Lipase-mediated regioselective modifications of macrolactonic sophorolipids

Article abstract of DOI:10.1016/j.tet.2017.02.043

Chemoenzymatic synthesis and modification of well-defined macrolactonic sophorolipid (SLML) analogues via a series of successive regioselective de-esterification/transesterification reactions is investigated. Of the lipases screened, Candida antartica lip

Full text of DOI:10.1016/j.tet.2017.02.043

Tetrahedron xxx (2017) 1e8
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Lipase-mediated regioselective modifications of macrolactonic
sophorolipids
,
*
Aliya Sembayeva a, Beniam Berhane b, Jason A. Carr a
a Nazarbayev University, Dept. of Chemistry, 53 Kabanbay Batyr Ave., Astana, 010000, Kazakhstan
b University of California Irvine, Dept. of Chemistry, 1102 Natural Sciences 2, Irvine, CA, 92697, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemoenzymatic synthesis and modification of well-defined macrolactonic sophorolipid (SLML) ana-
logues via a series of successive regioselective de-esterification/transesterification reactions is investi-
gated. Of the lipases screened, Candida antartica lipase- B (Novozyme-435) successfully deacylated the C-
60 acetoxy group of natural and peracylated SLMLs. Subsequent transesterification with acylating agents
(esters of fatty acids) was successful only with the C-60 deacetylated natural SLML providing an avenue to
well-defined analogues of varying amphilicity. The macrolactonic motif was essential for enzymatic
recognition of the sophorose rings of these complex glycolipids. In the absence of the lactonic motif, the
peracylated sophorose rings are not deacylated, rather the carboxyl end of the non-lactonic forms that
was preferentially transesterified. All macrolactonic derivatives were characterized by IR, 1H, 13C, 1H-1H
and 1H-13C NMR spectroscopy, as well as HRMS where applicable.
Received 21 December 2016
Received in revised form
9 February 2017
Accepted 20 February 2017
Available online xxx
Keywords:
Sophorolipids
Glycolipids
Lipases
© 2017 Elsevier Ltd. All rights reserved.
NMR spectroscopy
Lactone
Fatty acid esters
Biosurfactant
1. Introduction
Of the two major components, the free carboxylic acid form and
its derivatives have been more studied, perhaps because of their
Surface-active biosurfactant agents have attracted much in-
terests due to their unique structures, biodegradability, biochem-
ical and pharmacological properties such antimicrobial,1
anticancer,2,3 antivirus4 and anti-sepsis5 actions. Of these, the
sophorolipids (SLs) (Fig. 1) have attracted broad attention and have
been utilized for various applications. Sophorolipids are complex
stronger interfacial activity and hydrophilicity as compared to the
lactonic form. Regardless, modification of either form, to study their
interfacial functions and structure-activity relationships, is not a
trivial task. There are acid-sensitive glycosidic linkages and acid/
base-sensitive ester functionalities. Chemical modification of
sophorolipids usually requires a series of protection/de-protection
steps with limited selectivity, but enzymes provide a powerful
tool to selectively modify both forms of sophorolipids and their
derivatives.11 We have previously shown that lipases, namely
Candida antartica lipase (CAL-B), will only modify the carboxyl end
of the fatty-acid side chain of the non-lactonic forms of peracylated
sophorolipid derivatives. The sophorose glycosyl rings, even when
esterified, were unperturbed and unreactive to lipase hydrolysis
under reaction conditions similar to those described within.12
Herein, we have shown that when the macrolactonic motif is pre-
served, the sophorose rings are recognized by the enzyme, Candida
antartica lipase (CAL-B), and selective deacylation or acylation is
accomplished under mild conditions. Careful selection of the fatty
acid esters, the acylating agents, leads to carefully tailored sopho-
rolipids which could lead to analogues of varying levels of
amphilicity.
glycolipids comprised of the disaccharide sophorose (2-O-
glucopyranosyl- -glucopyranose) linked to a hydroxy fatty acyl
group by a glycosidic bond between the 10-anomeric position and
the or -1 carbon of a fatty acid. They are produced by yeast
b-D-
b-D
u
u
strains such as Candida bombicola,6 Candida apicola,7 Candida
bogoriensis,8 Candida batistae9 and Wickerhamilella domercqiae10
and are usually harvested as a mixture of glycolipids with varying
degrees of acetylation on the sophorose moiety. The predominant
forms of this mixture are the macrolactonic sophorolipid (SLML,
Fig. 1a) and the free carboxylic acid form (SLCA, Fig. 1b) that are
acetylated at both the 60- and 600- positions, or various combina-
tions thereof, on the sophorose rings.
* Corresponding author.
E-mail addresses: jacarr@nu.edu.kz, avogadro1998@gmail.com (J.A. Carr).
http://dx.doi.org/10.1016/j.tet.2017.02.043
0040-4020/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sembayeva A, et al., Lipase-mediated regioselective modifications of macrolactonic sophorolipids,
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2
A. Sembayeva et al. / Tetrahedron xxx (2017) 1e8
Fig. 1. The major components of sophorolipids: the macrolactonic form (1a) and the free acid form (1b), R1¼R2¼CH3C(O).
2. Results and discussion
propionated derivative (6, m/z 893.45 [M þ Na]þ) (Scheme 1,
Table 1). It is important to note that longer reaction times of up to
7e10 days had no effect on the yields of the 60-deacetylated
products formed.
