Enzymic synthesis, physicochemical, and cell activity of glucosyl ester derivatives based on N-fatty acyl amino acid

Article abstract of DOI:10.1007/s11696-018-0614-4

Series of glucosyl esters derivatives were synthesized by enzymatic acylation and some surface properties, and cell activity were calculated and tested. The antitumor activity in vitro against three cancer cells, human colon carcinoma (K562), human hepatoma (HepG2), and human breast adenocarcinoma (MCF-7) of compounds, were evaluated using MTT methods. The glucosyl esters could significantly displayed high anticancer activity. Thereinto, compounds 12 and 12′ showed higher inhibition effect to cancer cells than others. Several compounds were more active than control drug 5-FU. The structure–activity relationship analysis revealed that lipophilic properties might be essential parameter for their activity.

Full text of DOI:10.1007/s11696-018-0614-4

Chemical Papers
https://doi.org/10.1007/s11696-018-0614-4
ORIGINAL PAPER
Enzymic synthesis, physicochemical, and cell activity of glucosyl ester
derivatives based on N‑fatty acyl amino acid
Dong An · Dexin Feng1
1
Received: 22 February 2018 / Accepted: 10 October 2018
©
Institute of Chemistry, Slovak Academy of Sciences 2018
Abstract
Series of glucosyl esters derivatives were synthesized by enzymatic acylation and some surface properties, and cell activity
were calculated and tested. The antitumor activity in vitro against three cancer cells, human colon carcinoma (K562), human
hepatoma (HepG2), and human breast adenocarcinoma (MCF-7) of compounds, were evaluated using MTT methods. The
glucosyl esters could signiꢀcantly displayed high anticancer activity. Thereinto, compounds 12 and 12′ showed higher inhibi-
tion eꢁect to cancer cells than others. Several compounds were more active than control drug 5-FU. The structure–activity
relationship analysis revealed that lipophilic properties might be essential parameter for their activity.
Keywords Amino acid · Antitumor cell lines · Glucosyl ester
Introduction
researched by many pharmaceutical companies and vast
amount of money and research are pumped into cancer
drug development. Carbohydrate fatty acid esters are not
only surfactant but also an important research direction for
developing anticancer drugs (Riva et al. 1988; Liang et al.
2012; Yang and Huang 2012). There were large amount of
literature on research for antimicrobial, antiinꢂammatory,
and antitumor activities of sugar ester derivatives (Pinazo
et al. 2016). The research on inhibition of tumor cells pro-
liferation indicated that the activity of sugar esters may have
relationship between glucose and fatty acyl chain.
Candidate compounds for screening drug were provided
through some appropriate method which is selected to
modify active molecular structure for changing its biologi-
cal activity (Arami et al. 2017; Kaapu et al. 2015; Wang
et al. 2012; Yonehara et al. 2015). Such as, antitumor and
antiviral activity of glucose phosphate derivatives have been
applied to clinical research (Sampaio et al. 2012; Song et al.
2
017). Development of antitumor drugs is the duty of medi-
cal workers. In the new drug research procedure, ꢀnding an
active leading compound is the foundation of all the research
work (Hanan et al. 2011; Pragya and Singh 2016; Guo et al.
Sugar esters can be synthesized by esteriꢀcation of car-
bohydrates and acylated compounds under chemical or
enzymatic catalysis at present (Behnia et al. 2016; Zhen and
Zhang 2014; Hu et al. 2016). The enzymic esteriꢀcation
reaction of long-chain fatty acid and carbohydrate alcohol
could be proceed under modest conditions, adopting mul-
tireaction, high regioselectivity to get target compounds,
that are great advantage over the chemical catalysis method
2
013).
Anticancer drug preparation synthesized from small mol-
ecule compounds had become an attractive strategy. New
potential anticancer reagents’ development becomes urgent
need (Pranee and Natcha 2017; Tian et al. 2016; Ali et al.
2
016). Therefore, some antitumor drugs are continually
(
Mallakpour and Behranvand 2015; Christensen et al. 2014;
Zhang et al. 2013; Wei et al. 2016).
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11696-018-0614-4) contains
supplementary material, which is available to authorized users.
*
Dexin Feng
fengdexin@126.com
1
CAS Key Laboratory of Biobased Materials, Qingdao
Institute of Bioenergy and Bioprocess Technology, Chinese
Academy of Sciences, Qingdao, China
Vol.:(0
1
123456789)
3

Chemical Papers
Experimental
(0.05 g), 10.0 mL t-butanol, molecular sieve (0.02 g), and
0
.5 mL of deionized water as enzyme activator was added
Materials and methods
to a 50 mL round-bottom ꢂask in order. The reaction was
reacted at 55 °C for 8 h. The reaction was followed by TLC
(Vethyl acetate:Vmethanol =2:1). After cooling reaction mixture to
room temperature, the lipase and activated molecular sieve
were ꢀltered from the reaction mixture. The ꢀltrate was
diluted with ethyl acetate (30 mL) and washed successively
with aqueous saturated sodium thiosulphate (Na2S2O3)
(2 × 20 ml) and aqueous saturated sodium bicarbonate
(NaHCO3) solutions (2 × 20 ml). The organic layer was
then dried with sodium sulfate (Na2SO4) and concentrated in
vacuo to give the crude product, and the residue was puriꢀed
by column chromatography (ethyl acetate:methanol=4:1)
to obtain white waxy solid 11 (0.75 mmol, 0.31 g, 75.1%
yield). (Scheme 1).
