Synthesis and antibacterial profiles of targeted triclosan derivatives

Article abstract of DOI:10.1016/j.ejmech.2018.10.053

There is an ongoing urgent need for new targeted antibacterial compounds with novel mechanisms of action for the treatment of infections caused by bacteria that are resistant to currently available materials. Since the expression of glycosidase enzymes within bacteria is unequally distributed, glycoside derivatives of antibacterial agents offer potential as targeted prodrugs for bacterial infections. Herein we report the synthesis and characterisation of four α-D-glycopyranosides and three β-D-glycopyranosides of the broad antibacterial agent triclosan, in generally good synthetic yields, and with excellent purities. Each glycoside was analysed to determine its ability to inhibit the growth of a wide range of Gram-negative and Gram-positive organisms, including many of clinical significance. All of the triclosan glycosides that were synthesized demonstrated antibacterial activity against many of the organisms that were examined. For example, β-galactoside (3a) and α-arabinoside (3c) had MIC values of 0.5 μg/ml for several strains of S. aureus and S. haemolyticus. The triclosan glycosides were also generally found to be more water soluble and much more selective than the underivatized triclosan, making them ideal both for the targeted inhibition of bacterial growth and as agents for the selective recovery of bacteria from mixed cultures. In the latter case, two Bacillus strains could be identified from various strains of Bacillus and Staphylococcus after inoculation onto Nutrient Agar No. 2 with 0.25 μg/ml triclosan-α-D-glucopyranoside (3e). This glucoside may, therefore, be of use for the isolation and identification of the food-poisoning organism Bacillus cereus.

Full text of DOI:10.1016/j.ejmech.2018.10.053

Accepted Manuscript
Synthesis and antibacterial profiles of targeted triclosan derivatives
Gemma L. Howse, Richard A. Bovill, Peter J. Stephens, Helen M.I. Osborn
PII:
S0223-5234(18)30923-1
DOI:
https://doi.org/10.1016/j.ejmech.2018.10.053
EJMECH 10839
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 July 2018
Accepted Date: 22 October 2018
Please cite this article as: G.L. Howse, R.A. Bovill, P.J. Stephens, H.M.I. Osborn, Synthesis and
antibacterial profiles of targeted triclosan derivatives, European Journal of Medicinal Chemistry (2018),
doi: https://doi.org/10.1016/j.ejmech.2018.10.053.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Synthesis and Antibacterial profiles of Targeted Triclosan
Derivatives
Gemma L. Howse,a Richard A. Bovill,b# Peter J. Stephens,b Helen M. I. Osborna#
a Reading School of Pharmacy, University of Reading, Whiteknights, Reading. RG6 6AD. UK
b ThermoFisher Scientific, Wade Road, Basingstoke, Hampshire. RG24 8PW. UK
#
To whom correspondence should be addressed. Richard Bovill : Richard.bovill@thermofisher.com;
Helen Osborn : h.m.i.osborn@reading.ac.uk
Abstract
There is an ongoing urgent need for new targeted antibacterial compounds with novel mechanisms of
action for the treatment of infections caused by bacteria that are resistant to currently available materials.
Since the expression of glycosidase enzymes within bacteria is unequally distributed, glycoside derivatives
of antibacterial agents offer potential as targeted prodrugs for bacterial infections. Herein we report the
synthesis and characterisation of four α-D-glycopyranosides and three β-D-glycopyranosides of the broad
antibacterial agent triclosan, in generally very good synthetic yields, and with excellent purities. Each
glycoside was analysed to determine its ability to inhibit the growth of a wide range of Gram-negative and
Gram-positive organisms, including many of clinical significance. All of the triclosan glycosides that were
synthesized demonstrated antibacterial activity against many of the organisms that were examined. For
example, β-galactoside (3a) and α-arabinoside (3c) had MIC values of 0.5 µg/ml for several strains of S.
aureus and S. haemolyticus. The triclosan glycosides were also generally found to be more water soluble
and much more selective than the underivatized triclosan, making them ideal both for the targeted inhibition
of bacterial growth and as agents for the selective recovery of bacteria from mixed cultures. In the latter case,
two Bacillus strains could be identified from various strains of Bacillus and Staphylococcus after inoculation
onto Nutrient Agar No. 2 with 0.25 µg/ml triclosan-α-D-glucopyranoside (3e). This glucoside may,
therefore, be of use for the isolation and identification of the food-poisoning organism Bacillus cereus.
Keywords: antibacterial, prodrug, selective recovery
Introduction
The need for new antibacterial compounds to treat human bacterial infections has never been more
important. The introduction of antibiotics into clinical use represented one of the most significant milestones
in medicine, and allowed huge advances in areas such as organ transplantation, major surgeries and cancer

chemotherapy. However, this extensive use, alongside their widespread misuse, has contributed to the
ACCEPTED MANUSCRIPT
spread of antibiotic resistance. Strains of bacteria demonstrating widespread resistance to antibiotics
(“Multi-drug-resistant”, “Pan-drug-resistant”) are being isolated at an alarming rate (1). It is estimated that
by 2050, 10 million lives a year and a cumulative 100 trillion USD of economic output are at risk due to the
rise of drug-resistant infections. Even today, 700,000 people die of resistant infections every year (2). In a
recent WHO study it was found that very few new antibiotics are in the clinical pipeline and most of those
are modifications of existing classes to which resistant mechanisms are arising. Very few potential treatment
options were identified for those antibiotic-resistant infections posing the greatest threat to health, including
drug-resistant tudeculosis which kills around 250,000 people each year (3).
Triclosan (TCS; 5-chloro-2-[2,4-dichlorophenoxy]phenol) (Figure 1) also known as irgasan is a
synthetic, non-ionic, bis-aryl antimicrobial which has been shown to have anti-fungal and anti-viral
properties (4-9). It is non-toxic to humans but has a broad-spectrum of activity inhibiting all commonly
encountered pathogens. Its mechanism of antibacterial action at higher concentrations is towards the cell
wall but at lower concentrations it inhibits fatty acid synthesis at the enoyl-acyl carrier protein reductase
(FabI) step (10-13). Fatty acid synthesis is essential in bacteria and inhibition causes disruption of the cell
membrane and cell breakdown (14-16).
Whilst triclosan has been extensively used as a disinfectant and has become widely used in many food
preparation and household products, its low solubility at physiological pH has limited its antibacterial usage
to topical treatments. Derivatizing the hydroxyl group, which is an essential functional group for
antibacterial activity, to form a glycoside would be expected to improve its aqueous solubility and enhance
its uptake into bacterial cells via active carbohydrate transport mechanisms (17-19) whilst also limiting its
toxicity to those organisms that are able to remove the sugar using the appropriate glycosidase enzyme.
Since glycosidases are unevenly distributed throughout bacterial taxa (20-24) these targeted bacterial growth
inhibitors would offer the advantage of not disturbing the natural bioflora of the patient. The triclosan
glycosides would also be useful as selective agents in bacterial isolation media, hence allowing more easy
ideintification of bacterial pathogens in food and clinical samples.
The aim of this investigation was therefore to synthesise a range of novel α- and β-linked triclosan
glycosides with enhanced aqueous solubility and targeted antibacterial properties compared with triclosan.
Figure 1

Results and Discussion
ACCEPTED MANUSCRIPT
Triclosan-β-D-galactopyranoside (3a), β-D-glucopyranoside (3b) and α-D-arabinopyranoside (3c)
were each synthesized in two steps as summarised in Scheme 1. The first step involved reaction of the
bromide donor with triclosan (1) in the presence of 1M NaOH (1.06 eq.) to yield, selectively, the desired
trans-linked protected glycopyranosides (2a-2c) in generally good yields. These were then deprotected by
treatment with MeOH and catalytic K2CO3 (0.1 eq.) to afford the required triclosan glycosides (3a-3c) in
very good to excellent yields (25).
Scheme I. i) 45 % HBr in AcOH, 0 °C, 1.5-3 h: ii) triclosan (1), 1M NaOH, H2O/ acetone, r.t, 18 h: iii)
MeOH, K2CO3, r.t, 2-17.5 h.
Synthesis of the thermodynamically favoured α-galacto, gluco and mannopyranosides, and β-
arabinopyranoside (2d, 2e, 2f, 2g respectively), was achieved via glycosidation of the acetate donors under
thermodynamic conditions. Removal of the acetyl protecting groups was again achieved using catalytic
amounts of potassium carbonate (0.1 eq.) in the presence of methanol to afford the triclosan glycosides (3d,
3e, 3f, 3g ) in generally excellent yields.
Scheme II. i) triclosan (1), BF3.OEt2, CH2Cl2, 0°C- r.t, 18 h: ii) MeOH, K2CO3, r.t, 3h.
13
The structures of the synthesized intermediates and final compounds were confirmed by 1H and C
NMR spectroscopy and mass spectrometry. In all cases free triclosan and unreacted sugars could not be
detected in the final products using HPLC analysis and purities were ≥97%.

