The (5Z)-5-Pentacosenoic and 5-Pentacosynoic Acids Inhibit the HIV-1 Reverse Transcriptase

Article abstract of DOI:10.1007/s11745-015-4064-2

The natural fatty acids (5Z)-5-pentacosenoic and (9Z)-9-pentacosenoic acids were synthesized for the first time in eight steps starting from either 4-bromo-1-butanol or 8-bromo-1-butanol and in 20-58 % overall yields, while the novel fatty acids 5-pentacosynoic and 9-pentacosynoic acids were also synthesized in six steps and in 34-43 % overall yields. The ?⁵ acids displayed the best IC_50's (24-38 μM) against the HIV-1 reverse transcriptase (RT) enzyme, comparable to nervonic acid (IC₅₀ = 12 μM). The ?⁹ acids were not as effective towards HIV-RT with the (9Z)-9-pentacosenoic acid displaying an IC₅₀ = 54 μM and the 9-pentacosynoic acid not inhibiting the enzyme at all. Fatty acid chain length and position of the unsaturation was important for the observed inhibition. None of the synthesized fatty acids were toxic (IC₅₀ > 500 μM) towards peripheral blood mononuclear cells. Molecular modeling studies indicated the structural determinants underlying the biological activity of the most potent compounds. These results provide new insights into the structural requirements that must be present in fatty acids so as to enhance their inhibitory potential towards HIV-RT.

Full text of DOI:10.1007/s11745-015-4064-2

Lipids
DOI 10.1007/s11745-015-4064-2
COMMUNICATION
The (5Z)‑5‑Pentacosenoic and 5‑Pentacosynoic Acids Inhibit the
HIV‑1 Reverse Transcriptase
Lizabeth Giménez Moreira1 · Elsie A. Orellano1 · Karolyna Rosado1 ·
Rafael V. C. Guido2 · Adriano D. Andricopulo2 · Gabriela Ortiz Soto3 ·
José W. Rodríguez3 · David J. Sanabria‑Ríos4 · Néstor M. Carballeira1
Received: 7 July 2015 / Accepted: 24 August 2015
© AOCS 2015
Abstract The natural fatty acids (5Z)-5-pentacosenoic and
(9Z)-9-pentacosenoic acids were synthesized for the first
time in eight steps starting from either 4-bromo-1-butanol or
8-bromo-1-butanol and in 20–58 % overall yields, while the
novel fatty acids 5-pentacosynoic and 9-pentacosynoic acids
were also synthesized in six steps and in 34–43 % overall
yields. The ∆5 acids displayed the best IC50’s (24–38 µM)
against the HIV-1 reverse transcriptase (RT) enzyme, com-
parable to nervonic acid (IC50 = 12 µM). The ∆9 acids were
not as effective towards HIV-RT with the (9Z)-9-pentacose-
noic acid displaying an IC50 = 54 µM and the 9-pentacosy-
noic acid not inhibiting the enzyme at all. Fatty acid chain
length and position of the unsaturation was important for the
observed inhibition. None of the synthesized fatty acids were
toxic (IC50 > 500 µM) towards peripheral blood mononuclear
cells. Molecular modeling studies indicated the structural
determinants underlying the biological activity of the most
potent compounds. These results provide new insights into
the structural requirements that must be present in fatty acids
so as to enhance their inhibitory potential towards HIV-RT.
Keywords Fatty acids · Human immunodeficiency virus ·
Pentacosenoic acid · Reverse transcriptase · Synthesis
Abbreviations
ABTS
2,2′-Azino-bis(3-ethylbenzothiazoline-6-sul-
fonic acid)
DIG
DMSO
DTT
FA
Digoxigenin
Dimethyl sulfoxide
Dithiothreitol
Fatty acids
GC-MS Gas chromatography-mass spectrometry
HAART Highly active-antiretroviral therapy
Electronic supplementary material The online version of this
article (doi:10.1007/s11745-015-4064-2) contains supplementary
material, which is available to authorized users.