Candida cylindracea lipase (CRL), porcine pancreas lipase
(
PPL), immobilized thermophilic fungal lipase (TLIM),
Novozym 435, and Lipozyme 435 were all purchased from
Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Solvent
evaporation was performed under reduced pressure using
Rotatory Evaporator unless otherwise stated. Commercially
available grades of organic solvents of adequate purity were
used in all the reactions. NMR were recorded in DMSO-d6
on a Jeol 500 MHz instrument using TMS as internal stand-
ard. Mass spectra were recorded on a Shimadzu PQ-5000
GC–MS apparatus. Column chromatography was performed
on ꢂash silica gel. All reactions were monitored using thin-
layer chromatography (TLC) on silica gel plates.
1
H NMR (500 MHz, DMSO): δ/ppm 8.16 (t, 1H,
J=6.5 Hz, NH), 5.14 (s, 1H, C –OH), 4.88 (s, 1H, C –OH),
4
3
4
.79 (s, 1H, C –OH), 4.46 (s, 1H, H–1), 4.34 (d, 1H,
2
Sample preparation N‑fatty acyl amino acids
J=11.5 Hz, H-6b), 4.21–4.19 (m, 1H, H-5), 4.03–3.97 (m,
1
H, CH), 3.50–3.44 (m, 1H, H-6a), 3.38–3.36 (m, 1H, H-2),
N-fatty acyl amino acids 7–10 and 7′–10′ were synthesized
using chemical method from glycine and l-valine with four
respective fatty acyl chloride adopting our previous report
3.20 (s, 3H, OCH ), 3.16–3.14 (m, 1H, H-3), 3.02–2.99 (m,
3
1H, H-4), 2.01 (t, 2H, J=6.0 Hz, CH ), 1.42–1.40 (m, 2H,
2
CH ), 1.22–1.19 (m, 3H, CH ). 1.18 (s, 12H, 6 × (CH )),
2
3
2
1
3
(
An et al. 2015).
0.86 (t, 3H, J=6.5 Hz, CH ); C NMR (125 MHz, DMSO):
3
Glycine 1 (1.46 g, 10 mmol) was dissolved in mixed sol-
δ/ppm 173.20 (NHC=O), 169.86 (C=O), 95.23, 78.37 (C-5),
76.66 (C-3), 72.88 (C-2), 69.79 (C-4), 60.90 (C-6), 35.56,
31.76, 29.52,29.47, 29.37, 29.06, 25.67, 22.55, 14.35;
vent of 40 mL 1 mol/L NaOH solution and acetone (vol-
ume ratio 4:6), pH about 9. Palmitoyl chloride 6 (2.54 g,
+
1
3.37 mmol) was added slowly under 0–5 °C; meanwhile,
ESI–MS: C H N O , m/z: 428 [M+Na] .
2
0
38
2
7
sodium hydroxide solution (1 mol/L) was added slowly drop
by drop to adjust the pH of the resulting mixture about 9.
The mixture was continued to react at 25 °C for 3 h. The
reaction mixture was added hydrochloric acid solution
The Methyl 6-O-(N-fatty acyl amino acid)-d-glucosyl
esters 12–14′ were synthesized according to the above
preparation method, respectively.
Methyl 6-O-(N-dodecanoyl-glycyl)-d-glucosyl ester
(12): treatment of 8 (1.5 mmol, 0.41 g) and methyl α-d-
glucopyranoside (1 mmol) as described for 11 to aꢁord
(
1 mol/L) to regulate pH to 1–2 and some solid precipitated
out from solution. Then, the crude product was ꢀltered and
washed with plenty of deionized water and petroleum ether,
successively. Filter cake was the white powder solid product
N-palmitoyl glycine 10 (2.17 g, 78.5%).
1
white oil product 12 (0.78 mmol, 0.35 g, 78.7% yield); H
NMR (500 MHz, DMSO): δ/ppm 8.19 (d, 1H, J=6.0 Hz,
NH), 5.17 (s, 1H, C –OH), 4.91 (s, 1H, C –OH), 4.82 (s, 1H,
4
3
1
m.p.: 121–122 °C; H NMR (500 MHz, DMSO): δ/
C –OH), 4.53–4.52 (m, 1H, H-1), 4.42–4.40 (m, 1H, H-6b),
2
ppm 12.45 (s, 1H, COOH), 8.07 (t, 1H, NH), 3.69 (d, 2H,
4.28–4.25 (m, 1H, H-5), 4.10–4.06 (m, 1H, CH), 3.54–3.52
J=6.0 Hz, NCH ), 2.09–2.07 (m, 2H, CH ), 1.46 (m, 2H,
(m, 1H, H-6a), 3.50–3.45 (m, 1H, H-2), 3.26 (s, 3H, OCH ),
2
2
3
CH ), 1.27 (s, 24H, C H ), 0.84 (t, 3H, J=6.5 Hz, CH ).
3.22–3.20 (m, 1H, H-3), 3.07–3.05 (m, 1H, H-4), 2.09–2.07
2
12 24
3
Other compounds were synthesized by above method as
white solid and their structure conꢀrmed were provided in
supporting information.
(m, 2H, CH ), 1.48–1.47 (m, 2H, CH ), 1.27–1.26 (m, 3H,
2
2
CH ), 1.24 (s, 16H, 8×(CH )), 0.86 (t, 3H, J=7.0 Hz, CH );
3
2
3
1
3
C NMR (125 MHz, DMSO): δ/ppm 173.08 (NHC=O),
72.02 (C=O), 100.26, 73.63, 72.21, 70.94, 70.76, 69.98,
4.64, 57.66, 39.62, 35.33, 31.73, 29.37, 29.06, 25.76,
2.55, 19.45, 18.63, 14.38; ESI–MS: C H N O , m/z: 456
1
6
2
General enzymatic synthesis of glucosyl esters
2
2
42
2
7
11–14′
+
[
M+Na] .