The Minimum Inhibitory Concentrations (MICs) for triclosan and its glycosides against a wide range
ACCEPTED MANUSCRIPT
of Gram-negative and Gram-positive bacteria, including many of clinical significance, were generated using
the Bioscreen system. Data is summarized in Table 1 (Gram-negative organisms) and Table 2 (Gram-
positive organisms) below.
Tables 1 and 2 MIC (µg/ml) of triclosan glycosides for a range of Gram-negative and Gram-
positive organisms in Nutrient Broth.
Gram-negative
organism
No. of
Triclosan
(1)
β-gal
β-glu
α-ara
α-gal
α-glu
α-man
β-ara
strains
(3a)
(3b)
(3c)
(3d)
(3e)
(3f)
(3g)
Aer. hydrophila
Cit. freundii
Cr. sakazakii
Ent. aerogenes
Ent. cloacae
E. coli
1
1
8
0.5
256
8
>256
64
128
8
>256
64
256
>256
64
>256
64
4
8
1
0.5
8
64
8
128
128
32
128
32
128
128
16
1
0.5
8
64
8
8
1
0.5
0.5
0.5-32
2
16
0.5
0.5-4
1
1
8
8
0.1-0.5
0.5
8-64
16
16-64
32
2-8
1
4-64
16
8-32
32
E. hermanii
Hafnia alvei
Kleb. aerogenes
Kleb.
1
1
0.1
0.5
4
4
0.5
4
16
1
4
4
1
0.5
32
32
2
32
32
1
0.1
0.5
4
0.5
16
0.5
4
1
1
4
4
Pr. mirabilis
Pr. vulgaris
Ps. aeruginosa
Salmonella
1
0.5
32
2
16
32
1
0.5
8
64
16
64
4
32
128
>256
32-64
>256
1
32
>256
2-16
>256
>256
16-32
>256
>256
2-8
>256
>256
32-64
>256
>256
2-8
>256
>256
16-64
>256
11
1
0.5-1
64
Ser. marcescens
Underiv.
triclosan
Gram-positive
organism
No. of
β-gal
β-gluc
α-ara
α-gal
α-gluc
α-man
β-ara
strains
(3a)
(3b)
(3c)
(3d)
(3e)
(3f)
(3g)
(1)
1
1
1
1
8
3
1
1
1
1
2
256
32
256
256
16
128
32
>256
256
128
32
128
256
1
256
128
256
256
2-8
4-8
8
128
128
256
256
4 - 8
8 - 16
128
64
B. cereus
1
8
B. subtilis
128
>256
0.5
0.5
0.5
8
128
256
0.5
0.5
0.5
8
>256
>256
8 - 16
8 - 32
16
Entero. faecalis
Entero. faecium
S. aureus
4
>256
4 - 8
8 - 16
16
0.01
0.01
0.01
0.01
4
1
S. epidermidis
S. haemolyticus
S. saprophyticus
Str. agalactiae
Str. pneumoniae
1
64
64
4
32
256
128
256
128
128
64
>256
128
128
32
128
64
128
64
1

1
1
1
4
128
32
16
256
32
32
32
Str. pyogenes
Str. viridans
ACCEPTED MANUSC>R2I5P6T
256
256
128
128
128
128
From Tables 1 and 2 it can be seen that all of the triclosan glycosides that were synthesized
demonstrated antibacterial activity against many of the organisms that were examined. In particular, the α-
arabinoside (3c) and β-galactoside (3a) both had MICs of 0.5 µg/ml for several organisms including the
pathogenic staphylococcal species S. aureus and S. haemolyticus. It is well recognised that Methicillin
Resistant Staphylococcus aureus (MRSA) can be difficult to treat and often last resort compounds, that may
have toxic side-effects, are therefore used (26, 27). That S. aureus was one of the most sensitive organisms
to the triclosan glycosides is therefore particularly significant from a clinical perspective. Moreover,
although various resistance mechanisms are involved for bacteria, resistance in MRSA is mainly due to the
production of high levels of β-lactamase (28, 29). Currently, drugs with toxic side-effects are often used
(30-32) and hence antibiotics such as those described herein that have a different mode of activity are likely
to be particularly valuable.
From Tables 1 and 2 it can also be seen that in contrast to the broad toxicity exhibited by the parent
triclosan, the toxicities of the triclosan glycosides varied considerably according to the organism. As an
example, the MIC for triclosan was 0.5 µg/ml for eight Gram-negative organisms but MICs for the α-
arabinoside (3c) for these organisms was from 0.5 to 16 µg/ml. This presumably is a reflection of
differences in glycosidase activity and/ or transport rates of the arabinoside into the cell, and reinforces the
hypothesis that derivatisation of triclosan as glycosides is a useful strategy for enhancing selectivity.
Moreover, since the solubility of the triclosan glycosides in growth media and buffers at physiological pH is
much improved compared to underivatised triclosan, which is reflected in the calculated cLogP values for
the hexose glycosides of 3.02 compared to 4.80 for triclosan (33), it should be possible to use the β-
galactoside (3a) and the α-arabinoside (3c) in peroral and parenteral treatments of MRSA infections rather
than only in topical applications, as is the current case for triclosan.
Generally if an organism was resistant to free triclosan it was also resistant to glycosides formed from
it, indicating that any resistance is probably due to rapid efflux of triclosan, or its inability to cause
disruption to fatty acid biosynthesis rather than poor transport into the cell. Thus, strains of Aeromonas
hydrophila, Bacillus cereus, Enterococcus faecium, Ps. aeruginosa, S. marcescens, Streptococcus
agalactiae and Streptococcus viridans all demonstrated MIC of 2 or greater for triclosan and 128 or greater
for the glycosides. An exception was Enterococcus faecalis with a MIC of 16µg/ml for the β-glucoside (3b);
only one doubling dilution higher than that for free triclosan.
As the activities of the triclosan glycosides were much more bacterial selective than the
underivatized triclosan, this also demonstrates their excellent potential for enriching food or clinical

specimens for pathogens prior to instrumental, moleular or traditional means of identification. For example,
ACCEPTED MANUSCRIPT
the results in Table 2 demonstrate that the addition of triclosan α-arabinoside (3c) in selective enrichment
broth at 1.0 µg/ml would allow recovery of Salmonellae but inhibit the growth of many organisms that
outgrow it such as strains of Enterobacter cloacae, Escherichia hermanii, Escherichia coli, Hafnia alvei,
Klebsiella pneumoniae and Staphylococcus. Triclosan α-mannoside (3f) at 64 µg/ml also demonstrated
inhibition profiles that would be particularly useful for the selective recovery of Cronobacter sakazakii
which is of particular concern when present in infant formula milk (34, 35). To determine whether such
differences in toxicity of the triclosan glycosides could indeed be used in selective isolation plates as well as
in broth cultures, MIC values were also determined in Nutrient Agar. Pleasingly, results were essentially the
same as those obtained in broth (data not shown). As an example, to demonstrate the effectiveness of
triclosan glycosides in isolation plates, recovery of Bacillus cereus in the presence of strains of
staphylococci was investigated. Bacillus cereus is an important foodborne pathogen in humans causing
severe nausea, vomiting, and diarrhoea (36). It is often present in samples with large numbers of other
organisms and so can be difficult to isolate. PEMBA medium containing polymyxin B and cycloheximide
with egg yolk precipitation as a presumptive identification is usually used (37). The Bacillus and
Staphylococcus strains were inoculated onto Nutrient Agar plates with and without 8 µg/ml triclosan-α-D-
glucopyranoside (3e). Although all strains grew well on Nutrient Agar only the Bacillus cereus strain could
be recovered on plates containing the inhibitor (Figure 2) and its growth was not affected by free triclosan
that may have been released from dead staphylocococcal cells; presumably amounts were too low or did not
permeate through the agar.
Figure 2. Nutrient agar plates inoculated with strains of Bacillus and Staphylococcus. Left, without and
right with 8µg/ml triclosan-α-D-glucopyranoside (3e). Only the 2 Bacillus strains could be recovered on
plates containing the inhibitor.

