Fluorogenic Phospholipids as Head Group-Selective Reporters of

Fluorogenic Phospholipids as Head Group-Selective Reporters of Phospholipase A Activity Tyler M. Rose and Glenn D. Prestwich* Department of Medicinal Chemistry and Center for Cell Signaling, University of Utah, 419 Wakara Way, Suite 205, Salt Lake City, Utah 84108

T

he PLA are an ever-expanding family of enzymes, both in numbers and newly recognized roles in cell signaling. The PLA2 (phospholipase A2) superfamily in mammals consists of over 19 enzymes (1 ), which are broadly divided into three groups: cytosolic (cPLA2), secretory (sPLA2), and Ca2+-independent (iPLA2). PLA2 isozymes catalyze the hydrolysis of sn-2 position acyl chains of phospholipids, while PLA1 catalyze sn-1 cleavage. All PLA reactions generate a free fatty acid and a lysophospholipid, and each product has the potential to mediate cellular responses. Arachidonic acid, for example, can be a precursor for pro-inflammatory eicosanoids (1 ). Lysophospholipids such as LPA (lysophosphatidic acid), known to be generated by the action of PLA1 or PLA2, often in concert with a PLD (phospholipase D) (2 ) or lysoPLD/ATX (3 ), are associated with a variety of cellular events (4 ). The widespread distribution of PLA in the human body and the bioactive nature of their cleavage products have implicated these enzymes in many human diseases, including autoimmune (5 ) and cardiovascular diseases (6 ), neurological disorders (7 ), and cancer (8 ). To explore the effects of particular PLA isozymes in cell physiology, it is important to understand their spatiotemporal activation, diacyl group selectivity, and head group selectivity. For example, PLA isozymes have been reported with selectivities for PA (9 ), PE (10, 11 ), PC (12–14 ), PS (15, 16 ), and PG (17, 18 ), but information regarding the biological significance of the head group selectivity is limited. Furthermore, the pathophysiology of PLA activity in various diseases has made these enzymes important targets for isoform-specific drug development. In this case, rapid, sensitive, highthroughput, and real-time fluorescence-based activity www.acschemicalbiolog y.o rg

A B S T R A C T PLA (phospholipases A) are important mediators of cell signaling, generating bioactive fatty acids and LPLs (lysophospholipids). PLA products having different head groups can initiate vastly different types of signaling. Fluorogenic analogues of the PLs (phospholipids) PA (phosphatidic acid), PC (phosphatidyl choline), PE (phosphatidylethanolamine), and PG (phosphatidylglycerol) were synthesized as PLA substrates for rapidly determining in real time the influence of head group modifications on cell signaling both in vitro and in cells. Enzymeassisted remodeling of the sn-2 position of the diacylglyceryl moiety with cobra venom PLA2 and transphosphatidylation with a particular PLD (phospholipase D) were central steps in the preparation of these enzymatic probes. The resulting fluorogenic Dabcyl- and BODIPY-containing PL analogues, DBPA, DBPC, DBPE, and DBPG, were used in mixed micelle assays to determine PLA2 kinetics. Next, the assays were used to determine the Xi(50) value of a common PLA2 inhibitor. Finally, the head group selectivities of a series of commercially available PLA2 enzymes were readily established using the DBPLs (Dabcyl-BODIPY PLs) as substrates.

*To whom correspondence should be addressed. E-mail: [email protected].

Received for review November 11, 2005 and accepted January 27, 2006. Published online March 3, 2006. 10.1021/cb5000014 CCC: $33.50 © 2006 American Chemical Society

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a

O HO P HO O O N

HO

OH

a) HOOC(CH2)5NHDabcyl (1), O carbonyldiimidazole, DMSO N b) DMSO, DBU, 40 oC

O P

O

O

O

O

O

O cobra venom PLA2, HO P boric acid buffer (pH 8.5), O OHO o MeOH, 38 C

O

O

N

2

NH NH

O

O

NH O

N N

N N

3

N N N

HOOC(CH2)11NHBoc (3 Eq), TPSNT, O methylimidazole, CH2Cl2 HO P O

N

N

O

O

O

O

a) 20% TFA in CH2Cl2 b) DMF:1.0 M TEAB, pH 8.4 (3:2) C5-BODIPY(FL), SE

O

N NH

O HO P O O O

NH

O HO P O O O

O

O

O

N NH O

4

N N

NH

O

N

a) Peanut PLD (240 U), buffer: CHCl3 (7:3), 40 oC O O [buffer = 0.1 M NaOAc, HO P O HO O 0.1 M CaCl2, pH 5.6] O b) 20% TFA in Ch2Cl2 c) DMF:1.0 M TEAB, pH 8.5 (3:2), C5-BODIPY(FL), SE