HIV
IC50
Human immunodeficiency virus
Inhibitory concentration for half-life maximal
inhibition
* Néstor M. Carballeira
nestor.carballeira1@upr.edu
NA
Nervonic acid
NNRTI Non-nucleoside reverse transcriptase inhibitors
1
Department of Chemistry, University of Puerto Rico, Rio
Piedras Campus, PO Box 23346, San Juan, PR 00931-3346,
USA
NRTI
PBMC
PDC
p-TSA
RT
Nucleoside reverse transcriptase inhibitors
Peripheral blood mononuclear cells
Pyridinium dichromate
p-Toluenesulfonic acid
Reverse transcriptase
2
Laboratório de Química Medicinal e Computacional, Centro
de Pesquisa e Inovação em Biodiversidade e Fármacos,
Instituto de Física de São Carlos, Universidade de São Paulo,
Avenida João Dagnone 1100, Jardim Santa Angelina, São
Carlos, SP 13563-120, Brazil
THF
Tetrahydrofuran
3
Department of Microbiology and Immunology, Universidad
Central del Caribe School of Medicine, PO Box 60327,
Bayamón, PR 00960, USA
Introduction
4
Faculty of Science and Technology, Inter American
The potential of fatty acids to combat infectious dis-
eases such as malaria, tuberculosis, and fungal infections
University of Puerto Rico, Metropolitan Campus, PO
Box 191293, San Juan, PR 00919, USA
1 3

Lipids
continues to be a focus of recent research activity [1]. Fatty
acids are important in a diversity of pathological condi-
tions and have been postulated as possible drug candi-
dates against viruses, bacteria, fungi, and cancerous cells
by inducing death responses [1–6]. Numerous fatty acids,
mainly of bacterial origin, also inhibit DNA polymerase
and topoisomerase I, and display antimalarial and antiviral
activity [1, 7–11].
The HAART regime currently used is not potent enough
to completely suppress virus replication to the point of
eradication due to the development of drug-resistance
[13, 25]. It is still necessary to develop new less cytotoxic
antiretrovirals for the treatment of this disease. Natural
products from various marine sources have been known
to possess diverse biological activities including antiviral
activity [11, 31].
The World Health Organization (WHO) considers the
human immunodeficiency virus (HIV) infection pan-
demic [12]. In 2012 alone, around 2.3 million adults
and children were infected with HIV, bringing the total
population living with HIV to 35.3 million, mostly on the
African continent [12, 13]. Even after more than twenty
years of research, a HIV vaccine has yet to be discovered
and current research focuses on the search for novel anti-
HIV agents [14]. There are more than twenty antiretro-
viral drugs approved for clinical use today [15], but due
to their long-term use in drug therapies these drugs must
be relatively nontoxic [16]. This implies that our present
arsenal to combat the disease is limited due to adverse
effects and toxicities that normally arise from long-term
use coupled to the emergence of drug resistance [14].
Since HIV-1 can acquire drug resistance to any single
inhibitor quite easily, multiple drugs are typically used
simultaneously [14].
Nervonic acid (NA) is a cis-15-tetracosenoic acid found
in the sphingolipids of white matter in human brain where
it plays an important role in the biosynthesis of nerve cell
myelin [32]. NA is also abundantly found in the spotted
seal, king salmon and beluga whale. NA is used to treat
adrenoleukodystrophy and multiple sclerosis where the
decreased level of nervonic acid in sphingolipids causes
demyelination [32]. This FA possesses great inhibitory
activity against several enzymes involved in replication
[33–36]. Mizushina and co-workers discovered that NA
was a potent non-competitive inhibitor of HIV-1 RT with
an IC50 of 4.8 μM and almost complete inhibition (more
than 80 %) at 8 μM [36]. Modification of the carboxyl
group of NA to a carboxyl ester results in the loss of inhibi-
tory activity thus indicating that the carboxyl group in NA
is important for RT inhibition [33]. Therefore, the carboxyl
group and length of the alkyl chain in NA play crucial roles
in HIV-RT inhibition.
