Enzyme mediated-transesterification of verbascoside and evaluation of antifungal activity of synthesised compounds

Article abstract of DOI:10.1080/14786419.2014.984181

Enzymatic acylation of verbascoside, a polyhydroxylated natural product, has been reported in this study using five different commercial lipases and taking p-nitrophenyl alkanoates as acyl donors. Out of these enzymes, the immobilised Candida antarctica lipase B was found as the enzyme of choice. Mono-and di-acylated products were formed, with mono as major product indicating high regioselective nature of such transformations. A series of acyl esters of verbascoside have been synthesised by this enzymatic transesterification methodology. The lipophilicity of the synthesised analogues was also checked. The analogues were further subjected to synergistic antifungal activity with amphotericin B (AmB) against Candida albicans. Fourfold reduction in minimum inhibitory concentration of AmB was observed with few synthesised analogues such as verbascoside 4″-octanoate (3b), verbascoside 4″-palmitate (3d) and verbascoside 4″,4′-dipalmitate (4d) at a concentration of 0.5 g/mL.

Full text of DOI:10.1080/14786419.2014.984181

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Enzyme mediated-transesterification
of verbascoside and evaluation of
antifungal activity of synthesised
compounds
ab
c
c
Jabeena Khazir , Intizar Ali , Inshad Ali Khan & Halmuthur
d
Mahabalarao Sampath Kumar
a
Medicinal Chemistry Division, Indian Institute of Integrative
Medicine (IIIM), Canal Road, Jammu Tawi 180001, India
b
Department of Chemistry, University of Pretoria, Pretoria 0028,
Click for updates
South Africa
c
Clinical Microbiology Division, Indian Institute of Integrative
Medicine (IIIM), Canal Road, Jammu Tawi 180001, India
d
NPC Division, Indian Institute of Chemical Technology,
Hyderabad 500007, India
Published online: 01 Dec 2014.
To cite this article: Jabeena Khazir, Intizar Ali, Inshad Ali Khan & Halmuthur Mahabalarao Sampath
Kumar (2015) Enzyme mediated-transesterification of verbascoside and evaluation of antifungal
activity of synthesised compounds, Natural Product Research: Formerly Natural Product Letters,
29:8, 727-734, DOI: 10.1080/14786419.2014.984181
To link to this article: http://dx.doi.org/10.1080/14786419.2014.984181
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Natural Product Research, 2015
Vol. 29, No. 8, 727–734, http://dx.doi.org/10.1080/14786419.2014.984181
Enzyme mediated-transesterification of verbascoside and evaluation of
antifungal activity of synthesised compounds
ab
Jabeena Khazir *, Intizar Ali , Inshad Ali Khan and Halmuthur Mahabalarao Sampath Kumar
c
c
d
a
Medicinal Chemistry Division, Indian Institute of Integrative Medicine (IIIM), Canal Road, Jammu Tawi
b
80001, India; Department of Chemistry, University of Pretoria, Pretoria 0028, South Africa; Clinical
c
1
Microbiology Division, Indian Institute of Integrative Medicine (IIIM), Canal Road, Jammu Tawi 180001,
d
India; NPC Division, Indian Institute of Chemical Technology, Hyderabad 500007, India
(
Received 9 July 2014; final version received 28 October 2014)
OH
OH
HO
O
OH
HO
O
OH
OH
OH
HO
RO
Lipases
O
O
O
OH
OH
OH
O
OH
OR
OH
O
O
O
O
O
O
OH
Verbascoside
OH
O
R =
n
n= 0, 5, 11, 13
Enzymatic acylation of verbascoside, a polyhydroxylated natural product, has been
reported in this study using five different commercial lipases and taking p-nitrophenyl
alkanoates as acyl donors. Out of these enzymes, the immobilised Candida antarctica
lipase B was found as the enzyme of choice. Mono- and di-acylated products were
formed, with mono as major product indicating high regioselective nature of such
transformations. A series of acyl esters of verbascoside have been synthesised by this
enzymatic transesterification methodology. The lipophilicity of the synthesised
analogues was also checked. The analogues were further subjected to synergistic
antifungal activity with amphotericin B (AmB) against Candida albicans. Fourfold
reduction in minimum inhibitory concentration of AmB was observed with few
0
0
00
synthesised analogues such as verbascoside 4 -octanoate (3b), verbascoside 4 -
0
0
0
palmitate (3d) and verbascoside 4 ,4 -dipalmitate (4d) at a concentration of 0.5 mg/mL.
