A new class of antimicrobial biosurfactants: Quaternary ammonium sophorolipids

Article abstract of DOI:10.1039/c5gc00120j

New synthetic pathways are proposed for the production of a broad range of innovative sophorolipid amines and sophorolipid quaternary ammonium salts starting from microbially produced sophorolipids. The selective formation of an intermediate sophorolipid

Full text of DOI:10.1039/c5gc00120j

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A new class of antimicrobial biosurfactants:
quaternary ammonium sophorolipids†
Cite this: DOI: 10.1039/c5gc00120j
a
a
b
b
a
E. I. P. Delbeke, B. I. Roman, G. B. Marin, K. M. Van Geem and C. V. Stevens*
New synthetic pathways are proposed for the production of a broad range of innovative sophorolipid
amines and sophorolipid quaternary ammonium salts starting from microbially produced sophorolipids.
The selective formation of an intermediate sophorolipid aldehyde proved to be a key synthetic step of the
new derivatives. The sophorolipid quaternary ammonium salts were evaluated for their antimicrobial
activity against Gram-negative and Gram-positive test strains. Minimum inhibitory concentration (MIC)
values were determined for the active compounds. Derivatives with an octadecyl group on the nitrogen
atom proved to be more active than the antibiotic gentamicin sulfate against all tested Gram-positive
strains. The results show great promise for modified sophorolipids in the medical sector.
Received 19th January 2015,
Accepted 15th April 2015
DOI: 10.1039/c5gc00120j
www.rsc.org/greenchem
building blocks in a synthetic pathway. This will reduce the
number of required steps to obtain the desired compounds
Introduction
In our modern society, the concept of sustainability gains and will additionally contribute to the sustainability of both
1
more interest from day to day. Indeed, over the last decades, process and end product. Target compounds for this ‘green
the transition towards a bio-based economy has initiated. The synthesis’ approach should preferably possess the desired pro-
significance of renewable resources for the chemical industry perties or biological activities which the traditionally obtained
is gradually increasing because they are considered good competing molecules lack.
alternatives for fossil resources, whose supplies are limited
In this light, sophorolipids are very useful building blocks
2
and which have a major environmental impact. At present for chemical derivatization. They occur in a lactonic form or
already more than 8% of all the chemicals produced in Europe an open acid form (Scheme 1). These glycolipids are produced
3
are based on renewable resources. Most of these renewable by micro-organisms from renewable resources through fermen-
4
resources are directly used as biomaterials such as paper, plas- tation. The yeast Starmerella bombicola is the preferred produ-
−
1
tics, textile and fibres or as biochemicals in paint, detergents, cer, with output of around 400 g L and a production price of
and cosmetics. However, the incorporation of renewable 2 to 5€ kg−
1 4a
.
Due to the presence of a hydrophilic carbo-
resources in high value added products is mostly based on the hydrate head and a hydrophobic lipid tail, sophorolipids
same concept as the one adopted for fossil resources, i.e. start- possess surface-active properties classifying them as biosurfac-
ing from small hydrocarbon building blocks. Traditionally, tants. They are low foaming surfactants which can be used for
fossil and renewable resources are broken down into base hard surface cleaning and automatic dishwashing rinse aid
5
chemicals which are then used for the synthesis of the desired applications. Nevertheless, application of sophorolipids in
products. Using this methodology, the cost of renewable based the detergent sector is limited because they have a competitive
pharmaceuticals outweighs the cost of their fossil counter- disadvantage compared to synthetic surfactants in terms
parts, giving renewable resources a competitive disadvantage of production cost. Therefore, it is desirable to look for
for the synthesis of these high value products. Obviously the
business case would be far more interesting if renewable
resources with a complex structure would be directly used as
a
SynBioC, Department of Sustainable Organic Chemistry and Technology, Ghent
University, Coupure Links 653, 9000 Ghent, Belgium.
E-mail: Chris.Stevens@UGent.be
b
LCT, Department of Chemical Engineering and Technical Chemistry, Ghent
University, Technologiepark 914, 9052 Ghent, Belgium
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
Scheme 1 Microbial produced sophorolipid lactone 1 and sophorolipid
acid 2.
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Scheme 2 Retrosynthetic scheme for sophorolipid quaternary
ammonium salts 3.
other application areas which are economically more attractive,
e.g. in the medical sector. Sophorolipids feature beneficial bio-
logical activities such as anticancer, anti-HIV, sperm-immobi-
6
lizing and antibacterial activity. Recently, the self-assembly
properties of sophorolipids have been described, demonstrat-
ing the formation of nanostructures with supramolecular chira-
lity. Until now, sophorolipid production is restricted to only
Scheme 3 Chemical modification towards sophorolipid aldehyde 5.
