2-Methoxylated FA Display Unusual Antibacterial Activity Towards Clinical Isolates of Methicillin-Resistant Staphylococcus aureus (CIMRSA) and Escherichia coli

Article abstract of DOI:10.1007/s11745-017-4262-1

The naturally occurring (6Z)-(±)-2-methoxy-6-hexadecenoic acid (1) and (6Z)-(±)-2-methoxy-6-octadecenoic acid (2) were synthesized in 7–8 steps with 38 and 13% overall yields, respectively, by using an acetylide coupling approach, which made it possible to obtain a 100% cis-stereochemistry for the double bonds. In a similar fashion, the acetylenic analogs (±)-2-methoxy-6-hexadecynoic acid (3) and (±)-2-methoxy-6-octadecynoic acid (4) were also synthesized in 6–7 steps with 48 and 16% overall yields, respectively. The antibacterial activity of acids 1–4 was determined against clinical isolates of methicillin-resistant Staphylococcus aureus (ClMRSA) and Escherichia coli. Among the series of compounds, acid 4 was the most active bactericide towards CIMRSA displaying IC_50s (half maximal inhibitory concentrations) between 17 and 37?μg/mL, in sharp contrast to the 6-octadecynoic acid, which was not bactericidal at all. On the other hand, acids 1 and 3 were the only acids that displayed antibacterial activity towards E. coli., but 1 stood out as the best candidate with an IC₅₀ of 21?μg/mL. The critical micelle concentrations (CMCs) of acids 1–4 were also determined. The C18 acids 2 and 4 displayed a five-fold lower CMC (15–20?μg/mL) than the C16 analogs 1 and 3 (70–100?μg/mL), indicating that 4 exerts its antibacterial activity in a micellar state. None of the studied acids were inhibitory towards S. aureus DNA gyrase discounting this type of enzyme inhibition as a possible antibacterial mechanism. It was concluded that the combination of α-methoxylation and C-6 unsaturation increases the bactericidal activity of the C16 and C18 FA towards the studied bacterial strains. Acids 1 and 4 stand out as viable candidates to be used against E. coli and CIMRSA, respectively.

Full text of DOI:10.1007/s11745-017-4262-1

Lipids
DOI 10.1007/s11745-017-4262-1
ORIGINAL ARTICLE
2‑Methoxylated FA Display Unusual Antibacterial Activity
Towards Clinical Isolates of Methicillin‑Resistant Staphylococcus
aureus (CIMRSA) and Escherichia coli
Néstor M. Carballeira1 · Nashbly Montano1 · Christian Morales1 · Joseph Mooney2 ·
Xiomara Torres2 · Dakeishla Díaz2 · David J. Sanabria‑Rios2
Received: 28 March 2017 / Accepted: 30 April 2017
© AOCS 2017
Abstract The naturally occurring (6Z)-( )-2-methoxy-
6-hexadecenoic acid (1) and (6Z)-( )-2-methoxy-6-
octadecenoic acid (2) were synthesized in 7–8 steps with
38 and 13% overall yields, respectively, by using an
acetylide coupling approach, which made it possible to
obtain a 100% cis-stereochemistry for the double bonds.
In a similar fashion, the acetylenic analogs ( )-2-meth-
oxy-6-hexadecynoic acid (3) and ( )-2-methoxy-6-oc-
tadecynoic acid (4) were also synthesized in 6–7 steps
with 48 and 16% overall yields, respectively. The anti-
bacterial activity of acids 1–4 was determined against
clinical isolates of methicillin-resistant Staphylococcus
aureus (ClMRSA) and Escherichia coli. Among the series
of compounds, acid 4 was the most active bactericide
towards CIMRSA displaying IC50s (half maximal inhibi-
tory concentrations) between 17 and 37 μg/mL, in sharp
contrast to the 6-octadecynoic acid, which was not bac-
tericidal at all. On the other hand, acids 1 and 3 were the
only acids that displayed antibacterial activity towards E.
coli, but 1 stood out as the best candidate with an IC50
of 21 μg/mL. The critical micelle concentrations (CMCs)
of acids 1–4 were also determined. The C18 acids 2 and
4 displayed a fve-fold lower CMC (15–20 μg/mL) than
the C16 analogs 1 and 3 (70–100 μg/mL), indicating that
4 exerts its antibacterial activity in a micellar state. None
of the studied acids were inhibitory towards S. aureus
DNA gyrase discounting this type of enzyme inhibition
as a possible antibacterial mechanism. It was concluded
that the combination of α-methoxylation and C-6 unsatu-
ration increases the bactericidal activity of the C16 and
C18 FA towards the studied bacterial strains. Acids 1 and
4 stand out as viable candidates to be used against E. coli
and CIMRSA, respectively.
Keywords Antibacterial · Escherichia coli · Methicillin-
resistant · Methoxylated fatty acids · Staphylococcus
aureus · Synthesis
Abbreviations
ClMRSA
Clinical isolates of methicillin-resistant
Staphylococcus aureus
CMC
DCM
DMI
DMSO
FA
Critical micelle concentration
Dichloromethane
1,3-Dimethyl-2-imidazolidinone
Dimethyl sulfoxide
Fatty acid(s)
GC/MS
IC50
MIC
Gas chromatography-mass spectrometry
Half maximal inhibitory concentration
Minimum inhibitory concentration
Pyridinium chlorochromate
p-Toluenesulfonic acid
Relaxed deoxyribonucleic acid
Standard error of the mean
Super coiled deoxyribonucleic acid
Trypticase Soy Broth
PCC
PTSA
Rel DNA
SEM
SC DNA
TSI
* Néstor M. Carballeira
nestor.carballeira1@upr.edu
1
Department of Chemistry, University of Puerto Rico, Rio
Piedras Campus, PO Box 23346, 00931-3346 San Juan, PR,
USA
THF
Tetrahydrofuran
TMSCN
Trimethylsilyl cyanide
2
Faculty of Science and Technology, Inter American
University of Puerto Rico, Metropolitan Campus, PO
Box 191293, 00919 San Juan, PR, USA
UPLC-MS Ultra high performance liquid chromatogra-
phy-mass spectrometry
1 3

Lipids
spectrometer. The samples were diluted in 99.8% chlo-
roform-d (CDCl3) and the solvent signals at 7.26 (1H)
and 77.0 (13C) ppm were used as internal standards for
hydrogen and carbon, respectively. Mass spectral data
were acquired on a GC–MS (Agilent 5977E MS Chem-
Station) equipped with a 30 × 0.25 mm (flm 0.25 μm)
special performance capillary column (HP-5MS) of poly-
methylsiloxane cross-linked with 5% phenyl methylpoly-
siloxane [4]. IR spectra were measured neat on a Bruker
Tensor 27 FT-IR spectrometer. High-resolution mass
spectral data were acquired using a quadrupole time-of-
fight mass spectrometer (Q-TOF, Synapt, G2-S, Waters)
with electrospray ionization in either negative or positive
ion mode as previously described [4].
Introduction
Despite the fact that the antibacterial activity of FA (fatty
acids) has been amply studied [1], the bactericidal activ-
ity of the less ubiquitous α-methoxylated FA has been less
documented in the literature. The α-methoxylated FA are a
group of mainly marine FA, which have been isolated from
many different species of Caribbean sponges [2]. These FA
can range between 14 and 28 carbon atoms and can possess
either the iso/anteiso methyl branching and/or double bond
unsaturations at C-6 in the shorter chain analogs [2] or at C-5
and C-9 in the longer chain analogs, among other variants [3,
4]. The stereochemistry at the chiral center of the naturally
occurring acids has been established as R [2].
The little research reported so far tends to indicate that
the α-methoxy functionality imparts to a FA biophysical
properties that increases their toxicity towards leukemia
and neuroblastoma cell lines [5, 6], fungitoxicity towards C.
albicans and C. neoformans [7, 8], and bactericidal activity
towards Gram-positive bacteria (Staphylococcus aureus and
S. faecalis) as well as toxicity towards mycobacteria (Myco‑
bacterium tuberculosis H37Rv) [9, 10]. The reasons behind
these rather interesting differences seems to be speculative
at this point, which could range from changes in the lipophi-
licity (logP) and pH of the acids to different rates of micelle
formation. In order to get more insight into the properties
behind the antibacterial potential of these rather peculiar
compounds, we explored the antibacterial profle of the two
naturally occurring (6Z)-( )-2-methoxy-6-hexadecenoic
acid (1) and (6Z)-( )-2-methoxy-6-octadecenoic acid
(2) together with their corresponding acetylenic analogs
( )-2-methoxy-6-hexadecynoic acid (3) and ( )-2-meth-
oxy-6-octadecynoic acid (4). In this study we chose pri-
marily antibacterial profles against clinical isolates of
methicillin-resistant S. aureus (ClMRSA). Compounds 1
and 2 are ideal to be compared to the corresponding non
methoxylated analogs since it has been well established
in the literature that 16:1 isomers, in particular the 6- and
9-hexadecenoic acids, are toxic towards S. aureus while the
18:1 isomers, such as the 6- and 9-octadecenoic acids, are
relatively nontoxic to some S. aureus strains [11]. In this
work we also present the frst total synthesis for 2, a natural
product previously identifed by us in the Caribbean sponge
Spheciospongia cuspidifera [12] as well as a third genera-
tion synthesis for 1, initially isolated from the same sponge.
Synthesis
5‑Heptadecyn‑1‑ol (6)
To a stirred solution of 0.34 g (1.9 mmol) of 1-tridecyne
(5) in 5.0 mL of dry THF was added 0.70 mL (7.6 mmol)
of nBuLi (2.5 M) in hexane at 0 °C under an argon atmos-
phere. The mixture was stirred at 0 °C for 45 min, and
then 1.70 mL (10.3 mmol) of HMPA (Caution! HMPA
is toxic and safety precautions should be taken handling
this solvent) was added to the reaction mixture followed
by the addition of 0.90 g (3.8 mmol) of 2-(4-bromobu-
toxy)tetrahydro-2H-pyran and the solution was stirred for
24 h. After that time, the reaction mixture was quenched
with water, and the organic crude was washed with a
brine solution (2 × 10 mL), diethyl ether (2 × 10 mL),
dried over MgSO4, fltered and the solvent evaporated in
vacuo. Then, to the extracted product was added 20 mL
of methanol followed by catalytic amounts (0.1 M) of
PTSA and stirred for 12 h at 35 °C. The reaction mix-
ture was then neutralized with a sodium bicarbonate
solution (2 × 15 mL) and extracted with diethyl ether
(2 × 15 mL), dried over MgSO4, fltered, and evaporated
in vacuo. The crude product was purifed using silica gel
column chromatography while eluting with hexane/ether
(9:1). The heptadec-5-yn-1-ol (6) [13] was obtained as
a colorless oil 0.34 g (1.3 mmol) for a combined 71%
yield for the two steps. IR (NaCl) νmax: 3355 (O–H),
2928, 2852, 1462, 1377, 1330, 1065, 717 cm−1; 1H NMR
(CDCl3, 300 MHz) δ (ppm) 3.67 (2H, t, J = 6.3 Hz, H
1), 2.18-2.11 (4H, m), 1.67 (2H, m, H-2), 1.56–1.48 (4H,
m), 1.27 (16H, br s, –CH2–), 0.87 (3H, t, J = 6.7 Hz, –
CH3); 13C-NMR (CDCl3, 75 MHz) δ (ppm) 80.7 (s), 79.7
(s), 62.5 (t, C-1), 31.9 (t), 31.8 (t), 29.7 (t), 29.6 (t), 29.5
(t), 29.3 (t), 29.2 (t), 29.1 (t), 28.9 (t), 25.3 (t, C-4), 22.7
(t), 18.7 (t), 18.5 (t), 14.1 (q, C-17); GC/MS (70 eV) m/z
(relative intensity): 252 (M+, 1), 234 (M+–H2O, 1), 231
Materials and Methods
Instrumentation
1
All compounds were analyzed by H NMR (300 MHz)
and 13C NMR (75 MHz) using a Bruker DPX-300
1 3

