Inhibition of Escherichia coli glycosyltransferase MurG and Mycobacterium tuberculosis Gal transferase by uridine-linked transition state mimics

Graphical abstract

Abstract

Glycosyltransferase MurG catalyses the transfer of N-acetyl-d-glucosamine to lipid intermediate I on the bacterial peptidoglycan biosynthesis pathway, and is a target for development of new antibacterial agents. A transition state mimic was designed for MurG, containing a functionalised proline, linked through the α-carboxylic acid, via a spacer, to a uridine nucleoside. A set of 15 functionalised prolines were synthesised, using a convergent dipolar cycloaddition reaction, which were coupled via either a glycine, proline, sarcosine, or diester linkage to the 5′-position of uridine. The library of 18 final compounds were tested as inhibitors of Escherichia coli glycosyltransferase MurG. Ten compounds showed inhibition of MurG at 1 mM concentration, the most active with IC₅₀ 400 μM. The library was also tested against Mycobacterium tuberculosis galactosyltransferase GlfT2, and one compound showed effective inhibition at 1 mM concentration.

1. Introduction

Glycosyltransferase enzymes are involved in a wide range of biosynthetic pathways responsible for the formation of polysaccharides, glycoproteins, glycolipids and glycosylated natural products.¹ The glycosyl transfer reaction involves transfer of a monosaccharide from a nucleotide mono/diphosphate donor to an acceptor alcohol, usually with inversion of stereochemistry at the anomeric centre, and the transition state for the reaction is thought to possess oxonium ion character,¹ as shown in Figure 1.

Figure 1.

Glycosyl transfer reaction, showing presumed transition state, transition state mimic, and retrosynthetic disconnection.

Although a number of glycosyltransferases are potentially interesting targets for chemotherapeutic intervention, there are relatively few documented inhibitors for glycosyltransferases,^2–5 compared with many examples of glycosidase enzyme inhibitors.⁶ Known inhibitors of glycosyltransferases in general contain the nucleoside found in the donor substrate: synthetic inhibitors include analogues of UDP-galactose in which the diphosphate linkage has been replaced by methylenediphosphate,² or the ring oxygen replaced by a methylene group,³ or the glycosidic linkage replaced by a hydroxymethylene linkage.⁴ C-Glycosides have also been prepared, containing a linker to a uridine nucleoside, as inhibitors of chitin synthase.⁵

Enzymes of the bacterial peptidoglycan biosynthetic pathway are well-established targets for antibacterial action. A lipid-linked cycle of reactions is responsible for transformation of cytoplasmic precursor UDPMurNAc-l-Ala-γ-d-Glu-m-DAP-d-Ala-d-Ala into peptidoglycan, via a series of lipid-linked reactions,⁷ shown in Figure 2. The first step, catalysed by translocase MraY, is the reaction of UDPMurNAc-pentapeptide with undecaprenyl phosphate to form lipid intermediate I: this reaction is inhibited by several nucleoside-containing natural products, upon which some structure–activity studies have been undertaken.⁸ The second step, catalysed by glycosyltransferase MurG, is the reaction of lipid intermediate I with UDPGlcNAc to form lipid intermediate II. Escherichia coli MurG is an extrinsic membrane protein,⁹ for which a crystal structure has been solved, in complex with UDPMurNAc.¹⁰ The E. coli enzyme has been overexpressed,¹¹ and is able to accept synthetic lipid I analogues containing shortened prenyl chains.^12,13 Using a fluorescent binding assay, several small molecule inhibitors of MurG have been identified by screening of combinatorial libraries.¹⁴ The active compounds are structurally unrelated to the enzyme substrates, and it is not known exactly how they bind to the enzyme.

Figure 2.

Reactions of the lipid-linked cycle of bacterial peptidoglycan biosynthesis catalysed by MraY and MurG.

Examination of the MurG structure reveals that there is are a series of specific interactions with the uracil base, and hydrogen-bonding interactions with the GlcNAc C-3 and C-4 hydroxyl groups, but no electrostatic interactions with the diphosphate bridge (see Fig. 3A).¹⁰ In order to prepare a substrate-based inhibitor for MurG, we have therefore designed a cyclic mimic for the oxonium ion transition state, linked via an uncharged spacer to a uridine nucleoside, shown in Figure 1. Adjacent to the GlcNAc binding site is a large cavity, lined with hydrophobic residues, therefore some members of the inhibitor set included an aromatic substituent able to bind to this site.

Figure 3.

(A) Binding of UDPGlcNAc to E. coli MurG, showing twisted substrate conformation. (B) Example of docked inhibitor structure, showing positioning of proline substituent

In this paper, we report the synthesis and screening of a set of transition state analogues using this design. The functionalised proline transition state mimic is readily assembled using a 1,3-dipolar cycloaddition sequence developed by Grigg and co-workers,¹⁵ which allows the convergent assembly of analogues containing a range of R₁ groups (see Fig. 1). We have included a range of hydrogen-bond acceptor groups in the analogue set (methoxy-aryl substituents, ethane-1,2-diol substituent), in order to interact with the MurG active site. This approach could be used to inhibit other UDP-sugar glycosyltransferases, therefore we also report the screening of the inhibitor set against Mycobacterium tuberculosis galactosyl transferase.^16,17

2. Results

2.1. Docking of transition state analogues into E. coli MurG active site

Several of the transition state analogue structures were energy minimised, and docked into the E. coli MurG active site (PDB file 1NLM) using eHiTS software.¹⁸ Analogues containing a glycine linker were found to be a suitable length to fit into the MurG active site, as shown in Figure 3B. Binding of the substrate UDPGlcNAc requires a twisted conformation at the GlcNAc-phosphate glycosidic linkage, in order to access the GlcNAc binding site, as shown in Figure 3A, and in several cases the docked proline substituent was found not to lie in the GlcNAc binding site. Therefore, conformationally flexible linkers were also included in the inhibitor collection, containing N-methyl glycine, proline amide linkers, or ester linkages, as described below.

2.2. Preparation of substituted prolines via 1,3-dipolar cycloaddition

Grigg and co-workers have previously demonstrated that imines formed between amino acid methyl esters and a range of aryl and alkyl aldehydes can undergo a 1,3-dipolar cycloaddition with N-phenyl maleimide,¹⁵ or with dimethyl maleate or dimethyl fumarate,¹⁹ to give a single major diastereoisomer product in each case. Using the imine formed between benzaldehyde and l-alanine methyl ester, cycloaddition reactions with N-phenyl maleimide were found to proceed with variable yield and product purity using the thermal cyclisation method,¹⁹ or using a one-pot acid-catalysed procedure also reported.¹⁹

A procedure involving 0.1 equiv of silver(I) acetate, carried out using the benzaldehyde imine of l-alanine isopropyl ester,²⁰ was found to proceed in 40–88% yield and with high product purity after chromatography, however, subsequent hydrolysis of the isopropyl ester was found in our hands to be problematic. This latter procedure was found to work well using the benzaldehyde imine of l-alanine benzyl ester, to give cycloadduct 2a with N-phenyl maleimide in 59% yield after flash chromatography. Benzyl ester 2a was debenzylated in quantitative yield by hydrogenation using 10% palladium on carbon to give free acid 3a, as shown in Scheme 1. Dipolar cycloadditions under the same reaction conditions with dimethyl fumarate and dimethyl maleate gave the benzyl esters 4a and 6a in 69% and 73%, respectively, which were debenzylated in 94% and 96% yield, respectively, to give free acids 5a, and 7a, as shown in Scheme 1.

Scheme 1.

Preparation of substituted prolines via 1,3-dipolar cycloaddition. Reagents and conditions: (a) AgOAc, KOH, toluene, rt, 24 h; (b) H₂, Pd/C.

In order to prepare enzyme inhibitors containing hydrogen bond acceptors in the sidechain R, imines of l-alanine benzyl ester were prepared from d-glyceraldehyde acetonide, o-anisaldehyde, 2,3-dimethoxybenzaldehyde, and 3,4-dimethoxybenzaldehyde. Dipolar cycloadditions with l-alanine benzyl ester, using the above conditions, proceeded well in each case to give a single major diastereoisomer, with yields of cycloadducts 2a–e, 4a–e, and 6a–e in most cases in the range 50–75%, the highest yield being 96% for cycloadduct 2b. Debenzylation proceeded smoothly, in most cases in 90–100% yield, to give the free acids 3a–e, 5a–e, and 7a–e, as shown in Scheme 1.

In the case of diethyl fumarate cycloadducts 4a–e, the major diastereomer obtained was the endo-isomer, as found by Casas et al.,²⁰ with smaller amounts of the exo-diastereoisomer (epimer at C-3′′/C-4′′). Casas et al. reported a 94:6 ratio of endo:exo products, using the benzaldehyde imine of alanine iso-propyl ester;²⁰ using the corresponding benzyl ester, we observed a 97:3 ratio of endo:exo products. For compounds with aryl R groups, only small amounts of the exo-diastereoisomer were formed (4a 3%; 4b not detected; 4c 16%; 4d 13%); in the case of 4e, the exo-isomer was formed as 40% of the isolated product. With the exception of 4e, the minor exo-diastereoisomer was not observed in subsequent coupled products, following purification.

2.3. Coupling of glycine linker

Since docking studies had indicated that a glycine linker was a suitable length for binding to MurG, each of the acids 3a–e, 5a–e, and 7a–e was coupled with glycine benzyl ester, using either carbodi-imide EDCI in the presence of hydroxybenzotriazole (HOBt), or uronium coupling agent HATU, in the presence of HOBt, which in most cases proceeded in 60–90% yield. Debenzylation was achieved by hydrogenation using 10% palladium on carbon, in high yield, as illustrated in Scheme 2.

Scheme 2.

Coupling of glycine linker. Reagents and conditions: (a) H₂NCH₂CO₂Bn, HATU, DIPEA, THF, 0 °C; (b) H₂, Pd/C. R = Ph (a), 2-MeOPh (b), 2,3-(MeO)2Ph (c), 3,4-(MeO)₂Ph (d), (isopropylidene)ethane-1,2-diol (e).

In the case of the dimethyl maleate cycloadducts 7a–e, the coupled products were found to contain variable amounts of a bicyclic product, in which the nitrogen atom of the glycine linker had cyclised onto the cis-substituted methyl ester to form a cyclic imide. No cyclisation was observed in compound 10e containing an aliphatic sidechain R, whereas compounds containing aromatic sidechains had cyclised to the extent of 50% for 10a/11a, 67% for 10b/11b, and 100% for 10c/11c and 10d/11d. The bicyclic compounds were not separable by silica chromatography from the uncyclised material, so compounds 10a/11a and 10b/11b were taken forward as mixtures.