The reverse transcriptase (RT) is a multifunctional
enzyme that plays a key role in HIV-1 replication [16–
22]. It possesses distinct DNA polymerase and RNase
H activities, which are used to convert the viral RNA
genome into double-stranded linear proviral DNA that
are subsequently integrated into the host genome [16–
24]. Since RT inhibitors prevent the RNA-DNA conver-
sion, a vital role of the viral life cycle, it is no wonder
that more than half the currently approved HIV-1 antiret-
rovirals are RT inhibitors [21]. There are two classes
of RT inhibitors, namely nucleoside/nucleotide reverse
transcriptase inhibitors (NRTI) and non-nucleoside
reverse transcriptase inhibitors (NNRTI) [15, 25–27].
Together they are the backbone of the highly active-
antiretroviral therapy (HAART) regime [24], the stand-
ard treatment that usually combines two NRTI with one
NNRTI or with one protease inhibitor (PI) [14, 16, 24].
However, over the years, NNRTI have become more
popular than the PI [21].
Even though the NNRTI are a chemically heterogene-
ous group of compounds, they possess a common mode
of binding and they all bind to the same site on the RT
enzyme [28, 29]. This binding site is located in the palm
sub-domain between two beta-sheets of the p66 subunit
about 10 Å away from the DNA polymerase catalytic site
and approximately 60 Å from the RNase H active site of
the p66 subunit [24, 28, 30].
Since nervonic acid is a potent HIV-1 RT inhibitor, other
monounsaturated long chain fatty acids could be poten-
tial HIV-1 RT inhibitors as well. The pentacosenoic acids
are a minor class of lipids found in a wide range of organ-
isms including bacteria and marine sponges [37]. The (5Z)-
5-pentacosenoic acid (2a) is naturally found in the marine
sponge Pseudaxinella cf. lunaecharta and in the bacterium
Mycobacterium tuberculosis [38, 39]. On the other hand,
the (9Z)-9-pentacosenoic acid (2b), which is more ubiqui-
tous, was identified in various sponges including the marine
sponges Dysidea fragilis, Desmapsamma anchorata, Geod-
inella robusta, Spheciospongia cuspidifera and Hymeniaci-
don sanguinea as well as in the bacterium Mycobacterium
tuberculosis [38, 40–44]. These long-chain monounsaturated
fatty acids have the potential to be RT inhibitors and our aim
was to explore them as such. However, since the isolation and
purification of these lipids from natural sources, in reasonable
quantities, is an extremely difficult task, they must be synthe-
sized if we are to explore their full biological potential.
In this work, we synthesized, for the first time, the natu-
rally occurring fatty acids 2a and 2b as well as the alkynoic
analogs 5-pentacosynoic (1a), 9-pentacosynoic (1b), and
2-pentacosynoic acids. Their HIV-RT inhibitory activity
was determined and compared to other shorter chain ana-
logs. Molecular modeling studies indicated the structural
basis underlying the inhibitory activities of the most potent
compounds.
1 3

Lipids
8.0. Single optimized conformation of each molecule
was energetically minimized employing the Tripos force
field [47] and the Powell conjugate gradient algorithm
[48] with a convergence criterion of 0.05 kcal/mol/Å and
Gasteiger–Hückel charges [49]. Molecular docking and
scoring protocols were carried out with GOLD 5.1 (Cam-
bridge Crystallographic Data Centre, Cambridge, UK)
[50]. To investigate the binding mode of 1a and 2a GOLD
5.1 default parameters were used. The coordinates for
HIV-1 RT solved at 2.8 Å (PDB ID 2HMI) [51] were used
during the molecular modeling investigation. Hydrogen
atoms were added in standard geometry using the GOLD
5.1 wizard. Histidine, glutamine, and asparagine residues
within the binding site were manually checked for possible
flipped orientation, protonation, and tautomeric states by
the GOLD side chain wizard. The binding cavity of HIV-1
RT was defined as all the amino acid residues encompassed
within a 20 Å radius sphere centered on the three dimen-
sional coordinates (−4.029; 122.631; 11.880) of the side
chain OH of Tyr188 in the p51 subunit. For each ligand the
docking protocols were repeated twenty-five times. The
ASP (Astex Statistical Potential) scoring function and vis-
ual inspection were employed to select the representative
conformation for each inhibitor.