Methyl 6-O-(N-myristyl-glycyl)-d-glucosyl ester
13): Treatment of 9 (1.5 mmol, 0.45 g) and methyl α-d-
Methyl 6-O-(N-decanoyl-glycyl)-d-glucosyl ester (11): The
mixture of methyl d-glucopyranoside (0.19 g, 1 mmol),
N-fatty glycine 7 (0.36 g, 1.5 mmol), the Lipozyme 435
(
glucopyranoside (1 mmol) as described for 11 gave oily
1
product 13 (0.69 mmol, 0.31 g, 69.2% yield); H NMR
1
3

Chemical Papers
Scheme 1 General synthetic route of glucosyl esters
(
500 MHz, DMSO): δ/ppm 8.22 (d, 1H, J = 6.0 Hz, NH),
.14 (s, 1H, C –OH), 4.89 (s, 1H, C –OH), 4.79 (s, 1H,
α-d-glucopyranoside (1 mmol) to give oily product 11′
1
5
(0.57 mmol, 0.26 g, 57.3% yield); H NMR (500 MHz,
4
3
C –OH), 4.46 (s, 1H, H-1), 4.34 (d, 1H, J=11.5 Hz, H-6b),
DMSO): δ/ppm 8.10 (d, 1H, J=8.0 Hz, NH), 5.12 (s, 1H,
OH), 4.88 (s, 1H, OH), 4.77 (s, 1H, OH), 4.49 (d, 1H,
J=3.0 Hz, H-1), 4.40–4.38 (m, 1H, H-6b), 4.27–4.23 (m,
1H, H-5), 4.02–3.93 (m, 1H, CH), 3.53–3.51 (m, 1H, H-6a),
2
4
.21–4.19 (m, 1H, H-5), 4.02–3.89 (m, 1H, CH), 3.54–3.51
(
m, 1H, H-6a), 3.34–3.31 (m, 1H, H-2), 3.24 (s, 3H, OCH ),
3
3
2
.20–3.16 (m, 1H, H-3), 3.01–2.97 (m, 1H, H-4), 2.02 (t,
H, J=7.0 Hz, CH ), 1.41–1.39 (m, 2H, CH ), 1.21–1.19
3.36–3.34 (m, 1H, H-2), 3.23 (s, 3H, OCH ), 3.17–3.15 (m,
2
2
3
(
m, 3H, CH ), 1.18 (s, 20H, 10 × (CH )), 0.84–0.86 (m,
1H, H-3), 3.04–3.00 (m, 1H, H-4), 2.07–2.03 (m, 2H, CH ),
3
2
2
1
3
3
H, CH ); C NMR (125 MHz, DMSO): δ/ppm 173.24
1.60–1.56 (m, 1H, CH), 1.51–1.44 (m, 2H, CH ), 1.44–1.42
3
2
(
NHC=O), 169.54 (C=O), 95.22, 78.41, 76.68, 72.93, 69.82,
(m, 2H, CH ), 1.23–1.21 (m, 12H, 6 × (CH )), 0.85–0.78
2
2
1
3
6
2
0.83, 35.56, 31.75, 29.51, 29.52, 29.42, 29.29, 29.17,
(m, 9H, CH , 2 × CH ); C NMR(125 MHz, DMSO): δ/
3 3
9.06, 25.65, 22.56, 14.41; ESI–MS: C H N O , m/z: 484
ppm 173.43 (NHC=O), 171.13 (C=O), 97.15 (C-1), 79.42
(C-5), 76.73 (C-3), 73.77 (C-2), 70.38 (C-4), 61.79 (C-6),
56.67, 46.22, 36.36, 34.47, 33.71, 31.89, 29.62, 29.33,
29.22, 28.79, 28.41, 28.26, 25.62, 24.74, 22.69, 22.32,
2
4
46
2
7
+
[
M+Na] .
Methyl 6-O-(N-palmitoyl-glycyl)-d-glucosyl ester
14): treatment of 10 (0.49 g, 1.5 mmol) and methyl
(
+
α-d-glucopyranoside (1 mmol) to give oily product 14
17.26, 14.18; ESI–MS: C H N O , m/z: 470 [M+Na] .