ACCEPTED MANUSCRIPT
Conclusions
Chemical derivatization of the antibacterial agent triclosan was optimised to afford four novel
triclosan-α-D-glycopyranosides and three novel triclosan-β-D-glycopyranosides in generally very good
yields, and excellent purities. The abilities of the glycosides to inhibit the growth of a broad range of gram-
positive and gram-negative bacterial organsisms, including many of clinical and industrial relevance, were
probed and many were shown to be potent, selective, antibacterial agents. For example, β-galactoside (3a)
and α-arabinoside (3c) had MIC values of 0.5 µg/ml for several strains of S. aureus and S. haemolyticus.
Since Methicillin Resistant Staphylococcus aureus can be difficult to treat, and last resort compounds that
may have toxic side-effects are often used, this is a significant advance. In addition, since antibacterial
resistance in many cases is due to the production of high levels of β-lactamase, antibiotics with a different
mode of activity such as those described herein are highly beneficial. Moreoever, as the glycoside
derivatives prepared herein have increased aqueuous solubilities compared with free triclosan, as
demonstrated by calculated logP values, this now provides opportunities for triclosan to be used perorally
for the treatment of systemic infections.
Since the novel glycosides were designed to be hydrolysed to afford triclosan in a bacteria dependent
manner, based on the glycosidase profiles of the bacteria, their application for the selective recovery of
bacteria from mixed bacterial populations was also explored. The activity of the triclosan glycosides was
indeed generally found to be much more selective than the underivatized triclosan, and this was clearly
demonstrated when considering the profiles of triclosan α-arabinoside (3c) and triclosan α-mannoside (3f).
For the former, inclusion in selective recovery media at 1.0 µg/ml would allow recovery of Salmonella by
inhibition of the growth of many of organisms that compete with Salmonella, such as strains of Enterobacter
cloacae, Escherichia hermanii, Escherichia coli, Hafnia alvei, Klebsiella pneumoniae and Staphylococcus.
Triclosan α-mannoside (3f) also demonstrated inhibition profiles that would be particularly useful for
selective recovery, and hence identification, of Cronobacter sakazakii which is of particular concern when
present in infant formula milk. In general, results from broth cultures were replicated in selective isolation
plates that used Nutrient Agar No. 2. This was exemplified by recovery of two Bacillus strains from various
strains of Bacillus and Staphylococcus after inoculation onto NA2 with 0.25 µg/ml triclosan-α-D-
glucopyranoside (3e). This glucoside is, therefore, of use for the isolation of the food-poisoning organism
Bacillus cereus.
Taken together, these results therefore demonstrate that glycoside derivatives of triclosan offer
considerable advantages as antibacterial agents compared with triclosan itself, specifically enhanced
aqueous solubilities, targeted antibacterial profiles, and a mechanism of action that is complementary with
those of existing antibacterial agents. These features have also allowed additional impact to be realised
through the selective recovery of bacteria from mixed bacterial populations.

ACCEPTED MANUSCRIPT
Acknowledgements
We would like to express our gratitude to the BBRSC (BB/E527812/1) and Thermo Fisher Scientific
for their assistance in providing funding for Gemma Howse
Materials and methods
Analytical
High Performance Liquid Chromatography (Agilent 1100 series) was used to determine the purity of the
synthesised triclosan-α/β-D-glycopyranosides. A Primsep-B column (weak anion exchange, Hichrom, UK)
was used and eluted with 0.1% formic acid and acetonitrile with a flow rate of 1.5 ml/min. Detection of
triclosan (1) and triclosan-α/β-D-glycopyranosides was determined to be optimal at 275 nm. Calibration
curves of both triclosan (1) and the triclosan-α/β-D-glycopyranosides (dissolved in H2O: Acetonitrile, 1:1)
were constructed using a concentration range of 7.8 µg/ml to 1000µg/ml and used to determine the
concentration of any triclosan (1) impurity.
NMR spectra were recorded on a Bruker DPX 400 MHz instrument running ICON NMR 2.1 under
TOPSPIN 1.3 at the University of Reading (Reading, UK). NMR spectra were analysed using MestreNova
v.8.1.4-12489 software (Mestrelab, Santiago de Compostela, Spain). Samples were analysed in either
deuterated chloroform (CDCl3) or dimethyl sulfoxide (DMSO-d6). Chemical shifts are quoted in parts per
million using the abbreviations: s, singlet, d, doublet, at, apparent triplet, as, apparent singlet m, multiplet.
All coupling constants are quoted to the nearest 0.5 Hz.
Synthesis (25)
General procedure A: Glycosylation with 1M NaOH
Triclosan (1) (1.06 eq.) was suspended in water (10.4 ml per mmol), 1M NaOH (1.06 eq) was then added.
To this was added acetone (5.8 ml per mmol). Separately, acetobromo-derivative (1 eq.) was dissolved in
acetone (6.2 ml per mmol). This solution was then added in one portion to the stirred solution of triclosan
and sodium hydroxide and then stirred at room temperature for 18 hours. The reaction was then extracted
with CH2Cl2, dried (MgSO4), and concentrated in vacuo. The crude product was then purified by column
chromatography (eluent 3:2 hexane/ethyl acetate).
Synthesis
of
O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-5-chloro-2-(2,4-
dichlorophenoxy)phenol (2a). Following general procedure A: Triclosan (1) (1.01 g, 3.5 mmol) was
20°C
reacted with acetobromogalactose (1.36 g, 3.3 mmol) to yield (2a) as a clear oil (1 g, 49 %). [α]D
-5.1 (c
13.9, CHCl3). 1H NMR (400 MHz, CDCl3), 1.79 (3H, s, OAc), 1.90 (3H, s, OAc), 2.02 (3H, s, OAc), 2.09
(3H, s, OAc), 3.61-4.39 (3H, m, H5, H6', H6), 4.96-5.04 (2H, m, H1, H3), 5.31 (1H, dd, J 10.5, 8.0 Hz, H2),
5.36 (1H, dd, J 3.5, 1.0 Hz, H4), 6.64 (1H, d, J 9.0 Hz, H18), 6.74 (1H, d, J 8.5 Hz, H9), 6.96 (1H, dd, J 8.5,

