Synthesis and antifungal properties of α-methoxy and α-hydroxyl substituted 4-thiatetradecanoic acids

Article abstract of DOI:10.1016/j.chemphyslip.2007.06.218

4-Thiatetradecanoic acid exhibited weak antifungal activities against Candida albicans (ATCC 60193), Cryptococcus neoformans (ATCC 66031), and Aspergillus niger (ATCC 16404) (MIC = 4.8-12.7 mM). It has been demonstrated that α-methoxylation efficiently blocks β-oxidation and significantly improve the antifungal activities of fatty acids. We examined whether antifungal activity of 4-thiatetradecanoic acid can be improved by α-substitution. The unprecedented (±)-2-hydroxy-4-thiatetradecanoic acid was synthesized in four steps (20% overall yield), while the (±)-2-methoxy-4-thiatetradecanoic acid was synthesized in five steps (14% overall yield) starting from 1-decanethiol. The key step in the synthesis was the hydrolysis of a trimethylsilyloxynitrile. In general, the novel (±)-2-methoxy-4-thiatetradecanoic acid displayed significantly higher antifungal activities against C. albicans (ATCC 60193), C. neoformans (ATCC 66031), and A. niger (ATCC 16404) (MIC = 0.8-1.2 mM), when compared with 4-thiatetradecanoic acid. In the case of C. neoformans the (±)-2-hydroxy-4-thiatetradecanoic acid was more fungitoxic (MIC = 0.17 mM) than the α-methoxylated analog, but not as effective against A. niger (MIC = 5.5 mM). The enhanced fungitoxicity of the (±)-2-methoxy-4-thiatetradecanoic acid, as compared to decylthiopropionic acid, might be the result of a longer half-life in the cells due to a blocked β-oxidation pathway which results in more time to exert its toxic effects. Thus, these novel fatty acids may have applications as probes to study fatty acid metabolic routes in human cells.

Full text of DOI:10.1016/j.chemphyslip.2007.06.218

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Chemistry and Physics of Lipids 150 (2007) 82–88
Synthesis and antifungal properties of ␣-methoxy and
␣-hydroxyl substituted 4-thiatetradecanoic acids
Nestor M. Carballeiraa,∗, Rosann O’Neilla, Keykavous Parangb
´
a
Department of Chemistry, University of Puerto Rico, P.O. Box 23346, San Juan 00931-3346, Puerto Rico
b
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy,
University of Rhode Island, Kingston, USA
Received 26 April 2007; received in revised form 18 June 2007; accepted 19 June 2007
Available online 28 June 2007
Abstract
4-Thiatetradecanoic acid exhibited weak antifungal activities against Candida albicans (ATCC 60193), Cryptococcus neoformans
(ATCC 66031), and Aspergillus niger (ATCC 16404) (MIC = 4.8–12.7 mM). It has been demonstrated that ␣-methoxylation effi-
ciently blocks ␤-oxidation and significantly improve the antifungal activities of fatty acids. We examined whether antifungal activity
of 4-thiatetradecanoic acid can be improved by ␣-substitution. The unprecedented ( )-2-hydroxy-4-thiatetradecanoic acid was syn-
thesized in four steps (20% overall yield), while the ( )-2-methoxy-4-thiatetradecanoic acid was synthesized in five steps (14%
overall yield) starting from 1-decanethiol. The key step in the synthesis was the hydrolysis of a trimethylsilyloxynitrile. In general,
the novel ( )-2-methoxy-4-thiatetradecanoic acid displayed significantly higher antifungal activities against C. albicans (ATCC
60193), C. neoformans (ATCC 66031), and A. niger (ATCC 16404) (MIC = 0.8–1.2 mM), when compared with 4-thiatetradecanoic
acid. In the case of C. neoformans the ( )-2-hydroxy-4-thiatetradecanoic acid was more fungitoxic (MIC = 0.17 mM) than the
␣-methoxylated analog, but not as effective against A. niger (MIC = 5.5 mM). The enhanced fungitoxicity of the ( )-2-methoxy-4-
thiatetradecanoic acid, as compared to decylthiopropionic acid, might be the result of a longer half-life in the cells due to a blocked
␤-oxidation pathway which results in more time to exert its toxic effects. Thus, these novel fatty acids may have applications as
probes to study fatty acid metabolic routes in human cells.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Antifungal; Decylthiopropionic acid; Methoxylated fatty acids; Synthesis; Thia fatty acids
1. Introduction
cylglycerols and cholesterol and to induce in hepatocytes
peroxisomal ␤-oxidation more efficiently than normal
fatty acids (Hovik et al., 1990; Skrede et al., 1995).
