
enzyme in an unreactive state which eventually leads to bacterial cell wall disruption and cell death. 6 However, L-AEP is ineffective as an antibacterial agent due to its poor uptake by bacteria. Attempts to enhance the uptake of the agent by bacteria have been reported, for example L-AEP has been linked to an amino acid, to form a dipeptide analogue, to allow active uptake into the bacterial cell via a peptide permease uptake system. Once inside the cell the peptide bond is cleaved by an appropriate amido-hydrolase, releasing the toxic L-AEP moiety. 7 Examples of systems modified in this way were first reported by Roche in the 1970's with L-alanyl-AEP being brought to the market as alafosfalin 8 . However, alafosfalin was found to suffer from a number of major drawbacks including the occurrence of mutants defective in the peptide permease mechanism (around 1 in 10 6 ) and the rapid development of resistant populations. 9 Moreover, peptidase enzymes are widespread within serum and the gastrointestinal tract and hydrolysis of the peptide derivatives occurred before the agent reached the target site, limiting its applications within humans and animals to the treatment of urinary tract infections. 9a In addition, it was observed that uptake of the compound into the bacterial cell was competitively inhibited by other peptides.
To overcome these limitations, alternative and more effective uptake and release systems are required. In this paper we present our studies that have investigated glycoside derivatives of L-AEP for inhibiting the growth of a range of bacteria. We hypothesise that linking L-AEP to a glycoside will produce L-AEP glycosides that are readily taken into the bacterial cell via active carbohydrate transport mechanisms. 10, 11, 12 Once inside the cell, it is hypothesized that glycosidase enzymes will release the active antibacterial agent, via hydrolysis of the anomeric linkage, as illustrated in Scheme 1. Since different bacteria contain different glycosidase enzymes it is envisaged that selective inhibition can be effected by careful selection of the appropriate L-AEP glycoside. 11 Importantly, glycosidases are much more specific and less widespread in the human body, particularly in the gut, than peptidases, and they have already proved invaluable for localized hydrolysis of prodrugs within a number of therapeutic strategies.
13

Scheme 1. Proposed Transport and Delivery Mechanism
The first four targets of interest, specifically the N-linked (1) and O-linked (2) glucosides and galactosides, required the direct attachment of L-AEP to glucose or galactose, as illustrated in Figure 1 .
Figure 1. N-Linked and O-linked glucoside and galactoside targets
The N-linked derivative (1b) was accessed in 2 steps (Scheme 2), firstly by the reaction of L-AEP (3) with 2,3,4,6-tetra-O-acetyl-α-D-galactopyranoside bromide (4) to give the galactoside (5) which was purified by ion exchange column chromatography. This was then deprotected using Zémplen conditions to yield the N-linked derivative (1b) in 58% overall yield. The successful formation of the -anomer was evident from 1 H NMR spectroscopy, where a coupling constant of 10 Hz between H1 and H2 was consistent with an axial-axial arrangement of protons at C-1 and C-2. Synthesis of the glucose analogue (1a) was also attempted but as derivative (1a) proved to be extremely unstable, possibly due to a competing Amadori reaction, its synthesis was not investigated further. (16a) and (16b) in a similar manner to that reported for entry to (12a) and (12b).
Scheme 5: Synthesis of urea glycosides (16a) and (16b) i) triphosgene, DCM, aq.NaHCO3,
For the final series of compounds two different spacer groups were incorporated between the carbohydrate and the AEP moiety. The extended spacer group concept has previously been used in conjunction with peptide carbohydrate conjugates. 20 Access to the derivatives required synthesis of (18a) and ( The synthesized AEP-glycosides were then assessed to determine their ability to inhibit bacterial growth using a panel of Gram-negative bacteria. 22 Growth of organisms was examined in 2 different types of media; a defined version (DM1; formulation based on that of Atherton et al. 23 ) containing amino acids rather than peptides, and a complex medium (Nutrient Broth Number 2, NB2; "Oxoid"; Thermofisher Scientific, Basingstoke, UK).
Inhibitory activity was compared with hydrolysis of the equivalent chromogenic onitrophenyl glycoside (ONP; 1mg/ml in DM1 or NB2) to determine if the presence of the glycosidase hydrolyzing enzyme correlated with toxicity. Compound (1b) was too unstable to reliably bioassay. Compounds (2a) and (2b), (16a) and (16b), and (20a) and (20b) did not demonstrate any inhibitory activity in DM1 or NB2 even when added at a concentration of 1mg/ml. Pleasingly, carbamates (12a) and (12b) 26 did inhibit the growth of a range of bacterial species, with lowest minimum inhibitory concentrations (MICs) being <0.75 mg/l.
The inhibitory activity of glucoside (12a) was less at pH 7.2 when organisms were tested in NB2 than in DM1 for the strains of C.sakazakii, Ps.aeruginosa and S.marcescens (Table 2 ).
This mirrors the toxicity of AEP-peptides that are also reduced by complex media due to reduced uptake of the substrate by competitive inhibition. 24 This would imply that the carbamate glucoside (12a) is also taken into the cell via the peptide permease systems rather than solely via glycoside transport mechanisms. The addition of 0.5% glucose to DM1 at pH 7.2 increased the toxicity of (12a) for E.aerogenes and K. pneumoniae but decreased it for Ps.aeruginosa. For the former, glucose may provide energy for active uptake into the cell. For the latter, glucose may repress uptake and hydrolysis by carbon catabolite repression. Decreasing the pH of DM1 from 7.2 to 6.9 increased the toxicity of (12a) for E. aerogenes and S. marcescens strains. An increased toxicity as pH decreases has also been observed for AEP-dipeptides. 25 With the exception of the strain of Ps.aeruginosa, organisms that hydrolysed o-nitrophenyl-β-D-glucoside (ONP-β-Glu) were more susceptible to (12a) than those that did not, suggesting that release of free AEP by the glycosidase enzyme is required for toxicity, which is in line with our hypothesis.
Growth of 5 organisms was examined in the presence of 2mg/ml of (12b) in DM1 without glucose, and with and without the gratuitous inducer iso-propyl-β-D-galactoside (IPTG) at 1mg/ml (Table 3) . Cronobacter sakazakii and K.pneumoniae grew without IPTG but were inhibited when it was added. Surprisingly, E.aerogenes hydrolysed o-nitrophenyl-β-Dgalactoside and it does, therefore, have mechanisms for uptake and hydrolysis of galactosides, but its growth was unaffected by galactoside (12b). dd, J = 8.0, 1.5 Hz, m, 

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