2.5 Hz, H10), 7.07 (1H, dd, J 9.0, 2.5 Hz, H17), 7.22 (1H, d, J 2.5 Hz, H12), 7.37 (1H, d, J 2.5 Hz, H15).
ACCEPTED MANUSCRIPT
13C NMR (100 MHz, CDCl3), 20.3 (OAc), 20.5 (OAc), 20.6 (OAc), 20.6 (OAc), 61.7 (C6), 66.9 (C4), 68.2
(C2), 70.6 (C3), 71.4 (C5), 100.1 (C1), 119.0 (C18), 119.8 (C12), 121.1 (C9), 124.2 (C10), 125.1 (ArQ),
127.9 (C17), 128.6 (ArQ), 129.8 (ArQ), 130.4 (C15), 144.4 (ArQ), 148.1 (ArQ), 151.4 (ArQ)168.9 (OAc),
169.9 (OAc), 170.1 (OAc), 170.4 (OAc). IR νmax/cm-1 (thin film), 3085 (C-H arene), 2971 (C-H), 1757
(C=O), 1589 (C=C), 1367 (C-O), 1064 (C-C), 736 (C-C), 643 (C-Cl). FTMS (ESI, M+Na), 641.0338
C26H25O11Cl3Na, req. 641.0355.
Synthesis of O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-5-chloro-2-(2,4-dichlorophenoxy)phenol
(2b). Following general procedure A: Triclosan (1) (1.1 g, 3.8 mmol) was reacted with acetobromoglucose
20°C
(1.47 g, 3.6 mmol) to yield (2b) as a clear oil (1 g, 45 %). [α]D
-6.26 (c 4.6, CHCl3). 1H NMR (400
MHz, CDCl3), 1.86 (3H, s, OAc), 2.00 (3H, s, OAc), 2.05 (3H, s, OAc), 2.12 (3H, s, OAc), 3.92-3.97 (1H,
m, H5), 4.20-4.24 (2H, m, H6', H6), 5.10 (1H, app. t. J 8.0 Hz, H4), 5.13-5.18 (2H, m, H1, H2/3), 5.24-5.35
(1H, m, H2/3), 6.71 (1H, d, J 9.0 Hz, H18), 6.86 (1H, d, J 8.5 Hz, H9), 7.05 (1H, dd, J 8.5, 2.5 Hz, H10),
7.14 (1H, dd, J 9.0, 2.5 Hz, H17), 7.28 (1H, d, J 2.5 Hz, H12), 7.44 (1H, d, J 2.5 Hz, H15). 13C NMR (100
MHz, CDCl3), 20.2 (OAc), 20.4 (OAc), 20.4 OAc), 20.6 (OAc), 62.0 (C6), 68.25 (C4), 70.7 (C2/3), 72.2
(C5), 72.4 (C2/3), 99.26 (C1), 118.7 (C18), 119.0 (C12), 121.4 (C9), 124.1 (C10), 124.9 (ArQ), 127.8
(C17), 128.5 (ArQ), 129.8 (ArQ), 130.3 (C15), 144.0 (ArQ), 148.1 (ArQ), 151.0 (ArQ), 168.8 (OAc), 169.3
(OAc), 169.9 (OAc), 170.4 (OAc). IR νmax/cm-1 (thin film), 3069 (C-H arene), 2961 (C-H), 1746 (C=O),
1594 (C=C), 1364 (C-O), 731 (C-C), 651 (C-Cl). FTMS (ESI, M+Na), 641.0334 C26H25O11Cl3Na, req.
641.0355.
Synthesis of O-(2,3,4-tri-O-acetyl-α-D-arabinopyranosyl)-5-chloro-2-(2,4-dichlorophenoxy)phenol
(2c). Following general procedure A: Triclosan (1) (8.1 g, 28 mmol) was reacted with acetobromoarabinose
1
(9.03 g, 26 mmol) to yield (2c) as a clear oil (1.78 g, 13 %). [α]D20°C +11 (c 0.9, CHCl3). H NMR (400
MHz, CDCl3), 1.95 (3H, s, OAc), 1.99 (3H, s, OAc), 2.05 (3H, s, OAc), 3.72 (1H, dd, J 13.0, 2.5 Hz, H5'),
4.09-4.15 (1H, m, H5), 5.10-5.12 (2H, m, H1, H2/H3), 5.27-5.31 (2H, m, H2/H3, H4), 6.73 (1H, d, J 9.0 Hz,
H18), 6.79 (1H, d, J 8.5 Hz, H9), 7.03 (1H, dd, J 8.5, 2.5 Hz, H10), 7.16 (1H, dd, J 9.0, 2.5 Hz, H17), 7.26
(1H, d, J 2.5 Hz, H12), 7.46 (1H, d, J 2.5 Hz, H15). 13C NMR (100 MHz, CDCl3), 20.5 (OAc), 20.6 (OAc),
20.9 (OAc), 21.1 (OAc), 60.4 (C5), 66.7 (C2/C3/C4), 68.7 (C2/C3/C4), 69.2 (C2/C3/C4), 99.4 (C1), 119.2
(C18), 120.0 (C12), 121.0 (C9), 124.2 (C10), 128.0 (C17), 130.5 (C15). IR νmax/cm-1 (thin film), 1748
(C=O), 1636 (C=C), 1474 (C=C), 1371 (C-O), 1227 (C-O). FTMS (+ p APCI, M+Na) 569.0141
C23H21O9Cl3Na req. 569.0143. FTMS (ESI, M+Na) 569.0141 C23H21O9Cl3Na req. 569.0143.
General procedure B: Glycosylation with BF3.OEt2
Under argon, the per-O-acetylated carbohydrate (1 eq.) was dissolved in anhydrous CH2Cl2. To this was
added triclosan (1) (1.06 eq.). The reaction was then cooled to 0 °C and BF3.OEt2 (3 eq.) was then added.
The reaction was then allowed to warm to room temperature and stirred for 18 hours. Water was then added
to quench the reaction, which was then stirred for a further 15 minutes. The reaction was then diluted with

CH2Cl2, washed with water, brine, dried (MgSO4), filtered and concentrated in vacuo. The crude product
ACCEPTED MANUSCRIPT
was then purified by column chromatography (eluent 3:2 hexane/ethyl acetate) to yield the desired product.
Synthesis
of
O-(2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl)-5-chloro-2-(2,4-
dichlorophenoxy)phenol (2d). Following general procedure B: 1,2,3,4,6-Penta-O-acetyl-β-
D-
galactopyranoside (2 g, 5.12 mmol) was reacted with triclosan (1) (1.57 g, 5.43 mmol) in the presence of
1
BF3.OEt2 (1.9 ml, 15.4 mmol) to yield (2d) as a clear oil (400 mg, 13 %). [α]D20°C +23 (c 1.1, CHCl3). H
NMR (400 MHz, CDCl3), 1.91 (3H, s, OAc), 1.99 (3H, s, OAc), 2.03 (3H, s, OAc), 2.15 (3H, s, OAc), 4.09-
4.14 (2H, m, H6', H6), 4.28 (1H, app.t, J 7.0 Hz, H5), 5.18-5.25 (2H, m, H2, H3), 5.45-5.46 (1H, m, H4),
5.80 (1H, d, J 3.0 Hz, H1), 6.77 (1H, d, J 9.0 Hz, H18), 6.89 (1H, d, J 8.5 Hz, H9), 7.04 (1H, dd, J 8.5, 2.5
13
Hz, H10), 7.17 (1H, dd, J 9.0, 2.5 Hz, H17), 7.25 (1H, d, J 2.5 Hz, H12), 7.50 (1H, d, J 2.5 Hz, H15). C
NMR (100 MHz, CDCl3), 20.4 (OAc), 20.5 (OAc), 20.5 (OAc), 20.9 (OAc), 61.7 (C6), 67.2
(C2/C3/C4/C5), 67.4 (C2/C3/C4/C5), 67.6 (C2/C3/C4/C5), 67.6 (C2/C3/C4/C5), 95.8 (C1), 119.1 (C18),
119.3 (C12), 121.2 (C9), 123.9 (C10), 125.2 (ArQ), 127.9 (C17), 128.9 (ArQ), 129.8 (ArQ), 130.7 (C15),
144.7 (ArQ), 147.1 (ArQ), 151.6 (ArQ), 169.5 (OAc), 170.0 (OAc), 170.3 (OAc), 170.3 (OAc). IR νmax/cm-1
(oil), 1757 (C=O), 1472 (C=C), 1370 (C-O), 1095 (C-O), 829 (C-H), 727 (C-Cl). FTMS (ESI, M+Na)
641.0356 C26H25O11Cl3Na req. 641.0355.
Synthesis of O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-5-chloro-2-(2,4-dichlorophenoxy)phenol
(2e). Following general procedure B: 1,2,3,4,6-Penta-O-acetyl-β-D-glucopyranside (500 mg, 1.28 mmol)
was reacted with triclosan (1) (393 mg, 1.36 mmol) in the presence of BF3.OEt2 (0.5 ml, 3.84 mmol) to yield
1
(2e) as a clear oil (103 mg, 13 %). [α]D20°C +5.3 (c 0.5, CHCl3). H NMR (400 MHz, CDCl3) 1.94 (3H, s,
OAc), 2.01 (3H, s, OAc), 2.05 (3H, s, OAc), 2.06 (3H, s, OAc), 3.88 (1H, ddd, J 10.0, 5.5, 2.0 Hz, H5), 4.03
(1H, dd, J 12.0, 2.0 Hz, H6'), 4.17 (1H, dd, J 12.0, 5.5 Hz, H6), 4.92 (1H, dd, J 10.0, 3.5 Hz, H2), 5.02 (1H,
dd, J 10.0, 9.5 Hz, H4), 5.16 (1H, app.t, J 9.5 Hz, H3), 5.73 (1H, d, J 3.5 Hz, H1), 6.71 (1H, d, J 9.0 Hz,
H18), 6.97 (1H, d, J 8.5 Hz, H9), 7.06 (1H, dd, J 8.5, 2.5 Hz, H10), 7.15 (1H, dd, J 9.0, 2.5 Hz, H17), 7.21
(1H, d, J 2.5 Hz, H12), 7.54 (1H, d, J 2.5 Hz, H15). 13C NMR (100 MHz, CDCl3), 20.5 (2 x OAc), 20.6 (2 x
OAc), 61.8 (C6), 68.1 (C4), 68.3 (C5), 69.8 (C3), 70.2 (C2), 94.7 (C1), 118.4 (C12), 118.4 (C18), 121.9
(C9), 123.9 (C10), 127.8 (C17), 131.1 (C15). IR νmax/cm-1 (oil), 1746 (C=O), 1599 (C=C), 1409 (C=C),
1248 (C-O), 1023 (C-O), 849 (C-H), 812 (C-H), 757 (C-Cl). FTMS (ESI, M+Na) 641.0356 C26H25O11Cl3Na
req. 641.0355.
Synthesis
of
O-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl)-5-chloro-2-(2,4-
dichlorophenoxy)phenol (2f). Following general procedure B: 1,2,3,4,6-penta-O-acetyl-α/β-
D-
mannopyranoside (5.0 g, 13 mmol) was reacted with triclosan (1) (3.99 g, 14 mmol) in the presence of
1
BF3.OEt2 (4.8 ml, 39 mmol) to yield (2f) as a clear oil (3.39 g, 42 %). [α]D20°C +27.7 (c 1.3, CHCl3). H
NMR (500 MHz, CDCl3), 1.98 (3H, s, OAc), 2.06 (3H,s OAc), 2.07 (3H, s, OAc), 2.15 (3H, s, OAc), 3.83
(1H, ddd, J 10.0, 6.5, 2.0 Hz, H5), 4.05 (1H, dd, J 12.0, 2.0 Hz, H6), 4.18 (1H, dd, J 12.0, 6.5 Hz, H6), 4.89
(1H, dd, J 10.0 3.5 Hz, H3), 5.18-5.23 (2H, m, H2, H4), 5.51 (1H, d, J 1.8 Hz, H1), 6.70 (1H, d, J 9.0 Hz,