NH N

F F B N N O

NH

DBPA

O N N

N N Boc

DBPC

O N N

b

required for efficient energy transfer. We demonstrate herein that these four DBPLs (Dabcyl-BODIPY PLs), specifically DBPA, DBPC, DBPE, and DBPG, are suitable for in vitro monitoring of PLA activity, including applications for inhibitor screening and head group selectivity studies.

O

N

4

O

Boc

O O

NH N

O

NH N

F F B N N

Figure 1. Enzyme-assisted route to DBPC (a) and DBPA (b). See Methods for experimental details.

assays are desirable which would be compatible with isolated enzymes or suitable for cell-based assays. PLA activity has traditionally been monitored using radiometric, titrametric, or chromatographic endpoint analyses (19 ), all of which are time-consuming and often do not permit real-time monitoring. Chromogenic assays (20 ) allow real-time monitoring of PLA in vitro, but fail for in situ applications. Recently, fluorogenic PLA probes based on a PC skeleton have been developed by our group (21 ), as well as by others (22, 23). These fluorogenic probes provide rapid, sensitive, realtime monitoring of PLA activity in vitro and in situ. We describe here the synthesis and evaluation of fluorogenic PLA probes with four different head groups, PA, PC, PE, and PG, by enzyme-assisted organic synthesis. Each fluorogenic substrate contains the same diacylglyceryl moiety, in which the sn-1-acyl chain contains an attached fluorescence quencher (Dabcyl, also known as p-methyl red), and the sn-2 acyl chain contains an appended BODIPY fluorophore. Intramolecular FRET (fluorescence resonance energy transfer) to the Dabcyl group quenches BODIPY fluorescence until PLAmediated substrate cleavage; then, a fluorophore is released when the lysolipid and fatty acid moieties are separated and the intermolecular distance exceeds that 84

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RESULTS AND DISCUSSION The significance of head group selectivity among PLA enzymes is one aspect of their crucial roles in cell signaling that has not been studied in great detail. To address this unmet need, we synthesized fluorogenic phospholipid analogue probes having different head groups using an enzyme-assisted synthetic route. The resulting probes were validated using real-time continuous-monitoring in vitro assays. In addition, these substrates are being employed to identify spatiotemporal regulation of PLA activity in living cells. Enzyme-Assisted Synthesis of DBPC and DBPA. The original synthesis of DBPC (21 ) was modified to improve yields and permit access to a variety of phospholipid head groups. The new enzyme-assisted synthetic route is shown in Figure 1a and was based on a route used to prepare photoactive phosphatidic acid derivatives (24 ). Initial attempts to condense Dabcyl-linked aminohexanoic acid (1) with PC-glycerol using DCC/DMAP conditions resulted in poor yields of a mixture of monoacyl and diacyl products. Other unsuccessful esterification conditions included use of Sc(OTf)3 as a catalyst (25 ), elevation of reaction temperature, and conversion of the fatty acid to an acid chloride. Finally, acyl imidazole chemistry, previously shown to be effective at acylating PC-appended glycerol (26 ), provided the desired diacyl product 2 in acceptable yield (Figure 1a). After removal of the sn-2 fatty acid with cobra venom and re-esterification in high yield, the carbamate 4 was cleaved and the resulting primary amine was conjugated with C5–BODIPY(FL), SE (Molecular Probes) in 78% yield to give DBPC as the final product. This new synthesis generates a DPBC that has a diacylglycerol moiety with more appropriately matched distances between the glyceryl backbone and the appended quencher and fluor. Treatment of intermediate 4 with peanut PLD yielded its PA analogue, which was deprotected with TFA and then condensed with the active ester C5–BODIPY(FL), SE to give DBPA. This revised route, using PLA2 and PLD as synthetic reagents, provided w w w. a c s c h e m i ca l biology.org

