Discovery of Hydrolysis-Resistant Isoindoline N -Acyl Amino Acid Analogues that Stimulate Mitochondrial Respiration

Article abstract of DOI:10.1021/acs.jmedchem.8b00029

N-Acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. We found that administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure, indicating that this pathway might be useful for treating obesity and associated disorders. We report the full account of the synthesis and mitochondrial uncoupling bioactivity of lipidated N-acyl amino acids and their unnatural analogues. Unsaturated fatty acid chains of medium length and neutral amino acid head groups are required for optimal uncoupling activity on mammalian cells. A class of unnatural N-acyl amino acid analogues, characterized by isoindoline-1-carboxylate head groups (37), were resistant to enzymatic degradation by PM20D1 and maintained uncoupling bioactivity in cells and in mice.

Full text of DOI:10.1021/acs.jmedchem.8b00029

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Discovery of Hydrolysis-resistant Isoindoline N-Acyl Amino
Acid Analogs that Stimulate Mitochondrial Respiration
Hua Lin, Jonathan Z Long, Alexander M. Roche, Katrin J. Svensson, Florence Dou, Mi Ra Chang,
Timothy Strutzenberg, Claudia Ruiz, Michael D Cameron, Scott J. Novick, Charles M. Berdan, Sharon
Louie, Daniel K. Nomura, Bruce M. Spiegelman, Patrick R. Griffin, and Theodore M. Kamenecka
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00029 • Publication Date (Web): 13 Mar 2018
Downloaded from http://pubs.acs.org on March 13, 2018
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Discovery of Hydrolysis-resistant Isoindoline N-Acyl Amino
Acid Analogs that Stimulate Mitochondrial Respiration
Hua Lin1, ‡, Jonathan Z. Long2, ‡, Alexander M. Roche3, Katrin J. Svensson2, Florence Dou3, Mi Ra
Chang1, Timothy Strutzenberg1, Claudia Ruiz1, Michael D. Cameron1, Scott J. Novick1, Charles M.
Berdan4, Sharon Louie4, Daniel K. Nomura4, Bruce M. Spiegelman3, Patrick R. Griffin1, Theodore M.
Kamenecka1*
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1Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL 33458
2Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305
3Departments of Cancer Biology and Cell Biology, DanaꢀFarber Cancer Institute and Harvard Medical School, Boston, MA
02215
4Departments of Chemistry, Molecular and Cell Biology, and Nutritional Sciences and Toxicology, University of California
Berkeley, Berkeley, CA 94720
ABSTRACT: Nꢀacyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1ꢀindependent respiꢀ
ration. We found that administration of Nꢀacyl amino acids to mice improves glucose homeostasis and increases energy expenditure
indicating that this pathway might be useful for treating obesity and associated disorders. We report the full account of the synthesis
and mitochondrial uncoupling bioactivity of lipidated Nꢀacyl amino acids and their unnatural analogs. Unsaturated fatty acid chains
of medium length and neutral amino acid head groups are required for optimal uncoupling activity on mammalian cells. A class of
unnatural Nꢀacyl amino acid analogs, characterized by isoindolineꢀ1ꢀcarboxylate head groups (37), were resistant to enzymatic degꢀ
radation by PM20D1 and maintained uncoupling bioactivity in cells and in mice.
INTRODUCTION
cose homeostasis. Mechanistically, Nꢀacyl amino acids directꢀ
ly act on isolated mitochondria to increase respiration, potenꢀ
tially by interacting with the SLC25 family of inner mitochonꢀ
drial transporters11.
The Nꢀacyl amino acids are a class of endogenous lipid meꢀ
tabolites with pleotropic bioactivities1. Specific members of
this metabolite class include Nꢀoleoyl serine, Nꢀarachidonoyl
glycine, and Nꢀoleoyl glycine, which have been shown to regꢀ
ulate bone remodelin2, pain sensation3, and food intake4, reꢀ
spectively. At a molecular level, these lipids can act as ligands
for ion channels5 (e.g., TRPV1) or Gꢀprotein coupled recepꢀ
tors6 (e.g., GPR92). To date, over fifty distinct species of Nꢀ
acyl amino acids have been detected in mammalian tissues7.