In particular, tetradecylthioacetic acid is also known to
have beneficial antioxidant properties, to lower hyper-
glycemia, and displays a positive effect on insulin levels
(Dyroy et al., 2006). On the other hand, 4-thia fatty
acids inhibit ␤-oxidation (or it occurs rather slowly) in
peroxisomes and mitochondria thereby inducing hep-
atic lipidosis when administered to rats, i.e., the hepatic
levels of triacylglycerols are increased (Hvattum et al.,
1992). The difference between the 3- and 4-thia fatty
Thia fatty acids with the sulfur atom at the 3 and
4 positions have been of considerable interest for their
effects on the metabolism of fatty acids (Dyroy et al.,
2006; Skrede et al., 1997). The 3-thia fatty acids have
beenreportedtodecreasetheserumconcentrationoftria-
∗
Corresponding author. Tel.: +787 764 0000x4791;
fax: +787 756 8242.
E-mail address: nmcarballeira@uprrp.edu (N.M. Carballeira).
0009-3084/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2007.06.218

N.M. Carballeira et al. / Chemistry and Physics of Lipids 150 (2007) 82–88
83
2. Materials and methods
acids can be ascribed as to the position of the sul-
fur atom in the fatty acyl chain; the 3-thia fatty acids
cannot be ␤-oxidized and are rather metabolized by
␻-oxidation (Gudbrandsen et al., 2005). On the other
hand, the 4-thia fatty acids can be ␤-oxidized once, but
the resulting metabolites (alkylthioacrylyl-CoA) accu-
mulate in the mitochondria thereby inhibiting fatty acid
oxidation (Hvattum et al., 1992).
Despite much of the above studies on the metabolism
of thia fatty acids in humans nothing is known as to their
antifungal properties. However, earlier work by Gor-
don and collaborators (Kishore et al., 1993) indicated
that the myristoyl-CoA:protein N-myristoyltransferase
(NMT) of the yeast Saccharomyces cerevisiae, an
enzyme that catalyzes the transfer of myristic acid
(C14:0) from myristoyl-CoA to the ␣-amino nitrogen
of glycine, has a greater affinity for 4-thiatetradecanoic
acid than for the 3-thiatetradecanoic acid (Kishore et al.,
1993). Moreover, when the authors compared human
NMT versus S. cerevisiae NMT they found that the
4-thiatetradecanoic acid has a greater affinity for the
S. cerevisiae NMT than for the human NMT. On the
other hand, the 3-thiatetradecanoic acid had almost the
same affinity for both types of NMTs (Kishore et al.,
1993).
Our studies showed that 4-thiatetradecanoic acid
has weak antifungal activities against Candida albi-
cans (ATCC 60193), Cryptococcus neoformans
(ATCC 66031), and Aspergillus niger (ATCC 16404)
(MIC = 4.8–12.7 mM). We have previously shown that
␣-methoxylation of tetradecanoic acid can significantly
improve the antifungal activities of tetradecanoic acid
(Carballeira et al., 2005). We investigated whether
␣-substitution of 4-thiatetradecanoic acid can improve
the antifungal activities of 4-thiatetradecanoic acid by
inhibition of ␤-oxidation or fungi NMT.