2
3
444
2
7
1
(
0.66 mmol, 0.30 g, 65.8% yield); H NMR (500 MHz,
Methyl 6-O-(N-dodecanoyl-valine acyl)-d-glucosyl
ester (12′): treatment of 8′ (1.5 mmol, 0.47 g) and methyl
α-d-glucopyranoside (1 mmol) to give oily product 12′
DMSO): δ/ppm 8.23 (t, 1H, J= 6.0 Hz, NH), 5.36 (s, 1H,
OH), 5.28 (s, 1H, OH), 5.11 (s, 1H, C –OH), 5.02 (m, 1H,
2
1
H-1), 4.62 (s, 1H, CH), 4.28–4.25 (m, 1H, CH), 4.10–4.07
(0.8 mmol, 0.38 g, 79.3% yield); H NMR (500 MHz,
(
m, 1H, CH), 3.55–3.53 (m, 1H, CH), 3.52–3.47 (m, 1H,
DMSO): δ/ppm 8.08 (d, 1H, J = 7.5 Hz, NH), 5.11 (s,
CH), 3.26 (s, 3H, OCH ), 3.23–3.20 (m, 1H, H-3), 3.09–3.07
1H, C –OH), 4.86 (s, 1H, C –OH), 4.76 (s, 1H, C –OH),
3
4
3
2
(
m, 1H, CH), 2.11–2.09 (m, 2H, CH ), 1.49–1.47 (m, 2H,
4.47 (d, 1H, J = 3.0 Hz, H-1), 4.40–4.37 (m, 1H, H-6b),
2
CH ), 1.27–1.26 (m, 3H, CH ), 1.24 (s, 24H, 12 ×(CH )),
4.28–3.49 (m, 1H, H-6a), 3.35–3.33 (m, 1H, H-2), 3.22 (s,
2
3
2
1
3
0
.88–0.86 (m, 3H, CH ); C NMR (125 MHz, DMSO): δ/
3H, OCH ), 3.16–3.14 (m, 1H, H-3), 3.04–3.01 (m, 1H,
3
3
ppm 173.24 (NHC=O), 169.54 (C=O), 95.25 (C-1), 78.42
H-4), 2.08–2.04 (m, 2H, CH ), 1.61–1.55 (m, 1H, CH),
2
(
C-5), 76.71 (C-3), 72.93 (C-2), 69.82 (C-4), 60.90 (C-6),
1.52–1.46 (m, 2H, CH ), 1.44–1.42 (m, 2H, CH ), 1.22–1.19
2
2
3
5.56, 31.76, 29.52, 29.47, 29.42, 29.29, 29.17, 29.06, 25.65,
(m, 16H, 8×(CH )), 0.84–0.83 (m, 3H, CH ), 0.82–0.80 (m,
2
3
+
13
2
2.56, 14.41; ESI–MS: C H N O , m/z: 512 [M+Na] .
3H, CH ). 0.79–0.77 (m, 3H, CH ); C NMR (125 MHz,
3 3
2
6
50
2
7
Methyl 6-O-(N-decanoy l-valine acyl)-d-glucosyl
DMSO): δ/ppm 173.90 (NHC=O), 171.10, 95.14 (C-1),
ester (11′): treatment of 7′ (1.5 mmol, 0.43 g) and methyl
76.69 (C-5), 72.80 (C-3), 69.77 (C-2), 60.83 (C-4), 56.60
1
3

Chemical Papers
(
C-6), 35.34, 31.65, 30.18, 29.28, 29.11, 28.91, 25.75,
9.35, 18.74, 18.04 14.35; ESI–MS: C H N O , m/z: 498
where n represents the number of species at the air–water
1
interface whose concentration changes with surfactant con-
2
5
48
2
7
+
−1
[
M+Na] .
centration, R is the gas constant [8.314 J/(mol. K) ], T is
Methyl 6-O-(N-myristyl-valine acyl)-d-glucosyl ester
13′): treatment of 9′ (1.5 mmol, 0.51 g) and methyl
the absolute temperature (K), and (dγ/dlgc) is the slope in
the surface tension isotherm.
(
α-d-glucopyranoside (1 mmol) to give oily product 13′
The areas per molecule (Amin) at air/water interface were
calculated from the equation:
1
(
0.60 mmol, 0.32 g, 62.3% yield); H NMR (500 MHz,
DMSO): δ/ppm 8.07–8.05 (m, 1H, NH), 5.11 (s, 1H,
ꢀ
ꢁ−1 ,
C –OH), 4.86 (s, 1H, C –OH), 4.76 (s, 1H, C –OH), 4.47
(2)
4
3
2
Amin
= 1∕ N ꢀ
A max
(
d, 1H, J=3.0 Hz, H-1), 4.40–4.37 (m, 1H, H-6b), 4.27–4.23
where N is the Avogadro constant. pC20 was the value of
(
m, 1H, H-5), 4.01–3.90 (dd, J = 11.5, 6.0 Hz, 1H, CH),
A
the logarithm of surfactant concentration required to reduce
3
.51–3.49 (m, 1H, H-6a), 3.36–3.34 (m, 1H, H-2), 3.22
the surface tension of water by 20 mN/m.
(
s, 3H, OCH ), 3.16–3.14 (m, 1H, H-3), 3.04–3.00 (m,
3
The eꢁectiveness of surfactants adsorption was also cal-
culated using the following equation:
ꢀcmc = ꢁ − ꢁcmc,
1
H, H-4), 2.07–2.03 (m, 2H, J= 7.0 Hz, CH ), 1.61–1.55
2
(
m, 1H, CH), 1.51–1.44 (m, 2H, CH ), 1.44–1.42 (m, 2H,
2
CH ), 1.20–1.17 (m, 20H, 10×(CH )), 0.83–0.82 (m, 6H,
2
2
0
(3)
cmc
2
×CH ). 0.81–0.80 (m, 3H, CH ). 0.79–0.78 (m, 3H, CH );
3 3 3
where γ is the surface tension of water at 25 °C and γ is
1
3
0
C NMR (125 MHz, DMSO): δ/ppm 73.90 (NHC=O),
71.10 (C=O), 95.14 (d, C-1), 76.69 (C-5), 72.80 (C-3),
9.77 (C-2), 60.83 (C-4), 56.60 (C-6), 35.34, 31.65, 30.18,
the surface tension value of the measured solution at CMC.
1
6
2
9.28, 29.11, 28.91, 25.75, 19.35, 18.74, 18.04 14.35;
Stability study
+
ESI–MS: C H N O , m/z: 526 [M+Na] .
2
7
52
2
7
Methyl 6-O-(N-palmitoyl-valine acyl)-d-glucosyl ester
The compounds 12′ were set as examples to discuss series
methyl d-glucosyl esters stability in various pH 3.0–10.0
aqueous solutions and rat plasma. The glucosyl esters were
dissolved in buꢁer solution which was prepared with sodium
bicarbonate and acetic acid and stand for 0–8 h. Similarly,
the methanol solution of compound 12′ was diluted by fresh
rat plasma and preserved in refrigerator (below 2–5 °C) to
keep quality.
