Synthesis of mycolic acid biosurfactants and their physical and surface-active properties

Article abstract of DOI:10.1007/s11746-005-5170-8

Five mycolic acids [2-alkyl-3-hydroxy FA: R ₁C*H(OH)C*HR₂COOH] were synthesized using acyl chlorides with alkyl chains of different lengths (total carbon numbers of mycolic 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) βlactone ring cleavage under alkaline conditions. The yields of C₁₂-, C₁₆-, C₂₀-, C ₂₄-, 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, cyclohexane, and diesel oil. The results showed that C ₁₂-mycolic acid was the best emulsifier for diesel oil, C ₁₆-mycolic acid was the best emulsifier for n-tetradecane and n-hexadecane, and C₂₀-mycolic 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 detergent/cleaning material industries, for example, in oil spill cleanup, oil recovery, textiles, pharmaceuticals, and cosmetics. Copyright

Full text of DOI:10.1007/s11746-005-5170-8

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
181
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182
M. LEE ET AL.
ketene dimer was obtained in high yield. The physical proper-
HPLC analysis. All the products obtained were analyzed
ties of the alkyl ketene dimer were confirmed by TLC (ethyl by HPLC before structural analysis. The HPLC analyses were
acetate/n-hexane = 1:20 vol/vol), FTIR spectrometry, MS, and performed with an HPLC system (consisting of a processor,
1H NMR spectrometry. The 1H NMR spectra were recorded in pump, UV detector, column oven, and an autosampler),
CDCl3 with a Fourier transform-NMR spectrometer using equipped with a Waters Atlantis dC18 reversed-phase column
tetramethylsilane as an internal standard.
(150 × 3.9 mm, 5 µm; Waters, Milford, MA). Five hundred
The second step was selective reduction of the alkyl ketene microliters of the properly diluted reaction mixture was in-
dimer by hydrogenation by adding 100 g of the dimer and 0.2 jected. Methanol (100%) was used as eluent at 30°C with a
g of 10% palladium/charcoal (Sigma, St. Louis, MO) to 800 flow rate of 0.5 mL/min. Products were detected using a UV
mL of an ethyl acetate/ethanol (3:1 vol/vol) mixture. This mix- detector (215 nm).
ture was stirred for 3 or 4 h under H2 and then purified using a
Surface tension measurement of mycolic acids. The surface
column packed with silica gel (Silica gel 60; Merck, Darm- tension properties of the synthetic mycolic acids were mea-
stadt, Germany). This step produced the β-lactone in high sured in an alkaline solution (pH 9.5) at various concentrations.
yield. The physical properties of the β-lactone were confirmed The solution was prepared by dissolving mycolic acid in 0.01
by analyses using TLC (ethyl acetate/hexane = 1:20), FTIR M NaOH. It was then titrated with 0.001 M NaOH to attain a
spectrometry, MS, and 1H NMR spectrometry.
pH value between 10 and 12. Surface tension was determined
The final step was the cleavage of β-lactone to make my- at 20°C with a Thermo Cahn tensiometer supported by the
colic acids. This was done by adding 50 g of the β-lactone and WinDCA32 software suite.
NaOH (1.5 molar equiv) to 600 mL of an ethanol/water (3:1
Measurement of emulsification activity. To estimate the
vol/vol) mixture. This mixture was stirred for 3 or 4 h and acid- emulsification activity, 5 mL of n-tetradecane, n-hexadecane,
ified with 6 N HCl, and then purified using a column packed cyclohexane, or diesel oil was added to 5 mL of alkaline water
with silica gel. The mycolic acids were obtained in high yields. (pH 9.5) containing the synthetic mycolic acids at various con-
The physical properties of the final products were confirmed centrations (10, 100, and 1,000 mg/L) in graduated test tubes.
by analyses using TLC (methylene chloride/ethanol = 45:1 These tubes were then vortexed for 2 min and allowed to stand
vol/vol), FTIR spectrometry, MS, and 1H NMR spectrometry. at 30°C. The emulsion stability was measured after incubation
Table 1 summarizes the properties and yields (both in grams for 24 h. The emulsification index (E24%) was calculated by
and percent yield) of the intermediates and products obtained dividing the height of the emulsion layer by the total height of
for the five mycolic acids synthesized.
the mixture and multiplying by 100.