It was our expectation that the ␣-methoxy substitution
would essentially block the only ␤-oxidation pathway
left for the 4-thia fatty acid thus allowing for only ␻-
oxidation to be the most likely catabolic pathway for
this novel fatty acid, which could render the ␣-methoxy
4-thia fatty acid metabolically similar to a 3-thia fatty
acid and thus become a good tool for further lipid
metabolic studies. Furthermore, based on our previous
findings (Carballeira et al., 2005), it was expected that
the ␣-methoxy-4-thia fatty acid to be a better antifungal
compound than the parent 4-thia fatty acid. Therefore,
in this work we present the first total synthesis for a
2-methoxy-4-thia fatty acid, namely the ( )-2-methoxy-
4-thiatetradecanoic acid (1), and report on its antifungal
properties and compare it to other analogs of similar
chain lengths.
2.1. Instrumentation
1H NMR (300 MHz) and 13C NMR (75 MHz) were
recorded on a Bruker DPX-300 spectrometer. 1H NMR
chemical shifts are reported with respect to inter-
nal (CH3)4Si (77.0 ppm), 13C NMR chemical shifts
are reported in parts per million relative to CDCl3
(77.0 ppm). GC–MS analyses were recorded at 70 eV
using a Hewlett Packard 5972A MS equipped with a
30 m × 0.25 mm special performance capillary column
(HP-5MS) of polymethyl siloxane crosslinked with 5%
phenyl methylpolysiloxane. High-resolution mass spec-
tral data was performed at the Emory University Mass
Spectrometry Center on a thermo LTQ-FTMS using flow
injection analysis.
2.2. 2-(2-Thiadodecanoyl)-1,3-dioxolane (2)
Into a two necked 100 mL round-bottomed flask
equipped with a magnetic stirrer and a condenser was
added NaH (0.54 g, 23.0 mmol) and diluted with THF
(10 mL). The solution was kept under argon. The tem-
perature was lowered to 0 ◦C and the 1-decanethiol
(4.6 mL, 23.0 mmol) was slowly added to the solution.
The reaction mixture was warmed to room temperature
in a period of 35 min, then cooled to 0 ◦C and the 2-(2-
bromomethyl)-1,3-dioxolane (1.5 mL, 15.0 mmol) was
added. The reaction was refluxed for 5 h and extracted
with ether (2× 15 mL), dried over Na2SO4, and filtered.
The solvent was removed in vacuo affording 3.59 g of
2-(2-thiadodecanoyl)-1,3-dioxolane (2) (92% yield). 1H
NMR (CDCl3) δ 5.04 (1H, t, J = 4.6 Hz, H-2), 4.00 (2H,
AAꢀ BBꢀ, –OCH2–), 3.88 (2H, AAꢀ BBꢀ, –OCH2–), 2.70
(2H, d, J = 4.6 Hz, H-1ꢀ), 2.60 (2H, t, J = 7.4 Hz, H-3ꢀ),
1.57 (2H, quintet, J = 7.4 Hz, H-4ꢀ), 1.35 (2H, m, H-5),
1.25 (12H, brs, –CH2–), 0.86 (3H, t, J = 7.0 Hz, –CH3–);
13C NMR (CDCl3) δ 104.3 (d, C-2), 65.2 (t), 35.4 (t),
32.9 (t), 31.9 (t), 29.6 (t), 29.51 (t), 29.47 (t), 29.3 (t),
29.2 (t), 28.8 (t), 22.6 (t), 14.1 (q, CH3); GC–MS (70 eV)
m/z (relative intensity) 260 (M+, 2), 187 (1), 87 (1),
86 (1), 83 (1), 75 (1), 74 (3), 73 (100), 69 (2), 67 (1),
61 (3), 60 (1), 59 (1), 57 (2), 56 (1), 55 (5); HRMS
(ESI) Calcd for C14H27O2S [M − H]+ 259.1726, found
259.1724.