(
14′): treatment of 10′ (1.5 mmol, 0.55 g) and methyl α-d-
glucopyranoside (1 mmol) to give light yellow soil 14′
1
(
0.57 mmol, 0.30 g, 57.6%); H NMR (500 MHz, DMSO):
δ/ppm 8.05 (d, 1H, J=7.5 Hz, NH), 5.08 (s, 1H, OH), 4.84
s, 1H, OH), 4.76 (s, 1H, OH), 4.46–4.43 (m, 1H, H-1),
.40–4.37 (m, 1H, H-6b), 4.26–4.23 (m, 1H, H-5), 4.00–3.89
m, 1H, CH), 3.48–3.46 (m, 1H, H-6a), 3.32–3.30 (m, 1H,
(
4
(
H-2), 3.20 (s, 3H, OCH ), 3.16–3.14 (m, 1H, H-3), 3.04–3.00
3
(
m, 1H, H-4), 2.07–2.03 (m, 2H, J=7.0 Hz, CH ), 1.61–1.55
2
Cell culture
(
m, 1H, CH), 1.51–1.44 (m, 2H, CH ), 1.44–1.42 (m, 2H,
2
CH ), 1.22–1.19 (m, 24H, 12×(CH )), 0.84–0.83 (m, 6H,
2
2
The K562, HepG2, and MCF7 cell lines were cultured in
DMEM medium, supplemented with 10% fetal bovine serum
1
3
2
× CH ). 0.81–0.76 (m, 3H, CH ); C NMR (125 MHz,
3
3
DMSO): δ/ppm 173.24 (NHC=O), 169.54 (C=O), 95.25
(
FBS), 100 U/mL penicillin, and 100 U/mL streptomycin
(
C-1), 78.42, 76.70, 72.93, 69.82, 60.90, 35.56, 31.76, 29.52,
solution. The cells were grown at 37 °C under an atmosphere
2
9.42, 29.11, 29.291, 29.17, 29.06, 25.65, 22.56, 14.41;
of 5% CO incubator (Hua et al. 2015).
+
2
ESI–MS: C H N O , m/z: 554 [M+Na] .
2
9
56
2
7
Surface tension measurements
Antitumor activity in vitro
The surface tension of glucosyl diesters’ aqueous solutions
was measured for three times to achieve good repeatability
with Du Nouy ring method at 15–35 °C. The CMC (Criti-
cal Micelle Concentration) value was determined from the
break point on the curve of surface tension vs surfactant
concentrations.
All compounds 11–14′ were dissolved in DMSO of mini-
mum quantity to prepare 10.0 mg/mL stock solution and
stored less than 20 °C, respectively. The prepared solution
was diluted to required concentration using saline before
test antitumor activity. The antitumor activity in vitro was
performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) assay which is based on
the reduction of soluble MTT into a blue–purple formazan
product, mainly by mitochondrial reductase activity inside
living cells. The activity was evaluated against three diꢁer-
ent cancer cell lines: K562, HepG2, and MCF-7 cell lines,
The surface excess concentration (Γmax) was calculated
from Gibbs equation:
ꢀ
= −(1∕2.303nRT)(dꢁ∕d lg c),
(1)
max
1
3

Chemical Papers
using control drug 5-ꢂuorouracil (5-FU) for comparison
and DMSO as blank control (Ye and Hayes Douglas 2016).
Cultured primary cancer cells in logarithmic growth
phase were seeded into a 96-well cell culture plate with
Results and discussion
Synthesis at various temperatures
5
cell density of 2 × 10 cells/mL in the presence of 0.1 mL
Eꢁects of temperature on lipase activity to synthesis glu-
cosyl ester in t-butanol at reaction time 8 h was optimized,
taking glycine series compound 12 and l-valine series com-
pound 12′ as example. As shown in Fig. 1, the highest yield
78.7% and 79.3% was obtained at 55 °C. The structural sta-
bility of lipase was decreased probably and enzyme activity
was impacted by solvent t-butanol under higher temperature
which is also often deleterious to enzymes. Other similar
experiments were performed at 55 °C based on the experi-
mental exploration results.
RPMI 1640 in 5% CO incubator for 24 h at 37 °C, and
2
allowed to adhere for 12 h, while suspended cells were
seeded just before drug addition. The medium was then
removed and refilled with fresh medium-containing
graded concentrations of sample after the original cul-
ture medium was removed and continued to incubate for
3
days. Next, MTT stock solution (20 μL, 5 mg/mL, prep-
aration in phosphate-buꢁered saline) was added to each
well. The incubation continued for 4 h at 37 °C and DMSO
(
100 μL/well) was introduced into each well to dissolve the
formazan crystals after medium sucked out. The culture
plate shocked at 150 rpm for 10 min placed in microporous
oscillator.
Synthesis at various time
Eꢁects of the reaction time on yield of compound 12 and
12′ as example had also been studied. As can be seen from
Fig. 2, the yields were increased and decreased gradually
with reaction time from 5 to 10 h. The highest yield 78.7%
and 79.3% of 12 and 12′ was obtained under optimal 8 h,
respectively. The results showed that the increasing speed
of conversion rate slowed down when the reaction time was
up to 8 h. The reversible enzymic reaction took place prob-
The optical density (OD) of each well was measured
at a wavelength of 550 nm in a 96-well microtiter plate
reader. The IR % (inhibition rate) % was calculated
using OD mean values by the formula, inhibition rate
(
%) = [(OD
− OD
)/OD ] × 100%. The IC50
sample AC
control
value was obtained from ꢀtting nonlinear regression model
parameters relevant to S-fold on Graph pad Prism Version
5
.0 program in which the diꢁerent concentrations of IR %
ably under by-product H O increased and Lipozyme 435
2
values were input.
inactivation was further produced as the time prolonged.