2.4. Synthesis of uridine-containing analogues

Attempts to couple acid 8a to the 5′-hydroxyl group of 2′,3′-isopropylidene-uridine, using a range of coupling methods, gave none of the desired ester-linked product after chromatography. In several reactions a new product was observed in the reaction mixture, using thin-layer chromatography, which was not observed after work-up and chromatography, suggesting that an ester linkage in this series of compounds may be unstable. Therefore, a 5′-amino, 5′-deoxyuridine derivative 12 containing TBDMS 2′-O and 3′-O protecting groups was prepared, as shown in Scheme 3. As observed previously,²¹ we found that in the presence of the 2′,3′-isopropylidene protecting group, a 5′-amino substituent was prone to intramolecular cyclisation onto the uracil base, but when re-protected as the 2′,3′-OTDBMS derivative, was quite stable.

Scheme 3.

Preparation of 5′-amino,2′,3′-OTBDMS uridine. Reagents and yields: (a) TsCl, pyr, 49%; (b) NaN₃, DMF, 50 °C, 100%; (c) CF₃COOH, 95%; (d) TBSCl, imidazole, DMF, 81%; (e) H₂, Pd/C, 100%.

Coupling of acid 8a with 12 was found to proceed in 56% yield using HATU, in the presence of HOAt, to give the amide product, which was deprotected to give 10a. The same procedure was used to couple the other analogues (see Scheme 4). After deprotection, the final products were purified by semi-preparative reverse phase HPLC. In the case of 13b, two diastereoisomers were separated by HPLC.

Scheme 4.

Coupling to 5′-amino, 5′-deoxyuridine. Reagents and conditions: (a) 9, HATU, HOAt, DIPEA, THF, 0 °C; (b) CF₃COOH, H₂O/CH₂Cl₂. R = Ph (a), 2-MeOPh (b), 2,3-(MeO)₂Ph (c), 3,4-(MeO)₂Ph (d), ethane,1-2-diol (e).

The cyclisation of the nitrogen atom of the glycine linker to form a bicyclic imide was once again observed in the reaction products after coupling: for the dimethyl maleate adducts 10, all of the reaction products 16a–d contained only a bicyclic imide. Cyclisation was also observed to varying extents for the dimethyl fumarate adducts 9b (partial cyclisation, products 14b and 15b separated by HPLC) and 9e (product 15e wholly cyclised), but not in the case of 9a (product 14a). The slower cyclisation of the dimethyl fumarate adducts can be explained by formation of the less favourable trans-fused bicyclic imide in these cases. Compounds 9e and 15e contained 40% of the C-3″/C-4″ epimer arising from the earlier cycloaddition reaction, whereas compounds 9a and 9b (and hence 14a, 14b, and 15b) contained only a single diastereoisomer.

2.5. Synthesis of compounds containing conformationally flexible linkers

Since the earlier docking studies had indicated that the conformation of the bound UDPGlcNAc substrate contained a twisted conformation at the GlcNAc-phosphate glycosidic linkage, several analogues were synthesised containing conformationally flexible linkers. Coupling of acid 5c with sarcosine (N-methyl glycine) benzyl ester, using the HATU/HOBt coupling used above, gave the unexpected reaction product 17, in which the benzyl ester had undergone an intramolecular reaction with the endocyclic nitrogen atom. This reaction was unexpected, since earlier studies had found that the endocyclic nitrogen of derivatives 2a–e was extremely unreactive towards acylation or alkylation reactions, however, such a cyclisation would be assisted by the presence of an N-methyl amide group. Similar intramolecular reactions were observed using acids 3c, 7c, and 5b,

Therefore, methylation of the endocyclic nitrogen of derivative 2b was undertaken, in order to prevent intramolecular cyclisation. Derivative 2b was methylated in 38% yield using dimethyl sulphate using the method of Prashad et al.,²² however, after debenzylation, coupling of the free acid with sarcosine benzyl ester was unsuccessful. Therefore, compound 2f lacking the α-methyl substituent was prepared by dipolar cycloaddition of the o-anisaldehyde imine of glycine benzyl ester with N-phenyl maleimide in 86% yield, followed by debenzylation. N-Methylation of 2f using the method of Prashad et al.²² proceeded in 35% yield, and subsequent debenzylation proceeded in quantitative yield. Coupling with sarcosine benzyl ester, using HATU/HOBt, gave the desired amide product in 49% yield, which was debenzylated to give 18 (see Scheme 5). Coupling with 5′-amino, 5′-deoxy, 2′,3′-OTBDMS uridine 12 was carried out on a small scale, using the above method, and after deprotection and HPLC purification gave the sarcosine-linked analogue 19.

Scheme 5.

Synthesis of analogues containing N-substituted linkers. Reagents and conditions: (a) Me₂SO₄, NaH, THF/H₂O, 35%; (b) H₂, Pd/C, 100%; (c) H₂NCH₂CO₂Bn, HATU, HOBt, DIPEA, THF, 0 °C, 49%; (d) H₂, Pd/C, 100%; (e) 9, HATU, HOAt, DIPEA, THF, 0 °C; (f) CF₃COOH, H₂O/CH₂Cl₂, 31% over two steps.

Several compounds containing an l-proline linker were also prepared, which would possess a low energy barrier for cis/trans-amide bond interconversion. Acid 3b was coupled to l-proline benzyl ester using HATU/HOBt in 91% yield, and debenzylated in 92% yield. Coupling with uridine derivative 12, followed by deprotection, gave analogue 20a. Using the same procedure, proline-containing analogues 20b–d were prepared. Mixtures of cis- and trans-amide rotamers were observed for 20a–d by NMR spectroscopy (Scheme 6).

Scheme 6.

Analogues containing proline linker. Compound 20a, R₁ 2-MeOPh, R₂/R₃N-phenylsuccinimide; 20b, R₁ 2-MeOPh, R₂ β-CO₂Me, R₃ β-CO₂Me; 20c, R₁ 2-MeOPh, R₂ β-CO₂Me, R₃ α-CO₂Me; 20d, R₁ ethane-1,2-diol, R₂/R₃N-ethylsuccinimide.

Two compounds containing a diester linker were also prepared. Alcohol 21 was prepared by reduction of the isopropyl ester 2g with DIBAL at −78 °C, in 26% yield. The 5′-O-succinyl uridine derivative 22 was prepared by treatment of 2′,3′-isopropylidene-uridine with succinic anhydride, while the 5′-O-malonyl derivative 23 was prepared by HATU/HOBt coupling of benzyl malonate with 2′,3′-isopropylidene-uridine, followed by debenzylation. Coupling of alcohol 21 with acid 22 was achieved using carbodi-imide EDCI, in the presence of DMAP, in 42% yield, and deprotected using aqueous trifluoroacetic acid, to give the succinyl diester 24. Coupling of alcohol 21 with acid 23 using the same methods gave the malonyl diester 25, as shown in Scheme 7.

Scheme 7.

Synthesis of analogues containing ester linkages. Reagents and conditions: (a) DIBAL, THF, −78 °C, 26%; (b) EDCI/DMAP or HATU, THF, 0 °C; (c) CF₃COOH/H₂O, 16–24% over two steps.

2.6. Assay of compounds as inhibitors of E. coli MurG

Recombinant E. coli MurG-C-His₆ was purified from E. coli strain C43 transformed with a pET3a vector containing the murG gene.¹² From 2 l of cell culture, enzyme was purified by Ni²⁺ affinity HisTrap FPLC, yielding 19 mg of homogeneous protein. UDPMurNAc-pentapeptide was prepared from Bacillus subtilis W23, as previously described.²³ MurG was assayed using a coupled radiochemical assay, as described by Zawadzke et al.,²⁴ in which lipid intermediate I is generated in situ by membranes containing translocase MraY, and is then converted in the presence of [³H]-UDPGlcNAc and MurG to give radiolabelled lipid intermediate II, which is extracted into n-butanol and quantitated by scintillation counting. A time-course in the absence of inhibitor revealed that the product release was linear for 15–20 min, therefore, assays were recorded over 15 min. Treatment with 20 μM ramoplanin resulted in >90% inhibition, consistent with literature data.²⁵

Eighteen compounds were tested as inhibitors of MurG, each at 1 mM final concentration. The results are shown in Table 1. Ten of the 18 compounds showed inhibition of MurG, the highest levels of inhibition by compounds 13b (85% inhibition) and 14b (32% inhibition). Selected compounds were then assayed at variable concentrations, and in each case, concentration-dependent inhibition was observed. Compounds 14b, 15e, and 20 each gave IC₅₀ values of >5 mM, while compound 16d showed IC₅₀ 4 mM, and compound 13b gave an IC₅₀ value of 400 μM.

Table 1.

Percentage inhibition of Escherichia coli MurG and Mycobacterium tuberculosis galactosyl transferase by compounds 13–25, at 1 mM concentration

	R1	R2	R3	linker	E. coli MurG	Mt GTase
13a	Ph	N-Phenyl succinimide		Gly	NT	NT
13b	2-MeOPh	N-Phenyl succinimide		Gly	85	0
13c	2,3-(MeO)2Ph	N-Phenyl succinimide		Gly	0	0
13e	Ethane-1,2-diol	N-Phenyl succinimide		Gly	0	0
14a	Ph	-CO2Me	α–CO2Me	Gly	0	NT
14b	2-MeOPh	-CO2Me	α–CO2Me	Gly	32	0
15b	2-MeOPh	-CO2Me	Bicyclic	Gly	22	0
15e	Ethane-1,2-diol	-CO2Me	Bicyclic	Gly	27	NT
16a	Ph	-CO2Me	Bicyclic	Gly	0	80
16b	2-MeOPh	-CO2Me	Bicyclic	Gly	0	0
16c	2,3-(MeO)2Ph	-CO2Me	Bicyclic	Gly	0	NT
16d	3,4-(MeO)2Ph	-CO2Me	Bicyclic	Gly	26	0
19	2-MeOPh	N-Phenyl succinimide		Sar	31	NT
20a	2-MeOPh	N-Phenyl succinimide		Pro	0	0
20b	2-MeOPh	-CO2Me	β–CO2Me	Pro	0	0
20c	2-MeOPh	-CO2Me	α–CO2Me	Pro	22	0
20d	Ethane-1,2-diol	N-Ethyl succinimide		Pro	0	0
24	2-MeOPh	N-Phenyl succinimide		C3	17	0
25	2-MeOPh	N-Phenyl succinimide		C4	15	0

Linkers: glycine (Gly), sarcosine (Sar), proline (Pro), C-3 diester (C3), C-4 diester (C4). NT, not tested. Assays carried out as described in Section 4.

These compounds were tested for antibacterial activity against Gram-negative strains E. coli and Pseudomonas putida, and Gram-positive strains Micrococcus luteus and B. subtilis. Compound 13b showed 50% growth inhibition of M. luteus at 100 μg/ml, but no effects upon the other strains.