However, the full spectrum of biological functions for this
class of endogenous lipids remain incompletely characterized.
To more fully understand how Nꢀacyl amino acids stimulate
respiration, we herein disclose a full account of our structureꢀ
activity relationship (SAR) studies of Nꢀacyl amino acids and
their uncoupling bioactivity. The uncoupling activity of this
metabolite class is largely restricted to those with neutral amiꢀ
no acid head groups and unsaturated fatty acyl chains of mediꢀ
um length. By exploring unnatural analogs of these metaboꢀ
lites, we identified the proline derivative Nꢀoleoylꢀisoindolineꢀ
1ꢀcarboxylate 37 as a semisynthetic Nꢀacyl amino acid analog
with exceptional uncoupling activity. 37 possessed uncoupling
activity in cells and in mice, but unlike the natural metabolites,
was entirely resistant to enzymatic hydrolysis by PM20D1.
We have recently identified a novel function for several Nꢀ
acyl amino acids, including Nꢀoleoyl leucine, Nꢀoleoyl pheꢀ
nylalanine, and Nꢀarachidonoyl glycine. These compounds can
uncouple mitochondrial respiration, stimulating respiration on
isolated mitochondria in both cells and mice8. Uncoupling
oxidative respiration makes ATP production less efficient
resulting in increased metabolic rate, and loss of energy
RESULTS AND DISCUSSION
To determine the uncoupling activity of the Nꢀacyl amino acid
analogs, we tested their effect on cellular respiration of C2C12
mouse myoblast cells. Because these compounds show a timeꢀ
dependent effect on changes in respiration, we standardized
our assay results by reporting the maximal stimulation of resꢀ
piration as compared to DMSOꢀcontrol. Further standardizing
our respiration assays, all compounds were assayed at 50 µM
(see also Supplemental Figure 1). This is below the critical
micelle concentration for these analogs (see Supplemental
Figure 2)12. Initially, we restricted the scope to oleic acidꢀ
conjugated amino acids. As previously reported, C18:1ꢀPhe
through heat production9,10
. Key to this discovery was the
annotation of the circulating factor peptidase M20 domain
containing 1 (PM20D1) as a dominant enzyme that regulates
Nꢀacyl amino acid levels in vivo. Genetically increased circuꢀ
lating PM20D1 by adenoꢀassociated virus (AAV) delivery to
the liver augments circulating Nꢀacyl amino acids, leading to
increased respiration and blunted weight gain on high fat diet
in mice. Direct administration of Nꢀacyl amino acids to mice
by intraperitoneal (IP) injection also produces increased whole
body energy expenditure, with weight loss and improved gluꢀ
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Journal of Medicinal Chemistry
possess potent uncoupling bioactivity8, stimulating respiration
Page 2 of 7
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to ~160% of baseline levels (Table 1, compound 1). Amino
acid stereospecificity was not observed in this uncoupling
bioactivity, as both C18:1ꢀDꢀPhe and C18:1ꢀLꢀPhe stimulated
cellular respiration equally well (Table 1, compound 2). Conꢀ
sistent with our previous report, the amino acid head group
carboxylate was absolutely required for activity: removal of
the carboxylate moiety entirely abolished uncoupling activity
(Table 1, compound 3). Next, by varying the amino acid head
group, we found that amino acids containing neutral side
chains (e.g., Leu, Ile, Gln, Pro, Trp, compounds 4 – 8) were
potent uncouplers, but those compounds with charged side
chains (e.g., Lys, Tyr, Glu, compounds 9 – 11) were unable to
induce respiration (Table 1). Some tolerance for backbone
steric modifications were acceptable on the amino acid side.
For instance, inclusion of homoglycine or the dipeptide Glyꢀ
Gly head group preserved uncoupling bioactivity (Table 1,
compounds 12 and 13).
