ORCID IDs Leah M. Seebald 0000-0001-8890-8985 Pouya Haratipour 0000-0003-0181-8958 Michaela R. Jacobs 0009-0005-6063-5010 Hannah M. Bernstein 0000-0003-0871-0376 Boris A. Kashemirov 0000-0001-7175-9960 Charles E. McKenna 0000-0002-3540-6663 Barbara Imperiali 0000-0002-5749-7869

Nucleoside diphosphate sugar (NDP-sugar) substrates are involved in a variety of essential cellular processes and serve key roles as glycosyl and phosphoglycosyl donor substrates for the biosynthesis of complex glycoconjugates.1 An important strategy for glycoconjugate assembly involves a membrane-associated initiation step catalyzed by a phosphoglycosyl transferase (PGT). PGTs mediate the transfer of a phosphosugar fro m an NDP-sugar substrate to a membrane-anchored polyprenol phosphate (Pren-P) acceptor ( Figure 1). 2 The resulting PGT product, a Pren-PP-sugar, is then further elaborated by stepwise addition of sugars from NDP-sugar donors, catalyzed by the sequential action of a set of glycosyl transferases (GTs). 3 There is emerging interest in PGTs due in part to the surprising mechanistic and structural dichotomy between the two known PGT superfamilies.4 These superfamilies include either a polytopic or a monotopic functional domain,5 and, although the different PGTs catalyze chemically equivalent transformations, thus far, it has been shown that polytopic PGTs (polyPGTs) proceed throug h a ternary complex mechanism,6-8 and monotopic PGTs (monoPGTs) invoke a ping-pong mechanism ( Figure 1AB). 9 Although polyPGTs are observed across all domains of life, extensive bioinformatics analyses have revealed that the monoPGT superfamily is exclusive to prokaryotes.10, 11 The biological importance of PGTs in the first step of glycoconjugate assembly in bacterial pathogens and symbionts highlights the significance of structural and mechanistic studies to provide new insight into the determinants of ligand specificity and the key drivers of catalysis as the foundation for inhibitor and chemical probe discovery.

Progress in our understanding of the structures and mechanisms of PGTs is challenged by the fact that these enzymes are integral membrane proteins. To date, structures of two polyPGTs have been reported. These are MraY (Aquifex aeolicus) 12 the essential PGT in the biosynthesis of the bacterial cell wall peptidoglycan and the human GlcNAc-1-P-transferase or GPT (DPAGT1), which catalyzes the initiating step in the dolichol pathway for N-linked protein glycosylation.8, 13, 14 The structures are intriguing as several show complexes of the polyPGTs with bisubstrate analogues of natural product origin (e.g. tunicamycin and muraymycin D2). 12, 14, 15 The polyPGT superfamily features a conserved Gly-Xaa-Xaa-Asp-Asp (GXXDD) motif with the conserved adjacent asparti c acid residues proposed to be required for substrate orientation and essential for activity in these and other polyPGTs.16,17 Although many of the inhibitor- and substrate-bound complexes lack the Mg2+ co-factor, two structures of the human GPT with tunicamycin14 and the UDP-GlcNAc/Mg2+ complex13 provide insight into polyPGT-small molecule binding.

For the monoPGT superfamily, there is a single X-ray crystal structure of PglC (from Campylobacter concisus). 18 Structural and mechanistic studies support a mechanism involving a catalytic Asp-Glu (DE) dyad, wherein the aspartic acid carboxylate attacks at the ³-phosphat e of the NDP-sugar substrate forming a covalent phosphosugar intermediate.9 In the structure, the essential Mg2+ cofactor is coordinated to the Asp carboxyl group and a phosphate ion.18 In this case, the two-step ping-pong mechanism of the monoPGTs precludes determination of the structure of the Michaelis complex in the presence of the NDP-sugar substrate bound and Mg2+, as the covalent intermediate forms even in the absence of the Pren-P acceptor substrate. At first glance, the GXXDD motif of the polyPGT superfamily appears to bear resemblance to the Asp-Glu (DE) catalytic dyad in the monoPGT family. However, based on the crystal structures of the PGTs, the geometry of these motifs are quite distinct; the conserved adjacent aspartic acid residues of the polyPGTs are presented on a canonical ³-helix, 19 whereas the DE catalytic-dyad of the monoPGT superfamily has side chains with co-facial positioning along the 310 helix that scaffolds the active site (Figure 1, inserts). 18 Despite the biological significance of PGTs, the mechanistic tools available for probing these crucial enzymes in glycoconjugate assembly are limited. Furthermore, many of the existing small molecule probes are derived from complex nucleoside natural products, which are mostly proposed to bind to the polyPGTs as bisubstrate analogues.15, 20 As a complement to the natural product inhibitors and the synthetic analogues that they inspire,21 non-cleavable NDP-sugar CXY-bisphosphonate analogues (NBPs) can potentially provide insight into the s tructure and function of enzymes, such as the PGTs, that act on NDP-sugar substrates. Importantly, NBP-sugars could provide critical information for structure determination,22, 23 as inhibitor scaffolds for identifying structure-activity relationships (SAR), 21 including inhibitor binding modes24 and, in the case of monoPGTs, as leads for agents with antibiotic activity.

