Total synthesis and biological evaluation of (5Z,9Z)-5,9-hexadecadienoic acid, an inhibitor of human topoisomerase I

Article abstract of DOI:10.1021/np0202576

The naturally occurring (5Z,9Z)-5,9-hexadecadienoic acid was synthesized stereochemically pure in six steps starting with commercially available 1,5-hexadiyne. The title compound was antimicrobial against the Gram-positive bacteria Staphylococcus aureus (MIC 80 μM) and Streptococcus faecalis (MIC 200 μM), but inactive against Gram-negative bacteria such as Pseudomonas aeruginosa. In addition, the (5Z,9Z)-5,9-hexadecadienoic acid completely inhibits human topoisomerase I at a concentration of 800 μM, while 5,9-hexadecadiynoic acid and hexadecanoic acid do not inhibit topoisomerase I (>1000 μM). This comparison reveals that the cis double bond geometry in the title compound is required for topoisomerase I inhibition. Moreover, these results suggest that the antimicrobial activity of (5Z,9Z)-5,9-hexadecadienoic acid against either S. aureus or S. faecalis could be a result, at least in part, of the inhibitory activity of the acid against topoisomerases.

Full text of DOI:10.1021/np0202576

J . Nat. Prod. 2002, 65, 1715-1718
1715
Tota l Syn th esis a n d Biologica l Eva lu a tion of (5Z,9Z)-5,9-Hexa d eca d ien oic Acid ,
a n In h ibitor of Hu m a n Top oisom er a se I
Ne´stor M. Carballeira,* J ose´ E. Betancourt, Elsie A. Orellano, and Fernando A. Gonza´lez
Department of Chemistry, University of Puerto Rico, P.O. Box 23346, San J uan, Puerto Rico 00931-3346
Received J une 7, 2002
The naturally occurring (5Z,9Z)-5,9-hexadecadienoic acid was synthesized stereochemically pure in six
steps starting with commercially available 1,5-hexadiyne. The title compound was antimicrobial against
the Gram-positive bacteria Staphylococcus aureus (MIC 80 µM) and Streptococcus faecalis (MIC 200 µM),
but inactive against Gram-negative bacteria such as Pseudomonas aeruginosa. In addition, the (5Z,9Z)-
5,9-hexadecadienoic acid completely inhibits human topoisomerase I at a concentration of 800 µM, while
5,9-hexadecadiynoic acid and hexadecanoic acid do not inhibit topoisomerase I (>1000 µM). This
comparison reveals that the cis double bond geometry in the title compound is required for topoisomerase
I inhibition. Moreover, these results suggest that the antimicrobial activity of (5Z,9Z)-5,9-hexadecadienoic
acid against either S. aureus or S. faecalis could be a result, at least in part, of the inhibitory activity of
the acid against topoisomerases.
Fatty acids with the ∆5,9 diunsaturation are an interest-
ing group of lipids with a variety of reported biological
activities. Among these compounds the long-chain ∆5,9 fatty
acids (C27-C30), typical of marine sponges, are known
inhibitors of human topoisomerase I.1 For example, (5Z,9Z)-
5,9-heptacosadienoic acid effectively inhibits human topo-
isomerase I with an IC50 of 0.9 µM.1 Some of these fatty
acids are also cytotoxic inasmuch as a mixture of (5Z,9Z)-
23-methyl-5,9-tetracosadienoic acid and (5Z,9Z)-22-methyl-
5,9-tetracosadienoic acid are cytotoxic against mouse
Ehrlich carcinoma cells with an ED50 of 1.8 µg/mL and
show a hemolytic effect on mouse erythrocytes.2 On the
other hand, the short-chain ∆5,9 fatty acids (C16-C19) are
antimicrobial against Gram-positive bacteria. For example,
the branched fatty acid (5Z,9Z)-14-methyl-5,9-pentadeca-
dienoic acid, first isolated from the Caribbean gorgonian
Eunicea succinea, displays antimicrobial activity against
the Gram-positive bacteria Staphylococcus aureus (MIC
240 µM) and Streptococcus faecalis (MIC 160 µM), but is
inactive against Gram-negative bacteria.3 These findings
