Chemical probes for profiling fatty acid-associated proteins in living cells

Article abstract of DOI:10.1016/j.bmcl.2008.09.083

Chemical probes appended with reactive electrophiles afford powerful tools for profiling discrete protein families in living cells. Herein, we have synthesized cell-permeable chemical probes that target fatty acid-associated proteins. These fatty acid-bas

Full text of DOI:10.1016/j.bmcl.2008.09.083

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5982–5986
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chemical probes for profiling fatty acid-associated proteins in living cells
*
Anuradha Raghavan, Guillaume Charron, James Flexner, Howard C. Hang
The Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, 1230 York Avenue, Box 250, New York, NY 10065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2008
Revised 22 September 2008
Accepted 22 September 2008
Available online 30 September 2008
Chemical probes appended with reactive electrophiles afford powerful tools for profiling discrete protein
families in living cells. Herein, we have synthesized cell-permeable chemical probes that target fatty
acid-associated proteins. These fatty acid-based chemical probes contain acyloxymethylketone or
fluorophosphonate functional groups and an alkyne click chemistry tag for visualization of covalently
modified proteins by in-gel fluorescence scanning. Our fatty acid-based chemical probe affords new tools
to evaluate the activity/expression of lipid-associated proteins that should facilitate their functional
characterization and inhibitor discovery.
Keywords:
Click chemistry
Chemical probe
Fatty acid
Published by Elsevier Ltd.
Mechanism-based probe
Activity-based probe
Bioorthogonal labeling
Fatty acids control diverse biological processes in microbes and
animals. The metabolism of fatty acids provides a principle carbon
source for energy in cells, the mis-regulation of which is at the
heart of metabolic disorders. Besides being a source of nutrients,
the composition of fatty acids inside cells influences basic pro-
cesses such as vesicle trafficking and protein activity.1,2 Moreover,
the covalent attachment of fatty acids onto proteins directly
modulates their subcellular localization and signaling properties.3
While some key functions of fatty acids have emerged, the
mechanisms that link lipid metabolism to discrete cellular pro-
cesses is less clear. New tools for fatty acid-associated proteins
(transporters, chaperones and enzymes) in cells should facilitate
their characterization in physiology and disease.
Chemical probes appended with detection tags have provided
new opportunities to analyze complex proteomes.4–6 These mech-
anism- or activity-based probes covalently label specific protein
families and provide excellent tools to measure the expression
and/or activity of proteins in complex mixtures by virtue of the
appended detection tag.4–6 The selectivity of these chemical probes
towards various protein families is determined by the reactive
functional groups chosen for covalent labeling, and the surround-
ing chemical scaffold.4–6 A variety of chemical probes have been
generated using covalent electrophilic traps for enzyme classes
with active-site nucleophiles such as serine hydrolases, cysteine
proteases, glycosidases, kinases, phosphatases and other protein
families.4–6 For enzyme families without reactive nucleophiles,
photocrosslinking groups have been appended onto chemical
scaffolds to target proteases7–11 and histone deacetylases.12,13
Non-directed chemical probes have also been generated to label
unanticipated protein activities.14 Furthermore, these chemical
probes also label amino acid residues beyond the active site of
enzymes and provide unique pharmacological tools to profile
diverse enzyme/protein families.15–17 Attachment of detection tags
such as biotin onto chemical probes enables visualization and
identification of labeled proteins with streptavidin reagents. Alter-
natively, the introduction of fluorophores onto chemical probes
provides quantitative means for profiling the expression/activity
of protein families by in-gel fluorescence scanning.4–6 In addition
to profiling specific protein families in different cell types, mecha-
nism- or activity-based probes have provided powerful tools for
annotating the function of uncharacterized gene products, classifi-
cation of small molecule inhibitor specificities and even revealed
novel regulatory mechanisms of enzymes.4–6
The application of chemical probes to living cells should provide
new insight into the function of protein families not apparent in
cell lysates (Fig. 1A). Direct attachment of detection tags to chem-
ical probes has enabled the labeling of specific proteins in cell
lysates, but the pharmacological properties of biotin or fluorescent
dyes often precludes their passive diffusion into cells and also
influences specificity of protein labeling.5,6 The emergence of
bioorthogonal chemical reactions such as the Staudinger ligation
and Huisgen [3 + 2] cycloaddition or click chemistry (Fig. 1B)
allows the installation of detection tags onto chemical probes after
protein labeling in living cells (Fig. 1A).18 For example, the intro-
duction of alkynes or azides onto various chemical probes enabled
the labeling of diverse protein families in living cells.19–22 Cell
lysates can then be prepared and reacted with phosphine- or
* Corresponding author. Fax: +1 212 327 7276.
E-mail address: hhang@mail.rockefeller.edu (H.C. Hang).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.09.083