LꢀTyr
105 ± 13
106 ± 7
10
11
LꢀGlu
Glyꢀ
Gly
12
13
206 ± 32
185 ± 10
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Homoꢀ
glycine
aRespiration in C2C12 cells is shown as maximal increases
versus basal oligomycinꢀtreated respiration, which is normalꢀ
ized to 100%. Data are shown as means ± SEM, n=3ꢀ6/group;
bstimulation of respiration (baseline = 100%).
Table 1. Structure and uncoupling bioactivity of compounds
with different amino acid head groups.a
For the fatty acid side chain, we observed striking structural
requirements regarding length and type of unsaturation for
uncoupling bioactivity. By sequentially stepping through inꢀ
creasing fatty acid chain lengths, we found that fatty acyl
chains that were too short (e.g., < C12:0) or too long (e.g.,
>C20:0) possessed no uncoupling activity (Table 2, comꢀ
pounds 14 – 18). Within the “medium” fatty acyl chain range,
specific types of unsaturation potently improve the respiration
response. For instance, we observed a stepꢀwise increase in
uncoupling activity from C18:0ꢀPhe < C18:1ꢀPhe < C18:2ꢀPhe
(compound 16 vs. 1 vs. 19) that, surprisingly, drops off entireꢀ
ly with C18:3ꢀPhe (compound 20). Other specific olefin perꢀ
turbations also underscored the importance of specific types
and locations of unsaturation. For instance, compared to cisꢀ
∆9ꢀC18:1ꢀPhe (compound 1), transꢀ∆9ꢀC18:1ꢀPhe (Table 2,
compound 21) olefin shows essentially no uncoupling bioacꢀ
tivity. Similarly, cisꢀ∆6ꢀC18:1ꢀPhe and cisꢀ∆11ꢀC18:1ꢀPhe
(Table 3, compound 22 and 23) were all inferior to the parent
compound 1.
Head
group
b
Cmpd
1
R
%
LꢀPhe
161 ± 12
2
3
4
DꢀPhe
n/a
173 ± 9
104 ± 4
178 ± 13
Given the strong influence of unsaturation on the acyl chain
length, we systematically increased unsaturation of C20:0ꢀPhe
(compound 17), a parent compound that initially does not posꢀ
sess uncoupling activity. Again, increasing unsaturation augꢀ
ments uncoupling bioactivity with an apparent optimal unsatuꢀ
ration at C20:4 (Table 2, compounds 24 – 26). A similar stratꢀ
egy to increase unsaturation also converted inactive C22:0ꢀPhe
(compound 18) into a potent uncoupler (C22:6ꢀPhe, compound
27). The specific requirement for unsaturation is reminiscent
of cannabinoid receptor ligands, in which only arachidonoylꢀ
but not other fatty acylꢀcontaining lipids can agonize the reꢀ
ceptor13. The unsaturation specificity observed in this Nꢀacyl
amino acid series (Table 2) is consistent with the shape comꢀ
plementarity imposed by interactions with proteinaceous facꢀ
tors that mediate uncoupling. Taken together, our data map out
the optimal requirements for uncoupling responses within the
Nꢀacyl amino acid class: an uncharged carboxylateꢀcontaining
head group, a medium fatty acid chain length, and specific
sites of unsaturation along the acyl chain.
LꢀLeu
5
6
LꢀIle
167 ± 14
164 ± 4
LꢀGln
7
8
9
LꢀPro
LꢀTrp
LꢀLys
182 ± 7
147 ± 9
108 ± 4
Table 2. Structure and uncoupling bioactivity of compounds
with different fatty acid side chains.a
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Fatty
acid
b
Cmpd
R
%
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29
104 ± 3
97 ± 5
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8
14
15
C12:0
C16:0
96 ± 4
180 ± 20
aRespiration in C2C12 cells is shown as maximal increases
versus basal oligomycinꢀtreated respiration, which is normalꢀ
ized to 100%. Data are shown as means ± SEM, n=3ꢀ6/group;
bstimulation of respiration (baseline = 100%).