clearly demonstrate the biomedical potential of marine ∆5,9
fatty acids. Therefore, in an effort to expand our present
understanding of the bioactivity of ∆5,9 fatty acids we have
undertaken the total synthesis and biological evaluation
of the shortest ∆5,9 fatty acid known, namely, (5Z,9Z)-5,9-
hexadecadienoic acid (1). We were particularly interested
in studying the antimicrobial and topoisomerase I inhibi-
tory activities of 1 so as to gain more insight into the
structural features required for bioactivity, since these
bioactivities have not been explored for 1. A working
hypothesis is that the short-chain ∆5,9 fatty acids are
antimicrobial because they inhibit the bacterial topo-
isomerases of the Gram-positive organisms, in addition to
other possible mechanisms of action, such as DNA gyrase
inhibition or changes in membrane fluidity.4-6
meric 5Z/5E mixture cannot be used for topoisomerase I
inhibition studies since it is known that trans fatty acids,
such as elaidic acid (9t-18:1), do not inhibit topoisomerase
I (IC50 > 1000 µM), while cis fatty acids such as oleic acid
(9c-18:1) do inhibit the enzyme (IC50 31 µM).10 Therefore,
herein we report the first 100% stereoselective synthesis
for (5Z,9Z)-5,9-hexadecadienoic acid (1) together with its
antimicrobial and topoisomerase I inhibitory activities.
The synthesis of (5Z,9Z)-5,9-hexadecadienoic acid (1)
started with commercially available 1,5-hexadiyne (50% in
pentane), which was coupled with 1-bromohexane and
n-BuLi, affording a 3:1 mixture of 1,5-dodecadiyne and the
dialkylated adduct, which were separated by fractional
distillation using a Bu¨chi glass oven (Scheme 1). The
isolated 1,5-dodecadiyne was then alkylated with (4-
bromobutoxy)-tert-butyldimethylsilane (prepared from com-
mercially available 4-bromobutanol) and n-BuLi, resulting
in a 35% isolated yield of 1-(tert-butyldimethylsilyloxy)-5,9-
hexadecadiyne. Deprotection of the silyl group with tet-
rabutylammonium fluoride (TBAF) and oxidation of the
alcohol with pyridinium chlorochromate (PCC) resulted in
a combined 77% yield of 5,9-hexadecadiynal. The aldehyde
was then oxidized with NaClO2 in t-BuOH, affording the
5,9-hexadecadiynoic acid in an 81% isolated yield. Final
hydrogenation over Lindlar’s catalyst yielded the stereo-
chemically pure (5Z,9Z)-5,9-hexadecadienoic acid (1) in an
80% isolated yield. No double-bond isomerization was
observed. The total overall yield in this six-step synthesis
starting from 1,5-hexadiyne was 8%.
The (5Z,9Z)-5,9-hexadecadienoic acid (1) displayed mod-
erate antimicrobial activity against the Gram-positive
bacteria Staphylococcus aureus (MIC 80 µM) and Strepto-
coccus faecalis (MIC 200 µM). This fatty acid was not active
against the Gram-negative bacteria Pseudomonas aerugi-
nosa and Escherichia coli. It is important to mention that
hexadecanoic acid (16:0) showed no activity (MIC > 400
µM) against any of these four microorganisms. Therefore,
the double bonds in 1 are required for the antimicrobial
activity.
(5Z,9Z)-5,9-Hexadecadienoic acid (1) is a rare fatty acid
first isolated from the cellular slime mold Dictyostelium
discoideum,7 but later identified in a few marine sponges
such as Chondrilla nucula.8 There is only one four-step
synthesis reported for 1, but it yielded a 10:1 mixture of
(5Z,9Z)- and (5E,9Z)-5,9-hexadecadienoic acids.9 This iso-
In an effort to broaden the scope of the biological
potential of the parent (5Z,9Z)-5,9-hexadecadienoic acid (1),
we studied its inhibitory activity against human topo-
isomerase I (from freshly extracted human placenta). We
* To whom correspondence should be addressed. Tel: (787)-764-0000,
ext. 4791. Fax: (787)-756-8242. E-mail: ncarball@upracd.upr.clu.edu.