Materials and Methods
Synthesis of the Fatty Acids
The synthetic experimental procedures, the analytical and
the spectral data of the products are presented in the sup-
porting information.
Reverse Transcriptase Colorimetric Assay
The Reverse Transcriptase Colorimetric Assay Kit was
purchased from the Roche Diagnostics Corporation, Indi-
anapolis, Indiana. The assay was carried out according to
the procedure described in Roche’s Reverse Transcriptase
Colorimetric Assay Kit manual with some minor modifica-
tions [45, 46]. First a 1000-μM stock solution of each of
the fatty acids in dimethyl sulfoxide (DMSO) was prepared
(final concentration of DMSO = 1 %). The stock solutions
were sonicated for 1 h, and then each stock solution was
further diluted to final concentrations of 100, 10, 1, 0.1 and
0.01 μM with Lysis buffer. The recombinant HIV-1-RT
was briefly dissolved in Tris-buffer solution [50 mM Tris,
80 mM KCl, 2.5 mM DTT, 0.75 mM EDTA and 0.5 % Tri-
ton X-100, pH 7.8] to reach a final concentration of 2 ng/
μL. The HIV-1-RT enzyme solutions with the experimen-
tal agents were incubated for 60 min at 37 °C. A 20 µL
aliquot of a reaction mixture [incubation buffer: 50 mM
Tris-buffer, containing 319 mM KCl, 33 mM magnesium
chloride, and 11 mM DTT; nucleotides: 50 mM Tris–HCl
at pH 7.8 with DIG-dUTP, biotin-dUTP and dTTP; and
template: template/primer hybrid poly (A) × oligo (dT)15]
was added and incubated for 60 min at 37 °C. The reactions
were transferred to microplate modules coated with strepta-
vidin and incubated for 60 min at 37 °C. After the incuba-
tion the plate was washed 5 times with the kit’s washing
buffer followed by the addition of 200 μL of peroxidase-
labeled antibody against digoxigenin (anti-DIG-POD) and
incubated for 60 min at 37 °C. After that, the wells were
washed five times with the kit’s 250 µL washing buffer
and incubated with 200 µL of ABTS substrate solution for
about 10 min at rt (25 °C) at 250 rpm. The absorbance was
taken at 405 nm (with reference wavelength at 490 nm) on
a microplate reader (MRX II; Dynex Technologies, Chan-
tilly, VA). The IC50 values were calculated using the Prism
Software (Graphpad, San Diego, CA) from titration curves
generated from the experimental values.
Cytotoxicity
The cytotoxicity of the unsaturated fatty acids against
peripheral blood mononuclear cells (PBMC) was tested
as described by Sanabria-Ríos et al. [52]. Briefly, PBMC
were cultured in a culture medium supplemented with
interleukin-2 (IL-2). These cells were seeded into a 96-well
microplate (20,000 cells/200 µL/well) and fatty acids were
added to the cell cultures. The final concentrations of fatty
acids ranged from 5 to 500 µM. The cells were incubated
at 37 °C for 3 days in a humidified 5 % CO2 incubator. The
cytotoxicity of the cells was evaluated by the MTT assay.
