Fluorous enzymatic synthesis of phosphatidylinositides

Article abstract of DOI:10.1039/c4cc00022f

A fluorous tagging strategy coupled with enzymatic synthesis is introduced to efficiently synthesize multiple phosphatidylinositides, which are then directly immobilized on a fluorous polytetrafluoroethylene (PTFE) membrane to probe protein-lipid interactions.

Full text of DOI:10.1039/c4cc00022f

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Fluorous enzymatic synthesis of
phosphatidylinositides†‡
Cite this: Chem. Commun., 2014,
50, 2928
Weigang Huang,a Angela Proctor,b Christopher E. Sims,b Nancy L. Allbrittonbc and
Qisheng Zhang*a
Received 2nd January 2014,
Accepted 28th January 2014
DOI: 10.1039/c4cc00022f
www.rsc.org/chemcomm
A fluorous tagging strategy coupled with enzymatic synthesis is intro- derivatives,5 efficient synthesis of various PIs remains a technical
duced to efficiently synthesize multiple phosphatidylinositides, which challenge.
are then directly immobilized on a fluorous polytetrafluoroethylene
(PTFE) membrane to probe protein–lipid interactions.
Using enzymes as catalysts in organic synthesis has long been
an alternative method to traditional organic synthesis.6 This
approach has not been extended to PI synthesis although multiple
Phosphatidylinositol (PtdIns) is a membrane-bound lipid that enzymes that catalyze the formation of various PIs from PtdIns are
features a hydrophilic inositol head unit linked with a hydrophobic well studied.7 The highly hydrophilic nature of the inositol phos-
diacylglycerol (DAG) through a phosphate group.1 Three of the five phates head group further makes it difficult to separate the PIs
hydroxyls in inositol undergo dynamic phosphorylation–dephos- from the enzymatic reaction mixtures containing inorganic salts.
phorylation processes to generate the seven known phosphatidyl- Utilizing highly fluorinated (fluorous) tags to assist separation of
inositides (PIs). Due to their interactions with a wide range of enzymatic products from mixtures over fluorous media8 has also
effector proteins,2 PIs are one family of the most versatile signaling been explored. For example, kinetic resolution of a fluorous ester
molecules that regulate many cellular processes such as cell pro- has been carried out in a fluorous triphasic separative reaction
liferation and vesicle transport.3 Abnormal levels of PIs have been to generate pure products without chromatography.9 Recently,
associated with multiple diseases including cancer and neuro- fluorous tagged oligosaccharides have been used as enzymatic
degenerative diseases.4 However, the detailed mechanisms by which substrates in Nimzyme assays to detect enzymatic activities in cell
PIs regulate different diseases are largely unknown, partly because lysates.10 However, these developments are focused on one-step
of the difficulty in generating PI derivatives as cellular probes.
enzymatic transformation and further applications of the products
PIs and their derivatives are notorious for their structural com- are not explored. We introduce here ‘‘fluorous enzymatic synthesis’’
plexity, with seven stereogenic centers and the hydroxyl groups (Fig. 1) where tandem enzymatic reactions are used to generate
around the inositol head unit having similar reactivity. Most of the multiple probes after purification through fluorous solid phase
synthetic strategies require selective protection and deprotection of extraction (FSPE).8a These probes can then be used as enzyme
the hydroxyl groups, and usually take more than 15 steps to reporters, or be directly immobilized on a fluorous surface to form
synthesize one PI.5 The synthetic efforts are daunting when multiple a microarray11 to investigate protein-small molecule interactions.
PIs are targeted. In addition, PIs contain both the highly hydrophilic
PtdIns(4,5)P2 is the most well-studied PI and functions as a
inositol phosphate head group and highly hydrophobic aliphatic substrate of multiple enzymes including phosphoinositide 3-kinase
side chains, making them difficult to purify from the reaction (PI3K) and phospholipase C (PLC).12 To validate ‘‘fluorous enzymatic
mixtures. Despite elegant work from several groups on developing
novel methods and convergent strategies to prepare PIs and their
a Division of Chemical Biology and Medicinal Chemistry, University of North
Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. E-mail: qszhang@unc.edu;
Fax: +1-919-966-0204; Tel: +1-919-966-9687
b Department of Chemistry, University of North Carolina at Chapel Hill, USA
c Department of Biomedical Engineering, University of North Carolina at Chapel
Hill, USA
† Dedicated to Professor Dennis P. Curran on the occasion of his 60th birthday.
Fig. 1 Schematic illustration of ‘‘Fluorous Enzymatic Synthesis’’. The
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/
enzymatic products can be directly immobilized on a fluorous surface.
c4cc00022f
2928 | Chem. Commun., 2014, 50, 2928--2931
This journal is ©The Royal Society of Chemistry 2014