Keywords: transesterification; verbascoside; acylation; lipophilicity; antifungal
activity
1
. Introduction
Phenylethanoid glycosides constitute an important class of natural products possessing wide
range of biological activities (Fu et al. 2008). Verbascoside (Scarpati & Delle 1993) also named
as acteoside was first isolated from the flowers of Syringa vulgaris (Birkofer et al. 1968); it is a
phenylethanoid glycoside and is widely distributed in plants such as Ligstrum purpurascens
(Wong et al. 2001), Callicarpa dichotoma (Koo et al. 2006; Lee et al. 2006), Cistanche
deserticola and Boschniakia rossica (Wu et al. 2006), Scrophularia ningpoenis (Huang et al.
*
Corresponding author. Email: jabina.khazir@gmail.com
q 2014 Taylor & Francis

7
28
J. Khazir et al.
2
008) and Rehmannia glutinosa (Li et al. 2006). It is a highly water soluble polyphenolic
molecule. In vitro and in vivo pharmacological studies have shown that verbascoside possesses
various pharmacological activities, such as anti-inflammatory (Diaz et al. 2004), hepatoprotec-
tive (Xiong et al. 1999), anti-apoptotic, antioxidative (Chiou et al. 2004), anticancer (Kyung et al.
2
007) and neuroprotective (Sheng et al. 2002; Koo et al. 2006; Li et al. 2008). Verbascoside and
its analogues have also been reported to effectively scavenge 1,1-diphenyl-2-picrylhydrazyl and
nitric oxide in vitro (Chen et al. 2009; Liu et al. 2013)
Besides the above-mentioned biological properties of verbascoside, it is also reported that
verbascoside exhibits synergistic antifungal activity with antifungal drug amphotericin B (AmB)
against various pathogenic Candida species (Ali et al. 2011). AmB, an antifungal antibiotic used
for the treatment of invasive fungal infections from the 1950s till date, has various drawbacks
and has become imperative to search for safe and more-effective chemotherapeutic approaches
for the treatment of invasive fungal infections. Combination therapy has been found to be an
important strategy, as synergistic interactions can potentially increase antifungal efficacy,
reduce toxicity, cure faster, prevent the emergence of resistance and provide broader-spectrum
antifungal activity than monotherapy regimens (Richardson 2005). While exploring verbasco-
side for antifungal activity, when synergistically used along with AmB it potentiated the
membrane disruption activity of AmB against a wide range of pathogenic strains of Candida
albicans, Cryptococcus neoformans and Aspergillus fumigatus resulting in a strong fungicidal
effect (Ali et al. 2011).
Recently, verbascoside has been found to show a very low oral bioavailability (BA) of 0.12%
Wu et al. 2012; Qia et al. 2013). It also shows a high aqueous solubility owing to its
(
polyhydroxylated structure. However, a proper hydrophilic–lipophilic balance is very important
for sustained release of a molecule in the biological system. In order to increase the structural
diversity, and to improve physical and chemical properties for better pharmacological and
pharmacokinetic attributes, acylation of natural and synthetic organic compounds is a common
synthetic protocol (Singh et al. 2013). Therefore, transesterification was chosen for selective
acylation of verbascoside with acyl groups of varying side chains to improve its BA and
subsequent antifungal potentiating effect. Catalysis of the transesterification reaction is
generally carried by chemical and enzymatic methods. Currently, the chemical transesterifica-
tion methods are the major industrial approach, however; due to its obvious drawbacks, such as
formation of non-specific products and unwanted by-products and a limited choice of reagents
needed to accomplish a selective acylation, it is challenging (Richardson 2005). Enzymatic
acylation of polyhydroxylated compounds has been proved to be one of the most attractive
alternatives to the conventional chemical methods due to its high selectivity and simplicity
(
Richardson 2005). Enzyme catalysed reactions offer a highly efficient process under mild
conditions, diminish undesired side reactions, and facilitate more pure product recovery.