TABLE 1
Volumes, Yields, and Properties of the Intermediates and Products for Five Mycolic Acids
Starting material
Hexanoyl chloride
Octanoyl chloride
Decanoyl chloride
Lauroyl chloride
Stearoyl chloride
Step 1
Step 2
Step 3
IUPACa
(E)-3-Butyl-4-
pentylidene-
oxetan-2-one
100 g (0.74 mol)
58 g
(E)-4-Heptylidene-
3-hexyl-
oxetan-2-one
100 g (0.61 mol)
69 g
(E)-4-Nonylidene-
3-octyl-
oxetan-2-one
100 g (0.52 mol)
69 g
(E)-3-Decyl-4-
undecylidene-
oxetan-2-one
100 g (0.45 mol)
74 g
90%
0.70
White solid
(E)-3-Heptadecylene-
3-hexadecyl-
oxetan-2-one
100 g (0.30 mol)
90 g
Initial
Final
Yield
72%
0.70
90%
0.70
90%
0.70
White solid
90%
0.70
White solid
Rf valueb
Phase
Yellowish-white liquid
Yellowish-white liquid
IUPACa
2-Butyl-octanoic
2-Hexyl-decanoic
2-Octyl-
dodecanoic
β-lactone
100 g (0.32 mol)
90 g
90%
0.58
White solid
2-Decyl-
tetradecanoic
β-lactone
100 g (0.27 mol)
90 g
90%
0.58
White solid
2-Hexadecyl-
eicosanoic
β-lactone
100 g (0.19 mol)
90 g
90%
0.58
White solid
β-lactone
β-lactone
Initial
Final
100 g (0.51 mol)
100 g (0.39 mol)
95 g
95%
0.58
90 g
90%
0.58
Yield
Rf valueb
Phase
Yellowish-white wax
Yellowish-white wax
IUPACa
2-Butyl-3-
hydroxyoctanoic
acid
2-Hexyl-3-
hydroxydecanoic
acid
2-Octyl-3-
hydroxydodecanoic
acid
2-Decyl-3-
hydroxytetradecanoic
acid
2-Hexadecyl-3-
hydroxyeicosanoic
acid
Initial
Final
50 g (0.25 mol)
51 g
50 g (0.20 mol)
48 g
50 g (0.16 mol)
47 g
50 g (0.125 mol)
43.3 g
50 g (0.09 mol)
45 g
Yield
95%
0.48
90%
0.48
90%
0.48
White solid
90%
0.48
White solid
90%
0.48
White solid
Rf valueb
Phase
Yellowish-white wax
Yellowish-white wax
aIUPAC, IUPAC names of the intermediates and products for five mycolic acids; Initial, the reaction volumes of starting materials and intermediates; Final,
the final volumes of intermediates and products.
bThe Rf values of intermediates and products were confirmed by TLC (ethyl acetate/n-hexane = 1:20 vol/vol).
JAOCS, Vol. 82, no. 3 (2005)

SYNTHESIS OF MYCOLIC ACID BIOSURFACTANTS
183
The product structure in each step was analyzed and deter-
mined by the 1H NMR spectra. In the first synthesis step, the
1H NMR (CDCl3) signals of (E)-4-heptylidene-3-hexyloxetan-
2-one observed included the hydrogens of the methyl carbons
of (E)-4-heptylidene-3-hexyloxetan-2-one (t, 6H, CH3: 0.90
ppm), the hydrogens of the alkyl chain carbons of (E)-4-hep-
tylidene-3-hexyloxetan-2-one (br, 16H, CH2: 1.29–1.44 ppm),
the hydrogen of the carbon next to the α-carbon of (E)-4-hep-
tylidene-3-hexyloxetan-2-one (q, 2H, CH2CH2CHC=O: 1.76
ppm), the hydrogens of the carbon next to the double bonded
γ-carbon (q, 2H, CH2CH2CH=C: 2.12 ppm), the hydrogen of
the α-carbon next to the carbonyl group (dd, 1H, CH2CHCO:
SCHEME 1
RESULTS AND DISCUSSION
Synthesis and characterization of mycolic acids. The mycolic 3.93 ppm), and the hydrogen of the double bonded γ-carbon of
acid containing 16 total carbons was synthesized in three steps (E)-4-heptylidene-3-hexyloxetan-2-one (t, 1H, CH2CH=C:
with octanoyl chloride as the starting material. The first step was 4.69 ppm) (data not shown). In the second synthesis step, the
the dimerization of octanoyl chloride to make (E)-4-heptylidene- 1H NMR (CDCl3) signals of 2-hexyl-decanoic β-lactone in-
3-hexyloxetan-2-one. The second step was selective reduction cluded the hydrogens (t, 6H, CH3) of the methyl carbons of 2-