A. Raghavan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5982–5986
5983
Figure 1. Chemical probes targeted at fatty acid-associated proteins in living cells. (A) Administration of fatty acid-based chemical probes to live cells, followed by click
chemistry/in-gel fluorescence detection of labeled proteins. (B) CuI-mediated Huisgen [3 + 2] cycloaddition or click chemistry. CuSO4, tris(2-carboxyethyl)phosphine (TCEP)
and tris-(triazolyl)benzyl amine (TBTA) reagents for click chemistry. (C) Fatty acid-based chemical probes. (D) Detection tags for click chemistry/in-gel fluorescence scanning.
azide-functionalized detection tags for the visualization of specifi-
cally targeted proteins.19–22 This two-step protein labeling and
detection approach has even allowed the imaging of the cysteine
protease cathepsin B within the endocytic vacuoles of primary
macrophages infected with intracellular bacterial pathogens such
as Salmonella typhimurium.21 Application of mechanism-based
probes to living cells therefore affords new opportunities to ana-
lyze the function of specific protein families in vivo. Herein we
describe the synthesis and characterization of cell-permeable
chemical probes targeted at fatty acid-associated proteins in living
cells and the detection of labeled proteins using click chemistry/
in-gel fluorescence scanning.
di-2,6-trifluoromethybenzoate in the presence of potassium fluo-
ride to afford the azide/alkyne-modified fatty acid-AOMKs (1–4)
(Scheme 1). The alkyne-modified phosphonates were generated by
reaction of the terminal alkynyl–alkyl bromide with trimethylphos-
phine to yield the 16-carbon alkynyl-dimethylphosphonate (5)
(Scheme 1). Partial hydrolysis of compound 5 followed by fluorina-
tion with diethylaminosulfur trifluoride (DAST) afforded the 16-car-
bon alkynyl-methylfluorophosphonate (alk-14-MFP, 6) (Scheme 1).
With the fatty acid-based chemical probes in hand, we charac-
terized the ability of the AOMKs (1–4) and phosphonates (5 and
6) to selectively label proteins in living cells. The compounds
(1–6) were incubated in HeLa cells, a commonly used tumor cell
To explore the diversity of fatty acid-associated proteins in
cells, we designed azide/alkyne-modified fatty acid-based chemical
probes functionalized with reactive electrophiles (acyloxymethylk-
etone (AOMK) or fluorophosphonate) to target enzymes or proteins
that would utilize or bind fatty acids as well as fluorescent detection
tags for click chemistry analysis of covalently labeled proteins
(Fig. 1C). Fatty acid-based chemical probes containing the AOMK
functionality were synthesized to specifically target lipid-associated
proteins with reactive cysteines (1–4) (Fig. 1C). Chemical probes
containing the AOMK moiety have been shown to react with cys-
teine residues of proteases.23,24 We also synthesized alkyne-modi-
fied fatty acid derivatives bearing alkyl- and fluorophosphonate
groups that should target nucleophilic serine residues (5 and 6)
(Fig. 1C).25,26 The synthesis of the AOMK probes proceeded by isobu-
tylchloroformate activation of azide/alkyne-fatty acids followed by
reaction with diazomethane generated in situ (Scheme 1). The
corresponding chloromethylketones formed were then reacted with
line, at 50 lM for 1 h. Cell lysates were then prepared and reacted
with azide/alkyne-rhodamine detection tags (az-rho or alk-rho)
under standard click chemistry conditions,20 separated by gel elec-
trophoresis and analyzed by in-gel fluorescence scanning. Cells
were lysed with buffer containing high detergent to maximize the
recovery of proteins from various cellular compartments and in
the presence of protease inhibitor cocktail to minimize
post-lysis labeling of proteins with chemical probes. A number of
proteins are labeled with the fatty acid AOMKs and phosphonate
probes (Fig. 2). The profile of polypeptides selectively targeted by
azide- and alkynyl-fatty acid AOMKs were similar, but the non-spe-
cific labeling of proteins with the alk-rho detection tag is more pro-
nounced (Fig. 2). We therefore proceeded with the alkyne-modified
chemical probes and az-rho for optimal click chemistry detection.
While the fatty acid AOMK probes have some overlap in the labeled
proteins, the shorter chain (1 and 3) and long chain (2 and 4) fatty
acid AOMK probes also labeled unique sets of proteins (Fig. 2).
Scheme 1. Synthesis of fatty acid-based chemical probes. Reagents and conditions: (A) acyloxymethylketones. i—(a) IBCF, NMM, CH2Cl2. (b) CH2N2. (c) HCl/acetic acid (48–
50%). ii—KF, bis-2,6-trifluoromethybenzoic acid (35–45%). (B) Phosphonates. iii—P(OMe)3.(73%). iv—TMSBr, CH2Cl2. v—DAST, 0 °C (49%).