2.3. 3-Thiatetradecanal (3)
Into a 100 mL round-bottomed flask equipped
with a magnetic stirrer was added the dioxolane 2
(2.0 g, 8.0 mmol) diluted with 20 mL of a solution of

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N.M. Carballeira et al. / Chemistry and Physics of Lipids 150 (2007) 82–88
water–acetone (1:1). Amberlyst 15® (excess) was added
to the solution which was stirred at 60 ◦C for 24 h. The
reaction mixture was filtered, extracted with ethylac-
etate (2× 20 mL) and dried over Na2SO4. The solvent
was removed in vacuo affording 1.05 g (60% yield) of
solution was stirred overnight at room temperature
followed by 2 h at 65 ◦C. The reaction mixture was
washed with water, extracted with ether (2× 10 mL),
and dried over Na2SO4. The solvent was removed in
vacuo affording 0.26 g (50% yield) of ( )-2-hydroxy-4-
thiatetradecanoic acid (5) as a white solid. mp 84–86 ◦C;
1H NMR (CDCl3) δ 4.40 (1H, dd, J = 4.2 Hz, J = 6.3 Hz,
H-2), 3.05 (1H, dd, J = 4.2 Hz, J = 14.2 Hz, H-3), 2.90
(1H, dd, J = 6.3 Hz, J = 14.2 Hz, H-3), 2.55 (2H, t,
J = 7.2 Hz, H-5), 1.58 (2H, quintet, J = 7.2 Hz, H-6), 1.35
(2H, m), 1.25 (12H, brs, –CH2–), 0.87 (3H, t, J = 5.9 Hz,
–CH3); 13C NMR (CDCl3) δ 176.6 (s, C-1), 69.8 (d),
36.2 (t), 32.9 (t), 31.9 (t), 29.5 (t), 29.3 (t), 29.2 (t), 28.8
(t), 22.7 (t), 14.1 (q); HRMS (ESI) Calcd for C13H25O3S
[M − H]+ 261.1535, found 261.1527.
1
3-thiatetradecanal (3). H NMR (CDCl3) δ 9.45 (1H,
t, J = 3.5 Hz, CHO), 3.17 (2H, d, J = 3.5 Hz, H-2), 2.42
(2H, t, J = 7.4 Hz, H-4), 1.55 (2H, quintet, J = 7.4 Hz, H-
5), 1.34 (2H, m, H-6), 1.25 (12H, brs, –CH2–), 0.87 (3H,
t, J = 6.9 Hz, –CH3); 13C NMR (CDCl3) δ 193.9 (d, C-1),
41.4 (t), 31.8 (t), 31.6 (t), 30.9 (t), 29.5 (t), 29.4 (t), 29.2
(t), 29.1 (t), 28.6 (t), 22.6 (t), 14.1 (q, CH3); GC–MS
(70 eV) m/z (relative intensity) 216 (M+, 16), 188 (6),
187 (44), 173 (25), 171 (3), 143 (4), 129 (18), 115 (7),
112 (3), 101 (16), 99 (3), 97 (23), 96 (4), 95 (3), 89 (5),
88 (5), 87 (28), 85 (5), 84 (8), 83 (57), 82 (7), 81 (6), 77
(3), 76 (6), 75 (7), 71 (10), 70 (14), 69 (54), 68 (8), 67
(12), 65 (2), 63 (4), 62 (4), 61 (90), 60 (26), 57 (32), 56
(18), 55 (100).
2.6. Methyl ( )-2-hydroxy-4-thiatetradecanoate
(5a)
Was prepared from 5 to confirm GC purity by means
of the reaction of the acid with methanolic HCl fol-
lowed by purification using column chromatography on
Si gel eluting with hexane/ether (9:1). GC–MS (70 eV)
m/z (relative intensity) 276 (M+, 11), 258 (4), 226 (2),
217 (8), 198 (2), 189 (5), 188 (13), 187 (100), 184 (5),
173 (10), 171 (7), 146 (3), 135 (2), 119 (5), 118 (8), 103
(7), 97 (28), 95 (3), 89 (12), 87 (18), 83 (67), 71 (16), 69
(55), 67 (11), 61 (90), 59 (18), 57 (40), 56 (12), 55 (97).