16
17
18
C18:0
C20:0
C22:0
148 ± 12
95 ± 8
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We also surveyed unnatural amino acid head groups. We were
intrigued by the potent uncoupling observed with C18:1ꢀPro
(compound 7) and noted that this derivative with a cyclic head
group is the most structurally distinct from the other Nꢀacyl
amino acids tested. We therefore synthesized and tested a vaꢀ
riety of unnatural proline and homoproline Nꢀacyl amino acid
derivatives with oleate as a fatty acyl chain. Testing these unꢀ
natural analogs in cellular respiration assays revealed that they
all stimulated uncoupling to a maximum of ~150ꢀ240% of
DMSO control (Table 4, compounds 30 – 35). Derivatization
of these isoindoline head groups with linoleoyl instead of
oleoyl acyl groups also produced potent uncouplers (Table 4,
compounds 36 and 37). These analogs 30 – 37 demonstrate
that the uncoupling bioactivity of Nꢀacyl amino acids can exꢀ
tend beyond structures containing only the proteinogenic amiꢀ
no acids.
100 ± 3
19
20
C18:2
C18:3
218 ± 10
109 ± 12
transꢀ
C18:1
21
22
23
107 ± 3
110 ± 12
115 ± 8
∆6ꢀ
C18:1
∆11ꢀ
C18:1
Table 4. Structure and uncoupling bioactivity of compounds
with unnatural prolineꢀ and homoprolineꢀdrived head groups.a
24
25
26
27
C20:1
C20:4
C20:5
C22:6
103 ± 3
200 ± 12
259 ± 43
166 ± 11
Fatty
acid
b
Cmpd
30
R
%
CO2H
C18:1
174 ± 3
N
aRespiration in C2C12 cells is shown as maximal increases
versus basal oligomycinꢀtreated respiration, which is normalꢀ
ized to 100%. Data are shown as means ± SEM, n=3ꢀ6/group;
bstimulation of respiration (baseline = 100%).
31
32
C18:1
C18:1
187 ± 19
216 ± 17
Given the structure activity relationships we observed by
simply varying the two sides of the amide bond, we next exꢀ
plored an alternative bond linkage between the fatty acid and
amino acid to identify those that may possess superior properꢀ
ties as compared to the naturally occurring metabolites, with
the ultimate goal of developing improved mitochondriaꢀ
uncoupling analogs that might be useful for the treatment of
obesity and metabolic disease14,15. Towards this end, we synꢀ
thesized two urea analogs of C18:1ꢀGly and C18:1ꢀPhe that
we hypothesized might be less metabolically labile compared
to the natural amides, and tested their effects on cellular respiꢀ
ration. Both urea analogs failed to induce respiration on cells,
demonstrating a potential preference for the presence of an
amide bond in the bioactivity of this class of compounds (Ta-
ble 3).
33
C18:1
240 ± 24
34
35
C18:1
C18:1
190 ± 12
146 ± 5
Table 3. Structure and uncoupling bioactivity of unnatural
ureaꢀcontaining Nꢀacyl amino acid analogs.a
b
Cmpd
Structure
%
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37
C18:2
C18:2
185 ± 18
190 ± 12
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aRespiration in C2C12 cells is shown as maximal increases
versus basal oligomycinꢀtreated respiration, which is normalꢀ
ized to 100%. Data are shown as means ± SEM, n=3ꢀ6/group;
bstimulation of respiration (baseline = 100%).
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Nꢀacyl amino acids can be hydrolytically inactivated through
cleaveage by PM20D1, a bidirectional amidase that cleaves
fatty acid amides to liberate free fatty acids and amino acids8.