With these opportunities in mind, we describe here the modular syntheses of a series of uridine 5'bisphosphonate (CXY-UBP) and uridine 5'-bisphosphon ate-N-acetylglucosamine (GlcNAc-CXY-UBP) analogues (Figure 2) in which the central diphosphate (P-O-P) oxygen i s replaced by a substituted methylene (P-CXY-P), wherein X/Y = H/H, F/F, Cl/Cl, CH3/CH3, (S)-H/F and ( R)-H/F. The ³,³-CXY- and ³,³-bisphosphonate analogue s of deoxynucleotides25-29 have been previously used as probes of nucleic acid polymerase structure and function.30-33 In addition to introduction of a non-hydrolyzable bisphosphonate mimicking the natural diphosphate, the CXY substitution allows variation in such properties as POH/PO3 acidity, P-O/P-C bond lengths, P-O-P/P-C-P bond angles,34 and the effect of steric perturbation adjacent to the CXY group.35 Finally, for BPs where X b Y, as in CHF, the individual diastereomeric analogues will have nearly equivalent POH/PO3 acidity but different orientations of the C-X and C-Y substituents within the binding site. Herein, the PGT substrate analogues are applied in comparative inhibition studies of representative members of the two PGT superfamilies.

We developed two synthetic routes to the nucleotide analogues in this study (Figure 2, 1-11). The routes differ in the initial conjugation partner for the bisphosphonate moiety (uridine or GlcNAc). Scheme 1A, exemplified by the synthesis of ³-GlcNAc-CH 2 (11) (11% overall yield), benefits from the use of unpro tected uridine but requires two demethylation steps due to the use of trimethyl ester bisphosphonate. In contrast, Scheme 1B is more modular, facilitating diversification of the bisphosphonate components, and affords higher overall yields. Scheme 1B was utilized to synthesize five R-CXY-UBPs (R=H, CYX: CCl2 (1), CF 2 (3), ( R/S)-CHF ( 6), C(CH 3) 3 (9), CH 2 (10) (71-90% overall yields) and five GlcNAc-CXY-UBP derivatives ³-GlcNAc-CCl 2 (2), ³-GlcNAc-CF 2 (4), ³-GlcNAc-CF2 (5), ³GlcNAc-(S)-CHF (7), ³-GlcNAc-( R)-CHF (8) (~25% overall yields). All compounds were charact erized by 31P NMR, 1H NMR, 13C NMR, 19F NMR (for CHF and CF2 analogues), COSY, HSQC, and high-resolution mass s pectrometry (HRMS). To assign the CHF stereocenter in ³-GlcNAc- (S)-CHF (7) and ³-GlcNAc-( R)-CHF (8), a modified synthesis incorporating a sterochemically-defined bisphosphonate intermediate derivatized with a chiral auxiliary was deployed (Scheme 2).

Scheme 1A begins with peracetyl-GlcNAc, 12, which is converted to the corresponding oxazoline (13) following known procedures.36 Compound 13, in the presence of trimethyl methylenebis(phosphonate) ( 14) resulted in the bisphosphonate ester 15. The bisphosphonate ester (15) was symmetrically di-demethylated with sodium iod ide to obtain the intermediate 16, which was converted to 17 by passage through DOWEX H+. The Mitsunobu coupling of 17 and uridine (18), which favored attack at the less sterically hind ered ³-P atom provided intermediate 19, which was then demethylated with DIPEA/PhSH to yield 20.37, 38 The O-acetyl groups in 20 were removed with aqueous ammonium hydroxide solution to afford the desired product ³-GlcNAc-CH 2 (11).