Lipids
(1), 217 (4), 215 (4), 203 (2), 189 (1), 179 (3), 173 (1),
165 (1), 161 (1), 151 (2), 149 (2), 139 (1), 137 (9), 135
(5), 123 (11), 121 (5), 111 (4), 109 (25), 107 (8), 105 (4),
97 (16), 96 (16), 95 (78), 93 (22), 91 (21), 83 (24), 81
(91), 79 (48), 77 (26), 71 (5), 69 (24), 67 (100), 65 (17),
57 (26), 55 (62).
(relative intensity): 349 (M+, 1), 334 (M+-CH3, 4), 264 (1),
259 (1), 234 (1), 223 (2), 209 (9), 208 (12), 194 (9), 181
(3), 174 (3), 168 (7), 157 (2), 155 (7), 152 (3), 146 (5), 135
(4), 133 (10), 129 (11), 121 (7), 119 (32), 118 (13), 116
(18), 109 (8), 105 (10), 101 (11), 95 (20), 93 (20), 92 (43),
91 (26), 84 (27), 81 (24), 79 (41), 75 (61), 73 (C3H9Si+,
100), 69 (14), 67 (36), 65 (10), 59 (15), 57 (28), 55 (47).
5‑Heptadecynal (7)
Methyl ( )‑2‑Hydroxy‑6‑Octadecynoate (9)
To a stirred solution of 0.40 g (1.86 mmol) of pyridin-
ium chlorochromate (PCC) and 20.0 mL of dry DCM
was added 0.31 g (1.24 mmol) of 6 at rt under an argon
atmosphere and the reaction was left stirring for 24 h. The
product was obtained by fuorisil column chromatography
purifcation eluting with diethyl ether. The 5-heptadecynal
(7) was obtained as colorless oil (0.26 g, 1.02 mmol) for
an 82% yield. IR (NaCl) νmax: 2958, 2931, 2852 (CHO),
To a solution of 0.29 g (0.84 mmol) of 8 and 12.3 mL
(152 mmol) of 2-methyltetrahydrofuran (2-MeTHF)
was added 4.9 mL (159 mmol) of concentrated HCl and
refuxed for 24 h. Then, the solvent was removed in vacuo
and the crude product was washed with ether (2 × 10 mL),
a brine solution (2 × 10 mL), and concentrated. The methyl
ester was obtained by esterifcation of the crude product
with HCl/MeOH while refuxing for 3 h. The ester was
purifed using silica gel column chromatography eluting
with hexane/ether (7:3), affording 9 as colorless oil 0.11 g
1
2213, 1718 (C=O), 1674, 1462, 722, 634 cm−1; H NMR
(CDCl3, 300 MHz) δ (ppm) 9.78 (1H, t, J = 1.2 Hz, –
CHO), 2.56 (2H, dt, J = 7.3, 1.2 Hz, H-2), 2.22–2.12
(4H, m), 1.81 (2H, m), 1.45 (2H, m), 1.26 (16H, br s, –
CH2–), 0.87 (3H, t, J = 6.7 Hz, –CH3); 13C NMR (CDCl3,
75 MHz) δ (ppm) 202.2 (d, CHO), 81.6 (s), 78.6 (s), 42.8
(t, C-2), 31.9 (t), 29.7 (t), 29.6 (t), 29.5 (t), 29.3 (t), 29.1 (t),
29.0 (t), 28.9 (t), 22.7 (t), 21.5 (t), 18.7 (t), 18.2 (t), 14.1
(q, C-17); GC/MS (70 eV) m/z (relative intensity): 250
(M+, 1), 209 (1), 191 (1), 175 (1), 165 (1), 151 (6), 147 (3),
137 (10), 133 (7), 123 (14), 119 (16), 111 (16), 110 (29),
109 (20), 107 (12), 106 (10), 105 (11), 98 (6), 97 (15), 96
(16), 95 (50), 93 (31), 92 (47), 91 (36), 83 (40), 82 (68), 81
(52), 79 (77), 77 (29), 69 (26), 67 (85), 65 (24), 57 (28), 55
(100).
1
(0.36 mmol) for a 44% yield for the two steps. H NMR
(CDCl3, 300 MHz) δ (ppm); 4.20 (1H, m, H-2), 3.78 (3H,
s –OCH3), 2.77 (1H, d, J = 5.3 Hz, –OH), 2.19–2.12 (4H,
m), 1.88 (2H, m), 1.64 (2H, m), 1.45 (2H, m), 1.26 (16H,
brs, –CH2–), 0.87 (3H, t, J = 6.7 Hz, –CH3); 13C NMR
(CDCl3, 75 MHz) δ (ppm) 175.6 (s, C-1), 80.9 (s), 79.2
(s), 70.1 (d, C-2), 52.5 (q, –OCH3), 33.5 (t), 31.9 (t, C-3),
29.6 (t), 29.0 (t), 29.5 (t), 29.3 (t), 29.2 (t), 29.1 (t), 28.9 (t),
24.4 (t), 22.7 (t, C-4), 18.7 (t), 18.5 (t), 14.1 (q, –CH3); GC/
MS (70 eV) m/z (relative intensity): 310 (M+, 6), 251 (M+–
C2H3O2, 6), 233 (M+–C2H3O2–H2O, 6), 221 (4), 197 (4),
169 (C9H13O3+, 19), 156 (40), 151 (21), 135 (20), 128 (12),
123 (20), 121 (18), 111 (14), 109 (28), 105 (12), 103 (28),
97 (31), 95 (33), 90 (C3H6O3+, 65), 81 (55), 79 (57), 77
(28), 73 (39), 69 (47), 67 (70), 59 (33), 57 (100), 55 (99).
2‑[(Trimethylsilyl)oxy]‑Octadec‑6‑Ynenitrile (8)
To a stirred solution of 7 (0.26 g, 1.0 mmol) in dry
DCM (10 mL) at 0 °C was added trimethylsilyl cya-
nide (TMSCN) (0.19 g, 1.9 mmol) followed by catalytic
amounts (0.025 M) of trimethylamine under an argon
atmosphere. The mixture was left stirring under argon
for 3 h. Then, the solvent was removed in vacuo and the
crude product was washed with water (2 × 10 mL), die-
thyl ether (2 × 10 mL), dried over MgSO4, fltered, and
evaporated in vacuo affording 0.29 g (0.84 mmol) of 8 as
an oil in an 82% yield. The product was used as such for
the next step without further purifcation. 1H NMR (CDCl3,
300 MHz) δ (ppm) 4.43 (1H, t, J = 6.4 Hz, H-2), 2.20 (4H,
m), 1.63 (2H, m), 1.56 (2H, m), 1.45 (2H, m), 1.25 (16 H,
brs, –CH2–), 0.87 (3H, t, J = 6.7 Hz, –CH3), 0.18 (9H, s,
–Si(CH3)3); 13C NMR (CDCl3, 75 MHz) δ (ppm) 120.4 (s,
–CN), 61.1 (d, C 2), 31.9 (t), 29.7 (t), 29.7 (t), 29.6 (t), 29.5
(t), 29.3 (t), 29.2 (t), 29.1 (t), 28.9 (t), 26.9 (t), 24.7 (t), 22.6
(t), 14.1 (q, –CH3), 0.4 (q, –Si(CH3)3); GC/MS (70 eV) m/z
( )‑2‑Methoxy‑6‑Octadecynoic Acid (4)
To a stirred solution of NaH (0.02 g, 0.61 mmol) in dry
THF (3.0 mL) and under argon was added a solution of
0.05 g (0.17 mmol) of 9 in THF (2.0 mL). After 10 min
at rt, the solution temperature was lowered to 0 °C and
0.04 mL (0.61 mmol) of methyl iodide was added drop-
wise. Then, HCl (conc) was added to the solution until the
pH was acidic (pH = 1–2). The crude was extracted with
diethyl ether (2 × 10 mL), dried over MgSO4, and evap-
orated in vacuo. The product was purifed using silica gel
column chromatography frst eluting with hexane/ether
(9:1) and then with diethyl ether affording the methyl
( )-2-methoxy-6-octadecynoate. To obtain 4, a solution
of KOH/MeOH (1.5 M) (8.0 mL) and the methyl ester was
stirred for 2 h at 60 °C. After this time, the solvent was
removed in vacuo and hexane (15.0 mL) and HCl (3.0 mL)
1 3