H18), 7.07-7.8 (2H, m. H9, H10), 7.16 (1H, dd, J 9.0, 2.5 Hz, H17), 7.26-7.27 (1H, m, H12), 7.49 (1H, d, J
ACCEPTED MANUSCRIPT
13
2.5 Hz, H15). C NMR (126 MHz, CHCl3), 20.3 (OAc), 20.4 (OAc), 20.5 (OAc), 20.5 (OAc), 62.0 (C6),
65.5 (C2/C4), 68.2 (C3), 68.6 (C2/C4), 69.3 (C5), 95.9 (C1), 117.5 (C18/C12), 117.9 (C18/C12), 122.4
(C9/C10), 123.6 (C9/C10), 124.4 (ArQ), 127.7 (C17), 128.1 (ArQ), 130.2 (ArQ), 130.8 (C15), 143.6 (ArQ),
146.5 (ArQ), 152.0 (ArQ), 169.0 (OAc), 169.4 (OAc), 169.6 (OAc), 170.2 (OAc). IR νmax/cm-1 (oil), 1746
(C=O), 1595 (C=C), 1475 (C=C), 1222 (C-O), 1045 (C-O), 829 (C-H), 822 (C-H), 812 (C-H), 608 (C-Cl).
FTMS (ESI, M+Na) 569.0150 C23H21O9Cl3Na req. 569.0143.
Synthesis of O-(2,3,4-tri-O-acetyl-β-
(2g). Following general procedure B: 1,2,3,4-tetra-O-acetyl-α/β-
α/β- -arabinofuranose (5.26 g, 17 mmol) was reacted with triclosan (1) (5.2 g, 18 mmol) in the presence of
D-arabinopyranosyl)-5-chloro-2-(2,4-dichlorophenoxy)phenol
D
-arabinpyranose/1,2,3,4-tetra-O-acetyl-
D
BF3.OEt2 (6.29 ml, 51 mmol) to yield (2g) as a clear oil (4.15 g, 45 %). [α]D20°C -53.6 (c 1.2, CHCl3). H
NMR (400 MHz, CDCl3), 1.93 (3H, s, OAc), 2.01 (3H, s, OAc), 2.13 (3H, s, OAc), 3.74 (1H, dd, J 13.0, 2.0
Hz, H5’), 3.96 (1H, app. d, J 12.5 Hz, H5), 5.17 (1H, dd, J 11.0, 3.5 Hz, H3), 5.25 (1H, dd, J 11.0, 3.5, H2),
5.32-5.33 (1H, m, H4), 5.81 (1H, d, J 3.5 Hz, H1), 6.74 (1H, d, J 9.0 Hz, H18), 6.96 (1H, d, J 8.5 Hz, H9),
7.03 (1H, dd, J 8.5, 2.5 Hz, H10), 7.15 (1H, dd, J 9.0, 2.5 Hz, H17), 7.23 (1H, d, J 2.5 Hz, H12), 7.49 (1H,
d, J 2.5 Hz, H15). 13C NMR (100 MHz, CDCl3), 20.4 (OAc), 20.5 (OAc), 20.7 (OAc), 61.4 (C5), 66.8 (C3),
67.52 (C2), 68.5 (C4), 95.9 (C1), 118.4 (C18/C12), 118.5 (C18/C12), 121.8 (C9), 123.5 (C10), 124.8 (ArQ),
127.6 (C17), 128.4 (ArQ), 130.2 (ArQ), 130.6 (C15), 144.2 (ArQ), 147.5 (ArQ), 151.9 (ArQ), 169.4 (OAc),
169.9 (OAc), 170.2 (OAc). IR νmax/cm-1 (oil), 1745 (C=O), 1599 (C=C), 1495 (C=C), 1248 (C-O), 1095 (C-
O), 862 (C-H), 800 (C-H), 709 (C-Cl). FTMS (ESI, M+Na) 569.0150 C23H21O9Cl3Na req. 569.0143.
General procedure C: Deprotections
1
Under argon, the protected sugar (1eq) was dissolved in anhydrous MeOH (1 ml per mmol). K2CO3 (0.1
eq) was then added. The reaction was then stirred until it was deemed to be complete as evidenced by TLC
analysis. Amberlite IR-120 (plus) resin was then added and the reaction was stirred for a further 30 minutes.
The resin was then filtered off and the filtrate concentrated in vacuo to yield the desired product.
Synthesis of triclosan-β-D-galactopyranoside (3a). Following general procedure C: O-(2,3,4,6-tetra-O-
acetyl-β- -galactopyranosyl)-5-chloro-2-(2,4-dichlorophenoxy)phenol (2a) (715 mg, 1.15 mmol) was
D
reacted with potassium carbonate (16 mg, 0.115 mmol) to yield (3a) as an amorphous foam (429 mg, 83 %).
20°C
[α]D
-28.29 (c 0.35, MeOH). 1H NMR (400 MHz, MeOD), 3.59 (1H, dd, J 10.0, 3.0 Hz, H3), 3.66-3.80
(4H, m, H6', H6, H2, H5), 3.91 (1H, app. d, J 3.0 Hz, H4), 4.98 (1H, d, J 8.0 Hz, H1), 6.85 (1H, d, J 9.0 Hz,
H18), 6.87 (1H, d, J 8.5 Hz, H9), 7.02 (1H, dd, J 8.5 Hz, 2.5 Hz, H10), 7.22 (1H, dd, J 9.0, 2.5 Hz, H17),
13
7.38 (1H, d, J 2.5 Hz, H12), 7.49 (1H, d, J 2.5 Hz, H15). C NMR (100 MHz, MeOD), 62.3 (C6), 70.1
(C4), 72.1 (C2/5), 74.9 (C3), 77.1 (C2/5), 103.0 (C1), 119.3 (C12), 120.9 (C18), 122.5 (C9), 124.0 (C10),
126.2 (ArQ), 129.4 (C17), 129.62 (ArQ), 131.2 (C15), 131.26 (ArQ), 145.5 (ArQ), 150.6 (ArQ), 153.4
(ArQ). IR νmax/cm-1 (thin film), 3391 (OH), 1652 (C=C), 1449 (C=C), 1026 (C-O), 668 (C-Cl). FTMS (ESI,
M+Na), 472.9914 C18H17O7Cl3Na, req. 472.9932.