The second route (Scheme 1B) begins with activation of 29,39- O-isopropylidene uridine (21) as the 59-tosyl ester 22,39 followed by reaction with the tris(tetrabutylammonium) salt of the selected methylene-bisphosphonates (23a-e) and then ion exchange to afford 24a-e. The 29,39-isopropylidene protecting group was then removed under acidic conditions to obtain five H-CXY-UBPs: H-CCl2 (1), H-CF 2 (3), H-( R/S)-CHF ( 6), H-C(CH 3) 3 (9), H-CH 2 (10). Selected H-CXY-UBP products (1, 3, 6) were reacted with 13 to afford O-acetyl protected GlcNAc-CXY-UBPs (25ac). In summary, the reaction of 1 with 13 at 45 °C, provided 25a³. Under similar conditions, UBP 3 provided 25ba. In addition, when 3 was subjected to modified conditions, including higher temperatures, the glycosidic linkage was epimerized to the ³-anomer at C-1 designated as 25bb.36 Finally, the H-(R/S)-CHF mixture, 6*, was converted to 25c³* by reaction with 13 using the standard 45 °C conditions and individual (R/S)-CHF diastereomers were separated by RP-HPLC. O-acetyl deprotection of the GlcNAc under basic conditions produced ³-GlcNAc-CCl 2 (2), ³-GlcNAc-CF 2 (4), b-GlcNAc-CF2 (5), and the individual stereoisomer ³-GlcNAc-( S)-CHF (7) and ³-GlcNAc-( R)-CHF (8). For 7 and 8, the O-acetylated GlcNAc substituent was crucial to this separation as the precursor H-(R/S)-CHF (6) could not be resolved by RP-HPLC, and thus was st udied as an epimeric mixture 6*. Isolation of each Oacetylated GlcNAc containing CHF-epimer (25c³) was carried out by RP-HPLC at the penultimate step in Scheme 1B, by separating 25c³*. This purification yielded individual, but not yet stereochemically-defined, epimers of S25c³ and R-25c³, primed for the final synthetic step.25 Compound 9 was not converted to the corresponding ³GlcNAc-C(CH3) 2 derivative due to the weak inhibition of the PGTs observed for this UBP precursor. The CHF-bisphosphonate intermediate (23c) is prochiral, thus, the product mono- and di-phos phonate esters with uridine and GlcNAc, such as 7 and 8, were obtained as a 1:1 mixture of diastereomers. These related isomers could be cleanly separated by preparative HPLC. However, as presented later, the diastereomeric ³-GlcN Ac-(S/R)CHF compounds showed different inhibitory activity with the mono- and polyPGTs thus motivating elucidation of the absolute stereochemistry at the CHF chiral center. To accomplish this, we prepared CHF bisphosphonate derivatives modified with an enantiopure chiral auxiliary, (R)-(+)-³-ethylbenzylamine, and a photolabile protecting group (26) that allowed the correlation of chromatographic e lution and NMR properties with absolute stereochemistry as defined by X-ray crystallographic analysis (Scheme 2, shown for a selected isomer). 25 The chiral auxiliary was removed from 26 under acidic conditions producing 27, which was then coupled to the tosylactivated uridine isopropylidine (22) to give 28. Photochemical deprotection at 365 nm yielded 29, followed by uridine deprotection (to 30) and coupling to the GlcNAc oxazoline ( 13) afforded the acetyl protected ³- and ³nucleotide analogues 31³ and 31³. After deacetylation of 31³, chromatographic analysis was conducted as in the initial synthesis of 7 and 8, and the HPLC retention times correlated ³-GlcNAc- (S)-CHF (7) as the (S)-isomer and ³GlcNAc-(R)-CHF (8) as the ( R)-isomer. Detailed NMR analysis was also carried ou t to support the absolute assignment of configuration (S186 and S187)