Results and Discussion
Synthesis
The 5- and 9-pentacosynoic acids 1a and 1b were pre-
pared according to Scheme 1. The synthesis of 1a and 1b
started with the protection of either commercially avail-
able 4-bromo-1-butanol (3a) or 8-bromo-1-octanol (3b)
utilizing 3,4-dihydro-2H-pyran (DHP) in chloroform with
catalytic amounts of p-toluenesulfonic acid (p-TSA) at rt
for 3 h, affording 4a or 4b in 93–99 % yields. The alkyne
coupling of 4a or 4b with (trimethylsilyl)acetylene using
n-BuLi in THF-HMPA at −78 °C, afforded silanes 5a or
5b in 92–94 % yields. The removal of the trimethylsilyl
Molecular Modeling
The 3D structures of the inhibitors 1a and 2a were con-
structed using standard geometric parameters provided
by the molecular modeling software package SYBYL
1 3

Lipids
Scheme 1 Synthesis of the pentacosynoic acids 1a and 1b, and the
pentacosenoic acids 2a and 2b. (i) DHP, p-TSA, CHCl3, 3 h; (ii)
n-BuLi, (trimethylsilyl)acetylene, HMPA, THF, −78 °C; (iii) TBAF,
THF, 0 °C; (iv) 1-bromononadecane or 1-bromopentadecane, n-BuLi,
HMPA, THF, −78 °C to 0 °C, 24 h; (v) p-TSA, MeOH, 45 °C, 3 h;
(vi) PDC, DMF, rt, 24 h; (vii) H2, Pd/C (10 %), quinoline, hexane;
(viii) PDC, DMF, rt, 24 h
protecting group in 5a or 5b was achieved with tetrabu-
tylammonium fluoride (TBAF) in THF, which resulted in
the terminal alkynes 6a or 6b in 98–99 % yields. A sec-
ond acetylenic coupling of 6a with 1-bromononadecane
or 6b with 1-bromopentadecane using n-BuLi in THF-
HMPA at −78 to 0 °C (the temperature was increased in
order to increase the solubility of the bromo alkane which
was added slowly for a period of 1 h) resulted in the
alkynes 7a or 7b in 60–81 % yields. The deprotection of
either 7a or 7b with p-TSA in methanol at 45 °C yielded
the desired 5-pentacosyn-1-ol (8a) or 9-pentacosyn-1-ol
(8b) in 83–99 % yields. Subsequent oxidation of 8a or
8b with pyridinium dichromate (PDC) in dimethylforma-
mide (DMF) afforded the 5-pentacosynoic acid (1a) or the
9-pentacosynoic acid (1b) in 59–80 % yields. The overall
yields for these six-step syntheses were 43 % for 1a and
34 % for 1b.
The natural products (5Z)-5-pentacosenoic acid (2a)
and (9Z)-9-pentacosenoic acid (2b) were also synthesized
as described in Scheme 1. This divergent synthetic proce-
dure used the corresponding alkynols 8a and 8b obtained
in the previous synthesis as starting points. Therefore, the
synthesis of 2a or 2b started from the previously synthe-
sized 8a or 8b, which were hydrogenated utilizing Lind-
lar’s conditions (hydrogen gas, 10 % Pd/C and quinoline
in hexane) in order to obtain the (5Z)-5-pentacosen-1-ol
(9a) or the (9Z)-9-pentacosen-1-ol (9b) in 70–97 % yields.
The pentacosenols 9a or 9b were oxidized with PDC in
DMF at rt resulting in either 2a in an 83 % yield or in 2b
in a 66 % yield. The synthesis of 2a was completed with
an overall yield of 58 % for the 8 steps (last two steps hav-
ing a combined yield of 81 %), while the synthesis of 2b
was completed with an overall yield of 20 %. This is the
first total synthesis for either 2a or 2b, which permits the
full characterization of these fatty acids that were previ-
ously identified in nature by only gas chromatographic
means [38–44].
HIV‑RT Inhibitory Studies
As mentioned above our goal was to assess the natural fatty
acids 2a and 2b, as well as their acetylenic analogs 1a and
1b as potential inhibitors of the DNA polymerase, reverse
transcriptase (RT). In this regard, we modified an existing
protocol for screening fatty acid candidates using a non-
radioactive colorimetric assay method, which assesses the
activity of HIV-1 RT [45, 46]. The colorimetric enzyme
immunoassay quantitatively determines the retroviral
reverse transcriptase activity by measuring the incorpora-
tion of digoxigenin- and biotin-labeled dUTP into DNA.
The DNA molecule labeled with biotin nucleotides binds
to the streptavidin coated MP module, followed by the
binding of the peroxidase-labeled digoxigenin antibody
molecule to the DNA molecule. Then, the 2,2′-azino-bis(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS) is added. The
peroxidase catalyzes the cleavage of ABTS resulting in a
colored product. The absorbance is directly correlated to
1 3

Lipids
the amount of DNA synthesized, thus it is proportional to
the level of RT activity.
or C20) ∆6 acetylenic fatty acid was tested, as exempli-
fied by the natural fatty acids 6-heptadecynoic or 6-icosy-
noic acids [53], no inhibition of the HIV-1 RT enzyme was
observed. Therefore, we can say that both fatty acid chain
length and the triple bond position are directly related to
the effectiveness of inhibition against the HIV-1 RT.