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A. Raghavan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5982–5986
These data demonstrate that different profiles of proteins are
targeted depending on the chain length of fatty acid AOMK probes.
In comparison to the AOMKs, the dimethylfluorophosphonate
probe (5) did not label proteins under conditions of the assay,
whereas the methylfluorophosphonate analog (6) targeted a large
number of proteins (Fig. 2). The differential reactivity of the dial-
kylphosphonates compared with fluorophosphonates in our exper-
iments is consistent with their observed inhibitory activities with
serine hydrolases in vitro.27 Time- and dose-dependent analysis
of the fatty acid-based probes in cells demonstrates that the AOMK
probes (alk-12-AOMK 3 and alk-14-AOMK 4) protein labeling is
D(OMe)-fmk did not reduce the labeling of any proteins targeted
by the fatty acid-based chemical probes (3, 4 or 6) (Supplementary
Fig. 2). These competitive labeling experiments in cells suggest
that the fatty acid AOMKs (3 and 4) label cysteine residues on
target proteins that are distinct from known cysteine proteases,
whereas alk-14-MFP (6) reacts primarily with proteins containing
nucleophilic serine residues. Interestingly, the fatty acid-based
probes (3, 4 and 6) appear to target discrete sets of proteins that
are not entirely blocked by known cysteine and serine hydrolase
inhibitors.
The generality of the fatty acid-based chemical probes (3, 4 and 6)
was determined with a variety of cell lines. Compounds (3, 4 and 6)
were incubated with a panel of cell lines and the profiles of selec-
tively labeled proteins were evaluated. In general, unique profiles
of proteins were labeled in different cell types for all three chem-
ical probes (Fig. 3). Direct comparison of the fatty acid AOMKs (3
and 4) revealed both discrete and overlapping profiles of polypep-
tides labeled in various cell types, even though these two fatty
acid-based chemical probes only differ in structure by two methy-
lene units. The profile of proteins targeted by the fatty acid AOMKs
(3 and 4) also varies between cell types (Fig. 3), suggesting that
these compounds do not just target abundant house keeping pro-
teins but may indeed label specific proteins associated with the
function of individual cell types. The profile of proteins targeted
by the alk-14-MFP (6) in the different cell types was significantly
broader than the AOMKs (Fig. 3). Between the various cell types,
the alk-14-MFP (6) labeled overlapping as well as discrete sets of
proteins (Fig. 3). In general, our experiments with alk-14-MFP (6)
are consistent with other fluorophosphonate probes reported,29,30
although the exact chemical scaffold and cell types examined are
different. Collectively, these experiments demonstrate that our
fatty acid-based chemical probes (3, 4 and 6) function in a variety
of cell types and label a diverse profile of proteins, which depends
upon the fatty acid chain length and the reactive electrophile of the
probe.
To determine the reactivity of our chemical probes with a
fatty acid-associated protein or enzyme, we evaluated the label-
ing of cytoplasmic phospholipase A2 in living cells. Cytoplasmic
phospholipase A2 (cPLA2) is a broadly expressed serine hydrolase
that cleaves fatty acids such as arachidonic acid from glyceroli-
pids and is associated with cellular differentiation, cytotoxicity
as well as inflammation.31 HeLa cells were incubated with DMSO
or our fatty acid-based chemical probes (3, 4 and 6), lysed and
analyzed for cPLA2 labeling after immunoprecipitation with
specific anti-cPLA2 polyclonal sera. While the fatty acid AOMK
probes (3 and 4) do not covalently react with cPLA2, the fluor-
optimal at 50
ylfluorophosphonate probe (6) protein labeling saturates at
approximately 20 M and labels proteins after 10 min of incubation
lM and 30 min (Supplementary Fig. 1). The meth-
l
with cells (Supplementary Fig. 1). These experiments demonstrate
our fatty acid-based chemical probes label unique profiles of
proteins in cells and that the alkyne-modified probes (3, 4 and 6)
provide superior visualization of labeled proteins using click chem-
istry/in-gel fluorescence scanning as compared to azide-modified
probes.
To survey the protein labeling selectivity of our fatty acid-based
probes, we performed competition experiments in cells with com-
pounds that react with nucleophilic cysteine and serine residues
on proteins or hydrolytic enzymes. Reactive amino acid residues
on proteins were blocked with either N-ethyl maleimide (NEM)
or broad-spectrum cysteine protease inhibitors27: E-64 (cathep-
sins) and z-VAD(OMe)-fmk (caspases). Alternatively, nucleophilic
serine residues were blocked with phenylmethylsulfonylfluoride
(PMSF) or phospholipase A2 inhibitors28: methyl arachidonyl fluor-
ophosphonate (MAFP) or bromoenol lactone (BEL). HeLa cells were
pretreated with cysteine and serine reactive compounds for 1 h
and subsequently incubated with our fatty acid-based probes
(3, 4 and 6), harvested and evaluated for selective protein labeling
(Supplementary Fig. 2). The majority of proteins targeted by fatty
acid AOMK (4) were blocked by NEM (Supplementary Fig. 2B),
whereas the profile of polypeptides labeled with fatty acid AOMK
(3) was only partially inhibited and altered by NEM (Supplemen-
tary Fig. 2A). Proteins targeted by the fatty acid AOMKs (3 and 4)
were not blocked by serine reactive compounds such as PMSF or
MAFP (Supplementary Fig. 2A and B). Interestingly, pre-incubation
of cells with BEL did not compete with protein labeling by the
AOMKs (3 and 4), but altered the profile of proteins that were tar-
geted. The competition experiments with alk-14-MFP (6), demon-
strated that NEM, PMSF, MAFP and BEL partially abrogated protein
labeling and also altered the profile of targeted proteins (Supple-
mentary Fig. 2C). The cysteine protease inhibitors E-64 and z-VA-
Figure 2. Analysis of fatty acid-based chemical probes in cells. Approximately 20
l
g of cell lysate was loaded in each lane. Fluorescently labeled proteins were visualized with
Amersham Biosciences Typhoon 9400 variable mode imager with ex/em 532/580 nm.