of the (E)-4-heptylidene-3-hexyloxetan-2-one to produce 2- hexyl-decanoic β-lactone (0.87 ppm), the hydrogens (br, 22H,
hexyl-decanoic β-lactone. The third step was the cleavage of the CH2) of the alkyl-chain carbons of 2-hexyl-decanoic β-lactone
2-hexyl-decanoic β-lactone to make 3-hydroxy-2-hexyldecanoic (1.12–1.91 ppm), the hydrogen (m, 1H, CH2CHC=O) of the α-
acid. Scheme 1 shows the scheme for the synthesis of mycolic carbon (3.60 ppm), and the hydrogen (m, 1H, CHCH-O) of the
acids (where C* indicates chiral carbons). The mycolic acids β-carbon of 2-hexyl-decanoic β-lactone (4.51 ppm) (data not
thus obtained consist of the mixture of approximately equal shown). Finally, the 1H NMR (CDCl3) signals of the mycolic
amounts of syn-type and anti-type isomers. With regard to the acid 3-hydroxy-2-hexyldecanoic acid include the hydrogens (t,
stereochemistry of natural mycolic acid, on the other hand, there 6H, CH3) of the methyl carbons (0.87 ppm), the hydrogens (br,
are four different possible arrangements at two chiral carbon 28H, CH2) of the alkyl-chain carbons (0.880–1.733 ppm), the
atoms of the mycolic acids, i.e., 2R/3R, 2R/3S, 2S/3R, and 2S/3S. hydrogen (m, 1H, CHCHC=O) of the α-carbon (2.476 ppm),
Natural mycolic acids usually show the same stereochemistry as and the hydrogen (m, 1H, CH2CH(OH)CH) of the β-carbon
2R/3R formed at the chiral carbon positions. Therefore, it can be (3.860 ppm) of the mycolic acid (Fig. 1). However, 1H NMR
postulated that natural mycolic acids consist of only the anti-type spectral data did not show that the syn- and anti-diastereomers
isomer, although this was not demonstrated by our experiments. of C16 mycolic acid had slightly different shifts.
FIG. 1. 1H NMR spectrum of 2-hexyl-3-hydroxydecanoic acid. A sample of 10 mg of the material in 0.6 mL of
CDCl3 was used. Frequency: 300 MHz; internal standard: tetramethylsilane; 0.87 ppm: t, 6H, CH3; 0.880–1.733
ppm: br, 28H, CH2; 2.476 ppm: m, 1H, CHCHC=O; 3.860 ppm: m, 1H, CH2CH(OH)CH.
JAOCS, Vol. 82, no. 3 (2005)

184
M. LEE ET AL.
FIG. 2. FTIR spectrum of 2-hexyl-3-hydroxydecanoic acid. The FTIR spectrum of 2-hexyl-3-hydroxydecanoic acid
was measured in a liquid state (chloroform). Absorption band at 3,376 cm−1: O–H stretching vibration; absorption
band at 3000–2850 cm−1: C–H stretching in CH3 and CH2; absorption band at 1,707 cm−1: vibration of the keto
group.
Figure 2 is an FTIR spectrum of 2-hexyl-3-hydroxy-
decanoic acid in the liquid state. Absorption bands were ob-
served at 3,376 cm−1 (O–H stretching vibration) and 1,707 cm−1
(the vibration of the keto group), which determined the exis-
tence of a hydroxyl group and a keto group in the structure of
the mycolic acid.
cients (ε) of the mycolic acids using a UV spectrophotometer
(Cary 1 Bio; Varian, Palo Alto, CA): ε = 88.68 M−1cm−1 at 211
nm for C12-, 249.02 M−1cm−1 at 215 nm for C16-, 161.51 M−1
cm−1 at 212 nm for C20-, 231.88 M−1cm−1 at 214 nm for C24-,
and 256.37 M−1cm−1 at 202 nm for C36-mycolic acid. Figure 3
shows the fragmentation pattern of C16-mycolic acid. It was
identified by comparing the fragmentation pattern and the
M.W. (MS 254 M − H2O and MS 272). Figure 4 shows the
peak for C16-mycolic acid obtained by HPLC, which appeared
at 3.40 min. However, it did not show separate peaks for the
syn- and anti-diastereomers of C16-mycolic acid.
