Fluorous enzymatic synthesis is used to synthesize multiple phosphatidylinositides, which are directly immobilized on a microarray to probe protein–lipid interactions

Allbritton, Nancy L.

Huang, Weigang

Proctor, Angela

Sims, Christopher E.

Zhang, Qisheng

PubMed

Carolina Digital Repository

Fluorous Enzymatic Synthesis of PhosphatidylinositidesWeigang Huanga, Angela Proctorb, Christopher E. Simsb, Nancy L. Allbrittonb,c, andQisheng ZhangaQisheng Zhang: qszhang@unc.eduaDivision of Chemical Biology and Medicinal Chemistry, University of North Carolina at ChapelHill, Chapel Hill, NC 27599, USA. Fax: 919-966-0204; Tel: 919-966-9687bDepartment of ChemistrycDepartment of Biomedical EngineeringAbstractA fluorous tagging strategy coupled with enzymatic synthesis is introduced to efficientlysynthesize multiple phosphatidylinositides, which are then directly immobilized on a fluorouspolytetrafluoroethylene (PTFE) membrane to probe protein-lipid interactions.Phosphatidylinositol (PtdIns) is a membrane-bound lipid that features a hydrophilic inositolhead unit linked with a hydrophobic diacylglycerol (DAG) through a phosphate group.[1]Three of the five hydroxyls in inositol undergo dynamic phosphorylation/dephosphorylationprocesses to generate the seven known phosphatidylinositides (PIs). Due to their interactionswith a wide range of effector proteins,[2] PIs are one family of the most versatile signalingmolecules that regulate many cellular processes such as cell proliferation and vesicletransport.[3] Abnormal levels of PIs have been associated with multiple diseases includingcancer and neurodegenerative diseases.[4] However, the detailed mechanisms by which PIsregulate different diseases are largely unknown, partly because of the difficulty in generatingPI derivatives as cellular probes.PIs and their derivatives are notorious for their structural complexity, with seven stereogeniccenters and the hydroxyl groups around the inositol head unit having similar reactivity. Mostof the synthetic strategies require selective protection and deprotection of the hydroxylgroups, and usually take more than 15 steps to synthesize one PI.[5] The synthetic efforts aredaunting when multiple PIs are targeted. In addition, PIs contain both the highly hydrophilicinositol phosphate head group and highly hydrophobic aliphatic side chains, making themdifficult to purify from the reaction mixtures. Despite elegant work from several groups ondeveloping novel methods and convergent strategies to prepare PIs and their derivatives,[5]efficient synthesis of various PIs remains a technical challenge.© The Royal Society of ChemistryCorrespondence to: Qisheng Zhang, qszhang@unc.edu.†Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here].See DOI: 10.1039/b000000x/Dedicated to Professor Dennis P. Curran on the occasion of his 60th birthdayNIH Public AccessAuthor ManuscriptChem Commun (Camb). Author manuscript; available in PMC 2015 March 18.Published in final edited form as:Chem Commun (Camb). 2014 March 18; 50(22): 2928–2931. doi:10.1039/c4cc00022f.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptUsing enzymes as catalysts in organic synthesis has long been an alternative method totraditional organic synthesis.[6] This approach has not been extended to PI synthesisalthough multiple enzymes that catalyze the formation of various PIs from PtdIns are wellstudied.[7] The highly hydrophilic nature of the inositol phosphates head group furthermakes it difficult to separate the PIs from the enzymatic reaction mixtures containinginorganic salts. Utilizing highly fluorinated (fluorous) tags to assist separation of enzymaticproducts from mixtures over fluorous media[8] has also been explored. For example, kineticresolution of a fluorous ester has been carried out in a fluorous triphasic separative reactionto generate pure products without chromatography.[9] Recently, fluorous taggedoligosaccharides have been used as enzymatic substrates in Nimzyme assays to detectenzymatic activities in cell lysates.[10] However, these developments are focused on one-step enzymatic transformation and further applications of the products are not explored. Weintroduce here “fluorous enzymatic synthesis” (Fig. 1) where tandem enzymatic reactionsare used to generate multiple probes after purification through fluorous solid phaseextraction (FSPE)[8a]. These probes can then be used as enzyme reporters, or be directlyimmobilized on a fluorous surface to form a microarray[11] to investigate protein-smallmolecule interactions. PtdIns(4,5)P2 is the most well-studied PI and functions as a substrateof multiple enzymes including phosphoinositide 3-kinase (PI3K) and phospholipase C(PLC).