First total synthesis and antileishmanial activity of (Z)-16-methyl-11- heptadecenoic acid, a new marine fatty acid from the sponge Dragmaxia undata

Article abstract of DOI:10.1016/j.chemphyslip.2010.11.006

The first total synthesis for the (Z)-16-methyl-11-heptadecenoic acid, a novel fatty acid from the sponge Dragmaxia undata, was accomplished in seven steps and in a 44% overall yield. The use of (trimethylsilyl)acetylene was key in the synthesis. Based on a previous developed strategy in our laboratory the best synthetic route towards the title compound was first acetylide coupling of (trimethylsilyl)acetylene to the long-chain protected 10-bromo-1-decanol followed by a second acetylide coupling to the short-chain 1-bromo-4- methylpentane, which resulted in higher yields. Complete spectral data is also presented for the first time for this recently discovered fatty acid and the cis double bond stereochemistry of the natural acid was established. The title compound displayed antiprotozoal activity against Leishmania donovani (IC ₅₀ = 165.5 ± 23.4 μM) and inhibited the leishmania DNA topoisomerase IB enzyme (LdTopIB) with an IC₅₀ = 62.3 ± 0.7 μM.

Full text of DOI:10.1016/j.chemphyslip.2010.11.006

Chemistry and Physics of Lipids 164 (2011) 113–117
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
First total synthesis and antileishmanial activity of
(Z)-16-methyl-11-heptadecenoic acid, a new marine fatty acid from the sponge
Dragmaxia undata
Néstor M. Carballeiraa,∗, Nashbly Montanoa, Gabriel A. Cintróna, Carmary Márqueza,
Celia Fernández Rubiob, Christopher Fernández Pradab, Rafael Balan˜a-Fouceb
a Department of Chemistry, University of Puerto Rico, P.O. Box 23346, San Juan Puerto Rico 00931-3346, USA
b Department of Biomedical Sciences, University of León, Campus de Vegazana s/n, 24071 León, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The first total synthesis for the (Z)-16-methyl-11-heptadecenoic acid, a novel fatty acid from the sponge
Dragmaxia undata, was accomplished in seven steps and in a 44% overall yield. The use of (trimethylsi-
lyl)acetylene was key in the synthesis. Based on a previous developed strategy in our laboratory the best
synthetic route towards the title compound was first acetylide coupling of (trimethylsilyl)acetylene to
the long-chain protected 10-bromo-1-decanol followed by a second acetylide coupling to the short-chain
1-bromo-4-methylpentane, which resulted in higher yields. Complete spectral data is also presented for
the first time for this recently discovered fatty acid and the cis double bond stereochemistry of the natural
acid was established. The title compound displayed antiprotozoal activity against Leishmania donovani
(IC50 = 165.5 23.4 ␮M) and inhibited the leishmania DNA topoisomerase IB enzyme (LdTopIB) with an
IC50 = 62.3 0.7 ␮M.
Received 14 October 2010
Received in revised form
18 November 2010
Accepted 24 November 2010
Available online 1 December 2010
Keywords:
Dragmaxia undata
Fatty acids
Leishmaniasis
(Z)-16-methyl-11-heptadecenoic acid
Sponges
© 2010 Elsevier Ireland Ltd. All rights reserved.
Synthesis
Topoisomerase IB
1. Introduction
acid. However, acid 1 was identified in D. undata by means of
mass spectrometry on the corresponding methyl ester and pyrro-
Marine organisms continue to yield interesting and unprece-
dented phospholipid fatty acids with no counterpart in terrestrial
organisms. One such class of fatty acids is the iso methyl-branched
octadecenoic acids, from which only a handful number of com-
pounds have been reported. To the best of our knowledge, the
only known examples of this type of fatty acids are the (Z)-16-
methyl-8-heptadecenoic acid, also synthesized for the first time by
our group (Carballeira and Pagán, 2001), and the (Z)-16-methyl-
6-heptadecenoic acid, which were identified in a Micrococcus
bacterium isolated from Lake Pomorie in Bulgaria (Carballeira et al.,
2000). Just recently, Osorno and coworkers identified the new
fatty acid (Z)-16-methyl-11-heptadecenoic acid (1) in the phos-
pholipids (1.1% relative abundance) of the Colombian Caribbean
sponge Dragmaxia undata, collected at 21 m depth in Punta de Betin,
Santa Marta, Colombia (Rodríguez et al., 2010). This rather unusual
fatty acid is the iso methyl-branched octadecenoic acid with the
closest double bond to the ␻ end of the chain, i.e., a (n−6) fatty
lidide derivative and the identification by GC–MS relied on minute
amounts from a complex mixture of 30 fatty acids. Therefore, it was
not possible to elucidate if the double bond stereochemistry of the
naturally occurring fatty acid was cis or trans.
