosa virulence, yet few studies have addressed αPMM/PGM inhibition from this important Gram-negative bacterial human pathogen. Four structurally different α-D-glucopyranose 1-phosphate (αG1P) derivatives including 1-C-fluoromethylated analogues (1−3), 1,2-cyclic phosph(on)ate analogues (4−6), isosteric methylene phosphono analogues (7 and 8), and 6-fluoro-αG1P (9), were synthesized and assessed as potential time-dependent or reversible αPMM/PGM inhibitors. The resulting kinetic data were consistent with the crystallographic structures of the highly homologous Xanthomonas citri αPGM with inhibitors 3 and 7−9 binding to the enzyme active site (1.65−1.9 Å). These structural and kinetic insights will enhance the design of future αPMM/PGM inhibitors.

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INTRODUCTION α-Phosphomannomutase/phosphoglucomutases (αPMM/ PGM), encoded by the algC gene from Pseudomonas aeruginosa,1 is a member of the α-D-phosphohexomutases superfamily, and plays important roles in alginate, B-band lipopolysaccharide (LPS), rhamnolipid, and LPS-core Psl and Pel (two critical biofilm of matrix exopolysaccharides) biosynthesis.1,2 These polysaccharides are crucial to the formation of the bacterial cell wall and capsule, and therefore αPMM/PGM3 is implicated in bacterial pathogenesis of additional notorious human pathogens, including Mycobacterium tuberculosis4 and Neisseria gonorrhoeae.5,6 These roles make this enzyme a potential antibacterial target. Mannoseconfigured inhibitors are particularly attractive to clinical applications, due to the absence of the αPMM/PGM subgroup in humans. αPMM/PGM catalyzes the reversible intramolecular phosphoryl transfer between 1- and 6-position of either α-Dmannopyranose 1-phosphate (αM1P) or α-D-glucopyranose 1phosphate (αG1P).7 It is known that αPMM/PGM has equally high substrate specificity for αM1P and αG1P. Mechanistically (Figure 1), the catalytic cycle of αPMM/ PGM is initiated by a phosphate group transfer from a conserved phosphoserine to the 6-OH group of αG1P, with production of an intermediate α-glucose 1,6-bisphosphate (αG16BP) and a dephosphoserine residue (step one). © XXXX American Chemical Society

Figure 1. Reaction catalyzed by αPMM/PGM represented by the substrate αG1P.

Reorientation of αG16BP by a 180° flip orients the 1-phospho group in the vicinity of the serine residue, allowing for the second phosphoryl group to transfer to the dephosphoenzyme and release the product G6P, thus regenerating the active phosphoenzyme (step two).7,8 Early inhibitors of rabbit muscle αPGM included galactose 1,6-bisphosphate (Gal16BP),9 fructose 2,6-bisphosphate,10 glycosyl phosphofluoridates, and cyclic phosphates,11 nojirimycin 6-phosphate,12 and 6-vanadate-αG1P.13 These inhibitors were all investigated to provide insight into the rabbit Received: May 15, 2019 Published: July 2, 2019 A

DOI: 10.1021/acs.joc.9b01305 J. Org. Chem. XXXX, XXX, XXX−XXX

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The Journal of Organic Chemistry muscle αPGM reaction mechanism. Although P. aeruginosa αPMM/PGM shares the same reaction mechanism with rabbit muscle αPGM, their protein sequences are less than 25% identical.14 The structure and catalytic mechanism of P. aeruginosa αPMM/PGM have been well studied,7 but inhibitory studies are rare.14,15 As a first step toward designing lead compounds to inhibit αPMM/PGM from P. aeruginosa, we proposed four different scaffolds (Figure 2) to probe αG1P function in αPMM/PGM

attempted to install a phosphate group at the anomeric position of 10 using a P(V) reagent (diphenyl phosphoryl chloride)17 and nBuLi in tetrahydrofuran (THF) at 0 °C. However, this only led to recovery of unreacted starting material 10. Screening conditions using alternative bases (Et3N, pyridine, or tBuMgCl) at either 0 °C or in refluxing THF were unsuccessful. This presumably occurred due to the strong electronegativity and/or bulkiness of the trifluoromethyl substituent in 10, rendering the hydroxyl group chemically inert to P(V) phosphorylation. Subsequently, compound 13, containing a smaller and less electronegative substituent group (CH3), was then subjected to the above conditions.18 However, no C1 phosphorylated product was observed. Instead, a ring-opened product 14 (Table 1) was isolated in 47% yield when using N-methyl imidazole as both solvent and base. We next chose to explore the phosphorylation of 10−13 using alternative P(III) chemistry involving phosphorylation with dibenzyl N,N-diethylphosphoramidite (DDP) and subsequent oxidation with tert-butyl hydroperoxide.19 When C1fluoromethylated analogues 10−12 (Table 1) were subjected to this two-step protocol, the expected corresponding C1 phosphates 15−17 were isolated in moderate to excellent yields exclusively as α anomers. The α-anomeric configurations were determined by analysis of one-dimensional (1D) 1H−19F heteronuclear Overhauser effect spectroscopy (HOESY) data. For example, a correlation between the fluorine atom and H2 was observed for compound 15 (Figure 3). By contrast, subjecting 13, the methyl derivative, to the above two-step conditions did not give the expected phosphate and instead produced tetrazole adduct 18 (38% isolated yield) as an inseparable anomeric mixture (β/α = 4.8, as determined by 1H NMR). The anomeric stereochemistry of 18 was determined by the observation of strong correlations of H1β with H4β and H6β for the β-anomer and of H1α with H3α for the α-anomer in the 1D NOESY spectra (Figure S1). The C2− N2′ linkage was confirmed by the absence of a correlation signal between C2 and H5′ in the HMBC spectra and also by chemical shift of C5′ (153 ppm) being consistent with similar isomers.20 It is interesting that fluorine incorporation resulted in dramatically different reactivities on structurally similar compounds (10−13). A possible explanation for this behavior is that fluorine may be stabilizing the resultant intermediates by preventing phosphite group departure to generate a postulated oxocarbenium intermediate that could subsequently react with excess 1H-tetrazole. Finally, hydrogenolysis of 15 with Pd/C in iPrOH−EtOAc followed by neutralization with NH4OH provided product 1 in excellent yield (Scheme 1). Subjecting the difluoromethylated compound 16 to the above debenzylation conditions without neutralization, however, furnished a mixture of the expected product 2 and a cyclic phosphate 4, in a ratio of 4.4:1, as determined by 1H NMR

Figure 2. Designed αG1P analogues to probe αPMM/PGM function.

catalysis, including (i) C1-fluoroalkylated αG1P analogues1−3 with varying-sized substitutions at the anomeric position, (ii) α-D-glucopyranose 1,2-cyclic phosph(on)ate 4−6 featuring the presence of an electrophilic functionality, (iii) nonhydrolyzable isosteric methylenephospho G1P analogues 7 and 8, and (iv) 6-fluorinated αG1P 9. It was hypothesized that analogues 1−8 could potentially be turned over or react with the phosphoenzyme during step one and thus inhibit or inactivate the enzyme. Herein, we report the chemical synthesis of analogues 1−9 (Figure 2) and evaluate their enzymatic and Xray structural data with αPMM/PGM.