2.7. Assay of compounds against M. tuberculosis galactosyltransferase

The arabinogalactan cell wall polymer of M. tuberculosis is biosynthesised through the action of a series of glycosyltransferases,¹⁶ several of which use UDP-sugar substrates, and therefore might be inhibited by this class of compound. We have characterised a UDP-galactose-dependent galactosyltransferase activity GlfT2, which can be assayed using specific neoglycolipid acceptors in the presence of membranes and Galf(β1→6)Galf-O-C₈ resulting in excellent [¹⁴C]Galf incorporation from UDP-[¹⁴C]Galp, following endogenous conversion to UDP-[¹⁴C]Galf and transferase activity (Fig. 4).¹⁷ TLC/autoradiography (Fig. 4) demonstrated the enzymatic conversion of the acceptor to both a trisaccharide product [¹⁴C]Gal to the 5′-OH of Galf(β1→6)Galf-O-C₈ and a second, slower migrating band (Fig. 4) which based on relative migration profiles would be anticipated to be a tetrasaccharide product resulting from further elongation of the above trisaccharide precursor at the 6′OH consistent with the alternating linkage pattern of arabinogalactan. The complete chemical characterisation of the enzymatically synthesised products along with the identity of the galactosyltransferase gene product were previously reported.¹⁷

Figure 4.

(A) TLC/autoradiogram of reactions products produced through the inclusion of acceptor Galf(β1-6)Galf-O-C₈, mycobacterial membranes and UDP[¹⁴C]Galf. Lane 1, no acceptor; lane 2, 0.4 mM acceptor; and lane 3, 0.4 mM acceptor and 1 mM 16a. Assay carried out as described in Section 4.

Assays were carried out in the presence of 14 of the compounds, at 0.5–1 mM concentration, as described in Table 1. Compound 16a showed effective inhibition galactosyltransferase activity in the assay (Fig. 4).

3. Discussion

In this paper, we describe the synthesis and assay of a new family of glycosyltransferase transition state analogues, in which a uridine nucleoside is attached, via a variable length spacer, to a substituted proline analogue, which acts as an oxonium ion mimic. Using the 1,3-dipolar cycloaddition synthetic methodology developed by Grigg and co-workers,^19,20 followed by a range of coupling methods, we have prepared a library of 19 analogues. One unexpected side-reaction was the intramolecular cyclisation of 10 to 11, resulting in a number of bicyclic analogues.

Assay of the compound library as inhibitors of recombinant E. coli glycosyltransferase MurG revealed that 10 compounds showed some level of inhibition at 1 mM concentration, the highest activity being shown by compound 13b (IC₅₀ 400 μM). Most compounds showing inhibition of MurG contained a 2-methoxyphenyl R₁ substituent, whilst no compound containing a phenyl R₁ substituent inhibited MurG, consistent with a favourable hydrogen-bonding interaction between the 2-methoxy substituent and the MurG active site. Although the compounds were able to bind to MurG, the potency of inhibition was not as high as expected for binding of a transition state mimic, therefore it is suspected that the compounds do not adopt the desired conformation in the active site.

The crystal structure of E. coli MurG in complex with UDPGlcNAc shows that there is a significant twist in the conformation of the bound substrate between the diphosphate bridge and the anomeric centre. In order to encourage potential inhibitors to bind in such a conformation, a number of compounds containing conformationally flexible linkers were synthesised. Although compound 31 containing a sarcosine linker did bind to MurG, it bound no more effectively than the glycine analogue 13b, whilst compounds 20a–d containing a proline linker did not bind at all, implying that the MurG active site is unable to accommodate a more bulky substituent at this position. Compounds 24 and 25 containing a diester linker showed weak activity, but no improvement upon 13b.

This set of compounds represents a new set of ligands for MurG, and the first inhibitors related in structure to the natural substrates. This strategy could in principle be used to design inhibitors for other glycosyltransferase enzymes that use a UDP-sugar substrate. Screening of this set of compounds against M. tuberculosis galactosyltransferase GlfT2 revealed that compound 16a (which did not inhibit MurG) acted as an inhibitor for this enzyme. This is the first inhibitor that has been identified for this enzyme, which is a potential target for the development of new anti-TB drugs. Therefore, this strategy could be used to design inhibitors for other glycosyltransferase enzymes of therapeutic importance.

4. Experimental

4.1. Materials

d-Glyceraldehyde acetonide was prepared from d-mannitol using literature procedures,^26,27 via bis-acetonide protection with SnCl₂ and 2,2-dimethoxypropane, followed by oxidative cleavage by NaIO₄. Di-tert-butyldimethylsilyl-5′-amino-5′-deoxyuridine (12) was prepared (see Scheme 3) by sodium azide displacement of 5′-tosyl-2′,3′-isopropylidene uridine, as described by Wang et al.,²⁸ followed by deprotection of the acetonide as described by Winans and Bertozzi,²⁹ followed by TBDMS protection and hydrogenation, as described by Dempcy et al.³⁰ UDPMurNAc-l-Ala-γ-d-Glu-m-DAP-d-Ala-d-Ala was either prepared by the procedure previously described,²³ or was purchased from the BACWAN synthesis facility (University of Warwick).

4.2. General procedure for 1,3-dipolar cycloaddition reaction to form 2a–g, 4a–e, 6a–e, hydrogenation to form 3a–g, 5a–e, 7a–e

Imines 1a–e were formed using the method of Casas et al.²⁰ l-Alanine benzyl ester p-tosylate (0.50 g, 1.48 mmol), aldehyde (1.48 mmol), sodium carbonate (0.16 g, 1.48 mmol) were suspended in water (30 ml) and stirred vigorously at room temperature for 20 h. The mixture was extracted with ethyl acetate (3 × 30 ml), dried (Na₂SO₄) and then concentrated in vacuo to yield the crude imino acyl ester, which was used immediately. The 1,3-dipolar cycloaddition was carried out using the method of Casas et al.²⁰: The imino ester 1a (est 0.26 g, 0.97 mmol), N-phenyl maleimide (0.19 g, 1.07 mmol), and silver(I) acetate (16 mg, 0.09 mmol) were suspended in toluene (15 ml). Potassium hydroxide (5 mg, 0.09 mmol) was then added and the mixture was stirred vigorously at room temperature for 24 h. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate and percolated through a plug of silica gel, eluting with ethyl acetate. The filtrate was concentrated and the resulting material was purified by flash chromatography (eluent 3:2, hexane/ethyl acetate) to yield a white solid, 2a (0.25 g, 0.57 mmol, 59%).

Data for 2a: Melting point 183–184 °C. ¹H NMR (CD₃CN, 400 MHz) δ 7.49–7.09 (15H, m, Ar-H), 5.27 (1H, d, J = 12.0 Hz, CHHPh), 5.18 (1H, d, J = 12.0 Hz, CHHPh), 5.02–4.98 (1H, m, H5′′), 3.86 (1H, dd, J = 9.5, 9.5 Hz, H4′′), 3.65 (1H, d, J = 9.5 Hz, H3′′), 2.84–2.83 (1H, m, NH1′′), 1.69 (3H, s, 2′′-CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ (not all quaternary C’s seen) 138.7, 136.2, 128.8, 128.3, 128.1, 127.9, 127.9, 127.6, 127.3, 126.6, 66.9, 66.5, 61.1, 55.2, 49.4, 22.7; m/z (ESI, +ve ion) 479.0 (MK)⁺, 463.1 (MNa)⁺, 441.2 (MH)⁺; ν_max 3335, 1726, 1702 cm⁻¹; HRMS calcd for C₂₇H₂₄N₂O₄ (M+H⁺) 441.1814; found 441.1818. Data for 4a: colourless oil (0.34 g, 0.82 mmol, 69%). ¹H NMR (CDCl₃, 400 MHz) δ 7.43–7.23 (10H, m, Ar-H), 5.33 (1H, d, J = 12.5 Hz, CHHPh), 5.25 (1H, d, J = 12.5 Hz, CHHPh), 4.83 (1H, d, J = 9.5 Hz, H5′′), 4.02 (1H, d, J = 9.5 Hz, H3′′), 3.88 (1H, dd, J = 9.5, 9.5 Hz, H4′′), 3.61 (3H, s, CO₂CH₃), 3.17 (3H, s, CO₂CH₃), 1.42 (3H, s, 2′′-CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 172.4, 171.9, 171.4, 139.2, 135.5, 128.6, 128.4, 128.3, 127.9, 127.7, 127.3, 67.6, 67.2, 62.9, 53.7, 52.5, 52.1, 51.5, 21.3; m/z (ESI, +ve ion) 434.1 (MNa)⁺, 412.2 (MH)⁺; ν_max 2950, 1728 cm⁻¹; HRMS calcd for C₂₃H₂₅NO₆ (M+H)⁺ 412.1760; found 412.1777. Data for 6a: colourless oil (0.26 g, 0.63 mmol, 73%). ¹H NMR (CDCl₃, 400 MHz) δ 7.38–7.25 (10H, m, Ar-H), 5.26 (1H, d, J = 12.0 Hz, CHHPh), 5.15 (1H, d, J = 12.0 Hz, CHHPh), 4.63 (1H, d, J = 6.5 Hz, H5′′), 3.46–3.42 (4H, m, H4′′ and CO2CH3), 3.28 (1H, d, J = 7.0 Hz, H3′′), 3.25 (3H, s, CO2CH3), 1.71 (3H, s, 2′′-CH3); ¹³C NMR (CDCl3, 100 MHz) δ 174.1, 171.1, 170.7, 137.4, 135.4, 128.8, 128.5, 128.4, 128.3, 127.8, 126.8, 68.4, 67.6, 63.7, 57.7, 53.1, 51.8, 51.3, 28.6; m/z (ESI, +ve ion) 450.1 (MK)⁺, 434.1 (MNa)⁺, 412.2 (MH)⁺; ν_max 3032, 2949, 1729 cm⁻¹; HRMS calcd for C₂₃H₂₅NO₆ (M+H)⁺ 412.1760; found 412.1768. Data for 2b–g, 4b–e, 6b–e in Supplementary data.

4.3. Method for hydrogenation

The benzyl ester (0.20 g, 0.45 mmol) was dissolved in THF (20 ml), and 10 mol % of 10% palladium on carbon was added. The solution was stirred under a hydrogen atmosphere at room temperature for 3 h. The solution was then filtered through Celite and concentrated in vacuo to yield the deprotected acid.