10.1021/np0202576 CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/21/2002

1716 J ournal of Natural Products, 2002, Vol. 65, No. 11
Notes
Sch em e 1a
a
(i) n-BuLi, 1-bromohexane, THF, -78 °C; (ii) n-BuLi, (4-bromobutoxy)-tert-butyldimethylsilane, THF-HMPA, -78 °C; (iii) TBAF, THF, rt; (iv) PCC,
CH2Cl2, rt; (v) NaClO2, t-BuOH, 48 h, rt; (vi) H2, Lindlar.
where the fatty acids bind. Such a similarity has been
established for other systems. For example, it is also known
that cis-unsaturated fatty acids (C18 or higher) inhibit
mammalian DNA polymerase â and human DNA topo-
isomerase II.11 In fact, computer modeling analysis has
revealed that the amino acids Thr, His, Leu, and Lys form
a pocket, in both enzymes, where the fatty acid molecule
binds.11 Such a model could also be operative in other
topoisomerases such as I and IV. It is possible that these
5,9
∆
fatty acids may also have practical applications in the
synthesis of novel lipidic prodrugs aimed at antibiotic-
resistant S. aureus.12
Exp er im en ta l Section
F igu r e 1. Agarose gel stained with ethidium bromide showing the
inhibitory effect of (5Z,9Z)-5,9-hexadecadienoic acid at 800 and 1000
µM. OC DNA stands for open circular DNA and SC DNA for
supercoiled DNA.
Gen er a l Exp er im en ta l P r oced u r es. IR spectra were
recorded on a Nicolet 600 FT-IR spectrophotometer. 1H and
13C NMR were recorded on a General Electric QE-300 or
Bruker DPX-300 spectrometer. 1H NMR chemical shifts are
reported with respect to internal Me4Si, and 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 ChemStation equipped with a 30
m × 0.25 mm special performance capillary column (HP-5MS)
of polymethyl siloxane cross-linked with 5% phenylmethyl-
polysiloxane.
found that acid 1 completely inhibits topoisomerase I at a
concentration of 800 µM (Figure 1). However, the synthetic
precursor 5,9-hexadecadiynoic acid did not show any
inhibition of topoisomerase I (>1000 µM). This comparison
confirms that the cis double bond geometries in 1 are
essential for effective topoisomerase I inhibition. It is also
important to mention that hexadecanoic acid does not
inhibit topoisomerase I, even at concentrations as high as
1,5-Dod eca d iyn e. Into a 100 mL round-bottom flask,
equipped with a magnetic stirrer, was placed 1.0 g (12.8 mmol)
of 1,5-hexadiyne (50% in pentane, AlfaAesar) and 75 mL of
dry THF under a nitrogen atmosphere. The temperature was
then lowered to -78 °C, and n-butyllithium (2.5 M, 4.6 mL,
11.5 mmol) in hexane was slowly added. After 45 min 1-bro-
mohexane (1.8 mL, 12.8 mmol) was added in 5 mL of HMPA.
Stirring was continued for 24 h, and the reaction mixture was
finally quenched with ice water. The organic products were
extracted with diethyl ether (3 × 15 mL), and the organic layer
was dried over Na2SO4. The solvent was removed in vacuo,
affording a 3:1 mixture of the monoalkylated versus the
dialkylated adducts. The 1,5-dodecadiyne (0.89 g, 43% yield)
was obtained pure by fractional distillation: IR (neat) νmax
3313, 2956, 2932, 2851, 2204, 1460, 1379, 1259, 1078, 797
2000 µM.10 However, long-chain ∆5,9 fatty acids (C27-C28
)
are more effective topoisomerase I inhibitors (IC50’s 1-3
µM) than 1, implying that carbon chain length also plays
an important role in topoisomerase I inhibition.1
In summary, we have developed the first 100% stereo-
selective synthesis for (5Z,9Z)-5,9-hexadecadienoic acid (1),
utilizing as the key step the selective double alkylation of
1,5-hexadiyne. We have shown that 1 is moderately
antimicrobial against Gram-positive bacteria, such as S.
aureus, but the double bonds in 1 are required for the
activity. In addition, 1 inhibits human topoisomerase I at
high concentrations, whereas the corresponding 5,9-di-
alkynoic acid does not. This indicates that the cis-unsat-
urations in 1 are required for topoisomerase I inhibition.