A. Raghavan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5982–5986
5985
Figure 3. Profile of proteins targeted by fatty acid-based chemical probes in various cell types. Fatty acid probes 3, 4 and 6 were administered at 50 mM for 1 h. Cell lysates
were prepared and analyzed for selective protein labeling as described in Figure 2. HeLa, cervical cancer cells; DC2.4, murine monocytes; Jurkat, human T cell lymphoma; NIH
3T3, murine fibroblasts; RAW 264.7, murine macrophages.
ophosphonate probe alk-14-MFP (6) effectively labeled cPLA2 at
the expected molecular weight (ꢀ96 kDa) as visualized by click
chemistry/in-gel fluorescence scanning and Western blot analysis
of cPLA2 protein (Fig. 4). These results demonstrate that our
chemical probes can efficiently label fatty acid-associated pro-
teins/enzymes in cells.
Our studies with the fatty acid-based chemical probes describe
progress towards new chemical tools for targeting lipid-associ-
ated proteins in living cells. The global identification of specific
proteins labeled by our chemical probes is currently underway
and should afford important insight into the reactivity profile of
AOMKs and fluorophosphonate group in cells. These studies
should complement the analysis of other electrophilic chemical
probes17,5 as well as fatty acid chemical reporters that target
fatty-acylated proteins.32 The ability to profile the expression/
activity of fatty acid-associated proteins/enzymes in living cells
should provide new opportunities to dissect their functions in
physiology and disease.
Acknowledgments
The authors thank Prof. Ben Cravatt and Prof. Dale Boger for the
opportunity to participate in this issue honoring Prof. Cravatt as
one of the 2008 Tetrahedron Young Investigators. This work was
supported by The Rockefeller University and Irma T. Hirschl/Mon-
ique Weil-Caulier Trust. A.R. thanks the New York Community
Trust-Heiser Grant for a post-doctoral fellowship.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.083.
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cells. HeLa lysates and cPLA2 immunoprecipitates were analyzed by click chemis-
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(lower panel).

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