Synthesis and biological analysis of novel glycoside derivatives of L-AEP, as targeted antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2016.05.052

To develop targeted methods for treating bacterial infections, the feasibility of using glycoside derivatives of the antibacterial compound L-R-aminoethylphosphonic acid (L-AEP) has been investigated. These derivatives are hypothesized to be taken up by b

Full text of DOI:10.1016/j.bmcl.2016.05.052

Accepted Manuscript
Synthesis and biological analysis of novel glycoside derivatives of L-AEP, as
targeted antibacterial agents
Richard Bovill, Philip G. Evans, Gemma L. Howse, Helen M.I. Osborn
PII:
S0960-894X(16)30538-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.05.052
BMCL 23911
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
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19 April 2016
16 May 2016
18 May 2016
Please cite this article as: Bovill, R., Evans, P.G., Howse, G.L., Osborn, H.M.I., Synthesis and biological analysis
of novel glycoside derivatives of L-AEP, as targeted antibacterial agents, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.05.052
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Synthesis and biological analysis of novel glycoside derivatives of L-AEP, as targeted
antibacterial agents
Richard Bovilla, Philip G. Evansb, Gemma L. Howseb and Helen M.I. Osbornb*
aThermofisher Scientific., Wade Road, Basingstoke, Hampshire. RG24 8PW, UK
bReading School of Pharmacy, University of Reading, Whiteknights, Reading. RG6 6AD, UK
Abstract To develop targeted methods for treating bacterial infections, the feasibility of using
glycoside derivatives of the antibacterial compound L-R-aminoethylphosphonic acid (L-AEP)
has been investigated. These derivatives are hypothesized to be taken up by bacterial cells via
carbohydrate uptake mechanisms, and then hydrolysed in situ by bacterial borne glycosidase
enzymes, to selectively afford L-AEP. Therefore the synthesis and analysis of ten glycoside
derivatives of L-AEP, for selective targeting of specific bacteria, is reported. The ability of
these derivatives to inhibit the growth of a panel of Gram-negative bacteria in two different
media is discussed. -Glycosides (12a) and (12b) that contained L-AEP linked to glucose or
galactose via a carbamate linkage inhibited growth of a range of organisms with the best
MICs being <0.75 mg/ml; for most species the inhibition was closely related to the hydrolysis
of the equivalent chromogenic glycosides. This suggests that for (12a) and (12b), release of
L-AEP was indeed dependent upon the presence of the respective glycosidase enzyme.
Graphical abstract

With the emergence of ‘super bugs’ such as MRSA, and the ever-increasing resistance of
bacteria to many of the available antibacterial treatments, it is evident that new initiatives are
needed to find alternative methods to treat bacterial infections.1 One such strategy is to target
processes that are unique to bacteria as a way of developing selective antibacterial agents.2 In
this regard, the bacterial enzyme alanine racemase which catalyses the interconversion of L-
alanine to D-alanine is a useful target enzyme, since it is ubiquitous within bacteria, but is not
present within humans.3 Inhibitors of L-alanine racemase are well documented,4 for example
L-R-aminoethylphosphonic acid (L-AEP)5 forms a stable external aldimine which locks the
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 alafosfalin8. 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 106) 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 1H 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.
Scheme 2: Synthesis of N-linked galactoside (1b) i) H2O, Et3N, rt. ii) DOWEX ion exchange
column. iii) NaOMe, MeOH, 30 minutes, (1b) 58 %.
In order to synthesize the O-linked derivatives (2a) and (2b), the amine functionality of L-
AEP (3) was first protected as a benzoyl carbamate by reaction with benzyl chloroformate at
0 °C while keeping the pH at around 9.5 via addition of NaOH (4 M) as required. The Cbz- L-
AEP
(6)
was
then
reacted
with
2,3,4,6-tetra-O-benzyl-α-D-glucopyranose
trichloroacetimidate (7a) or 2,3,4,6-tetra-O-α-D-galactopyranose trichloroacetimidate (7b) to
give the protected O-linked derivatives (8a) and (8b) (Scheme 3). These compounds were
then de-O-benzylated via hydrogenation to give the O-linked targets (2a) in 89 % yield and
(2b) in 92 % yield.