Lipids
were added to the solution. The crude was washed with
a brine solution (1 × 10 mL), diethyl ether (1 × 10 mL),
dried over MgSO4, fltered, and evaporated in vacuo. The
product was purifed using fuorisil column chromatogra-
phy eluting with diethyl ether affording 4 as a colorless oil
H, brs, –CH2–), 0.87 (3H, t, J = 6.7 Hz, –CH3); 13C-NMR
(CDCl3, 75 MHz) δ (ppm) 130.4 (d), 129.2 (d), 62.9 (t,
C-1), 32.4 (t, C-3), 31.9 (t), 29.7 (t), 29.67 (t), 29.64 (t),
29.56 (t), 29.4 (t), 29.3 (t), 29.2 (t), 27.2 (t), 26.9 (t), 25.8
(t, C-4), 22.7 (t, C-17), 14.11 (q, –CH3); GC/MS (70 eV)
m/z (relative intensity): 254 (M+, 1), 236 (M+-H2O, 5), 208
(3), 179 (1), 165 (1), 152 (3), 151 (2), 1137 (7), 123 (12),
109 (24), 97 (29), 96 (24), 95 (58), 83 (44), 82 (100), 81
(65), 79 (17), 71 (18), 69 (38), 68 (65), 67 (81), 65 (4), 57
(36), 55 (56).
0.04 g (0.14 mmol) and in a 78% yield. IR (NaCl) νmax
:
3500–2500 (–OH), 3440, 2922, 2852, 2213, 1733 (C=O),
1
1459, 1380, 1262, 1171, 1124, 803, 717 cm−1; H NMR
(CDCl3, 300 MHz) δ (ppm) 3.83 (1H, m, H-2), 3.43 (3H,
s, –OCH3), 2.18-2.11 (4H, m), 1.84 (2H, m, H-3), 1.61
(2H, m), 1.45 (2H, m), 1.24 (16H, br s, –CH2–), 0.86 (3H,
t, J = 6.4 Hz, –CH3); 13C NMR (CDCl3, 75 MHz) δ (ppm)
177.8 (s, C-1), 81.1 (s), 79.7 (d, C-2), 79.0 (s), 58.2 (q, –
OCH3), 31.9 (t), 29.6 (t), 29.5 (t), 29.4 (t), 29.1 (t), 29.0 (t),
28.9 (t), 28.6 (t), 28.5 (t), 24.5 (t), 22.7 (t), 18.7 (t), 18.4 (t),
14.1 (q, –CH3); UPLC-HRMS (negative ion mode) Calcd
for C19H33O3 [M–H]+ 309.2430, found 309.2439.
(Z)‑5‑Heptadecenal (11)
To a stirred solution of 0.2 g (0.92 mmol) of PCC and
15.0 mL of dry DCM under argon was added 0.16 g
(0.61 mmol) of 10 at rt for 24 h. After that time the prod-
uct was obtained by fuorisil column chromatography elut-
ing with diethyl ether. The (Z)-5-heptadecenal (11) was
obtained as a colorless oil 0.15 g (0.58 mmol) for a 95%
yield. IR (NaCl) νmax: 2960, 2928, 2852 (CHO), 1712
(C=O), 1450, 729, 639 cm−1; 1H NMR (CDCl3, 300 MHz)
δ (ppm) 9.75 (1H, t, J = 1.7 Hz, CHO), 5.36-5.31 (2H,
m, –CH=CH–), 2.42 (2H, dt, J = 7.3, 1.7 Hz, H-2), 2.05
(4H, m), 1.68 (2H, m), 1.24 (18H, brs, –CH2–), 0.86 (3H,
t, J = 6.7 Hz, –CH3); 13C NMR (CDCl3, 75 MHz) δ (ppm)
202.7 (d, CHO), 130.2 (d), 129.7 (d), 43.3 (t, C 2), 31.9 (t),
29.7 (t), 29.64 (t), 29.56 (t), 29.34 (t), 29.32 (t), 29.2 (t),
28.9 (t), 27.2 (t), 27.1 (t), 22.7 (t), 22.0 (t, C-4), 14.1 (t, –
CH3); GC/MS (70 eV) m/z (relative intensity): 252 (M+, 1),
234 (2), 208 (6), 180 (2), 167 (1), 152 (3), 149 (1), 138 (4),
124 (7), 123 (5), 121 (3), 111 (13), 109 (12), 107 (3), 98
(67), 97 (26), 96 (40), 95 (24), 84 (24), 83 (35), 82 (47), 81
(43), 79 (29), 69 (37), 67 (81), 57 (38), 55 (100).
Methyl ( )‑2‑Methoxy‑6‑Octadecynoate
GC/MS (70 eV) m/z (relative intensity): 324 (M+, 1), 292
(M+-CH3OH, 1), 281 (1), 265 (M+-C2H3O2, 1), 249 (1),
233 (M+–C2H3O2–H2O, 17), 209 (4), 195 (5), 184 (9), 165
(7), 159 (1), 151 (28), 149 (11), 139 (14), 135 (38), 129
+
(34), 123 (27), 121 (50), 119 (19), 109 (50), 104 (C4H8O3
,
100), 97 (49), 95 (80), 91 (53), 81 (85), 79 (92), 71 (62), 69
(35), 67 (74), 59 (10), 57 (28), 55 (49).
(Z)‑5‑Heptadecen‑1‑ol (10)
Into a 50-mL two-necked round-bottomed fask contain-
ing 0.15 g (0.39 mmol) of palladium in activated carbon
(Lindlar’s catalyst) and 0.89 mL of quinoline were added
a solution of 6 (0.28 g, 1.11 mmol) and 1.7 mL of dry hex-
ane. One of the two necks was capped with rubber septa
and the other was connected via tygon tubing to a 25 mL
graduated pipet ending in a 150 mL beaker with distilled
water. While stirring at rt 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 a balloon flled
with hydrogen and attached to the hose barb-to-luer lock
adapter with stopcock and a needle. The reaction mixture
consumed 27.2 mL of hydrogen during 1 h. The mixture
was fltered and the solvent removed in vacuo. The prod-
uct was purifed under vacuum distillation (Kugelrohr) by
removing impurities and quinoline at 130 °C/3 mmHg. The
(Z)-5-heptadec-en-1-ol (10) was obtained as a colorless
(Z)‑2‑[(Trimethylsilyl)oxy]‑Octadec‑6‑Enenitrile (12)
To a stirred solution of 11 (0.14 g, 0.54 mmol) in dry
DCM (12.0 mL) at 0 °C and under argon was added
0.11 mL (0.82 mmol) of TMSCN followed by cata-
lytic amounts (0.025 M) of triethylamine. The mixture
was stirred for 3 h under argon. Then, the solvent was
removed in vacuo and the crude was washed with water (2
× 10 mL), diethyl ether (2 × 10 mL), dried over MgSO4,
fltered and evaporated in vacuo, affording 12 (0.12 g,
0.33 mmol) as a dark oil in a 61% yield. The product
was used as such for the next step without further puri-
1
fcation. H NMR (CDCl3, 300 MHz) δ (ppm); 5.34 (2H,
m, –CH=CH–), 4.38 (1H, t, J = 6.5 Hz, H-2), 2.12 (2H,
m), 2.01 (4H, m), 1.46 (2H, m), 1.25 (18H, brs, –CH2–),
0.86 (3H, t, J = 6.7 Hz, –CH3), 0.19 (9H, s, –OSi(CH3)3);
13C NMR (CDCl3, 75 MHz) δ (ppm); 130.1 (d), 129.4 (d),
119.9 (s, –CN), 61.3 (d, C-2), 31.9 (t), 29.73 (t), 29.65 (t),
29.63 (t), 29.5 (t), 29.3 (t), 29.3 (t), 29.15 (t), 29.12 (t),
oil 0.16 g (0.61 mmol) for a 55% yield. IR (NaCl) νmax
:
3384 (O–H), 3008 (=C–H), 2922, 2852, 1462, 1374, 1062,
717 cm−1; 1H-NMR (CDCl3, 300 MHz) δ (ppm) 5.63-5.32
(2H, m, –CH=CH–), 3.64 (2H, t, J = 6.5 Hz, H-1), 2.13-
1.99 (4H, m), 1.58 (2H, m), 1.44 (2H, m, H-3), 1.25 (18
1 3