Data for 3a¹⁶: White solid (0.16 g, 0.45 mmol, 100%). ¹H NMR (CD₃OD, 400 MHz) δ 7.46–7.14 (10H, m, Ar-H), 4.97 (1H, d, J = 9.5 Hz, H5′′), 3.84 (1H, dd, J = 7.5, 9.5 Hz, H4′′), 3.56 (1H, d, J = 7.5 Hz, H3′′), 1.68 (3H, s, Me); ¹³C NMR (CD₃OD, 100 MHz), 175.2, 172.1, 172.0, 138.1, 133.5, 129.9, 129.6, 129.5, 129.2, 128.5, 127.9, 66.9, 63.6, 57.2, 51.7, 23.8; m/z (ESI, +ve ion) 395.2 (MNa₂)⁺, 389.0 (MK)⁺, 373.1 (MNa)⁺, 351.1 (MH)⁺, 305.2 (MH−CO₂H)⁺; ν_max 2964, 1707 cm⁻¹. Data for 5a: white solid (0.21 g, 0.65 mmol, 96%). Melting point 198–200 °C (dec). ¹H NMR (CD₃CN, 400 MHz) δ 7.32–7.16 (5H, m, Ar-H), 4.87 (1H, d, J = 9.0 Hz, H5′′), 3.72 (1H, d, J = 7.5 Hz, H3′′), 3.65 (1H, dd, J = 7.5, 9.0 Hz, H4′′), 3.61 (3H, s, CO₂CH₃), 3.08 (3H, s, CO₂CH₃), 1.30 (3H, s, 2′′-CH₃); ¹³C NMR (CD₃CN, 100 MHz) δ (quaternary C’s not seen) 127.9, 127.5, 127.1, 62.3, 52.8, 51.9, 51.6, 50.9, 20.1; m/z (ESI, +ve ion) 344.1 (MNa)⁺, 322.1 (MH)⁺. Data for 7a: white solid (0.18 g, 0.57 mmol, 94%). Melting point 200–202 °C (dec). ¹H NMR (CD₃OD, 400 MHz) δ 7.44–7.39 (5H, m, Ar-H), 5.10 (1H, d, J = 7.5 Hz, H5′′), 3.83 (1H, dd, J = 7.5, 7.5 Hz, H4′′), 3.74 (3H, s, CO₂CH₃), 3.55 (1H, d, J = 7.5 Hz, H3′′), 3.32 (3H, s, CO₂CH₃), 1.81 (3H, s, 2′′-CH₃); ¹³C NMR (CD₃OD, 100 MHz) δ (not all quaternary C’s seen) 171.6, 134.1, 130.2, 130.0, 128.0, 72.4, 62.7, 56.0, 52.9, 52.0, 51.9, 25.1; m/z (ESI, +ve ion) 344.1 (MNa)⁺, 322.1 (MH)⁺, 276.2 (MH−CO₂H)⁺; ν_max 3006, 2951, 1743, 1724, 1655 cm⁻¹; HRMS calcd for C₁₆H₁₉NO₆ (M+H)⁺ 322.1291; found 322.1280. Data for 3b–g, 5b–e, 7b–e given in Supplementary data.

4.4. General procedure for coupling of glycine linker to form 8a–c,e, 9a–e, 10a,b,e, 11a–d

4.4.1. Method for EDCI coupling

The cycloadduct 3a (0.10 g, 0.29 mmol) was dissolved in dry DCM (5 ml) and cooled to 0 °C. EDCI·HCl (60 mg, 0.32 mmol) was then added followed by HOBt (49 mg, 0.32 mmol). The mixture was stirred for 10 min at 0 °C and then a solution of glycine benzyl ester tosylate (0.11 g, 0.32 mmol) and triethylamine (44 μl, 0.32 mmol) in dry DCM was added. The mixture was stirred at 0 °C for 2 h and was then allowed to warm to room temperature and stirred for a further 24 h. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate (10 ml) and water (10 ml). The organic phase was washed with brine (3 × 10 ml), dried (MgSO₄) and concentrated in vacuo to yield the crude product. The product was purified by flash chromatography (1:1, hexane/ethyl acetate) to yield the coupled benzyl ester as a white solid (0.11 g, 0.23 mmol, 79%). The benzyl ester (0.10 g, 0.20 mmol) was dissolved in methanol (10 ml) and 10 mol % of 10% palladium on carbon was added. The solution was stirred under a hydrogen atmosphere at room temperature for 3 h. The solution was then filtered through Celite and concentrated in vacuo to yield 8a as a white solid (80 mg, 0.2 mmol, 100%). Melting point 149–153 °C. ¹H NMR (CD₃OD, 400 MHz) δ 7.29 (2H, d, J = 7.5 Hz, Ar-H), 7.18–7.15 (2H, m, Ar-H), 7.09–7.07 (1H, m, Ar-H), 7.05–7.00 (2H, m, Ar-H), 6.91–6.87 (3H, m, Ar-H), 4.87 (1H, d, J = 6.0 Hz, H5′′), 4.14 (1H, d, J = 17.5 Hz, NHCHH of glycine linker), 4.08 (1H, d, J = 17.5 Hz, NHCHH of glycine linker), 3.64 (1H, dd, J = 6.0, 8.5 Hz, H4′′), 3.52 (1H, d, J = 8.5 Hz, H3′′), 1.59 (CH₃); ¹³C NMR (CD₃OD, 100 MHz) δ 179.1, 176.6, 170.7, 170.7, 138.5, 137.5, 129.5, 129.4, 129.1, 127.9, 125.7, 122.2, 68.8, 68.7, 56.8, 55.2, 40.7, 23.1; m/z (ESI, +ve ion) 430.1 (MNa)⁺, 408.1 (MH)⁺, 392.2 (MH–Me)⁺; v_max 3301, 1701, 1697 cm⁻¹; HRMS (LSIMS, +ve ion) calcd for C₂₂H₂₁N₃O₅ (M+H)⁺ 408.1559; found 408.1564.

4.4.2. Method for HATU coupling

The cycloadduct 3b (0.11 g, 0.29 mmol) was dissolved in dry THF (10 ml) and cooled to 0 °C. HATU (0.14 g, 0.38 mmol) was then added followed by HOAt (52 mg, 0.38 mmol) or HOBt. The mixture was stirred for 10 min at 0 °C and then DIPEA (75 mg, 0.58 mmol) was added, followed by glycine benzyl ester tosylate (0.11 g, 0.32 mmol) and dry THF (10 ml). The mixture was stirred at 0 °C for 2 h and then allowed to warm to room temperature and stirred for a further 24 h. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate (10 ml) and water (10 ml). The organic phase was washed with brine (3 × 10 ml), dried (MgSO₄) and concentrated in vacuo to yield the crude product. The product was purified by flash chromatography (4:1, ethyl acetate/hexane) to yield the coupled benzyl ester as a white solid (0.15 g, 0.29 mmol, 99%). The benzyl ester (0.13 g, 0.25 mmol) was dissolved in methanol (10 ml) and 10 mol % of 10% palladium on carbon was added. The solution was stirred under a hydrogen atmosphere at room temperature for 3 h. The solution was then filtered through Celite and concentrated in vacuo, to give 8b as a white amorphous solid (0.10 g, 0.23 mmol, 93%). ¹H NMR (CD₃OD, 400 MHz) δ 7.68–7.66 (0.6H, d, J = 7.5 Hz, Ar-H), 7.36–6.81 (8.4H, m, Ar-H), 5.15–5.13 (1H, m, H5′′), 4.20 (0.4H, d, J = 17.0 Hz, NHCH₂ of glycine linker), 4.14 (0.4H, d, J = 17.0 Hz, NHCH₂ of glycine linker), 4.03–3.95 (1H, m, NHCH₂ of glycine linker, H4′′), 3.91–3.83 (4.2H, m, OCH₃, NHCH₂ of glycine linker, H4′′), 3.66 (0.4H, d, J = 9.0 Hz, H3′′), 3.43 (0.6H, d, J = 8.0 Hz, H3′′), 1.69 (1.2H, s, 2′′-CH₃), 1.65 (1.8H, s, 2′′-CH₃); ¹³C NMR (CD₃OD, 100 MHz) δ 176.9, 176.0, 159.0, 158.0, 138.3, 133.5, 130.4, 129.9, 129.6, 129.6, 128.0, 127.6, 125.9, 122.5, 121.5, 121.4, 111.3, 68.4, 63.9, 57.9, 56.6, 56.3, 56.2, 56.1, 52.7, 48.5, 42.5, 23.0, 22.8; m/z (ESI, +ve ion) 482.1 (MNa₂)⁺, 460.1 (MNa)⁺, 438.2 (MH)⁺, 395.2 (MH−CO₂H)⁺; v_max 3374, 3303, 2944, 1701, 1685, 1654 cm⁻¹; HRMS (LSIMS, +ve ion) calcd for C₂₃H₂₃N₃O₆ (M+H)⁺ 438.1665; found 438.1680.

Data for 9a: white solid, 56 mg (EDCI coupling 57%, deprotection 71%). Melting point 144–146 °C. ¹H NMR (CD₃OD, 400 MHz) δ 7.30 (2H, d, J = 8.5 Hz, Ar-H), 7.20–7.13 (3H, m, Ar-H), 4.87 (peak hidden behind D₂O, H5′′), 3.88 (2H, s, NHCH₂ of glycine linker), 3.73 (1H, dd, J = 8.5, 8.5 Hz, H4′′), 3.62–3.61 (4H, m, H3′′ and CO₂CH₃), 3.04 (3H, s, CO₂CH₃), 1.30 (3H, s, 2′′-CH3); ¹³C NMR (CD3OD, 100 MHz) δ 177.2, 173.2, 173.2, 173.0, 141.9, 129.1, 128.8, 128.7, 67.8, 63.4, 54.3, 53.8, 52.7, 52.0, 42.2, 21.3; m/z (ESI, +ve ion) 417.1 (MK)⁺, 401.1 (MNa)⁺, 379.1 (MH)⁺; v_max 3345, 3302, 1719, 1658 cm⁻¹; HRMS calcd for C₁₈H₂₂N₂O₇ (M+H)⁺ 379.1505; found 379.1516.

