Identification, library synthesis and anti-vibriosis activity of 2-benzyl-4-chlorophenol from cultures of the marine bacterium Shewanella halifaxensis

Article abstract of DOI:10.1016/j.bmcl.2016.05.002

Summer Gut Syndrome (SGS) is caused by various Vibrio bacterial species and can have negative effects on aquaculture farms worldwide. In New Zealand, SGS is caused by Vibrio harveyii infecting King Salmon (Oncorhynchus tshawytscha). To find leads for the prevention of SGS, we screened the inhibitory effects of 16 strains of Shewanella upon V. harveyii growth in competitive solid phase cultures. The detailed investigation of Shewanella halifaxensis IRL548 revealed 2-benzyl-4-chlorophenol (1), a known, commercially available antibacterial agent, as the major bioactive component. Synthesis of a small library of congeners to confirm the natural product identity and to provide a structure-activity relationship for the observed activity was also completed. Compound 1 exhibits moderate activity against two pathogenic microorganisms.

Full text of DOI:10.1016/j.bmcl.2016.05.002

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification, library synthesis and anti-vibriosis activity of
2-benzyl-4-chlorophenol from cultures of the marine bacterium
Shewanella halifaxensis
Sarah L. Moore a,y, Lucile Berthomier a,y, Chriselle D. Braganza a, Joanna K. MacKichan b, Jason L. Ryan c,
Gabriel Visnovsky d, Robert A. Keyzers a,
⇑
a Center for Biodiscovery, and School of Chemical and Physical Sciences, Victoria University of Wellington, PO BOX 600, Kelburn, Wellington 6140, New Zealand
b Center for Biodiscovery, and School of Biological Sciences, Victoria University of Wellington, PO BOX 600, Kelburn, Wellington 6140, New Zealand
c Integrated Bioactive Technologies Group, Callaghan Innovation, Gracefield, PO Box 31310, Lower Hutt 5040, New Zealand
d Chemical and Process Engineering, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Summer Gut Syndrome (SGS) is caused by various Vibrio bacterial species and can have negative effects
on aquaculture farms worldwide. In New Zealand, SGS is caused by Vibrio harveyii infecting King Salmon
(Oncorhynchus tshawytscha). To find leads for the prevention of SGS, we screened the inhibitory effects of
16 strains of Shewanella upon V. harveyii growth in competitive solid phase cultures. The detailed inves-
tigation of Shewanella halifaxensis IRL548 revealed 2-benzyl-4-chlorophenol (1), a known, commercially
available antibacterial agent, as the major bioactive component. Synthesis of a small library of congeners
to confirm the natural product identity and to provide a structure–activity relationship for the observed
Received 13 March 2016
Revised 2 May 2016
Accepted 3 May 2016
Available online xxxx
Keywords:
Chlorophene
New Zealand
Shewanella
Chlorophenol
Probiotic
activity was also completed. Compound
microorganisms.
1
exhibits moderate activity against two pathogenic
Ó 2016 Published by Elsevier Ltd.
Significant recent advances in aquaculture, especially in con-
trolling dietary sources,1,2 have made the production of large vol-
umes of fish possible, although the high density of individuals
maintained within a cage make the population susceptible to
widespread and potentially catastrophic infection by pathogenic
microrganisms.2 In New Zealand, King (also known as Chinook)
Salmon (Oncorhynchus tshawytscha) are a valuable aquaculture
commodity farmed at various locations around the country. During
the warmer summer months, Salmon with suppressed immune
systems caused by diets enriched in non-animal based proteins,
along with overcrowding, can result in intestinal imbalance of
microbiota.3 This in turn can lead to infection by Vibrio harveyii,
the pathogen responsible for Summer Gut Syndrome (SGS), also
known as vibriosis.4 SGS presents symptoms such as vomiting,
diarrhea, loss of appetite and if left untreated, could be fatal. Con-
sequently, SGS is a large potential risk factor for the local King Sal-
mon aquaculture industry.
poration of preventative antibiotics into fish-feed economically or
environmentally viable. One alternative route to prevent SGS
would be the inclusion of a suitable probiotic microorganism into
the Salmon’s fish-feed. Bacteria of the genus Shewanella are
Gram-negative facultative anaerobes found from many marine
environments. Species of Shewanella have already been investi-
gated as potential probiotics for both sole5,6 and abalone7 aquacul-
ture, with improved resistance and a reduction of mortality noted
for treated abalone challenged with V. harveryii.7 Recent work by
Linington and coworkers has indicated that the gut microflora of
commercial fish species could be useful reservoirs of new micro-
bial strains,8 therefore we investigated multiple strains of She-
wanella from the guts of several fish species caught in New
Zealand waters. Solid-phase co-culture of 16 Shewanella species
with V. harveyi indicated eight inhibited Vibrio growth. Investiga-
tion of four of these strains resulted in the isolation of fifteen
diketopiperazines, indole and 3-carboxyindole,9 although none of
these appeared to be responsible for the observed Vibrio inhibition.