2.1. Regioselective hydrolysis and screening of lipases, solvent and
temperature
That the 60 position of the sophorose ring was hydrolyzed
selectively is a testament to the remarkable selectivity of bio-
catalysts, even on the peracylated sophorolipids. Otto et al.14 has
shown that deacetylation does occur primarily at the 60 position
when 1a is incubated with Candida rugosa lipase, more likely
because the 600 position is more sterically inaccessible to the en-
zyme's active site given that it is tucked between the fatty acid side
chain and the first glucosyl ring of these complex molecules. This
was more recently demonstrated by Ju15 who was able to deace-
tylate compound 1a using MML (Mucor michei lipase) as the most
effective biocatalyst as well as other biocatalysts including CAL-B
and Gross16 who was able achieve the 60 deacetylation using
lipase from Candida rugosa and 600 deacetylation with cutinase from
Humicola insolens. All reported modifications of native SLML (1a) to
date utilized buffer as the solvent of choice to realize the deacyla-
tion of either the 60 or 600 acetates, however, because of the very
limited solubility of derivatized SLMLs, careful screening of hy-
drolytic reactions in non-toxic organic solvents was necessary in
keeping with the “green” chemistry involved. More importantly,
we have demonstrated that for peracylated macrolactonic de-
rivatives to be recognized by lipases, the lactonic motif is an
essential requirement for enzymatic recognition of the glycon head.
Without this important structural factor, the enzyme CAL-B only
recognizes the aglyconic tail as in modification of the peracylated
non-lactonic derivatives.12
In order to determine the best lipase for regio-selective hydro-
lysis, reactions were modeled using compound 1a incubated with
lipases from different sources: Candida antartica B (CAL-B), Pseu-
domonas cepecia, AS “Amano”, AY “Amano” 30 SD-K, and “Amano”
M. The experiments were conducted in dry solvents namely THF,
acetonitrile, toluene or cyclohexane as the organic medium carried
out in tightly sealed N2-flushed vials containing a solution of 1a
with n-butanol in the dry solvents and incubated with the different
lipases. The choice of solvents reflected those considered to be mid-
to low polar solvents as it is known that more polar ones may
denature or inactivate enzymes.13 The reactions were repeated
with temperature variations from 40 ꢀC to 70 ꢀC in order to
determine the optimal temperature for highest substrate conver-
sion and were allowed to proceed for up to 72 h.
From the modeled reactions only CAL-B and PS-30 seemed to
offer any activity, with CAL-B in dry acetonitrile or THF at 60 ꢀC
having noticeably greater activity, giving the highest yield of the 60-
deacetylated substrate, 4 (Scheme 1, m/z 669.35 [M þ Na]þ), in 50%
yield (Table 1). Lower yields were observed when the modeled
reaction was done in solvents other than acetonitrile or THF, or at
temperatures lower or higher than 60 ꢀC. This can be attributed to a
decrease in solubility of the substrate as the solvent's dielectric
constant decreases, thereby decreasing the catalytic turnover rate
(kcat) or denaturation of the enzyme leading to a decrease in activity
with temperature variations. No other activity, hydrolytic or
otherwise, was observed with the other enzymes that were
screened, indicating that the glycolipids were unfit substrates for
these biocatalysts. Therefore, CAL-B was chosen as the lipase of
choice to carry out further studies on the peracylated derivatives, 2
(m/z 879.40 [M þ Na]þ) and 3 (m/z 935.46 [M þ Na]þ). The per-
acetylated and propionated substrates were formed by reacting 1a
with acetic anhydride and propionic anhydride respectively with a
catalytic amount of DMAP in dry THF. Subjecting compounds 2 and
3 to CAL-B hydrolysis by incubating the substrates in dry acetoni-
trile with n-butanol for 3 days resulted in highly selective hydro-
lysis of the 60-acetoxy group of these peracylated derivatives. The
yields obtained were 52% for the 60-deacetylated peracetylated
derivative (5, m/z 837.39 [M þ Na]þ) and 51% for the 60-deacetylated
2.2. Synthesis of fatty acid esters by lactone ring opening
In our effort to synthesize sophorolipid analogues of varying
levels of amphilicity, fatty-acid esters of various chain lengths were
synthesized by the ring-opening of lactones. Fatty-acid esters were
chosen as the acylating agents of choice as it is known the CAL-B
has high activity for these substrates.17 Apart from the ring-
opening of cyclopentadecanolide, the ring-opening of
g- and d-
lactones was no trivial task. For cyclopentadecanolide, the ring-
opening was accomplished by dissolving the substrate in meth-
anol with a catalytic amount of pTSA and the mixture brought to
reflux overnight. Excess solvent was then removed in vacuo, the
product taken up and extracted in DCM and water to yield the
Please cite this article in press as: Sembayeva A, et al., Lipase-mediated regioselective modifications of macrolactonic sophorolipids,
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A. Sembayeva et al. / Tetrahedron xxx (2017) 1e8
3
Scheme 1. Synthesis of SLML derivatives and lipase hydrolysis of SLML 1a and its derivatives. (i) Novozym-435, n-butanol, dry THF at 60 ꢀC (ii) acetic anhydride or propionic
anhydride, pyridine, DMAP, dry THF, 3 h; (iii) Novozym-435, n-butanol, dry acetonitrile at 60 ꢀC.
Table 1
imidazole in DCM which, after purification, yielded methyl 15-
((tert-butyldiphenylsilyl)oxy)pentadecanoate (8) as a colorless oil
in 55% yield (Scheme 2).18
Unfortunately, the ring-opening of g- and d-lactones was not as
trivial as that of cyclopentadecanolide. This was mainly because
entropy favors the formation of the lactones under those condi-
Solvent and temperature effect on the yield of lipase catalyzed reactions after 72 h.
Other lipases excluded as they were unreactive toward the substrates.
Compound code Temperature (ꢀC) Lipase Solvent
Isolated yield (%)
0
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
2
40
50
40
50
60
40
50
60
70
40
50
60
70
50
60
60
60
CAL-B cyclohexane
CAL-B cyclohexane 12
CAL-B toluene
CAL-B toluene
CAL-B toluene
CAL-B THF
CAL-B THF
CAL-B THF
30
36
38
34
46
49
35
42
47
51
43
5
tions. In order to prepare TBDPS-protected esters from g- and d-
lactones, the following general reaction conditions were employed.