The bisphosphonate analogues were assessed for inhibition of representative members of each of the two PGT superfamilies. For the monoPGT analysis we screened two PglC orthologs; one from Campylobacter jejuni (PglC (Cj), which is a human food-borne enteropathogen that is a significant cause of gastroenteritis worldwide40 and the other from C. concisus (72% sequence homology with Cj). 41 The PglC (Cc) was included as it is the target of the only successful monoPGT X-ray structure determination,18 however as the orthologs showed similar trends and as the majority of the mechanistic studies had been carried out with PglC (Cj), 9 we pursued detailed analysis of the Cj ortholog. The PglCs from Campylobacter catalyzes the first membrane-committed step in the biosynthesis of N-linked glycoproteins.42 The monoPGT data are compared with that obtained for the polyPGT WecA from Thermotoga maritima (WecA (Tm)) .6 WecA is an integral membrane protein that initiates the O-antigen and enterobacterial common antigen biosynthesis pathways by catalyzing the transfer of GlcNAc-1-phosphate to undecaprenyl phosphate (Und-P) to produce Und-PP-Gl cNAc.17, 43 WecA natively uses UDP-GlcNAc as substrate. Biochemical analysis has shown that the biochemically-preferred substrate for the Campylobacter PglCs is UDPN,N»-diacetylbacillosamine (UDP-diNAcBac) (Fig. S1). 3 However, for these studies, N-acetylglucosamine (GlcNAc) was included in the structures of the UBP analogues rather than diNAcBac to simplify and expedite synthesis. GlcNAc shares structural features with diNAcBac, including similar stereochemistry and the C2-N-acetamido moiety. UDP-GlcNAc is accepted as a substrate by the PglC enzymes included in this analysis although in general the Km values are significantly higher (~20-fold) than th ose for UDP-diNAcBac.

Using the Promega UMP/CMP-GloTM system, PGT steady-state kinetics were studied under initial linear rate conditions with <10% UDP-diNAcBac turnover. The UMP/CMP-GloTM assay quantifies PGT activity through detection of released UMP by enzyme-mediated conversion of the UMP by-product to a luciferase substrate that can be monitored by luminescence. In all cases, a correction to account for any off-target inhibition of the UMP GloTM assay reagent enzymes was included in all analyses.44 Due to off-target interactions with assay components when analyzing the simple (non-glycosylated) UBP an alogues, inhibition studies were also carried out applying an 5 orthogonal radioactivity-based assay which monitors transfer of a radiolabeled-phosphosugar from UDP-[3H]diNAcBac to the unlabeled Und-P acceptor; substrate conversion is quantified by scintillation counting after liquid/liquid extraction.3

The simple UBP analogues including 1 (H-CCl2), 3 (H-CF2), 6 (H-(R/S)-CHF), 9 (H-C(CH3) 3), and 10 (H-CH2), ( Figure 2, R=H), were first screened at 100 µM with the PGT en zymes to identify the bisphosphonate bridging atoms that conferred the best binding (Figure S2). Compound ( 3) H-CF 2 demonstrated modest inhibition towards each PGT, with 33% ± 6% inhibition of PglC (Cj), 14% ± 11% inhibition of PglC ( Cc), and 19% ± 10% inhibition of WecA ( Tm). Inhibition by the other UBP analogues was negligible and in some cases the limitations of the UMP-Glo assay precluded conclusive interpretation at the R=H UBP stage.