It is very likely that acid 1 was biosynthesized by a symbiotic
bacterium within D. undata, thus implying that having the complete
mass spectral data of 1 at hand will be useful to microbiologists
seeking to characterize 1 in bacterial samples. The latter statement
is backed up by the fact that acid 1 is a two-carbon chain exten-
sion of the (Z)-14-methyl-9-pentadecenoic acid, an acid that has
been identified in several bacterial sources and most recently in
a Streptomyces sp. B6007 originating from mangrove sediment in
Papua New Guinea (Stritzke et al., 2004). Of particular interest in
this context is also the report by Jung and collaborators that iso and
anteiso fatty acids from a Streptomyces sp. strain KM86-9B displayed
topoisomerase I inhibitory activity (Lee et al., 1998). In fact, we
have also shown that the acid (Z)-14-methyl-9-pentadecenoic acid
inhibits the human placenta DNA topoisomerase I enzyme at con-
centrations of 500 ␮M (Carballeira et al., 2007), while the marine
fatty acid (Z)-17-methyl-13-octadecenoic acid inhibits the leish-
mania DNA topoisomerase IB enzyme at concentrations of 50 ␮M
∗
Corresponding author. Tel.: +1 787 764 0000×4791; fax: +1 787 756 8242.
E-mail address: nmcarballeira@uprrp.edu (N.M. Carballeira).
0009-3084/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2010.11.006

114
N.M. Carballeira et al. / Chemistry and Physics of Lipids 164 (2011) 113–117
(Carballeira et al., 2009). The latter report indicated that these iso
monounsaturated fatty acids do have antiprotozoal potential.
It then becomes of interest to explore both the antileishmanial
and the topoisomerase I inhibitory activity of the new acid 1, but
it will remain unexplored until enough material becomes avail-
able for such studies. However, by applying our previous developed
synthetic methodology for unsaturated iso methyl-branched fatty
acids (Carballeira et al., 2009) to the synthesis of 1 enough mate-
rial for such studies could be obtained. Therefore, in order to
unequivocally conclude the total characterization of 1, i.e., deter-
mine the cis or trans double bond stereochemistry, as well as to
study its antileishmanial activity we are reporting herein the first
total synthesis for the (Z)-16-methyl-11-heptadecenoic acid (1).
This synthesis was accomplished following a synthetic sequence
consisting of seven steps and using (trimethylsilyl)acetylene as the
key reagent in the synthesis (Scheme 1). We also report that acid
1 displays antiprotozoal activity towards Leishmania donovani pro-
mastigotes by a mechanism that probably involves inhibition of the
leishmania DNA topoisomerase IB enzyme.
eluting with hexane/ether (9:1). The silylated alkyne 3 (Banaszak
et al., 2009) was obtained as a colorless oil 2.43 g (7.16 mmol) for a
97% yield.