[12] To validate “fluorous enzymatic synthesis”, we designed the fluorousPtdIns(4,5)P2 derivative 1 with the fluorous tag at the sn-2 position. The long alkyl chainwas used to ensure similar hydrophobicity and membrane localization as endogenousPtdIns(4,5)P2 and to minimize the effect of the fluorous group on the head unit, where mostmetabolic reactions take place. The fluorophore was also added at the sn-1 position forsensitive monitoring of subsequent reactions. To synthesize 1 (Scheme 1), the fluorinatedacid 2 was generated by the radical addition of the according perfluorinated iodide C6F13Iwith undec-10-enoic acid followed by reduction with lithium aluminum hydride.[13]Coupling of 2 with the alcohol 3 and subsequent removal of the p-methoxybenzyl (PMB)protective group provided 4 in 90% yield. The alcohol in 4 was then phosphorylated andcoupled with the inositol head group 5,[5a] and the resulting intermediate was oxidized witht-BuOOH to generate 6. Next, both benzyloxycarbonyl (Cbz) and benzyl (Bn) groups wereremoved by hydrogenolysis while the methoxymethyl (MOM) group was removed bytreatment with trimethylsilyl bromide (TMSBr) followed by methanolysis. The fullydeprotected 7 was produced in 81% yield. Selective coupling of the terminal amine in 7 withN-hydroxysuccinimide (NHS) ester of fluorescein 8 provided the fluorous, fluorescentPtdIns(4,5)P2 derivative 1. The critical micelle concentration (CMC) of 1 was measured as17 μM (Fig. S1), similar to that of the endogenous PtdIns(4,5)P2 suggesting that the fluorous1 is a good mimic as the endogenous PtdIns(4,5)P2.[14]To investigate whether the tagged PtdIns(4,5)P2 derivative worked as the enzyme substrate,the fluorous 1 was treated with purified PI3K, a kinase that phosphorylates endogenousPtdIns(4,5)P2 to form the corresponding PtdIns(3,4,5)P3 under standard PI3K reactionconditions.[7a] The reactions were monitored by fluorescent detection of both PtdIns(4,5)P2and PtdIns(3,4,5)P3 on TLC (Fig. 2). The starting material was cleanly converted to theproduct in 6 h under standard assay conditions. Likewise, when treated with PLC, anothermetabolic enzyme that utilizes PtdIns(4,5)P2 as a substrate, fluorous 1 was completelyHuang et al. Page 2Chem Commun (Camb). Author manuscript; available in PMC 2015 March 18.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptconverted to the product DAG without any indication of formation of side products (Fig. 2).In cellular systems, PIs work as both starting materials for and products of multipleenzymatic reactions. To demonstrate that such complexity can also be recapitulated in thefluorous enzymatic synthesis, the tandem sequence of enzymatic reactions wereinvestigated. The fluorous PtdIns(3,4,5)P3 generated from the PI3K reaction followed byFSPE was directly used in a reaction catalyzed by phosphatidylinositol 3,4,5-triphosphate 5-phosphatase 2 (SHIP2)[7c] to produce the corresponding PtdIns(3,4)P2. As measured byTLC, the reaction generated a single product within 6 h. After FSPE, the fluorousPtdIns(3,4)P2 was further subjected to dephosphorylation by phosphatase and tensinhomolog (PTEN)[7d] to yield the corresponding PtdIns(4)P. Again, only the desired productwas produced which was further purified through FSPE (Fig. 2).Both 31P NMR (Fig. 3) and MS (Fig. S2) were used to demonstrate the efficiency of FSPEpurification. As described above, fluorous PtdIns(4,5)P2 was first treated with PI3K and theresulting product was used as the substrate in SHIP2 reaction. To purify a product throughFSPE, the reaction mixture was added to a column packed with fluorous silica. The columnwas first eluted with 20% and then 60% MeOH in water. As shown in Figure 3, the 31PNMR of the reaction mixture is dominated by ATP, the phosphate donor for thephosphorylation, and its hydrolyzed product ADP, with little signal for the PtdIns(3,4,5)P3product before FSPE. In contrast, after FSPE, the 60% MeOH fraction gave only resonancescorresponding to PtdIns(3,4,5)P3. These results suggest that FSPE efficiently enriches thedesired fluorous product. Such enrichment is also critical for the MS analysis. No molecularion was detected for PtdIns(3,4,5)P3 before