Incorporation of a fluorous diazirine group into phosphatidylinositol 4,5-bisphosphate to illustrate its interaction with ADP-ribosylation factor 1

Article abstract of DOI:10.1039/c2ob25276g

Phosphatidylinositides are one family of the most versatile signaling molecules in cells, yet how they interact with different proteins to regulate biological processes is not well understood. Towards a general strategy to identify phosphatidylinositide-protein interactions, a fluorous diazirine group has been incorporated into phosphatidylinositol 4,5-bisphosphate (PIP ₂). The modified PIP₂ was effectively cleaved by phospholipase C, one signaling protein that utilizes PIP₂ as its endogenous substrate. Upon light illumination, the PIP₂ probe effectively crosslinks with small GTPase ADP-ribosylation 1 to form a complex, suggesting that the probe might be suitable to identify PIP₂- interacting proteins on the proteome level. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob25276g

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Incorporation of a fluorous diazirine group into phosphatidylinositol
4,5-bisphosphate to illustrate its interaction with ADP-ribosylation factor 1†
Weigang Huang,a Wei Sun,a Zhiquan Song,a Yanbao Yu,b Xian Chenb and Qisheng Zhang*a
Received 6th February 2012, Accepted 1st June 2012
DOI: 10.1039/c2ob25276g
traditional affinity pulldown approaches likely fail to identify
many PI-interacting proteins.
Phosphatidylinositides are one family of the most versatile
signaling molecules in cells, yet how they interact with
different proteins to regulate biological processes is not well
understood. Towards a general strategy to identify phospha-
tidylinositide–protein interactions, a fluorous diazirine group
has been incorporated into phosphatidylinositol 4,5-bispho-
sphate (PIP2). The modified PIP2 was effectively cleaved by
phospholipase C, one signaling protein that utilizes PIP2 as
its endogenous substrate. Upon light illumination, the PIP2
probe effectively crosslinks with small GTPase ADP-ribosy-
lation 1 to form a complex, suggesting that the probe might
be suitable to identify PIP2-interacting proteins on the pro-
teome level.
Fluorous molecules have seen increased applications in
addressing various biological problems.7,8 The fundamental
principle behind these applications is that fluorous molecules
tend to preferentially partition into the fluorocarbon enriched
phase of a bi-phase or tri-phase system.9–11 Some examples of
the recent advancement include fluorous proteomics and metab-
olite enrichment,12,13 fluorous small molecule microarrays,14–16
and fluorous delivery agents.17,18 These successes prompt us to
couple fluorous chemistry with photocrosslinking to identify
interacting proteins of endogenous PIs. As illustrated in Fig. 1A,
the fluorous affinity group Rf is incorporated into a substrate to
crosslink with its interacting proteins. The crosslinked proteins
or protein complexes are digested with proteases along with
other proteins in the cells. The resulting peptide mixture will
then be separated through fluorous solid phase extraction (FSPE)
and the fluorinated peptides will be enriched and analyzed.
Because the enrichment process is on peptides instead of pro-
teins, this approach will only identify fluorous group labeled
interacting proteins, and with high sensitivity. This approach is
simple and cost effective compared to other bioreagents, and
orthogonal in biological systems. In addition, the fluorous tags
are chemically inert and stable in tandem mass spectrometric
analysis.12
Phosphatidylinositol 4,5-bisphosphate (PIP2) is one of the
seven endogenous PIs and is a substrate or regulator for a
number of important signaling proteins such as PI3K, phospho-
lipase C (PLC), and ion channels. To illustrate the application of
the fluorous photoaffinity labeling approach, we designed probe
1 (Fig. 1B) that incorporates a fluorous diazirine19 unit into the
side chain of PIP2. In this design, the PIP2 unit functions as the
substrate for PIP2-interacting proteins while the diazirine func-
tions as the photoaffinity labeling group. As a control, the non-
fluorous PIP2 analog 2 (Fig. 1B) was also generated.