View Article Online
Communication
ChemComm
Scheme 1 Synthesis of the fluorous substrate PtdIns(4,5)P2.
synthesis’’, we designed the fluorous PtdIns(4,5)P2 derivative 1 with
the fluorous tag at the sn-2 position. The long alkyl chain was used
to ensure similar hydrophobicity and membrane localization as
endogenous PtdIns(4,5)P2 and to minimize the effect of the fluorous
group on the head unit, where most metabolic reactions take place.
The fluorophore was also added at the sn-1 position for sensitive
monitoring of subsequent reactions. To synthesize 1 (Scheme 1), the
Fig. 2 Enzymatic synthesis with PI3K, PLC, SHIP2, and PTEN. The reaction
progression was monitored by TLC.
fluorinated acid 2 was generated by the radical addition of the be recapitulated in the fluorous enzymatic synthesis, the tandem
according perfluorinated iodide C6F13I with undec-10-enoic acid sequence of enzymatic reactions were investigated. The fluorous
followed by reduction with lithium aluminum hydride.13 Coupling PtdIns(3,4,5)P3 generated from the PI3K reaction followed by FSPE
of 2 with the alcohol 3 and subsequent removal of the p-methoxy- was directly used in a reaction catalyzed by phosphatidylinositol
benzyl (PMB) protective group provided 4 in 90% yield. The alcohol 3,4,5-triphosphate 5-phosphatase 2 (SHIP2)7c to produce the
in 4 was then phosphorylated and coupled with the inositol head corresponding PtdIns(3,4)P2. As measured by TLC, the reaction
group 5,5a and the resulting intermediate was oxidized with generated a single product within 6 h. After FSPE, the fluorous
t-BuOOH to generate 6. Next, both benzyloxycarbonyl (Cbz) and PtdIns(3,4)P2 was further subjected to dephosphorylation by
benzyl (Bn) groups were removed by hydrogenolysis while the phosphatase and tensin homolog (PTEN)7d to yield the corres-
methoxymethyl (MOM) group was removed by treatment with ponding PtdIns(4)P. Again, only the desired product was produced
trimethylsilyl bromide (TMSBr) followed by methanolysis. The fully which was further purified through FSPE (Fig. 2).
deprotected 7 was produced in 81% yield. Selective coupling of
Both 31P NMR (Fig. 3) and MS (Fig. S2, ESI‡) were used to
the terminal amine in 7 with N-hydroxysuccinimide (NHS) ester demonstrate the efficiency of FSPE purification. As described
of fluorescein 8 provided the fluorous, fluorescent PtdIns(4,5)P2 above, fluorous PtdIns(4,5)P2 was first treated with PI3K and the
derivative 1. The critical micelle concentration (CMC) of 1 was resulting product was used as the substrate in SHIP2 reaction. To
measured as 17 mM (Fig. S1, ESI‡), similar to that of the endogenous purify a product through FSPE, the reaction mixture was added to
PtdIns(4,5)P2 suggesting that the fluorous 1 is a good mimic as the a column packed with fluorous silica. The column was first
endogenous PtdIns(4,5)P2.14
eluted with 20% and then 60% MeOH in water. As shown in
To investigate whether the tagged PtdIns(4,5)P2 derivative Fig. 3, the 31P NMR of the reaction mixture is dominated by ATP,
worked as the enzyme substrate, the fluorous 1 was treated with the phosphate donor for the phosphorylation, and its hydrolyzed
purified PI3K, a kinase that phosphorylates endogenous PtdIns(4,5)P2 product ADP, with little signal for the PtdIns(3,4,5)P3 product
to form the corresponding PtdIns(3,4,5)P3 under standard PI3K before FSPE. In contrast, after FSPE, the 60% MeOH fraction gave
reaction conditions.7a The reactions were monitored by fluorescent only resonances corresponding to PtdIns(3,4,5)P3. These results
detection of both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 on TLC (Fig. 2). suggest that FSPE efficiently enriches the desired fluorous
The starting material was cleanly converted to the product in 6 h product. Such enrichment is also critical for the MS analysis.
under standard assay conditions. Likewise, when treated with PLC, No molecular ion was detected for PtdIns(3,4,5)P3 before FSPE
another metabolic enzyme that utilizes PtdIns(4,5)P2 as a substrate, while the major signal in the MS spectrum after FSPE was the
fluorous 1 was completely converted to the product DAG without any enzymatic product PtdIns(3,4,5)P3 (Fig. S2, ESI‡). A similar
indication of formation of side products (Fig. 2). In cellular systems, phenomenon was observed for the SHIP2 reaction: in both
PIs work as both starting materials for and products of multiple 31P NMR or MS spectra, the desired reaction product was only
enzymatic reactions. To demonstrate that such complexity can also detected after FSPE (Fig. 3 and Fig. S2, ESI‡).
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 2928--2931 | 2929