The optimum reaction time was 8 h for the esteriꢀcation.
Fig. 1 Inꢂuence of reaction temperature on the yield of glucosyl esters 12 and 12′. [reaction conditions: methyl α-d-glucopyranoside (1 mmol),
N-fatty acyl glycine (1.5 mmol), 10% (w/w of substrates) Lipozyme 435, 10% (w/w reaction mixture) molecular sieves, and reaction time 8 h]
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Fig. 2 Inꢂuence of reaction time on the yield of glucosyl esters 12 and 12′. [reaction conditions: methyl α-d-glucopyranoside (1 mmol), N-fatty
acyl glycine (1.5 mmol), 10% (w/w of substrates) Lipozyme 435, 10% (w/w reaction mixture) molecular sieves, and reaction temperature 55℃]
been used eꢃciently on esteriꢀcation in much literature.
The catalytic eꢃciency of Lipozyme 435 selected for our
reaction was relatively better than Novozyme 435 in our
study.
Surface activity
The CMC values were conꢀrmed by lgc-γ distribution curve
as shown in Fig. 4. The surface tension was decreased as the
compound concentration increased until smooth. The CMC
values of 11–14 were 3.26, 3.11, 2.37, and 1.618 mmol/L,
and 11′–14′ were 2.70, 2.41, 1.93, and 1.75 mmol/L at
2
5 °C, respectively. The CMC values of glucosyl esters
1
1–14′ were decreased as the hydrophobic chains increased.
The valine structure had a slight eꢁect on CMC compared
to glycine.
Fig. 3 Diꢁerent lipases eꢁects on the yield of glucosyl esters 12 and
1
2′. (the reaction conditions: 1 mmol methyl α-d-glucopyranoside
Surface activity parameters at 25 °C are listed in Table 1.
with 1.5 mmol N-fatty acyl amino acid 8 or 8′ catalyzed by 10%
lipase at 55℃ in 10 mL t-butanol and 1 mL water for 8 h)
The CMC and γ
were decreased and the pC20 values
CMC
were increased as the fatty acyl chain length increased in the
same amino acid series glucosyl esters.
Eꢀect of diꢀerent enzyme for the esteriꢁcation
The positive Γmax values indicated positive adsorption of
glucosyl esters in solution. The Γ
values of compounds
max
Diꢁerent lipases like PPL Lipozyme 435, Novozyme 435,
CRL, and TLIM have been chosen to catalyze the esteri-
11–14 and 11′–14′ were 1.95, 1.84, 1.64, and 1.56 and 1.73,
2
1.55, 1.48, and 1.32 μmol/m , respectively, which accounted
ꢀ
cation between methyl α-d-glucopyranoside and N-fatty
for the concentration on the surface layer were higher than
that in the solution. The Amin were aꢁected by molecular
structure of glucosyl esters, especially amino acid and fatty
acyl structure. The Amin values of glucosyl esters 11–14′
were 0.85, 0.90, 1.01, and 1.06 and 1.04, 1.10, 1.17, and
acyl amino acid under certain reaction environment. The
diꢁerent catalytic eꢁect is shown in Fig. 3. It was indi-
cated that compound 12 or 12′ was got poor esteriꢀcation
yield 11.4% and 13.9% or 12.8% and 15.3% under CRL
and PPL as catalyst, respectively. Fortunately, the higher
yield 78.7%, 73.2% and 79.3%, 70.7% was given using
Lipozyme 435 and Novozyme 435, respectively, which had
2
1.25 nm , respectively. The Γmax values were increased and
Amin values were increased as the fatty acyl chain length
increased.
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Chemical Papers
Fig. 4 γ-lgc curve of glucose esters at 25℃. Surface activity parameters at 25℃ are listed in Table 1. The CMC and γ
were decreased and the
CMC
pC20 values were increased as the fatty acyl chain length increased in the same amino acid series glucosyl esters
Table 1 Surface active
Γmax (μmol/m2)
Amin (nm2)
Compounds CMC (mmol/L) γcmc (mN/m) πcmc (mN/m) Pc20
parameter of compounds 11–14′
aqueous solution at 25°C
1
1
1
1
1
1
1
1
1
3.26
3.11
2.37
1.61
2.70
2.41
1.93
1.75
39.79
38.06
37.97
37.31
37.55
37.07
36.22
35.52
32.21
33.94
34.03
34.69
34.45
34.93
35.78
36.48
2.84 1.95
0.85
0.90
1.01
1.06
0.96
1.07
1.12
1.25
2
3.15 1.84
3.48 1.64
3.55 1.56
3.94 1.73
3.98 1.55
4.13 1.48
4.17 1.32
3
4
1′
2′
3′
4′
Stability study in vitro
The stability results of compound 12′ are shown in Fig. 4
in diꢁerent pH solutions and rat plasma at diꢁerent time.
The concentration kept relative stability in pH 4–7 aqueous
solutions. It was tended downward and ꢀnally kept stabil-
ity in pH 8–10 aqueous solutions with time prolonging. On
the contrary, the concentration was decreased sharply in rat
plasma, which might be the ester bond of compound 12′
breaking by enzyme hydrolytic in plasma. Therefore, these
methyl d-glucosyl esters had certain stability in vitro, and
could be completely metabolized in vivo based on the above
analysis (Fig. 5).