To develop the probes, we then investigated modification of 3 with GlcNAc featuring either ³- or ³- anomer linkages (³-GlcNAc-CF 2 (4) and ³-GlcNAc-CF2 (5)). The inclusion of both anomers was valuable for determining whether the GlcNAc-UBPs bound in a substrate-like mode, as the PGTs in this analysis are known to act on the ³anomers of bacterial UDP-sugars. Inhibition studies at 100 µM with PglC (Cj) and WecA ( Tm), comparing UBP HCF2 (3) with ³-GlcNAc-CF 2 (4), and ³-GlcNAc-CF 2 (5) are illustrated in Figure 3A. With PglC (Cj), comparison of HCF2 (3) with ³-GlcNAc-CF 2 (4) showed that incorporation of ³-GlcNAc into the bi sphosphonate analogue enhances binding and increases inhibition from 33% ± 6% to 76% ± 13%. In contrast, ³-GlcNAc-CF 2 (5) shows < 5% inhibition under identical conditions, confirming that the inclusion of a sugar with the native stereochemistry promotes more efficient binding. Inhibition of PglC (Cj) by 3 and 4 is concentration dependent (Fig. 3B), and the IC 50 of 4 for PglC (Cj) was determined to be 32 µM ± 5.3 µM ( Fig. 3C) under assay conditions with competing UDP-diNAcBa c at 20 µM. In contrast, ³-GlcNAc-CF 2 (4) showed minimal inhibition of the polyPGT WecA ( Tm) relative to the parent UBP (3) ( Fig. 3A), consistent with the hypothesis that the UBP-CXY moiety supports a different mode of binding for polyPGTs and highlighting the catalytic divergence between PGT superfamilies.8, 9 The micromolar IC50 of a-GlcNAc-CF2 (4) prompted an investigation into the specific effec t of the fluorine atoms in the asymmetric environment of the PGT active sites. The contribution from each fluorine atom in the CXY moiety was parsed out using stereochemically-defined UBP analogues with the R substituent as ³-GlcNAc, namely: ³-GlcNAc-( S)-CHF (7) and ³-GlcNAc-( R)-CHF (8). Figure 3D shows the comparison of the percent inhibition of ³-GlcNAc-( S)-CHF (7) and ³-GlcNAc-( R)-CHF (8) at 100 µM towards PglC ( Cj) and WecA ( Tm). The results demonstrate a strong preference for the (S)-CHF stereochemistry of ³-GlcNAc-( S)-CHF (7) for PglC (Cj) (52% ± 10% inhibition) vs (R)-CHF of ³-GlcNAc-( R)-CHF (8) (<2% inhibition). In contrast, WecA ( Tm) demonstrates distinct binding preferences for these UBP probes, with ³-Gl cNAc-(R)-CHF (8) showing very slightly preferential inhibition of ³-GlcNAc-( R)-CHF (8) relative to ³-GlcNAc-( S)-CHF ( 7) ( Fig. 3D). Notably, with respect to PglC ( Cj), the percent inhibition of ³-GlcNAc-( S)-CHF (7) (52% ± 10% inhibition) is not additive with total percent inhibition from ³GlcNAc-CF2 (4) (76% ± 13%), suggesting additional physicochemica l contributions, for example effects on the pKas, from the more electronegative CF2 moiety that may drive binding beyond a specific interaction of the C-F moiety with the enzyme.

Due to the improved binding of GlcNAc-modified UBPs, such as 4, relative to the simple UBP analogues, we investigated PGT inhibition with analogues having a methylene bridge, CXY = H/H. The BP analogue H-CH 2 (10), with no R substituent, shows very limited inhibition of PglC (Cj) and WecA ( Tm). However, comparison of H-CH 2 (10) with the ³-GlcNAc modification (11), reinforces that the combination of CXY moiety wi th an a-linked sugar also supports a substrate-like binding mode, in this case, for a polyPGT. ³-GlcNAc-CH 2 (11) is the best inhibitor of WecA (Tm) with 48% ± 7% inhibition, compared to 28% ± 12% i nhibition with PglC (Cj) at 100 µM. The IC50 value of ³-GlcNAc-CH 2 (11) with WecA ( Tm) was found to be 41 µM ± 10 µM (Figure S3).

Overall, the inhibition of PglC and WecA by H-CCl2 (1), ³-GlcNAc-CCl 2 (2), H-CF 2 (3), ³-GlcNAc-CF 2 (4), ³-GlcNAc(S)-CHF (7), ³-GlcNAc-( R)-CHF (8), H-CH 2 (10), and ³-GlcNAc-CH 2 (11) are graphed and compared ( Figure S4). The relative affinities of all the UBP-sugar analogues are summarized in Figure 4.

The synthetic chemistry towards bisphosphonate analogues of NDP-sugars presented here is modular and can be adapted to prepare many other nucleotide-sugar mimics with tunable P-CXY-P bisphosphonate moieties, different nucleobases and various carbohydrates or carbohydrate bioisosteres.