2.4. ( )-2-Trimethylsilyloxy-
4-thiatetradecanonitrile (4)
Into a 100 mL round-bottomed flask equipped with
a magnetic stirrer was added the 3-thiatetradecanal (3)
(1.48 g, 7.0 mmol) diluted in dichloromethane (10 mL).
The temperature was lowered to 0 ◦C and trimethylsi-
lyl cyanide (1.19 mL, 9.0 mmol) was added. After 1 min
three drops of Et3N were added and the solution was
stirred at 0 ◦C for 3 h. The solution was extracted with
ether (2× 15 mL), dried over Na2SO4 and the solvent
removed in vacuo. The reaction afforded 1.64 g (74%
yield) of nitrile 4 as a viscous oil, which was used for the
next step without further purification. 1H NMR (CDCl3)
δ 4.46 (1H, t, J = 6.7 Hz, H-2), 2.84 (2H, d, J = 6.7 Hz, H-
3), 2.64 (2H, t, J = 7.4 Hz, H-5), 1.58 (2H, m, H-6), 1.37
(2H, m), 1.26 (12H, brs, –CH2–), 0.87 (3H, t, J = 6.4 Hz,
–CH3), 0.2 (9H, s, –Si(CH3)3); 13C NMR (CDCl3) δ
119.3 (d, C-2), 62.8 (t), 37.5 (t), 33.3 (t), 31.9 (t), 29.6 (t),
29.5 (t), 29.4 (t), 29.3 (t), 29.2 (t), 28.7 (t), 22.7 (t),14.1
(q),−0.38 (q); GC–MS (70 eV) m/z (relative intensity)
315 (M+, 4), 300 (3), 189 (5), 188 (13), 187 (100), 144
(2), 133 (6), 117 (4), 97 (13), 87 (3), 85 (4), 84 (5), 83
(29), 81 (2), 75 (9), 73 (22), 71 (6), 70 (2), 69 (17), 61
(30), 59 (4), 57 (13), 55 (22).
2.7. ( )-2-Methoxy-4-thiatetradecanoic acid (1)
Into a 50 mL round-bottomed flask was placed
NaH (0.13 g, 5.0 mmol) diluted in THF (5 mL).
Then 2-hydroxy-4-thiatetradecanoic acid (5) (0.20 g,
0.76 mmol) in 5 mL of THF, was added to the reaction
mixture. The reaction was stirred for 20 min, then cooled
to 0 ◦C and iodomethane (0.5 mL, 6.0 mmol) was slowly
added. The reaction was stirred for 4 h at room tempera-
ture, and then HCl(conc) was added to make the solution
acidic. The reaction mixture was extracted with ether
(2× 10 mL), dried over Na2SO4, and the solvent was
removed in vacuo. The crude product was purified using
silica gel column chromatography and eluting first with
hexane/EtOAc (9:1) to remove the impurities, followed
by hexane/EtOAc (7:3) to obtain 0.15 g (70% yield) of
the ( )-2-methoxy-4-thiatetradecanoic acid (1) as a vis-
1
cous oil. H NMR (CDCl3) δ 3.98 (1H, dd, J = 4.4 Hz,
J = 6.9 Hz, H-2), 3.48 (3H, s, –OCH3), 2.95 (1H, dd,
J = 4.4 Hz, J = 14.0 Hz, H-3), 2.85 (1H, dd, J = 6.9 Hz,
J = 14.0 Hz, H-3), 2.59 (2H, t, J = 7.4 Hz, H-5), 1.55
(2H, quintet, J = 7.4 Hz, H-6), 1.34 (2H, m), 1.26 (12H,
brs, –CH2–), 0.87 (3H, t, J = 6.4 Hz, –CH3); 13C NMR
2.5. ( )-2-Hydroxy-4-thiatetradecanoic acid (5)
Into a 50 mL round-bottomed flask equipped with
a magnetic stirrer was added the nitrile 4 (0.60 g,
2.0 mmol) diluted in 5.0 mL of concentrated HCl. The

N.M. Carballeira et al. / Chemistry and Physics of Lipids 150 (2007) 82–88
85
(CDCl3) δ 175.9 (s, C-1), 80.7 (d, C-2), 58.7 (q, OCH3),
34.1 (t), 33.2 (t), 31.9 (t), 29.53 (t), 29.49 (t), 29.3 (t),
29.2 (t), 29.0 (t), 28.8 (t), 22.6 (t), 14.1 (q, CH3); GC–MS
(70 eV) m/z (relative intensity) 276 (M+, 1), 248 (1), 247
(3), 246 (21), 231 (1), 189 (1), 188 (2), 187 (13), 185 (1),