MTT assay
The anticancer activity in vitro of compounds 11–14′ against
three human cancer cell lines including K562, HepG2, and
MCF-7 at 48 h and 72 h was evaluated by MTT assay,
respectively. The inhibition rate (IR %) values of series
Fig. 5 Stability of compound 12′ in aqueous buꢁer solutions
(pH=3–10) and rat plasma in vitro
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Fig. 6 Inhibition rate (IR %) of diꢁerent concentrations 11–14′ on K562 for 48 h and 72 h
Fig. 7 Inhibition rate (IR %) of diꢁerent concentrations 11–14′ on HepG2 for 48 h and 72 h
glucosyl esters and 5-FU are shown in Figs. 6, 7, and 8, and
the half inhibitory concentration (IC ) is listed in Table 2.
parameter for aꢁecting activity. Thereinto, compounds with
dodecanoyl and glycine structure had a better inhibiting
eꢁect on three cancer cells sample than other compounds.
Only compound 12′ (IR %=74.82 and 75.43 μM) has a
better activity than positive control 5-FU (IR %=67.26 and
70.23 μM) on K562 inhibition for 48 h and 72 h. Compound
12 (IR % = 69.66 and 75.43 μM) showed a better activity
against MCF7 cells than 5-FU (IR %=67.14 and 75.20 μM)
for 48 h and 72 h. However, no glucosyl esters showed a bet-
ter activity against HepG2 cells than 5-FU in concentration
of 50 and 100 μM.
5
0
MTT assay showed that glucosyl esters derivatives 11–14′
exhibited good anticancer activities to inhibit three cancer
cells proliferation in a dose- and time-dependent fashion. All
compounds had weak activity at experiment concentration
of 25 μM. However, the activity was enhanced as concen-
tration up to 100 μM. The highest inhibition rate could be
reached 75.43%, 49.92%, and 79.60% on K562, HepG2, and
MCF-7 cells for 72 h, respectively. Inhibiting proliferation of
K562 and MCF-7 cells increased gradually as time increased
from 48 to 72 h. The structure–activity relationship revealed
that amino acid and fatty acyl structure might be essential
Overall, compounds 11–14′ showed better inhibitory
activities against K562 and MCF7 cell proliferation than
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Fig. 8 Inhibition rate (IR %) of diꢁerent concentrations 11–14′ on MCF7 for 48 h and 72 h
Table 2 IC50 of 11–14′ on
human cancer K562, HepG2,
and MCF7 cells
IC50 (μM)a
Samples
48 h
72 h
K562
HepG2
MCF-7
K562
HepG2
MCF-7
1
1
1
1
1
1
1
1
1
77.24
62.35
72.42
74.60
70.92
55.83
61.57
63.12
115.24
102.43
117.53
120.21
114.73
103.19
117.82
125.37
73.44
70.31
72.82
75.46
87.30
82.29
112.15
119.27
73.84
56.89
69.80
70.65
62.53
49.89
56.31
58.47
117.27
103.45
121.32
129.56
117.12
109.25
118.49
130.59
68.85
62.31
65.92
66.38
75.74
70.28
105.36
107.58
2
3
4
1′
2′
3′
4′
aAll data are the means of at least three values measured in two independent plates
HepG2 cell. Two glucosyl ester groups 11–14 and 11′–14′
showed the same rule for human cancer cell lines inhibition.
It could be found that there was weak relation between inhib-
itory activity and amino acid structure according to the IC
compounds have good surface properties. Most glucosyl esters
derivatives exhibited high anticancer activity against three dif-
ferent cell lines; more importantly, several compounds were
more active than the control drug 5-Fu. All compounds had a
better inhibitory activity on K562 than the HepG2 and MCF-7
cells. The best IC50 values of compounds were 49, 103, and
62.31 μM for K562, HepG2, and MCF-7, respectively. The
structure–activity relationship analysis revealed that amino
acid and fatty acyl chain of glucosyl esters structure could
aꢁect the anticancer activity. The synthesized compounds may
have the potential as anticancer agents to inhibit tumor growth.
5
0
values. In addition, glucosyl esters with dodecanoyl groups
(
12 and 12′) had stronger activity than other compounds on
inhibiting the growth three human cancer cell lines based on
the IC values in Table 2.
5
0
Conclusions
We synthesized some compounds containing amino acid and
glucose structure by enzyme catalysis to develop new low-
cytotoxicity anticancer agents. The single-esteriꢀcation can be
obtained at 55 °C for 8 h, and the ratio of methyl d-glucopyra-
noside and the N-fatty acyl amino acid was 1:1.5. All target
References
Ali HM, Koza G, Davies C (2016) The synthesis of single enantiom-
ers of trans-alkene containing mycolic acids and related sugar
1
3

Chemical Papers
esters. Tetrahedron 72:7143–7158. https://doi.org/10.1016/j.tetle
t.2009.10.009
Pragya TR, Singh SN (2016) Plant secondary metabolism linked gly-
cosyltransferases: an update on expanding knowledge and scopes.