The relative inhibitory properties of the UBP-sugar analogues with the two PGT superfamilies are illustrated graphically in Figure 4. In general, although the simple uridine 5'-bisphosphonates (CXY-UBP) showed limited inhibition of both PGT superfamilies, the initial analysis with these analogues served as a guide to prioritize further elaboration into GlcNAc-modified nucleoside analogues. Elaboration of the CF2-UBP with an a-linked GlcNAc provides the best inhibitor of the monoPGT PglC (Cj) (IC 50 32 µM ± 5.3 µM, Fig. 3C). The improvement in inhibition contrasts the complete loss of activity with the b-linked GlcNAc analog, supporting that substrate mimicry is essential for binding. This observation highlights opportunities for improving monoPGT inhibition, for example through integration of diNAcBac, which is the carbohydrate in the native PglC substrate in place of GlcNAc. Although UDP-GlcNAc is a far poorer substrate for PglC than UDP-diNAcBac with a KM value ~20 fold higher, the manipulation of diNAcBac is synthetically more challenging and there are no commercial sources for this rare sugar or any of its derivatives. Alternatively, the modular synthetic approach readily enables synthesis of modified UBP conjugates that include moieties that substitute for the sugar. For example, in glycan-binding proteins recognition of sugar substrates is often promoted by electrostatic interactions wherein an electropositive C2H in carbohydrates interacts with electron-rich aromatic amino acids yielding CH2à interactions.45 Thus a carbohydrate substitute could mimic these CH-à interactions while also affording simplified synthetic routes. When occupying the sugar binding site, aryl groups can act as sugar bioisosteres by participating in similarly favorable à - à stacking interactions.

As illustrated in Figure 4, the mono- and polyPGTs show distinct preferences for different UBP-sugar analogues and although GlcNAc-CF2-UBP (4) is the preferred inhibitor for the monoPGT PglC ( Cj), GlcNAc-CH 2-UBP (11) is the preferred inhibitor for the polyPGT WecA (Tm). Both GlcNAc-CCl 2-UBP (2) and GlcNAc(C(CH 3) 2)-UBP ( 9) show very weak inhibition of the PGTs, however, we do not explicitly discuss these analogues in the trends due to the additional steric burden imposed at the bridging site of the bisphosphonate. Analysis of the trends for WecA (11>8>7>4), show that there is a correlation between the inc reasing basicity of the bisphosphonate moiety and the strength of inhibitor binding. This trend becomes evident when referencing the pKa values of the CXY-BP moieties: CF2-BP has the lowest pKa values, corresponding to the lowest relative basicity in its anionic forms, while CH2-BP and C(CH3) 2-BP have the highest pKa values and therefore the highest relative basicity. The IC50 of 11 for WecA (Tm) is 41 µM ± 10 µM (Fig. S3). The preferential binding to 11 suggests that the primary factor responsible for GlcNAc-UBP analogue binding is the structural Mg2+ cofactor, which may principally be responsible for the stability of the ternary complex by playing a role in substrate binding and orientation. Specifically, increasing the basicity of the bisphosphonate moiety will strengthen the ionic interactions between the phosphonate oxygen anions and the Mg2+. The P2C bonds of bisphosphonates are ca. 0.2 A longer than the P3O bonds in pyrophosphate, however this is compensated by the P³2C2P´ angles, that are more acute than the P³2O2P´ angle of phosphates, resulting in similar distances between P³ and P´ atoms in pyrophosphate and bisphosphonates. This conclusion, based on a study of dNTP ³,³-bisph osphonates,34 should be valid for P,P9 diesters (data not shown). Thus, these structural differences should n ot significantly alter the Mg2+ coordination geometry.

Analysis of the structures of GPT (the human polyPGTs), which also features the conserved DD motif ( Fig. S190) and uses UDP-GlcNAc as substrate, either in the presence of a UDP-GlcNAc-Mg2+ complex,13 or tunicamycin, a potent bisubstrate analog inhibitor14 (Fig. S191A-D) provide some insight. The structures show the UDP -GlcNAcMg2+ and tunicamycin coinciding in the bound state, but do not show the Mg2+ bound to the conserved aspartates to support a role in substrate orientation, but not a direct role in catalysis. This rationale is both consistent with its known function17 and our previously reported results where a similar correlation was observed between the basicity of bisphosphonate analogues and the binding affinity to Pol ³.46 In contrast to the polyPGT, PglC (Cj) inhibition follows a different trend ( 4>7>11>8), with the highest inhibition (IC50 32 µM ± 5.3 µM, Fig. 3C) observed with the least basic analogue ( 4). This alludes to the possibility that the role of Mg2+ in the monoPGT system is most important for catalysis in the ping-pong mechanism and plays a less significant role in substrate orientation. In proteins, such as Pol ³, catalytic Mg2+ is known to exhibit a lower affinity to the protein in the active site compared to structural Mg2+.46 This suggests that the substrate orientations in complex with PglC are nonsuperimposable and ground state conformations of this complex are mainly defined by protein-ligand interactions.