Lipids
28.9 (t), 27.1 (t), 24.6 (t), 22.7 (t), 14.1 (q, –CH3), 0.45 (q,
–OSi(CH3)3); GC/MS (70 eV) m/z (relative intensity): 351
(M+, 3), 336 (M+–CH3, 3), 238 (2), 210 (4), 169 (6), 168
(7), 155 (6), 135 (16), 129 (16), 121 (10), 109 (6), 101
(17), 95 (19), 93 (14), 84 (28), 81 (25), 79 (31), 75 (53),
73 (C3H9Si+, 100), 69 (26), 67 (51), 59 (14), 57 (40), 55
(83).
this time, the solvent was removed in vacuo and hexane
(12.0 mL) and HCl (2.0 mL) were added to the solution.
The crude was washed with a brine solution (1 × 10 mL),
diethyl ether (1 × 10 mL), dried over MgSO4, fltered,
and evaporated in vacuo. The product was purifed using
a fuorisil column chromatography eluting with diethyl
ether affording the (6Z)-( )-2-methoxy-6-octadecenoic
acid (2) as a colorless oil 0.045 g (0.14 mmol) in a 90%
yield. IR (NaCl) νmax: cm−1; 3500–2500 (–OH), 3438,
2955, 2925, 2852, 1733 (C=O), 1459, 1377, 1286, 1121,
1074 cm−1; 1H NMR (CDCl3, 300 MHz) δ (ppm) 5.34 (2H,
m, –CH=CH–), 3.79 (1H, m, H-2), 3.43 (3H, s, –OCH3),
2.01 (4H, m), 1.75 (2H, m), 1.49 (2H, m), 1.25 (18H, brs,
–CH2–), 0.86 (3H, t, J = 6.7 Hz, –CH3); 13C NMR (CDCl3,
75 MHz) δ (ppm) 177.4 (s, C-1), 130.7 (d), 128.7 (d), 80.0
(d, C-2), 58.3 (q, –OCH3), 31.9 (t, C-3), 29.72 (t), 29.68 (t),
29.66 (t), 29.63 (t), 29.5 (t), 29.45 (t), 29.34 (t), 29.32 (t),
27.2 (t), 26.7 (t), 24.9 (t), 22.6 (t, C-17), 14.11 (q, –CH3);
UPLC-HRMS (negative ion mode) Calcd for C19H35O3
[M-H]+ 311.2586, found 311.2595.
Methyl ( )‑2‑Hydroxy‑6Z‑Octadecenoate (13)
To a solution of 0.12 g (0.33 mmol) of 12 and 4.9 mL
(60.4 mmol) of 2-MeTHF was added concentrated HCl
(2.0 mL, 64.7 mmol) and the reaction was refuxed for
24 h. The solvent was then removed in vacuo and the crude
product was washed with ether (2 × 10 mL), a brine solu-
tion (2 × 10 mL), and concentrated. Then, the product was
obtained by esterifcation of the crude using HCl/methanol
while refuxing for 3 h. The product was purifed using sil-
ica gel column chromatography eluting with hexane/ether
(7:3) affording 13 as a colorless oil 0.07 g (0.21 mmol) in
a 64% yield for both steps. IR (NaCl) νmax: 3364 (O–H),
2945, 2922, 2854, 1739 (C=O), 1462, 1374, 1124 cm−1
;
2‑(5‑Pentadecyn‑1‑oxy)Tetrahydro‑2H‑Pyran (14)
1H NMR (CDCl3, 300 MHz) δ (ppm) 5.34 (2H, m, –
CH=CH–), 4.19 (1H, m, H-2), 3.78 (3H, s, –OCH3), 2.05
(2H, m), 1.78 (2H, m), 1.62 (2H, m), 1.43 (2H, m), 1.25
(18H, brs, –CH2–), 0.87 (3H, t, J = 6.7 Hz, –CH3); 13C
NMR (CDCl3, 75 MHz) δ (ppm) 175.8 (s, C-1), 130.7 (d),
128.8 (d), 70.3 (d, C-2), 52.5 (q, –OCH3), 31.9 (t), 29.73
(t), 29.69 (t), 29.67 (t), 29.63 (t), 29.55 (t), 29.34 (t), 29.32
(t), 27.2 (t), 26.9 (t), 24.8 (t, C-4), 22.7 (t, C-17), 14.1 (q,
–CH3); GC/MS (70 eV) m/z (relative intensity): 312 (M+,
7), 281 (M+–CH3O, 1), 263 (1), 253 (M+–C2H3O2, 44),
234 (4), 221 (1), 208 (6), 180 (2), 151 (4), 140 (6), 129 (4),
127 (12), 121 (11), 111 (19), 109 (22), 103 (22), 97 (33),
95 (43), 90 (C3H6O3+, 100), 83 (35), 81 (42), 69 (31), 67
(45), 57 (30), 55 (46).
Into a 50-mL round-bottomed fask, was added (0.70 g,
3.8 mmol) of 2-(5-hexyn-1-yloxy) tetrahydro-2H-pyran,
6.0 mL of dry THF, and the mixture was cooled at 0 °C.
To the cooled solution was added 2.9 mL (5.8 mmol) of
n-BuLi (2 M in hexane) under argon, and the reaction was
stirred for 15 min. To the stirring solution was added drop-
wise 3.0 mL of 1,3-dimethyl-2-imidazolidinone (DMI)
and 1.1 mL (5.8 mmol) of 1-bromononane. After 20 min
the cold bath was removed and the reaction was left stir-
ring for 24 h. The reaction mixture was then washed with
a brine solution (2 × 10 mL), extracted with diethyl ether
(4 × 15 mL), dried over MgSO4 and fltered. The solvent
of the fltered solution was removed in vacuo and the
crude product was purifed using silica gel column chro-
matography eluting 14 with hexane/ether (9:1). The fnal
product 14 (1.1 g, 3.4 mmol) was obtained as a colorless
oil in a 90% yield. IR νmax(neat): 2922, 2853, 1454, 1351,
(6Z)‑( )‑2‑Methoxy‑6‑Octadecenoic Acid (2)
To a stirred solution of NaH (0.013 g, 0.54 mmol) in dry
THF (2.0 mL), under argon, was added a solution of 0.05 g
(0.16 mmol) of 13 in THF (3.0 mL). After 10 min at rt, the
solution temperature was lowered to 0 °C and 0.04 mL
(0.64 mmol) of methyl iodide was added dropwise. The
reaction was left to stir for 3 h. After that, HCl (conc) was
added to the solution until the pH was acidic (pH = 1–2).
The crude was extracted with diethyl ether (2 × 10 mL),
dried over MgSO4, and evaporated in vacuo. The product
was purifed using silica gel column chromatography frst
eluting with hexane/ether (9:1) and then with diethyl ether
affording the methyl (6Z)-( )-2-methoxy-6-octadecenoate.
To obtain 2, a solution of KOH/MeOH (1.5 M) (6.0 mL)
and the methyl ester was stirred for 2 h at 60 °C. After
1
1200, 1119, 1033, 905, 868, 813 cm−1; H NMR (CDCl3,
300 MHz) δ (ppm) 4.57 (1H, t, J = 3.5 Hz), 3.89-3.36 (2H,
m), 3.52-3.36 (2H, m), 2.21-2.09 (4H, m), 1.86-1.26 (25H,
m), 0.87 (3H, t, J = 6.7 Hz, –CH3); 13C-NMR (CDCl3,
75 MHz) δ (ppm) 98.9 (d), 80.7 (s), 80.0 (s), 67.2 (t), 62.4
(t), 32.0 (t), 30.9 (t), 29.6 (t), 29.42 (t), 29.30 (t), 29.1 (t),
29.0 (t), 26.1 (t), 25.7 (t), 22.8 (t), 19.8 (t), 18.9 (t), 18.8 (t),
14.2 (q). GC/MS (70 eV) m/z (relative intensity): 308 (M+,
0.01), 263 (1), 235 (8), 224 (1), 195 (1), 181 (6), 167 (1),
151 (1), 138 (2), 123 (2), 111 (6), 109 (5), 101 (6), 95 (19),
85 (C5H9O+, 100), 67 (16), 55 (12); UPLC-HRMS (posi-
tive mode) Calcd for C20H37O2 [M + H]+ 309.2794, found
309.2817.
1 3