Dynamic regulation of the composition of cellular membranes,
particularly the phosphatidylinositide (PI) family of lipids, is
fundamental to many biological processes such as cell prolifera-
tion and migration.1,2 The enzymes that regulate PI metabolism,
such as phosphatidylinositol-3-kinase (PI3K) and phosphatase
and tensin homolog (PTEN), have been directly linked to a
number of diseases including cancer and diabetes.3 Various PIs
are not only integral components of cell membranes, but also
one of the most versatile endogenous signaling molecules due to
their dynamic interactions with a wide array of proteins. Under-
standing how different PIs interact with their binding proteins is
thus highly significant because potential therapeutic intervention
of PI-signaling could be designed.4 Indeed, unbiased pulldown
experiments have been carried out to identify interacting proteins
of various PIs on the proteome level.4–6 In these experiments,
phosphatidylinositides are immobilized on solid supports or lipo-
somes as baits to capture their interacting proteins through non-
covalent interactions. However, the binding affinity of PIs with
proteins is typically at the low μM range. In addition, some
PI-interacting proteins such as protein kinase B (PKB, aka Akt)
have low abundance in the cells. These features make the
The synthesis of the fluorous probe 1 started with compound
3 (Scheme 1), which was synthesized from glycerol according to
the literature.20 Coupling of 3 with heptanoic acid in the pres-
ence of dicyclohexylcarbodiimide (DCC) and 4-dimethylamino-
pyridine (DMAP) at 0 °C generated 4 in 98% yield. Removal of
the p-methoxybenzyl (PMB) group in 4 with 2,3-dichloro-
5,6-dicyanobenzoquinone (DDQ) in wet CH2Cl2 gave 5 without
any detectable migration of the acyl group from the C2- to the
aDivision of Chemical Biology and Medicinal Chemistry, University of
North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
E-mail: qszhang@unc.edu
bDepartment of Biochemistry and Biophysics, University of North
Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob25276g
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Fig. 1 (A) Fluorous photoaffinity labeling approach to identify small molecule interacting proteins. (B) Chemical structures of PIP2–fluorous diazir-
ine 1 and a non-fluorous PIP2 analog 2.
Scheme 1 Synthesis of the fluorous diazirine–PIP2 analog 1.
C3-position. Reaction of diacylglycerol 5 with dibenzyl diisopro-
pylphosphoramidite, which was protonated by weakly acidic
tetrazole to facilitate the subsequent nucleophilic addition,
provided the phosphorylation reagent in 92% isolated yield. This
reagent was not stable, and was used immediately after prepa-
ration for coupling to the protected inositol phosphate precursor
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6.21 Upon oxidation with t-butylperoxide, the phosphate 7 was
synthesized in 81% yield. Removal of the methoxymethyl
(MOM) protective group with trimethylsilyl bromide (TMSBr)
in CH2Cl2 followed by hydrogenolysis to remove the benzyl and
carboxybenzyl (Cbz) groups then provided 8 in 80% yield. The
terminal amine at the sn-1 position in 8 then coupled with
the fluorous diazirine 9, which was synthesized as described
in the literature,19 to generate the PIP2 probe 1. The PIP2 deriva-
tive 2 was synthesized in a similar sequence of reactions.
Under physiological conditions, PIP2 can be hydrolyzed by
phospholipase C (PLC) isozymes to generate second messengers
inositol trisphosphate (IP3) and diacylglycerol (DAG). To
demonstrate that the fluorous PIP2 derivative 1 retains functions
of PIP2, we tested its capacity as a PLC substrate and analyzed
the reaction mixtures by liquid chromatography-mass spec-
trometry (LC-MS). When probe 1 was incubated with purified,
wild type PLC-δ1, a new peak was observed (Fig. 2) and later
confirmed as the diacylglycerol analog 10 (Fig. S1†) by mass
spectrometry. In contrast, when the catalytically inactive mutant
PLC-δ1 (E341A) was used, no formation of 10 was observed
indicating that the cleavage of 1 is PLC-dependent. These results
suggest that the fluorous PIP2 derivative 1 still functions as an
effective substrate for PLC.
Small GTPase ADP-ribosylation factor 1 (ARF1) has been
implicated as a PIP2-interacting protein.5 Accordingly, we have
expressed and purified ARF1 with the N-terminal 17 amino acid
residues deleted as described in the literature.22 To carry out the
crosslinking experiment, the probe 1 (40 μM) was first incubated
with purified ARF1 (20 μM) at 4 °C for 30 min. The complex of
1 and ARF1 was then irradiated at 365 nm for 10 min and the
reaction mixture was subsequently analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). As
shown in Fig. 3A, new bands were formed on the gel suggesting
that the crosslinking took place. In addition, these newly-formed
Fig. 2 Hydrolysis of fluorous PIP2 derivative 1 by PLC-δ1.
Fig. 3 Fluorous diazirine–PIP2 analog 1 crosslinked with ARF1 in the presence or absence of 2 and analyzed by SDS-PAGE (A) and Western blot
(B, C).