To a solution of a lactone dissolved in ethanol, 1.1 molar equivalents
of NaOH was added and the reaction heated at 80ꢀ for 30 min, then
stirred at ambient temperature for 2 h. After removal of the solvent
in vacuo, the salt formed was dissolved in DMF followed by the slow
addition of ethyl iodide and the reaction allowed to stir at room
temperature for 5 days. After extraction with ethyl acetate and
water, the ester was dissolved in DCM followed by addition of
TBDPSCl with a catalytic amount of imidazole and the reaction
allowed to stir for an additional 2 days. Extraction followed by
purification via column chromatography yielded products (10, 12)
as colorless oils with yields ranging from 60% to 70% (Scheme 3).19
CAL-B THF
CAL-B CH3CN
CAL-B CH3CN
CAL-B CH3CN
CAL-B CH3CN
PS-30 CH3CN
PS-30 CH3CN
CAL-B CH3CN
CAL-B CH3CN
8
52
51
3
substrate, methyl 15-hydroxypentadecanoate,
7
(Scheme 2).
2.3. Lipase-mediated esterification of 60-deacetoxy SLMLs
Recognizing that the
u-hydroxymethylene group can diminish re-
action between the fatty-acid ester and the 60-deacetoxy sopho-
rolipids, the substrate 7 was protected with TBDPS by reacting 7
with tert-butyldiphenylsilyl chloride with a catalytic amount of
In order to ‘tailor’ the amphilicity of the 60-deacetylated SLMLs,
compounds 4, 5 and 6 were incubated with the fatty-acid esters
derived from the ring-opening of various lactones (Schemes 2 and
Scheme 2. Synthesis of methyl TBDPS pentadecanoate. (i) pTSA, methanol, reflux; (ii) TBDPSCl, imidazole, DCM.
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4
A. Sembayeva et al. / Tetrahedron xxx (2017) 1e8
Scheme 3. Synthesis of TBDPS-protected esters from
g
- and
d
-lactones. (i) NaOH, EtOH then EtI, DMF; (ii) TBDPSCl, imidazole, DCM.
3). In a typical reaction, Novozym-435 (CAL-B, immobilized prep-
aration from C. antartica) was incubated with the 60-deacetylated
compounds 4, 5 and 6 in 50 mL round bottom flasks using 1:1
substrate to lipase ratio (w/w). The transesterification was initially
carried out with 4 in dry solvents at 60 ꢀC in the presence of the
fatty-acid esters (8, 10, 12) and were allowed to incubate for a
period of 5 days. It was observed that only compound 8 responded
to the transesterification conditions employed to yield the lipo-
philic derivative 13 (Scheme 4, Table 2, [m/z 1147.67 [M þ Na]þ]) in
relatively low yields. Dry acetonitrile gave the highest yield at 39%
but a higher yield (61%) of the product was achieved when the
reaction was extended to 30 days. When the center of reactivity of
the fatty-acid esters became relatively hindered as seen in 10 and
12, the reactions failed altogether. It is known that the scissile fatty
acid binding site for CAL-B has high activity for short to medium
chain length fatty acids and decreasing activity for long-chain fatty
acids,17 so it came as a surprise to observe that no reaction occurred
when the shorter chain fatty acids (10, 12, Table 2) were incubated
with CAL-B and 4. It is suggested that the sterically bulky TBDPS
protecting groups so close to the ester moiety restricts access of
these substrates into the hydrophobic active site of CAL-B. Trans-
esterification failed altogether with the 60-deacetoxy peracylated
derivatives, 5 and 6, once again suggesting that the hydrophobic
pocket of the enzyme, which optimally fits the acyl moiety of the
substrates, experiences steric conflicts due to unfavorable van der
Waals interactions.
for 60-CH2- from 4.29 ppm to 3.80 ppm corresponds to the for-
mation of the 60 hydroxymethylene group. The resonances for the
600-CH2- and the 600 acetoxy group remained unperturbed reflecting
the selective hydrolysis of the 60-acetoxy group. The 13C NMR
spectrum of compound 4 along with its 13C DEPT 135 spectra
(Fig. 2), when compared to 1a, show an upfield shift of ~2.4 ppm for
the C-60, a downfield shift of ~1.2 ppm for C-50, and an upfield shift
of ~0.1 ppm for C-40. The effects of such alternating resonances are
known when sugars are deacylated at the hydroxymethylene po-
sitions, the
g
-effect. The resonances of the C-600, C-500, and C-400
remained relatively unperturbed between 1a and 4. Proton reso-
nances were established using additional 2D techniques. The other
derivatized macrolactonic products (5, 6 and 13) were unambigu-
ously characterized using 1H-1H COSY, HSQC and HMBC spectra.
The deacylated products, 5 and 6, and the esterified product, 13,
showed similar alternating resonance patterns upon close exami-
nation of their 13C NMR and 13C DEPT 135 spectra. In addition, a
mixture of equimolar amounts of 1a and 4 (Fig. 2) showed distinctly
resolved resonances for 1a at 63.8 (C-60), 73.8 (C-50) and 69.7 (C-40)
and for 4 at 61.3 (C-60), 75.0 (C-50) and 69.6 (C-40). Such observa-
tions led to the conclusion that CAL-B was an effective biocatalyst
for the selective hydrolysis of compounds 1a, 2, and 3 at the C-60
position or esterification of compound 4 at the C-60 position. Finally,
all compounds were carefully confirmed by HRMS analysis and the
data obtained show good agreement between experimental and
calculated values (see experimental section).
2.4. Structure determinations
3. Concluding remarks
Compound 1a was incubated with CAL-B at 60 ꢀC for 72 h with
excess n-butanol in dry acetonitrile successfully affording the
monoacetate 4 (Scheme 1) selectively hydrolyzing the C-60 acetate,
in 50% yield. The disappearance of a CH3- group in the 1H NMR
spectrum of 4 with the simultaneous upfield shift of two protons
Chemical modifications of sophorolipids, due to their
complexity, have always been a challenging undertaking because of
the hydrolysable glycosidic linkages, the sensitive ester moieties
located at the 60 and 600 positions (and the macrolactonic ester
formed between the 17-hydroxy oleic acid and the sophorose ring),
Scheme 4. CAL-B catalyzed transesterification of 60deacetylated macrolactonic sophorolipid (4).