FSPE while the major signal in the MS spectrumafter FSPE was the enzymatic product PtdIns(3,4,5)P3 (Fig. S2). A similar phenomenon wasobserved for the SHIP2 reaction: in both 31P NMR or MS spectra, the desired reactionproduct was only detected after FSPE (Fig. 3 and Fig. S2).Although cleavable linkers can be envisioned to avoid the trace of the fluorous tag on theenzymatic products, we chose to use non-cleavable fluorous tag for this work so that theenzymatic products could be directly immobilized on a fluorous surface for a small moleculearray. We chose to use Teflon as the fluorous surface because the fluorous array used in theliterature[11] is no longer commercially available. Equal molar amounts of the three fluoroustagged PIs, PtdIns(4,5)P2, PtdIns(3,4,5)P3, and PtdIns(3,4)P2, from the enzymatic reactionswere spotted on a perfluorinated Teflon membrane for immobilization. Because each PIcontains the same fluorophore fluorescein, the efficiency of loading was quantified byfluorescence 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 havesimilar capacity to immobilize on the Teflon membrane. When the quantity of theimmobilized lipid doubles, so does the fluorescence intensity. The membrane was thenincubated with biotin-conjugated antibody against PtdIns(3,4,5)P3. After washing withbuffer, the membrane was treated with Cy5-streptavidin and scanned for Cy5 signal(λex/λem = 633/670 nm) after washing with buffer (Fig. 4B). Only fluorous PtdIns(3,4,5)P3was detected by anti-PtdIns(3,4,5)P3. Similarly, both fluorous PtdIns(4,5)P2 andPtdIns(3,4,5)P3 were detected by anti-PtdIns(4,5)P2 (Fig. 4C) while anti-PtdIns(3,4)P2recognized fluorous PtdIns(3,4)P2 and PtdIns(4,5)P2 (Fig. 4D). These binding profiles areconsistent with the intrinsic affinity of each antibody with different endogenous PIs,[15]Huang et al. Page 3Chem Commun (Camb). Author manuscript; available in PMC 2015 March 18.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptsuggesting that the fluorous tag does not significantly interfere with the antibody-lipidinteractions.ConclusionsIn conclusion, a fluorous enzymatic synthesis strategy was developed to make multiple PIswhich can be directly immobilized onto a fluorous microarray. Our experiments clearlydemonstrate the advantage of the combination of fluorous tagging and enzymatic reactionsin making multiple PIs at both the synthesis and the separation stages. Although cleavablelinkers can be readily incorporated to remove the fluorous tag at the end of the enzymaticsyntheses, this work purposely leaves the tag intact after enzymatic reactions so that theproducts could be directly immobilized onto a fluorous microarray. Indeed, the antibodiesagainst PIs selectively recognize the fluorous-tagged lipids with the same binding profiles astheir 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 whosebiosynthetic enzymes are well characterized.Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.AcknowledgmentsWe thank Dr. John Sondek (University of North Carolina) for purified PLC enzyme. We also thank Dr. ZhiquanSong (University of North Carolina) for help with synthesis of the fluorous acid 2. This work was supported in partby the U.S. National Institutes of Health under GM098894 and CA139599.Notes and references1. Prestwich GD. Chem Biol. 2004; 11:619–637. [PubMed: 15157873]2. Lemmon MA. Nat Rev Mol Cell Biol. 2008; 9:99–111. [PubMed: 18216767]3. Di Paolo G, De Camilli P. Nature. 2006; 443:651–657. [PubMed: 17035995]4. a) Bunney TD, Katan M. 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Author manuscript; available in PMC 2015 March 18.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFig. 3.Efficiency of FSPE on product purification and characterization with 31P NMR.Huang et al. Page 8Chem Commun (Camb). Author manuscript; available in PMC 2015 March 18.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFig. 4.Selective binding profiles of fluorous lipids with antibodies on a fluorous membrane.Huang et al. Page 9Chem Commun (Camb). Author manuscript; available in PMC 2015 March 18.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptScheme 1.Synthesis of the fluorous substrate PtdIns(4,5)P2.Huang et al. Page 10Chem Commun (Camb). Author manuscript; available in PMC 2015 March 18.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

Fluorous enzymatic synthesis of phosphatidylinositides

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