HO

O HO P O OO

O O

O

HO

O HO P O OO

O O

PLD, glycerol 40-45 oC

O

O

4

N O

Boc

NH

NH

N N

Boc

5

NH

N N

NH

PLD, ethanolamine O O HO 40-45 oC O P O O H3N

O

O

N

Boc

N

O

NH

6

NH

N N

N

both DBPA and DBPC in eight steps overall. The combined yield for DBPC was 24%, an approximately 100‑fold increase over the totally synthetic route previously reported (21 ). DBPA was synthesized in 25% overall yield from intermediate 4. Synthesis of DBPE and DBPG by Transphosphatidylation. In the presence of excess alcohol, PLD can preferentially catalyze the alcoholysis of phosphatidylcholine in a process called transphosphatidylation (27–29 ). It was therefore envisaged that transphosphatidylation of intermediate 4 might yield PS, PE, and PG analogues, which could be processed further to give DBPS, DBPE, and DBPG. In our hands, the use of anhydrous conditions defined earlier (28 ) for transphosphatidylation gave little or no product regardless of nucleophile. Addition of nucleophile in buffer, or of buffer alone, was required to drive the PLD reaction to completion. This procedure, a modification of earlier studies (27 ), was followed using L-serine, ethanolamine, and glycerol as nucleophiles, and DBPC precursor 4 as electrophile. The method gave DBPG and DBPE precursors, 5 and 6, in good to excellent yields (Figure 2). Streptomyces sp. PLD(P) (Genzyme) was the only PLD that consistently catalyzed transphosphatidylation over hydrolysis. Other commercial PLD gave either no reaction or PA analogues. In the case of the L-serine reaction, the product distribution favored DBPA precursor upon buffer addition. Modifying the nucleophile to include variously protected forms of L-serine did not generate the desired DBPS. To eliminate the need for protection and deprotection of the phosphatidylethanolamine moiety, direct transwww.acschemicalbiolog y.o rg

phosphatidylation of DBPC was attempted with PLD using the same conditions as above. Unfortunately, these conditions resulted in decomposition of the starting material, with no DBPE product detected. With this knowledge, the routes shown in Figure 3 were used to convert precursors 5 and 6 to final products DBPG and DBPE. Using the described semienzymatic synthesis, we synthesized four fluorogenic PL analogues with different head groups from a common intermediate (4) in two to five steps. Chemical methods for head group introduction from modified diacylglyceryl precursors gave unacceptably low yields. Furthermore, chemical introduction of head groups before incorporation of acyl chains was rejected as inefficient, as it would require separate synthetic

Figure 2. Enzymatic (PLD) reactions leading to DBPE and DBPG precursors. Reagents and conditions are as follows: aqueous nucleophile (glycerol, or ethanolamine) was added incrementally, until a reaction occurred, to Amberlite IRC-50 ion exchange resin and Streptomyces sp. PLD(P) (Genzyme) in CHCl3 and stirred at 40–45 °C. See Methods for experimental details.

a HO

O HO P O OO

O

O

O

a) TFA:CH2Cl2 (1:1)

O HO P O OO

O O

b) DMF:1.0 M TEAB, pH 8.4 HO (1:1), C5 BODIPY(FL), SE

HO NH O

Boc

HO

DBPG

NH O

5 N N

NH

O

O

N N

NH

N

N

F F B N N

b H3N

O HO P O OO

O O

O

NH O

Boc

NH

Fmoc-Cl, dioxane: sat. NaHCO3 (2:1)

Fmoc

H N

O HO P O OO

6

O O

O

NH

7

O

N N Boc

N N

NH

N

a) TFA:CH2Cl2 (1:1)

Fmoc

N

O O HO P O H O O O N

b) DMF:1.0 M TEAB, pH 8.4 (1:1), C5 BODIPY(FL), SE

O

5% piperidine in DMF NH

O

O

NH

H3N

O HO P O OO

O

NH

8

DBPE

O

N N O N

F F B N N

O O

NH

N N N

F F B N N

Figure 3. Synthesis of DBPG from 5 (a) and of DBPE from 6 (b). See Methods for experimental details. VOL.1 NO. 2 • ACS CHEMICAL BIOLO GY