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CHEMICAL SYNTHESIS It was envisioned that 1-C-fluoroalkylated αG1P analogues could be accessed by chemical phosphorylation of corresponding fluoroalkylated hemiketals 10−13 (Table 1), which were Table 1. Two-Step Protocol for Phosphorylation of Compounds 10−13

substrate

R

product

yield (two-step) (%)

10 11 12 13

CF3 CF2H CH2F CH3

15 16 17 18

62 89 74 38

readily prepared starting from 2,3,4,6-tetra-O-benzyl-D-gluconolactone according to the reported procedures.16 We initially

Figure 3. Anomeric configuration assignment of 15 by 1D 1H−19F HOESY. B


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The Journal of Organic Chemistry Scheme 1. Preparation of Compounds 1−4 by Hydrogenolysis

Figure 4. Conversion of 2 to 4 over time monitored by 1H NMR (500 MHz, D2O).

control experiment without preincubation of αPMM/PGM with any of these compounds was performed for comparison. None of these compounds showed a time-dependent inhibition behavior toward αPMM/PGM (Figures S6−S8). A second time-dependent inhibition assay was also performed in the absence of G16BP, with the same result (Figure S10). The lack of time-dependent inhibition for these C-1 fluoromethylated αG1P analogues (1−3) is likely due to the phosphoenzyme failing to transfer a phosphoryl group to the primary hydroxyl, whereby inactive dephosphorylated αPMM/ PGM would be produced (Figure 1, step one). This observation is in accordance with discovery that glycosyl phosphofluoridates were reversible inhibitors rather than timedependent inactivators of αPGM from rabbit muscle.11 However, the observed lack of time-dependent inhibition of P. aeruginosa αPMM/PGM by the phosphono αG1P analogues 7 and 8 is contrary to the capability of rabbit muscle αPGM to turnover 7 to its 6-phosphoric acid derivative, albeit in a very poor efficiency (2 × 10−5 times of kcat/Km of αG1P).23 The reason why αPMM/PGM from P. aeruginosa fails to perform the same transformation of 7 or 8 is presumably due the low protein sequence similarity between the mammalian and bacterial enzymes. Examination of α-Glucopyranose 1,2-Cyclic Phosphate/Phosphonate Analogues (4−6) as Potential Mechanism-Based Inactivators of αPMM/PGM. The primary hydroxyl of three α-glucopyranose 1,2-cyclic phosphate/phosphonate analogues (4−6) could be potentially phosphorylated by αPMM/PGM (Figure 1, step one). The resulting 6-phospho cyclic phosph(on)ates could potentially dissociate from the enzyme, rebind with the cyclic phosph(on)ates adjacent to the nucleophilic Ser108, and bind covalently, thus inactivating the enzyme. A related cyclic phosphate, α-glucopyranose 1-phosphate-4,6-cyclicphosphate, was unable to inactivate αPGM from rabbit muscle.11 This was ascribed in part to the inactive six-membered 4,6-cyclic phosphate group; however, the presence of a more strained five-membered 1,2-cyclic phosphate/phosphonate, especially one containing an additional electron-withdrawing group (CF2H) at the anomeric position (5), might promote the expected nucleophilic attack by dephosphorylated Ser108. Unfortunately, none of these three compounds (4−6)

(Figure 4). Over approximately 3 weeks, 2 would spontaneously convert to 4. This result suggests that an acidic reaction media facilitates the formation of cyclic product 4. The structure of 4 was established by analysis of highresolution mass spectrometry data, 1D and 2D NMR spectra, including 1D 1H−19F HOESY spectra (Figures S2 and S3). As expected, hydrogenolysis of 16 using Pd(OH)2/C and sodium bicarbonate in iPrOH−EtOAc−H2O afforded compound 2 exclusively in 84% yield. Similarly, the presence of sodium bicarbonate was also important to achieve clean hydrogenolysis of 17. Unexpectedly, 3 was found to decompose by loss of the phosphate group over 3 months when stored at −20 °C whereas compounds 1 and 2 were stable under similar conditions. The cyclic G1P analogue 6 was prepared from the known compound 7 in two steps,21 involving acetylation of 7 with acetic anhydride and pyridine and a subsequent deacetylation of the resultant intermediate with Et3N/CH3OH/H2O (Figure S4). α-D-Glucopyranose 1,2-cyclic phosphate 5,22 isosteric methylene phosphono M1P analogue 8, and 6-fluoro-G1P analogue 917 were prepared following known procedures.

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EVALUATION AS INHIBITORS OF αPMM/PGM Investigation of Compounds 1−3, 7, and 8 as TimeDependent Inhibitors of αPMM/PGM. None of the synthesized compounds (1−9) were found to be substrates of αPMM/PGM at concentrations up to 0.2 mM and were thus evaluated as inhibitors. Compounds 1−3, 7, and 8 had the potential to be time-dependent inhibitors of αPMM/PGM; mechanistically, they could dephosphorylate the phosphoenzyme in the first phosphoryl transfer step to produce corresponding G16BP analogues and dephosphoenzyme. The corresponding G16BP analogues, however, could not dissociate, rebind in the opposite orientation, and rephosphorylate the resultant dephosphoenzyme. Therefore, to probe this mechanism of action, each analogue (1−3, 7, and 8) was independently preincubated with the phosphoenzyme form of αPMM/PGM in the presence of G16BP for periods of up to 20−40 min. Aliquots were taken at different incubation times and added to a reaction mixture consisting of Tris buffer, MgCl2, NAD+, G6PDH, ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), and αG1P (Figure S5).12 A C


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was demonstrated by increases in conversion of αG1P (250 μM) to G6P of 120, 129, and 145% when αPMM/PGM was kinetically assayed in the presence of 5 at concentrations of 100, 500, and 1000 μM, respectively (Figure S12). This acceleration in the reaction rate was not due to the hydrolysis of 5 to form αG1P, as no G6P product signal was observed by 31 P NMR analysis of incubated reaction mixtures of 5 with αPMM/PGM and MgCl2. The two isosteric αG1P analogues 7 and 8 were determined to inhibit αPMM/PGM, consistent with the observed initial reaction rate decline upon their incubation with αPMM/PGM during the time-dependent inhibition study. The inhibition of these compounds with αPMM/PGM is similar (7: 213 μM and 8: 184 μM), both approximately 10-fold weaker than that of the native substrate αG1P Km. The similarity in inhibition of 7 and 8 demonstrates the inherent ability of αPMM/PGM to equally accommodate gluco- and manno-configured hexoses. It also demonstrates that fluorine modification at C2 has a negligible effect on enzyme binding. However, these two compounds differ in the mode of enzyme inhibition, as observed by the Lineweaver−Burk plots (Figure 5). Compound 7 inhibits by binding the enzyme or enzyme−substrate complex (mixed inhibition), whereas compound 8 was determined to bind the enzyme active site (Figure S13) in competition with native αG1P (competitive inhibition). The 6fluorinated αG1P analogue 9 was found to be the most potent competitive inhibitor with a Ki value of 53 μM. This indicates that fluorine substitution at the 6-position abrogates phosphoryl transfer between the substrate and enzyme but does not prohibit tight binding to αPMM/PGM. X-ray Crystallographic Studies of αG1P Analogues with αPMM/PGM. To provide insight into interactions of the αG1P analogues with an enzyme in the αPMM/PGM family, we employed the related protein αPGM from the bacterium Xanthomonas citri (XcPGM). Crystals of XcPGM are highly amenable to form ligand complexes and diffract to high resolution.25 XcPGM shares 34% amino acid sequence identity with the P. aeruginosa enzyme, and active site residues in the two proteins are very highly conserved (Figure S13). Crystal structures of XcPGM as apoenzyme and in complex with αG1P and G16BP have been previously published [Protein Data Bank (PDB) IDs: 5BMN, 5BMP, and 5KLO].25 These structures were used for comparisons with the inhibitor complexes, as described below. Although XcPGM was described as an α-phosphoglucomutase, its amino acid sequence suggests it is more closely related to enzymes in the αPMM/PGM subgroup.7

displayed mechanism-based inactivation of αPMM/PGM upon incubation (Figure S9). One possible explanation for the lack of inactivation of αPMM/PGM by these three compounds is the inability of αPMM/PGM to phosphorylate the 6-OH group of these 1,2-cyclic phosphate/phosphonate analogues. Reversible Inhibition of Compounds (1−9) against αPMM/PGM. Compounds (1−9) at varying concentrations (0−2000 μM) were evaluated as potential reversible inhibitors of αPMM/PGM, with varying concentrations of the substrate αG1P. The production of G6P was monitored through a coupled assay using G6P dehydrogenase as described in the literature.24 Rate data were fitted to a Michaelis−Menten equation, and the inhibition constant (Ki) as well as the inhibition mode were determined by the Lineweaver−Burk (L−B) plot analysis (Figure S11). Neither the trifluoromethylated αG1P analogue 1 nor the difluoromethylated analogue 2 was found to inhibit αPMM/PGM (Table 2). By contrast, Table 2. Kinetic Inhibition Results of Compounds 1−9 against αPMM/PGM compound