Lipids
5‑Pentadecyn‑1‑ol (15)
2‑[(Trimethylsilyl)oxy]‑6‑Hexadecynenitrile (17)
Into a 50-mL round-bottomed fask, was added (0.90 g,
2.9 mmol) of 14, catalytic amounts of PTSA and 20 mL
of MeOH. The stirring solution was refuxed at 65 °C for
12 h. After the reaction time, the solvent was removed in
vacuo, the reaction crude was washed with a sodium bicar-
bonate (NaHCO3) saturated aqueous solution (2 × 10 mL),
extracted with diethyl ether (2 × 15 mL), dried over
MgSO4 and fltered. The solvent of the fltered solution
was removed in vacuo and the crude product thus obtained
was purifed using silica gel column chromatography elut-
ing 15 with hexane/ether (7:3). The pentadecynol (0.60 g,
2.7 mmol) was obtained as a colorless oil in a 93% yield.
IR νmax (neat): 3340 (–OH), 2922, 2853, 1456, 1377, 1332,
1057, 721 cm−1; 1H NMR (CDCl3, 300 MHz) δ (ppm) 3.68
(2H, t, J = 6.2 Hz, H-1), 2.21-2.09 (4H, m, H-4, H-7),
1.67–1.26 (19H, brs, –CH2–), 0.87 (3H, t, J = 0.87 Hz, –
CH3); 13C NMR (CDCl3, 75 MHz) δ (ppm) 80.9 (s), 79.9
(s), 62.7 (t, C-1), 32.04 (t), 32.03 (t), 29.6 (t), 29.4 (t),
29.3 (t), 29.0 (t), 25.5 (t), 22.8 (t), 18.9 (t), 18.7 (t), 14.2
(q, C-15). GC/MS (70 eV) m/z (relative intensity): 224
(M+, 0.01), 196 (M+-H2O, 0.01), 180 (3), 167 (1), 152 (3),
135 (4), 121 (6), 111 (29), 97 (54), 79 (100), 67 (41), 55
(36); UPLC-HRMS (positive mode) Calcd for C15H29O
[M + H]+ 225.2218, found 225.2240.
Into a 50-mL round bottomed fask and under argon was
added 0.45 g (2.0 mmol) of and 8.1 mL of dry DCM. The
stirring aldehyde solution was cooled to 0 °C, TMSCN
(0.3 mL, 2.3 mmol) was added followed by the addition
of catalytic amounts of triethylamine (0.03 mL, 2.2 mmol)
and the mixture was stirred for 12 h. Then, the solvent was
removed in vacuo, the reaction mixture was washed with a
brine aqueous solution (2 × 10 mL), extracted with diethyl
ether (3 × 15 mL), dried over MgSO4, and fltered. The sol-
vent was removed and the crude product was purifed using
silica gel column chromatography eluting the silyloxy nitrile
17 with hexane/ether (9:1), which was obtained as colorless
oil (0.52 g, 1.8 mmol) in an 88% yield. IR νmax (neat): 2954,
1
2924, 2854, 1256, 1254, 1110, 842, 752 cm−1; H-NMR
(CDCl3, 300 MHz) δ (ppm) 4.45 (1H, t, J = 6.5 Hz, H-2),
2.25-2.10 (4H, m, H-5, H-8), 1.94–1.87 (2H, m), 1.69-1.27
(19H, brs, –CH2–), 0.88 (3H, t, J = 6.5 Hz, –CH3), 0.21 (9H,
s, –Si(CH3)3); 13C NMR (CDCl3, 75 MHz) δ (ppm) 120.0
(s, C-1), 81.6 (s), 78.8 (s), 61.3 (d), 35.4 (t), 32.0 (t), 29.7
(t), 29.4 (t), 29.3 (t), 29.2 (t), 29.1 (t), 24.2 (t), 22.8 (t), 18.9
(t), 18.3 (t), 14.8 (q, CH3), 0. 24 (q, –Si(CH3)3). GC/MS
(70 eV) m/z (relative intensity): 321 (M+, 3), 306 (M+–CH3,
31), 293 (3), 278 (17), 264 (3), 250 (4), 236 (4), 232 (6), 222
(9), 208 (35), 194 (27), 181 (9), 168 (15), 155 (12), 133 (19),
129 (20), 119 (58), 107 (14), 92 (63), 73 (C3H9Si+, 100),
67 (30), 55 (26); UPLC-HRMS (positive mode) Calcd for
C19H36NOSi [M + H]+ 322.2566, found 322.2566.
5‑Pentadecynal (16)
Into a 50-mL round-bottomed fask and under argon was
placed 0.86 g (4.0 mmol) of PCC in 20 mL of dry DCM.
To the stirring solution was added 0.60 g (2.7 mmol) of 15
and the reaction was left stirring for 12 h. After this time,
the crude was fltered using silica gel and diethyl ether as
eluent. The solvent of the fltered solution was removed
in vacuo and the obtained yellowish liquid was purifed
using silica gel column chromatography eluting with hex-
ane/ether (9:1). The 5-pentadecynal (0.53 g, 2.4 mmol)
was obtained as a colorless oil in an 89% yield. IR νmax
(neat): 2953, 2922, 2853 (CHO), 2715 (CHO), 1710
Methyl ( )‑2‑Hydroxy‑6‑Hexadecynoate (18)
Into a 100-mL round-bottomed fask were placed 23 mL of
MeOH and 9 mL of HCl 12.1 M. To the stirred solution was
added 0.50 g (1.6 mmol) of the trimethylsilyl nitrile 17 and
the reaction was refuxed at 60–65 °C for 12 h. The solvent
was removed in vacuo, the reaction mixture was washed
with a brine aqueous solution (2 × 10 mL), extracted with
diethyl ether (3 × 15 mL), dried over MgSO4, and fltered.
The solvent was removed and the crude product was puri-
fed via silica gel column chromatography eluting with
hexane/ether (9:1) affording the methyl ester 18 (0.4 g,
1.3 mmol), which was obtained as a colorless oil in an
87% yield. IR νmax (neat): 3463 (–OH), 2953, 2923, 2853,
1736 (C=O), 1455, 1437, 1211, 1103, 996 cm−1; 1H-NMR
(CDCl3, 300 MHz) δ (ppm) 4.24–4.18 (1H, m), 3.79 (3H, s,
–OCH3), 2.71 (1H, s), 2.23–2.10 (4H, m, H-5, H-8), 1.97–
1.20 (19H, brs, –CH2–), 0.88 (3H, t, J = 0.88 Hz, –CH3);
13C NMR (CDCl3, 75 MHz) δ (ppm) 175.8 (s, C-1), 81.1
(s), 79.4 (s), 70.3 (d, C-2), 52.7 (q, –OCH3), 33.7 (t), 32.0
(t), 29.7 (t), 29.4 (t), 29.32 (t), 29.27 (t), 29.0 (t), 24.6 (t),
22.8 (t), 18.9 (t), 18.6 (t), 14.2 (q, –CH3). GC/MS (70 eV)
1
(C=O), 1456, 1389, 1378, 722 cm−1; H-NMR (CDCl3,
300 MHz) δ (ppm) 9.80 (1H, t, J = 1.5 Hz, –CHO), 2.59-
2.53 (2H, td, J = 7.5 and 1.5 Hz, H-2), 2.25-2.09 (4H,
m, H-4, H-7), 1.85-1.76 (2H, m), 1.51–1.26 (16H, brs, –
CH2–), 0.87 (3H, t, J = 6.7 Hz, –CH3); 13C NMR (CDCl3,
75 MHz) δ (ppm) 202.2 (d, C-1), 81.8 (s), 78.8 (s), 43.0
(t, C-2), 32.0 (t), 29.7 (t), 29.4 (t), 29.3 (t), 29.2 (t), 29.0
(t), 22.8 (t), 21.8 (t), 18.8 (t), 18.4 (t), 14.2 (q, C-15). GC/
MS (70 eV) m/z (relative intensity): 222 (M+, 1), 204 (1),
193 (M+-CHO, 1), 178 (2), 161 (4), 151 (11), 123 (27),
137 (19), 119 (30), 110 (40), 95 (67), 79 (100), 67 (87),
55 (69).
1 3