In addition, hexadecanoic acid is inactive in both anti-
microbial and topoisomerase I bioassays, thus confirming
that a specific structural geometry is needed for the
bioactivity. Moreover, when compared with the recent
literature, carbon chain length also plays an important role
in topoisomerase I inhibition, inasmuch as fatty acids with
long carbon chains (C27-C30) are more inhibitory than those
with short chains (C16).1
cm-1 1H NMR (CDCl3, 300 MHz) δ 2.35 (4H, m, H-3, H-4),
;
2.12 (2H, brt, J ) 6.9 Hz, H-7), 1.98 (1H, brt, J ) 2.4 Hz, H-1),
1.47-1.23 (8H, m, H-8, H-9, H-10, H-11), 0.86 (3H, t, J ) 7.0
Hz, H-12); 13C NMR (CDCl3, 75 MHz) δ 83.0 (s, C-2), 81.5 (s,
C-5), 78.0 (s, C-6), 68.9 (d, C-1), 31.3 (t, C-10), 28.9 (t), 28.4
(t), 22.5 (t), 19.1 (t), 18.8 (t), 18.6 (t), 14.0 (q, C-12); GC-MS
(70 eV) m/z 162 [M+] (0.1), 147 (1), 133 (6), 123 (6), 120 (2),
119 (11), 106 (10), 105 (29), 103 (5), 95 (5), 92 (25), 91 (100),
81 (26), 79 (26), 77 (18), 69 (7), 67 (26), 65 (17), 55 (17), 53
(14).
1-(ter t-Bu tyld im eth ylsilyloxy)-5,9-h exa d eca d iyn e. Into
a 50 mL round-bottom flask, equipped with a magnetic stirrer,
was placed 0.5 g (3.1 mmol) of 1,5-dodecadiyne in 25 mL of
tetrahydrofuran under a nitrogen atmosphere. The tempera-
ture was then lowered to -78 °C, and n-butyllithium (2.5 M,
1.2 mL, 3.1 mmol) in hexane was slowly added. After 45 min
(4-bromobutoxy)-tert-butyldimethylsilane (0.82 g, 3.1 mmol)
was added in 5 mL of HMPA. Stirring was continued for 24 h,
and the reaction mixture was finally quenched with ice water.
Before a precise correlation between antimicrobial activ-
ity and topoisomerase inhibition can be established, the
inhibitory effect of 1 against the topoisomerase IV from S.
aureus should be determined.4-6 Topoisomerase IV appears
to be the preferential antibacterial target for the quinolones
in Gram-positive organisms.4 It is possible that human
topoisomerase I and bacterial topoisomerase IV might
share a similar structural homology for a binding pocket

Notes
J ournal of Natural Products, 2002, Vol. 65, No. 11 1717
The organic products were extracted with diethyl ether (3 ×
15 mL), and the organic layer was dried over Na2SO4. The
solvent was removed in vacuo, and the crude product was
purified by fractional distillation, affording 0.38 g of the 1-(tert-
butyldimethylsilyloxy)-5,9-hexadecadiyne for a 35% yield. The
product was used for the next step without further purifica-
tion: 1H NMR (CDCl3, 300 MHz) δ 3.62 (2H, t, J ) 6.9 Hz,
H-1), 2.31 (4H, m, H-7, H-8), 2.15 (4H, m, H-4, H-11), 1.62-
1.26 (12H, m, H-2, H-3, H-12, H-13, H-14, H-15), 0.85 (12H,
brs, -Si-C(CH3)3 and -CH3), 0.03 (6H, s, Si-CH3); 13C NMR
(CDCl3, 75 MHz) δ 81.5 (s), 81.1 (s), 78.9 (s), 78.6 (s), 62.7 (t,
C-1), 31.8 (t, C-2), 31.3 (t, C-14), 28.9 (t), 28.5 (t), 25.9 (t), 25.3
(q, Si-C(CH3)3), 19.4 (t), 18.7 (t), 18.5 (t), 18.3 (t), 14.0 (q, C-16),
-5.4 (q, Si-CH3); GC-MS (70 eV) m/z 349 [M+] (0.1), 291 (6),
217 (3), 215 (7), 207 (1), 193 (3), 187 (2), 173 (2), 171 (2), 161
(2), 155 (2), 145 (11), 133 (5), 131 (19), 119 (19), 117 (13), 105
(8), 101 (6), 97 (6), 91 (17), 85 (3), 81 (6), 79 (9), 77 (9), 75
(100), 59 (10), 55 (10).