Ki (μM)

inhibition mode

1 2 3 4 5 6 7 8 9

NIa NIa 428 ± 17c 1075 ± 193 activator 521 ± 31 213 ± 51 184 ± 23 53 ± 5

NDb NDb noncompetitive competitive NDb noncompetitive mixed competitive competitive

a

No inhibition at the concentration of 2 mM. bNot determined. Standard error.

c

monofluoromethylated compound 3, with the smallest anomeric substitute, was found to be a weak noncompetitive inhibitor. 3 had a Ki value of 428 μM, an affinity approximately 20-fold weaker than that of the native substrate αG1P (Km = 20 μM). This result suggests that αPMM/PGM appears to tolerate small anomeric substitutes for the proton in αG1P. For the α-glucopyranose 1,2-cyclic phosphate/phosphonate analogues (4−6), the C1-difluoromethyl 1,2-cyclic phosphate analogue 4 was a very poor inhibitor with a Ki value of 1075 μM. The 1,2-cyclic phosphonate analogue 6 displayed weak inhibition toward αPMM/PGM (Ki = 521 μM). By contrast, α-glucopyranose 1,2-cyclic phosphate 5 was found to be a weak activator rather than an inhibitor of αPMM/PGM. This

Figure 5. Lineweaver−Burke plots demonstrating different modes of inhibition for 3 (noncompetitive), 7 (mixed), and 9 (competitive). Each plot is representative of one of three repetitive experimental results. D


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Figure 6. Structural analysis of inhibitors binding to αPMM/PGM. (A) A superposition of the crystal structures of the four inhibitor complexes. The enzyme is shown as a ribbon model, and the inhibitors, as stick models. Colors are as follows: (3), pink; (7), cyan; (8), green; and (9), white. (B) An overview of the core enzyme−inhibitor contacts in the XcPGM active site. Positions of novel substituents for compounds 3 and 7−9 are highlighted by green boxes. (C) A close-up view of the active site showing the similarity between the four inhibitor complexes [colors as in (A)] and the XcPGM complex with G1P (black). PDB codes: 6MNV, 6MLH, 6MLW, and 6MLF, respectively.

High-resolution structures (1.65−1.9 Å) were obtained for XcPGM in complex with four inhibitors (compounds 3 and 7− 9) (Figure 6A). Overall, there was a correspondence between those compounds with more potent inhibition (Table 2) and our ability to obtain crystal structures with bound ligand. Polder omit maps revealed excellent ligand electron density for all four structures (Figure S14). The binding of each inhibitor produces a conformational change to XcPGM, relative to apoenzyme. Specifically, an active site loop spanning residues 415−421 closes around the ligand, essentially creating a lid over the active site. The inhibitors are deeply buried in the active site cleft, with less than 20% of their surface area exposed to solvent. It is convenient to consider the binding modes of the inhibitors relative to the interactions of XcPGM with its substrate αG1P. Enzyme−substrate interactions include conserved contacts to both the phosphate group and the O3/O4 hydroxyls (Figure 6B). Residues that interact with the phosphate group include Arg414, Arg423, Asn417, Ser416, and Thr418. Contacts with the O3 and O4 sugar hydroxyls are made by Glu320 and Ser322, both of which are also highly conserved in αPMM/PGMs. Arg280 makes contacts with both O2 and O3 but is presumably less important to binding, since both glucose and mannose-based substrates are processed in the active site.26 The four tight-binding inhibitors differ from αG1P at only several positions, none of which perturb these core enzyme−ligand interactions (Figure 6C and Table 3). Thus, in principal, it should be possible for the inhibitors to maintain most, if not all, of these critical interactions with the enzyme. On the other hand, inhibitors with more significant structural differences from αG1P, such as 1,2-cyclic phosphate compounds (4−6) and substituted methyl derivatives (1 and 2), bound poorly, likely due to their inability to fully utilize

Table 3. Details of Protein−Inhibitor Hydrogen Bonds in the XcPGM Complexes ligand atom O1P O2P

O3P

O2 O3

O4

protein residue and atoma

G1P

3

7

8

9

5BMP

6MNV

6MLH

6MLW

6MLF

2.88 3.03b 3.23 2.69 2.53 2.80b 3.27b

3.14 3.09b 3.15b 3.28 2.97 2.52 2.39 3.07

3.19 2.87b 2.91b 2.92 2.75

3.00b 3.14 2.40 3.01b 2.99 2.90

Phosphate Contacts (Å) R414 NH1 3.04 3.11 2.98 R423 NE 2.87 2.84 2.77 R414 NH2 2.67 3.05b 2.75 S416 OG N417 N 2.83 2.80 2.85 S416 OG 2.53 2.67 2.61 T418 OG1 2.52 2.62 2.52 R423 NH1 3.04 3.07 3.03 Sugar Hydroxyl Contacts (Å) R280 NH1 2.55 2.74b 2.78b R280 NH2 2.97 2.79 2.93 E320 OE2 2.59 2.59 2.63 S322 OG 2.61 2.79 2.73 H303 N 3.14 3.11 3.06 E320 OE1 2.84 2.60c 2.69

a

Protein residue numbers correspond to the XcPGM sequence from UniProt ID Q8PGN7 (without His tag). Atom names are from PDB ID 5BMP; atoms that correspond spatially were used for other structures. bFurther by >0.2 Å compared with 5BMP. cCloser by >0.2 Å compared with 5BMP.

these core interactions with the enzyme. Details of the individual complexes are discussed below. Among the C1 fluoroalkylated compounds, only compound 3 was characterized by X-ray crystallography (Figure 7A). This compound binds similar to αG1P, with enzyme contacts to E


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modification appears to be easily accommodated at this position, consistent with the potent competitive inhibition by this compound. In all cases, there is a significant volume of active sites between the C6 and the nucleophilic serine and catalytic Mg2+. This observation may provide insight into the design of more potent inhibitors.

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CONCLUSIONS Three new C1 fluoromethylated αG1P analogues (1−3) were synthesized using P(III) chemistry. The requirement for one or more fluorine atoms on the methyl substituent to furnish the products was evident by the isolation of a novel glycosyl tetrazole from the nonfluorinated methyl derivative. Three αglucopyranose 1,2-cyclic phosph(on)ate analogues (4−6) were also chemically prepared. Collectively, a series of nine structurally related analogues of αG1P were evaluated as substrates or inhibitors of αPMM/PGM. None of these compounds were substrates, and no time-dependent inhibition was observed. Instead, a series of reversible inhibitors with different modes of inhibition was discovered. The lack of binding by the C1 trifluoro and difluoro derivatives relative to the monofluoro derivative demonstrates a high degree of discrimination by the αPMM/PGM active site between a fluorine and hydrogen substituent. Different inhibition modes were observed with compound 7 showing mixed inhibition, whereas the C2 epimeric compound 8 demonstrated competitive inhibition. The 6-fluorinated αG1P analogue 9 was determined to be a potent competitive inhibitor of αPMM/PGM, revealing that fluorine modification at C6 position of αG1P does not have a negative effect on binding to αPMM/PGM. Crystal structures of XcPGM in complex with inhibitors 3 and 7−9 were obtained. The four tight-binding inhibitors are bound to the XcPGM active site and maintain the core interaction observed with the αG1P substrate and consequently closely adopt the same binding site. All of these inhibitors indicated that there is significant volume of active sites between C6 and the serine nucleophile and accordingly these insights will help facilitate the design of more potent αPMM/PGM inhibitors.