Scheme 3: Synthesis of O-linked glucoside and galactoside (2a) and (2b) respectively i) H2O,
NaOH, Et2O, benzyl chloroformate, 0 °C, pH ~9. ii) Et2O, 3 Å molecular sieves. iii) EtOH,
H2O, H2, Pd/C, (2a) 89 %, (2b) 92 %.
The third targets of interest contained L-AEP linked to the sugar via a carbamate moiety (12a,
12b). It was hypothesized that such products would be more stable than the N-linked
derivatives (1) but still able to release L-AEP upon hydrolysis. Thus carbamate linkages have
previously been used within glycosidase labile prodrugs14 for example within a range of
delivery systems, for example for cancer.15 In order to access the carbamate derivatives (12a
and 12b) as -linked glycosides, optimization of methodology was required.16 By varying the
reaction conditions, as outlined in Table 1, the use of hydroxy benzotriazole and 4-
nitrobenzoylchloroformate with either the glucose (9a) or galactose derivative (9b) in the
presence of a participating solvent proved optimum for affording the pure -4-nitrobenzyl
carbonate intermediates (10a and 10b) in good yield (Table 1, Scheme 4).

The carbonates were then reacted with L-diethyl aminoethylphosphonate in the presence of
Hunig’s base and HOBt to give the pure -carbamate linked products (11a) and (11b) in good
yield. Deprotection of the diethyl phosphonate to afford the final targets (12a) and (12b) was
conveniently combined with removal of the acetate groups by using 33% ammonia solution
after addition of methanol.
Table 1. Preparation of β-4-nitrobenzyl carbonate glycoside
Experiment
Conditions
Et3N, DCM, rt, 17h
Outcome
Mixture of anomers 1:1
Mixture  1:2
1
2
3
4
5
Et3N, DCM, 0°C, 1.5h
DABCO, DCM, DMAP, 0°C, 2h
DABCO, THF, DMAP, 0°C, 2h
DABCO, THF, HOBt, 0°C, 2h
Mixture  2:1
Mixture  1:0.8
-anomer only
Scheme 4: Synthesis of carbamate glycosides (12a) and (12b) i) p-NO2C6H4OCOCl,
DABCO, HOBt, THF, 0°C, 2h, (10a) 45%, (10b) 68% ii) DIPEA, HOBt, THF, 16h, (11a)
80%, (11b) 59% iii) TMSBr, DCM, 20h then NH4OH, MeOH, 3-4h then DOWEX ion-
exchange chromatography (12a) 80%, (12b) 91%

In an extension to this strategy, urea linked derivatives were also prepared as outlined in
Scheme 5. Initially the glucose-1--amine (13a)17 was converted to the isocyanate (14a) in a
one pot biphasic reaction originally reported by Nowick for the synthesis of peptides18 and
later employed by Ichikawa19 with carbohydrates. This was then used for entry to (15a) and
(15b), and then (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,
30-45 min. ii) DCM, overnight, (15a) 60% (15b) 21% iii) TMSBr, DCM, 16h then NH4OH,
MeOH, 2-3h then DOWEX ion-exchange chromatography (16a) 74%, (16b) 70%
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 (18b)21 with subsequent reaction of the alcohols with the
isocyanate (17). The derivatives (19a) and (19b) were then deprotected under standard

conditions and purified by ion-exchange chromatography to afford the aromatic carbamate
linked AEP derivatives (20a) and (20b). For this series of compounds, epimeric mixtures
resulted for (19a), (19b), (20a) and (20b), due to the use of a racemic AEP derivative when
forming isocyanate (17).
Scheme 6: Synthesis of extended carbamate glucosides (20a) and (20b) i) Boc2O, DMAP,
DCM or toluene, 0°C, 1h. ii) Et3N, overnight, iii) TMSBr, DCM, 16h then NH4OH, MeOH,
3-4h then DOWEX ion-exchange chromatography, (20a) 35% (20b) 40%
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 o-