The binding of 4 to PglC supports that the CF2 group is the best replacement of the oxygen atom in the parent UDP-sugar. However, this does not give information on the contribution of each C-F bond and whether there may be specific interactions with the target enzyme, or, if the effect is principally one of modulating the electronic and structural properties of the analog relative to the parent UDP-GlcNAc. To address this issue, we investigated the individual effects of the diastereotopic C-F bonds in 7 and 8 by assessing the inhibitory activity with stereochemically-defined a-GlcNAc-CHF-UBPs. The significant difference between (S)-CHF ( 7) and ( R)-CHF ( 8) (7>>8) is noteworthy. We considered two hypotheses. Firs t, that the C-F in 7 may form a specific interaction upon binding to PglC. In this case, a possible amino acid candidate is PglC residue Lys59 ( Fig. 1B). Lys59 is a catalyticallyessential residue, which in the current mechanistic proposal, based on the structure (PDB: 5WL7), woul d be close to the diphosphate oxygen to protonate the UMP leaving group upon formation of the covalent intermediate.9, 18 Alternatively, the (S)-CHF ( 7) and ( R)-CHF ( 8) isomers might preferentially adopt different solu tion state conformations. The solution-state conformations of UDP-GlcNAc, (S)-CHF ( 7), and ( R)-CHF ( 8) in water in the presence of MgCl2 were assessed by nuclear Overhauser effect (NOE) an alysis (SI NMR summary and Figs. S188 and S189). Under these conditions, all three compounds have uracil protons in an observable NOE range (< 5 Å) to many of the GlcNAc protons, indicating a collapsed structure. The key difference between these three structures arises with the interproton distances between uracil and the N-acetyl protons on the GlcNAc. In UDPGlcNAc and (S)-CHF ( 7) the N-acetyl protons are within range to observe clear NOEs with the uracil C6 proton. The (R)-CHF ( 8) also has the acetyl group protons within range, a lbeit with a much weaker signal. This difference is significant enough to conclude that 7 and 8 have different favored conformations in solution, and that 7 behaves similarly to UDP-GlcNAc in solution. A possible outcome of conformational differences between 7 and 8 may be that Mg2+-bound 7, like UDP-GlcNAc, may be preorganized into a favorable state for binding to the monoPGT leading to improved affinity relative to 8. In contrast to the results with PglC, there is no significant difference between the inhibitory activity of 7 and 8 with WecA. Future structural and computational studies will be applied to further investigate the observed variations in binding to the PGT superfamilies.

Nucleoside diphosphate sugar (NDP-sugar) substrates feature in innumerable cellular processes and there is considerable interest in the development and study of non-hydrolyzable analogues for applications in structural biology, mechanistic enzymology, and as leads for inhibitor development. In this study we have focused on phosphoglycosyl transferases, which catalyze the first membrane-committed step in many glycoconjugate assembly pathways. PGTs play pivotal roles in initiating production of diverse glycans that are essential for bacterial survival and pathogen-host interactions. In bacteria, PGTs and their NDP-sugar substrates are far more varied than in eukaryotes, reflecting the greater diversity of glycoconjugates in unicellular microorganisms and the potential for selective antibiotic and probe development.

We have presented the synthesis and biochemical analysis of a panel of uridine 5'-bisphosphonate (CXY-UBP) and uridine 5'-bisphosphonate-N-acetylglucosamine (GlcNAc-CXY-UBP) analogues of th e UDP-sugar substrates of phosphoglycosyl transferases. The analogues feature a central substituted methylene group that can be tuned to modify the steric and electronic properties of the bridging bisphosphonate. The two PGT superfamilies are differentiated by mechanism and involve either - the direct attack by UndP on the b-phosphate of a UDP-sugar for the polyPGTs, or attack by the conserved aspartic acid residue on the UDP-sugar for the monoPGTs. The polyPGT and monoPGT superfamilies are also distinguished by highly divergent 3D structures. Together, the studies presented here underscore the mechanistic dichotomy of the PGT superfamilies and provide a pathway towards selective inhibition of either the prokaryotic monoPGT superfamily or the polyPGT superfamily found across domains of life. As the monoPGTs are exclusively prokaryotic,11 these enzymes represent potential new targets for the development of antibiotic and antivirulence agents due to the pivotal roles played by the complex glycoconjugates that are biosynthesized in pathways that are initiated by monoPGTs in bacteria.