2.4. 1-[(Tetrahydropyran-2-yl)oxy]dodec-11-yne (4)
To a mixture of 2.35 g (6.93 mmol) of 3 and 12.0 mL of dry THF
was added dropwise 6.93 mmol of tetrabutylammonium fluoride
(1 M) at 0 ◦C. After 24 h at room temperature the reaction was
quenched with HCl (2 M), and the organic layer was washed with
brine (1 × 20 mL), ether (1 × 20 mL), dried over MgSO4, filtered, and
evaporated in vacuo. The crude product was purified using silica
gel column chromatography eluting with hexane/ether (9:1). The
alkyne 4 was obtained as a colorless oil 1.66 g (6.22 mmol) for a 90%
yield. IR (neat) ꢀmax 3312, 2926, 2854, 1466, 1441, 1352, 1323, 1259,
1200, 1136, 1121, 1078, 1026, 988, 904, 870, 814, 721, 627 cm−1
;
1H NMR (CDCl3, 500 MHz) ı 4.55 (1H, t, J = 2.7 Hz), 3.84 (1H, m),
3.71 (1H, m), 3.49 (1H, m), 3.36 (1H, m), 2.16 (2H, dt, J = 7.1, 2.6 Hz,
H-10), 1.92 (1H, t, J = 2.6 Hz, H-12), 1.81 (2H, m), 1.70 (2H, m), 1.53
(6H, m, –CH2–), 1.27 (12H, m, –CH2–); 13C NMR (CDCl3, 125 MHz)
␦ 98.80 (d), 84.74 (s, C-11), 68.01 (d, C-12), 67.64 (t, C-1), 62.29
(t), 30.75 (t), 29.71 (t, C-2), 29.48 (t), 29.41 (t), 29.39 (t), 29.04 (t),
28.70 (t), 28.45 (t), 26.19 (t, C-3), 25.48 (t), 19.66 (t, C-10), 18.36 (t);
GC–MS (70 eV) m/z (relative intensity) 266 (M+, 1), 237 (1), 225 (1),
211 (1), 195 (1), 165 (1), 149 (1), 135 (1), 123 (1), 109 (4), 107 (2),
101 (27), 97 (2), 95 (11), 93 (4), 91 (2), 86 (6), 85 (100), 84 (10), 81
(19), 79 (9), 67 (21), 57 (9), 56 (17), 55 (28). HRMS (APCI) Calcd. for
2. Materials and methods
2.1. Instrumentation
1H NMR (500 MHz) and 13C NMR (125 MHz) were recorded
on a Bruker DRX-500 spectrometer. 1H NMR chemical shifts are
reported with respect to internal (CH3)4Si, 13C NMR chemical shifts
are reported in parts per million relative to CDCl3 (77.0 ppm).
Gas chromatography/mass spectrometry (GC/MS) analyses were
recorded at 70 eV using either a Hewlett Packard 5972A MS
ChemStation or an Agilent 5975C MS ChemStation coupled to an
Agilent 7890A GC where both instruments were equipped with a
30 m × 0.25 mm special performance capillary column (HP-5MS) of
polymethyl siloxane crosslinked with 5% phenyl methylpolysilox-
ane. IR spectra were recorded on a Nicolet Magna 750 FT-IR
spectrophotometer (Thermo-Nicolet, Madison, WI, USA). High res-
olution mass spectral data was performed at the Emory University
Mass Spectrometry Center on a thermo linear ion trap-Fourier
transform mass spectrometer (LTQ-FTMS) using atmospheric pres-
sure chemical ionization (APCI) as the probe.
C
19H35O2 [M+H]+ 267.2682, found 267.2681.
2.5. 16-Methyl-1-[(tetrahydropyran-2-yl)oxy]heptadec-
11-yne (5)
To a solution of 1.54 g (5.77 mmol) of 4 and 11.2 mL of THF at
0 ◦C, 1.87 mL (20.2 mmol) of n-BuLi (2.5 M) in hexane was added
while stirring under an argon atmosphere. After 45 min, 2.87 mL
of HMPA was added. After additional 15 min, 2.56 mL (17.3 mmol)
of 1-bromo-4-methylpentane was added to the reaction mixture
and the reaction was left stirring for 24 h at room temperature.
After this time the reaction mixture was quenched with water and
the organic product was extracted with ether (2 × 25 mL), dried
over MgSO4, filtered, and evaporated in vacuo. The crude prod-
uct was purified using silica gel column chromatography eluting
with hexane/ether (9:1). The product 5 was obtained as colorless
oil 2.02 g (5.75 mmol) for a 99% yield. IR (neat) ꢀmax 2926, 2854,
1466, 1441, 1383, 1366, 1352, 1259, 1200, 1128, 1121, 1078, 1032,
2.2. 10-Bromo-[(tetrahydropyran-2-yl)oxy]decane (2)
To 2.00 g of 10-bromo-1-decanol in 15 mL of chloroform (CHCl3)
was added dropwise 16.9 mmol of 3,4-dihydro-2H-pyran (DHP)
and catalytic amounts of p-toluenesulphonic acid (pTSA). The reac-
tion mixture was stirred for 24 h at room temperature. The organic
layer was washed with water (2 × 20 mL), NaHCO3 (2 × 20 mL),
CHCl3 (1 × 20 mL), and dried over MgSO4, filtered, and evaporated
in vacuo. The crude product was purified using silica gel column
chromatography eluting with hexane/ether (9:1). The pure prod-
uct 2 (Banaszak et al., 2009) was obtained as a colorless oil 2.35 g
(7.32 mmol) for a 100% yield.