Synthesis of triclosan-β-
D
-glucopyranoside (3b). Following general procedure C: O-(2,3,4,6-tetra-O-
ACCEPTED MANUSCRIPT
acetyl-β-
D
-glucopyranosyl)-5-chloro-2-(2,4-dichlorophenoxy)phenol (2b) (500 mg, 0.81 mmol) was reacted
with potassium carbonate (11 mg, 0.081 mmol) to yield (3b) as an amorphous foam (258 mg, 71 %).
[α]D20°C -21 (c 0.5, MeOH). 1H NMR (400 MHz, MeOD), 3.27-3.53 (4H, m, H2, H3, H4, H5), 3.70 (1H, dd,
J 12.0, 5.5 Hz, H6'), 3.88 (1H, dd, J 12.0, 2.0 Hz, H6), 5.04 (1H, d, J 8.0 Hz, H1), 6.85 (1H, d, J 9.0 Hz,
H18), 6.88 (1H, d, J 8.5 Hz, H9), 7.03 (1H, dd, J 8.5, 2.5 Hz, H10), 7.22 (1H, dd, J 9.0, 2.5 Hz, H17), 7.36
(1H, d, J 2.5 Hz, H12), 7.49 (1H, d, J 2.5 Hz, H15). 13C NMR (100 MHz, MeOD), 62.4 (C6), 71.1
(C2/3/4/5), 74.8 (C2/3/4/5), 78.0 (C2/3/4/5), 78.3 (C2/3/4/5), 102.3 (C1), 119.3 (C12), 120.9 (C18), 122.5
(C9), 124.1 (C10), 126.2 (ArQ), 129.4 (C17), 129.6 (ArQ), 131.2 (C15), 131.3 (ArQ), 145.5 (ArQ), 150.4
(ArQ), 153.4 (ArQ). IR νmax/cm-1 (thin film), 3411 (OH), 1652 (C=C), 1450 (C=C), 1031 (C-O), 758 (C-C).
FTMS (ESI, M+Na), 472.9924 C18H17O7Cl3Na, req. 472.9932.
Synthesis of triclosan-α-D-arabinopyranoside (3c). Following general procedure C: O-(2,3,4-tri-O-
acetyl-α-D-arabinopyranosyl)-5-chloro-2-(2,4-dichlorophenoxy)phenol (2c) (1.47 g, 2.69 mmol) was reacted
20°C
with potassium carbonate (37 mg, 0.269 mmol) to yield (3c) as an amorphous foam (1.195 g, 99 %). [α]D
1
+17.6 (c 1.1, MeOH). H NMR (400 MHz, MeOD), 3.63 (1H, dd, J 8.5, 3.5 Hz, H3), 3.71 (1H, dd, J 12.5,
2.0 Hz, H5'), 3.74 (1H, dd, J 8.5, 6.5 Hz, H2), 3.86-3.89 (1H, m, H4), 3.93 (1H, dd, J 12.5, 3.5 Hz, H5), 5.00
(1H, d, J 6.5 Hz, H1), 6.84 (1H, d, J 9.0 Hz, H18), 6.92 (1H, d, J 8.5 Hz, H9), 7.06 (1H, dd, J 8.5, 2.5 Hz,
H10), 7.25 (1H, dd, J 9.0, 2.5 Hz, H17), 7.32 (1H, d, J 2.5 Hz, H12), 7.53 (1H, d, J 2.5 Hz, H15). 13C NMR
(100 MHz, MeOD), 66.8 (C5), 69.1 (C4), 72.0 (C2), 73.9 (C3), 102.7 (C1), 119.5 (C12), 120.6 (C18), 122.6
(C9), 124.1 (C10), 126.3 (ArQ), 129.3 (C17), 129.8 (ArQ), 131.2 (C15), 145.7 (ArQ), 150.2 (ArQ), 153.3
(ArQ). IR νmax/cm-1 (solid), 3359 (O-H), 1595 (C=C), 1471 (C=C), 1223 (C-C), 1067 (C-C), 1011 (C-C),
906 (C-H), 778 (C-Cl). FTMS (ESI, M+Na) 442.9824 C17H15O6Cl3Na, req.442.9826.
Synthesis of triclosan-α-D-galactopyranoside (3d). Following general procedure C: O-(2,3,4,6-tetra-O-
acetyl-α-D-galactopyranosyl)-5-chloro-2-(2,4-dichlorophenoxy)phenol (2d) (432 mg, 0.7 mmol) was reacted
20°C
with potassium carbonate (10 mg, 7 x 10-5 mol) to yield (3d) as an orange oil (166 mg, 37 %). [α]D
1
+88.6 (c 1.3, MeOH). H NMR (400 MHz, MeOD), 3.56 (1H, dd, J 10.0, 3.5 Hz, H3), 3.65-3.66 (2H, m,
H6', H6), 3.74-3.78 (1H, m, H5), 3.85 (1H, dd, J 3.5, 1.5 Hz, H4), 3.92 (1H, dd, J 10.0, 3.5 Hz, H2), 5.34
(1H, d, J 3.5 Hz, H1), 3.92 (1H, d, J 9.0 Hz, H18), 7.01 (1H, d, J 8.5 Hz, H9), 7.07 (1H, dd, J 8.5, 2.5 Hz,
H10), 7.23 (1H, dd, J 8.5, 2.5 Hz, H12), 7.43 (1H, d, J 2.5 Hz, H12), 7.53 (1H, d, J 2.5 Hz, H15). 13C NMR
(100 MHz, MeOD), 62.4 (C6), 69.7 (C2), 70.7 (C4), 71.2 (C3), 73.8 (C5), 100.0 (C1), 119.4 (C12), 119.8
(C18), 123.3 (C9/C10), 124.0 (C9/C10), 125.6 (ArQ), 129.3 (C17), 131.3 (C15), 131.7 (ArQ), 145.4 (ArQ),
150.0 (ArQ x 2), 153.8 (ArQ). IR νmax/cm-1 (solid), 3358 (O-H), 2935 (C-H), 1569 (C=C), 1491 (C=C),
1259 (C-C), 1073 (C-C), 949 (C-C), 866 (C-H), 754 (C-Cl). FTMS (ESI, M+Na) 472.9930 C18H17O7Cl3Na,
req. 472.9932.

Synthesis of triclosan-α-
D
-glucopyranoside (3e). Following general procedure C: O-(2,3,4,6-tetra-O-
ACCEPTED MANUSCRIPT
acetyl-α-D-glucopyranosyl)-5-chloro-2-(2,4-dichlorophenoxy)phenol (2e) (345 mg, 0.56 mmol) was reacted
with potassium carbonate (8 mg 5.6 x 10-5 mol) to yield (3e) as an amorphous foam (250 mg, 99 %).
[α]D20°C +7.9 (c 12.5, MeOH). 1H NMR (400 MHz, MeOD), 3.44 (1H, dd, J 10.5, 8.5 Hz, H3/H4), 3.56-3.72
(5H, m, H6', H6, H2/H3/H4/H5), 5.59 (1H, d, J 3.0 Hz, H1), 6.83 (1H, d, J 9.0 Hz, H18), 6.94 (1H, d, J 8.5
Hz, H9), 7.05 (1H, dd, J 8.5, 2.5 Hz, H10), 7.23 (1H, dd, J 9.0, 2.5 Hz, H17), 7.44 (1H, d, J 2.5 Hz, H12),
7.50 (1H, d, J 2.5, H15). 13C NMR (100, MeOD), 62.1 (C6), 70.9 (C2/C3/C4/C5), 73.1 (C2/C3/C4/C5), 74.7
(C2/C3/C4/C5), 75.0 (C2/C3/C4/C5), 100.4 (C1), 119.8 (C12), 120.2 (C18), 122.8 (C9), 124.8 (C10), 125.9
(ArQ), 129.4 (C17), 131.3 (C15), 131.4 (ArQ), 145.8 (ArQ), 150.2 (ArQ), 153.5 (ArQ). IR νmax/cm-1 (solid),
3356 (O-H), 2938 (C-H), 1569 (C=C), 1471 (C=C), 1224 (C-C), 1056 (C-C), 863 (C-H), 783 (C-Cl). FTMS
(ESI, M+Na) 472.9929 C18H17O7Cl3Na, req.472.9932.
Synthesis of triclosan-α-D-mannopyranoside (3f). Following general procedure C: O-(2,3,4,6-tetra-O-
acetyl-α-D-mannopyranosyl)-5-chloro-2-(2,4-dichlorophenoxy)phenol (2f) (381 mg, 0.6 mmol) was reacted
20°C
with potassium carbonate (8.5 mg, 0.06 mmol) to yield (3f) as an amorphous foam (246 mg, 89 %). [α]D
1
+41.9 (c 0.82, MeOH). H NMR (400 MHz, MeOD), 3.46-3.76 (6H, m, H2, H3, H4, H5, H6, H6’), 5.45
(1H, d, J 1.5 Hz, H1), 6.75 (1H, d, J 9.0 Hz, H18), 7.06-707 (2H, m, H9, H10), 7.21 (1H, dd, J 9.0, 2.5 Hz,
13
H17), 7.48 (1H, d, J 2.5 Hz, H12), 7.51 (1H, d, J 2.5 Hz, H15). C NMR (100 MHz, MeOD), 62.5 (C6),
68.0 (C2/C3/C4/C5), 71.6 (C2/C3/C4/C5), 72.2 (C2/C3/C4/C5), 75.8 (C2/C3/C4/C5), 101.2 (C1), 119.2
(C18), 119.5 (C12), 123.6 (C10/C9), 124.2 (C10/9), 125.4 (ArQ), 129.3 (ArQ), 129.3 (C17), 131.3 (C15),
131.8 (ArQ), 145.0 (ArQ), 149.8 (ArQ), 153.9 (ArQ). IR νmax/cm-1 (solid), 3325 (O-H), 2933 (C-H), 1597
(C=C), 1490 (C=C) 1258 (C-O), 1098 (C-C), 1000 (C-C), 751 (C-Cl). FTMS (ESI, M+Na) 472.9930
C18H17O7Cl3Na, req.472.9932.
Synthesis of triclosan-β-D-arabinopyranoside (3g). Following general procedure C: O-(2,3,4-tri-O-
acetyl-β-
D
-arabinopyranosyl)-5-chloro-2-(2,4-dichlorophenoxy)phenol (2g) (3.5g, 6.4 mmol) was reacted
20°C
with potassium carbonate (88 mg, 0.64 mmol) to yield (3g) as an amorphous foam (2.37 g, 88 %). [α]D
-
56.4 (c 0.85, MeOH). 1H NMR (400 MHz, MeOD), 3.61 (1H, dd, J 12.5, 2.0 Hz, H5’), 3.63 (1H, dd, J 10.0,
3.5 Hz, H3), 3.76 (1H, dd, J 12.5, 1.0 Hz, H5), 3.81-3.82 (1H, m, H4), 3.95 (1H, dd, J 10.0, 3.5 Hz, H2),
5.64 (1H, d, J 3.5 Hz, H1), 6.78 (1H, d, J 9.0 Hz, H18), 6.99 (1H, d, J 8.5 Hz, H9), 7.05 (1H, dd, J 8.5, 2.5
13
Hz, H10), 7.20 (1H, dd, J 9.0, 2.5 Hz, H17), 7.35 (1H, d, J 2.5 Hz, H12), 7.50 (1H, d, J 2.5 Hz, H15). C
NMR (100 MHz, MeOD), 65.6 (C5), 69.7 (c2), 70.5 (C4), 70.6 (C3), 100.6 (C1), 119.5 (C12), 119.8 (C18),
123.4 (C9), 124.1 (C10), 125.6 (ArQ), 129.3 (C17), 129.5 (ArQ), 131.3 (C15), 131.7 (ArQ), 145.5 (ArQ),
150.0 (ArQ), 153.7 (ArQ). IR νmax/cm-1 (solid), 3360 (O-H), 2931 (C-H), 1596 (C=C), 1224 (C-O), 1076 (C-
C), 999 (C-C), 756 (C-Cl). FTMS (ESI, M+Na) 442.9824 C17H15O6Cl3Na, req.442.9826.
Bacterial strains. All strains were from the American Type Culture Collection (ATCC University
Boulevard, Manassas, VA, USA) except as indicated NCTC (National Collection of Type Cultues, Public