7
a few derivatives. Consequently, the variation in their surface-
active properties is limited and their biological activities have not
yet been optimized. Chemical modification offers the opportunity
to extend the limited set of microbial derivatives. In this paper,
chemical modification pathways are evaluated which increase the
applicability of sophorolipids for medical applications.
The double bond was cleaved through ozonolysis in MeOH,
and reductive work-up with NaBH(OAc)
3
furnished the perace-
tylated sophorolipid aldehyde 7 and methyl 9-oxononanoate
1
1
side product 8 (Scheme 3). At first, ozonolysis was attempted
in CH Cl , followed by reductive work-up with dimethyl
2
2
At present, chemical and enzymatic modification of sophoro-
lipids have been mostly limited to the sugar head or the lipid
sulfide. Analysis of the reaction mixture demonstrated the
presence of the desired peracetylated sophorolipid aldehyde 7,
but methyl 9-oxononanoate 8 could not be detected. Instead,
only the presence of a stable 1,2,4-trioxolane (ozonide) inter-
mediate was observed. The presence of high concentrations of
residual ozonides after work-up with dimethyl sulfide has
8
tail. Cleavage of the double bond, however, has only been
described for the synthesis of short-chained sophorolipid
9
acids or in ring-opening cross-metathesis reactions. It was
never included in a synthetic pathway for the production of a
functionalized building block towards modified derivatives.
This is surprising because such modification would result in
the formation of sophorolipid analogues with shorter chain
lengths, which would be better soluble in water.
Therefore in this work, a series of chemical modification
pathways is described for sophorolipids towards quaternary
ammonium salt derivatives (Scheme 2). First, an ozonolysis
reaction is performed on sophorolipids in order to transform
the double bond into a reactive site. In a second key step, a
nitrogen functionality is incorporated. Further modification
leads to a library of sophorolipid quaternary ammonium salts,
which are evaluated as antimicrobial compounds.
1
2
already been described by Dussault et al. Therefore, reaction
conditions were changed towards the use of the more environ-
3
mental friendly NaBH(OAc) . Besides, isolation of methyl
9
-oxononanoate 8 is most desirable for a green synthetic
pathway. This side product could be used as a valuable build-
ing block for other applications. Direct ozonolysis of the diace-
tylated sophorolipid lactone
1 was also evaluated. An
intermediate dialdehyde was obtained after ozonolysis, but
transesterification to the desired sophorolipid aldehyde was
not possible. Analysis of the reaction mixture demonstrated
the presence of aldol condensation product 9 formed from
methyl 9-oxononanoate indicating the non-compatibility of
the aldehyde function with alkaline reaction conditions
(
Scheme 4).
Results and discussion
Synthesis of the sophorolipid aldehyde intermediate
Care should be taken during the ozonolysis reaction due to
the formation of unstable ozonides and peroxides, and the use
A chemical modification pathway for sophorolipids was develo- of methanol as a solvent. The reaction is performed in a taped
ped starting from the major microbial product, i.e. the diace- washing flask on a small scale of 10–15 g peracetylated
1
0
tylated sophorolipid lactone 1. This lactone was transformed
into sophorolipid methyl ester 5 and, subsequently, into pera-
8
c,d
cetylated analogue 6 according to literature procedures.
Cleavage of the double bond through ozonolysis, offering an
aldehyde functionality, would create opportunities for the
incorporation of nitrogen. These nitrogen containing deriva-
tives can then easily be transformed into cationic surfactants.
Scheme 4 Observed aldol condensation product 9.
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sophorolipid methyl ester 6 at −78 °C. Ozone is generated from Table 2 Quaternization towards peracetylated sophorolipid quaternary
ammonium salts 11
dry air and is purged through the reaction mixture at a flow of
2
.15 L min− and a concentration of 6.2 mg L . For large
1
−1
scale reactions, use of microreactor equipment should be
1
3
considered.
The purification of the sophorolipid aldehyde 7 proved to
be a challenge due to the presence of saturated sophorolipids
from the fermentation process and degradation products from
the ozonolysis reaction. Multiple column chromatography
purifications were evaluated, giving the highest yield and
purity for automated flash chromatography with a gradient
elution of ethyl acetate and hexane. A sodium bisulfite
addition reaction was performed after the chromatography
purification to improve the purity but resulted in a very low
yield.
3
Entry
10
R I
Time (h)
Yield (%)
1
2
3
4
5
6
7
8
9
10a
10b
10c
10c
10d
10e
10f
Methyl iodide
Methyl iodide
Methyl iodide
Butyl iodide
Methyl iodide
Methyl iodide
Methyl iodide
Methyl iodide
Butyl iodide
18
18
18
48
18
18
18
18
48
11a
11b
11c
11d
11e
11f
11g
11h
11i
91
89
96
94
Quant.