984, 905, 870, 814, 743 cm−1 1H NMR (CDCl3, 500 MHz) ı 4.56 (1H,
;
t, J = 2.7 Hz), 3.85 (1H, m), 3.72 (1H, m), 3.49 (1H, m), 3.37 (1H, m),
2.11 (4H, m, H-10, H-13), 1.81 (2H, m), 1.69 (2H,m), 1.51 (7H, m,
–CH2–), 1.26 (16H, m, –CH2–), 0.86 (6H, d, J = 6.6 Hz, –CH(CH3)2);
13C NMR (CDCl3, 125 MHz) ı 98.79 (d), 80.22 (s), 80.19 (s), 67.64
(t, C-1), 62.27 (t), 38.18 (t, C-15), 30.75 (t), 29.72 (t, C-2), 29.51 (t),
29.46 (t), 29.44 (t), 29.12 (t), 28.81 (t), 27,61 (d, C-16), 27.04 (t, C-3),
26.20 (t), 25.48 (t), 22.54 (q, C-17, C-18), 19.65 (t), 18.98 (t, C-13),
18.71 (t, C-10); GC–MS (70 eV) m/z (relative intensity) 350 (M+, 1),
294 (1), 279 (1), 277 (1), 265 (1), 221 (1), 207 (1), 195 (1), 165 (1),
151 (1), 149 (2), 138 (1), 137 (2), 135 (3), 123 (6), 121 (5), 111 (3),
109 (27), 107 (6), 101 (24), 96 (12), 95 (31), 93 (9), 86 (6), 85 (100),
84 (20), 83 (18), 82 (20), 81 (34), 79 (15), 69 (41), 68 (12), 67 (32),
57 (13), 56 (14), 55 (46). HRMS (APCI) Calcd. for C23H43O2 [M+H]+
351.3257, found 351.3255.
2.3. 12-(Trimethylsilyl)-1-[(tetrahydropyran-2-yl)oxy]dodec-
11-yn (3)
To a stirred solution of 3.04 mL (22.0 mmol) of (trimethylsi-
lyl)acetylene in 9.5 mL of dry THF and under argon was added
2.36 mL (25.6 mmol) of n-BuLi (2.5 M) in hexane at −78 ◦C. After
2 min, 3.36 mL of HMPA and 2.35 g (7.32 mmol) of 2 was added to
the reaction mixture, maintaining the temperature at −78 ◦C. After
24 h the reaction mixture was quenched with water. The organic
product was extracted with brine solution (2 × 20 mL), diethyl ether
(2 × 20 mL), dried over MgSO4, filtered, and evaporated in vacuo.
The product was purified using silica gel column chromatography
2.6. 16-Methylheptadec-11-yn-1-ol (6)
To a mixture of methanol (20.0 mL) and 1.94 g (6.36 mmol) of
5 was added catalytic amounts of pTSA and the reaction mix-
ture was stirred at 35 ◦C for 48 h. After this time the organic

N.M. Carballeira et al. / Chemistry and Physics of Lipids 164 (2011) 113–117
115
Scheme 1. Synthesis of (Z)-16-methyl-11-heptadecenoic acid (1).
13C NMR (CDCl3, 125 MHz) ␦ 129.91 (d), 129.86 (d), 62.30 (t, C-1),
38.64 (t, C-15), 38.78 (t, C-2), 29.74 (t), 29.58 (t), 29.54 (t), 29.51 (t),
29.41 (t), 29.28 (t), 27.87 (d, C-16), 27.54 (t, C-13), 27.46 (t, C-10),
27.18 (t, C-14), 25.72 (t, C-3), 22.60 (q, C-17, C-18); GC–MS (70 eV)
m/z (relative intensity) 268 (M+, 1), 250 (4), 223 (1), 208 (1), 195
(1), 151 (3), 149 (2), 137 (7), 135 (5), 125 (5), 123 (18), 121 (6), 111
(17), 109 (37), 98 (15), 97 (37), 96 (54), 95 (70), 83 (55), 82 (82),
81 (65), 80 (10), 79 (15), 70 (29), 69 (100), 68 (41), 67 (63), 65 (4),
57 (48), 56 (69), 55 (96). HRMS (APCI) Calcd. for C18H37O [M+H]+
269.2839, found 269.2838.