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bands were competed off in the presence of soluble PIP2 deriva-
tive 2 (Fig. 3A). The proteins on the SDS-PAGE gel were then
transferred to a polyvinylidene fluoride (PVDF) membrane and
probed with an antibody against PIP2 (Fig. 3B). The newly-
formed bands showed a strong signal on the Western blot. When
the membrane was further probed with an antibody against
ARF1, both pure ARF1 and the new bands showed a strong
signal (Fig. 3C). Taken together, these results demonstrate that
the new bands contain both PIP2 and ARF1, and are the desired
crosslinking products.
In conclusion, we have developed a fluorous diazirine-conju-
gated PIP2 derivative as a novel probe to identify PIP2-interact-
ing proteins. The diazirine group will enable photoaffinity
labeling of the interacting proteins while the fluorous tag will
enrich peptides derived from the crosslinked proteins for analy-
sis. These features will likely make the new probe suitable for
identification of low abundant, low affinity interacting proteins.
Although the fluorous photoaffinity group is attached to the sn-1
position of PIP2 in this work, it could also potentially be linked
through the sn-2 position or the inositol head group. In the long
term, similar techniques should be able to apply to identification
of interacting proteins of other endogenous PIs and possibly
other families of small molecules.
2.99 (brs, 1H), 2.35 (m, 4H), 1.44–1.68 (m, 6H), 1.23–1.40 (m,
8H), 0.88 (t, J = 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
δ 173.41, 173.40, 156.45, 136.60, 128.51, 128.09, 72.05, 66.63,
62.19, 61.44, 40.82, 34.28, 33.87, 31.42, 29.62, 28.73, 26.11,
24.88, 24.44, 22.47, 14.02; ESI-HRMS for [M + Na]+
C24H37NO7Na: calcd 474.2468, found 474.2472.
Compound 7. A solution of 5 (150 mg, 0.33 mmol) in anhy-
drous CH2Cl2 (1.5 mL) was added dropwise under argon to a
flask that contained 1-(benzyloxy)-N,N,N′,N′-tetraisopropylpho-
sphinediamine (0.66 mmol) and tetrazole (24 mg, 0.34 mmol) in
anhydrous CH2Cl2 (1.5 mL). The mixture was stirred at room
temperature for 2 h before the solvent was removed by vacuum.
The resulting residue was purified by flash column chromato-
graphy (hexane–ethyl acetate–triethyl amine = 100 : 20 : 3) to
give the phosphoramidite intermediate as a colorless oil. The so-
formed phosphoramidite (0.28 mmol) was dissolved in anhy-
drous CH2Cl2 and added to the solution of compound 6
(120 mg, 0.14 mmol) and 1H-tetrazole (39 mg, 0.56 mmol) in
anhydrous CH2Cl2 (1.0 mL). The reaction mixture was stirred at
room temperature overnight before t-BuOOH (5.5 M, 0.25 mL,
1.4 mmol) was added at −40 °C. The reaction mixture was then
warmed to room temperature gradually and the solvents were
removed. The residue was purified by column chromatography
(hexane–acetone = 2 : 1) over silica to provide 7 (187 mg, 81%
from compound 5) as a colorless oil. 1H NMR (CDCl3,
400 MHz) δ 7.20–7.40 (m, 25H), 4.82–4.55 (m, 15H),
4.65–4.80 (m, 3H), 4.62 (dd, J = 6.7, 6.9 Hz, 1H), 4.55
(d, J = 7.0 Hz, 1H), 4.02–4.48 (m, 8H), 3.54 (m, 1H), 3.39 (con-
formation 1) and 3.36 (conformation 2) (s, 3H), 3.32 (confor-
mation 1) and 3.28 (conformation 2) (s, 3H), 3.24 (conformation
1) and 3.23 (conformation 2) (s, 3H), 3.18 (q, J = 6.6 Hz, 2H),
2.22–2.34 (m, 4H), 1.40–1.68 (m, 6H), 1.16–1.38 (m, 8H),
0.88 (t, J = 6.9 Hz, 1H); 13C NMR (CDCl3, 75 MHz) δ 173.0,
172.9 and 172.8 (1C), 156.6, 136.8, 135.9, 135.8, 135.7, 135.5,
128.9, 128.8, 128.59, 128.56, 128.55, 128.52, 128.49, 128.44,
128.11, 128.07, 127.99, 98.9, 97.6, 97.0, 96.9, 78.9, 77.5, 77.2,
76.8, 76.5, 75.9, 74.7, 74.6, 74.5, 70.0, 69.8, 69.5, 69.3, 66.6,
65.80, 65.75, 65.57, 61.6, 56.74, 56.72, 56.69, 56.0, 55.8, 40.9,
34.1, 34.0, 33.9, 33.8, 31.5, 29.8, 29.7, 28.8, 26.2, 24.8, 24.4,
22.5, 14.1; 31P NMR (CDCl3, 162 MHz) δ −0.18 (2P),
−0.42 and −0.47 (1P); ESI-HRMS for [M + H]+ C71H93-
NO24P3: calcd 1436.5300; found 1436.5282.