Data for 10a/11a: white solid, 0.16 g (EDCI coupling 32%, deprotection 97%), containing 10a and bicyclic 11a in a 1:1 molar ratio. ¹H NMR (CD₃OD, 400 MHz) δ 7.48 (2H, d, J = 7.5 Hz, Ar-H) 7.22 (3H + 5H, m, Ar-H), 5.01 (1H, d, J = 6.5 Hz, H5′′ 11a), 4.95 (1H, d, J = 7.0 Hz, H5′′ 10a), 4.26 (1H, d, J = 17.5 Hz, NHCHH of glycine linker 11a), 4.18 (1H, d, J = 17.5 Hz, NHCHH of glycine linker 11a), 4.02 (1H, d, J = 18.0 Hz, NHCHH of glycine linker 10a), 3.97 (1H, d, J = 18.0 Hz, NHCHH of glycine linker 10a), 3.75 (1H, dd, J = 6.5, 8.0 Hz, H4′′ 11a), 3.70 (3H, s, CO₂CH₃ 11a), 3.56 (2H, m, H3′′ 11a and H4′′ 10a), 3.46 (1H, d, J = 7.0 Hz, H3′′ 10a), 3.18 (3H, s, CO₂CH₃ 10a), 3.13 (3H, s, CO₂CH₃ 11a), 1.73 (3H, s, 2′′-CH₃ 11a), 1.65 (3H, s, 2′′-CH₃ 11a); ¹³C NMR (CD₃OD, 100 MHz) δ 178.7, 176.4, 172.9, 172.6, 172.5, 170.5, 138.5, 137.4, 129.4 129.4, 128.5, 127.8, 68.0, 67.9, 63.3, 56.8, 55.7, 53.7, 53.2, 52.8, 52.2, 51.9, 50.0, 42.4, 40.7, 27.2, 22.42; m/z (ESI, +ve ion) 401.1 (M(10a)Na)⁺, 379.1 (M(10a)H)⁺, 347.1 (M(11a)H)⁺; v_max 3328, 2989, 1746, 1711 cm⁻¹; HRMS (LSIMS, +ve ion) calcd for C₁₈H₂₂N₂O₇ (M_a+H)⁺ 379.1497; found 379.1505. Data for 8c, 8e, 9b–e, 10b/11b, 10e, 11c–d given in Supplementary data.

4.5. General procedure for uridine coupling and deprotection

The cycloadduct-linker (8b, 70 mg, 0.16 mmol) was dissolved in dry THF (10 ml) and cooled to 0 °C. HATU (80 mg, 0.21 mmol) was then added followed by HOAt (28 mg, 0.21 mmol). The mixture was stirred for 10 min at 0 °C and then DIPEA (40 mg, 0.32 mmol) was added followed by 5′-amino, 5′-deoxy-2′, 3′-O-bis(tert-butyldimethylsilyl) uridine (12, 80 g, 0.18 mmol). The mixture was stirred at 0 °C for 2 h and then allowed to warm to room temperature and stirred for a further 48 h. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate (10 ml) and water (10 ml). The organic phase was washed with brine (3 × 10 ml), dried (MgSO₄) and concentrated in vacuo to yield the crude product. The product was purified by flash chromatography (1:1 hexane/ethyl acetate, 100% ethyl acetate) to yield the coupled product as a pure white solid, (50 mg, 0.06 mmol, 36%) (1:1 ratio of diastereomers a and b). Melting point 174–179 °C. ¹H NMR (CDCl₃, 400 MHz) δ 8.10 (1H, br t, J = 11.4 Hz, NHCH₂ a of glycine linker), 7.99–7.95 (1H, m, NHCH₂ b of glycine linker), 7.46 (1H, br d, J = 8.5 Hz, H6 b), 7.31 (1H, d, J = 9.0 Hz, H6 a), 7.29–6.71 (9H + 9H, m, Ar-H a and b), 5.74–5.49 (2H + 2H, m, H5 a and b, H1′ a and b), 5.11–5.08 (1H, m, H5′′ a), 5.00–4.99 (1H, m, H5′′ b), 4.23–3.04 (10H + 10H, m, H2′ a and b, H3′ a and b, H4′ a and b, CH₂ 5′ a and b, OCH₃ a and b, H4′′ a and b, H3′′ a and b), 1.64 (3H, s, 2′′-CH₃ a), 1.59 (3H, s, 2′′-CH₃ b), 0.83, 0.81 (2 × 9H, s, C(CH₃)₃ a), 0.77, 0.73 (2 × 9H, s, C(CH₃)₃ b), 0.04, 0.03, 0.02, −0.02, −0.04, −0.06, −0.07, −0.11 (8 × 3H, s, Si-CH₃ a and b); ¹³C NMR (CDCl₃, 100 MHz) δ 177.6, 177.5, 175.3, 173.4, 172.7, 168.5, 167.9, 167.5, 157.9, 156.0, 142.4, 141.4, 136.1, 131.3, 129.2, 128.9, 128.6, 128.5, 126.6, 126.4, 126.2, 125.9, 125.1, 124.6, 124.3, 121.0, 120.9, 120.5, 120.1, 110.2, 109.9, 102.7, 102.3, 90.5, 89.8, 86.8, 85.5, 84.7, 73.9, 73.7, 73.6, 72.9, 72.6, 66.4, 62.5, 62.4, 55.6, 55.4, 54.5, 42.2, 41.9, 41.6, 41.5, 25.8, 25.8, 0.97, −4.5, −4.7, −4.8; m/z (ESI, +ve ion) 913.4 (MNa)⁺, 891.4 (MH)⁺, 779.4 (M−Ur)⁺, 647.3 (M−2 × TBS–Me)⁺, 474.2 (MNa−uracil–ribose–2 × TBS), 452.2 (MH⁺−uracil–ribose–2 × TBDMS); v_max 3313, 2931, 2856, 1707 cm⁻¹; HRMS (LSIMS, +ve ion) calcd for C₄₄H₆₂N₆O₁₀Si₂ (M+H)⁺ 891.4144; found 891.4137.

The 5′-cycloadduct-aminoacyl-2′,3′-O-bis(tert-butyldimethylsilyl) uridine compound (80 mg) was dissolved in a mixture of DCM, water and TFA in a 4:2:3 ratio, respectively. The mixture was stirred at room temperature for 24 h and was then diluted with water (10 ml) and DCM (10 ml). The aqueous portion was extracted with DCM (3 × 10 ml) and then concentrated in vacuo to yield the crude product. The product was purified by reverse phase HPLC on a Phenomenex Synergi 4u fusion-RP 80A column (250 × 10.0 mm, 4 μ) using a water/ethanol gradient (0–100% ethanol over 35 min, 3.5 ml/min). Isomer 13b.1 (32 mg, 0.04 mmol, 46%) was eluted at 19.45 min and 13b.2 (29 mg, 0.04 mmol, 42%) was eluted at 21.17 min.

Compound 13b.1: Melting point 197–200 °C.¹H NMR (CD₃OD, 500 MHz) δ 7.61 (1H, d, J = 8.0 Hz, H6), 7.55 (1H, d, J = 7.5 Hz, Ar-H), 7.53–7.36 (4H, m, Ar-H), 7.23–7.17 (2H, m, Ar-H), 7.12–7.06 (2H, m, Ar-H), 5.75 (1H, d, J = 4.5 Hz, H1′), 5.71 (1H, d, J = 8.0 Hz, H5), 5.46 (1H, d, J = 10.0 Hz, H5′′), 4.38–3.97 (7H, m, H2′, H3′, H4′, H4′′, H3′′, NHCH₂ of glycine linker), 3.83 (3H, s, OCH₃), 3.57 (2H, d, J = 5.0 Hz, CH₂ 5′), 1.97 (3H, s, 2′′-CH₃); ¹³C NMR (CD₃OD, 125 MHz) δ 174.3, 173.1, 172.4, 170.0, 169.0, 167.3, 164.7, 157.5, 156.4, 150.8, 141.6, 141.3, 136.5, 131.7, 130.9, 130.7, 128.6, 128.4, 126.2, 126.1, 124.9, 121.2, 120.7, 120.3, 118.3, 110.9, 110.6, 101.7, 101.5, 90.5, 90.1, 82.4, 82.3, 73.4, 70.8, 70.5, 69.4, 67.4, 62.8, 55.1, 55.0, 54.3, 48.2, 48.1, 43.2, 41.4, 40.7, 40.4, 24.9, 19.7; m/z (ESI, +ve ion) 701.2 (MK)⁺, 685.2 (MNa)⁺, 663.2 (MH)⁺; v_max 3345, 1668, 1179, 1130 cm⁻¹; HRMS (micrOTOF, +ve ion) calcd for C₃₂H₃₅N₆O₁₀ (M+H)⁺ 663.2409; found 663.2424.

Compound 13b.2: Melting point 160–164 °C. ¹H NMR (CD₃OD, 500 MHz) δ 7.60 (0.5H, d, J = 8.0 Hz, H6), 7.56–7.51 (1H, m, Ar-H), 7.46 (0.5H, d, J = 8.0 Hz, H6), 7.41–7.36 (1H, m, Ar-H), 7.23–7.12 (2H, m, Ar-H), 7.12–7.05 (4H, m, Ar-H), 6.99–6.93 (1H, m, Ar-H), 5.78–5.77 (1H, m, H5, H1′), 5.75 (0.5H, d, J = 4.5 Hz, H1′), 5.68–5.67 (1.5H, m, H5, H5′′), 4.36 (0.5H, d, J = 16.5 Hz, NHCHH of glycine linker), 4.25 (1.5H, m, NHCHH of glycine linker), 4.20–4.17 (1H, m, H4′′), 4.14 (0.5H, dd, J = 4.5, 5.5 Hz, H2′), 4.11 (0.5H, dd, J = 4.5, 5.5 Hz, H2′), 4.07–4.05 (1H, m, H3′′), 4.02–3.95 (5H, m, H3′, H4′, OCH₃), 3.53 (2H, m, CH₂ 5′), 1.97 (3H, s, 2′′-CH₃); ¹³C NMR (CD₃OD, 125 MHz) δ 173.1, 172.8, 172.4, 169.1, 168.9, 167.3, 167.1, 164.6, 164.4, 156.4, 150.9, 150.8, 141.7, 141.3, 136.5, 136.5, 130.7, 128.4, 128.3, 126.3, 126.2, 125.0, 124.9, 120.9, 120.7, 120.3, 118.3, 110.6, 101.7, 101.7, 90.7, 90.2, 82.4, 82.1, 73.4, 73.3, 70.9, 70.6, 67.4, 67.4, 62.9, 62.8, 55.0, 52.9, 49.4, 49.2, 41.4, 41.1, 40.5, 40.4, 19.7, 19.6; m/z (ESI, +ve ion) 701.2 (MK)⁺, 685.3 (MNa)⁺, 663.2 (MH)⁺; v_max 3309, 1663, 1176, 1139 cm⁻¹; HRMS (mircrOTOF, +ve ion) calcd for C₃₂H₃₅N₆O₁₀ (M+H)⁺ 663.2421; found 663.2409.