Given the unusual cyclic structures of 30 – 37, we hypotheꢀ
sized that these analogs might have lower rates of hydrolysis,
and therefore higher stability. We therefore incubated purified,
recombinant murine PM20D1 with a canonical Nꢀacyl amino
acid substrate, Nꢀoleoyl phenylalanine, or one of the unnatural
Nꢀacyl amino acids. We observed >90% hydrolysis of Nꢀ
oleoyl phenylalanine to oleic acid in our assay conditions,
demonstrating that this enzyme efficiently cleaves naturally
occurring Nꢀacyl amino acids. Remarkably, all unnatural Nꢀ
acyl amino acids tested were completely resistant to PM20D1ꢀ
mediated hydrolysis (Fig. 1a). Consistent with these observaꢀ
tions, when endogenously occurring Nꢀacyl amino acids or
unnatural analogs were incubated with mouse liver microꢀ
somes, the unnatural analogs also maintained exceptionally
long halfꢀlives (Fig. 1b): as a group, the average halfꢀlife of
the natural Nꢀacyl amino acids was 8 min, whereas the average
for Nꢀacyl analogs with unnatural head groups was 64 min.
The tertiary amide structures in 30ꢀ37 may also contribute to
improved metabolic stability relative to the secondary amides
tested. These data therefore demonstrate that changing the
amide head group can alter the downstream metabolism of Nꢀ
acyl amino acid analogs.
Fig. 1. (A) Amount of the indicated compound remaining in
the presence of PBS or recombinant, murine PM20D1 after
incubation at 37ºC for 1 h. Data are shown as means ± SEM, n
= 3/group. *** P < 0.001. (B) Halfꢀlife (min) of the indicated
compound after incubation in mouse liver microsomes. “Natuꢀ
ral” indicates endogenously present Nꢀacyl amino acid whereꢀ
as “unnatural” indicates an Nꢀacyl amino acid analog with a
nonꢀproteinogenic head group.
Lastly, we tested the uncoupling bioactivity of unnatural Nꢀ
acyl amino acid analogs in mice. Towards this end, we adoptꢀ
ed a previously used assay in which mice rendered obese by
high fat diet feeding (dietꢀinduced obesity, DIO) are given Nꢀ
acyl amino acids daily by IP administration. Synthetic chemiꢀ
cal uncouplers such as 2,4ꢀdinitrophenol have been shown to
blunt weight gain in this model15, and administration of Nꢀacyl
amino acid led to weight loss8. For these experiments, we seꢀ
lected compound 37 because this compound showed excellent
uncoupling bioactivity (Table 4) and resistance to hydrolysis
(Fig. 1). DIO mice (initial weight, 46.8 ± 0.6 g) were treated
with increasing doses of compound 37 (5, 10, and 25
mg/kg/day IP) daily for 7 consecutive days (Fig. 2a). Though
no differences in weight were observed at 5 mg/kg vs. vehicleꢀ
treated mice, both 10 and 25 mg/kg doses of compound 37 led
to doseꢀdependent blunting of body weight gain (net weight
change, 0.2 ± 0.2 g and –1.2 ± 0.6 g, respectively, vs. 1.1 ± 0.1
g for vehicleꢀtreated mice, P < 0.05 for each comparison).
Food intake was not statistically significantly reduced in mice
at these higher doses (Fig. 2b). However, ad lib blood glucose
2 h after the last compound injection was significantly lower
in mice treated with 25 mg/kg compound 37 versus vehicleꢀ
treated mice (172 ± 7 mg/dl versus 204 ± 7 mg/dl, respectiveꢀ
ly, Fig. 2c). These data therefore indicate that unnatural Nꢀacyl
amino acid 37 maintains uncoupling bioactivity in vivo.
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reacted with amino esters 44 in the presence of DIPEA. The
1
2
3
resulting urea esters 45 were hydrolyzed with LiOH to provide
the desired urea analogs 28 or 29 in good yields (using Genꢀ
eral procedure C) (Scheme 2).
4
5
6
Scheme 1. Synthesis of Natural and Unnatural NꢀAcyl Amino
Acid Analogsa
7
8
9
10
11
12
13
14
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21
22
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Scheme 2. Synthesis of Urea Analogsa
aReaction conditions: (a) oxalyl chloride, DMF, DCM; (b)
K2CO3, acetone/H2O or DIPEA, DCM; (c) NH4OH, THF; (d)
LiAlH4, THF; (e) CDI, DCM; (f) DIPEA, DCM; (g) LiOH,
then HCl.