5,9-Hexa d eca d iyn -1-ol. Into a 35 mL round-bottom flask,
equipped with a magnetic stirrer, was placed 0.38 g (1.08
mmol) of 1-(tert-butyldimethylsilyloxy)-5,9-hexadecadiyne in
20 mL of THF. To this solution was added dropwise 1.1 mL
(1.08 mmol) of tetrabutylammonium fluoride (1 M) in THF.
After 24 h the reaction mixture was poured into H2O and
extracted with diethyl ether (3 × 15 mL). The organic extracts
were washed with brine, dried over Na2SO4, and concentrated
in vacuo, affording 0.2 g of 5,9-hexadecadiyn-1-ol for a 78%
yield. The product was used for the next step without further
purification: 1H NMR (CDCl3, 300 MHz) δ 3.62 (2H, t, J )
6.3 Hz, H-1), 2.30 (4H, m, H-7, H-8), 2.14 (4H, m, H-4, H-11),
1.93 (1H, brs, OH), 1.65-1.26 (12H, m, H-2, H-3, H-12, H-13,
H-14, H-15), 0.87 (3H, t, J ) 7.1 Hz, -CH3); 13C NMR (CDCl3,
75 MHz) δ 81.2 (s), 80.6 (s), 79.2 (s), 78.6 (s), 62.3 (t, C-1),
31.7 (t, C-2), 31.3 (t, C-14), 28.9 (t), 28.4 (t), 25.6 (t), 25.1 (t),
22.5 (t), 19.4 (t), 18.6 (t), 18.4 (t), 14.0 (q, C-16); GC-MS (70
eV) m/z 234 [M+] (0.1), 205 (4), 203 (3), 191 (3), 189 (7), 175
(11), 163 (17), 149 (27), 145 (15), 137 (8), 135 (12), 133 (16),
131 (29), 121 (15), 119 (50), 117 (49), 115 (17), 110 (20), 107
(20), 105 (64), 103 (11), 95 (15), 93 (40), 91 (100), 81 (33), 79
(62), 77 (51), 67 (47), 65 (27), 57 (14), 55 (51).
5,9-Hexa d eca d iyn a l. To a stirred solution of 5,9-hexadec-
adiyn-1-ol (0.2 g, 0.8 mmol) in 15 mL of dichloromethane was
slowly added pyridinium chlorochromate (0.2 g, 0.8 mmol) at
room temperature. After 24 h the reaction mixture was filtered
through Florisil and washed with diethyl ether (50 mL). After
evaporation of the solvent 0.2 g of 5,9-hexadecadiynal was
obtained for a 99% yield. The product was used for the next
step without further purification: 1H NMR (CDCl3, 300 MHz)
δ 9.78 (1H, t, J ) 1.2 Hz, H-1), 2.57 (2H, dt, J 2,1 ) 1.2 Hz and
J 2,3 ) 7.2 Hz, H-2), 2.30 (4H, m, H-7, H-8), 2.21 (2H, brt, J )
6.8 Hz, H-4), 2.11 (2H, brt, J ) 7.0 Hz, H-11), 1.79 (2H, q, J
) 6.9 Hz, H-3), 1.52-1.24 (8H, m, H-12, H-13, H-14, H-15),
0.86 (3H, t, J ) 7.1 Hz, -CH3); 13C NMR (CDCl3, 75 MHz) δ
202.1 (s, C-1), 81.2 (s, C-6), 80.1 (s, C-9), 79.6 (s, C-5), 78.5 (s,
C-10), 42.7 (t, C-2), 31.3 (t, C-14), 28.9 (t), 28.5 (t), 22.5 (t),
21.3 (t), 19.35 (t), 19.32 (t), 18.7 (t), 18.1 (t), 14.0 (q, C-16);
GC-MS (70 eV) m/z 231 [M+ - 1] (1), 217 (1), 204 (3), 203 (6),
189 (7), 175 (20), 162 (9), 161 (28), 149 (5), 147 (24), 143 (9),
136 (5), 133 (31), 131 (21), 129 (18), 119 (45), 117 (51), 115
(20), 105 (63), 103 (12), 95 (10), 93 (23), 91 (100), 81 (38), 79
(62), 77 (37), 69 (10), 67 (40), 65 (33), 55 (51).