89
Quant.
98
10g
10g
Quant.
Synthesis of a quaternary ammonium salt library
Next, a nitrogen functionality was introduced through reduc-
tive amination of the aldehyde function of 7 with a variety of
secondary amines (Table 1). Complete conversion to the result-
ing sophorolipid tertiary amines 10 was obtained, encompass-
ing methyl, butyl, benzyl and octadecyl groups. Purification of
sophorolipid tertiary amines 10 was necessary to avoid purifi-
cation of sophorolipid quaternary ammonium salts 11 in the
next step, but resulted in low yields.
Table 3 Transesterification towards sophorolipid quaternary ammonium
salts 10
Subsequently, the sophorolipid tertiary amines 10 were qua-
ternized with alkyl iodides in pressure vials to obtain the
desired peracetylated sophorolipid quaternary ammonium
salts 11 (Table 2). In a final step, the sugar head group of the
quaternary ammonium salts 11 was deprotected to obtain the
water soluble sophorolipid quaternary ammonium salts 3
Entry
11
Yield (%)
1
2
3
4
5
6
7
8
9
11a
11b
11c
11d
11e
11f
11g
11h
11i
3a
3b
3c
3d
3e
3f
3g
3h
3i
Quant.
88
Quant.
Quant.
Quant.
Quant.
99
(Table 3).
97
66
Evaluation of the antimicrobial activity
The antimicrobial activity of peracetylated sophorolipid
aldehyde 7, peracetylated sophorolipid amines 10a, 10b, 10c,
1
0d, 10f and 10g, peracetylated sophorolipid quaternary
ammonium salts 11 and deprotected sophorolipid quaternary LMG 2095, and the Gram-positive strains Staphylococcus aureus
ammonium salts
was evaluated. The Gram-negative LMG 8064 and Bacillus subtilis LMG 13579 were chosen as test
strains Escherichia coli LMG 8063 and Klebsiella pneumoniae strains. The bioassay was carried out in 96-well microtiter
3
−
1
plates at a concentration of approximately 0.5 mg mL of
4
−1
the test compound and 10 CFU mL test bacteria. None of
the test compounds showed significant growth inhibition
of Escherichia coli LMG 8063 and Klebsiella pneumoniae
LMG 2095. Compounds 11b, 11c, 11d, 11e, 11f, 11g, 11h,
Table 1 Reductive amination towards sophorolipid amines 10
11i, 3b, 3h and 3i showed significant growth inhibition of
Staphylococcus aureus LMG 8064 and Bacillus subtilis LMG 13579.
For the active compounds, the minimum inhibitory con-
centration (MIC) was determined against a test panel of four
Gram-positive bacterial strains, namely Staphylococcus aureus
LMG 8064, Enterococcus faecium LMG 11397, Bacillus subtilis
LMG 13579 and Streptococcus pneumoniae LMG 16738. The MIC
1
2
Entry
R R NH
Yield (%)
1
2
3
4
5
6
7
Dimethylamine
N-Methylbutylamine
Dibutylamine
N-Methylbenzylamine
N-Butylbenzylamine
Dibenzylamine
10a
10b
10c
10d
10e
10f
38
52
53 value is considered as the lowest concentration of the test com-
40
49
pound for which a lack of visible bacterial growth is observed.
This bioassay was carried out in 96-well microtiter plates at a
48
1
N-Methyloctadecylamine
10g
39 concentration range between 100 and 2.5 µg mL− or 1000 and
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Table 4 MIC values (µg mL− ) for the active compounds and the antibiotic gentamicin sulfate
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1
Gentamicin
sulfate
1
1b
11c
11d
11e
500
>1000
1000
1000
11f
11g
50
>100
50
11h
11i
3b
3h
3i
S. aureus
E. faecium
B. subtilis
>100
>100
>100
>100
>100
>100
25
>100
>100
>100
>100
>100
>100
>100
>100
10
10
10
10
10
10
10
10
>100
>100
>100
>100
5
5
5
5
5
5
5
5
5
10
5
S. pneumoniae
100
100
25
Table 5 MIC values (µM) for the active compounds and the antibiotic gentamicin sulfate
Gentamicin
sulfate
1
1b
11c
11d
11e
489
977
977
11f
11g
11h
11i
3b
3h
3i
S. aureus
E. faecium
B. subtilis
>101
>101
>101
>101
>97
>97
24
>93
>93
>93
>93
>94
>94
>94
>94
45
>91
45
8
8
8
8
8
8
8
8
>144
>144
>144
>144
6
6
6
6
5
5
5
5
10
21
10
52
S. pneumoniae
97
>977
91
µg mL− of the test compounds for respectively strong or weak lipid quaternary ammonium salts have been evaluated for
1
5
4
−1
inhibitors and 10 CFU mL test bacteria. MIC values for the their antimicrobial activity against the Gram-negative strains
active compounds are given in Table 4 together with MIC values Escherichia coli LMG 8063 and Klebsiella pneumoniae LMG
for the antibiotic gentamicin sulfate. Microscopic analysis in 2095, and the Gram-positive strains Staphylococcus aureus LMG
addition to the determination of the MIC values revealed that 8064 and Bacillus subtilis LMG 13579. Eight of the peracety-
lysis of the cells occurred at the active concentrations.