Figure 7. Close-up view of inhibitors bound in the active site of XcPGM. Location and nature of introduced substituent atoms relative to G1P are labeled. Each inhibitor is superimposed with G1P (black lines) from the XcPGM−substrate complex to show the similar mode of binding. (A)−(D) are for compounds 3, 7, 8, and 9, respectively; colors are the same as in Figure 6. Orientations vary slightly to optimize the view of introduced atoms. Red spheres are the location of discrete water molecules within each complex.

both the phosphate group and to the O3/O4 hydroxyls. A few small structural rearrangements occur in other residues of the protein, apparently to accommodate the CH2F substituent, resulting in adjustments in the length of several hydrogen bonds relative to the G1P complex (Table 3). The only atoms in the active site near the fluorine are two water molecules; the closest protein atoms (to the methyl carbon) are >4 Å away. The observed noncompetitive rather than competitive inhibition profile of this compound based on the crystal structures is potentially due to the ability of this inhibitor to bind both the enzyme and enzyme−substrate complex under the kinetic assay condition where Ser108 was phosphorylated by G16BP. The two nonhydrolyzable isosteric analogues, 7 and 8, also bind quite similarly to G1P (Figure 7B,C). Neither the phosphonate modification nor 2-fluoro substitution appears to cause any perturbation of the core enzyme−substrate interactions, except for the necessary loss of interaction with O2 in the 8 complex. The phosphonate carbons are >3.5 Å away from atoms of the protein. In the 7-complex, the enzyme−ligand hydrogen bonds are nearly indistinguishable from the αG1P complex (Table 3). In the case of 8, a number of adjustments in hydrogen bond lengths occur, including to the nearby O3 hydroxyl (Table 3). The 2-fluoro substituent is ∼3 Å from several water molecules; the closest protein atoms (∼3.3 Å away) are the side chains of Ser322 and His324. The observed binding modes are generally consistent with the competitive inhibition by compound 8 and mixed inhibition by compound 7. In the complex with compound 9, binding of the inhibitor utilizes the core enzyme−ligand interactions (Figure 7D), although several hydrogen bonds have altered bond lengths (Table 3). Few protein atoms are nearby: only the side chain His324 is within 4 Å of the 6-fluoro group. Thus, the fluorine

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EXPERIMENTAL SECTION

Chemical Synthesis. All chemicals and reagents were purchased from commercial sources and used as received, unless otherwise noted. Syntheses that required anhydrous conditions were performed under an inert atmosphere of dried high-purity nitrogen. Highperformance liquid chromatography grade methanol was employed where stated. Glassware was dried overnight in an oven set at 120 °C and assembled under a stream of inert gas. Analytical thin-layer chromatography (TLC) was performed using silica gel 60 F254 precoated glass plates; compound spots were visualized by ultraviolet light at 254 and/or by charring after treatment with vanillin stain. Flash chromatography was performed with silica gel 60 (230−400 mesh). Lyophilization of samples was carried out using a freeze-dryer. NMR spectra were recorded on a Bruker 300 or 500 MHz spectrometer. All 1H, 13C, 19F, and 31P chemical shifts are reported in ppm using tetramethylsilane (0.00 ppm) or the solvent signal [CDCl3 (1H 7.26 ppm; 13C 77.16 ppm) with D2O (1H 4.79 ppm)] as the internal reference or MeOD (13C 49.50 ppm in D2O) or 85% aqueous (aq) H3PO4 (31P 0.00 ppm) or CF3COOH (19F, −76 ppm) as an external reference. Splitting patterns are indicated as follows: br, broad; s, singlet; d, doublet; t, triplet; at, apparent triplet; q, quartet; and m, multiplet. All coupling constants (J) are reported in hertz (Hz). Mass spectra were recorded using ion trap [electrospray ionization time-of-flight (ESI-TOF)] instruments. F


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The Journal of Organic Chemistry General Procedure I for Phosphorylation and the Subsequent Oxidation. To a solution of ketol (1 mmol) and 1Htetrazole (290 mg, 4.2 mmol) in anhydrous THF was added dropwise commercially available 85% dibenzyl N,N-diethylphosphoramidite (0.8 mL, 2.3 mmol) at room temperature under N2. The clear reaction mixture was stirred at room temperature until the starting material disappeared, as determined by TLC (1−2 h). Upon completion, it was diluted with Et2O (50 mL) and then quenched with cold aqueous saturated NaHCO3 solution (10 mL). The mixture was extracted with Et2O (3 × 15 mL) and washed with H2O (2 × 5 mL) and brine (5 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The resultant crude product was used directly in the next step without further purification. To a solution of the above crude product in THF was added dropwise 70 wt % tBuOOH (2.3 mL, 17 mmol) at −10 °C. This reaction solution was then allowed to stir at room temperature until the starting material was consumed, as determined by TLC (1−2 h). The reaction was then diluted with EtOAc (70 mL), quenched with cold aqueous saturated Na2S2O3 solution (10 mL), and then stirred at 0 °C for 20 min. The water phase was extracted with EtOAc (3 × 15 mL). The combined organic layers were washed with H2O (2 × 5 mL), brine (5 mL), dried over (MgSO4), and concentrated in vacuo to give crude product residue, which was purified on a silica gel column. General Procedure II for Hydrogenolysis Debenzylation. Method a: To a solution of perbenzylated phosphate compound (0.1 mmol) in iPrOH−EtOAc (3 mL, 5:1, v/v) was added Pd/C (15 mg). The suspension was degassed under vacuum and saturated with H2 in a 1 atm balloon. This reaction mixture was stirred at room temperature until the starting material disappeared as monitored by TLC. Upon completion, the catalyst was filtered off and the filtrate was concentrated under reduced pressure. The resultant residue was dissolved in millipore water (3 mL) and neutralized carefully with concentrated NH4OH solution to adjust its pH to 7. The aqueous solution was then concentrated down and lyophilized to give the final product. Method b: To a solution of perbenzylated phosphate compound (0.1 mmol) in iPrOH−EtOAc−H2O (6 mL, 9:3:0.5, v/v) was added Pd(OH)2/C (12 mg) and NaHCO3 (25 mg, 0.3 mmol). The suspension was degassed under vacuum, saturated with H2, and shaken in a Parr Shaker at 60 psi pressure. Upon completion, the catalyst was filtered off and the filtrate was concentrated under reduced pressure. The resultant residue was dissolved in millipore water (3 mL) and neutralized carefully with concentrated NH4OH solution to adjust its pH to 7. The aqueous solution was concentrated down and lyophilized to give the final product. 1-Deoxy-3,4,5,7-tetra-O-benzyl- D -gluco-2-heptulose 6(Diphenyl)phosphate (14). To a solution of 13 (100 mg, 0.18 mmol) in N-methyl imidazole (5 mL) was added dropwise diphenyl phosphoryl chloride (60 μL, 0.27 mmol) at room temperature. The mixture was allowed to stir until the starting material disappeared, as monitored by TLC. Upon completion, the reaction was quenched with sat. NH4Cl solution (1 mL) and the solvent was evaporated under reduced pressure. The resultant residue was then extracted with EtOAc (3 × 5 mL) and water (2 × 3 mL). The combined organics were washed with brine (2 mL), dried over MgSO4, filtered, and concentrated to give a colorless oil, which was subjected to silica gel column chromatography purification (n-hexane/EtOAc = 6:1) to afford compound 14 (66 mg, 47%) as colorless oil. Data for 14: 1H NMR (500 MHz, CDCl3) δ 7.40−7.17 (m, 30H), 5.05 (dt, J = 4.5 and 9.0 Hz, 1H), 4.68−4.65 (m, 2H), 4.59−4.54 (m, 3H), 4.52−4.44 (m, 3H), 4.21 (t, J = 5.3 Hz, 1H), 4.05 (t, J = 5.4 Hz, 1H), 4.02−4.01 (m, 1H), 3.99 (dd, J = 3.5 and 11.0 Hz, 1H), 3.83 (dd, J = 5.0 and 10.9 Hz, 1H), 2.04 (s, 3H). 31P{1H} NMR (202 MHz, CDCl3) δ −12.5. 13C{1H} NMR (125 MHz, CDCl3) δ 208.5, 150.84, 150.78, 150.7, 138.3, 137.8, 137.4, 130.0, 129.9, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 128.05, 128.0, 127.9, 127.7, 125.5, 125.47, 120.5, 120.4, 120.38, 120.3, 83.4, 80.4, 79.6 (d, J = 5 Hz), 79.2 (d, J = 6.3 Hz), 74.9, 74.8, 73.5 (d, J = 3.8 Hz), 69.0, 27.9 1-Deoxy-1-trifluoro-3,4,5,7-tetra-O-benzyl-α-D-gluco-2-heptulose 2-Dibenzyl Phosphate (15). Following the general procedure I,