Experimental
1
Synthesis of probe 1
16-(4-Methoxyphenyl)-3,10-dioxo-1-phenyl-2,11,15-trioxa-4-
azahexadecan-13-yl heptanoate (4). The mixture of compound 3
(518 mg, 1.12 mmol), heptanoic acid (172 μL, 1.22 mmol),
DCC (322 mg, 1.56 mmol) and DMAP (76 mg, 0.62 mmol) in
anhydrous CH2Cl2 (20 mL) was stirred at room temperature
overnight. After removal of the solvent, the residue was purified
by column chromatography (hexane–ethyl acetate = 3 : 1) over
silica to yield 4 (627 mg, 98%) as a colorless oil. 1H NMR
(CDCl3, 300 MHz) δ 7.26–7.36 (m, 5H), 7.22 (d, J = 8.2 Hz,
2H), 6.86 (d, J = 8.6 Hz, 2H), 5.22 (m, 1H), 5.08 (s, 2H),
5.01 (brs, 1H), 4.45 (d, J = 11.4 Hz, 2H), 4.34 (dd, J = 11.8,
3.7 Hz, 1H), 4.15 (dd, J = 11.6, 6.8 Hz, 1H), 3.78 (s, 3H),
3.54 (d, J = 5.2 Hz, 2H), 3.16 (q, J = 6.7 Hz, 2H), 2.28 (m, 4H),
1.41–1.65 (m, 6H), 1.22–1.36 (m, 8H), 0.88 (t, J = 6.6 Hz, 3H);
13C NMR (CDCl3, 75 MHz) δ 173.1, 173.0, 159.3, 156.4,
136.7, 129.7, 129.3, 128.4, 128.03, 128.01, 113.8, 72.9, 70.0,
67.8, 66.5, 62.8, 55.2, 40.8, 34.3, 33.8, 31.4, 29.6, 28.7, 26.1,
24.9, 24.4, 22.5, 14.0; ESI-HRMS for [M + Na]+ C32H45-
NO8Na: calcd 594.3043, found 594.3042.
Compound 8. Freshly distilled TMSBr (0.6 mL) was added to
a solution of compound 7 (20 mg, 13.9 μmol) in anhydrous
CH2Cl2 (0.6 mL) at 0 °C under argon. The reaction mixture was
stirred at room temperature for 1 h and the solvents were then
removed under vacuum for 1 h. The residue was redissolved in
MeOH (1.0 mL) and the resulting solution was stirred at room
temperature for 1 h. After removal of the solvent, the residue
was dried under vacuum for another 1 h. The residue was again
redissolved in MeOH (3.0 mL) and 10% Pd/C (10 mg) was
added. The mixture was stirred under H2 at room temperature for
8 h before the Pd/C was filtered off. The filtrate was concen-
trated, washed with cold CHCl3 three times (0.9 mL in total),
and dried under vacuum to yield 8 (10 mg, 80%) as a white
foamy solid. 1H NMR (CD3OD, 400 MHz) δ 5.24 (m, 1H),
4.50 (dd, J = 9.2, 18.4 Hz, 1H), 4.39 (m, 1H), 3.95–4.26
1-(6-Benzyloxycarbonylaminhexanoyloxy)-3-hydroxypropan-
2-yl heptanoate (5). To the solution of 4 (314 mg, 0.55 mmol)
in wet CH2Cl2 (18 mL) was added DDQ (262 mg, 1.16 mmol).
The reaction mixture was stirred at room temperature for 4 h, and
then washed with 10% NaHCO3 and saturated NaCl, dried over
MgSO4 and concentrated under vacuum. The resulting residue
was purified by column chromatography (hexane–ethyl acetate =
2 : 1) over silica to generate 5 (199 mg, 80%) as a colorless oil.