Data for 14a: White solid (16 mg, coupling 31%, deprotection 74%) isolated as a 1:1 ratio of diastereoisomers a and b). HPLC retention time 19.77 min. ¹H NMR (D₂O, 400 MHz) δ 7.70 (1H + 1H, d, J = 8.0 Hz, H6 a and b), 7.55–7.53 (3H + 3H, m, Ar-H a and b), 7.47–7.45 (2H + 2H, m, Ar-H a and b), 5.91 (1H + 1H, d, J = 8.0 Hz, H5 a and b), 5.82 (1H + 1H, d, J = 4.0 Hz, H1′ a and b), 5.48 (1H + 1H, d, J = 10.0 Hz, H5′′ a and b), 4.46 (1H + 1H, dd, J = 10.0, 10.0 Hz, H4′′ a and b), 4.39 (1H + 1H, dd, J = 4.0, 4.0 Hz, H2′ a and b), 4.27 (1H + 1H, d, J = 10.0 Hz, H3′′ a and b), 4.21–4.16 (3H + 3H, m, NHCHH of glycine linker a and b, H3′ a and b, H4′ a and b), 4.09 (1H + 1H, d, J = 17.0 Hz, NHCHH of glycine linker a and b), 3.92 (3.5H + 3.5H, s, CO₂CH₃ a and b, CHH 5′ a and b), 3.88–3.87 (0.5H + 0.5H, m, CHH 5′ a and b), 3.56 (1H + 1H, dd, J = 7.5, 14.5 Hz, CHH 5′ a and b), 3.35 (3H + 3H, s, CO₂CH₃ a and b), 1.84 (3H + 3H, s, 2′′-CH₃ a and b); ¹³C NMR (D₂O, 100 MHz) δ 170.7, 170.7, 169.7, 166.2, 166.2, 151.3, 141.9, 132.1, 129.9, 129.2, 127.2, 101.8, 90.6, 81.9, 73.4, 70.4, 68.8, 61.3, 53.7, 52.6, 52.5, 50.0, 43.4, 40.9, 17.9; m/z 642.1 (MK)⁺, 626.2 (MNa)⁺, 604.3 (MH)⁺; v_max 3293, 2980, 1662 cm⁻¹; HRMS calcd for C₂₇H₃₃N₅O₁₁ (M+H)⁺ 604.2255; found 604.2242.

Data for 16a: White solid (62 mg, coupling 33%, deprotection 75%) isolated as 1:1 ratio of diastereoisomers a and b. HPLC retention time 20.21 min. ¹H NMR (CD₃OD, 500 MHz) δ 7.65–7.63 (1H + 1H, 2 × overlapping d, J = 7.5 and 7.5 Hz, H6 c and d), 7.42–7.36 (5H + 5H, m, Ar-H c and d), 5.79 (1H + 1H, d, J = 4.5 Hz, H1′ c and d), 5.77 (1H, d, J = 7.5 Hz, H5 c or d), 5.73 (1H, d, J = 8.0 Hz, H5 c or d), 5.32–5.29 (1H + 1H, 2 × overlapping d, J = 7.5 and 7.5 Hz, H5′′ c and d), 4.33–4.17 (3H + 3H, m, NCH₂ c and d, H2′ c and d), 4.05–3.99 (2H + 2H, m, H3′ c and d, H4′ c and d), 3.94–3.90 (1H + 1H, m, H4′′ c and d), 3.81 (1H, d, J = 9.5 Hz, H3′′ c or d), 3.79 (1H, d, J = 9.5 Hz, H3′′ c or d), 3.64–3.56 (2H + 2H, m, CH₂ 5′ c and d), 3.25 (3H, s, CO₂CH₃ c or d), 3.24 (3H, s, CO₂CH₃ c or d), 1.80 (3H + 3H, s, 2′′-CH₃ c and d); ¹³C NMR (CD₃OD, 125 MHz) δ 176.2, 176.0, 175.4, 175.2, 172.7, 172.6, 168.8, 168.8, 166.2, 152.4, 143.6, 143.3, 134.9, 134.6, 130.2, 130.1, 129.9, 129.8, 128.6, 127.8, 103.2, 103.1, 92.2, 92.1, 83.9, 83.9, 74.7, 74.7, 72.2, 72.1, 68.3, 68.3, 68.0, 54.8, 52.8, 52.7, 49.5, 49.3, 42.5, 42.4, 42.1, 41.9, 21.4, 21.3; m/z (ESI, +ve ion) 610.1 (MK)⁺, 594.2 (MNa)⁺, 572.2 (MH)⁺; v_max 3294, 2956, 1665, 1560 cm⁻¹; HRMS (LSIMS, +ve ion) calcd for C₂₆H₂₉N₅O₁₀ (M+H)⁺ 572.1993; found 572.1997. Data for ¹³C, 13e, 14b, 15e, 16b–d given in Supplementary data.

4.6. Preparation of N-methylated sarcosine-linked compound 19

A solution of 2f (0.43 g, 0.91 mmol) and water (3.40 μl, 0.21 mmol) in dry THF (4 ml) was added dropwise over a period of 20 min to a suspension of sodium hydride (82 mg, 3.40 mmol) in dry THF (6 ml), whilst a temperature of 0 °C was maintained. The mixture was stirred for 10 min and then dimethyl sulfate (0.29 ml, 3.06 mmol) was added dropwise at 0 °C. The mixture was stirred for 3 h and then allowed to warm to room temperature and stirred for a further 21 h. The reaction mixture was quenched by the addition of 30% ammonium hydroxide (5 ml) over a period of 10 min, maintaining a temperature of below 20 °C, and stirring was continued for a period of 1 h. The mixture was diluted with toluene (20 ml) and water (10 ml). The organic phase was separated and washed with water (10 ml) and concentrated in vacuo. The product was purified by flash chromatography (4:1, hexane/ethyl acetate) to yield the N-methylated product as a pure white solid (0.31 g, 0.31 mmol, 38%). Melting point 108–111 °C. ¹H NMR (CDCl₃, 400 MHz) δ 7.46 (1H, dd, J = 1.5, 7.5 Hz, Ar-H), 7.27–7.15 (9H, m, Ar-H), 6.88–6.81 (4H, m, Ar-H), 5.21 (1H, d, J = 13.0 Hz, CHHPh), 5.17 (1H, d, J = 13.0 Hz, CHHPh), 4.46 (1H, d, J = 10.0 Hz, H5′′), 3.82 (3H, s, OCH₃), 3.66 (1H, dd, J = 8.0, 10.0 Hz, H4′′), 3.36 (1H, d, J = 8.0 Hz, H3′′), 2.21 (3H, s, N1′′-CH₃), 1.45 (3H, s, 2′′-CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 174.9, 173.9, 171.7, 158.1, 136.1, 131.7, 129.0, 128.9, 128.5, 128.4, 128.3, 128.1, 127.1, 126.2, 125.4, 120.6, 110.4, 70.1, 67.1, 62.3, 55.6, 54.9, 46.5, 34.4, 15.6; m/z (ESI, +ve ion) 523.1 (MK)⁺, 507.2 (MNa)⁺, 485.2 (MH)⁺; v_max 2958, 2834, 1738, 1712 cm⁻¹; HRMS (LSIMS, +ve ion) calcd for C₂₉H₂₈N₂O₅ (M+H)⁺ 485.2076; found 485.2057. This benzyl ester was deprotected by hydrogenation (100% yield), coupled with glycine benzyl ester (49% yield), and further deprotected by hydrogenation (100% yield), as described above for compounds 8a–e, to give compound 18 (32 mg). ¹H NMR (CD₃OD, 400 MHz) δ 7.61–6.91 (9H, m, Ar-H), 4.69–4.61 (1H, m, H5′′), 4.24–4.06 (1H, m, H2′′), 3.88–3.67 (7H, m, OCH₃, H4′′, H3′′, CH₂ of sarcosine linker), 3.34 (3H, s, NCH₃ of sarcosine linker), 2.18 (3H, s, N1′′-CH₃); ¹³C NMR (CD₃OD, 100 MHz) δ not all quaternary C’s were seen) 177.1, 176.4, 159.3, 133.6, 130.3, 130.1, 129.9, 129.6, 129.6, 129.5, 127.5, 111.4, 69.4, 66.6, 56.0, 51.1, 46.9, 40.3, 36.6; m/z (ESI, +ve ion) 496.1 (MNa₂–H)⁺, 490.1 (MK)⁺, 474.2 (MNa)⁺, 452.2 (MH)⁺. Coupling to uridine derivative 12 and deprotection was carried out as described above, to give compound 19 as a white solid (9 mg, 9%). HPLC retention time 20.43 min. A mixture of cis and trans N-methyl amide rotamers was observed by NMR spectroscopy. ¹H NMR (CD₃OD, 500 MHz) δ7.72–7.30 (7.5H, m, Ar-H, H6), 7.21–7.13 (2H, m, Ar-H), 7.00 (0.5H, m, Ar-H), 5.83–5.73 (1.5H, m, H5, H1′), 5.41–5.38 (0.5H, m, H1′, H5′′), 5.27–5.19 (1H, m, H5′′), 4.66–3.49 (13H, m, H2′, H3′, H4′, CH₂ 5′, H2′′, H5′′, H3′′, OCH₃, N(CH₃)CH₂), 3.40–3.38 (2H, m, NCH₃ of sarcosine linker), 3.15 (1H, s, NCH₃ of sarcosine linker), 2.94–2.93 (2H, m, N1′′-CH₃), 2.27–2.26 (1H, m, N1′′-CH₃); ¹³C NMR (CD₃OD, 125 MHz) δ 171.3, 165.4, 164.7, 157.9, 157.1, 150.8, 132.4, 131.6, 128.8, 128.7, 128.3, 128.0, 127.5, 126.6, 126.1, 125.9, 122.1, 111.5, 110.0, 101.9, 101.6, 101.5, 91.6, 90.8, 90.4, 89.5, 82.9, 82.6, 82.5, 82.1, 81.9, 73.6, 73.3, 72.9, 72.1, 71.6, 71.1, 70.9, 70.8, 70.5, 68.1, 65.1, 55.6, 55.4, 54.6, 51.6, 51.5, 51.3, 45.7, 44.6, 41.2, 40.7, 40.4, 40.3, 38.9, 38.6, 35.6, 35.3, 34.9, 34.6; m/z (ESI, +ve ion) 715.1 (MK)⁺, 699.2 (MNa)⁺, 677.3 (MH)⁺; v_max 3350, 1713, 1661, 1167, 1130 cm⁻¹. HRMS (MicrOTOF, +ve ion) calcd for C₃₃H₃₇N₆O₁₀ (M+H)⁺ 677.2566; found 677.2580.

4.7. Preparation of proline-linked compounds

Cycloadducts 3b, 5b, and 5b were coupled with l-proline benzyl ester, and deprotected by hydrogenation, using the methods described above for compound 8, to give the l-prolyl adducts. These compound were coupled with 5′-amino-uridine derivative 12, and deprotected as described above for compounds 13–16.