In the case of the monoenoic fatty acids 2a and 2b,
changing the position of the double bonds from ∆5 to ∆9
in the C25 acyl chain had only some minor effect on the
inhibitory activity towards HIV-RT (Table 1). For example,
acid 2a displayed an IC50 of 38 1 μM against HIV-1 RT,
In this study, RT inhibitory assays were performed in
duplicate in a range of 0.01–1000 μM for each acid along
with a positive control (no inhibitor of HIV-1 RT), negative
control (no HIV-1-RT enzyme), inhibitor control (nervonic
acid instead of experimental agent) and solvent control
(DMSO instead of experimental agent). Our results first
confirmed that DMSO can be used as solvent for the exper-
imental agents at maximal and final concentration of 2 %
with no effect on the activity of the HIV-1 RT. The results
of the experimental agents were compared with a positive
control, nervonic acid (NA), which according to our results
displayed an IC50 of 12 1 μM. The latter result supports
previous studies indicating that NA inhibits HIV-1 RT in
the micromolar range [36].
while acid 2b displayed an IC50 of 54
1 µM. It seems
that the olefinic acid 2b sits better into the HIV-RT active
site than the acetylenic acid 1b, being the cis double bond
stereochemistry critical for the inhibition.
It is known that the degree of unsaturation in fatty acids
affects its inhibitory activity towards DNA polymerase
enzymes, such as the DNA polymerase β [33–35]. Since
HIV-1 RT shares structural similarities with the DNA pol
β enzyme [36], this would imply, as we have shown, that
the unsaturation should also affect the inhibitory activity of
fatty acids towards the RT enzyme. Our results do reveal
a dependency between the degree and site of unsaturation
and the inhibitory activity of these fatty acids, e.g., ∆5-
25:1 versus 25:0. Moreover, the position of the triple bond
unsaturation was also important for the inhibition since a
triple bond at C-5 was more effective than a triple bond at
either C-2 or C-9. Our results also show that the longer the
fatty acid chain length (C25 vs. either C20 or C17) the greater
its inhibitory activity. This coincides with previous studies
with the HIV-1 RT as well as with DNA pol β, where the
long-chain fatty acids had greater inhibitory activity against
these enzymes [33].
As starting point for our fatty acid structural studies, we
used commercially available palmitic acid (16:0) as well
as pentacosanoic acid (25:0). Our results revealed that the
saturated (C16 or C25) as well as other shorter chain ∆6
acetylenic fatty acids (C17 or C20) were the least potent of
the acids tested (IC50 > 1000 μM). Among the acetylenic
fatty acids evaluated (Table 1), acid 1a was the best inhibi-
tor of HIV-1 RT with an IC50 of 24 1 μM. Surprisingly,
in our hands, acid 1b was not inhibitory towards the HIV-
RT enzyme. When the position of the triple bond in the C25
acyl chain was changed from ∆5 to ∆2, a decrease in the
inhibitory activity towards HIV-1 RT was also observed, as
exemplified by an IC50 of 566
1 μM displayed by the
2-pentacosynoic acid. Moreover, when a shorter chain (C17
Table 1 The reverse transcriptase (RT) inhibitory activity of the
tested fatty acids
Experimental agents
IC50 (μM)a
Molecular Modeling Studies
Palmitic acid (16:0)
>1000
>1000
>1000
6-Heptadecynoic acid (17:1)b
6-Icosynoic acid (20:1)b
2-Pentacosynoic acidc
To better understand the relationships between fatty acid
chain length, degree of unsaturation and inhibitory activ-
ity, we modeled the binding modes of acids 1a and 2a to
the p51 subunit of HIV-1 RT. Both fatty acids tested herein
have similar molecular interactions in the HIV-RT binding
pocket as those exhibited by nervonic acid [36] (Fig. 1a, b).