2.24 (2H, brt, J ) 6.9 Hz, H-4), 2.13 (2H, brt, J ) 6.9 Hz, H-11),
1.80 (2H, q, J ) 7.0 Hz, H-3), 1.48-1.27 (8H, m, H-12, H-13,
H-14, H-15), 0.88 (3H, t, J ) 7.0 Hz, -CH3); 13C NMR (CDCl3,
75 MHz) δ 179.5 (s, C-1), 81.3 (s, C-6), 80.0 (s, C-9), 79.5 (s,
C-5), 78.6 (s, C-10), 32.7 (t, C-2), 31.3 (t, C-14), 28.9 (t), 28.5
(t), 23.7 (t), 22.5 (t), 19.40 (t), 19.37 (t), 18.7 (t), 18.1 (t), 14.0
(q, C-16); GC-MS (70 eV) m/z 248 [M+] (0.1), 219 (4), 213 (5),
205 (8), 191 (13), 187 (4), 177 (17), 175 (21), 161 (11), 159 (11),
145 (24), 135 (13), 133 (29), 131 (58), 129 (12), 124 (12), 119
(64), 117 (100), 115 (22), 105 (93), 97 (10), 95 (16), 91 (98), 81
(41), 79 (70), 77 (53), 69 (21), 67 (49), 65 (32), 57 (13), 55 (69).
(5Z,9Z)-5,9-Hexa d eca d ien oic Acid . Into a 15 mL round-
bottom flask, equipped with a magnetic stirrer and 10 mL of
dry hexane, were placed 0.015 g (0.04 mmol) of 5,9-hexadec-
adiynoic acid, 0.2 equiv of quinoline, and 0.010 g of Lindlar’s
catalyst. After a couple of purging cycles, hydrogen was added
until a volume equivalent to the initial amount of alkynes was
consumed. After filtration and removal of the solvent in vacuo,
0.012 g (80% yield) of the previously reported (5Z,9Z)-5,9-
hexadecadienoic acid was obtained.9
An tiba cter ia l Activity. Antibacterial activity against P.
aeruginosa (ATCC 27853), E. coli (ATCC 25922), S. aureus
(ATCC 25923), and S. faecalis - group D (ATCC 29212) was
determined following National Committee of Clinical Labora-
tory Standards (NCCLS).15 A 200 µL solution of the hexadeca-
dienoic acid in Mueller-Hinton broth was inoculated with 105
colony-forming units in a 96-well plate. The minimal inhibitory
concentration (MIC) was determined after an overnight incu-
bation of the fatty acid and the microorganisms at 37 °C. The
MIC was determined by observing the highest dilution of the
fatty acid that inhibited growth when compared to an un-
inoculated chemical-control well. The generated data were
taken from at least three separate experiments in duplicate.
DNA Top oisom er a se I Assa y. The enzyme activity of
topoisomerase I was assessed with a Topoisomerase I Drug
Screening Kit (TopoGEN, Inc., Columbus, OH) using topo-
isomerase I from human placenta (1 unit relaxes 0.5 µg of DNA
in 15 min at 37 °C) and 0.25 µg of supercoiled pHOT1 plasmid
DNA. Fatty acids were dissolved in DMSO and were tested at
a final concentration of 1000, 800, 500, 100, and 10 µM.