reaction of 10 (600 mg, 0.987 mmol) with dibenzyl N,Ndiethylphosphoramidite (0.8 mL, 2.27 mmol) and 1H-tetrazole (270 mg, 3.9 mmol) in THF (5 mL) and a second reaction with t BuOOH (1.6 mL, 16.8 mmol) in THF (5 mL) gave phosphate 15 (530.3 mg, colorless oil) in 62% overall yield after chromatography purification (n-hexane/EtOAc = 5). Data for 15: 1H NMR (500 MHz, CDCl3) δ 7.36−7.21 (m, 30H), 5.13−4.98 (m, 4H), 4.89−4.76 (m, 5H), 4.67−4.60 (m, 2H), 4.51− 4.48 (m, 1H), 4.28−4.26 (m, 1H), 4.07 (t, J = 10.0 Hz, 1H), 3.92 (dd, J = 10.0 and 5.0 Hz, 1H), 3.89 (t, J = 10.0 Hz, 1H), 3.77 (dd, J = 11.7 and 2.9 Hz, 1H), 3.57 (dd, J = 11.7 and 1.5 Hz, 1H). 19F{1H} NMR (470 MHz, CDCl3), δ −78.6. 31P{1H} NMR (202 MHz, CDCl3), δ −9.0. 13C{1H} NMR (125 MHz, CDCl3), δ 138.4, 138.3, 138.1, 137.7, 135.8, 135.71, 135.66, 128.62, 128.57, 128.55, 128.49, 128.4, 128.1, 128.0, 127.93, 127.88, 127.81, 127.79, 127.74, 121.5 (q, 1 JC‑1,F = 287.5 Hz, C-1), 100.3 (dq, 2JC‑2,F = 32.3 Hz, 2JC‑2,P = 11.0 Hz, C-2), 82.1, 79.2 (d, 4JC‑3,F = 6.3 Hz, C-3), 76.8, 75.9, 75.7, 75.6, 75.1, 73.5, 70.1, 67.4. High-resolution mass spectrometry (HRMS) (ESITOF): m/z: calcd for [M + Na]+ C49H48F3NaO9P, 891.2880; found 891.2899. 1-Deoxy-1-trifluoro-α-D-gluco-2-heptulose 2-Phosphate (1). Following the general procedure II (method a), global deprotection of 15 (76 mg, 0.088 mmol) with Pd/C (15 mg) provided compound 1 (30.7 mg, 100%) as a colorless solid. Data for 1: 1H NMR (500 MHz, D2O) δ 4.10 (t, J = 9.5 Hz, 1H), 3.93 (t, J = 9.2 Hz, 1H), 3.82−3.72 (m, 3H), 3.50 (t, J = 9.5 Hz, 1H). 19 1 F{ H} NMR (470 MHz, D2O) δ −82.4 (s). 31P{1H} NMR (202 MHz, D2O) δ 2.5 (s). 13C{1H} NMR (125 MHz, D2O) δ 122.2 (q, 1 JC‑1,F = 286 Hz, C-1), 94.3 (dq, 2JC‑2,F = 30.6 Hz, 2JC‑2,P = 6.4 Hz, C2), 73.4, 73.3 (d, 3JC‑3.F = 5.1 Hz, C-3), 72.9, 68.7, 60.1, HRMS (ESITOF): m/z: [M − H]− calcd for C7H11F3O9P, 327.0098; found 327.0099. 1-Deoxy-1,1-difluoro-3,4,5,7-tetra-O-benzyl-α-D-gluco-2-heptulose 2-Dibenzyl Phosphate (16). Following the general procedure I, reaction of 11 (300 mg, 0.51 mmol) with dibenzyl N,Ndiethylphosphoramidite (375 μL, 1.1 mmol) and 1H-tetrazole (135 mg, 1.9 mmol) in THF (8 mL) and following a second reaction with t BuOOH (1.2 mL, 8.7 mmol) in THF (8 mL) produced 16 (386 mg, colorless oil) in 89% overall yield after chromatography purification (n-hexane/EtOAc = 5). Data for 16: 1H NMR (500 MHz, CDCl3) δ 7.48−7.29 (m, 30H), 6.67 (t, 2JH‑1,F = 54.0 Hz, 1H, H-1), 5.19−5.15 (m, 4H), 4.96−4.92 (m, 5H), 4.77−4.70 (m, 2H), 4.62−4.59 (m, 1H), 4.18 (dd, J = 10.0 and 1.5 Hz, 1H), 4.11−4.05 (m, 2H), 3.92 (t, J = 9.8 Hz, 1H), 3.80 (dd, J = 11.5 and 3.4 Hz, 1H), 3.65 (dd, J = 11.5 and 1.5 Hz, 1H). 19 1 F{ H} NMR (470 MHz, CDCl3) δ −131.4 (d, 2JFa,Fb = 286.7 Hz), −134.6 (d, 2JFa,Fb = 286.7 Hz). 19F NMR (470 MHz, CDCl3) δ −131.4 (dd, 2JFa,Fb = 286.7 Hz, 2JFa,H‑1 = 53.8 Hz), −134.6 (dd, 2JFb,Fa = 286.7 Hz, 2JFb,H‑1 = 55.2 Hz), 31P NMR (202 MHz, CDCl3) δ −6.1 (s). 31P{1H} NMR (202 MHz, CDCl3) δ −6.1 (s). 13C{1H} NMR (125 MHz, CDCl3) δ 138.7, 138.5, 138.4, 138.1, 136.1, 136.0, 135.9, 128.91, 128.89, 128.82, 128.76, 128.73, 128.7, 128.5, 128.4, 128.3, 128.23, 128.16, 128.13, 128.0, 127.98, 112.5 (t, 1JC‑1,F = 248.7 Hz, C1), 101.8 (dt, 2JC‑2,F = 25.5 Hz, 2JC‑2,P = 7.7 Hz, C-2), 82.6, 78.4 (d, 3 JC‑3,F = 6.4 Hz, C-3), 77.23, 76.1, 75.8, 75.4, 75.0, 73.8, 70.1 (d, J = 4.1 Hz), 68.0, 65.4. HRMS (ESI-TOF): m/z: calcd for [M + Na]+ C49H49F2NaO9P, 873.2974; found 873.2976. 1-Deoxy-1,1-difluoro-α-D-gluco-2-heptulose 2-Phosphate (2) and 1-Deoxy-1,1-difluoro-α-D-gluco-2-heptulose 2,3-Diphosphate (4). Following the general procedure II (method a), global deprotection of 16 (73 mg, 0.086 mmol) with Pd/C (14 mg) gave a mixture of 2 and 4 in a 4.4:1 ratio, which was completely converted to 4 after approximately 3 weeks. Data for 4: 1H NMR (500 MHz, D2O) δ 5.90 (t, 2JH‑1,F = 54.3 Hz, 1H), 3.98 (t, J = 9.2 Hz, 1H), 3.88 (t, J = 9.2 Hz, 1H), 3.82−3.72 (m, 3H), 3.46 (t, J = 9.4 Hz, 1H). 19F{11H} NMR (470 MHz, D2O) δ −133.1 (d, 2JFa,Fb = 282 Hz), −142.0 (2JFa,Fb = 282 Hz). 19F NMR (470 MHz, D2O) δ −133.1 (dd, 2JFa,Fb = 282 Hz, 2JFa,H = 54 Hz, 1F), −142.0 (dd, 2JFb,Fa = 282 Hz, 2JFb,H = 55 Hz, 1F). 31P{1H} NMR (202 G