Furthermore, enzymes are proteins, and as such they are completely biodegradable and
environment friendly (Singh et al. 2009). As a result, this method has been extensively used for
the esterification of carbohydrates, nucleosides and other natural products. The use of lipases as
biocatalysts for selective acylation of polyhydroxylated natural products such as flavonoids,
saponins and other polyphenolic compounds have been extensively studied (Svendsen 1994;
Akimoto et al. 1999; Ferrero & Gator 2000; Kontogianni et al. 2001; Riva 2002; Park et al. 2003;
Intra et al. 2004; Gonzalez-Sab ´ı n et al. 2011). Lipases accept a wide array of complex molecules
as substrates and catalyse reactions with high enantio- and regioselectivity. Keeping in view the
tremendous biological activity and potentiating fungicidal effect of verbascoside on AmB
against wide range of invasive fungal pathogens, this study was aimed to synthesise a focused
library of selectively acylated analogues of verbascoside with different HLB by employing
commercially available lipases for the evaluation of synergistic antifungal activity with AmB
against C. albicans (ATCC 9002).

Natural Product Research
729
2
2
. Results and discussion
.1. Chemical acylation
Chemical acylation of verbascoside was carried through its reaction with two anhydrides, namely
acetic anhydride and butyric anhydride in dry DCM/DIMAP (dichloro methane/dimethylamino
pyridine) (Liljeblad et al. 2010). Acylation through this method resulted in peracylated products
which were characterised by spectral analysis. These peracylated compounds when studied for
change in lipophilicity using Schrodinger software were found to show drastic increase in C log P
(
Table S1). Thus, direct chemical acylation of verbascoside was not appropriate, due to lack of
suitable reagents and protocols to discriminate among specific hydroxyl groups of the same
molecule and to acylate regioselectively one over several hydroxyl groups present on the scaffold.
Furthermore, this molecule being highly sensitive to acidic and thermal conditions, the acid-
sensitive protection–deprotection strategies cannot be employed while designing its regioselective
acylation. Therefore, we switched towards the enzymatic transesterification using various
commercial lipases and a suitable labile ester as acyl transfer agent.
2
.2. Enzymatic acylation
Enzymes are attractive bio-catalysts due to their cost-effective production, stereo- and
regioselectivity, simple and fast reaction and rational alteration of properties. Therefore initially,
transesterification of the verbascoside was studied using a panel of commercially available
lipases and p-nitropalmitate was used as an acyl donor.
Five commercial lipases, namely Candida rugosa lipase, lipase acrylic resin from Candida
antarctica (lipase B), lipase from wheat germ, Candida cylindracea lipase and porcine
pancreatic lipase, were used in this study to investigate the regioselective acylation of
verbascoside using p-nitrophenyl palmitate in dry organic solvent. These commercial enzymes
were screened to evaluate their potential in terms of selectivity and yields.