Lipids
m/z (relative intensity): 282 (M+, 0.1), 264 (M+-H2O, 0.1),
239 (2), 223 (M+-C2H3O2, 21), 205 (M+-C2H3O2 -H2O, 5),
183 (3), 169 (C9H13O3+, 100), 156 (34), 151 (14), 141 (7),
124 (46), 113 (42), 109 (50), 97 (51), 90 (C3H5O3+, 35), 81
(52), 67 (54), 55 (48); UPLC-HRMS (positive mode) Calcd
for C17H31O3 [M + H]+ 283.2273, found 283.2292.
was obtained. Into a 50-mL round-bottomed fask, under
argon, was added 0.16 g (6.7 mmol) of sodium hydride
(NaH), 15 mL of dry THF and 0.53 g (1.86 mmol) of
the 2-hydroxy methyl ester. After 15 min of stirring
the suspension it was cooled to 0 °C and then 0.41 mL
(6.6 mmol) of methyl iodide was added. After stirring
the reaction for 4 h, it was washed with a bicarbonate
(NaHCO3) saturated aqueous solution (1 × 15 mL) and
with diethyl ether (2 × 15 mL). The aqueous solution was
acidifed using concentrated HCl and then extracted with
diethyl ether (3 × 15 mL). The solvent was removed and
the crude product was purifed via silica gel column chro-
matography eluting acid 1 with hexane/ether (7:3). The
α-methoxylated acid 1 (0.33 g, 1.2 mmol) was obtained
as colorless oil in a 62% yield, with spectral data similar
to the one previously reported [9].
( )‑2‑Methoxy‑6‑Hexadecynoic Acid (3)
Into a 50-mL round-bottomed fask was added 0.09 g
(3.8 mmol) of NaH, under argon, to 15 mL of dry THF
together with 0.30 g (1.1 mmol) of 18. After 15 min of stir-
ring the suspension at rt the reaction was cooled to 0 °C
and then 0.23 mL (3.7 mmol) of methyl iodide were added.
The reaction was stirred for 4 h, and then washed with a
sodium bicarbonate (NaHCO3) saturated aqueous solution
(1 × 15 mL), as well as with diethyl ether (2 × 15 mL).
The aqueous solution was acidifed with concentrated HCl,
extracted with diethyl ether (3 × 15 mL), dried over MgSO4,
and fltered. The solvent was removed and the crude product
was purifed using silica gel column chromatography elut-
ing 3 with hexane/ether (7:3), which was obtained as color-
less oil (0.26 g, 0.9 mmol) in an 87% yield. IR νmax (neat):
3500–2500 (–OH), 2921, 2853, 1719 (C=O), 1457, 1194,
1117, 940, 719 cm−1; 1H-NMR (CDCl3, 300 MHz) δ (ppm)
3.85–3.81 (1H, dd, J = 4.8 and 7.1 Hz, H-2), 3.44 (3H, s,
–OCH3), 2.20–2.10 (4H, m, H-5, H-8), 1.89–1.27 (21H, brs,
–CH2–), 0.88 (3H, t, J = 6.7 Hz, –CH3). 13C NMR (CDCl3,
75 MHz) δ (ppm) 176.9 (s), 81.2 (s), 79.9 (d), 79.2 (s), 58.4
(q, –OCH3), 32.0 (t), 31.5 (t), 29.6 (t), 29.4 (t), 29.30 (t),
29.28 (t), 29.0 (t), 24.6 (t), 22.8 (t), 18.9 (t), 18.6 (t), 14.2
(q, –CH3). HRMS (TOFMS) Calcd for C17H33O3 [M + H]+
305.2093, found 305.2052.
(6Z)‑6‑Octadecenoic and 6‑Octadecynoic Acids
The syntheses of these acids followed procedures previ-
ously reported [13]. The acetylenic coupling between com-
mercially available 1-tridecyne and the 2-[(5-bromopentyl)
oxy]tetrahydro-2H-pyran using n-BuLi in THF-HMPA
at 0 °C followed by the deprotection of the pyranyl group
using methanol and PTSA afforded the 6-octadecyn-1-ol in
a 75% yield. The alkynol was hydrogenated under Lindlar’s
conditions, as described above, resulting in the (6Z)-6-oc-
tadecen-1-ol in a 70% yield. The oxidation of the alkenol
with PDC in DMF resulted in the formation of the known
(6Z)-6-octadecenoic acid in a 45% yield. The overall yield
for the four-step synthesis was 24%.
The 6-octadecynoic acid was prepared from the 6-octa-
decyn-1-ol by means of PDC oxidation in DMF, which
resulted in a 75% yield of the acid. The overall yield for the
three-step synthesis was 56%. The purity of both acids was
confrmed by 13C-NMR spectroscopy.
Methyl ( )‑2‑Methoxy‑6‑Hexadecynoate
GC/MS (70 eV) m/z (relative intensity): 296 (M+, 0.01),
264 (M+-CH3OH, 2), 237 (M+-C2H3O2, 56), 205 (23), 184
(10), 167 (5), 152 (24), 137 (25), 135 (34), 129 (30), 121
(52), 111 (55), 104 (C4H8O3+, 86), 93 (89), 81 (95), 79
(100), 77 (30), 71 (67), 67 (81), 55 (53).
( )‑2‑Methoxyhexadecanoic Acid
Was synthesized from the catalytic hydrogenation of 3 in
hexane with H2 and 10% Pd/C and the purity of the fnal
product was confrmed by 13C-NMR spectroscopy as well
as comparison of the MS spectral data with the one previ-
ously reported [12].
(6Z)‑( )‑2‑Methoxy‑6‑Hexadecenoic Acid (1)
Into a 50-mL round-bottomed fask a 5–10% Lindlar
catalyst/dry hexane solution was prepared as described
for 10 above. To the stirring solution was added 0.60 g
(2.1 mmol) of 18. After 5 min of stirring the suspension,
hydrogen was added. The reaction was stirred for 12 h,
and then the reaction mixture was fltered and extracted
with a 5% HCl aqueous solution (4 × 15 mL), washed
with diethyl ether (3 × 15 mL), dried over MgSO4, and
fltered. The solvent was removed and the crude product
Antibacterial Activity
Microorganisms
Staphylococcus aureus (ATCC 29213), Escherichia coli
(ATCC 25922), and Methicillin-resistant S. aureus (MRSA,
1 3

Lipids
ATCC 43300) were obtained from the American Type Cul-
ture Collection (Manassas, VA). Clinical isolates of MRSA
(CIMRSA) were kindly donated by a community hospi-
tal in San Juan, Puerto Rico. Stock cultures were kept on
blood Trypticase Soy Agar (TSA with 5% sheep blood,
Remel and Oxoid Microbiology Products, Lenexa, KS).
Subcultures were incubated for 18-24 h on TSA at 37 °C.
Suspension cultures were prepared by inoculation of single
colonies in 5 mL Trypticase Soy Broth (TSB, BD Diag-
nostic Systems, Franklin Lakes, NJ). Prior to preparation
of susceptibility assays, bacteria cells were re-suspended in
TSB (not centrifuged) and visually standardized by using a
0.5 McFarland standard solution, which provided an equiv-
alent concentration of 1.0 × 108 CFU/mL.
Reaction was stopped by adding 4 µL of electrophoresis
universal stop and loading buffer following electrophoresis
in a 1% agarose gel (70 V/105 min). To visualize the reac-
tion products, the gel was stained with ethidium bromide
for 25 min and destained for 10–15 min in distilled water.
DNA bands were detected and visualized in a Min BIS bio-
imaging system (model 241016P1, Israel).
CMC Determination
Determination of CMC was performed as described by
Sanabria-Ríos [14]. The FA, in either 95% ethanol or 100%
DMSO, depending on the FA solubility, and starting from
a stock solution of 1.0 × 105 µg/mL, were serially diluted
(0.1–1000 µg/mL) in 1X phosphate-buffered saline (1X
PBS, HyClon, Utah, USA) with Rhodamine 6G at a con-
centration of 2.5 × 10−6 M. The wavelength of maximum
absorption (525 nm) for each dilution was determined in a
Thermo Scientifc Genesys 10S UV–Vis spectrophotometer
(Cambridge, United Kingdom) and plotted as a function
of FA concentration. The CMC value was described as the
point at which the wavelength of maximum absorption frst
deviated from linearity.
Susceptibility Testing
Susceptibility testing was carried out as described by San-
abria-Ríos et al. [14]. Flat-bottomed microplate wells were
previously inoculated with a 10 µL TSB solution containing
4–5 × 105 colony-forming units (CFU). Into these wells,
dissolved in 100% DMSO, the FA were serially diluted
with sterile TSB (stock solutions of 1.0 × 105, 1.0 × 104,
and 1.0 × 103 µg/mL). For the spectrophotometrical anal-
yses at 620 nm both a positive control well (containing
the bacterial inoculated TSB but not the FA solution) and
a negative control well (containing only TSB) were used.
The minimum inhibitory concentration (MIC) was deter-
mined as the concentration at which the FA prevented tur-
bidity in the well after incubation for 18–24 h at 37 °C.
Turbidity was determined at 620 nm in a spectrophotom-
eter at 25 °C for those samples with optical densities higher
than 0.6–1.0. The IC50 values were calculated by plotting
dose–response curves [15]. The dose–response curves were
analyzed using the biostatistics software GraphPad Prism®
(v 5.0, San Diego, CA). The IC50 was defned as the con-
centration in which the FA inhibited 50% of the bacterial
growth.
Results
Synthesis of the α‑methoxylated FA
The synthesis of the α-methoxylated acids 1–4 followed
a synthetic procedure previously reported by us for other
similar analogs [6]. The ( )-2-methoxy-6-octadecynoic
acid (4) was prepared starting with the acetylenic cou-
pling of commercially available 1-tridecyne with 2-(4-bro-
mobutoxy)tetrahydro-2H-pyran using n-Buli in THF-
HMPA at 0 °C, which after the deprotection of the pyranyl
group using methanol and p-toluenesulfonic acid (PTSA)
afforded the desired 5-heptadecyn-1-ol (6) in a 71% yield
(Scheme 1). Alkynol 6 was then oxidized with Corey’s
pyridinium chlorochromate (PCC) in dichloromethane
(DCM) affording the 5-heptadecynal (7) in an 82% yield.
Then, aldehyde 7 was reacted with trimethylsilyl cyanide
(TMSCN) in DCM and catalytic amounts of triethyl-
amine (Et3N) at 0 °C resulting in an 82% yield of nitrile
8. Nitrile 8 was then transformed into the desired methyl
ester 9 in two steps. First, the hydrolysis of 8 in 2-meth-
yltetrahydrofuran (2-MeTHF) at 60 °C for 24 h afforded
the ( )-2-hydroxy-6-octadecynoic acid, which was esteri-
fed in HCl/methanol resulting in the methyl ester 9 in a
combined 44% yield for both steps. The methylation of
9 with sodium hydride and methyl iodide in tetrahydro-
furan (THF) followed by an acidic workup afforded the
( )-2-methoxy-6-octadecynoic acid (4) in a 78% yield.
S. aureus DNA Gyrase Inhibitory Tests
Staphylococcus aureus DNA gyrase inhibitory tests were
performed as described Sanabria-Ríos et al. [15]. Briefy,
the enzyme activity of DNA gyrase was assessed using
DNA gyrase from S. aureus (1 unit will supercoil 0.05 µg
of DNA in 60 min at 37 °C) and 0.20 µg of relax pHOT1
plasmid DNA. FA were dissolved in 50% DMSO obtain-
ing a stock solution of 2.5 × 104 µg/mL and tested at dif-
ferent concentrations that ranged from 1.9 to 1000 µg/mL.
Reactions (fnal volume 20 µL) were carried out for 60 min
at 37 °C, after which 2 µL of 10% sodium dodecyl sulfate
(SDS) was added. Bound protein was digested by incuba-
tion with proteinase K (0.05 mg/mL) for 30 min at 37 °C.
1 3