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give an inseparable anomeric mixture 18 (62.3 mg, 38%, β/α = 4.8:1) as a colorless oil. Data for 18 (β/α anomeric mixture): 1H NMR (500 MHz, MeOHd4) δ 8.75 (s, 0.23H, H5′α), 8.72 (s, 0.91H, H5′β), 7.36−7.09 (m, 23H, 4 × −OCH2C6H5), 6.93−6.91 (m, 2H, −OCH2C6H5), 4.89 (s, 2H, −OCH2Ph, β), 4.87−4.86 (m, 0.32H, −OCH2Ph, α), 4.84−4.82 (m, 2H, −OCH2Ph, β), 4.80−4.78 (m, 0.26H, −OCH2Ph, α), 4.75− 4.73 (m, 0.24H, −OCH2Ph, α), 4.64−4.73 (m, 1H, −OCH2Ph, β), 4.61−4.58 (m, 2H, H3β, −OCH2Ph), 4.52 (d, 2J = 12.0 Hz, 1H, −OCH2Ph, β), 4.51 (d, 2J = 12.0 Hz, 0.27H, −OCH2Ph, α), 4.44 (d, 2 J = 12.0 Hz, 1H, −OCH2Ph, β), 4.43 (d, 2J = 12.0 Hz, 0.23H, −OCH2Ph, α), 4.16 (d, 2J = 11.0 Hz, 1H, −OCH2Ph, β), 4.0 (t, 3J = 9.3 Hz, 1H, H4β), 3.95 (d, 3J = 9.4 Hz, 0.3H, H3α), 3.92−3.89 (m, 1H, H6β), 3.82 (t, 3J = 9.4 Hz, 1H, H5β), 3.72 (dd, 3J = 11.3 and 4.4 Hz, 1H, H7β), 3.67 (dd, 3J = 11.2 and 1.5 Hz, 1H, H7β), 3.56 (dd, 3J = 11.1 and 1.5 Hz, 0.22H, H7α), 2.17 (s, 3H, H1β), 2.11 (s, 0.63H, H1α). 13C{1H} NMR (125 MHz, MeOH-d4), δ 153.8 (C5′β), 153.1 (C5′α), 139.9 (α), 139.8 (β), 139.6 (α), 139.56 (β), 139.4 (β), 139.36 (α), 139.2 (α), 139.1 (β), 129.6, 129.5, 129.45, 129.4, 129.2, 129.1, 129.0, 128.9, 129.87, 129.8, 129.7, 128.1, 96.4 (C2α), 96.2 (C2β), 84.9 (C4α), 84.8 (C4β), 84.1 (C3α), 83.9 (C3β), 79.4 (C5β), 79.2 (C5α), 77.2 (C10α), 76.8 (C10β), 76.5 (C9α), 76.4 (C11α), 76.2 (C9β), 76.16 (C11β), 76.14 (C6β), 76.0 (C6α), 74.5 (C8β), 69.9 (C7β), 69.8 (C7α), 26.5 (C1α), 17.2 (C1β). HRMS (ESI-TOF) m/z: calcd for [M + Na]+ C36H38NaO5N4, 629.2734; found 629.2727. 3,4,6-O-Triacetyl-1′-deoxy-1′-methylene-α-D-glucopyranose 1,2Cyclic Phosphonate (7a). To a solution of 7 (67 mg, 0.1 mmol) in pyridine (1 mL) was added Ac2O (1 mL), and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated at 50 °C under high vacuum, and the residue was then purified by silica gel column chromatography [dichloromethane (DCM)/MeOH, 20:1] to give product 7a (36 mg, 38%) as an orange sirup. Data of 7a: 1H NMR (500 MHz, CD3OD), δ (ppm) 2.21−1.93 (m, 2H), 2.04 (s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 4.12−4.06 (m, 2H), 4.31 (dd, 3J = 12.6 and 6.5 Hz, 1H), 4.37 (dd, 3J = 12.2 and 5.1 Hz, 1H), 4.85 (m, 1H), 4.95 (t, 3J = 9.3 Hz, 1H), 5.37 (t, 3J = 8.4 Hz, 1H). 13C NMR (125 MHz, CD3OD), δ (ppm) 19.2, 19.4, 19.4, 23.2 (d, 1JCP = 120.0 Hz), 62.0, 67.9, 70.13, 73.5, 75.3, 72.8 (d, 3JCP = 11.1 Hz), 171.6, 171.9, 172.5. 31P{1H} NMR (202 MHz, CD3OD), δ (ppm) 32.90. HRMS (ESI-TOF−) m/z: calcd for [M − H]− C13H18O10P 365.0643; found 365.0645. 1′-Deoxy-1′-methylene-α-D-glucopyranose 1,2-Cyclic Phosphonate (6). Compound 7a (36 mg, 0.1 mmol) was dissolved in triethylamine/H2O/CH3OH (1:3:7, 5 mL). The resultant mixture was stirred at room temperature overnight. After evaporation of triethylamine and CH3OH in vacuo, the remaining aqueous solution was washed with DCM (15 mL × 2) and then concentrated to give product 6 (23 mg, 97%) as a white powder. Data of 6: 1H NMR (500 MHz, D2O), δ (ppm) 1.24 (t, 3JHH = 7.3 Hz, 9H, N(CH2CH3)3), 2.05−1.88 (m, 2H), 3.16 (q, 3JHH = 7.3 Hz, 6H, N(CH2CH3)3), 3.80−3.34 (m, 6H), 4.13−4.06 (m, 1H). 13C NMR (125 MHz, D2O), δ (ppm) 8.31 (N(CH2CH3)3), 22.22 (d, 1JCP = 115.0 Hz), 46.7 (N(CH2CH3)3), 60.5, 68.4, 72.7 (d, 2JCP = 13.1 Hz), 73.8, 74.1, 78.3. 31P NMR (202 MHz, D2O), δ (ppm) 36.6. HRMS (ESI-TOF) m/z: calcd for [M − H]− C7H12O7P 239.0326; found 239.0329. Time-Dependent Inhibition Assay in the Presence of G16BP. Assays were conducted in 96-well plates in a final volume of 200 μL containing 50 mM Tris buffer pH 7.4, 1.5 mM MgCl2, 0.9 mM NAD+, 1.0 mM DTT, 0.5 U/mL G6PDH, 2 μM G16BP, 150 μM αG1P, 0.6 μM αPGM/PMM, and 0−600 μM inhibitors. Stock solution I contains a mixture of 100 mM Tris buffer pH 7.4, 3 mM MgCl2, 4 μM αG16P, 2.0 mM DTT, 1.2 μM αPGM/PMM, and 0− 600 μM inhibitor (1−8). During preparation of the stock solution I, αPGM/PMM and inhibitors were the last two components added. Upon addition of αPGM/PMM, the resultant mixture was incubated for 2−3 min, followed by addition of the inhibitors. Stock solution II is a mixture of 300 μM αG1P, 1.8 mM NAD+, and 1.0 U/mL G6PDH. Reactions were initiated by addition of 100 μL of the stock