CONCLUSIONS
Here we provide a detailed account of the synthesis and strucꢀ
ture activity relationships of the Nꢀacyl amino acids and their
uncoupling activity on mammalian cells. Our data reveal the
structural requirements for the uncoupling bioactivity of this
class of endogenous metabolites, which include a strict reꢀ
quirement for neutral carboxylateꢀcontaining head groups,
medium chain fatty acids with specific unsaturation, and an
amide bond connecting the two. Through exploration of unꢀ
natural Nꢀacyl amino acid analogs, we identify Nꢀacyl isoindoꢀ
line 37 as a potent, hydrolysisꢀresistant compound that disꢀ
plays uncoupling bioactivity in cells and in vivo. Projecting
forward, compound 37 or other hydrolysisꢀresistant Nꢀacyl
amino acid analogs may serve as useful probes for understandꢀ
ing the full spectrum of bioactivities associated with the Nꢀ
acyl amino acid family of lipids. Such compounds may also
serve as leads for alternative structural classes that may have
widened therapeutic windows or altered beneficial and adverse
profiles compared with the more “classical” chemical uncouꢀ
plers such as 2,4ꢀdinitrophenol.
Fig. 2. (AꢀC) Total change in body weight (A), food intake
(B), and ad lib blood glucose (C) of dietꢀinduced obese mice
(19 weeks high fat diet) after 7 days daily treatment with the
indicated dose (IP) of compound 37. Prior to compound adꢀ
ministration, mouse weights were not different between the
groups (average weight across all groups, 46.8 ± 0.6 g). Data
are shown as means ± SEM, n=5/group. *, P < 0.05; **, P <
0.01 for the indicated dose versus vehicleꢀtreated mice.
CHEMISTRY
Compounds were synthesized as depicted in Scheme 1 followꢀ
ing wellꢀknown procedures. Commercially available acid
chlorides 39 (using General procedure A), or those generated
from acid 38 with oxalyl chloride (using General procedure
B), were coupled with the corresponding natural or unnatural
amino acids 40 to provide Nꢀacyl amino acid analogs 1ꢀ27 and
30ꢀ37 (Scheme 1). Oleoyl chloride reacted with ammonia
hydroxide to give primary amide 41, which was reduced with
LiAlH4 to afford primary amine 42. Imidazole intermediate 43
was obtained by treatment of amine 42 with CDI, which then
EXPERIMENTAL SECTION
All solvents and chemicals were reagent grade. Unless otherꢀ
wise mentioned, all reagents and solvents were purchased
from commercial vendors and used as received. Flash column
chromatography was carried out on a Teledyne ISCO Comꢀ
biFlash Rf system using prepacked columns. Solvents used
include hexane, ethyl acetate (EtOAc), dichloromethane and
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Journal of Medicinal Chemistry
methanol. Purity and characterization of compounds were
Page 6 of 7
mediate 45 (75ꢀ80%). Step 5: To a solution of intermediate 45
in THF and H2O (1:1) was added LiOH (5 eq.). The mixture
was stirred at room temperature for 3 hours. The mixture was
then acidified with HCl (1M) until pH<3. Then the mixture
was extracted with ethyl acetate, washed with brine, dried
(Na2SO4) and concentrated to give the desired compound 28 or
29 (94ꢀ96%).