Lipids
Scheme 1 Synthesis of ( )-2-methoxy-6-octadecynoic acid (4). (i)
2-(4-bromobutoxy)tetrahydro-2H-pyran, n-BuLi, THF-HMPA, 0 °C,
24 h; (ii) PTSA, MeOH, 35 °C, 12 h; (iii) PCC, CH2Cl2, 24 h, rt; (iv)
TMSCN, Et3N, CH2Cl2, 0 °C, 3 h; (v) HCl (conc.) 2-MeTHF, 60 °C,
24 h; (vi) HCl, MeOH, refux, 3 h; (vii) NaH, THF, CH3I, 0 °C, 3 h;
(viii) KOH/MeOH (1.5 M), 60 °C, 2 h
The overall yield for the seven-step synthetic sequence was
16% and 4 is a novel FA.
For the synthesis of the (6Z)-( )-2-methoxy-6-
hexadecenoic acid (1) and ( )-2-methoxy-6-hexadecyn-
oic acid (3) a different strategy was pursued in terms of
hydrogenating the alkyne late in the synthetic sequence
when aiming for 1 and thus saving several synthetic steps
in between (Scheme 3). The synthesis of 3 started from
commercially available 2-(5-hexyn-1-yloxy) tetrahydro-
2H-pyran, which was coupled with 1-bromononane using
n-BuLi in THF-DMI at 0 °C and after deprotection of
the pyranyl group using methanol and PTSA, the desired
5-pentadecyn-1-ol (15) was obtained in a combined 84%
yield for these two steps. The oxidation of alkynol 15
using Corey’s PCC in DCM resulted in an 89% yield
of the 5-pentadecynal (16). In an analogous fashion as
the two syntheses above, aldehyde 16 was reacted with
TMSCN in DCM and catalytic amounts of Et3N result-
ing in an 88% yield of the nitrile 17. Hydrolysis of 17
in HCl/MeOH at 60-65 °C for 12 h afforded the methyl
ester 18 in an 85% yield. The methylation of 18 with
sodium hydride and methyl iodide in THF followed by
the acidic workup afforded the ( )-2-methoxy-6-hexa-
decynoic acid (3) in an 87% yield. The overall yield for
this six-step synthetic sequence was 48% and acid 3 is a
novel FA.
The (6Z)-( )-2-methoxy-6-octadecenoic acid (2), a
natural product whose synthesis has not been reported
before, started from alkynol 6, which was catalytically
hydrogenated under Lindlar’s conditions, resulting in a
55% yield of the (Z)-5-heptadecen-1-ol (10) as shown in
Scheme 2. Characterization of the cis alkenol 10 by 13C
NMR confrmed the signals of the two sp2 carbons that
resonated at δ 130.4 and 129.2 ppm. The oxidation of the
alkenol 10 using Corey’s PCC in DCM resulted in a 95%
yield of the (Z)-5-heptadecenal (11). Then aldehyde 11
was reacted with TMSCN in DCM and catalytic amounts
of Et3N at 0 °C that yielded the nitrile 12 in a 61% yield.
As with the synthesis of 4 described above, nitrile 12 was
transformed into the desired ester 13 in two steps. First,
the acid hydrolysis of 12 in 2-MeTHF afforded the (6Z)-
( )-2-hydroxy-6-octadecenoic acid, and after esterifca-
tion using HCl/MeOH the desired ester 13 was obtained
in a combined 64% yield for the last two steps. The
fnal acid 2 was obtained after methylation of 13 with
sodium hydride and methyl iodide in THF, following an
acidic workup, resulting in the (6Z)-( )-2-methoxy-6-
octadecenoic acid (2) in a 90% yield. The overall yield
for this eight-step synthetic sequence (starting from 5)
was 13%, and this is the frst reported total synthesis for
2 [12].
Our improved synthesis for the (6Z)-( )-2-
methoxy-6-hexadecenoic acid (1) took advantage of
the synthetic route for 3 by starting with the methyl
Scheme 2 Synthesis of (6Z)-( )-2-methoxy-6-octadecenoic acid (2).
(i) H2, Pd/C (10%), quinoline, hexane, rt; (ii) PCC, CH2Cl2, 24 h, rt;
(iii) TMSCN, Et3N, CH2Cl2, 0 °C, 3 h; (iv) HCl (conc.) 2-MeTHF,
60 °C, 24 h; (v) HCl, MeOH, refux, 3 h; (vi) NaH, THF, CH3I, 0 °C,
3 h; (vii) KOH/MeOH (1.5 M), 60 °C, 2 h
1 3

Lipids
Scheme 3 Synthesis of ( )-2-methoxy-6-hexadecynoic acid (3) and
the (6Z)-( )-2-methoxy-6-hexadecenoic acid (1). (i) n-BuLi, THF-
DMI, 1-bromononane, 0 °C, 24 h; (ii) MeOH, PTSA, 65 °C, 12 h;
(iii) PCC, DCM, 12 h; (iv) TMS-CN, TEA, DCM, 0 °C; (v) HCl/
MeOH, 60 °C, 12 h (vi) NaH/THF, MeI, 0 °C, 4 h, then HCl; (vii) H2,
Lindlar catalyst, hexane, 12 h; (viii) NaH/THF, MeI, 0 °C, 4 h, then
HCl
( )-2-hydroxy-6-hexadecynoate (18), which was cata-
lytically hydrogenated under Lindlar’s conditions, fol-
lowed by methylation with sodium hydride/methyl
iodide in THF, and after the acidic workup the (6Z)-( )-
2-methoxy-6-hexadecenoic acid (1) was obtained in a
64% yield for the last two steps. The overall yield for
this seven-step synthetic sequence was 36%. The advan-
tage of using this acetylide coupling approach over our
previous methodology, which utilized a Wittig approach
[9], is that a 100% cis stereochemistry was obtained for
the C-6 double bond in 1, while with the Wittig approach
a 10:1 mixture of the Z/E isomers was obtained [9]. This
is important for our aims since there is a considerable
difference in the antibacterial activity of cis vs. trans FA
[11]. For example, the trans 16:1 Δ9 acid has a consid-
erably higher MIC than the cis 16:1 Δ9 acid towards S.
aureus [11].
Antibacterial Studies
The antibacterial studies performed herein for the
α-methoxylated acids 2 and 4 are presented in Table 1,
while the corresponding studies for 1 and 3 are shown in
Table 2. Assays were performed against clinical isolates of
methicillin-resistant S. aureus (CIMRSA) as well as against
one strain of E. coli (ATCC 25922) following the proce-
dures described in Materials and Methods. As expected
from previous studies with the 18:1Δ6 or 18:1Δ9 acids on
S. aureus RN4220 [11], the 6-octadecenoic and 6-octade-
cynoic acids were ineffective towards all the studied strains
of CIMRSA as well as against E. coli. However, the 6-octa-
decynoic acid did show some activity towards S. aureus
(ATCC 29213) but with a very high IC50 of 274 µg/mL. The
α-methoxylated octadecynoic acid 4 was the most bacteri-
cidal acid towards clinical isolates of methicillin-resistant
Table 1 Antibacterial activity
Microorganisms
MIC/IC50 SEM, μg/mL
Fatty acids
of the C18 unsaturated FA
against multi-drug resistant
bacteria
(
)-(4)
(
)-(2)
6-Octadecynoic acid
500/273.8 11.2
6-Octadecenoic acid
>1000
S. aureus
(ATCC 29213)
125/67.7 5.2
>1000
>1000
>1000
E. coli
>1000
>1000
(ATCC 25922)
ClMRSA I
ClMRSA II
62.5/32.2 3.5
62.5/37.0 1.6
31.3/17.7 1.2
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
MRSA
(ATCC 43300)
ClMRSA IX
31.3/21.3 0.9
>1000
>1000
>1000
Experiments were performed in triplicate (n = 3). E. coli experiments were done in sextuplicate (n = 6)
MRSA methicillin-resistant S. aureus, ClMRSA clinical isolates of MRSA, SEM standard error of the mean
1 3