The carboxylate group of these fatty acids interacts with
key amino acids such as Lys65, Lys66, Lys220 and Tyr232,
while their long alkyl chain establishes attractive hydro-
phobic interactions with the side chain of Lys104, Ile195,
and His235. The modeling studies also indicated that
shorter carbon chains would not be capable of stabilizing
the molecule within the binding site. An ideal length of 25
carbons is required to fulfill the binding site, thereby inhib-
iting the enzymatic activity. Furthermore, the small differ-
ence in the inhibitory activities between 1a and 2a might be
due to the cis-configuration of acid 2a preventing it from
566
24
1
5-Pentacosynoic acid (1a)
9-Pentacosynoic acid (1b)
(5Z)-5-Pentacosenoic acid (2a)
(9Z)-9-Pentacosenoic acid (2b)
Nervonic acid (NA)
1
>1000
38
54
12
1
1
1
Pentacosanoic acid (25:0)
2 % DMSO
>1000
>1000
a
RT inhibitory assays were performed in duplicate. The IC50 values
were calculated using Prism Software (Graphpad, San Diego, CA)
from titration curves generated from the experimental values
The synthesis of the shorter-chain ∆6 acetylenic fatty acids was
b
previously described [53]
c
Obtained from the reaction of 1-tetracosyne with CO2 using n-BuLi
in THF
1 3

Lipids
Fig. 1 Modeled binding modes of the fatty acids 1a (green) and 2a
(orange) in the p51 subunit of HIV-1 RT. a, b Inhibitors and residues
involved in the ligand-receptor binding are indicated as stick models,
protein structure is indicated as cartoon and electrostatic interactions
as yellow dashed lines. c, d View of the fatty acid binding site indicat-
ing the solvent accessible surface of p51 subunit and the spatial com-
plementarity of the inhibitors (sphere model) (color figure online)
fully occupying the HIV-RT hydrophobic cavity formed by
Lys104, Ile195 and His235 (Fig. 1c, d). It is also impor-
tant to note that the unsaturations constrain the alkyl chain
degrees of freedom, thereby orienting the carbon chain
toward the hydrophobic pocket close to Ile195, Lys104 and
His235. The latter can explain the fact that pentacosanoic
acid does not inhibit HIV-1 RT at all.
dose necessary for HIV-1 RT inhibition, both acids 1a and
2a would not be cytotoxic at their optimum inhibitory dose
concentrations of 24 and 38 μM, respectively.
Conclusions
In summary, we have shown that 2a, as well as its acety-
lenic analogue 1a, are inhibitors of the HIV-1 RT enzyme.
While other modes of action could be envisaged for acids
1a and 2a, the one presented herein was consistent with the
enzymatic inhibitory studies. These results open the door
to the synthesis of other structurally related analogs, which
can combine in a single molecule, favorable structural fea-
tures of both NA and 1a for a more efficient HIV-RT inhi-
bition. The first total synthesis for the naturally occurring
fatty acids 2a and 2b was also accomplished.
Cytotoxicity
The cytotoxicities of 1a, 1b, 2a, 2b as well as nervonic
acid were tested against peripheral blood mononuclear
cells (PBMC) isolated from healthy volunteers following a
methodology previously described by us [52]. In this assay
none of the synthetic fatty acids tested, including nervonic
acid, displayed significant toxicity at concentrations higher
than 500 µM. Comparing these results with the effective
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Acknowledgments Part of the work described herein was initially
supported by Award Number SC1GM084708 from the National Insti-
tutes of General Medical Sciences (NIGMS) of the NIH. K. Rosado
acknowledges the support of the UPR RISE program for an under-
graduate fellowship. We thank Dr. Fred Strobel (Emory University)
for the high resolution mass spectral data. We acknowledge the sup-
port of the National Center for Research Resources and the NIGMS
of the NIH through Grant Number 5P20 GM 103475-13, the PRC-
TRC Grant through the Grant Numbers U54 RR026139 and 8U54MD
007587-03, and the National Institute on Minority Health and Health
Disparities of the NIH through Grant Number 8G12MD007583-28
for the use of their research facilities for performing cytotoxicity tests
involving PBMC.
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