The substrate solubility was assayed in different solvents, owing to the poor solubility of
verbascoside in less polar solvents, dimethylformamide (DMF) was found to be suitable solvent
for carrying out the enzymatic reaction. For each lipases used, two acylated products of
verbascoside were formed, with mono-acylated in major yield (Scheme 1). It was very
interesting to note that all the five enzymes showed same specificity in transesterification;
however, they only differed in the percentage conversion of the reaction. While with soluble
OH
OH
HO
O
OH
OH
HO
O
OH
OH
3
"
"
O
R
NO2
HO 4"
RO
2
O
OH
5
"
1"
5'
4'
O
OH
9
" O
OH
OH
6
'
O
OH
OH
8
"
6
"
7"
8'
1'
2
'
Candida antartica lipase, dry DMF
O
3'
O
O
O
7'
O
O
1
Molecular sieves, 4 A°
OH
OH
3
a-3d
OH
O
O
O
HO
OH
OH
Where R=
RO
5
O
O
1
1
3
a, 4a
O
OH
OR
OH
3
b, 4b
3
c, 4c
O
O
O
O
OH
4a-4d
1
5
3
d, 4d
Scheme 1. Acylation of verbascoside using p-nitrophenyl alkanoate.

7
30
J. Khazir et al.
lipases, reaction conversion was lower ranging from 12% (porcine pancreatic lipase) to 21%
lipase from wheat germ); whereas higher conversion of approximately 50% was achieved with
(
resin-bound immobilised C. antarctica lipase B. Owing to the higher conversion rate, resin-
bound immobilised C. antarctica lipase B was the catalyst of choice for transesterification.
Various acylated products thus obtained were purified through column chromatography and
1
13
their structures determined by using H NMR, C NMR and mass spectra (MS). After
introducing various long fatty acid chains, change in lipophilicity was calculated using
Schrodinger software (Table S1).
Using C. antarctica lipase B, and different long-chain p-nitrophenyl alkanoates as acyl
donors, two types of products, mono- (3a–3d) and di-acylated (4a–4d) compounds were
observed with mono-acylated compounds in major yields (35%) indicating high regioselectivity
of such transformations. In all the cases, acylation was taking place on phenolic hydroxyls. The
1
structures of the compounds were confirmed by using H NMR in which a shift of the protons
attached to the carbon adjacent to the carbon whose hydroxyl was getting acylated was
0
0
1
observed, as in the case of ‘verbascoside 4 -butyrate’; H NMR signal at d 7.26 (J ¼ 8.50 Hz)
00
corresponding to H-5 of the caffeic acid moiety appeared downfield (0.45 ppm) compared with
its parent d 6.8 (J ¼ 8.5 Hz). Simultaneous little downfield shift was also observed in H-6
0
0
signal of caffeic acid moiety, which showed that acylation has taken place on the hydroxyl
0
0
00
0
attached to C-4 carbon of caffeic acid moiety. While in ‘verbascoside 4 ,4 -butyrate’, acylation
1
was taking place at two places. In one place, H NMR signal at d 6.4 (J ¼ 8.45 Hz)
0
corresponding to H-5 proton of hydroxyltyrosol was shifted downfield to d 6.8 (J ¼ 8.45 Hz)
0
which reveals that hydroxyl group attached to C-4 of hydroxyltyrosol has got acylated.
Simultaneous downfield shift was also observed in H-6 of hydroxyltyrosol. Another shift was
0
0
0
same as observed in mono-acylated analogues that are on C-4 hydroxyl of caffeic acid moiety.
Same pattern was found for other cases also in which same signal shifts were observed for
mono- and di-acylated products, and no shift in sugar signals was observed. Confirmation was
1
3
also obtained from C as additional peaks were observed at 170–175 ppm corresponding to the
additional carbonyls.
2
.3. Synergistic action of verbascoside analogues with AmB
Verbascoside and its analogues synthesised were assayed to test whether these possess
synergistic effect with AmB against ‘C. albicans.’ Interestingly, all the analogues showed a
promising potentiating AmB activity. It was observed that at lower concentration range of 1.5
and 0.75 mg/mL most of the derivative compounds were strongly potentiating the activity
of AmB than the parent verbascoside (Table 1). Few analogues such as verbascoside
00
0
0
0
0
0
4
-octanoate (3b), verbascoside 4 -palmitate (3d) and verbascoside 4 ,4 -palmitate (4d) with
increased lipophilicity showed fourfold reduction in minimum inhibitory concentration (MIC) of
AmB at a concentration of 1.5 mg/mL than parent verbascoside. These analogues could be
attractive prototypes that can be used to modulate the currently available antifungal antibiotics
with reduced toxicity.