Materials and methods including enzyme expression and purification, biochemical assays, detailed synthetic procedures and full structural characterization of all new compounds, summary of NOE studies, and sequence and structure comparison of polyPGTs.

* imper@mit.edu, mckenna@usc.edu

§Cancer Biology & Molecular Medicine Department, Beckman Research Institute of City of Hope National Medical Center, CA 91010, United States ³Haverford College, Department of Chemistry, Haverford, PA 19041, United States

!These authors contributed equally.

This work was supported the NIH GM039334 (B.I.), th e USC Dornsife Chemical Biology Training Program (P.H.) and the USC Bridge Institute (C.McK.).

The authors declare no competing financial interest.

The authors thank Christine Arbour (MIT) and Inah K ang (USC) for assistance with reviewing and editing the manuscript and Walt Massefski and Bruce Adams of the MIT Department of Chemistry Instrumentation Facility (DCIF) for assistance with NOE studies.

PGT, phosphoglycosyl transferase; monoPGT, monotopic PGT; polyPGT, polytopic PGT; NDP, nucleoside diphosphate; UDP, uridine diphosphate; BP, bisphosphonate; UBP, Uridine bisphosphonate; Cj, Campylobacter jejuni; Cc, Campylobacter concisus; Tm, Thermotoga maritima. GlcNAc, N-acetyl glucosamine; Und-P, undecaprenyl phosphate; diNAcBac diacetylbacillosamine; Pgl, protein glycosylation. MraY active site. PolyPGTs invoke a mechanism involving a ternary complex intermediate and feature a conserved Gly-Xaa-Xaa-Asp-Asp motif. B. Ribbon diagram of monoPGT PglC from Campylobacter concisus (PDB 5W7L), insert shows the close-up view of the PglC active site Asp-Glu catalytic dyad. MonoPGTs follow a ping-pong mechanism, wherein a covalent phosphoglycosyl-Asp intermediate is formed. acetylglucosamine analogues (GlcNAc-CXY-UBP) synthe sized and analyzed in this study. The diphosphate bridging oxygen of the UDP is replaced by a panel of substituted methylene groups (CXY; X/Y = F/F, Cl/Cl, ( R)-H/F, ( S)-F/H, H/H, CH3/CH3). Uridine is denoted as a circled <U=. The stereochemical assignments of 7 and 8 are (S)-CHF and (R)-CHF, respectively.

NO2 d

O O overnight (31% yield of 8³ and 8³). Note: The designation of chirality at the -CHF- center changes through the scheme due to the change in Cahn-Ingold-Prelog priority of the phosphate ester substituents. (42) Nothaft, H.; Scott, N. E.; Vinogradov, E.; Liu, X.; Hu, R.; Beadle, B.; Fodor, C.; Miller, W. G.; Li, J.; Cordwell, S. J.; et al. Diversity in the protein N-glycosylation pathways within the Campylobacter genus. Mol Cell Proteomics 2012, 11, 1203-1219. (43) Chung, B. C.; Mashalidis, E. H.; Tanino, T.; Kim, M.; Matsuda, A.; Hong, J.; Ichikawa, S.; Lee, S.-Y. Structural insights into inhibition of lipid I production in bacterial cell wall synthesis. Nature 2016, 533, 557-560. (44) Madec, A. G. E.; Schocker, N. S.; Sanchini, S.; Myratgeldiyev, G.; Das, D.; Imperiali, B. Facile solid-phase synthesis and assessment of nucleoside analogs as inhibitors of bacterial UDP-sugar processing enzymes. ACS Chem Biol 2018, 13, 2542-2550. (45) Kiessling, L. L.; Diehl, R. C. CH-pi Interacti ons in Glycan Recognition. ACS Chem Biol 2021, 16, 1884-1893. (46) Chamberlain, B. T.; Batra, V. K.; Beard, W. A. ; Kadina, A. P.; Shock, D. D.; Kashemirov, B. A.; McKenna, C. E.; Goodman, M. F.; Wilson, S. H. Stereospecific formation of a ternary complex of (S)-alpha,beta-fluorome thylenedATP with DNA pol beta. Chembiochem 2012, 13, 528-530.