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A. Sembayeva et al. / Tetrahedron xxx (2017) 1e8
5
Table 2
Solvent effects on yield of CAL-B catalyzed transesterification reactions of 60-deacetylated SLMLs with fatty acid esters.
Compound code
Temperature (ꢀC)
Duration (# of days)
Solvent
Isolated yield (%)
4 þ 8
4 þ 8
4 þ 8
4 þ 8
4 þ 8
4 þ 8
4 þ 8
4 þ 8
4 þ 10
4 þ 12
5 þ 8
6 þ 8
60
60
60
60
60
60
60
60
60
60
60
60
5
5
5
5
30
30
30
30
7
neata
THF
toluene
CH3CN
neata
THF
toluene
CH3CN
THF
CH3CN
THF
12
11
17
39
30
32
57
61
0
7
7
7
0
0
0
CH3CN
a
(In the absence of solvent).
Fig. 2. Dept-135 Spectra from 56 to 108 ppm for 1a, 4, and a mixture of 1a and 4 in CDCl3.
or the global presence of numerous hydroxyl groups. This study
represents a careful examination of the lipase-catalyzed modifica-
tion of sophorolipid macrolactone and its esterified derivatives.
Derivatization was accomplished without the need for protection/
deprotection steps in the preparation of the 60deacetylated mac-
rolactones. CAL-B (Novozym-435) was found to be the best bio-
catalyst to accomplish this highly selective process with dry
acetonitrile at 60 ꢀC being the most favorable conditions. To the
best of our knowledge, this is the first careful study of the limita-
tions of lipase modification of macrolactonic sophorolipids. The
reactions were found to be scalable, thus providing an avenue by
which macrolactonic derivatives can be chemo-enzymatically
synthesized in our ongoing investigations into their interfacial
functions. Since sophorolipids are known active therapeutic agents,
biological activities of all new compounds (2, 3, 4, 5, 6,13) are under
investigation and will be reported separately.
4. Experimental section
4.1. General methods
The NMR spectra were measured on a JEOL ECA-500 spec-
trometer at 500 MHz (1H), 125 MHz (13C) in CDCl3 at ambient
temperature. The 1H and 13C chemical shifts were referenced to the
internal standard, TMS, and values recorded on the ppm scale (
d). J
values are given in Hz. HRMS were recorded on a LCT Premier TOF
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6
A. Sembayeva et al. / Tetrahedron xxx (2017) 1e8
mass spectrometer from Waters. IR spectra were recorded on a
Nicolet IS5 FTIR spectrometer over a range of 400e4000 cmꢁ1. TLC
was carried out on Sigma Aldrich Fluka TLC plate with silica gel
matrix. For column chromatography, Fisher Scientific,
0.035e0.070 mm, 60A silica gel was used for flash chromatography.
Candida antartica lipase (CAL-B) was purchased from Sigma-Aldrich
and all other lipases were donated from Amano Enzyme, USA. The
sophorolipid macrolactone was donated by the United States
Department of Agriculture (USDA). For all reactions, HPLC solvents
were used where applicable or solvents were purified per estab-
lished procedures.
61.96, 62.22, 67.70, 68.54, 71.38, 71.89, 72.18, 72.50, 73.84, 76.61,
79.08, 99.51, 102.16, 129.42, 130.17, 170.38, 170.44, 171.81, 172.77,
173.02, 173.27, 173.48; HRMS (TOF-ESþ) m/z Calcd. For
(C46H72O18 þ Na)þ 935.4616, found: 935.4643.
4.3. Procedure for lipase catalyzed hydrolysis of macrolactonic
sophorolipids
4.3.1. 17-L-(20-O-
b-D-Glucopyranosyl-b-D-glucopyranosyl]oxy)-9-
ocyadecenoic acid 10-400-lactone 600-acetate (4)
A solution of the sophorolipid macrolactone (1a) (0.500 g,
0.73 mmol) in dry THF or acetonitrile (4 mL) was incubated with
Novozym-435 (0.250 g) and n-butanol (0.14 mL,1.5 mmol) in an N2-
flushed 10 mL vial at 60 ꢀC for 72 h. The enzyme was filtered over a
bed of celite, followed by washing with THF (2 ꢂ 3 mL). The filtrate
was concentrated in vacuo and the residue was subjected to puri-
fication via column chromatography using a gradient eluent of
ethyl acetate: petroleum ether (1:1) then ethyl acetate to afford the
60-deacetylated product as a white solid (4) (0.236 g, yield ¼ 50%).