Diketopiperazines have been suggested as quorum-sensing
metabolites in Shewanella baltica previously.10
Treating large numbers of farmed fish with antibiotics is not
easily controlled in a marine aquaculture setting, nor is the incor-
Detailed investigation of the liquid culture of Shewanella
halifaxensis strain IRL548,11 sourced from the guts of a Bluenose
fish, sequentially purified by various chromatographic processes
⇑
Corresponding author. Tel.: +64 4 463 5117; fax: +64 4 463 5241.
E-mail address: rob.keyzers@vuw.ac.nz (R.A. Keyzers).
Contributed equally to this project.
y
http://dx.doi.org/10.1016/j.bmcl.2016.05.002
0960-894X/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Moore, S. L.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.002

2
S. L. Moore et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
(Supplementary materials), led to the isolation of 2-benzyl-
4-chlorophenol (1) as the main bioactive component. To the best
of our knowledge, although 1 is a known antibacterial agent used
in hospital disinfectants,12 this is the first Letter of it as a natural
product. To confirm the structure of the compound and to probe
a possible structure–activity relationship (SAR), the synthesis of 1
and a small series of analogues was performed, and the bioactivity
of both the natural product and analogues assessed against three
pathogenic bacterial isolates. The results of the isolation, synthesis
and bioactivity profiling of 1 and analogues are presented herein.
A 20 L culture of S. halifaxensis IRL548 was grown using marine
medium under aerobic conditions. This culture was clarified by
centrifugation after which the supernatant was passed through a
column of HP-20 poly-(styrene-divinylbenzene) copolymer that
was subsequently eluted using 30%, 75% and 100% mixtures of
acetone in water. The 75% acetone elution was subsequently
re-suspended in methanol and filtered, with the methanol-soluble
portion being chromatographed using a stepped gradient of 50%
methanol in water to methanol using HP-20ss. The methanol
fraction was finally separated using C18 HPLC to yield compound
1 as a colorless solid.
The chemical formula of 1 (C13H11OCl) was determined from
the deprotonated molecule detected using high mass accuracy
ESI-MS (det. m/z 217.0434; calcd m/z 217.0426), and in particular
the presence of chlorine identified from the distinctive 3:1 M:M
+2 ratio of chlorine isotopomers. The structure of 1 was rapidly
determined from interpretation of comprehensive 1D and 2D
NMR data. In particular, only 11 13C resonances were detected,
10 of which were indicative of aromatic carbon signals, suggesting
the presence of either a mono- or para-disubstituted benzene ring
due to internal symmetry arguments. A monosubstituted benzene
ring was inferred from the characteristic doublet/triplet/triplet 1H
NMR multiplicities in a 2:2:1 integration ratio. This left both a
meta- and an ortho-coupled doublet, and an ortho,meta-coupled
doublet of doublet, and a highly deshielded methylene to be
assigned. Through the use of chemical shift arguments in conjunc-
tion with key HMBC correlations (Fig. 1), the structure of 1 could be
assigned as 2-benzyl-4-chlorophenol (Table 1). A thorough litera-
ture survey using several commercial databases (MarinLit,13
AntiBase,14 SciFinder Scholar15) could find no evidence that 1 has
been reported as a natural product previously although it is a
well-known synthetic material, having been synthesized as early
as 1932.16,17
1H (600 MHz) and 13C (150 MHz) NMR data of 2-benzyl-4-chlorophenol (1), CD3OD
Position
dC
dH
Multiplicity, J (Hz), rel. int.
1
2
3
4
155.1
131.3
130.9
124.8
127.8
117.1
36.4
141.9
129.9
129.3
127.0
—
—
—
—
6.91
—
6.98
6.73
3.89
—
7.20
7.25
7.16
d, (2.6), 1H
—
dd, (8.5, 2.6), 1H
d, (8.6), 1H
s, 2H
5
6
10
100
—
200/500
300/600
400
d, (7.4), 2H
t, (7.4), 2H
t, (7.1), 1H
OH
OH
OH
1
1
1"
1"
1"
1'
3"
3"
3"
1'
1'
ZnCl2
60°C
+
+
+
Br
Br
Br
+
5
3
3
3
Cl
Cl
Cl
1
2
OH
OH
1
OH
1
Cl
Cl
Cl
1'
ZnCl
Cl
Cl
60°C2
+
5
3
5
3"
3
1"
1'
1'"
1"
1""
3""
3"
1"
3
4
OH
OH
OH
OH
1
1
1"
3"
1
1'
ZnCl2 3"
60°C
1'
+
+
Cl
5 Cl
5
3
3
5
3
1'"
1""
1'
1"
3"
3""
5
6
7
Scheme 1. Library synthesis of benzylchlorophenol analogues from benzyl bromide
and various chlorophenols.