1
2
3
4
5
6
7
8
established by a combination of HPLC, TLC, mass spectromeꢀ
try, and NMR analyses. 1H and 13C NMR spectra were recordꢀ
ed on a Bruker Avance DPXꢀ400 (400 MHz) spectrometer and
were determined in chloroformꢀd or DMSOꢀd6 with solvent
peaks as the internal reference. Chemical shifts are reported in
ppm relative to the reference signal, and coupling constant (J)
values are reported in hertz (Hz). Thin layer chromatography
(TLC) was performed on EMD precoated silica gel 60 F254
plates, and spots were visualized with UV light or iodine stainꢀ
ing. Low resolution mass spectra were obtained using a Therꢀ
mo Scientific ultimate 3000/ LCQ Fleet system (ESI). High
resolution mass spectra were obtained using a Thermo Scienꢀ
tific EXACTIVE system (ESI). All test compounds were
greater than 95% pure as determined by qNMR on a Bruker
Avance DPXꢀ400 (400 MHz) spectrometer.
PM20D1 hydrolysis assays. 10 nmol of the indicated
compound was incubated in 100 ꢁl PBS (100 ꢁM initial subꢀ
strate). Reactions were initiated by the addition of PBS or
mPM20D1 (5 ꢁl). After 1 h at 37ºC, reactions were quenched
with 600 ꢁl of a 2:1 v/v mixture of chloroform and methanol
with 10 nmol d31ꢀpalmitate as an internal standard. The reacꢀ
tions were vortexed and the organic layer was transferred to a
sample vial for analysis by LCꢀMS. For separation of polar
metabolites, normalꢀphase chromatography was performed
with a Lunaꢀ5 mm NH2 column (50 mm × 4.60 mm, Phenomꢀ
enex). Mobile phases were as follows: Buffer A, acetonitrile;
Buffer B, 95:5 water/ acetonitrile with 0.1% formic acid or
0.2% ammonium hydroxide with 50 mM ammonium acetate
for positive and negative ionization mode, respectively. The
flow rate for each run started at 0.2 ml/min for 2 min, followed
by a gradient starting at 0% B and increasing linearly to 100%
B over the course of 15 min with a flow rate of 0.7 ml/min,
followed by an isocratic gradient of 100% B for 10 min at 0.7
ml/min before equilibrating for 5 min at 0% B with a flow rate
of 0.7 ml/min. MS analysis was performed with an elecꢀ
trospray ionization (ESI) source on an Agilent 6430 QQQ
LC−MS/MS. The capillary voltage was set to 3.5 kV, and the
fragmentor voltage was set to 100 V. The drying gas temperaꢀ
ture was 325 °C, the drying gas flow rate was 10 l/min, and
the nebulizer pressure was 45 psi. Monitoring of hydrolysis
starting materials and products was performed by scanning a
mass range of m/z 50ꢀ1200. Peaks corresponding to the liberꢀ
ated fatty acids (products) or the intact starting material was
integrated.
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General procedure A. To the amino acid (2 eq.) in aceꢀ
tone and water (0.1 M, 1:1, v/v) was added K2CO3 (3 eq.) and
o
acyl chloride (1 eq.) at 0 C. The reaction mixture was stirred
at room temperature overnight and was then acidified with
HCl (1 M) until pH<3. The mixture was extracted with ethyl
acetate, washed with brine, dried (Na2SO4) and filtered. The
filtrate was concentrated in vacuo, and the residue was puriꢀ
fied by column chromatography (silica gel, ethyl aceꢀ
tate/hexanes) to give the desired product.
General procedure B. To a solution of fatty acid (1 eq.) in
DCM was added oxalyl chloride (1.2 eq.) and one drop of
DMF at 0 oC. The reaction was aged at room temperature for 2
hours and then concentrated in vacuo. The crude residue was
dissolved in DCM and added to a suspension of amino acid
(1.5 eq.) and DIPEA (2 eq.) and DCM. The reaction mixture
was stirred at room temperature overnight and was then acidiꢀ
fied with 1.0M HCl to pH<3. The result mixture was extracted
with DCM, washed with brine, dried (Na2SO4), and concenꢀ
trated. The crude residue was purified by flash chromatogꢀ
raphy on silica gel to give the product.
General procedure C. Step 1: To a 0 oC solution of oleoyl
chloride (1 g) in THF (20 mL) was added ammonia hydroxide
(10 eq.). The mixture was allowed to warm to room temperaꢀ
ture and stirred for 3 hours. The mixture was filtered to give
the desired oleamide 41 as a colorless solid (796 mg, 86%).