176 (1), 175 (6), 174 (14), 173 (100), 171 (2), 161 (3),
143 (1), 140 (1), 129 (3), 120 (1), 119 (4), 112 (1), 111
(2), 107 (3), 106 (13), 105 (3), 101 (2), 97 (7), 96 (1), 91
(2), 90 (1), 89 (14), 88 (8), 87 (6), 85 (2), 84 (3), 83 (11),
82 (2), 81 (1), 79 (1), 75 (1), 73 (3), 71 (2), 70 (4), 69
(8), 68 (1), 67 (3), 61 (6), 60 (3), 59 (2), 57 (5), 56 (4),
55 (12). HRMS (ESI) Calcd for C14H27O3S [M − H]+
275.1675, found 275.1680.
not inhibitory to the organisms in the concentrations
tested.
2.11. Susceptibility testing
Microdilutions for control experiments with C. albi-
cans, A. niger, and C. neoformans were the modified
method of National Committee for Clinical Laboratory
Standards (NCCLS) method as described by Galgiani
(Galgiani, 1993) and by the more recent NCCLS
M27-A microdilution methods as described previously
(Carballeira et al., 2004; Nam et al., 2004). Dilutions
were prepared in 0.1 mL of SDB; the inocula were either
104 C. albicans or C. neoformans cells or A. niger spores.
The tubes were incubated for 24–28 h at 36 1 ◦C, and
turbidity was read visually. MICs were calculated in
comparison to growth control as the lowest concentra-
tion that shows inhibition for AMB, FLC, and the test
compounds.
2.8. 4-Thiatetradecanoic acid
Was obtained in a 75% yield from the reaction of
3-bromopropionic acid (1 g, 6 mmol) and decanethiol
(2.4 mL, 12 mmol) using NaH in THF and follow-
ing the procedure described by Skrede (Skrede et al.,
1997).
3. Results and discussion
A five-step synthesis for the ( )-2-methoxy-
4-thiatetradecanoic acid (1) was accomplished as
outlined in Scheme 1. This synthesis started with
commercially available 1-decanethiol that was cou-
pled with 2-(2-bromomethyl)-1,3-dioxolane under basic
conditions using NaH in dry THF affording the 2-
(2-thiadodecanoyl)-1,3-dioxolane (2) in a 92% yield.
The acetal in dioxolane 2 was removed by treatment
with acidic Amberlyst 15® (wet) ion-exchange resin
in acetone–water (1:1) for 24 h, which afforded the 3-
thiatetradecanal (3) in a 60% yield. The aldehyde 3
was then reacted with trimethylsilyl cyanide and tri-
ethylamine in dichloromethane at 0 ◦C affording the
( )-2-trimethylsilyloxy-4-thiatetradecanonitrile (4) in a
74% yield. Hydrolysis of the nitrile 4 was carried out
in the presence of concentrated HCl by stirring for 24 h
at room temperature followed by 2 h at 65 ◦C, which
resulted in a 50% yield of the desired ( )-2-hydroxy-4-
thiatetradecanoic acid (5). The hydrolysis of the nitrile
4 was the key step in this synthesis. The desired ␣-
methoxylated 4-thia fatty acid 1 was then obtained in
a 70% yield in the reaction of the ␣-hydroxy acid 5
with sodium hydride and iodomethane in tetrahydro-
furan (THF) which only afforded methylation at the
␣-hydroxy group and not at the carboxylic acid. The
overall yield for this five-step synthesis was 14%. Our
developed synthetic methodology can be applied to syn-
thesize other methoxylated 4-thia fatty acid analogs with
different chain lengths by just changing the length of the
starting 1-alkanethiol.