Chlorophenols are known to exert their antiseptic bioactivity by
interfering with the cell membrane, and by uncoupling of oxidative
phosphorylation.18 All seven synthesized compounds were
assessed for their bioactivity against Escherichia coli, Staphylococcus
aureus, and most importantly for the purposes of this project, Vibrio
harveyii. Although 1 is a known antibiotic compound, we could find
only one previous Letter of the bioactivity of 1 versus a species of
Vibrio (V. fischeri)19 hence it was of interest to assess the activity of
the compound against the more relevant pathogenic isolate.
All of the synthesized compounds were moderately active
against both E. coli and V. harveyii, although somewhat surpris-
ingly, only the dibenzylated analogues 2, 4, 6 and 7 were active
against the Gram positive pathogen S. aureus (Table 2). This result
was unexpected given the use of 1 as a clinical disinfectant within
hospitals and within antibacterial cosmetics and other personal
grooming products. Intriguingly, the natural product was also the
least active compound tested. No other clear SAR could be
determined.
To confirm the structure of 1 and to establish any potential SAR,
Friedel–Crafts alkylation of ortho, meta and para-chlorophenol with
benzyl bromide was used to provide material for biological evalu-
ation. The method of Klarmann et al.16 was used to prepare an
authentic standard of 1 for spectroscopic analysis from para-
chlorophenol. The other two possible regioisomers were also used
to provide simple analogues for comparison of bioactivity. All three
regioisomers also provided bis-benzyl adducts in addition to the
expected mono-addition products (Scheme 1). The spectroscopic
data of both natural and synthetic 1 were identical, confirming
the structure of the isolated metabolite (Figs. S1 and S2).
Table 2
Bioactivity of the synthetic benzylchlorophenol library
OH
Compound
IC50 (lM)
1
1"
3"
1'
S. aureus
E. coli
V. harveyii
3
5
1
2
3
4
5
6
7
NS
19.4
NS
13.9
NS
NS
54.9
35.6
50.3
35.6
50.3
50.3
35.6
41.2
24.3
38.9
29.1
38.9
36.6
24.3
1
Cl
OH
= COSY
= HMBC
Cl
6.5
NS = not sensitive.
Figure 1. Key COSY and HMBC correlations used to establish the structure of 1.
Please cite this article in press as: Moore, S. L.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.002

S. L. Moore et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Also commonly known as chlorophene, 1 is used clinically as a
medical disinfectant and within personal grooming products.12,20
To probe the biosynthesis of 1, attempts were made to culture
Shewanella halifaxensis IRL548 with NaBr instead of NaCl, to try
and promote production of a brominated analogue however the
bacteria would not grow with this salt replacement. To validate
that the isolated compound is in fact a true natural product and
was not the product of the highly unlikely and inadvertent con-
tamination of the culture, a repeat culture was prepared and the
same compound was detected by both NMR and MS analyses in
the culture supernatant (Fig. S18).
biosynthetic capacity to produce unusual bioactive compounds
and may be a valuable resource for both probiotics themselves,
and also for discovering new natural product leads for other
biotechnological uses.
Acknowledgments
Funding to carry out this research from Victoria University of
Wellington and Callaghan Innovation is gratefully acknowledged,
as are scholarships from VUW (S.L.M., C.D.B.) and the Graduate
Women of Wellington (S.L.M.).
Chlorophene has a wide range of observed bioactivities, includ-
ing antimicrobial properties, hence its use as a disinfectant. It has
been suggested, however, to be a carcinogen and possible fertility
disruptor20 and hence its widespread use where the potential for
human exposure, including within a food product, should be
limited. The production of 1 as a microbial metabolite is unex-
pected yet given its broad bioactivities, the ecological advantages
to S. halifaxensis in controlling its local microenvironment are
potentially high. Although the inhibition of V. harveyii by 1, deter-
mined by our study, is only modest in potency, this may be enough
to control local populations of the pathogen in vivo.