MHz, D2O) δ −0.9 (s). 13C{1H} NMR (125 MHz, D2O) δ 112.6 (t, 1 JC‑1,F = 247.5 Hz, C-1), 94.0 (dt, 2JC‑2,F = 21.9 Hz, 2JC‑2,P = 5 Hz, C2), 74.6 (d, J = 5.0 Hz), 72.7, 72.7, 69.0, 60.3. HRMS (ESI-TOF): m/ z: calcd for [M − H]− C7H10F2O8P, 291.0087; found 291.0094. Following the general procedure II (method b), global deprotection of 16 (53 mg, 0.06 mmol) with 20 wt % Pd(OH)2/C (20 mg) and NaHCO3 (16 mg, 0.19 mmol) provided compound 2 (16 mg, 84%) as a colorless solid. Data for 2: 1H NMR (500 MHz, D2O) δ 6.26 (t, 2JH‑1,F = 53.5 Hz, 1H, H-1), 3.92 (ddd, J = 10.2, 4.0, and 2.3 Hz, 1H), 3.78−3.74 (m, 2H), 3.66 (dd, J = 12.5 and 4.5 Hz, 1H), 3.54 (dd, J = 10.0 and 1.5 Hz, 1H), 3.34 (t, J = 10.0 Hz, 1H). 19F{11H} NMR (470 MHz, D2O) δ −131.5 (d, 2JFa, Fb = 282 Hz), −137.6 (2JFa,Fb = 282 Hz). 19F NMR (470 MHz, D2O) δ −131.5 (dd, 2JFa,Fb = 282 Hz, 2JFa,H = 54 Hz, 1F), −137.6 (dd, 2JFb,Fa = 282 Hz, 2JFb,H = 55 Hz, 1F). 31P{1H} NMR (202 MHz, D2O) δ −0.9 (s). 31P NMR (202 MHz, D2O) δ −0.9 (s). 13 C{1H} NMR (125 MHz, D2O) δ 112.8 (t, 1JC‑1,F = 247 Hz, C-1), 98.1 (dt, 2JC‑2,F = 20.5 Hz, 2JC‑2,P = 8.7 Hz, C-2), 74.2, 73.0, 70.2 (d, 3 JC‑3,F = 5.5 Hz, C-3), 68.6, 59.9. HRMS (ESI-TOF): m/z: calcd for [M − H]− C7H12F2O9P, 309.0192; found 309.0204. 1-Deoxy-1-monofluoro-3,4,5,7-tetra-O-benzyl-α-D-gluco-2-heptulose 2-Dibenzyl Phosphate (17). Following the general procedure I, reaction of 12 (505 mg, 0.88 mmol) with 1H-tetrazole (234 mg, 3.34 mmol), dibenzyl N,N-diethylphosphoramidite (710 μL, 2.02 mmol) in THF (10 mL) and following a second reaction with t BuOOH (2.1 mL, 15 mmol) in THF (10 mL) afforded phosphate 17 (543 mg, 74%) isolated as a colorless oil, through silica gel column chromatography (n-hexane/EtOAc = 6 + 0.5% (v/v) Et3N). Data for 17: 1H NMR (500 MHz, CDCl3) δ 7.40−7.23 (m, 30H), 5.10−4.80 (m, 11H), 4.67−4.64 (m, 2H), 4.54−4.52 (m, 1H), 4.12− 4.10 (m, 1H), 4.05 (t, J = 9.5 Hz, 1H), 3.89−3.83 (m, 2H), 3.75 (dd, J = 11.5 and 3.5 Hz, 1H), 3.56 (dd, J = 11.3 and 1.0 Hz, 1H). 19F NMR (470 MHz, CDCl3) δ −226.4 (t, 2JF,H = 46.7 Hz). 19F{11H} NMR (470 MHz, CDCl3) δ −226.4. 31P{1H} NMR (202 MHz, CDCl3) δ −5.9. 13C{1H} NMR (125 MHz, CDCl3) δ 138.4, 138.1, 138.0, 137.8, 135.7, 135.6, 128.5, 128.4, 128.3, 128.26, 127.9, 127.84, 127.82, 127.8, 127.7, 127.6, 127.5, 104.3 (dd, 2JC‑2,F = 20.6 Hz, 2JC‑2,P = 8.5 Hz, C-2), 82.0, 81.5 (d, 1JC‑1,F = 176.5 Hz, C-1), 78.3, 78.26, 75.5, 75.0, 74.2, 73.3, 69.5 (t, J = 4.5 Hz), 67.8. HRMS (ESI-TOF): m/z: calcd for [M + Na]+ C49H50FNaO9P, 855.3069; found 855.3069. 1-Deoxy-1-monofluoro-α-D-gluco-2-heptulose 2-Phosphate (3). Following the general procedure II (method b), global deprotection of 17 (72 mg, 0.087 mmol) with 20 wt % Pd(OH)2/C (12 mg) and NaHCO3 (22 mg, 0.26 mmol) provided compound 3 (22.5 mg, 90%) as a colorless solid. Data for 3: 1H NMR (500 MHz, D2O) δ 4.46 (dd, 2JH‑1a,F = 46.7 Hz, 3JH‑a,H‑1b = 9.8 Hz, 1H, H-1a), 4.31 (dd, 2JH‑1b,F = 46.7 Hz, 3 JH‑a,H‑1b = 9.8 Hz, 1H, H-1b), 3.81−3.75 (m, 2H), 3.72−3.68 (m, 2H), 3.46 (d, J = 9.7 Hz, 1H), 3.37 (t, J = 9.8 Hz, 1H). 19F NMR (470 MHz, D2O) δ −231.1 (t, 2JF, H = 46.6 Hz). 19F{1H} NMR (470 MHz, D2O) δ −231.1. 31P{1H} NMR (202 MHz, D2O) δ 1.3. 13 C{1H} NMR (125 MHz, D2O) δ 98.7 (dd, 2JC‑2,F = 18.8 Hz, 2JC‑2,P = 7.5 Hz, C-2), 82.2 (d, 1JC‑1,F = 171.3 Hz, C-1), 73.5, 73.1, 71.3, 69.8, 60.7. HRMS (ESI-TOF) m/z: calcd for [M − H]− C7H13FO9P, 291.0287; found 291.0293. 2′-Tetrazole 1-Deoxy-3,4,5,7-tetra-O-benzyl-β/α-D-gluco-2-heptuloside (18). To a solution of 13 (150 mg, 0.27 mmol) and 1Htetrazole (76 mg, 1.09 mmol) in anhydrous THF was added dropwise commercially available 85% dibenzyl N,N-diethylphosphoramidite (145 μL, 0.42 mmol) at room temperature under N2. The clear reaction mixture was stirred at room temperature until the starting material disappeared, as determined by TLC (1−2 h). Upon completion, it was diluted with Et2O (15 mL) and then quenched with cold aqueous saturated NaHCO3 solution (10 mL). The mixture was extracted with Et2O (3 × 5 mL) and washed with H2O (2 × 5 mL) and brine (5 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The resultant crude product was purified on a silica gel column (n-hexane/EtOAc = 8) to H