3
3
. Experimental
.1. Measurements
1
13
NMR ( H and C) spectra were recorded at 500 and 100 MHz on Bruker DPX200 instrument
Bruker, Billerica, MA, USA) in CD OD/DMSO/CDCl with TMS as internal standard for
(
3
3
protons and solvent signals as internal standard for carbon spectra. Chemical shift values were
expressed in d (ppm), and coupling constants were given in Hz. MS were recorded on MALDI
mass spectrometer. Melting points were recorded on Buchi Melting point apparatus D-545

Natural Product Research
731
Table 1. In vitro antifungal activity in of verbascoside derivatives screened for their synergistic activity
with AmB.
AmB
AmB
þ 50
AmB
þ 25
AmB
þ 12.5
AmB
þ 6.15
AmB
þ 3.12
AmB
AmB
þ 0.75
0.5
0.5
0.5
0.25
0.5
0.5
a
Compound
Acteoside
þ 0
0.5
þ 1.5
0.5
b
0.12
0.12
0.12
0.12
0.12
0.12
0.12
0.12
0.12
0.12
0.12
0.25
0.12
0.12
0.12
0.12
0.12
0.12
0.12
0.12
0.25
0.12
0.25
0.12
0.12
0.25
0.12
0.25
0.12
0.25
0.12
0.25
0.25
0.25
0.12
0.12
0.25
0.25
0.25
0.12
0.25
0.25
0.25
0.12
0.12
3
4
3
4
3
4
3
4
a
a
b
b
c
c
d
d
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.25
0.12
0.25
0.25
0.25
0.12
0.12
0.25
0.25
0.25
a
Concentration of verbascoside and its analogues in mg/mL.
MIC values.
b
(
Buchi, Postfach, Meierseggstrasse, Switzerland); IR spectra (KBr discs) were recorded on
Bruker Vector 22 instrument (Bruker, Billerica, MA, USA). Silica gel-coated aluminium plates
of thickness 0.25 mm (Merck) were used for TLC. Reagents and solvents used were LR grade.
The chromatograms were visualised under UV (254–366 nm) and MeOH/H SO .
2
4
3
.2. Experimental procedure for chemical and enzymatic transesterification of verbascoside
In a typical experimental procedure for chemical transesterification, a solution of verbascoside
0.112 g, 1 mmol) in dry DCM (10 mL) was taken, to which the anhydrides (acetic or butyric
(
anhydride, 4 mmol), DMAP (10 mg) were added and stirred for 24 h at ambient temperature.
After the completion of reaction, solvent was evaporated in vacuo and the crude product was
subjected to column chromatography (silica gel, 100–200 mesh, elution; n-chloroform/MeOH
(90:10) gradient) to obtain pure products. The pure products were characterised on the basis of
1 13
H NMR, C NMR and MS given in compound characterisation (supporting information).