nitrophenyl 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.
Table 2. MIC (mg/l) of (12a) (AEP-carbamate-β-D-glucoside) in
different growth media and at pH 6.9 and 7.2
DM1 +
Glucose NB2
(0.5g/l)
Colour
from
DM1
Organisms
ONP-β- (ATCC numbers)
Glu
pH 6.9 pH 7.2 pH 7.2 pH 7.2
+
+
-
E.aerogenes 13048
C.sakazakii 29544
<0.75 >1.0
<0.75 <0.75
1.0
<0.75
1.0
>1.0
1.0
K.pneumoniae 13882 >1.0
P.mirabilis 14253 >1.0
>1.0
>1.0
>1.0
>1.0
1.0
>1.0
1.0
-
Ps.aeruginosa 27853 <0.75 <0.75
S.enterica serovar
Typhimurium 14028
S.enterica serovar
-
>1.0
>1.0
>1.0
1.0
>1.0
>1.0
1.0
>1.0
>1.0
>1.0
-
>1.0
-
Virchow 5742
+
S.marcescens 13880
<0.75
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-D-
galactoside and it does, therefore, have mechanisms for uptake and hydrolysis of galactosides,
but its growth was unaffected by galactoside (12b).
Table 3. Growth of organisms in the presence of 2mg/ml 16b in DM1 pH 7.2 without glucose
and with and without IPTG
Growth
without IPTG
Growth with
IPTG
Colour from
ONP-β-Gal
Organisms (ATCC numbers)
+
+
+
+
+
+
-
+
+
+
-
E.aerogenes 13048
C sakazakii 29544
K.pneumoniae 13882
-
+
+
S.enterica serovar Typhimurium 14028
S.enterica serovar Virchow 5742
-
In summary, a series of ten AEP glycosides has been prepared. All were stable except the N-
linked glycosides (1a) and (1b). The carbamate compounds (12a) and (12b) demonstrated
inhibitory activity for the organisms that were examined with best MIC values of <0.75mg/l
being observed for some bacterial species. Inhibition, in nearly all cases, was closely related
to the hydrolysis of the equivalent chromogenic glycosides suggesting that release was

mediated by the glycosidase enzyme. This is in line with the hypothesis that glycoside
derivatives of antibacterial agents can facilitate selective, targeted delivery of antibacterial
agents, via their in situ hydrolysis by bacterial borne glycosidase enzymes.
Acknowledgements
The authors are grateful to Thermofisher Scientific and the BBSRC (BB/E527812/1) for their
financial support of this work.

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26. a) To 2,3,4,6-tetra-O-acetyl-D-glucose (9a) (6.82 g, 19.6 mmol) in dry THF (70 ml)
cooled to 0°C under argon, was added 1,4-diazabicyclo(2.2.2)octane (3.55 g, 31.6 mmol) and
the solution was stirred for 2 minutes before the addition of 1-hydroxybenzotriazole hydrate