View Article Online
ChemComm
Communication
Fig. 3 Efficiency of FSPE on product purification and characterization
with 31P NMR.
Although cleavable linkers can be envisioned to avoid the
trace of the fluorous tag on the enzymatic products, we chose
to use non-cleavable fluorous tag for this work so that the
enzymatic products could be directly immobilized on a
fluorous surface for a small molecule array. Teflon was selected
as the fluorous surface because the fluorous array used in the
literature11 is no longer commercially available. Equal molar
amounts of the three fluorous tagged PIs, PtdIns(4,5)P2,
PtdIns(3,4,5)P3, and PtdIns(3,4)P2, from the enzymatic reac-
tions were spotted on a perfluorinated Teflon membrane for
immobilization. Because each PI contains the same fluoro-
phore fluorescein, the efficiency of loading was quantified by
fluorescence scanning at the excitation/emission wavelengths
of 488/520 nm (Fig. 4A). Indeed, the intensities of the three
lipids were in the same range suggesting that they have similar
capacity to immobilize on the Teflon membrane. When the
quantity of the immobilized lipid doubles, so does the fluores-
cence intensity. The membrane was then incubated with biotin-
conjugated antibody against PtdIns(3,4,5)P3. After washing
with buffer, the membrane was treated with Cy5-streptavidin
and scanned for Cy5 signal (lex/lem = 633/670 nm) after
washing with buffer (Fig. 4B). Only fluorous PtdIns(3,4,5)P3
was detected by anti-PtdIns(3,4,5)P3. Similarly, both fluorous
PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were detected by anti-
PtdIns(4,5)P2 (Fig. 4C) while anti-PtdIns(3,4)P2 recognized
fluorous PtdIns(3,4)P2 and PtdIns(4,5)P2 (Fig. 4D). These
binding profiles are consistent with the intrinsic affinity of
each antibody with different endogenous PIs,15 suggesting
that the fluorous tag does not significantly interfere with the
antibody–lipid interactions.
Fig. 4 Selective binding profiles of fluorous lipids with antibodies on a
fluorous membrane.
microarray. Indeed, the antibodies against PIs selectively recog-
nize the fluorous-tagged lipids with the same binding profiles
as their non-tagged parents, suggesting that the fluorous tag
has little impact on their functions. This strategy should also be
applicable to other complex endogenous small molecules
whose biosynthetic enzymes are well characterized.
We thank Dr John Sondek (University of North Carolina)
for purified PLC enzyme. We also thank Dr Zhiquan Song
(University of North Carolina) for help with synthesis of the
fluorous acid 2. This work was supported in part by the U.S.
National Institutes of Health under GM098894 and CA139599.
Notes and references
1 G. D. Prestwich, Chem. Biol., 2004, 11, 619–637.
2 M. A. Lemmon, Nat. Rev. Mol. Cell Biol., 2008, 9, 99–111.
3 G. Di Paolo and P. De Camilli, Nature, 2006, 443, 651–657.