A typical experimental procedure for the enzymatic transesterification is illustrated in
Scheme 1. The reaction was initiated by dissolving the substrate (verbascoside, 0.1 mmol) in dry
organic solvent (DMF) in the presence of the lipase (C. antarctica lipase B, 20% weight
equivalent), an acyl donor ( p-nitrophenyl alkanoate, (0.2 mmol), pre-activated molecular sieves
˚
4 A) and left for shaking on digital shaker at 120 rpm. The reaction mixture was worked up in
(
H O and Et O. The organic layer was separated and evaporated in vacuo and the crude product
2
2
was subjected to column chromatography as described earlier. The acylated product was purified
by using column chromatography and the structure was determined by using MS and NMR
1
13
spectra ( H, C, and by comparison with spectra of their parent molecule) as shown in compound
characterisation S2. p-Nitro alkanoates were synthesised using known procedure involving
esterification reaction between p-nitrophenol (1 mmol), and corresponding acids (1.3 mmol) in
the presence of DCC (6.0 mmol) and DMAP (10 mg, catalytic amount) under dry conditions gave
the crude product. The crude product was purified by using column chromatography (silica gel,
6
0–120 mesh, eluent n-hexane/EtOAc gradient). Lipase from C. rugosa, porcine pancreas type
II, wheat germ lipase and C. antarctica were purchased from Sigma, and C. cylindracea lipase
from Fluka acrylic resin. All the commercial enzymes were used as such. Verbascoside was
procured from Natural Product Division of the institute (CSIR-IIIM, Jammu) and isolated from
its natural resource by known procedure (Steglich & H o¨ fle 1969). Enzymatic reactions were
carried out on IKA digital shaker at room temperature and 120 rpm.

7
32
J. Khazir et al.
3
.3. Bioassay for antifungal activity
The fungicidal effect of AmB in the presence and absence of verbascoside and its analogues was
performed against ‘C. albicans ATCC 90028’ as described previously (Bjorkling et al. 1991).
6
21
The adjusted inoculum suspension (,5 £ 10 CFU mL ) was diluted 10-fold in medium
containing AmB at a concentration of 0.25 £ MIC and in combination with increasing
concentrations of verbascoside and analogues ranging from 0.75 to 50 mg/mL. AmB alone at
4
£ MIC was used as a control. Culture flasks were incubated with an agitation of 200 rpm at
358C for 24 h, and the number of CFU was determined on Sabouraud dextrose agar plates with
lecithin and polysorbate 80 (Becton Dickinson, Becton Drive, NJ, USA) using a serial dilution
method. Plates were then incubated at 358C for 24–48 h. The lower limit of accurate and
2
1
reproducible detectable colony counts was 10 CFU mL . Synergy was defined as a 100-fold or
greater decrease in colony count at 24 h by the combination of agents with reference to the
starting inoculum and also when compared with the most active single agent. The MIC of AmB
was determined for C. albicans in the absence and presence of increasing concentrations of
verbascoside analogues as described previously. The synergistic interaction between AmB and
verbascoside analogues against ‘C. albicans’ was calculated based on the fractional inhibitory
concentration (FIC) index, obtained by adding the FICs of AmB and verbascoside analogues.
The FIC was calculated by dividing the MIC of the combination of AmB and verbascoside
analogues by the MIC of AmB or verbascoside analogues alone. All experiments were
conducted in duplicate three times on separate occasions.
4
. Conclusion
Selective acylation of verbascoside was achieved using resin-bound immobilised C. antarctica
lipase B in the presence of p-nitro butyrate, p-nitro octanoate, p-nitro myristylate and p-nitro
palmitate as acyl donors. Mono- and di-acylated products were formed which were subjected to
0
0
synergistic antifungal activity with AmB. The analogues such as verbascoside 4 -octanoate (3b),
0
0
00
0
verbascoside 4 -palmitate (3d) and verbascoside 4 ,4 -palmitate (4d) were found to reduce the
MIC of AmB to fourfold from 0.5 mM to 0.12 mM compared with the parent verbascoside in the
concentration range of 1.5–0.75 mg/mL.
Supplementary material
Supplementary material relating to this paper is available online, alongside Table S1.
Acknowledgements
The authors thank the Director, CSIR-Indian Institute of Integrative Medicine (IIIM), Jammu for providing
facilities to carry this work and for his constant support. Thanks are also due to Dr Parvinder Pal, Dr Qazi
Naveed and Dr S.D. Sawant for their support. Special thanks to Dr Syed Shafi and Dr K.A Suri for
providing the verbascoside. Thanks are also due to IIIM and University of Pretoria for providing Doctoral
and Postdoctoral fellowships to JK.
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