Synthesis of Mycolic Acid Biosurfactants and Their
Physical and Surface-Active Properties
Myungjin Leea,b, Hyung Sub Gwaka, Byeong Deog Parka, and Sung-Taik Leeb,*
aNeoPharm Co., Ltd., Daejeon 305-333, Republic of Korea, and bKorea Advanced Institute of Science
and Technology, Department of Biological Sciences, Daejeon 305-701, Republic of Korea
ABSTRACT: Five mycolic acids [2-alkyl-3-hydroxy FA:
R1C*H(OH)C*HR2COOH] were synthesized using acyl chlorides
with alkyl chains of different lengths (total carbon numbers of my-
colic acids, 12, 16, 20, 24, 36). The relationship between the
chemical structures of the mycolic acids and their surface-active
properties was determined. The acids were synthesized in three
steps: (i) dimerization of acyl chloride into alkyl ketene dimer, (ii)
selective reduction of C=C to C–C by hydrogenation, and (iii) β-
lactone ring cleavage under alkaline conditions. The yields of
C12-, C16-, C20-, C24-, and C36-mycolic acid were 72, 73, 73, 73,
and 73%, respectively. The critical micelle concentrations (CMC)
of C12-, C16-, and C20-mycolic acid were 2.2 × 10−4, 1.36 × 10−4,
and 7.4 × 10−5 M, respectively. As the carbon number increased,
the surface tension at the CMC value was also lower; the values
for C12-, C16-, and C20-mycolic acid were 46.54, 43.59, and
41.57 dyn/cm, respectively. The emulsifying activities of mycolic
acids were determined for n-tetradecane, n-hexadecane, cyclo-
hexane, and diesel oil. The results showed that C12-mycolic acid
was the best emulsifier for diesel oil, C16-mycolic acid was the
best emulsifier for n-tetradecane and n-hexadecane, and C20-my-
colic acid was the best emulsifier for cyclohexane. This study
showed that mycolic acids having surface-active properties can
be chemically synthesized for potential applications in the deter-
gent/cleaning material industries, for example, in oil spill
cleanup, oil recovery, textiles, pharmaceuticals, and cosmetics.
Paper no. J10882 in JAOCS 82, 181–188 (March 2004).
long-chain, β-hydroxy FA with a moderately long aliphatic
chain at the α-carbon atom (10). The total number of carbon
atoms varies from 30 to 86. They are produced by bacterial
species belonging to the genus Mycobacterium and to genera
of Nocardia, Rhodococcus, Corynebacterium, as well as other
species in the minor genera (e.g., Gordonia, Bacterionema, Mi-
cropolyspora, and Brevibacterium) (11). Mycolic acids usually
form part of the cell wall complex and are linked to the ara-
binogalactan–peptidoglycan matrix (12). However, mycolic
acids are also found as a lipid component of certain extracellu-
lar glycolipids produced by these bacteria, particularly follow-
ing growth on alkanes or related substrates. In these cases, the
mycolic acid usually is esterified with the hydroxyl groups of a
trehalose unit (13).
There are considerable variations in the FA chain. They may
contain a keto-, methoxy-, or methyl-group, and/or cyclo-
propane rings. Both cis and trans double bonds may occur
within the alkyl backbone chain itself, and the side-chain FA
also may contain a double bond.
Mycolic acids have previously been produced through dif-
ferent synthetic routes (14,15). In this paper, we report a new
synthesis pathway to obtain a high yield of mycolic acids using
various acyl chlorides as starting materials. The present study
also describes the yield of each step, characterization of each
mycolic acid, and determination of the properties of mycolic
acids as surface-active agents.
KEY WORDS: Biodegradation, biosurfactant, emulsification,
mycolic acid, surface tension, synthesis.
EXPERIMENTAL PROCEDURES
Biosurfactants are amphiphilic molecules produced by mi-
croorganisms with both hydrophobic and hydrophilic domains.
They are frequently produced in nature by microorganisms
growing on a water-immiscible substrate. By synthesizing a
surface-active agent, they can reduce the interfacial energy/ten-
sion through emulsification or solubilization of hydrophobic
substrates that otherwise are not bioavailable to them (1). Bio-
surfactants have a wide range of applications in various indus-
tries (2–4), including the environmental remediation of hy-
drophobic pollutants (5–9).
Mycolic acid synthesis and characterization. The mycolic
acids were synthesized in three steps from acyl chlorides with
alkyl chain lengths of 6, 8, 10, 12, and 18, respectively, as the
starting material.
The first step was the dimerization of acyl chloride to make
alkyl ketene dimer (16). This was accomplished by adding 100
g of acyl chloride to 800 mL of toluene and cooling to a tem-
perature below 10°C in an ice bath. Triethylamine (TEA, 1.1
molar equiv) was then slowly added from a dropping funnel
while stirring. After 1 h in the ice bath, the mixture was stirred
for about 3 to 4 h at room temperature. Water (250 mL) was
then added to the reaction mixture and the mixture was ex-
tracted with 1,000 mL of CH2Cl2, filtered using Na2SO4 to re-
move the water, and then dried at reduced pressure using a ro-
tary evaporator. It was then purified by flash column chroma-
tography on silica gel eluting with ethyl acetate/n-hexane (1:30
vol/vol) and dried at reduced pressure. In the end, the alkyl
Biosurfactants may be divided into four different groups: (i)
glycolipids, (ii) phospholipids, (iii) lipoproteins or lipopep-
tides, and (iv) polymeric biosurfactants. Mycolic acids are
*To whom correspondence should be addressed at Korea Advanced Institute
of Science and Technology, Department of Biological Sciences, Kuseong-
dong 373-1, Yuseong-gu, Daejeon, 305-701, Republic of Korea.
E-mail: e_stlee@kaist.ac.kr
Copyright © 2005 by AOCS Press
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JAOCS, Vol. 82, no. 3 (2005)