4 (a) T. D. Bunney and M. Katan, Nat. Rev. Cancer, 2010, 10,
342–352; (b) P. Popvics and A. J. Stewart, J. Alzheimer’s Dis., 2012,
30, 737–744; (c) G. F. Passos, et al., Brain, Behav., Immun., 2010, 24,
493–501.
5 (a) R. J. Kubiak and K. S. Bruzik, J. Org. Chem., 2003, 68, 960–968;
(b) Y. Xu, et al., J. Am. Chem. Soc., 2006, 128, 885–897;
(c) A. J. Morgan, S. Komiya, Y. Xu and S. J. Miller, J. Org. Chem.,
2006, 71, 6923–6931.
6 (a) N. J. Turner, Nat. Prod. Rep., 1994, 11, 1–15; (b) J. Grunewald and
M. A. Marahiel, Microbiol. Mol. Biol. Rev., 2006, 70, 121–146;
(c) S. M. Li, Nat. Prod. Rep., 2010, 27, 57–58; (d) J. Duchek,
D. R. Adams and T. Hudlicky, Chem. Rev., 2011, 111, 4223–4258;
(e) P. L. DeAngelis, J. Liu and R. J. Linhardt, Glycobiology, 2013, 23,
764–777.
7 (a) W. Huang, et al., Anal. Bioanal. Chem., 2011, 401, 1881–1888;
(b) X. Wang, et al., Biochemistry, 2012, 51, 5300–5306; (c) D. A. Annis,
et al., Comb. Chem. High Throughput Screening, 2009, 12, 760–771;
(d) T. Maehama and J. E. Dixon, J. Biol. Chem., 1998, 273,
13375–13378.
In conclusion, a fluorous enzymatic synthesis strategy
was developed to make multiple PIs which can be directly
immobilized onto a fluorous microarray. Our experiments
clearly demonstrate the advantage of the combination of fluor-
ous tagging and enzymatic reactions in making multiple PIs at
both the synthesis and the separation stages. Although cleava-
ble linkers can be readily incorporated to remove the fluorous
tag at the end of the enzymatic syntheses, this work purposely
leaves the tag intact after enzymatic reactions so that the
products could be directly immobilized onto a fluorous
8 (a) W. Zhang and D. P. Curran, Tetrahedron, 2006, 62, 11837–11865;
(b) I. T. Hovath and J. Rabai, Science, 1994, 266, 72–75; (c) A. Studer,
et al., Science, 1997, 275, 823–826; (d) B. Cornils, Angew. Chem., Int.
Ed. Engl., 1997, 36, 2057–2059; (e) Z. Luo, Q. Zhang, Y. Oderaotoshi
2930 | Chem. Commun., 2014, 50, 2928--2931
This journal is ©The Royal Society of Chemistry 2014

View Article Online
Communication
ChemComm
and D. P. Curran, Science, 2001, 291, 1766–1769; ( f ) M. Cametti,
et al., Chem. Soc. Rev., 2012, 41, 31–42.
7960–7964; (c) H. D. Edwards, S. K. Nagappayya and N. L. Pohl,
Chem. Commun., 2012, 48, 510–512.
9 Z. Luo, S. M. Swaleh, F. Theil and D. P. Curran, Org. Lett., 2002, 4, 12 M. P. Czech, Cell, 2000, 100, 603–606.
2585–2587.
13 B. N. Huang and W. Y. Huang, Acta Chim. Sin., 1981, 39,
10 T. R. Northen, et al., Proc. Natl. Acad. Sci. U. S. A., 2008, 105,
3678–3683.
481–484.
14 J. P. Walsh, R. Suen and J. A. Glomset, J. Biol. Chem., 1995, 270,
11 (a) K. S. Ko, F. A. Jaipuri and N. A. Pohl, J. Am. Chem. Soc., 2005, 127,
28647–28653.
13162–13163; (b) A. J. Vegas, et al., Angew. Chem., Int. Ed., 2007, 46, 15 www.echelon-inc.com.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 2928--2931 | 2931