Journal of Environmental Biotechnology
Vol. 17, No. 1, 69–72, 2017

Technical Note

Application of Bio-Based Organic Acid, d-Glyceric Acid:
Synthesis and Interfacial Property of Dihexanoyl Glycerate
Shun Sato1, Shota Nagata2, Dai Kitamoto1, Yutaka Takahashi2,
Yukishige Kondo2 and Hiroshi Habe1*,†1

Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST),
Tsukuba Central 5–2, 1–1–1, Higashi, Tsukuba, Ibaraki 305–8565, Japan2
Department of Industrial Chemistry, Faculty of Engineering, Tokyo University of Science,
1–3, Kagurazaka, Shinjuku, Tokyo 162–8601, Japan* TEL: +81–29–861–6247 FAX: +81–29–861–8326* E-mail: hiroshi.habe@aist.go.jp†
Present address: Environmental Management Research Institute, National Institute of Advanced Industrial Science andTechnology (AIST), 16–1, Onogawa, Tsukuba, Ibaraki 305–8569, Japan(Received: 22 December, 2016/Accepted: 11 January, 2017)
Key words: biorefinery, green surfactant, glycerol, glyceric acid, diacyl glycerate

Glycerol is produced as a by-product in the growing biodiesel
and oleochemical industries, and many projects are under way
to convert glycerol into various marketable and value-added
products 11,18). Glyceric acid (2,3-dihydroxypropanoic acid;GA) is a natural minor organic compound in specificplants 10,12), but can be mass-produced biotechnologicallyfrom glycerol 3,6,7,16). One of the significant advantages of
producing GA from glycerol using acetic acid bacteria isthat Acetobacter tropicalis provides a chiral skeleton in theC-2 of GA and produces enantiopure d-GA with 99% enantiomer excess (ee) 7). Therefore, applications of d-GA and its
derivatives should be developed to expand its commercial
production and application.
Biological activities of d-GA and those of GA derivatives,
such as diacyl GA and glucosyl GA have been reported 1,5,8,9,13).
In 2001, Lesová et al. reported that glyceric acids esterified
with long acyl chains (>C16) exhibit antitrypsin activity 9),
however, their hydrophobic nature, derived from the long acyl
chains, limits the number of studies that can be performedon their physical and biological properties. For example, we
previously synthesized diacyl GAs with acyl chain lengthsof C16 and C18 and investigated their biological properties 4,14,15), but the trypsin inhibitory efficiency of dioleoyl GA

Fig. 1.

was low, likely due to its low water solubility 4).
To improve water solubility of diacyl GAs, we recently
synthesized a diacyl GA with a shorter octanoyl group(C8) acyl chain and investigated its physical properties 17).
Synthesized dioctanoyl GA (diC8GA) was not soluble inwater, whereas its sodium salt (diC8GA-Na) was watersoluble and had surface tension-lowering properties; thecritical micelle concentration (CMC) was 0.82 mM, andsurface tension at the CMC was 25.5 mN/m 17). These data
support the usefulness of diacyl glycerates as new surfactants;
however, water solubility and interfacial properties of diacyl
glycerates with acyl chains other than C8 have not been
investigated. In this study, we synthesized more water-soluble
diacyl GA sodium salts with a shorter hexanoyl group (C6)acyl chain than that of diC8GA-Na (Fig. 1) and investigatedtheir surface-tension-lowering property.
d-GA with an ee of 99% was prepared from glycerolby oxidative fermentation using Acetobacter tropicalis
NBRC16470 3,7). The d-GA calcium salts were converted
to free acids using DOWEX cation exchange resin (Dow
Chemicals, Midland, MI, USA), as described previously 2).
Hexanoyl chloride were purchased from Wako Pure Chemicals(Osaka, Japan). d-GA was acylated in anhydrous acetone in

Synthesis of dihexanoyl glyceric acid (diC6GA).

Sato et al.

70

the presence of dimethylaminopyridine and triethylamine, as
described for diC8GA 17). A 3.04-mL aliquot of d-GA solution (10 mmol in acetone) was added to a 100-mL three-neckround-bottom flask and maintained at 0°C on ice to synthesize diC6GA. Anhydrous acetone (35.7 mL), triethylamine(3.04 mL, 22 mmol), and dimethylaminopyridine (87.3 mg,
0.7 mmol) were added to the solution with stirring, andthe flask was purged with dry air. Acylation was started bydrop-wise addition of hexanoyl chloride (1.35 mL, 33 mmol)over 30 min at 0°C. The solution was incubated at 0°C for
an additional 30 min. Reaction progress was monitored bythin layer chromatography (TLC). TLC was developed withchloroform : methanol (8 : 2), and the organic compounds
were visualized by heating at 120°C for 5 min with a 5%(w/v) phosphoric-molybdate solution in ethanol containing
5% (v/v) sulfuric acid and 0.6% phosphoric acid. Afterthe spot corresponding to GA disappeared, the reaction
mixture was filtered with no. 40 filter paper (GE Healthcare
UK Ltd., Little Chalfont, UK), and the filtrates were evaporated in vacuo. To the resulting material, 1 M HCl andethyl acetate (33 mL) were added, and the organic layer wasFig. 2.