extract was washed with a saturated solution of sodium bicar-
bonate (2 × 50 mL), ether (2 × 20 mL), dried over MgSO4, filtered,
and evaporated in vacuo. The product was purified using silica gel
column chromatography eluting with hexane/ether (9:1). The 16-
methylheptadec-11-yn-1-ol (6) was obtained as colorless oil 1.41 g
(5.31 mmol) for an 83% yield. IR (neat) ꢀmax 3317 (OH, broad), 2926,
2854, 1466, 1385, 1333, 1057, 721, 627 cm−1 1H NMR (CDCl3,
;
500 MHz) ı 3.61 (2H, t, J = 6.6, H-1), 2.11 (4H, m, H-10, H-13), 1.54
(2H, m), 1.45 (2H, m), 1.34 (2H, m, –CH2–), 1.26 (16H, m, –CH2–),
0.86 (6H, d, J = 6.6 Hz, –CH(CH3)2); 13C NMR (CDCl3, 125 MHz) ı
80.23 (s), 80.18 (s), 62.95 (t, C-1), 38.17 (t, C-15), 32.74 (t, C-2),
29.70 (t), 29.52 (t), 29.44 (t), 29.38 (t), 29.11 (t), 28.80 (t), 27.70
(d, C-16), 27.03 (t), 25.70 (t), 22.53 (q, C-17, C-18), 19.62 (t, C-13),
18.96 (t, C-10); GC–MS (70 eV) m/z (relative intensity) 266 (M+, 1),
251 (1), 210 (1), 191 (1), 167 (1), 163 (2), 149 (4), 135 (9), 123 (14),
121 (15), 111 (7), 110 (18), 109 (88), 107 (14), 97 (13), 96 (38), 95
(84), 94 (11), 93 (24), 91 (12), 83 (23), 82 (65), 81 (89), 80 (21),
79 (38), 77 (12), 70 (11), 69 (100), 68 (37), 67 (73), 65 (8), 57 (13),
56 (9), 55 (61). HRMS (APCI) Calcd. for C18H35O [M+H]+ 267.2682,
found 267.2682.
2.8. (Z)-16-Methyl-11-heptadecenoic acid (1)
A solution of 3.87 mmol of pyridinium dichromate (PDC) and
2.0 mL of dimethylformamide (DMF) was added under argon to a
solution of 0.21 g (0.775 mmol) of alcohol 7 and 5.0 mL of DMF,
and the reaction mixture was left stirring at room temperature
for 48 h. After this time, the reaction mixture was washed with
water (3 × 25 mL), ether (2 × 20 mL), dried over MgSO4, filtered, and
the solvent evaporated in vacuo. The crude product was purified
using florisil column chromatography eluting with ether. The (Z)-
16-methyl-11-heptadecenoic acid (1) was obtained as colorless oil
0.15 g (0.53 mmol) for a 68% yield. IR (neat) ꢀmax 3500–2500, 3005,
2924, 2854, 1709 (C O), 1464, 1414, 1385, 1366, 1119, 1099, 1024,
2.7. (Z)-16-Methylheptadec-11-en-1-ol (7)
Into a 50-mL two-necked round-bottomed flask were placed
dry hexane, 0.61 g (2.28 mmol) of alkyne 6, quinoline (1.8 mL), and
palladium in activated carbon (Lindlar’s catalyst). One of the two
necks was capped with a rubber septum and the other was con-
nected via tygon tubing to a 25-mL graduated pipet ending in a
150 mL beaker with distilled water. While stirring at room tem-
perature a 20 mL syringe with needle was used to withdraw air
from the system and draw water up into the graduated pipet to
the 0.0 mL mark. Hydrogen was then introduced into the system
using balloon filled with hydrogen attached to the hose barb-to-
luer lock adapter with stopcock and a needle. The reaction mixture
consumed 55.9 mL of hydrogen during 1 h. The mixture was filtered
and the solvent removed in vacuo. The product was purified under
vacuum distillation (Kugelrohr) by removing impurities at 130 ◦C/3
mmHg. The (Z)-16-methylheptadec-11-en-1-ol (7) was obtained as
colorless oil 0.55 g (2.07 mmol) for a 91% yield. IR (neat) ꢀmax 3335,
721 cm−1 1H NMR (CDCl3, 500 MHz) ı 5.35 (2H, m, H-11, H-12),
;
2.34 (2H, t, J = 7.4 Hz, H-2), 2.00 (4H, m, H-10, H-13), 1.64 (2H, m,
H-3), 1.53 (2H, m), 1.31 (12H, m, –CH2–), 1.17 (2H, m, H-15), 0.88
(6H, d, J = 6.6 Hz, –CH(CH3)2); 13C NMR (CDCl3, 125 MHz) ı 179.88
(s, C-1), 129.94 (d), 129.84 (d), 38.64 (t, C-15), 34.05 (t, C-2), 29.73
(t), 29.46 (t), 29.39 (t), 29.24 (t), 29.22 (t), 29.05 (t), 27.88 (d, C-16),
27.55 (t, C-13), 27.43 (t, C-10), 27.18 (t), 24.68 (t, C-3), 22.61 (q,
C-17, C-18). HRMS (APCI) Calcd. for C18H33O2 [M−H]+ 281.2486,
found 281.2488.