Data for 20a: The product was purified by HPLC using a water/methanol gradient and 20a was eluted at 17.89 min and concentrated to yield a white solid (25 mg; l-Pro coupling 91%, deprotection 100%, uridine coupling/deprotection 15%) isolated as a 1:1 ratio of diastereoisomers a and b. ¹H NMR (CD₃OD, 500 MHz) δ 7.66 (1H + 1H, d, J = 8.0 Hz, H6 a and b), 7.50–7.39 (5H + 5H, m, Ar-H a and b), 7.25–7.23 (2H + 2H, m, Ar-H a and b), 7.13–7.07 (2H + 2H, m, Ar-H a and b), 5.76–5.70 (2H + 2H, m, H1′ a and b, H5 a and b), 5.53 (1H + 1H, d, J = 10.0 Hz, H5′′ a and b), 4.44 (1H + 1H, dd, J = 7.5, 9.5 Hz, NCH of proline linker a and b), 4.29–4.16 (3H + 3H, m, H2′ a and b, H3′ a and b, H4′′ a and b), 4.15–3.95 (3H + 3H, m, NCH₂ of proline linker a and b, H4′ a and b), 3.77 (1H + 1H, m, H3′′ a and b), 3.74 (3H + 3H, s, OCH₃ a and b), 3.60–3.50 (2H + 2H, m, CH₂ 5′ a and b), 2.43–2.38 (1H + 1H, m, CH₂ of proline linker a and b), 2.17–2.12 (2H + 2H, m, CH₂ of proline linker a and b), 2.10 (3H + 3H, s, 2′′-CH₃ a and b), 1.94–1.86 (1H + 1H, m, CH₂ of proline linker a and b); ¹³C NMR (CD₃OD, 125 MHz) δ174.4, 174.3, 173.3, 173.1, 168.7, 166.1, 158.8, 152.4, 152.3, 143.6, 143.4, 133.2, 133.0, 132.7, 130.1, 129.9, 127.3, 123.1, 117.9, 112.8, 103.1, 103.0, 92.8, 83.9, 74.6, 72.4, 72.3, 64.7, 62.0, 56.9, 53.6, 51.6, 42.4, 30.7, 26.9, 21.3; m/z (ESI, +ve ion) 725.3 (MNa)⁺, 703.3 (MH)⁺; HRMS (ESI, +ve ion) calcd for C₃₅H₃₈N₆O₁₀ (M+H)⁺ 703.2722; found 703.2741. Data for 20b–d given in Supplementary data.

4.8. Preparation of ester-linked compounds 25 and 26

Cycloadduct isopropyl ester 2g (0.20 g, 0.47 mmol) was dissolved in dry THF (5 ml) and cooled to −78 °C under a nitrogen atmosphere. A 1 M solution of DIBAL-H in hexane (1.00 ml, 0.99 mmol) was added dropwise. The mixture was stirred at −78 °C for 1 h and was then quenched by the cautious addition of methanol (1 ml). The THF was removed in vacuo and the residue was dissolved in diethyl ether (10 ml). A saturated solution of potassium sodium tartrate (10 ml) was then added and the mixture was left to stir overnight until two layers had separated. The organic portion was washed with brine (2 × 10 ml), dried (MgSO₄) and concentrated in vacuo. The product was purified by flash chromatography (3:1, hexane/ethyl acetate) to yield alcohol 22 as a white crystalline solid (44 mg, 0.12 mmol, 26%). Melting point 104–107 °C. ¹H NMR (CDCl₃, 300 MHz) δ 7.78 (1H, d, J = 7.5 Hz, Ar-H), 7.53–7.50 (2H, m, Ar-H), 7.33–7.23 (3H, m, Ar-H), 7.16–7.11 (1H, m, Ar-H), 7.06–7.01 (1H, m, Ar-H), 6.86 (1H, d, J = 8.5 Hz, Ar-H), 5.04 (1H, dd, J = 2.0, 3.5 Hz), 4.90 (1H, d, J = 12.0 Hz), 4.67 (1H, br d, J = 9.5 Hz), 4.49 (1H, br d, J = 12.0 Hz), 3.81 (3H, s, OCH₃), 3.72 (1H, dd, J = 3.5, 5.5 Hz), 3.01 (1H, dd, J = 2.0, 5.5 Hz), 1.92 (2H, br s, NH₂), 1.39 (3H, s, 2′′-CH₃); ¹³C NMR (CDCl₃, 75 MHz) δ 171.9, 156.2, 137.2, 128.9, 128.7, 127.9, 127.0, 125.4, 120.9, 120.5, 109.2, 95.1, 88.1, 65.1, 57.1, 56.5, 55.2, 50.4, 19.5; m/z (ESI, +ve ion) 405.1 (MK)⁺, 389.1(MNa)⁺, 367.1 (MH)⁺, 349.1 (M–OH)⁺; v_max 3343, 2924, 1708, 1045, 753 cm⁻¹. HRMS (LSIMS, +ve ion) calcd for C₂₁H₂₂N₂O₄ (M+H)⁺ 367.1645; found 367.1658.

4.9. Malonyl diester 25

5′-Malonyl 2′,3′-isopropylidene uridine 23 was prepared by EDCI coupling of monobenzyl malonate with 2′,3′-isopropylidene uridine (23% yield), followed by hydrogenation (100% yield). Compound 23 (87 mg, 0.23 mmol) was dissolved in dry THF (5 ml) and cooled to 0 °C. HATU (128 mg, 0.34 mmol) was added and the mixture was stirred at 0 °C for 10 min. DIPEA (60 mg, 0.46 mmol) was added followed by alcohol 22 (100 mg, 0.26 mmol). The mixture was stirred at 0 °C for 4 h and was then allowed to warm to room temperature and stirred for 24 h. The solvent was removed in vacuo and the residue was partitioned between ethyl acetate and water. The organic portion was washed with saturated sodium bicarbonate (10 ml), brine (2 × 10 ml) and then dried (MgSO₄) and concentrated in vacuo. The crude yellow oil (90 mg) was dissolved in DCM (2 ml). Sixty-six percentage of TFA (3 ml) was then added and the mixture was stirred at room temperature for 24 h. DCM (5 ml) and water (5 ml) was then added and the mixture was allowed to separate. The aqueous phase was extracted with DCM (2 × 5 ml) and then concentrated in vacuo. The residue was then purified by HPLC using a water/methanol gradient and 25 was eluted at 24.80 min and concentrated to yield a white solid (29 mg, 0.11 mmol, 48%), isolated as a 1:1 ratio of diastereoisomers a and b. ¹H NMR (CD₃OD, 400 MHz) δ 9.97 (1H + 1H, s, NH3 a and b), 7.70 (1H, d, J = 8.0 Hz, H6 a or b), 7.60 (1H, d, J = 8.0 Hz, H6 a or b), 7.40–7.34 (2H + 2H, m, Ar-H a and b), 7.22–7.12 (5H + 5H, m, Ar-H a and b), 6.99–6.94 (1H + 1H, m, Ar-H a and b), 6.72–6.69 (1H + 1H, m, Ar-H a and b), 5.98 (1H, d, J = 10.5 Hz, H5′′ a or b), 5.96 (1H, d, J = 10.5 Hz, H5′′ a or b), 5.89–5.87 (1H + 1H, m, H1′ a and b), 5.76 (1H, d, J = 8.0 Hz, H5 a or b), 5.66 (1H, d, J = 8.0 Hz, H5 a or b), 4.78 (1H + 1H, d, J = 4.0 Hz), 4.36–4.06 (5H + 5H, m, H2′ a and b, H3′ a and b, H4′ a and b, CH₂ 5′ a and b), 4.02 (3H, s, OCH₃ a or b), 4.00 (3H, s, OCH₃ a or b), 3.70 (1H + 1H, d, J = 7.5 Hz, H3′′ a and b), 3.65 (1H + 1H, ddd, J = 3.0, 7.5, 10.5 Hz, H4′′ a and b), 3.25 (1H + 1H, dd, J = 3.0, 16.0 Hz, CHHOCO a and b), 3.02 (1H + 1H, dd, J = 3.0, 16.0 Hz, CHHOCO a and b), 1.71 (3H, s, 2′′-CH₃ a or b), 1.69 (3H, s, 2′′-CH₃ a or b); ¹³C NMR (CD₃OD, 125 MHz) δ 199.3, 199.2, 171.5, 169.0, 167.7, 166.0, 157.8, 152.3, 142.6, 142.3, 137.4, 131.1, 129.6, 128.5, 128.1, 126.1, 125.8, 124.7, 122.7, 112.3, 111.7, 103.2, 91.2, 91.1, 86.7, 86.7, 83.0, 82.9, 75.1, 75.0, 74.9, 72.1, 71.1, 65.6, 65.5, 59.2, 59.0, 57.3, 57.3, 56.5, 56.3, 48.9, 20.0; m/z (micrOTOF, +ve ion) 701.2 (MNa)⁺; v_max 3392, 1679, 1399 cm⁻¹; HRMS (micrOTOF, +ve ion) calcd for C₃₃H₃₄N₄O₁₂Na, 701.2065; found 701.2069.