(0.55 g, 4.1 mmol) followed by 4-nitrophenyl chloroformate (4.78 g, 23.7 mmol). The
mixture was stirred for 2 hours at 0°C and then filtered through a short pad of silica eluting
with hexane-EtOAc (1:1) to remove the white HCl salt precipitate formed. The product was
purified by flash column chromatography on silica eluting with hexane-EtOAc (3:2), to yield
the pure -glucoside (10a) (4.56 g, 45%) as a cream solid (Rf 0.47 hexane-EtOAc 1:1). m.p.
135-136°C dec; []20D -3.6° (c 1, CHCl3); 1H NMR (250MHz, CDCl3) : 8.30 (2H, d, J = 9.0
Hz, ArH), 7.44 (2H, d, J = 9.0 Hz, ArH), 5.70 (1H, d, J = 7.5 Hz, H-1), 5.35-5.16 (3H, m, H-
2,3 and H-4), 4.34 (1H, dd, J = 4.5, 12.5 Hz, H-6a), 4.18 (1H, dd, J = 2.0, 12.5 Hz, H-6b),
3.94 (1H, ddd, J = 2.0, 4.5, 9.5 Hz, H-5), 2.10 (3H, s, Ac), 2.10 (3H, s, Ac), 2.05 (3H, s, Ac),
13
2.02 (3H, s, Ac); C NMR (63MHz, CDCl3) : 170.88 (4°, Ac), 170.41 (4°, Ac), 169.69 (4°,
Ac), 169.49 (4°, Ac), 155.24 (4°, -O(CO)O), 151.28 (4°, ArC), 146.07 (4°, ArC), 125.76
(ArC), 122.01 (ArC), 96.24 (C-1), 73.28 (C-5), 72.81 (CH), 70.37 (CH), 67.79 (CH), 61.64
(C-6), 21.03 (CH3, Ac), 20.96 (CH3, Ac), 20.89 (CH3, Ac), 20.89 (CH3, Ac).
b) To 2,3,4,6-tetra-O-acetyl-D-galactose (9b) (8.32 g, 24 mmol) in dry THF (80 ml) cooled to
0°C under argon, was added 1,4-diazabicyclo(2.2.2)octane (4.08 g, 36.4 mmol) and the
solution was stirred for 2 minutes before the addition of 1-hydroxybenzotriazole hydrate (0.65
g, 4.8 mmol) followed by 4-nitrophenyl chloroformate (5.84 g, 28.9 mmol). The mixture was
stirred for 2 hours at 0°C and then filtered through a short pad of silica eluting with hexane-
EtOAc (1:1) to remove the white HCl salt precipitate formed. The product was purified by
flash column chromatography on silica eluting with hexane-EtOAc (3:2 then 1:1) to yield the
pure -galactoside (10b) (8.35 g, 68%) as a cream solid (Rf 0.50 hexane-EtOAc 1:1) m.p. 59-
1
62°C. []20 +10.5° (c 1, CHCl3); H NMR (250MHz, CDCl3) : 8.30 (2H, d, J = 9.5 Hz,
D
ArH), 7.42 (2H, d, J = 9.5 Hz, ArH), 5.66 (1H, d, J = 8.0 Hz, H-1), 5.47-5.43 (2H, overlapped
m, H-2 and H-4), 5.17-5.11 (1H, dd, J = 8.0, 1.5 Hz, H-3), 4.22-4.05 (3H, m, H-6 and H-5),
2.19 (3H, s, Ac), 2.11 (3H, s, Ac), 2.05 (3H, s, Ac), 2.02 (3H, s, Ac); 13C NMR (63MHz,
CDCl3) : 170.68 (4°, Ac), 170.49 (4°, Ac), 170.31 (4°, Ac), 169.74 (4°, Ac), 155.27 (4°, -
O(CO)O), 151.24 (4°, ArC), 146.08 (4°, ArC), 125.77 (ArC), 122.03 (ArC), 96.77 (C-1),
72.30 (C-5), 70.93 (C-3), 68.19 (CH), 66.94 (CH), 61.31 (C-6), 21.16 (CH3, Ac), 21.06 (CH3,
Ac), 20.99 (CH3, Ac), 20.86 (CH3, Ac).
c) To -glucoside (10a) (3.94 g, 7.7 mmol) in anhydrous THF (60 ml) with (R)-
aminodiethylphosphonate (1.83 g, 10.1 mmol) and 1-hydroxy benzotriazole hydrate (1.040 g,
7.7 mmol) under argon was added 3Å molecular sieves (5.7 g) followed by N,N’-
diisopropylethylamine (2.0 ml, 11.4 mmol) whereupon the solution turned yellow. After
stirring at room temperature for 16h the mixture was filtered through a short pad of silica
eluting with ethyl acetate. The nitrophenol byproduct (Rf 0.63 EtOAc) was removed by
extracting the product in DCM followed by washing of the organic layer by 1M NaOH until