2.8.1. Methyl (Z)-16-methyl-11-heptadecenoate
GC–MS (70 eV) m/z (relative intensity) 296 (M+, 5), 265 (15), 264
(25), 249 (3), 241 (11), 222 (11), 221 (5), 209 (10), 191 (8), 185 (2),
165 (4), 153 (4), 152 (5), 151 (6), 141 (6), 139 (7), 137 (9), 125 (13),
124 (10), 123 (15), 121 (6), 111 (24), 109 (18), 98 (27), 97 (46), 96
(34), 95 (31), 87 (40), 85 (10), 84 (34), 83 (53), 82 (21), 81 (31), 79
(11), 74 (54), 70 (22), 69 (100), 68 (19), 67 (36), 59 (22), 57 (32), 56
(50), 55 (98).
3005, 2924, 2853, 1464, 1383, 1366, 1119, 1057, 908, 733 cm−1
;
1H NMR (CDCl3, 500 MHz) ı 5.52 (2H, m, H-11, H-12), 3.62 (2H, t,
J = 6.6 Hz, H-1), 2.00 (4H, m, H-10, H-13), 1.55 (4H, m), 1.31 (16H,
m, –CH2–), 1.18 (2H, m, H-15), 0.87 (6H, d, J = 6.6 Hz, –CH(CH3)2);

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N.M. Carballeira et al. / Chemistry and Physics of Lipids 164 (2011) 113–117
2.9. Cell cultures
affording the corresponding dihydropyranyl protected alcohol 2 in
a 100% isolated yield (Scheme 1). Compound 2 was then submitted
to the first acetylide coupling reaction with the versatile reagent
(trimethylsilyl)acetylene using n-BuLi in THF-HMPA at −78 ◦C
affording the trimethylsilylacetylenic derivative 3 in a 97% yield.
Deprotection of the terminal trimethylsilyl group with tetrabuty-
lammonium fluoride (TBAF) afforded the terminal alkyne 4 in a 90%
yield. A second acetylide coupling with 1-bromo-4-methylpentane
using n-Buli, THF-HMPA at 0 ◦C resulted in the iso-branched alkyne
5 in a 99% isolated yield (Scheme 1). Deprotection of the dihydropy-
ranyl group in 5 with PTSA in methanol at 35 ◦C for 48 h afforded
the 16-methyl-11-heptadecyn-1-ol (6) in an 83% isolated yield. Cat-
alytic hydrogenation under Lindlar’s conditions of 6 resulted in
the (Z)-16-methyl-11-heptadecen-1-ol (7) in a 91% yield with a
100% cis stereochemistry for the double bond. Final oxidation of the
alcohol with pyridinium dichromate (PDC) in dimethylformamide
(DMF) resulted in the formation of the desired acid 1 in a 68% yield.
The overall total yield for the seven steps was 44%, which afforded
enough material for its complete spectral characterization.
L. donovani (MHOM/ET67/L82 strain) promastigotes were
propagated in
a completely defined medium 199, supple-
mented with 10% heat inactivated fetal calf serum (FCS) and
penicillin/streptomycin cocktail (containing 50 U/mL penicillin,
50 ␮g/mL streptomycin). The IC50 value was determined with
different concentrations of compound 1 dissolved in DMSO (rang-
ing from 3.9 to 1000 ␮M) added to cultures and cell population
assessed by Coulter.