TLC: Rf ¼ 0.1 (100% ethyl acetate); mp ¼ 109e111 ꢀC; IR (cmꢁ1):
4.2. Procedure for the preparation of peracylated SLMLs
4.2.1. 17-L-(20-O-
b-D-Glucopyranosyl-b-D-glucopyranosyl]oxy)-9-
ocyadecenoic acid 10-400-lactone 30,40,60,200,300,600- hexaacetate (2)
To a solution of 1.0 g of 1a (1.45 mmol) in dry THF (15 mL) was
added acetic anhydride (0.67 g, 6.54 mmol) and pyridine (0.52 g,
6.54 mmol) along with a catalytic amount of DMAP (100 mg). The
mixture was allowed to stir at ambient temperature for a period of
2 h. The reaction mixture was then concentrated under reduced
pressure, and the crude material was taken up in ethyl acetate
(25 mL) and washed first with a concentrated solution of sodium
bicarbonate (25 mL) then brine (25 mL). The organic layer was
dried over anhydrous sodium sulfate and then concentrated to
dryness which upon standing yielded a white waxy solid. Yield:
3364, 2927, 1744, 1072; 1H NMR (500 MHz, CDCl3,
d): 1.24 (d,
J ¼ 6 Hz, 3H, H-18), 1.30 (m, 15H, H-4-7 and 12e14,15), 1.44e1.52
(m, 2H, H-15, 16), 1.55e1.66 (m, 3H, H-3, 16), 1.98e2.04 (m, 4H, H-
8,11), 2.07 (s, 3H, (CO)CH3), 3.32e2.38 (m, 3H, H-2, H-50), 3.43e3.56
(m, 1H, H-30), 3.64e3.67 (m, 4H, H-20, 40, 200, 500), 3.75e3.87 (m, 4H,
H-17, 300, 60), 4.09e4.18 (m, 2H, H-600), 4.49 (d, J ¼ 6.5 Hz, 1H, H-10),
4.56 (d, J ¼ 7 Hz, 1H, H-100), 4.96e5.00 (t, 1H, J ¼ 9.5 Hz, H-400),
1.23 g, 99%. TLC: Rf
¼
0.62 (1:1 ethyl acetate: hexane);
mp ¼ 75e77 ꢀC; IR (cmꢁ1): 2921, 1746, 1221, 1034; NMR: 1H NMR
5.30e5.39 (m, 2H, H-9, 10); 13C NMR (125 MHz, CDCl3,
d): 20.79,
(500 MHz, CDCl3,
d
): 1.23 (d, J ¼ 5 Hz, 3H, H-18), 1.26e1.59 (m, 24H,
21.08, 24.77, 25.53, 26.92, 27.11, 28.24, 28.30, 28.39, 29.03, 29.65,
30.15, 34.31, 37.59, 61.25, 62.02, 69.18, 69.61, 72.51, 73.31, 75.01,
75.54, 75.75, 79.32, 82.39, 102.72, 104.24, 129.78, 129.90, 170.46,
H-3-8 and 11e16), 1.97 (s, 3H, (CO)CH3), 1.99 (s, 3H, (CO)CH3), 2.04
(s, 3H, (CO)CH3), 2.04 (s, 3H, (CO)CH3), 2.05 (s, 3H, (CO)CH3), 2.08 (s,
3H, (CO)CH3), 2.19e2.32 (m, 2H, H-2), 3.62e3.67 (m, 2H, H-50, 500),
3.70e3.76 (m, 2H, H-20, 17), 4.05e4.16 (m, 4H, H-60, 600), 4.47 (d,
J ¼ 8 Hz, 1H, H-10), 4.84e4.93 (m, 4H, H-100, 200, 300, 40), 5.09e5.19 (m,
2H, H-30, 400), 5.28e5.36 (m, 2H, H-9,10); 13C NMR (125 MHz, CDCl3,
173.11; HRMS (TOF-ESþ) m/z Calcd. for (C32H54O13
þ
Na)þ
669.3462, found: 669.3483.
4.3.2. 17-L-(20-O-
b-D-Glucopyranosyl-b-D-glucopyranosyl]oxy)-9-
d
): 20.57, 20.60, 20.69, 20.78, 21.07, 23.99, 25.15, 26.64, 27.30, 27.38,
ocyadecenoic acid 10-400-lactone 30,40,200,300,600- pentaacetate (5)
A solution of the 30,40,60,200,300,600-sophorolipid macrolactone
hexaacetate (2) (0.500 g, 0.58 mmol) in dry acetonitrile (4 mL) was
incubated with Novozym-435 (0.250 g) and n-butanol (0.35 mL,
3.8 mmol) in an N2-flushed 10 mL vial at 60 ꢀC for 72 h. The enzyme
was filtered over a bed of celite, followed by washing with aceto-
nitrile (2 ꢂ 3 mL). The filtrate was concentrated in vacuo and the
residue was subjected to purification via column chromatography
using ethyl acetate: petroleum ether (1:1) as the eluent to afford
the 60-deacetylated pentaacetate as a white solid (5) (0.246 g,
yield ¼ 52%). TLC: Rf ¼ 0.39 (50% ethyl acetate/50% petroleum
ether); mp ¼ 83e85 ꢀC; IR (cmꢁ1): 3524, 2925, 1746, 1221, 1035; 1H
27.71, 28.48, 30.28, 30.40, 33.49, 37.17, 62.00, 62.30, 67.77, 68.84,
71.40, 72.00, 72.52, 72.77, 74.04, 79.17, 99.63, 102.25, 129.58, 130.24,
169.36, 169.70, 169.96, 170.22, 170.49, 170.59, 171.96; HRMS (TOF-
ESþ) m/z Calcd. For (C42H64O18 þ Na)þ 879.3990, found: 879.3990.