Up to this point, 1H NMR and FTIR spectra for 2-hexyl-3-
hydroxydecanoic acid have been described. The 1H NMR and
FTIR spectra of the other mycolic acids, i.e., 2-butyl-3-hydroxy-
octanoic acid (C12-mycolic acid), 2-octyl-3-hydroxydo-
decanoic acid (C20-mycolic acid), 2-decyl-3-hydroxytetra-
decanoic acid (C24-mycolic acid), and 2-hexadecyl-3-hydroxy-
eicosanoic acid (C36-mycolic acid), showed values similar to
those of 2-hexyl-3-hydroxydecanoic acid (C16-mycolic acid)
because they had the same chemical structures and functional
groups (hydroxyl and carboxyl groups) except for the lengths
of the carbon chains. The 1H NMR and FTIR spectral data of
the other mycolic acids are tabulated in Table 2. There were no
unusual observations in the 1H NMR and FTIR spectra of the
other mycolic acids compared with those of 2-hexyl-3-hydroxy-
decanoic acid.
These mycolic acids can be obtained in high purity and po-
tentially at a relatively low cost. Since the synthesis of mycolic
acids is relatively simple, the yields were also high, at approxi-
mately 72 to 73% of the starting material (Table 2).
Surface-active properties of synthetic mycolic acids. The
surface tension of alkaline solutions (pH 9.5) containing C12-,
C16-, or C20-mycolic acid was measured at various concentra-
tions using a Thermo Cahn surface tensiometer at 20°C. The
C24- and C36-mycolic acids were partially soluble/dispersible
and insoluble in alkaline solution, respectively (Table 2). This
method allows a comparison of the surface-active properties of
different mycolic acids at their critical micelle concentration
(CMC) values. The surface tension–concentration plot is
shown in Figure 5. The surface tension of mycolic acids de-
creased with increasing concentrations and showed a well-
defined CMC (Fig. 5). The CMC values of C12-, C16-, and
Table 2 presents yields, m.p., and spectral data, i.e., FTIR,
1H NMR, UV, and mass spectra of the five mycolic acids. The
m.p. were 45–46°C for C20-, 54–55°C for C24-, and 68–70°C
for C36-mycolic acid. The C12- and C16-mycolic acids were
waxes at room temperature. The acids were analyzed by a mass
spectrophotometer. Table 2 also presents the extinction coeffi-
JAOCS, Vol. 82, no. 3 (2005)

SYNTHESIS OF MYCOLIC ACID BIOSURFACTANTS
185
TABLE 2
Yields, Physical Properties, and Spectral Dataa
A
B
C
D
E
Yield (%) (step)
m.p. (°C)
72
73
73
73
73
(80, 95, 95)
(90, 90, 90)
(90, 90, 90)
(90, 90, 90)
(90, 90, 90)
Wax
3391 (OH, br)
1707 (C=O)
Wax
3376 (OH, br)
1707 (C=O)
45–46
3368 (OH, br)
1690 (C=O)
54–55
3325 (OH, br)
1696 (C=O)
68–70
3301 (OH, br)
1693 (C=O)
IR (cm–1
)
NMR (CDCl3)
0.872–1.609 (m, 20 H) 0.880–1.733 (m, 28 H) 0.880–1.697 (m, 36 H)
0.880–1.705 (m, 44 H) 0.860–1.911 (m, 68 H
2.483
3.869
(m, 1 H)
(m, 1 H)
2.476
3.860
(m, 1 H)
(m, 1 H)
2.466
3.859
(m, 1 H)
(m, 1 H)
2.487
3.864
(m, 1 H)
(m, 1 H)
2.465
3.858
(m, 1 H)
(m, 1 H)
MS
198 (M − H2O)
88.68
254 (M − H2O)
249.02
310 (M − H2O)
161.51
366 (M − H2O)
231,88
534 (M − H2O)
256.37
ε (M−1cm−1
)
at 211 nm
100%
at 215 nm
100%
at 212 nm
100%
at 214 nm
Partially soluble or dispersible
at 202 nm
Insoluble
Water solubilityb
aA, 2-butyl-3-hydroxyoctanoic acid; B, 2-hexyl-3-hydroxydecanoic acid; C, 2-octyl-3-hydroxydodecanoic acid; D, 2-decyl-3-hydroxytetradecanoic acid; E,
2-hexadecyl-3-hydroxyeicosanoic acid; ε, extinction coefficient.