Murine macrophages (RAW 264.7) were grown at 37 ◦C in RPMI
1640 medium, supplemented with 10% FCS antibiotics (containing
50 U/mL penicillin, 50 ␮g/mL streptomycin). Toxicity IC50 values
were determined after 48 h incubation with different concentra-
tions of the compounds dissolved in DMSO (50–2000 ␮M) using
vital alamar-Blue® dye (Invitrogen, Carlsbad, CA, USA), according
manufacturer recommendations.
2.10. Purification of recombinant leishmanial TopIB
Acid 1 presented spectral data that confirmed its synthesis and
the structure of the natural acid. The iso methyl branching was eas-
ily identified by both 1H NMR and 13C NMR spectrometry as well as
by infrared spectroscopy (IR). In the 1H NMR spectra both terminal
isopropyl methyl groups appeared as a doublet at ı 0.88 ppm, while
in the 13C NMR both methyl groups resonated at ı 22.6 ppm. The IR
spectrum of 1 was also very characteristic since the terminal iso-
propyl group was observed as a doublet at 1385 and 1366 cm−1. The
presence of the olefinic hydrogens in 1 was easily detected in the 1H
NMR spectra by the olefinic signals at ı 5.35 ppm. The 13C NMR of
the olefinic region was most interesting, in particular the resonance
of the allylic methylene carbons that confirmed the cis double bond
stereochemistry. The C-10 allylic carbon resonated at ı 27.4 ppm,
while the C-13 allylic carbon was observed at ı 27.5 ppm, confirm-
ing the presence of the cis double bond (Gunstone et al., 1977). It
is well established that the allylic methylene carbons from cis dou-
ble bonds in fatty acids resonate in 13C NMR at around 27 ppm,
while the allylic methylene carbons from trans double bonds res-
onate at around 32 ppm (Gunstone et al., 1977). The cis double bond
stereochemistry was also further confirmed by IR since a strong
absorption at 721 cm−1 was observed for 1. As expected, the car-
boxylic acid carbonyl was observed in 13C NMR at ı 179.9 ppm and
Expression of a recombinant topoisomerase IB from L. donovani
(LdTopIB) in a topoisomerase IB-deficient Saccharomyces cerevisiae
strain has been described elsewhere (Villa et al., 2003). Purification
of recombinant LdTopIB was done according to Díaz-González et
al. (2008). Briefly, LdTopIB overexpressing yeasts were disrupted
with one freeze/thaw cycle at −80 ◦C, with the purpose of weaken-
ing the yeast wall; after lysis with 425–600 ␮m acid-washed glass
beads, the extracts were cleared by centrifugation at 15,000 × g for
30 min at 4 ◦C. The protein suspension was loaded onto a phospho-
cellulose (P-11) column, previously equilibrated as manufacturer
indications. LdTopIB was eluted at 4 ◦C with a discontinuous gradi-
ent of KCl (0.2, 0.4, 0.6, 0.8 and 1 M) in TEEG buffer, supplemented
with 0.1 mg/mL sodium bisulphite, 0.8 mg/mL NaF and the pro-
tease inhibitors cocktail. Active fractions were further loaded onto
a phenyl-sepharose column (Sigma-Aldrich, St Louis, US), eluted
with a discontinuous inverse gradient of ammonium sulphate (1,
0.8, 0.6, 0.4 and 0.2 M) and then concentrated by Microcon YM-30
(Millipore) before use.
2.11. DNA relaxation assays
in IR at 1709 cm−1
.
DNA topoisomerase IB activity was assayed by the relaxation
of negatively supercoiled plasmid DNA. The reaction mixture in a
total volume of 20 ␮l contained 0.2 ␮g of supercoiled pHOT plas-
mid, 10 mM Tris–HCl buffer pH 7.5, 5 mM MgCl2, 0.1 mM EDTA,
15 ␮g/mL bovine serum albumin, 50 mM KCl and various extracts
containing altered proteins or wild type enzyme, starting with 1
unit LdTopIB. The reaction mixtures were incubated for 30 min at
37 ◦C. The enzyme reactions were stopped by the addition of up
to 1% SDS – final concentration – and digested by 2 mg/mL pro-
teinase K with 1 h incubation to remove protein bonded to the
DNA fragment. The extent of plasmid DNA relaxation was assessed
by electrophoresis in a 1% agarose gel in 0.1 M Tris acetate EDTA
(TAE) buffer pH 8.0 at 2 V/cm for 14 h. The gels were visualized
under UV illumination after being stained with ethidium bromide
(0.5 mg/mL) and a posterior electrophoresis in the presence of
0.1 mg/mL ethidium bromide, in order to separate the nicked DNA
from the relaxed topoisomers. One unit of LdTopIB is defined as the
amount of purified protein able to relax 0.2 ␮g of pHOT supercoiled
DNA per 30 min at 37 ◦C.