Reactions (final volume 20 µL) were carried out for 30 min at
37 °C, after which 2 µL of 10% sodium dodecyl sulfate (SDS)
was added. Bound protein was digested by incubation with
proteinase K (final concentration 0.05 mg/mL) for 30 min at
37 °C. Reactions were stopped by adding 5 µL of electrophore-
sis loading buffer (0.25% bromophenol blue, 50% glycerol) and
then were electrophoresed in a 1% agarose gel (70 V/105 min).
To visualize the reaction products, the gel was stained with
0.5 µg/mL ethidium bromide for 45 min and destained for 30
min in distilled water. DNA bands were detected and quan-
titated in a Versa Doc imaging system (model 1000, Bio Rad).
Ack n ow led gm en t. This work was supported by a grant
from the National Institutes of Health under the SCORE
program (grant no. S06GM08102) and the RCMI program (G12
RR03641). We thank N. Herna´ndez-Alonso and J . L. Rodr´ıguez
(UPR-R´ıo Piedras) for technical assistance with the antimi-
crobial bioassays. We also thank C. Cruz (Faculty of General
Studies-UPR-R´ıo Piedras) for helpful discussions regarding the
dialkynoic fatty acids.
5,9-Hexa d eca d iyn oic Acid . To a stirred solution of 5,9-
hexadecadiynal (0.2 g, 0.8 mmol) in t-BuOH (20 mL) was added
a solution of NaClO2 (2.5 mmol) and NaHPO4 (3 mmol) in 3
mL of water over a period of 10 min. The pale yellow solution
was stirred at room temperature for 48 h. The mixture was
diluted with water and extracted with diethyl ether (3 × 15
mL). The combined extracts were washed with a saturated
brine solution, dried over Na2SO4, and concentrated. The
resulting product was purified by column chromatography on
silica gel using hexane-EtOAc (3:1), which afforded 0.17 g of
5,9-hexadecadiynoic acid for an 81% yield: IR (neat) νmax 2957,
2929, 2855, 2217, 1687, 1458, 1438, 1411, 1339, 1262, 1207,
Refer en ces a n d Notes
(1) Nemoto, T.; Yoshimo, G.; Ojika, M.; Sakagami, Y. Tetrahedron 1997,
53, 16699-16710.
(2) Makarieva, T. N.; Santalova, E. A.; Gorshkova, I. A.; Dmitrenok, A.
S.; Guzii, A. G.; Gorbach, V. I.; Svetashev, V. I.; Stonik, V. A. Lipids
2002, 37, 75-80.
(3) Carballeira, N. M.; Reyes, E. D.; Sostre, A.; Rodr´ıguez, A. D.;
Rodr´ıguez, J . L.; Gonza´lez, F. A. J . Nat. Prod. 1997, 60, 502-504.
(4) Takei, M.; Fukuda, H.; Kishii, R.; Hosaka, M. Antimicrob. Agents
Chemother. 2001, 45, 3544-3547.
(5) Hooper, D. C. Clin. Infect. Dis. 1998, 27, S54-63.
(6) Bearden, D. T.; Danzinger, L. H. Pharmacotheraphy 2001, 21, 224S-
232S.
1155, 1060, 1028, 918, 746, 729 cm-1
;
1H NMR (CDCl3, 300
MHz) δ 2.50 (2H, t, J ) 7.4 Hz, H-2), 2.32 (4H, m, H-7, H-8),

1718 J ournal of Natural Products, 2002, Vol. 65, No. 11
Notes
(7) Davidoff, F.; Korn, E. D. Biochem. Biophys. Res. Commun. 1962, 9,
54-58.
(11) Mizushino, Y.; Sugawara, F.; Lida, A,; Sakaguchi, K. J . Mol. Biol.
2000, 304, 385-395.
(12) Yamakawa, T.; Mitsuyama, J .; Hayashi, K. J . Antimicrob. Chemother.
2002, 49, 455-465.
(8) Carballeira, N. M.; Maldonado, L. Lipids 1986, 21, 470-471.
(9) Carballeira, N. M.; Emiliano, A.; Guzma´n, A. Chem. Phys. Lipids
1999, 100, 33-40.
(10) Suzuki, K.; Shono, F.; Kai, H.; Uno, T.; Uyeda, M. J . Enzymol. Inhib.
2000, 15, 357-366.
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