collected. The fraction was dried using anhydrous Na2SO4,
and the solvents were removed by evaporation. The crude oil
produced was purified using silica gel chromatography withhexane, hexane : ethyl acetate (8 : 2, v/v), and ethyl acetate.
Fractions containing diC6GA were combined, dried using
anhydrous Na2SO4, and concentrated in vacuo. The purified
diC6GA was obtained with a yield of 58.3%.
The resulting compound was characterized by nuclear
magnetic resonance (NMR) and liquid chromatography-mass
spectrometry (LC-MS). The 1H and 13C NMR spectra in
CDCl3 were recorded with a Bruker AV-400 NMR spectrometer (Bruker, Karlsruhe, Germany) at 27°C. 1H and13
C chemical shifts δ (ppm) were: 5.4 (t, 6H), 4.5 (m, 2H),
2.4 (m, 4H), 1.6 (quin, 4H), 1.3 (br, 12H), and 0.88 (t,6H) for 1H-NMR (Fig. 2A), and 173.31, 172.90 and 171.59(C=O), 69.72 (C2), 62.26 (C3), 33.8 (α-C), 24.33 (β-C),
22.27 (-CH2-), and 13.87 (-CH3) for 13C-NMR (Fig. 2B).
The 1H NMR spectrum showed diacylation of the hydroxyl
groups in GA, whereas the 13C NMR spectrum showed thepresence of three types of carbonyl groups. Collectively, thesedata confirmed synthesis of diC6GA.

(A) 400 MHz 1H and (B) 100 MHz 13C NMR spectra of dihexanoyl glyceric acid (diC6GA) in CDCl3.

Application of Bio-Based Organic Acid, d-Glyceric Acid: Synthesis and Interfacial Property of Dihexanoyl Glycerate

LC-MS was performed on a Shimadzu LC-MS 2020 system(Shimadzu, Otsu, Japan) equipped with a reverse-phase
Synergi 4-μm column (150×2.0 mm, Phenomenex, Torrance,
CA, USA). Samples were eluted in 0.1% (v/v) formic
acid : acetonitrile (15 : 85, v/v) at 0.2 mL/min, and thecolumn was kept at 40°C during analysis. Effluents were
ionized by electrospray-ionization and detected in negative
ion mode with an m/z range of 50–2,000. The synthesized
diC6GA was dissolved in a methanol : water (1 : 1) solution and converted to a sodium salt by neutralization withan equivalent volume of 1 M NaOH. The total ion current
chromatogram (TIC) from LC-MS revealed that diC6GA
was eluted at approximately 2.9 min, and [M-H]– (m/z=301)
and [2M-H]– (m/z=603) were the main ion forms with additional adduct ions (formate and proton adducts). TIC traces
showed a dominant peak at 2.9 min with no other peaks,
indicating the high purity of the diC6GA.
The diC6GA dissolved in methanol was converted to its
sodium salt by titration with NaOHaq 17). After the methanol
was evaporated, the resulting diC6GA-Na was recovered bylyophilization. Molecular weights of diC6GA and diC6GA-Na
were calculated from their chemical structures, and then thehydrophilic-lipophilic balance (HLB) values were derived
directly from the molecular structures according to Griffin’s
expression equation: HLB=20×[1–(mass of hydrophobic
part/total molecular mass)]. The data for HLB of diC6GA
and diC6GA-Na calculated from their chemical structures
were 10.59 and 11.17, respectively.
As diC6GA-Na was water-soluble and the resulting
aqueous 10 mM solution as well as the 200 mM solution wereclear, the surface tension of the aqueous solution containing
diC6GA-Na was measured at 25°C using the pendant drop
method with an automatic interfacial tensiometer (DM500,
Kyowa Interface Science, Niiza, Japan) and Drop ShapeAnalysis software (FAMAS v2.01, Kyowa Interface Science).
The CMC of the compound was calculated from thecross-point on the surface tension curve. The surface tension
value at CMC (γCMC) was also determined. As shown in
Figure 3, a decrease in the surface tension of diC6GA-Na
was observed with increasing concentration. The CMC of
diC6GA-Na was calculated as 2.92 mM from the cross-point

Fig. 3. Surface tension-concentration plot for dihexanoyl glyceric
acid sodium salt (diC6GA-Na).

71

of the surface tension plot. Generally, solubilization in water
increases as chain length of the acyl group becomes shorter;
therefore, the CMC value of diC6GA-Na was higher thanthat of diC8GA-Na (0.82 mM) 17). The γCMC value was
33.9 mN/m, although that of diC8GA-Na was 25.5 mN/m 17).
This result shows the potential of diC6GA-Na to be a new
green surfactant.
In summary, we synthesized dihexanoyl GA sodium salt
from GA and hexanoyl chloride for the first time. The
values of CMC and γCMC of diC6GA-Na were 2.92 mM
and 33.9 mN/m, respectively. Considering that the CMC
value of diC6GA (2.92 mM) is lower than that of commercially available synthetic surfactant, sodium dodecyl sulfate(SDS; CMC, 8.1 mM) 2), we can decrease the amount of
surfactant for use in surface-active applications. Also,
diC6GA-Na exhibited superior surface-tension-lowering
activity at CMC (33.9 mN/m) compared to that of SDS(γCMC, ca. 38 mN/m) 2). Although the previously synthesized
diC8GA-Na had superior surface-tension-lowering properties
in water compared to those of diC6GA-Na, development ofsome applications suitable for diC6GA-Na are now underway
in order to make use of the advantages of diC6GA-Na suchas higher water solubility.

Acknowledgments
This study was funded, in part, by KAKENHI
No. 25850064.
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