bWater solubility, 1 g mycolic acid was solubilized in 1,000 mL alkaline solution (pH 9.5) for water solubility and critical micelle concentration measure-
ments.
m/z
m/z
FIG. 3. Mass of 2-hexyl-3-hydroxydecanoic acid. M.W. of 2-hexyl-3-hydroxydecanoic acid: (A) MS 254 − H2O and
(B) MS 272; ionization mode: EI; ionization voltage: 70 eV; ion source temperature: 200°C.
JAOCS, Vol. 82, no. 3 (2005)

186
M. LEE ET AL.
FIG. 4. HPLC chromatogram of 2-hexyl-3-hydroxydecanoic acid. The peak for 2-hexyl-3-hy-
droxydecanoic acid appeared at 3.40 min (retention time). Injection volume: 500 µL; eluent:
100% methanol; temperature: 25°C; flow rate: 10 mL/min; HPLC column: Waters Atlantis
dC18 reversed-phase column (150 × 3.9 mm, 5 µm; Waters, Milford, MA); UV detector: 215
nm.
C20-mycolic acids were 2.2 × 10−4, 1.36 × 10−4, and 7.4 × 10−5
Emulsification activity. Low CMC values of C12-, C16-, and
M, respectively. As the carbon number increased, the CMC C20-mycolic acids indicate that they can effectively reduce the
value decreased. The surface tension at CMC also decreased surface tension. Therefore, as a measurement of their emulsifi-
(46.54, 43.59, and 41.57 dyn/cm for C12-, C16-, and C20-my- cation activities, the E24% of n-tetradecane, n-hexadecane, cy-
colic acid, respectively).
clohexane, and diesel oil was determined for each mycolic
FIG. 5. Surface tension vs. concentration curves of synthetic mycolic acids at 30°C. (G) 2-
Butyl-3-hydroxyoctanoic acid, (G) 2-hexyl-3-hydroxydecanoic acid, and (M) 2-octyl-3-hy-
droxydodecanoic acid. Critical micelle concentration values were: A (2-butyl-3-hydroxyoc-
tanoic acid), 2.2 × 10−4 M; B (2-hexyl-3-hydroxydecanoic acid), 1.36 × 10−4 M; and C (2-octyl-
3-hydroxydodecanoic acid), 7.4 × 10−5 M.
JAOCS, Vol. 82, no. 3 (2005)

SYNTHESIS OF MYCOLIC ACID BIOSURFACTANTS
187
FIG. 6. Comparison of emulsification activity (E24%) of the synthetic mycolic acids (MA; at 10, 100, and 1,000
mg/L) toward (A) n-tetradecane; (B) n-hexadecane; (C) cyclohexane; (D) diesel oil.
acid. Figure 6 shows the comparison of their E24% at 10, 100, with a chain length of 12 to 20 appeared to be the best surfac-
and 1,000 mg/L of each mycolic acid, respectively. The C16- tants because of their favorable HLB for the surface-active
mycolic acid showed higher emulsifying activity for n-tetra- properties. As the acids became longer and more lipophilic,
decane and n-hexadecane than did the C12- and C20-mycolic they became less soluble or insoluble in water and were poorer
acids at 10, 100, and 1,000 mg/L, while exhibiting a lower surfactants (Table 2). Being hydrophilic in nature, the hydroxyl
emulsifying activity for cyclohexane and diesel oil at 10 and functional group close to the carboxyl group renders the mole-
100 mg/L. The C12-mycolic acid showed higher emulsifying cule more hydrophilic, resulting in a more favorable HLB with
activity for diesel oil than did the C16- and C20-mycolic acids the large alkyl chains (17).