As several strains of Shewanella have already been trialed as
aquaculture probiotics,5–7 the use of S. halifaxensis IRL548 in a sim-
ilar role has precedence. Improved rates of survival by abalone
treated with S. colwelliana WA64 and S. olleyana WA65 challenged
with V. harveyii7 gives a promising outlook for the use of S. halifax-
ensis in New Zealand with farmed King Salmon. Such a use, how-
ever, must first be evaluated in light of the wide variety of
bioactivities associated with the compound although indications
are that 1 should not bioaccumulate in the fatty tissue of fish
and hence may pose minimal risk following human consumption,
especially as it exhibits only a low oral toxicity.20 The long-term
effect of exposing King Salmon to Shewanella halifaxensis IRL548
would need to be the first step in the evaluation of the probiotic
potential of this common bacteria.
Supplementary data
Supplementary data (full experimental details of the culture of
S. halifaxensis IRL548, isolation of 1, synthesis of 1–7 and NMR
spectra of all compounds) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.05.
002.
References and notes
1. De Santis, C.; Tocher, D. R.; Ruohonen, K.; El-Mowafi, A.; Martin, S. A. M.;
Dehler, C. E.; Secombes, C. J.; Crampton, V. Aquaculture 2016, 452, 169.
2. Dawood, M. A. O.; Koshio, S. Aquaculture 2016, 454, 243.
3. Green, T. J.; Smullen, R.; Barnes, A. C. Vet. Microbiol. 2013, 166, 286.
4. Diggles, B. K.; Hine, P. M.; Handley, S.; Boustead, N. C. A Handbook of Diseases
Important to Aquaculture in New Zealand; NIWA, 2002.
5. Tapia-Paniagua, S. T.; Vidal, S.; Lobo, C.; Prietoa-Álamo, M. J.; Jurado, J.; Cordero,
H.; Cerezuela, R.; Garc´ia de la Banda, I.; Esteban, M. A.; Balebona, M. C.;
Moriñigo, M. A. Fish Shellfish Immunol. 2014, 41, 209.
´
6. Garcia de la Banda, I.; Lobo, C.; Chabrillón, M.; León-Rubio, J. M.; Arijo, S.; Pazos,
G.; Lucas, L. M.; Moriñigo, M. A. Aquacult. Res. 2012, 43, 662.
7. Jiang, H.-F.; Liu, X.-L.; Chang, Y.-Q.; Liu, M.-T.; Wang, G.-X. Fish Shellfish
Immunol. 2013, 35, 86.
8. Sanchez, L. M.; Wong, W. R.; Riener, R. M.; Schulze, C. J.; Linington, R. G. PLoS
One 2012, 7, e35398.
9. Braganza, C.D. MSc Dissertation, Victoria University of Wellington, 2014.
10. Gu, Q.; Fu, L.; Wang, Y.; Lin, J. J. Agric. Food Chem. 2013, 61, 11645.
11. Ryan, J.; Farr, H.; Visnovsky, S.; Vyssotski, M.; Visnovsky, G. J. Microbiol.
Methods 2010, 82, 49.
In conclusion, a known, commercially available disinfectant
compound, 2-benzyl-4-chlorophenol (1) was isolated from a cul-
ture of Shewanella halifaxensis IRL548 as a natural product for the
first time. This bacterial strain had been selected for investigation
due to its inhibition of Vibrio harveyii, the pathogen responsible for
Summer Gut Syndrome in farmed New Zealand King Salmon, and
hence it may be a useful lead strain for inclusion as a probiotic.
Synthesis of 1 and analogues allowed for the bioassay testing of
the library, with all compounds showing activity against V. harveyii
and E. coli. This study reveals that species of Shewanella have
12. Yamarik, T. A. Int. J. Toxicol. 2004, 23, 1.
13. Royal Society of Chemistry, 2015.
14. Wiley-VCH, 2009.
15. Chemical Abstracts Service, 2015.
16. Klarmann, E.; Gates, L. W.; Shternov, V. A. J. Am. Chem. Soc. 1932, 54, 3315.
17. Huston, R. C.; Guile, R. L.; Chen, P. S.; Headley, W. N.; Warren, G. W.; Baur, L. S.;
Mate, B. O. J. Am. Chem. Soc. 1933, 55, 4639.
18. McDonnell, G.; Russell, A. D. Clin. Microbiol. Rev. 1999, 147.
19. Davoren, M.; Fogarty, A. M. Ecotoxicol. Environ. Safe 2005, 60, 203.
20. Committee for Risk Assessment, European Chemicals Agency Report, CLH-O-
0000001412-86-58/F, 2015.
Please cite this article in press as: Moore, S. L.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.002