Biotechnol Adv 5:714–739. https://doi.org/10.1016/j.biotechadv
.2016.03.006
An D, Zhao XH, Ye ZW (2015) Enzymatic synthesis and characteriza-
tion of galactosyl monoesters. Carbohydr Res 414:32–38. https://
doi.org/10.1016/j.carres.2015.05.011
Pranee I, Natcha W (2017) Lipase-catalyzed synthesis of sucrose
monoester: increased productivity by combining enzyme pretreat-
ment and non-aqueous biphasic medium. J Biotechnol 10:182–
190. https://doi.org/10.1016/j.jbiotec.2017.07.021
Arami SA, Mahdavi MJ, Molavi LL (2017) Apoptosis induction activ-
ity and molecular docking studies of survivin siRNA carried by
Fe3O4-PEG-LAC-chitosan-PEI nanoparticles in MCF-7 human
breast cancer cells. J Pharm Biomed 142:145–154. https://doi.
org/10.1016/j.jpba.2017.04.025
Riva S, Chopineau J, Kieboom APG et al (1988) Protease-catalyzed
regioselective esteriꢀcation of sugars and related compounds in
anhydrous dimethylformamide. J Am Chem Soc 110:584–589.
https://doi.org/10.1021/ja00210a045
Behnia I, Yuan ZS, Charpentier P, Xu CB (2016) Production of meth-
ane and hydrogen via supercritical water gasiꢀcation of renew-
able glucose at a relatively low temperature: eꢁects of metal cata-
lysts and supports. Fuel Process Technol 143:27–34. https://doi.
org/10.1016/j.fuproc.2015.11.006
Sampaio N, Nair DA, Rodrigues S (2012) Enzymatic synthesis of
sugar esters and their potential as surface-active stabilizers of
coconutmilk emulsions. Food Hydrocoll 27:324–331. https://doi.
org/10.1016/j.foodhyd.2011.10.009
Christensen DR, Calder PC, Houghton FD (2014) Eꢁect of oxygen ten-
sion on the amino acid utilisation of human embryonic stem cells.
Cell Physiol Biochem 33:237–246. https://doi.org/10.1159/00035
Song S, Zhu CL, Huo D (2017) A hybrid nanomaterial for the con-
trolled generation of free radicals and oxidative destruction of
hypoxic cancer cells. Angew Chem Int Ed 56:8801–8804. https
://doi.org/10.1002/anie.201702898
6
665
Guo YY, Luo J, Tan SW (2013) The applications of vitamin E TPGS
in drug delivery. Eur J Pharm Sci 49:175–186. https://doi.
org/10.1016/j.ejps.2013.02.006
Tian YX, Liu WR, Lu Y (2016) Naturally occurring cinnamic
acid sugar ester deriva-tives. Molecules 21:1–48. https://doi.
org/10.1016/S0031-9422(00)85108-0
Hanan M, Shoukry O, Mahran LG (2011) Novel sugar esters proni-
osomes for transdermal delivery of vinpocetine: preclinical and
clinical studies. Eur J Pharm Biopharm 77:43–55. https://doi.
org/10.1016/j.ejpb.2010.10.011
Wang YJ, Li PX, Wang S (2012) Anticancer peptidylarginine deimi-
nase (PAD) inhibitors regulate the autophagy ꢂux and the mam-
malian target of rapamycin complex 1 activity. J Biol Chem
287:25941–25953. https://doi.org/10.1074/jbc.M112.375725
Wei GF, Cui SS, Luan WJ (2016) Natural product-based design, syn-
thesis and biological evaluation of Albiziabioside A derivatives
that selectively induce HCT116 cell death[J]. Eur J Med Chem
113:92–101. https://doi.org/10.1016/j.ejmech.2015.12.034
Yang Z, Huang ZL (2012) Enzymatic synthesis of sugar fatty acid
esters in ionic liquids. Catal Sci Technol 2:1767–1775. https://
doi.org/10.1039/C2CY20109G
Hu GQ, He HB, Zhang W (2016) The transformation and renewal of
soil amino acids induced by the availability of extraneous C and
N. Soil Biol Biochem 96:86–96. https://doi.org/10.1016/j.soilb
io.2016.01.019
Hua SX, Huang RZ, Ye MY (2015) Design, synthesis and in vitro
evaluation of novel ursolic acid derivatives as potential antican-
cer agents. Eur J Med Chem 5:435–452. https://doi.org/10.1016/j.
ejmech.2015.03.051
Kaapu KJ, Ahti J, Tammela TL (2015) Sotalol, but not digoxin is asso-
ciated with decreased prostate cancer risk: a population-based
case-control study. Int J Cancer 137(5):1187–1195. https://doi.
org/10.1002/ijc.29470
Ye R, Hayes Douglas G (2016) Solvent free lipase-catalyzed synthesis
of technical-grade sugar esters and evaluation of their physico-
chemical and bioactive properties. Catalyst 6:1–13. https://doi.
org/10.3390/catal6060078
Liang JT, Zeng WT, Yao PJ, Wei YA (2012) Lipase-catalyzed regiose-
lective synthesis of palmitolyglucose ester in ionic liquids. Adv
Biol Chem 2:226–232. https://doi.org/10.4236/abc.2012.23027
Mallakpour SD, Behranvand VB (2015) Renewable and optically
active diacids containing amino acid as coupling agents for the
modiꢀcation of ZnO nanoparticles. Synth React Inorg Met Org
Nano Met Chem 45:1039–1044. https://doi.org/10.1080/15533
Yonehara A, Tanaka YK, Kulkeaw K (2015) Aloe vera extract sup-
presses proliferation of neuroblastoma cells in vitro. Anticancer
Res 35:4479–4486
Zhang F, Gao Y, Wu XK (2013) Regeneration performance of amino
acid ionic liquid (AAIL) activated MDEA solutions for CO
2
capture. Chem Eng J 223:371–378. https://doi.org/10.1016/j.
cej.2013.03.005
1
74.2013.862672
Zhen K, Zhang CZ (2014) Metabolic engineering of Escherichia coli
for production of 2-phenylethanol from renewable glucose. Appl
Biochem Biotechol 172:2012–2021. https://doi.org/10.1007/s1201
0-013-0659-3
Pinazo A, Manresa MA, Marques AM (2016) Amino acid-based sur-
factants: new antimicrobial agents. Adv Colloid Interface Sci
2
28:17–39. https://doi.org/10.1016/j.cis.2015.11.007
1
3