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The Journal of Organic Chemistry solution I to wells containing 100 μL of the stock solution II at various reaction time points within 20−40 min. The catalytic reaction rates were monitored at A340 over 42 s at 25 °C. The plots of the time course of reaction were made by the remaining enzyme activity (Y axis) against incubation time (X axis). Time-Dependent Inhibition Assay in the Absence of G16BP. Assays were conducted in 96-well plates in a final volume of 200 μL containing 50 mM Tris buffer pH 7.4, 1.5 mM MgCl2, 0.9 mM NAD+, 1.0 mM DTT, 0.5 U/mL G6PDH, 150 μM αG1P, 0.6 μM αPGM/ PMM, and 0−600 μM inhibitors. Stock solution I contains a mixture of 100 mM Tris buffer pH 7.4, 3 mM MgCl2, 2.0 mM DTT, compound 7, and 0.24 μM αPGM/PMM. αPGM/PMM was preincubated with G16BP at 4 °C for 3 h before addition to stock solution I as the last component.24 The excess of G16BP in the preincubation mixture was removed by centrifugation and was exchanged with 50 mM Tris buffer pH 7.5 three times with 10k Omega 24/pk spin filter. Stock solution II contains a mixture of 300 μM αG1P, 1.8 mM NAD+, and 1.0 U/mL G6PDH. Reactions were initiated by addition of 100 μL of the Stock solution I to wells containing 100 μL of the stock solution II at various reaction time points within 180 min. The catalytic reaction rates were monitored at A340 over 42 s at 25 °C. The plots of the time course of reaction were made by the remaining enzyme activity (Y axis) against incubation time (X axis). Kinetic Inhibition Assays of Inhibitors against αPMM/PGM. Assays were conducted in a final volume of 200 μL containing 50 mM Tris buffer pH 7.4, 1.5 mM MgCl2, 0.9 mM NAD+, 1 mM EDTA, 1 mM DTT, 0.5 U/mL G6PDH, 2 μM αG16P, αG1P (10−250 μM), 0.242 μM αPGM/PMM, and 0−2000 μM inhibitor (1−9). Reactions were initiated by addition of αPMM/PGM to the remaining reaction components and the reaction.27 Kinetic rates were monitored at A340 over 5−10 min at 25 °C. Assay of each compound was repeated three times. The linear portions of time progress curves were plotted using GraFit 5.0.4. Methods for Protein Expression and Purification. The gene for XcPGM was commercially synthesized (GenScript) and inserted into the pET-14B vector with an N-terminal tobacco etch virus (TEV) protease cleavage site and His6-affinity tag. The vector was transformed into Escherichia coli BL21(DE3) for recombinant expression. Cultures were grown at 37 °C in Luria−Bertani media, supplemented with 0.1 mg/mL ampicillin to an OD600 of 0.8−1.0. Prior to induction with isopropyl 1-thio-β-D-galactopyranoside (final concentration 0.4 mM), cultures were cooled at 4 °C for at least 30 min. Cells were induced for ∼18 h at 18 °C, and pellets were collected by centrifugation, flash frozen in liquid N2, and stored at −80 °C. For purification, frozen cell pellets were resuspended in buffer A (20 mM sodium phosphate, 0.3 M NaCl, pH 7.8) containing 14.4 mM β-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM tosyllysine chloromethyl ketone, 2 mM CaCl2, 2 mM MgSO4, and 10 μg/mL DNase. Cell lysis was performed with a French press, and the soluble fraction containing XcPGM was obtained through centrifugation. Protamine sulfate was added at 5 mg/g of cell pellet over 15 min, stirred for 30 min, and centrifuged. The supernatant was mixed with Ni2+ affinity resin (His-Select, Sigma), which had been previously equilibrated in buffer A, and incubated for 30 min on a two-way orbital rocker. The mixture was transferred into a gravitypacked column and washed with buffer A containing 5 mM imidazole, pH 7.8. Protein was eluted using buffer A supplemented with 250 mM imidazole, pH 7.8. The purified protein was dialyzed into a solution of 50 mM Tris−HCl, pH 8.0, with 0.3 M NaCl. TEV protease was added to the purified protein at 5% w/w. The mixture was incubated at room temperature for 4 h and left overnight at 4 °C. The mixture was then incubated with pre-equilibrated Ni2+ affinity resin for 30 min. Cleaved XcPGM was retrieved in the flow through and dialyzed into 20 mM Tris−HCl, pH 7.4, with 0.3 M NaCl and concentrated to ∼11 mg/mL. The purified protein was flash-frozen in liquid nitrogen and stored at −80° C. Total yield of protein from 1 L of cell culture was ∼80 mg.

The plasmid encoding the P. aeruginosa αPMM/PGM was transformed into E. coli BL21(DE3) for recombinant expression and overexpressed and purified following reported procedures.28 Crystallization and Complex Formation. Purified XcPGM was initially screened for crystallization via hanging drop vapor diffusion using previously published conditions,25 which did not yield data collection quality crystals. Several commercial screens were then utilized, including Morpheus 1 and Hampton Crystal Screen 1. Optimizations were set up around several hits, and final conditions of 22% poly(ethylene glycol) (PEG) 8000, 0.2 M MgCl2, 0.1 M N-(2hydroxyethyl)piperazine-N′-ethanesulfonic acid, and pH 7.5, were identified and used for all crystals described herein. Crystals typically grew overnight at 18 °C. Despite the different crystallization conditions, the XcPGM crystals reported here were isomorphous with those published previously (Table S1). Inhibitor complexes were obtained by soaking crystals with high concentrations of ligands. Ligand solutions at ∼20 mM were prepared in the crystallization buffer supplemented with cryoprotectant of 25− 30% PEG 3350. Crystals were removed from the drop, dipped quickly into the ligand solution, and immediately flash-cooled and stored in liquid N2.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.9b01305. Additional figures illustrating anomeric configuration assignments, time course of αPMM/PGM-catalyzed αG6P production in the presence of analogues (1−8) with and without G16BP, Michales−Menten and L−B plots of inhibitors (3−9) against αPMM/PGM, Sequence alignment of XcPGM and P. aeruginosa PMM/PGM, X-ray diffraction data collection and refinement, and NMR spectra (PDF) PDB file for 6MLF (PDB) PDB file for 6MLH (PDB) PDB file for 6MLW (PDB) PDB file for 6MNV (PDB) Accession Codes

PDB code for XcPGM with bound analogue 3 is 6MNV PDB code for XcPGM with bound analogue 7 is 6MLH PDB code for XcPGM with bound analogue 8 is 6MLW PDB code for XcPGM with bound analogue 9 is 6MLF Authors will release the atomic coordinates and experimental data upon article publication.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: (+1)573-882-6072. Fax: (+1)573-882-5635 (L.J.B.). *E-mail: [email protected]. Tel: (+1)902-494-7159. Fax: (+1)902-494-1396 (D.L.J.). ORCID

Lesa J. Beamer: 0000-0001-5689-200X David L. Jakeman: 0000-0003-3002-3388 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Canadian Institutes for Health Research, the Natural Sciences and Engineering Research Council of Canada, Canadian Glycomics network (Glyconet), the Canadian Institute of Health and Research (CIHR), and National I


Article


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Science Foundation (NSF) (MCB-1409898) are acknowledged for funding.

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PGM from Pseudomonas aeruginosa

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