Lipids
Table 2 Antibacterial activity
of the C16 unsaturated FA
against multi-drug resistant
bacteria
Microorganisms
MIC/IC50 SEM, μg/mL
Fatty acids
(
)-(3)
(
)-(1)
2-Methoxy-16:0a
250/92.9 4.0
6-Hexadecynoic acid
125/68.1 4.5
S. aureus
(ATCC 29213)
62.5/37.9 0.4
250/140.8 4.0
62.5/35.4 0.3
62.5/21.2 0.6
E. coli
>1000
>1000
(ATCC 25922)
ClMRSA I
ClMRSA II
250/151.3 1.3
250/121.5 5.8
62.5/38.2 2.0
62.5/31.3 0.5
125/80.6 4.3
62.5/30.0 0.9
250/155.5 15.2
ND
125/65.1 3.4
62.5/43.0 0.7
250/160.6 1.7
MRSA
250/146.1 0.7
(ATCC 43300)
ClMRSA IX
500/297.6 1.0
125/77.5 1.5
250/100.4 4.3
125/58.0 1.2
Experiments were performed in triplicate (n = 3). E. coli experiments were done in sextuplicate (n = 6)
MRSA methicillin-resistant S. aureus, ClMRSA clinical isolates of MRSA, ND not determined, SEM stand-
ard error of the mean
a
Palmitic acid did not display any antibacterial activity against these strains (IC50 SEM >1000 μg/mL)
S. aureus (CIMRSA) displaying MIC between 31 and
63 μg/mL and IC50′s between 17 and 37 μg/mL. In sharp
contrast to the results obtained for 4, the α-methoxylated
octadecenoic acid 2 was not effective at all against all the
studied strains, similar to what others have observed for the
6-octadecenoic acid against S. aureus [11] (Table 1). There-
fore, among the studied C18 FA, acid 4 stands out as the
best candidate against CIMRSA.
of the α-methoxylated acids. Previous studies relate the
biological activity of acids to their CMC as was dem-
onstrated for the lipoteichoic acids [16] and the 2-alky-
noic FA [14]. We have also shown that α-methoxylation
lowers the CMC of the 6-icosynoic acid from 500 µM
to 20–30 µM [6]. However, with just one single study,
there is no clear general picture as to what extend
α-methoxylation affects the CMC of a FA, and how this
is related to the FA chain length [6]. Therefore, the CMC
of the acids 1–4 were determined as previously described
in a solution of rhodamine 6G and 1X PBS buffer and
the obtained values are shown in Table 3. As expected
from their longer chain lengths, the C18 acids 2 and 4
displayed a fve-fold lower CMC (15–20 μg/mL) than
the C16 analogs 1 and 3 (70–100 μg/mL). These results
indicate that 4 mainly exerts its antibacterial activity in a
micellar state. When the CMC value of acid 4 was com-
pared to the CMC of the 6-octadecynoic acid (20–30 μg/
mL), it was observed that it was slightly lower than the
CMC of the 6-octadecynoic analog, but not as dramatic
The antibacterial studies for the C16 α-methoxylated
acids 1 and 3, displayed in Table 2, presented a rather dif-
ferent scenario. Most of the studied acids displayed some
sort of activity towards CIMRSA with MIC between 63
and 250 μg/mL and IC50′s between 38 and 298 μg/mL. In
this series both 1 and 3 were toxic to CIMRSA, but against
CIMRSA I and IX the α-methoxylated hexadecenoic acid
1 displayed a 4-fold better toxicity profle than its hexa-
decynoic analog 3. What was more surprising here is that
both 1 and 3 also displayed toxicity towards E. coli, with
MIC of 63 and 250 μg/mL, respectively, and IC50′s of 21
and 141 μg/mL, respectively, with acid 1 displaying a
4-sevenfold better toxicity profle than 3 towards E. coli.
Therefore, among the studied FA in the C16 series, acid 1
stands out as the best candidate against CIMRSA and E.
coli. However, between the C16 acid 1 and the C18 acid
4, the latter was more effective towards CIMRSA, while 1
was the most bactericidal towards E. coli. Important is the
fact that both 1 and 4 contained the α-methoxy functional-
ity in addition to a C-6 unsaturation in the acyl chain.
Table 3 Critical micelle concentration (CMC) of the 2-methoxylated
FA
CMC (μg/mL)
Fatty acids
(
(
)-(4)
)-(2)
15–18
15–20
20–30
90–100
70–90
10–50
6-Octadecynoic acid
(
(
)-(3)
)-(1)
Critical Micelle Concentration (CMC) Studies
2-Methoxy-16:0
The question as to why the α-methoxylated acids 1 and 4
are more effective towards the studied cell lines prompted
us to frst study the critical micelle concentrations (CMC)
The CMC determinations were done in triplicate (n = 3)
1 3

Lipids
a difference as the one obtained for the C20 series previ-
ously reported by us [6]. In any instance, it is evident that
α-methoxylation decreases the CMC of the parent FA, at
least in the C18 and C20 series.
of FA biosynthesis by inhibiting the FabI enzyme [22].
However, in the case of S. aureus RN4220 it was reported
that the C16 FA are toxic by a rapid membrane depolari-
zation mechanism coupled to the disruption of macromo-
lecular biosynthesis as well as the release of solutes and
low-molecular weight proteins from the cells [11]. We
found herein that the α-methoxylated acid 4 was the most
effective compound among those tested against CIMRSA.
It is evident that the combination of α-methoxylation and
a C-6 triple bond in the C18 structure augmented the
antibacterial effect as evidenced by the lack of toxic-
ity of 2, where the only difference between 2 and 4 is a
double bond versus a triple bond. The observed behavior
for acid 2 is similar to what is known for an 18:1Δ6 FA,
which is basically nontoxic to S. aureus RN4220 since it
is an acid that is readily used for phospholipid biosyn-
thesis given the fact that it is easily recognized by the
biosynthetic machinery of S. aureus [11]. Acid 4 seems
to be suffciently foreign to the bacterium that it is not
readily recognized by the S. aureus acyltransferase sys-
tem [11]. It is very likely for 4 to work by disruption of
the cytoplasmic membrane thereby releasing solutes and
low-molecular weight proteins from the cells in a sort
of a lysis mechanism of action. We should also mention
here that oleic acid has been reported to be marginally
toxic (MIC = 0.4 mM) towards other strains of S. aureus,
such as S. aureus 285 or S. aureus 503, presumably by a
mechanism involving inhibition of the enoyl-acyl carrier
protein reductase (FabI), which is present in S. aureus
and is essential for the bacterial FA biosynthetic machin-
ery [22]. However, our bacterial strains were suscepti-
ble towards the studied FA in a similar fashion as it was
reported for S. aureus RN4220 [11]. It is also important
to mention that acid 4 had a low CMC at 15–18 µg/mL,
while its antibacterial activity towards CIMRSA dis-
played IC50′s between 17 and 37 µg/mL (Table 3). This
implies that 4 might be interacting with the S. aureus
membrane in a micellar state and α-methoxylation is
helping in achieving lower CMCs since the correspond-
ing nonmethoxylated analog has a slightly higher CMC
(Table 3). This interaction with the bacterial membrane
can result in an increase of the membrane fuidity result-
ing in a distortion of the respiratory chain and alteration
of several membrane processes [23]. Since acid 4 is also
expected to be more acidic than 6-octadecynoic acid
(pKa 3.6 vs. pKa 4.7), it is also very likely that 4 might
be acting like a better protonophore once inside the cell.
This can only lead to the inability of the cell to control its
internal pH that can ultimately result in the disruption of
many enzymatic processes within S. aureus [23].
S. aureus DNA Gyrase Inhibitory Studies
Given that the S. aureus DNA gyrase is the target of several
antibacterial agents, such as the quinolone-based antibacteri-
als [17], it was of interest to study if the α-methoxylated FA 1
and 3 were inhibitory towards this topoisomerase II enzyme.
Our results show that both 1 and 3 were not inhibitory
towards the S. aureus DNA gyrase at concentrations as high
as 1000 μg/mL (Fig. 1). Therefore, the mechanism of toxic-
ity of these α-methoxylated FA towards S. aureus is different
from the mechanisms displayed by the more classic antibac-
terials such as the quinolones or fuoroquinolones [17].
Discussion
FA can exert their antibacterial activities by a myriad of
mechanisms that might include, among others, disruption
of the electron transport chain [18], interference with oxi-
dative phosphorylation [19], cell lysis with the concomi-
tant leakage of cell metabolites [20], formation of per-
oxidation or auto-oxidation products [21], and inhibition
In the case of the tested C16 methoxylated acids, the
α-methoxylated hexadecenoic acid 1 displayed the best
overall antimicrobial profle. Presumably, acid 1 is also
Fig. 1 Inhibitory effect of 1 and 3 on the supercoiling activity of S.
aureus DNA gyrase. Rel. DNA relaxed DNA, SC DNA supercoiled
DNA
1 3

Lipids
poorly metabolized by S. aureus, i.e., it is a poor sub-
strate for phospholipid biosynthesis and it accumulates for
a longer time in the medium, similar to the 16:1Δ6 acid,
since FA are normally not degraded by this bacterium [11].
Probably the unexpected result for 1 and 3 was their abil-
ity to be bactericidal towards E. coli (ATCC 25922), while
almost all of the studied FA, including the 6-hexadecy-
noic acid, were not active at all towards this Gram-nega-
tive bacterium. The combination of an unsaturation at C-6
and the α-methoxylation was important for the observed
activity since the acid 2-methoxy-16:0, which contains
the α-methoxylation but no double bond, was not effec-
tive towards E. coli. Apparently, acids 1 and 3 can fnd
their way more easily into the E. coli membrane, i.e., they
have the right functional groups (α-methoxylation and C-6
unsaturation) so as to traverse the outer membrane more
effciently via a FadL porin or a similar transport system
and once inside the cell create havoc [24]. Palmitic acid has
a slightly better FA binding affnity than oleic acid towards
FadL, while the affnity of myristic acid is nearly an order
of magnitude less towards FadL [25]. The α-methoxylated
acids 1 and 3 might interact more strongly with the amino
acids (such as arginine and lysine) known to be present at
the outside of the hydrophobic binding pockets of FadL
which are important for binding long-chain FA to the pro-
tein [26]. It is also interesting to point out that 1 (the best of
the two α-methoxylated acids against E. coli) displayed a
high CMC of 70–90 µg/mL, while its antibacterial activity
towards E. coli displayed an IC50 of 21 µg/mL (Table 3).
In contrast to 4, acid 1 is clearly interacting with the E.
coli membrane in a monomeric state. In any instance, acid
1 could be used in conjunction with known antibiotics by
serving as a debilitating agent that can disrupt the outer
membrane of E. coli, thus allowing the antibiotic to def-
nitely kill the Gram-negative bacteria.
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Acknowledgements Part of the work described herein was initially
supported by Award Number SC1 GM084708 from the National
Institute of General Medical Sciences (NIGMS) of the NIH. D.
Sanabria thanks the National Institute of General Medical Sciences
of the National Institutes of Health for an Institutional Develop-
ment Award (IDeA) Grant (# P20GM103475). N. Montano and C.
Morales acknowledge the support of the UPR RISE program (Grant
No. 5R25GM061151-15) for graduate fellowships. We thank William
O. Marrero for technical assistance. We are in debt to Dr. Mikhail Y.
Golovko and Svetlana A. Golovko of the COBRE Mass Spec Core
Facility at the Department of Basic Sciences of the University of
North Dakota for the high-resolution mass spectral data.
1 3

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