The methyl ester of the synthetic acid 1 was also prepared (HCl
in MeOH) and its mass spectrum (Fig. 1) and GC retention time
3. Results and discussion
The synthesis of 1 started with commercially available (Aldrich)
10-bromo-1-decanol, which was protected with 3,4-dihydro-2H-
pyran (DHP) in the presence of p-toluenesulphonic acid (pTSA)
Fig. 1. Mass spectrum (70 eV) of methyl (Z)-16-methyl-11-heptadecenoate.

N.M. Carballeira et al. / Chemistry and Physics of Lipids 164 (2011) 113–117
117
Fig. 2. Plots of percent (%) of living L. donovani promastigotes (A) and living percent of murine macrophages (B) versus concentrations (␮M) of the fatty acid 1. The calculated
therapeutic index (IC50/IC50) for fatty acid 1 is 3.7.
(ECL value) were compared to the ones reported for the natural
methyl ester of 1 (Rodríguez et al., 2010). The mass spectra and
GC retention times of both synthetic and natural methyl (Z)-16-
methyl-11-heptadecenoates were comparable. For example, in the
mass spectrum of the methyl ester of synthetic 1 both the M+−32
and M+−31 fragmentations peaks at m/z 264 (25%) and at m/z 265
(15%) can be easily seen (Fig. 1). In addition, a strong M+-55 peak
at m/z 241 (11%) as well as the typical McLafferty rearrangement
at m/z 74 (54%) were also observed (Rodríguez et al., 2010). The GC
retention times (ECL values) for both methyl esters (synthetic and
natural 1) were almost identical. For example, the natural methyl
ester of 1 was reported to have an ECL value of 17.46, while the
synthetic methyl ester of 1 was determined to have an ECL value of
17.47. These results clearly demonstrated the cis stereochemistry
of the double bond in the natural fatty acid 1.
biological studies tend to indicate that fatty acids such as 1 might
be good antiprotozoal lead compounds.
Acknowledgements
The project described was supported by Award Number
SC1GM084708 from the National Institutes of General Medical Sci-
ences of the NIH. G. Cintrón thanks the UPR RISE program for an
undergraduate fellowship. We thank Dr. Fred Strobel (Emory Uni-
versity) for the high resolution mass spectral data. This research
was also partially supported by a grant (Gr238) from Junta de
Castilla y León, AGL2009-11935 from MICINN, and the Tropical
Diseases Network (RICET) from Ministerio de Salud y Consumo
(Spain).
Aimed at exploring the biological activity of acid 1 we studied its
antiprotozoal activity towards L. donovani (the causative agent of
visceral leishmaniasis) and the inhibition of acid 1 of the leishmania
LdTopIB as a possible mechanism of action. We found acid 1 to be
cytotoxic to L. donovani at an IC50 of 165.5 23.4 ␮M, and to murine
macrophages at the higher value of IC50 = 604.0 107.9 ␮M (Fig. 2).
Moreover, acid 1 inhibits the LdTopIB with an IC50 = 62.3 0.7 ␮M.
These results tend to indicate that the toxicity of 1 towards L.
donovani could be due to the inhibition of its DNA topoisomerase
IB. This finding is interesting since it has recently been demon-
strated that there are substantial differences between the LdTopIB
and the human DNA topoisomerase I (Balan˜a-Fouce et al., 2006).
In this context one might be able to interfere with the proto-
zoan DNA topoisomerase IB without harming the mammalian
DNA topoisomerase IB. In fact, we have shown that the acid
(Z)-14-methyl-9-pentadecenoic acid, also a monounsaturated iso
methyl-branched fatty acid, inhibits the human DNA topoiso-
merase I but at the higher concentrations of 500 ␮M (Carballeira
et al., 2007). Therefore, the present findings corroborate our previ-
ous results that monounsaturated iso methyl-branched fatty acids
are more effective towards the LdTopIB than the human DNA topoi-
somerase I (Carballeira et al., 2009).
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