4.10. Succinyl diester 26

5′-Succinyl 2′,3′-isopropylidene uridine 24 was prepared by reaction of succinic anhydride with 2′,3′-isopropylidene uridine (40% yield). Compound 24 (35 mg, 0.09 mmol) was dissolved in dry THF (5 ml) and cooled to 0 °C. HATU (45 mg, 0.12 mmol) was then added. The mixture was stirred for 10 min at 0 °C and then DIPEA (23 mg, 0.18 mmol) was added followed by alcohol 19 (40 mg, 0.10 mmol). The mixture was stirred at 0 °C for 2 h and then allowed to warm to room temperature and stirred for a further 48 h. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate (10 ml) and water (10 ml). The organic phase was washed with brine (3 × 10 ml), dried (MgSO₄) and concentrated in vacuo to yield the crude product. The crude product was dissolved in a mixture of DCM, water and TFA in a 4:2:3 ratio, respectively. The mixture was stirred at room temperature for 24 h and was then diluted with water (10 ml) and DCM (10 ml). The aqueous portion was extracted with DCM (3 × 10 ml) and then concentrated in vacuo to yield the crude product. The product was purified by HPLC using a water/methanol gradient. Compound 26 was eluted at 22.49 min and concentrated to yield a white solid (8 mg, 0.01 mmol, 11%) as a 1:1 ratio of diastereoisomers a and b. ¹H NMR (CD₃OD, 500 MHz) δ 7.72–7.66 (3H + 3H, m, Ar-H a and b, H6 a and b), 7.53–7.43 (4H + 4H, m, Ar-H a and b), 7.31 (1H + 1H, dt, J = 1.5, 7.5 Hz, Ar-H a and b), 7.16 (1H + 1H, d, J = 7.5 Hz, Ar-H a and b), 7.12 (1H + 1H, d, J = 8.5 Hz, Ar-H a and b), 6.42 (1H + 1H, s), 6.00 (1H + 1H, dd, J = 6.5, 6.5 Hz), 5.86–5.85 (1H + 1H, m, H1′ a and b), 5.79–5.77 (1H + 1H, m, H5 a and b), 5.53 (1H + 1H, d, J = 6.5 Hz, H5′′ a and b), 4.50–4.43 (1H + 1H, m, CHH 5′ a and b), 4.36–4.31 (1H + 1H, m, CHH 5′ a and b), 4.24 (1H + 1H, dd, J = 4.5, 4.5 Hz, H2′ a and b), 4.18–4.14 (2H + 2H, m, H3′ a and b, H4′ a and b), 3.87–3.80 (2H + 2H, m, H4′′ a and b, H3′′ a and b), 3.77 (3H + 3H, s, OCH₃ a and b), 2.87–2.80 (4H + 4H, m, CH ₂ 1′′′ a and b, CH₂ 2′′′ a and b), 1.86 (3H + 3H, s, 2′′-CH₃ a and b); ¹³C NMR (CD₃OD, 125 MHz) δ 172.2, 170.4, 170.4, 170.1, 164.7, 157.9, 150.8, 141.1, 137.2, 131.5, 130.0, 128.7, 126.4, 122.2, 122.1, 121.5, 116.7, 111.5, 101.5, 98.3, 90.8, 90.4, 90.3, 81.5, 73.6, 73.6, 69.8, 64.7, 63.6, 55.3, 50.5, 49.2, 28.4, 28.1, 17.2; m/z (ESI, +ve ion) 731.1 (MK)⁺, 715.2 (MNa)⁺, 693.2 (MH)⁺; v_max 2972, 1674, 1130 cm⁻¹; HRMS (ESI, +ve ion) calcd for C₃₄H₃₆N₄O₁₂ (M+H)⁺ 693.2402; found 693.2412.

4.11. Purification of recombinant E. coli MurG

Freshly transformed E. coli C43 expressing E. coli murG from a pET3a vector were grown in 6L of LB medium supplemented with 100 μg/ml ampicillin, and induced with IPTG (1 mM) at OD₆₀₀ 0.5. The induced cell culture was grown for another 3 h and then the cells were centrifuged at 10,000 g for 10 min. The cell pellets were resuspended in 50 mM Tris–HCl pH 7.9 (50 ml) containing 2.5 mg/ml lysozyme, PMSF (0.2 mM), leupepsin (0.2 mM) and pepstatin (0.02 mM) were added and the suspension was sonicated. The suspension was centrifuged at 10,000g for 20 min and then the supernatant was transferred to fresh tubes and centrifuged at 50,000g for 1 h. The pellet was resuspended in 50 mM HEPES pH 7.6 (15 ml) containing 2 mM MgCl₂, 0.5% CHAPS, 10% glycerol, 0.5 M NaCl and 5 mM imidazole and stirred at 4 °C for 1 h. The suspension was then centrifuged again at 50,000g for 1 h, and the supernatant was retained. The pellet was suspended again in 15 ml of 50 mM HEPES pH 7.6, 2 mM MgCl₂, 0.5% CHAPS, 10% glycerol, 0.5 M NaCl and 5 mM imidazole and stirred at 4 °C for 1 h. The suspension was then centrifuged at 50,000g for 1 h and the supernatant was pooled with the supernatant from the previous centrifugation. The supernatant was loaded onto a Ni affinity HisTrap fast flow column (5 ml) equilibrated with 50 mM HEPES pH 7.6, 2 mM MgCl₂, 0.5% CHAPS, 10% glycerol, 0.5 M NaCl and 5 mM imidazole (Buffer A). The column was washed with 30 ml of Buffer A. The column was eluted with buffer solutions based upon Buffer A containing 50 mM imidazole (30 ml) and 250 mM imidazole (50 ml). MurG eluted with the 250 mM wash. The material was then loaded onto a Superdex 200 HR 16/60 column (Pharmacia Biotech) at a flow rate of 2 ml/min of 20 mM Tris–HCl pH 7.9, 150 mM NaCl, 50 mM EDTA, 4 mM DTT and 0.5% CHAPS. The protein eluted as a symmetrical peak at 180–250 ml. Fractions containing MurG were pooled and concentrated using Centricon tubes to a final volume of 4.5 ml. The protein concentration was estimated to be 4.15 mg/ml and the protein was seen to be homogeneous by a Coomassie blue-stained SDS–polyacrylamide gel. The purified enzyme was stored at −20 °C and was stable for at least three months.

4.12. Kinetic assays of E. coli MurG

A preparation of solubilised translocase MraY was prepared from Micrococcus flavus membranes (100 μl of 19 mg protein/ml stock) added to 150 μl of solubilisation buffer (50 mM Tris–HCl pH 7.5, 1 mM MgCl₂, 2 mM 2-mercaptoethanol, 0.5% CHAPS). The mixture was shaken at 4 °C for 30 min and was then centrifuged at 13,000 rpm for 30 min. The protein concentration of the supernatant was estimated by Biorad assay to be 1.5 mg/ml and was used directly in the radiochemical assay. Freshly solubilised MraY (12.5 μl) was added directly to undecaprenyl phosphate (0.25 μg). 12.5 μl of buffer (400 mM Tris–HCl pH 7.5, 100 mM MgCl₂) was added, followed by water (9 μl), UDP-MurNAc-pentapeptide solution (5 μl, 1 mM), MurG (1 μl, 110 μg protein/ml) and 5 μl of aqueous inhibitor solution. The mixture was incubated at 35 °C for 15 min, and then 5 μl of UDP-[³H]GlcNAc (10 μM, 500 mCi/mmol) was added, and the mixture was incubated for a further 15 min. The reaction was stopped by the addition of 50 μl of 6 M pyridinium acetate pH 4.6. Hundred microlitres of n-butanol were added, and the layers were mixed and separated by centrifugation. Hundred microlitres of the top n-butanol phase were removed and counted for radioactivity.

4.13. Antibacterial testing

The following procedure was performed using P. putida (ATCC 33015), M. luteus (ATCC 13513), E. coli BL21 and B. subtilis W23. A single colony of the bacterial strain was picked from an LB agar plate and grown in 10 ml of sterile LB medium overnight at 37 °C. 1 ml of this culture was then used to inoculate 50 ml of sterile LB medium at 2%. Hundred microlitres of this 2% culture was pipetted into each well of a 96-well plate. Hundred microlitres of sterile water or sterile aqueous inhibitor solution (200 μg/ml or 1 mg/ml) were then pipetted into each well and the 96-well plates were incubated at 37 °C. Bacterial growth was monitored by measuring the OD at 595 nm at 3, 6 and 24 h.

4.14. Preparation of M. smegmatis membrane and cell wall enzyme fractions

Liquid cultures of Mycobacterium smegmatis mc²155 were grown at 37 °C in Luria Bertoni (LB) broth medium (Difco) supplemented with 0.05% Tween 80, biomass harvested, washed with phosphate buffered saline (PBS) and stored at −20 °C until further use. M. smegmatis cells (10 g wet weight) were washed and re-suspended in 30 ml of buffer A, containing 50 mM MOPS (adjusted to pH 8.0 with KOH), 5 mM β-mercaptoethanol and 10 mM MgCl₂ at 4 °C and subjected to probe sonication (Soniprep 150, MSE Sanyo Gallenkamp, Crawley, Sussex, UK; 1 cm probe) for a total time of 10 min in 60 s pulses and 90 s cooling intervals between pulses. The sonicate centrifuged at 27,000g for 60 min at 4 °C. The resulting mycobacterial cell wall pellets were re-suspended in buffer A. Percoll (Pharmacia, Sweden) was added to yield a 60% suspension and centrifuged at 27,000g for 1 h at 4 °C. The upper, particulate diffuse cell wall enzymatically active (P60) band was collected and washed three times with buffer A and re-suspended in buffer A at a final protein concentration of 10 mg/ml. Membrane fractions were obtained by centrifugation of the 27,000g supernatant at 100,000g for 1 h at 4 °C. The supernatant was carefully removed and the membranes gently re-suspended in buffer A at a protein concentration of 20 mg/ml. Protein concentrations were determined using the BCA Protein Assay Reagent kit (Pierce Europe, Oud-Beijerland, Netherlands).

4.15. Galactosyltransferase assay

The reaction mixtures for assessing [¹⁴C]Gal incorporation consisted of UDP-[U-¹⁴C]Gal (Amersham Pharmacia Biotech, 327 mCi/mmol, 0.25 μCi, 10 μl), Galf(β1→6)Galf-O-C₈ acceptor (0.4 mM), ATP (1 mM, 5 μl), NADH (100 mM, 8 μl), membranes (250 μg, 12.5 μl) and the cell wall fraction (250 μg, 25 μl) in a final reaction volume of 80 μl. The reaction mixtures were then incubated at 37 °C for 1 h. A CHCl₃/CH₃OH (1:1, 533 μl) solution was then added to the incubation tubes and the entire contents centrifuged at 18,000g. The supernatant was recovered and dried under a stream of argon and re-suspended in C₂H₅OH/H₂O (1:1, 1 ml) and loaded onto a pre-equilibrated (C₂H₅OH/H₂O [1:1]) 1 ml Whatmann strong anion exchange (SAX) cartridge which was washed with 3 ml of ethanol. The eluate was dried and the resulting products partitioned between the two phases arising from a mixture of n-butanol (3 ml) and H₂0 (3 ml). The resulting organic phase was recovered following centrifugation at 3,500g and the aqueous phase was again extracted twice with 3 ml of n-butanol saturated water, the pooled extracts were back-washed twice with water saturated with n-butanol (3 ml). The n-butanol–saturated water fraction was dried and re-suspended in 200 μl of n-butanol. The total cpm of radiolabeled material extractable into the n-butanol phase was measured by scintillation counting using 10% of the labelled material and 10 ml of EcoScintA (National Diagnostics, Atlanta). The incorporation of [¹⁴C]Gal was determined by subtracting counts present in control assays (incubation of the reaction components in the absence of the compounds). Another 10% of the labelled material was subjected to thin-layer chromatography (TLC) in CHCl₃/CH₃OH/NH₄OH/H₂O (65:25:0.5:3.6) on aluminium backed Silica Gel 60 F₂₅₄ plates (E. Merck, Darmstadt, Germany). Autoradiograms were obtained by exposing TLCs to X-ray film (Kodak X-Omat) for 4–5 days. Competition based experiments were performed by mixing compounds together at various concentrations (acceptor, 0.4 mM with inhibitors at 0.5–1.0 mM) followed by thin-layer chromatography/autoradiography as described earlier to determine the extent of product formation.

Supplementary data

Supplementary data

Spectroscopic data for synthetic compounds.