Health England, Porton Down, Salisbury, UK), OCC (Oxoid Culture Collection (Thermofisher Scientific,
ACCEPTED MANUSCRIPT
Wade Road, Basingstoke, UK). Gram negative: Aeromonas hydrophila OCC 778, Citrobacter freundii
8090, OCC 851, Enterobacter aerogenes 13048, Enterobacter cloacae 13047, Cronobacter sakazakii
29544, Escherichia coli 8739, 10536, 11775, 25922, 35218, 700728, NCTC 13216, OCC 122, Escherichia
hermanii 33650, Hafnia alvei 13337, Klebsiella aerogenes NCTC 8167, Klebsiella pneumoniae 10031,
13883, Proteus mirabilis 12453, Proteus vulgaris OCC 195, Pseudomonas aeruginosa 27853, Salmonella
enterica subspecies enterica serotypes: Abony NCTC 6017, Arizonae OCC 706, Dublin OCC 627, Enteritidis
13076, Indiana OCC 597, Infantis OCC 2133, Nottingham NCTC 7832, Poona NCTC 4840, Pullorum OCC
273, Typhimurium 14028 and Virchow NCTC 5742, Serratia marcescens OCC 217. Gram positive: Bacillus
cereus 14579, Bacillus subtilis NCTC 10073, Enterococcus faecalis 29212, 33186, Enterococcus faecium
19434, Staphylococcus aureus 6538, 6538P, 9144, 25923, 29213, NCTC 13758, OCC 104, OCC 106,
Staphylococcus epidermidis 155, 12228, 14990, Staphylococcus haemolyticus OCC 2223, Staphylococcus
saprophyticus 15305, Streptococcus agalactiae OCC 182, Streptococcus pneumoniae 6305, 49619,
Streptococcus pyogenes 19615, Streptococcus viridans OCC 234.
MIC determination in broth growth medium. Triclosan and its glycosides were dissolved in ethanol
then amounts of these stock solutions diluted in Nutrient Broth (NB2; “Oxoid”, ThermoFisher, Basingstoke,
UK) to give concentrations of 0.01µg/ml to 128µg/ml for triclosan and 0.25µg/ml to 256µg/ml for its
glycosides. Volumes (180µl) were then added to wells of microtitre plates. Bacteria were grown overnight at
37oC in NB2 from plate inoculum. Cultures were diluted 1 in 104 in saline and 20µl amounts added to the
triclosan/ triclosan glycoside solutions. Once filled, plates were covered, placed in a Bioscreen C MBR
instrument (Oy Growth Curves Ab Ltd. Helsinki, Finland), incubated at 37oC and optical density (OD) of
each well measured every 15 minutes after agitation. The minimum inhibitory concentration (MIC) of a
compound was defined as the lowest concentration of inhibitor that was required to eliminate an increase in
OD after 18 hours incubation at 37oC.
MIC determination on agar plates. Amounts of triclosan glycoside solutions prepared as described
above were added to Nutrient Agar (NA2; “Oxoid”, ThermoFisher) and the media autoclaved for 15 minutes
at 121oC. Plates were poured, dried and stored at 4oC using within one week of pouring. Cultures were
prepared as above, diluted 1 in 102 in saline and inoculated onto plates using an “Oxoid” replicator system.
References
1. Docquier, J-D., and S. Mangani. 2018. An update on β-lactamase discovery and development. Drug
Res. Updates. 36: 13-29.
2. WHO. 2016. Tackling drug-resistant infections globally: final report and recommendations.
3. WHO. 2017. Antibacterial Agents in Clinical Development. An analysis of the antibacterial clinical
development pipeline, including tuberculosis. WHO/EMP/IAU/2017.12.

4. Bhargava, H. N., and P.A. Leonard. 1996. Triclosan: applications and safety. Am. J. Infect. Control.
ACCEPTED MANUSCRIPT
24:209-218.
5. Regos, J., and H.R. Hitz. 1974. Investigations on the model of action of triclosan, a broad spectrum
antibacterial agent. Zbl. Bakt-Int. J. Med. M. 226:390-401.
6. Regos, J., O. Zak, R. Solf, W.A. Vischer, and E.G. Weirich. 1979. Antimicrobial Spectrum of
Triclosan, a Broad-Spectrum Antimicrobial Agent for Topical Application. Dermatologica. 158:72-79.
7. Russell, A.D. 2002. Mechanisms of antimicrobial action of antiseptics and disinfectants: an
increasingly important area of investigation. J. Antimicrob. Chemother. 49:597-599.
8. Schweizer, H.P. 2001. Triclosan: a widely used biocide and its link to antibiotics. FEMS Microbiol.
Lett. 202:1-7.
9. Slater-Radosti, C., G. Van Aller, R. Greenwood, R. Nicholas, P.M. Keller, W.E. DeWolf, F. Fan,
D.J. Payne, and D.D. Jaworski. 2001. Biochemical and genetic characterization of the action of
triclosan on Staphylococcus aureus. J. Antimicrob. Chemother. 48:1-6.
10. Escalada, M.G., J.L. Harwood, J.Y. Maillard, and D. Ochs. 2005. Triclosan inhibition of fatty acid
synthesis and its effect on growth of E. coli and Ps. aeruginosa. J. Antimicrob. Chemother. 55:879-882.
11. Heath, R.J., N. Su, C.K. Murphy, and C.O. Rock. 2000. The enoyl-[acyl-carrier-protein] reductases
FabI and FabL from Bacillus subtilis. J. Biol. Chem. 275:40128-40133.
12. Hoang, T.T., and H.P. Schweizer. 1999. Characterization of Pseudomonas aeruginosa enoyl-acyl
carrier protein reductase: a target for the antimicrobial triclosan and its role in acylated homoserine
lactone synthesis. J. Bacteriol. 181:5489-5497.
13. McMurry, L. M., M. Oethinger, and S.B. Levy. 1998. Triclosan Targets Lipid Synthesis. Nature.
394:531-532.
14. Heath, R. J., Y.T. Yu, M.A. Shapiro, E. Olson, and C.O. Rock. 1998. Broad spectrum antimicrobial
biocides target the FABI component of fatty acid biosynthesis. J. Biol. Chem. 273:30316-30320.
15. Xu, H., T.J. Sullivan, J.I. Sekiguchi, T. Kirikae, I. Ojima, C.F. Stratton, W.M. Mao, F.L. Rock,
M.R.K. Alley, F. Johnson, S.G. Walker, and P.J. Tonge. 2008. Mechanism and Inhibition of saFabI,
the Enoyl Reductase from Staphylococcus aureus. Biochem. 47:4228-4236.
16. Zhang, Y.M., S.W. White and C.O. Rock. 2006. Inhibiting bacterial fatty acid synthesis. J. Biol.
Chem. 281:17541-17544.
17. a) Milner, S.J.; Carrick, C.T.; Kerr, K.G.; Snelling, A.M.; Thomas, G.H.; Duhme-Klair, A.K.;
Routledge, A. 2014. Probing bacterial uptake of glycosylated ciprofloxacin conjugates. Chembiochem.
15: 466-471; b) Jung, M.E.; Yang, E.C.; Vu, B.T.; Kiankarimi, M.; Spyrou, E.; Kaunitz, J. 1999.
Glycosylation of fluoroquinolones through directed and oxygenated polymethylene linkages as a sugar-
mediated active transport system for antimicrobials J. Med. Chem. 42: 3899-3909; c) Conners, S.B.;
Montero, C.I.; Comfort, D.A. ; Shockley, K.R.; Johnson, M.R.; Chhabra, S.R.; Kelly, R.M. 2005.
An expression-driven approach to the prediction of carbohydrate transport and utilization regulons in
the hyperthermophilic bacterium Thermotoga maritima. J. Bacteriol. 187: 7267-7282; d) Barrangou,