4.2.2. 17-L-(20-O-
b-D-Glucopyranosyl-b-D-glucopyranosyl]oxy)-9-
ocyadecenoic acid 10-400-lactone 60,600-diacetyl-30,40,200,300-
tetrapropionate (3)
To a solution of 1.0 g of 1a (1.45 mmol) in dry THF (15 mL) were
added propionic anhydride (0.83 mL, 6.54 mmol) and pyridine
(0.52 mL, 6.54 mmol) along with a catalytic amount of DMAP
(100 mg). The mixture was allowed to stir at ambient temperature
for a period of 2 h after which the reaction mixture was concen-
trated under reduced pressure, and the crude material taken up in
ethyl acetate (25 mL). The solution was washed with a concentrated
solution of sodium bicarbonate (25 mL) then brine (25 mL). The
organic layer was then dried over anhydrous sodium sulfate and
then concentrated to dryness which upon standing yielded a white
waxy solid. Yield: 1.30 g, 99%. TLC: Rf ¼ 0.65 (1:1 ethyl acetate:
hexane); mp ¼ 78 ꢀC; IR (cmꢁ1): 2930, 1744, 1161, 1027; NMR: 1H
NMR (500 MHz, CDCl3, d): 1.23e1.36 (m, 25H, H-4-8 and 11e16, 18),
1.53e1.62 (m, 2H, H-3), 1.99 (s, 3H, (CO)CH3), 2.05 (s, 3H, (CO)CH3),
2.06 (s, 3H, (CO)CH3), 2.08 (s, 3H, (CO)CH3), 2.09 (s, 3H, (CO)CH3),
2.21e2.29 (m, 2H, H-2), 3.44e3.47 (m, 1H, H-20), 3.51e3.55 (m, 1H,
H-50), 3.65e3.78 (m, 3H, H-17, 30, 500), 4.06e4.20 (m, 4H, H-600, 60),
4.48 (d, J ¼ 8 Hz, 1H, H-10), 4.82e4.89 (m, 1H, H-40), 4.97e5.02 (m,
2H, H-100, 300), 5.16e5.23 (m, 2H, H-200,400), 5.29e5.41 (m, 2H, H-9,
10); 13C NMR (125 MHz, CDCl3,
d): 20.57, 20.73, 20.83, 20.91, 24.44,
25.40, 26.57, 27.215, 27.78, 27.83, 28.20, 28.77, 29.34, 29.67, 29.72,
30.06, 30.17, 31.90, 33.97, 37.43, 61.52, 62.63, 67.46, 70.14, 71.53,
72.19, 72.44, 73.50, 78.86, 81.97, 100.45, 102.14, 129.73, 129.98,
170.12, 170.17, 170.40, 170.65, 170.74, 171.93; HRMS (TOF-ESþ) m/z
Calcd. for (C40H62O17 þ Na)þ 837.3885, found: 837.3874.
NMR (500 MHz, CDCl3, d): 0.98e1.07 (m, 12H, 4x (CO)CH2CH3), 1.18
(d, J ¼ 6 Hz, 3H, H-18), 1.21e1.52 (m, 24H, H-3-8 and 11e16), 2.00 (s,
3H, (CO)CH3), 2.04 (s, 3H, (CO)CH3), 2.15e2.33 (m, 10H, 4x (CO)
CH2CH3, H-2), 3.56e3.63 (m, 2H, H-500, 50), 3.67e3.70 (m, 2H, H-20,
17), 3.99e4.18 (m, 4H, H-60, 600), 4.43 (d, J ¼ 8 Hz, 1H, H-10), 4.79 (d,
J ¼ 8 Hz, 1H, H-100), 4.84e4.89 (m, 2H, H-200, 40), 5.05e5.16 (m, 3H,
H-300, 30, 400), 5.24e5.32 (m, 2H, H-9,10); 13C NMR (125 MHz, CDCl3,
4.3.3. 17-L-(20-O-
b-D-Glucopyranosyl-b-D-glucopyranosyl]oxy)-9-
ocyadecenoic acid 10-400-lactone-600-acetyl-30,40,200,300-
d
): 8.78, 8.80, 8.88, 20.57, 20.67, 20.99, 23.82, 25.06, 26.54, 27.05,
tetrapropionate (6)
27.21, 27.28, 27.47, 27.57, 28.31, 30.25, 30.26, 30.36, 33.28, 37.09,
A
solution of the 60,600-diacetyl-30,40,200,300-sophorolipid
Please cite this article in press as: Sembayeva A, et al., Lipase-mediated regioselective modifications of macrolactonic sophorolipids,
Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.02.043

A. Sembayeva et al. / Tetrahedron xxx (2017) 1e8
7
macrolactone tetrapropionate (3) (0.500 g, 0.55 mmol) in dry
acetonitrile (4 mL) was incubated with Novozym-435 (0.250 g) and
n-butanol (0.33 mL, 3.56 mmol) in an N2-flushed 10 mL vial at 60 ꢀC
for 72 h. The enzyme was filtered over a bed of celite, followed by
washing with acetonitrile (2 ꢂ 3 mL). The filtrate was concentrated
in vacuo and the residue was subjected to purification via column
chromatography using ethyl acetate: petroleum ether (1:1) as the
eluent to afford the 60-deacetylated 600-acetyl-30,40,200,300-tetrapro-
pionate as a light yellow solid (6) (0.244 g, yield ¼ 51%). TLC:
Rf ¼ 0.34 (50% ethyl acetate/50% petroleum ether); mp ¼ 92e95 ꢀC;
IR (cmꢁ1): 3510, 2939, 1744, 1161, 1029; 1H NMR (500 MHz, CDCl3,
layer was dried over anhydrous Na2SO4 and concentrated in vacuo
to afford the ethyl ester, 9, as a yellow liquid. The crude ester, 9, and
imidazole (0.5 g, 7.7 mmol) were dissolved in dry DCM (30 mL) and
TBDPSCl (1.8 mL, 7 mmol) was added dropwise to the solution at
room temperature. This mixture was allowed to stir for an addi-
tional 2 days after which the solvent was removed under reduced
pressure and the crude product was quenched with saturated NaCl
solution (30 mL) followed by extraction with diethyl ether
(2 ꢂ 20 mL). The organic layers were dried over anhydrous Na2SO4,
concentrated to dryness and the crude product purified via column
chromatography (5% ethyl acetate/95% petroleum ether) to give the
desired product (10) as a colorless oil (1.94 g, yield ¼ 65%).19 TLC:
Rf ¼ 0.65 (5% ethyl acetate/95% petroleum ether); IR (cmꢁ1): 3071,
d): 0.97e1.13 (m, 12H, 4x (CO)CH2CH3), 1.13e1.46 (m, 27H, H-3-8
and 11e16, 18), 1.99 (s, 3H, 600-(CO)CH3), 2.10e2.25 (m, 10H, 4x (CO)
CH2CH3, H-2), 3.52e3.59 (m, 3H, H-60a, 200,300), 3.60e3.65 (m, 4H, 20,
60b, 500, 17), 3.92e4.13 (m, 2H, H-600), 4.52 (d, J ¼ 7.5 Hz, 1H, H-10),
4.83e4.95 (m, 3H, H-100, 400, 40), 5.11e5.16 (m, 1H, H-50), 5.22e5.28
(m, 1H, H-30), 5.31e5.39 (m, 2H, H-9, 10); 13C NMR (125 MHz, CDCl3,
2931, 1734, 1105, 821, 700; 1H NMR (500 MHz, CDCl3,
d): 0.74e0.77
(t, 3H, J ¼ 7.5 Hz, 6.5 Hz, CH3), 1.06e1.13 (m, 11H, CH2, (CH3)3CSi),
1.21e1.24 (t, 3H, J ¼ 7.5 Hz, 7 Hz, CH3CH2O), 1.37e1.41 (m, 2H, CH2),
1.73e1.85 (m, 2H, CH2), 2.34e2.37 (m, 2H, CH2C(O)), 3.75e3.78 (m,
1H, SiOCH), 4.03e4.10 (m, 2H, OCH2), 7.36e7.44 (m, 6H, 4xCHAr),
d): 8.65, 8.74, 20.41, 20.51, 20.87, 23.65, 24.91, 26.41, 26.89, 26.95,
27.05, 27.08, 27.14, 27.30, 27.44, 28.16, 30.13, 30.17, 30.24, 33.11,
36.95, 61.82, 62.04, 67.59, 68.40, 71.25, 71.74, 72.04, 72.36, 73.77,
76.45, 78.90, 99.40, 102.00, 129.25, 130.04, 170.19, 171.65, 172.60,
172.84, 173.09, 173.29; HRMS (ESI) m/z Calcd. for (C44H70O17 þ Na)þ
893.4510, found: 893.4498.