the aqueous layer was no longer yellow. The DCM layer was dried (MgSO4), filtered and the
solvent removed in vacuo. The product was purified by flash column chromatography on
silica eluting with hexane-EtOAc (1:4) then EtOAc to give -glucoside (11a) (3.41 g, 80%)
1
as a white solid (Rf 0.39 EtOAc). m.p. 154-155°C. []20 -8.2° (c 1, CHCl3); H NMR
D
(250MHz, CDCl3) : 5.67 (1H, d, J = 8.5 Hz, H-1), 5.39 (1H, br, d, J = 10.0 Hz, NH), 5.22
(1H, app. t, J = 9.5 Hz, H-3), 5.19-5.07 (2H, m, H-4 and H-2), 4.31 (1H, dd, J = 4.5, 12.5 Hz,
H-6a), 4.19-4.01 (6H, m, H-6b, CH of AEP, 2 x OCH2CH3), 3.84 (1H, ddd, J = 2.0, 4.5, 10.0
Hz, H-5), 2.08 (3H, s, Ac), 2.03 (3H, s, Ac), 2.03 (3H, s, Ac), 2.02 (3H, s, Ac), 1.40 (3H, dd,
J = 7.5, 16.5 Hz, CH3 of AEP), 1.34 (3H, t, J = 7.0 Hz, OCH2CH3), 1.30 (3H, t, J = 7.0 Hz,
OCH2CH3); 13C NMR (63MHz, CDCl3) : 170.90 (4°, Ac), 170.38 (4°, Ac), 169.73 (4°, Ac),
169.56 (4°, Ac), 153.58 (4°, -O(CO)NH), 93.32 (C-1), 73.08 (C-3), 72.83 (C-5), 70.49 (C-H),
68.08 (C-H), 63.03 (d, J C,P = 4 Hz, OCH2CH3), 62.93 (d, J C,P = 4 Hz, OCH2CH3), 61.75 (C-
6), 43.72 (d, J C,P = 159 Hz, CH of AEP), 21.00 (CH3, Ac), 20.87 (CH3, Ac), 20.87 (CH3, Ac),
20.79 (CH3, Ac), 16.79 (OCH2CH3), 16.70 (OCH2CH3), 15.89 (CH3 of AEP).
d) To -galactoside (10b) (7.87 g, 15.3 mmol) in anhydrous THF (110 ml) with (R)-
aminodiethylphosphonate (3.33 g, 18.3 mmol) and 1-hydroxy benzotriazole hydrate (2.07 g,
15.3 mmol) under argon was added 3Å molecular sieves (8.8 g) followed by N,N’-
diisopropylethylamine (4.0 ml, 22.9 mmol) whereupon the solution turned yellow. After
stirring at room temperature for 16h the mixture was filtered through a short pad of silica
eluting with ethyl acetate. The nitrophenol byproduct (Rf 0.63 EtOAc) was removed by
extracting the product in DCM followed by washing of the organic layer by 1M NaOH until
the aqueous layer was no longer yellow. The DCM layer was dried (MgSO4), filtered and the
solvent removed in vacuo. The product was purified by flash column chromatography on
silica eluting with hexane-EtOAc (1:4) then EtOAc to give -galactoside (11b) (5.00g, 59%)
1
as a white solid (Rf 0.34 EtOAc). M.p. 129-132°C; []20 +14.6° (c 1, CHCl3); H NMR
D
(250MHz, CDCl3) : 5.66 (1H, d, J = 8.5 Hz, H-1), 5.48 (1H, br, d, J = 9.5 Hz, NH), 5.42
(1H, d, J = 3.5 Hz, H-4), 5.34-5.25 (1H, m, H-2), 5.10 (1H, dd, J = 3.5, 10.5 Hz, H-3), 4.19-
4.05 (8H, m, H-5, H-6, 2 x OCH2CH3 and CH of AEP), 2.16 (3H, s, Ac), 2.04 (6H, s, 2xAc),
1.99 (3H, s, Ac), 1.30 (3H, dd, J = 10.5, 16.5 Hz, CH3 of AEP), 1.32 (3H, t, J = 7.0 Hz,
OCH2CH3), 1.28 (3H, t, J = 7.0 Hz, OCH2CH3); 13C NMR (63MHz, CDCl3) : 170.63 (4°,
Ac), 170.26 (4°, Ac), 170.15 (4°, Ac), 169.71 (4°, Ac), 153.65 (4°, -O(CO)NH), 93.79 (C-1),
71.76 (C-5), 71.13 (C-3), 68.08 (C-2), 67.17 (C-4), 62.98 (d, J C,P = 4 Hz, OCH2CH3), 62.86
(d, J
= 4 Hz, OCH2CH3), 61.53 (C-6), 45.26 (d, J C,P = 158 Hz, CH of AEP), 21.02 (CH3,
C,P
Ac), 20.92 (CH3, Ac), 20.85 (CH3, Ac), 20.85 (CH3, Ac), 16.79 (OCH2CH3), 16.70
(OCH2CH3), 15.95 (CH3 of AEP).