Step 2: To a suspension of LiAlH4 (2 eq.) in THF was added
ASSOCIATED CONTENT
Supporting Information.
The Supporting information is available free of charge on the
ACS Publications website at DOI:
Full characterization data on all analogs including Molecular
Formula Strings, cell respiration and microsome stability assays,
PM20D1 generation, Seahorse experimentals, critical micelle
concentration measurements, and in vivo details.
o
oleamide in one portion at 0 C. The mixture was heated to
reflux overnight. Then the mixture was quenched with water
o
(3 drops) at 0 C, followed by 1 M NaOH solution and then
stirred at room temperature for 1 hour. The suspension was
filtered through celite. The filtrate was diluted with ethyl aceꢀ
tate, washed with water and brine, dried (Na2SO4) and concenꢀ
trated to give (Z)ꢀoctadecꢀ9ꢀenꢀ1ꢀamine 42 (726 mg, 95%)
which was used for the next step without further purification.
Step 3: To a solution of (Z)ꢀoctadecꢀ9ꢀenꢀ1ꢀamine 42 in DMF
AUTHOR INFORMATION
Corresponding Author
*Phone: 561ꢀ228ꢀ2207. Eꢀmail: kameneck@scripps.edu.
Author Contributions
o
was added DIEA (2 eq.) and CDI (1.5 eq.) at 0 C. The mixꢀ
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manuꢀ
script.
ture was stirred at room temperature overnight. The mixture
was then diluted with ethyl acetate, washed with NaHCO3,
brine, dried (Na2SO4) and concentrated. Purification of the
crude residue on silica gel afforded (Z)ꢀNꢀ(octadecꢀ9ꢀenꢀ1ꢀyl)ꢀ
1Hꢀimidazoleꢀ1ꢀcarboxamide 43 (795 mg, 81%). Step 4: To a
‡These authors contributed equally.
ORCID
Hua Lin: 0000ꢀ0002ꢀ0840ꢀ6553
solution
of
(Z)ꢀNꢀ(octadecꢀ9ꢀenꢀ1ꢀyl)ꢀ1Hꢀimidazoleꢀ1ꢀ
carboxamide 43 in DMF was added DIEA (2 eq.) and amino
ester 44. The mixture was stirred at room temperature overꢀ
night. Then the mixture was diluted with ethyl acetate, washed
with NaHCO3, brine, dried (Na2SO4) and concentrated in vacꢀ
uo. The crude residue was purified on silica gel to give interꢀ
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
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Page 7 of 7
Journal of Medicinal Chemistry
acids based on a targeted lipidomics approach. J. Lipid Res. 2010, 51
(1), 112ꢀ119.
This work was supported by grants from the National Institutes of
Health (DK31405, B.M.S.; DK105203, J.Z.L.; and
K99DK111916, K.J.S.).
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M. P.; Paulo, J. A.; Gygi, S. P.; Griffin, P. R.; Nomura, D. K.;
Spiegelman, B. M. The secreted enzyme PM20D1 regulates lipidated
amino acid uncouplers of mitochondria. Cell 2016, 166 (2), 424ꢀ435.
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Doulcier, A. M.; Bouillaud, F.; Ricquier, D. The biology of
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L. A.; Griffin, G.; Gibson, D.; Mandelbaum, A.; Etinger, A.;
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ABBREVIATIONS USED
UCP1, uncoupling protein 1; TRPV1, transient receptor potenꢀ
tial cation channel subfamily V member 1; GPR92, Gꢀprotein
coupled receptor 92; SLC25, solute carrier family 25; DMSO,
Dimethyl sulfoxide; CDI, Carbonyldiimidazole; DIPEA, N,Nꢀ
Diisopropylethylamine; DMF, Dimethylformamide; DCM,
Dichloromethane; THF, Tetrahydrofuran.
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TABLE OF
CONTENTS
GRAPHIC
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