R.; Azcarate-Peril, M.A.; Duong, T.; Conners, S.B.; Kelly, R.M.; Klaenhammer, T.R. 2006.
ACCEPTED MANUSCRIPT
Global analysis of carbohydrate utilization by Lactobacillus acidophilus using cDNA microarrays.
Proc. Natl. Acad. Sci. 103: 3816-3821; e) Bovill, R.; Evans, P.G.; Howse, G.L.; Osborn, H.M.I.
2016. Synthesis and biological analysis of novel glycoside derivatives of L-AEP, as targeted
antibacterial agents. Bioorg. Med. Chem. Lett. 26:3774-3779.
18. a) Cellier, M.; Fazackerley, E.; James, A.L.; Orenga, S.; Perry, J.D.; Turnbull, G.; Stanforth, S.P.
2014. Synthesis of 2-arylbenzothiazole derivatives and their application in bacterial detection. Bioorg.
Med. Chem. 22: 1250-1261; b) Orenga, S.; James, A.L.; Manafi, M.; Perry, J.D.; Pincus, D.H.
2009. Enzymatic substrates in microbiology. J. Microbiol. Methods 79: 139-155; c) Perry, J.D.;
Morris, K.A.; James A.L.; Oliver, M.; Gould, F.K. 2007. Evaluation of novel chromogenic
substrates for the detection of bacterial beta-glucosidase. J. Appl. Microbiol. 102: 410-415; d) Chilvers,
K.F.; Perry, J.D.; James, A.L.; Reed, R.H. 2001. Synthesis and evaluation of novel fluorogenic
substrates for the detection of bacterial beta-galactosidase J. Appl. Microbiol. 91: 1118-1130.
19. Fernandez, C.; Nieto, O.; Fontenla, J.A.; Rivas, R.; de Ceballos, M.L.; Fernandez-Mayoralas, A.
2003. Synthesis of glycosyl derivatives as dopamine prodrugs:interaction with glucose carrier GLUT-1.
Org. Biom. Chem. 1: 767-771.
20. Kämpfer, P., O. Rauhoff, and W. Dott. 1991. Glycosidase Profiles of Members of the Family
Enterobacteriaceae, J. Clin. Microbiol. 29:2877-2879.
21. Manafi, M., W. Kneifel, and S. Bascomb. 1991. Fluorogenic and Chromogenic Substrates Used in
Bacterial Diagnostics. Microbiol. Rev. 55:335-348.
22. Orenga, S., A.L. James, M. Manafi, J.D. Perry, and D.H. Pincus. 2009. Enzymatic substrates in
microbiology. J. Microbiol. Meth. 79:139-155.
23. Perry, J. D., and A.M. Freydiere. 2007. The application of chromogenic media in clinical
microbiology. J. Appl. Microbiol. 103:2046-2055.
24. Bascomb, S., and M. Manafi, M. 1998. Use of enzyme tests in characterization and identification of
aerobic and facultatively anaerobic gram-positive cocci. Clin. Microbiol. Rev. 11:318-30.
25. Bovill, R.A., and G. Howse. 2018. Triclosan derivatives and uses thereof. US9,908,912B2.
26. Choo, E. J. and Chambers, Henry F. 2016. Treatment of Methicillin-Resistant Staphylococcus aureus
Bacteremia. Infection & Chemotherapy. 48: 267-273.
27. Schentag JJ, Hyatt JM, Carr JR, Paladino JA, Birmingham MC, Zimmer GS, Cumbo TJ. 1998.
Genesis of methicillin-resistant Staphylococcus aureus (MRSA), how treatment of MRSA infections
has selected for vancomycin-resistant Enterococcus faecium, and the importance of antibiotic
management and infection control. Clin. Infect. Dis. 26: 1204–14.
28. Keith A. Rodvold, K.A. and K. W. McConeghy. 2014 Methicillin resistant Staphylococcus aureus
Therapy: Past, Present, and Future. Clin. Infect. Dis. 58:S20–S27.

29. K. Hiramatsu, Y. Katayama, M. Matsuo, T. Sasaki, Y. Morimoto, A. Sekiguchi, and T. Baba.
ACCEPTED MANUSCRIPT
2014. Multi-drug-resistant Staphylococcus aureus and future chemotherapy. J Infect. Chemother.
20:593-601.
30. Otto, M., B.N. Kreisworth, and H.F. Chambers. 2010. Community-associated methicillin-
resistant Staphylococcus aureus. Lancet. 375:1557-1568.
31. Hartman, B.J. and A. Tomasz. 1984. Low-affinity penicillin-binding protein associated with β-
lactam resistance in Staphylococcus aureus J. Bacteriol. 158:513-516.
32. Baek, K. T., A. Gründling, R.G. Mogensen, L. Thøgersen, A. Petersen, W. Paulander and D.
Frees. 2014. β-Lactam resistance in methicillin-resistant Staphylococcus aureus USA300 is increased
by inactivation of the ClpXP Protease. Antimicrob. Agent. Chemother. 58:4593–4603.
33. ChemBiodraw 14.0 PerkinElmer
34. Hunter, C.J., M. Petrosyan, H.R. Ford, N.V. Prasadarao. 2008. Enterobacter sakazakii: An
Emerging Pathogen in Infants and Neonates. Surg. Infect. 9:533–539.
35. Yan, Q.Q., O. Condell, K. Power, F. Butler, B.D. Tall, and S. Fanning. 2012. Cronobacter species
(formerly known as Enterobacter sakazakii) in powdered infant formula: a review of our current
understanding of the biology of this bacterium: A review of Cronobacter species. J. Appl. Microbiol.
113:1–15.
36. Kotiranta, A., K. Lounatmaa, and M. Haapasalo. 2000. Epidemiology and pathogenesis of Bacillus
cereus infections. Microbes Infect. 2:189–98.
37. Holbrook R. and J.M. Anderson. 1980. An improved selective and diagnostic medium for the
isolation of enumeration of Bacillus cereus in foods. Can. J. Microbiol., 26:753-759.

ACCEPTED MANUSCRIPT
Synthesis and Antibacterial profiles of Targeted
Triclosan Derivatives
Gemma L. Howse,a Richard A. Bovill,b# Peter J. Stephens,b Helen M. I.
Osborna#
a Reading School of Pharmacy, University of Reading, Whiteknights, Reading. RG6 6AD.
UK
b ThermoFisher Scientific, Wade Road, Basingstoke, Hampshire. RG24 8PW. UK
#
To whom correspondence should be addressed. Richard Bovill
:
Richard.bovill@thermofisher.com; Helen Osborn : h.m.i.osborn@reading.ac.uk
Highlights
•
Synthesis and characterisation of four α-D-glycopyranosides and three β-D-
glycopyranosides of the broad antibacterial agent triclosan, in generally very good
synthetic yields, and with excellent purities
•
•
•
All of the triclosan glycosides that were synthesized demonstrated antibacterial
activity against many of the organisms that were examined.
β-Galactoside (3a) and α-arabinoside (3c) had MIC values of 0.5 µg/ml for several
strains of S. aureus and S. haemolyticus.
Two Bacillus strains could be identified from various strains of Bacillus and
Staphylococcus after inoculation onto Nutrient Agar No. 2 with 0.25 µg/ml triclosan-
α-D-glucopyranoside (3e)