lated sophorolipid quaternary ammonium salts and three of
The lowest MIC value of 5 µg mL−1 was obtained with com- the deprotected sophorolipid quaternary ammonium salts
pounds 3h and 3i against all four Gram-positive test strains. showed inhibition against the Gram-positive strains, but not
−
1
Low MIC values of 10 µg mL were obtained with compounds against the Gram-negative strains. For the active compounds,
1h and 11i, also against all four Gram-positive test strains. their minimum inhibitory concentration (MIC) has been deter-
1
Interestingly, these activities lie in the same concentration mined against the four Gram-positive strains Staphylococcus
range as that of the antibiotic gentamicin sulfate. All four com- aureus LMG 8064, Enterococcus faecium LMG 11397, Bacillus
pounds perform as good or better as gentamicin sulfate subtilis LMG 13579 and Streptococcus pneumoniae LMG 16738.
against E. faecium and S. pneumoniae. Compounds 3h and 3i The best results are obtained with N,N-dimethyl,N-octadecyl-
even perform as good as gentamicin sulfate against S. aureus (8-L-[(2″,3′,3″,4′,4″,6′,6″-heptaacetoxy-2′-O-β-D-glucopyranosyl-
and B. subtilis. For better comparison, the MIC values were β-D-glucopyranosyl)-oxy])nonan-1-ammonium iodide 11h, N-
converted on the basis of their molecular weight (Table 5). On benzyl,N-methyl,N-octadecyl-(8-L-[(2″,3′,3″,4′,4″,6′,6″-heptaace-
this basis, we can conclude that compounds 3h, 3i, 11h and toxy-2′-O-β-D-glucopyranosyl-β-D-glucopyranosyl)-oxy])nonan-1-
1
1i are more active against all four Gram-positive test strains ammonium iodide 11i, N,N-dimethyl,N-octadecyl-(8-L-
than the antibiotic gentamicin sulfate. These results show [(2β-O-β-D-glucopyranosyl-β-D-glucopyranosyl)-oxy])nonan-1-
great promise for further evaluation of the biological activities ammonium iodide 3h and with N-benzyl,N-methyl,N-octadecyl-
of the active derivatives such as synergistic effects of multiple (8-L-[(2β-O-β-D-glucopyranosyl-β-D-glucopyranosyl)-oxy])nonan-1-
compounds against specific test strains or activities of specific ammonium iodide 3i. These four compounds are more active
compounds against multiple test strains. The active derivatives than the antibiotic gentamicin sulfate against all four Gram-
also offer opportunities for their use in medical applications positive test strains. These results show great promise for
such as the inhibition of biofilm formation. These sophoro- further evaluation of the biological activities of sophorolipid
lipid quaternary ammonium salts represent a new class of quaternary ammonium salts with an octadecyl group on the
antimicrobial surfactants.
nitrogen atom and their use for medical applications. These
sophorolipid quaternary ammonium salts represent a new
class of antimicrobial surfactants.
Conclusions
Microbial produced sophorolipids have been chemically modi-
fied into a series of novel sophorolipid amines and sophoro-
Acknowledgements
lipid quaternary ammonium salts via an intermediate The research leading to these results has received funding
sophorolipid aldehyde. The sophorolipid amines and sophoro- from the Long Term Structural Methusalem Funding by the
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Flemish Government (grant number BOF09/01M00409). The
authors gratefully acknowledge the company Ecover (Malle,
Belgium) and the InBio research group (Department of Bio-
chemical and Microbial Technology, Ghent University,
Belgium) for the delivery of the sophorolipid starting com-
pound, and the Laboratory for Microbiology (Ghent University,
Belgium) for the evaluation of the antimicrobial activities.
Bart I. Roman is a fellow of the Fund for Scientific Research –
Flanders (FWO-Vlaanderen).
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