2.9. Microorganisms
C. albicans ATCC 60193, A. niger ATCC 16404, and
C. neoformans ATCC 66031 were obtained from Amer-
ican Type Culture Collection, Manassas, Virginia. Stock
cultures were kept on Sabouraud dextrose agar (SDA;
Becton-Dickinson and Co., Sparks, MD). Subcultures
were prepared on SDA at 35–37 ◦C. Suspension cul-
tures were prepared by inoculation of single colonies
in 7 mL of normal saline solution. Prior to preparation
of susceptibility assays, yeast cells were resuspended
in normal saline to make a transmittance of 73–75%
at 530 nm. This transmittance provides a concentration
of 106 cells/mL for C. ablicans or C. neoformans and
106 spores/mL for A. niger in saline medium when com-
pared to the control tube. The media was Sabourad
dextrose broth (SDB; Becton Dickinson and Co., Sparks,
MD).
2.10. Chemicals and antifungal agents
Amphotericin B (AMB) was purchased from Acros,
New Jersey, USA and was kept as a 0.005 mM stock
in DMSO at 0 ◦C and used during 1 week of prepa-
ration. Fluconazole (FLC) was purchased from Medisa
Inc., New York, USA, or was provided from Vera Lab-
oratories Ltd., Hyderabad, India, and was kept as a
0.02 mM stock solution at 0 ◦C. Working dilutions were
made in SDB medium. The final maximum concentra-
tion of DMSO in the assays was 5% (v/v). DMSO was

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N.M. Carballeira et al. / Chemistry and Physics of Lipids 150 (2007) 82–88
Scheme 1. Synthesis of ( )-2-hydroxy-4-thiatetradecanoic acid (5) and ( )-2-methoxy-4-thiatetradecanoic acid (1).
The fungitoxicity of the novel ␣-substituted 4-thia
However, in the case of C. neoformans (ATCC 66031)
␣-hydroxylation (MIC = 0.17 mM) was about sevenfold
more effective than ␣-methoxylation (MIC = 1.24 mM)
in increasing the fungitoxicity of the 4-thia fatty acid.
In the case of A. niger (ATCC 16404) ␣-hydroxylation
did not improve significantly the antifungal activity
when compared to the parent analog. However, ␣-
methoxylation enhanced the antifungal activity towards
A. niger (MIC = 0.8 mM) approximately eightfold higher
than that of 4-thiatetradecanoic acid (MIC = 6.3 mM).
In Table 1 the previously reported antifungal activities
of ( )-2-methoxy-4-oxatetradecanoic acid (Carballeira
et al., 2005) have been given to compare the effect of
4-oxa substitution versus 4-thia substitution on antifun-
gal activities. With respect to C. neoformans (ATCC
66031) and A. niger (ATCC 16404) the ( )-2-methoxy-
4-oxatetradecanoic acid was a better antifungal fatty acid
fatty acids 5 and 1, as compared to 4-thiatetradecanoic
acid and other analogs, is presented in Table 1. It is
interesting to note that the 4-thiatetradecanoic acid is
quite benign (MIC = 5–12 mM) since it is less toxic to
the studied fungi than even myristic acid. As compared to
the parent 4-thiatetradecanoic acid both ␣-hydroxylation
and ␣-methoxylation increased the fungitoxicity of
the 4-thia fatty acid. In general, ␣-methoxylation was
more effective in increasing the antifungal activity
(MIC = 0.8–1.2 mM) of the 4-thiatetradecanoic acid
towards all the studied fungal strains (Table 1). How-
ever, there were some interesting differences between
the strains. For example, in the case of C. albicans
(ATCC 60193) ␣-hydroxylation and ␣-methoxylation
were equally effective in increasing the antifungal activ-
ity (MIC = 1.0–1.2 mM) of the 4-thiatetradecanoic acid.