at 10, 100, and 1,000 mg/L, but it showed a lower emulsifying
Since mycolic acids have two hydrophilic functional groups
activity for n-tetradecane, n-hexadecane, and cyclohexane than in the water and two hydrophobic chains, they can create lamel-
did the others at 10, 100, and 1,000 mg/L. The C20-mycolic lar-type surfactant aggregations. For this reason, they are struc-
acid showed higher emulsifying activity for cyclohexane than turally somewhat more complex than a linear amphiphile, and
did the C12- and C16-mycolic acids at 10, 100, and 1,000 mg/L, their packing orientations at the aqueous interface may be com-
and it also showed high emulsifying activity for n-tetradecane, plicated (18). This is analogous to the surface packing in some
n-hexadecane, and cyclohexane at 1,000 mg/L.
phospholiplids, which have good surfactant properties (19,20).
The CMC and emulsification activities of the synthetic my- Such a monomolecular-layer orientation at the air/water inter-
colic acids are sensitive to the length of the alkyl chains (Figs. face reduces the surface tension to less than 50 dyn/cm at the
5,6). This may be explained by the hydrophilic-lipophilic bal- CMC, probably because of the hindrance and repulsion be-
ance (HLB) of the acids. Longer mycolic acids tended to be tween the hydrocarbon chain of one molecule and that of the
lipophilic and shorter mycolic acids hydrophilic. Mycolic acids adjoining molecule.
JAOCS, Vol. 82, no. 3 (2005)

188
M. LEE ET AL.
9. Schippers, C., K. Gessner, T. Muller, and T. Scheper, Microbial
The new synthetic route to the mycolic acids presented here
Degradation of Phenanthrene by Addition of a Sophorolipid
Mixture, J. Biotechnol. 83:189–198 (2000).
10. Weete, J.D., Lipid Biochemistry of Fungi and Other Organisms,
Plenum Press, New York, 1980, pp. 75–90.
11. Brennan, P.J., Mycobacterium and Other Actinomycetes, in Mi-
crobial Lipids, edited by C. Ratledge, and S.C. Wilkinson, Aca-
demic Press, London. 1988, Vol. 1, pp. 203–298.
12. Minnikin, D.E., Lipids: Complex Lipids, Their Chemistry,
Biosynthesis, and Roles, in The Biology of the Mycobacteria,
edited by C. Ratledge and J.L. Stanford, Academic Press, Lon-
don, 1982, Vol. 1, pp. 95–184.
is relatively simple. They can be obtained in high purity and
potentially at a relatively low cost, and they show high emulsi-
fication properties toward several oils. These synthetic mycolic
acids thus have significant potential as new materials for use in
the removal of hydrocarbons in oil-contaminated environ-
ments, such as oil spills in the sea or soil. They also can be used
for in situ flushing of hydrocarbon contaminants in the soil and
for enhanced recovery of oil from oil fields.
13. Cooper, D.G., and J.E. Zajic, Surface Active Compounds from
Microorganisms, Adv. Appl. Microbiol. 26:229–256 (1980).
14. Gensler, W.J., I. Alam, R.S. Prasad, A.I. Radhakrishna, and A.P.
Chaudhuri, 3-Hydroxy-2-alkyl Carboxylic Acids Related to My-
colic Acid, Tetrahedron 35:2595–2600 (1979).
ACKNOWLEDGMENT
This work was supported by grants from the Ministry of Environ-
ment of Republic of Korea, which is gratefully acknowledged.
15. Utaka, M., H. Higashi, and A. Takeda, Asymmetric Reduction
of 3-Oxo-octadecanoic Acid with Fermenting Baker’s Yeast. An
Easy Synthesis of Optically Pure (+)-(2R,3R)-Corynomycolic
Acid, J. Chem. Soc., Chem.Commun.:1368–1369 (1987).
16. March, J., Advanced Organic Chemistry: Reactions, Mecha-
nisms, and Structure, Wiley-Interscience, New York, 1992, p.
855.
17. Cooper, D.G., J.E. Zajic, and C. Denis, Surface Active Proper-
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Monolayer Film by the Langmuir–Blodgett Technique and
Atomic Force Microscopy, Langmuir 16:7325–7330 (2000).
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(1996).
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[Received June 8, 2004; accepted February 7, 2005]
JAOCS, Vol. 82, no. 3 (2005)