7.66e7.69 (m, 4H, 4xCHAr); 13C NMR (125 MHz, CDCl3,
d): 13.93,
14.18, 19.37, 22.53, 27.03, 29.74, 30.97, 35.85, 60.17, 72.21, 127.38,
127.47, 129.44, 129.50, 134.16, 134.48, 135.86, 173.91.
4.4.3. Ethyl 5-((tert-butyldiphenylsilyl)oxy)pentanoate (12)
Valerolactone (1.0 g, 10 mmol) was added to a solution of NaOH
(0.44 g,11 mmol) dissolved in ethanol (90%, 23 mL) and the mixture
was stirred at 80ꢀ for 30 min then at ambient temperature for 2 h.
Excess solvent was then removed under reduced pressure to yield
the sodium salt quantitatively. Thereafter, ethyl iodide (1.4 mL,
17.2 mmol) was added dropwise to a solution of the sodium salt
(1.2 g, 8.6 mmol) in DMF (20 mL) and the solution was allowed to
stir at room temperature for an additional 5 days. The solution was
then taken up in ethyl acetate (30 mL) and washed with water. The
organic layer was dried over anhydrous Na2SO4 and then concen-
trated to dryness to afford the crude ethyl ester, 11, as a yellow
liquid. The crude ester, 11, and imidazole (0.6 g, 9.5 mmol) were
dissolved in dry DCM (30 mL) and TBDPSCl (2.2 mL, 8.6 mmol) was
added dropwise to the solution at room temperature. This mixture
was allowed to stir for an additional 2 days after which the solvent
was removed under reduced pressure and quenched with saturated
NaCl solution (30 mL) followed by extraction with diethyl ether
(2 ꢂ 20 mL). The organic layers were dried over anhydrous Na2SO4,
the solvent removed in vacuo and the crude product was purified
via column chromatography (5% ethyl acetate/95% petroleum
ether) to give the desired product (12) as a colorless oil (2.1 g,
yield ¼ 63%).19 TLC: Rf ¼ 0.28 (5% ethyl acetate/95% petroleum
ether); IR (cmꢁ1): 3071, 2931, 1734, 1108, 822, 700; 1H NMR
4.4. Procedure for the formation of TBDPS-protected hydroxy esters
4.4.1. Methyl 15-((tert-butyldiphenylsilyl)oxy)pentadecanoate (8)
Cyclopentadecanolide (2.0 g, 8.3 mmol) and para-toluene sul-
fonic acid (100 mg) were dissolved in methanol (25 mL) and the
mixture brought to reflux overnight. Excess solvent was removed
under reduced pressure and the resulting product was taken up
with DCM (40 mL) and washed with deionized water (10 mL). The
organic layer was collected, dried over anhydrous Na2SO4 and
concentrated
to
dryness
to
yield
methyl
15-
hydroxypentadecanoate (7) as a white, waxy solid (1.4 g, 63%
yield). Then, TBDPSCl (1.5 mL, 5.2 mmol) was added dropwise to a
solution of 7 (1.4 g, 5.2 mmol) and imidazole (0.4 g, 5.7 mmol)
dissolved in DCM (30 mL) and the resulting solution was allowed
additional stirring for 2 days at ambient temperature. Excess sol-
vent was then removed under reduced pressure and the crude
product was quenched with saturated NaCl solution (30 mL) and
extracted with diethyl ether (30 mL). The organic layer was dried
over anhydrous Na2SO4 and concentrated under reduced pressure.