e) To the -glucoside (11a) (2.71 g, 4.8 mmol) in anhydrous dichloromethane (50 ml) was
added trimethylsilylbromide (2.5 ml, 19.5 mmol) and the solution was stirred overnight (20 h)
at room temperature. Methanol (20 ml) was added and the mixture stirred for 15 minutes after
which time aq. ammonium hydroxide (20 ml) was added and the solvent removed under
reduced pressure at 50°C. Aq. Ammonium hydroxide (30 ml) was added again and the
mixture stirred at room temperature for 5 hours and the solvent removed under reduced
pressure at 50°C. The residue was purified by ion-exchange chromatography on Dowex
50WX2-400 resin eluting with water. The product was visualized clearly by TLC (Rf 0.20
propan-1-ol-EtOAc-AcOH-H2O 1:1:1:1). After removal of solvent, a deliquescent white solid
(12a) (1.36g, 80%) was obtained. The more stable ammonium salt was produced by re-
dissolving the white solid in a few drops of NH4OH prior to removal of the solvent under
1
reduced pressure. Data for (12a) H NMR (250MHz, D2O) : 5.22 (1H, d, J = 8.0 Hz, H-1),
3.87-3.67 (2H, m), 3.58-3.49 (2H, m), 3.42-3.20 (3H, m), 1.16 (3H, dd, J = 7.5, 16.0 Hz, CH3
of AEP); 13C NMR (63MHz, D2O) : 156.48 (-O(CO)NH), 95.37 (C-1), 76.73 (CH), 75.61
(CH), 72.18 (CH), 69.84 (CH), 60.66 (C-6), 46.39 (CH, d, J = 153 Hz, CH of AEP), 15.04
(CH3 of AEP).
f) To the -galactoside (11b) (4.44 g, 8.0 mmol) in anhydrous dichloromethane (70 ml) was
added trimethylsilylbromide (4.1 ml, 31.7 mmol) and the solution was stirred overnight (20 h)
at room temperature. Methanol (20 ml) was added and the mixture stirred for 10 minutes. Aq.
Ammonium hydroxide (20 ml) was added and the solvent removed under reduced pressure at
50°C. Aq. Ammonium hydroxide (30 ml) was added again and the mixture stirred at room
temperature for 3 hours and the solvent removed under reduced pressure at 50°C. The residue
was purified by ion-exchange chromatography on Dowex 50WX2-400 resin eluting with
water. The product was visualized clearly by TLC (Rf 0.22 propan-1-ol-EtOAc-AcOH-H2O
1:1:1:1). After removal of the solvent, a deliquescent white solid (12b) (2.40 g, 91%) was
obtained. The more stable ammonium salt was produced by re-dissolving the white solid in a
few drops of NH4OH and removal of the solvent under reduced pressure. Data for (12b) m.p
1
109-115°C dec; []20 +26.3° (c 1, H2O); H NMR (250MHz, D2O) : 5.18 (1H, d, J = 7.0
D
Hz, H-1), 3.88-3.46 (7H, m), 1.17 (3H, dd, J = 7.5, 16.0 Hz, CH3 of AEP); 13C NMR
(63MHz, D2O) : 156.17 (4°, -O(CO)NH), 95.92 (C-1), 76.07 (CH), 72.64 (CH), 69.83 (CH),
68.64 (CH), 61.08 (C-6), 44.97 (CH, d, J = 152 Hz, CH of AEP), 15.11 (CH3 of AEP).

Synthesis and biological analysis of novel glycoside derivatives of L-AEP, as targeted
antibacterial agents
Richard Bovill1, Philip G. Evans2, Gemma L. Howse2 and Helen M.I. Osborn2*
1Thermofisher Scientific., Wade Road, Basingstoke, Hampshire. RG24 8PW, UK
2Reading School of Pharmacy, University of Reading, Whiteknights, Reading. RG6 6AD, UK
Graphical abstract