1H NMR (CDCl3, 300 MHz) δ 7.31 (m, 5H), 5.05–5.17 (m,
3H), 4.33 (dd, J = 11.7, 4.1 Hz, 1H), 4.19 (dd, J = 12.1, 6.0 Hz,
1H), 3.71 (d, J = 5.1 Hz, 2H), 3.17 (q, J = 6.6 Hz, 2H),
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(m, 7H), 3.66 (m, 1H), 2.96 (t, J = 7.5 Hz, 2H), 2.30–2.46 (m,
4H), 1.55–1.76 (m, 6H), 1.26–1.50 (m, 8H), 0.91 (t, J = 7.1 Hz,
3H); 13C NMR (CD3OD, 101 MHz) δ 174.64, 174.60, 81.0,
79.8, 78.3, 72.5, 72.0, 71.6, 65.6, 63.1, 63.0, 40.6, 35.0, 34.5,
32.6, 29.8, 28.2, 26.8, 25.9, 25.3, 23.6, 14.4; 31P NMR
(CD3OD, 200 MHz) δ 2.19 (1P), 1.79 (1P), 0.33 (1P); MS (ESI)
m/z 718.1 [M − H]−. ESI-HRMS for [M + H]+ C22H45NO19P3:
calcd 720.1799; found 720.1798.
Goldberg (Memorial Sloan-Kettering) for providing the
expression vector for ARF1.
Notes and references
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Compound 1. A solution of NHS ester 9 (5.1 mg, 10.2 μmol)
in DMF (600 μL) was added to compound 8 (4.5 mg, 6.3 μmol)
in TEAB buffer (0.25 M, 600 μL). The reaction mixture was
stirred in the dark at room temperature overnight before the sol-
vents were removed under vacuum. The residue was purified
through fluorous solid phase extraction to yield 1 (3.9 mg, 52%)
as a white solid. 1H NMR (CD3OD, 400 MHz) δ 7.80 (d,
J = 8.4 Hz, 2H), 7.42 (d, J = 8.3 Hz, 2H), 5.13 (m, 1H), 4.35
(dd, J = 11.0, 3.4 Hz, 1H), 4.28 (dd, J = 17.7, 7.8 Hz, 1H),
3.94–4.19 (m, 4H), 3.84–3.94 (m, 3H), 3.52 (dd, J = 9.7,
2.6 Hz, 1H), 3.28 (t, J = 6.7 Hz, 2H), 2.18–2.30 (m, 4H),
1.42–1.62 (m, 6H), 1.24–1.38 (m, 8H), 0.78 (t, J = 7.0 Hz, 3H);
13C NMR (CD3OD, 125 MHz) δ 174.9, 174.8, 168.8, 138.0,
132.9, 129.5, 129.4, 80.5, 78.4, 77.8, 72.8, 72.5, 72.2, 72.1,
72.0, 65.0, 63.8, 59.8, 41.0, 35.2, 34.8, 32.7, 30.1, 29.9, 27.6,
26.0, 25.7, 23.6; 31P NMR (CD3OD, 162 MHz) δ 3.7 (1P),
3.0 (1P), 1.0 (1P); 19F NMR (CD3OD, 376 MHz) δ −80.7 (t,
J = 10.1 Hz), −108.8, −119.4, −121.2, −122.1, −125.6;
MS (ESI) m/z 1080.3 [M − H]−, 589.6 [M − 2H]2−. ESI-HRMS
for [M + H]+ C36H48F13N3O20P3: calcd 1182.1836; found
1182.1833.
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2
Crosslinking of ARF1 with probe 1
The protein ARF1 was expressed and purified as described
in the literature.22 A solution of ARF1 (20 μM) in the Tris
buffer (50 mM, pH 8.0, 60 μL) was incubated with the fluorous
diazirine 1 (40 μM) for 30 min at 4 °C. Subsequently, the reac-
tion mixture was illuminated at 365 nm at 4 °C for 10 min. For
competition experiments, PIP2 derivative 2 (4 mM) was added to
the mixture of ARF1 and probe 1. The reaction mixture was ana-
lyzed by SDS-PAGE and Western blot. The Western blot was
developed by using mouse anti-PIP2 (1 : 2000) and horseradish
peroxidase (HRP)-conjugated anti-mouse IgG (1 : 5000) or anti-
ARF1 (1 : 2000) and HRP-conjugated anti-rabbit IgG (1 : 5000).
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evaluation of glycero-D-manno-heptose binding to concanavalin A by
fluorous-tag assistance, Angew. Chem., Int. Ed., 2008, 47, 1707–1710.
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Acknowledgements
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complex reveals a role for coatomer in GTP hydrolysis, Cell, 1999, 96,
893–902.
We thank the University of North Carolina at Chapel Hill
for financial support of this work. We also thank Dr Jonathan
This journal is © The Royal Society of Chemistry 2012
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