Purification of the crude product was done via column chroma-
tography (5% ethyl acetate/95% petroleum ether) to afford methyl
15-((tert-butyldiphenylsilyl)oxy)pentadecanoate (8) as a colorless
oil (1.5 g, 55% yield).18 TLC: Rf ¼ 0.47 (5% ethyl acetate/95% petro-
leum ether); IR (cmꢁ1): 3071, 2924, 1740, 1110, 823, 700; 1H NMR
(500 MHz, CDCl3,
d): 1.13 (s, 9H, (CH3)3CSi), 1.27e1.30 (t, 3H,
J ¼ 7.5 Hz, 7 Hz, CH3CH2O), 1.64e1.69 (m, 2H, CH2), 1.78e1.82 (m,
2H, CH2), 2.33e2.37 (m, 2H, CH2), 3.72e3.75 (m, 2H, CH2), 4.14e4.19
(m, 2H, OCH2CH3), 7.40e7.46 (m, 6H, 6xCHAr), 7.72e7.79 (m, 4H,
(500 MHz, CDCl3, d): 1.06 (s, 9H, (CH3)3CSi), 1.26e1.31 (m, 20H,
10xCH2), 1.56e1.66 (m, 4H, 2xCH2), 2.30e2.34 (m, 2H, CH2C]O),
3.67e3.69 (m, 5H, OCH3,CH2OTBDPS), 7.38e7.44 (m, 6H, 6xCHAr),
4xCHAr); 13C NMR (125 MHz, CDCl3,
d): 14.15, 19.11, 21.37, 26.76,
7.69e7.71 (m, 4H, 4xCHAr); 13C NMR (125 MHz, CDCl3,
d
): 19.20,
31.82, 33.92, 60.06, 63.28, 127.54, 129.48, 133.79, 133.79, 134.73,
135.46, 173.53.
24.95, 25.75, 26.84, 29.15, 29.26, 29.38, 29.45, 29.61, 29.64, 3.56,
34.10, 51.44, 63.98, 127.54, 129.45, 134.13, 135.55, 174.34.
4.5. Procedure for CAL-B catalyzed transesterification reaction
4.4.2. Ethyl 4-((tert-butyldiphenylsilyl)oxy)octanoate (10)
g-Octanoic lactone (1.0 g, 7 mmol) was added to a solution of
4.5.1. 60-(15-((tert-butyldiphenylsilyl)oxy)pentadecanoyl)-17-L-(20-
NaOH (0.3 g, 7 mmol) dissolved in ethanol (90%) and the mixture
was stirred at 80ꢀ for 30 min then at ambient temperature for 2 h.
Excess solvent was then removed under reduced pressure to yield
the sodium salt quantitatively. Thereafter, ethyl iodide (1.1 mL,
14 mmol) was added dropwise to a solution of the sodium salt
(1.2 g, 7 mmol) in DMF (20 mL) and the solution was allowed to stir
at room temperature for an additional 5 days. The solution was
taken up in ethyl acetate and washed with water and the organic
O-b-D-glucopyranosyl-b-D-glucopyranosyl]oxy)-9-ocyadecenoic
acid 10-400-lactone 600-monoacetate (13)
A solution of the 60-deacetylated SLML (4) (0.2 g) in dry aceto-
nitrile (3 mL) was incubated with Novozym-435 (2:1 w/w, 0.1 g)
and methyl 15-((tert-butyldiphenylsilyl)oxy)pentadecanoate (8)
(0.2 g, 0.39 mmol)in an N2-flushed 10 mL vial at 60 ꢀC for 30 days.
The enzyme was filtered over a bed of celite, followed by washing
with acetonitrile (3 ꢂ 3 mL). The filtrate was concentrated in vacuo
Please cite this article in press as: Sembayeva A, et al., Lipase-mediated regioselective modifications of macrolactonic sophorolipids,
Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.02.043

8
A. Sembayeva et al. / Tetrahedron xxx (2017) 1e8
and the residue was subjected to purification via column chroma-
tography using ethyl acetate as the eluent to give 60-(15-((tert-
Ashby of the USDA for a sample of the macrolactonic sophorolipid.
butyldiphenylsilyl)oxy)pentadecanoyl)-600-acetyl
sophorolipid
macrolactone (13) as a yellow waxy solid (209 mg). Yield: 61%; TLC:
References
Rf ¼ 0.38 (100% ethyl acetate). IR (cmꢁ1): 3381, 2924, 2853, 1742,
1075, 701; 1H NMR (500 MHz, CDCl3,
d): 1.04 (s, 9H, (CH3)3CSi),
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1.24e1.36 (m, 39H, H-18, 18xCH2), 1.53e1.65 (m, 10H, 5xCH2),
2.00e2.04 (m, 2H, CH2), 2.08 (s, 3H, 600(CO)CH3), 2.32e2.38 (m, 4H,
2xCH2), 3.34e3.44 (m, 2H, H-50, 500), 3.52e3.56 (m, 2H, H-20, 200),
3.63e3.70 (m, 6H, H-300, 30, 40, 400, -CH2OTBDPS), 3.75e3.78 (m, 1H,
H-17), 4.13e4.21 (m, 2H, H-600), 4.26e4.32 (m, 1H, H-60a), 4.39e4.43
(m,1H, H-60b), 4.47 (d, J ¼ 7.5 Hz,1H, H-10), 4.56 (d, J ¼ 7.5 Hz,1H, H-
100), 5.31e5.39 (m, 2H, H-9, 10), 7.35e7.42 (m, 6H, 6xCHAr),
7.65e7.67 (m, 4H, 4xCHAr); 13C NMR (125 MHz, CDCl3,
d): 19.22,
20.78, 21.33, 24.72, 24.90, 25.05, 25.78, 26.87, 27.03, 27.22, 28.14,
28.34, 29.06, 29.15, 29.29, 29.39, 29.49, 29.63, 29.68, 29.77, 29.88,
32.59, 34.18, 34.31, 62.04, 63.15, 64.02, 69.75, 70.40, 72.89, 73.48,
74.06, 74.31, 75.43, 79.47, 80.68, 102.32, 103.06, 127.54, 129.45,
129.79, 130.00, 134.21, 135.57, 170.56, 173.27, 174.70; HRMS (TOF-
ESþ) m/z Calcd. for (C63H100O15Si þ Na)þ 1147.6730, found:
1147.6735.
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Acknowledgements
The authors gratefully recognize Sam Lazar of Amano Enzyme,
USA for his generous donation of samples of lipases and Dr. Richard
19. Ferris TJ, Went MJ. Forensic Sci Int. 2012;216(1):158e162.
Please cite this article in press as: Sembayeva A, et al., Lipase-mediated regioselective modifications of macrolactonic sophorolipids,
Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.02.043