Table 1
Antifungal activity (MIC values, mM)a against Candida albicans (SDB), Cryptococcus neoformans (SDB), and Aspergillus niger (SDB) at 35–37 ◦C
after 24–48 h
Compound
C. albicans ATCC 60193
C. neoformans ATCC 66031
A. niger ATCC 16404
14:0b
3.80c
12.66
1.04
1.90
4.75
0.17
1.24
0.11
3.80
6.33
5.53
0.83
0.11
0.22
1.50
<0.0009
>5
4-thia-14:0
2-OH-4-thia-14:0
2-OMe-4-thia-14:0
2-OMe-14:0b
2-OMe-4-oxa-14:0b
FLC
1.24
0.11c
3.50
0.08
>1.0
<0.0003
>5
0.002
<0.0003
>5
AMB
DMSO
The bold values are for the title compound 1.
a
The result is the average of three separate experiments. The upper limit of the standard error of the mean (S.E.M.) was 10%.
Antifungal data taken from Carballeira et al. (2005).
These MIC values were measured against C. albicans 14053, also taken from Carballeira et al. (2005).
b
c

N.M. Carballeira et al. / Chemistry and Physics of Lipids 150 (2007) 82–88
87
2-methoxy-4-oxatetradecanoic acid towards C. neofor-
mans and A. niger. In the case of C. albicans, 1 was a
betterantifungalagentthanthecorresponding4-oxaana-
log. This is not surprising since the 4-oxatetradecanoic
acid is known not to be effective against C. albicans
(Langer et al., 1992).
Thus, in addition to interesting antifungal activities,
␣-methoxylated fatty acid 1 may be used as a probe to
study fatty acid metabolic routes. Further mechanistic
and metabolic studies are underway to determine the
metabolism of 1.
(MIC = 0.08–0.22 mM) than 1 (MIC = 0.8–1.2 mM).
However, against C. albicans (ATCC 60193) the
( )-2-methoxy-4-thiatetradecanoic acid (1) was about
threefold more fungitoxic (MIC = 1.24 mM). Over-
all, the ( )-2-methoxytetradecanoic acid continues to
be the most effective antifungal fatty acid of the
series (MIC = 0.11 mM) against the three fungal strains
tested.
From our results it is clear that ␣-methoxylation
increases the fungitoxicity of 4-thiatetradecanoic acid
towards the studied fungal strains. The rationale
behind the increased fungitoxicity of 1 versus the 4-
thiatetradecanoic acid could be the result of several
factors. There are clear differences in the way acid
1 is metabolized by the fungi as compared to 4-
thiatetradecanoic acid. The presence of the additional
␣-methoxygroupshouldintroducesufficientstericeffect
at the ␣-position to delay the ␤-oxidation of acid 1,
thus leaving ␻-oxidation and sulfur oxidation as the
most likely metabolic pathways for 1. This steric block-
ade should increase the half-life of acid 1 in the cell
as compared to the half-life of the 4-thiatetradecanoic
acid, which can go through one cycle of ␤-oxidation.
Since 1 is expected to have longer half-life in the
fungal cells, it possibly gets incorporated more effi-
ciently into the membrane phospholipids of the fungi
and causes membrane disruption and eventual anti-
fungal activities (Hvattum et al., 1992). Presumably,
sulfur oxidation occurs more rapidly than methoxy
hydroxylation (removal of the ␣-methoxy group) or
␻-oxidation (Alterman et al., 1995), and metabolites
such as the 2-methoxydecylsulfoxypropionic acid and
2-methoxycarboxymethylsulfoxypropionic acid could
also be accumulating in the fungal cells (Hvattum et al.,
1992). Alternatively fatty acid 1 or its oxidized metabo-
lites may be inhibiting some key fatty acid metabolic
enzymes in the fungal cells or the NMT enzyme of some
of these fungi.
Acknowledgements
This work was supported by a grant from the SCORE
program of the National Institutes of Health (Grant No.
S06GM08102). R. O’Neill thanks the NIH-RISE pro-
gram for a graduate fellowship. We thank Fred Strobel
(Emory University) for the high resolution mass spectral
data.
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