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Figure 4. Concentration-dependent hydrolysis of DBPC (a) and of DBPA (b) by PLA2. An increasingly hyperbolic curve is observed for plots of initial velocity (RFU (relative fluorescence unit)/s) versus probe concentration (µM) as the fraction of probe (either DBPC (a), or DBPA (b)) in Triton X-100 is decreased. LysoMaxS PLA2 (0.3 U well–1) and bee venom PLA2 (0.5 U well–1), respectively, were used with DBPC and DBPA in these assays.

routes for each probe. Transphos phatidylation by PLD provided a mild, effective way to rapidly introduce head group diversity in our phospholipid probe design.

In vitro Activity Assays with DBPC and DBPA. In vitro enzyme assays with DBPC and DBPA produced linear fluorescence increases that were dependent on the concentrations of enzyme and probe (data not shown). These assays also revealed that predictable tracking of enzyme activity is predicated on the amount of detergent or phospholipid used as a carrier for the probe. As the fraction of probe in Triton X-100 micelles is decreased, the plots of initial velocity versus probe concentration become increasingly hyperbolic (Figure 4). At ≤0.2% (w/w) of DBPC in Triton (0.3 U well–1 LysoMaxS PLA2) and ≤0.03% (w/w) of DBPA in Triton (0.5 U well–1 bee venom PLA2), the data fit the Michaelis–Menten equation, and apparent Vmax and Km values can be determined at a given Xd. Regardless of whether the probes were dispersed in Triton micelles (Figure 4) or phospholipid vesicles (data not shown), a window of robust PLA activity was observed. Above a certain mole fraction of DBPL, the reported enzyme activity stopped following Michaelis– Menten kinetics; at too low a mole fraction of DBPL, the fluorescent signal fell below detection limits. Mole fractions of probe beyond an upper limit presumably yield disrupted aggregation states that are resistant to enzyme catalysis, resulting in lower-than-expected signal generation. Inhibitor Assay with DBPC. The Triton mixed micelle assay, despite its intrinsic limitations for obtaining primary kinetic parameters (30 ), is widely used to obtain relative kinetic information in inhibitor screens and is the basis for a well-developed chromogenic

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sPLA2 assay (31 ). Triton X-100 has the advantages of being commercially available and relatively inert and is frequently used for isolation of membrane-associated proteins. Triton/DBPC micelles provided a suitable matrix to allow an Xi(50) for thioether amide–PC (32 ) inhibition of PLA2 to be quantified (Figure 5). Increasing concentrations of thioether amide–PC were sonicated into Triton/DBPC mixed micelles and assayed in duplicate with bee venom PLA2 (Figure 5). The resulting Xi(50) was calculated to be 0.004, corre sponding to a thioether amide-PC concentration of 2 µM. This result correlates with that of a previous measurement of IC50, also 2 µM, for thioether amide–PC inhibition of cobra venom PLA2 (32 ). Head Group Selectivity Assays. The completed fluorogenic probes DBPA, DBPC, DBPE, and DBPG were used to experimentally determine the head group selectivities of a sampling of commercial PLA2 in Triton mixed micelles. Each fluorogenic substrate was assayed with LysoMaxS, bee venom, cobra venom, bovine pancreas, Streptomyces violaceoruber, and Human Type V PLA2 in TritionX-100 (reduced) micelles. Selectivities expressed as a percentage of the slope of the analogue showing the most activity (Figure 6) revealed several interesting trends. First, mammalian enzymes (bovine and human) preferred the PG head group, followed by PC > PE >> PA. Second, the venom and bacterial enzymes preferred the PC head group, followed by PG > PE >> PA. Third, only the venom and pancreatic enzymes significantly catalyzed DBPA hydrolysis. Previous reports on the head group selectivity of these or other closely related PLA2, as determined using a variety of assay methods, are summarized in Table 1. The degree of agreement between the listed head group selectivity studies is noteworthy, considering the contrasting methods used. Since the PLA head group preferences are conserved from assay platform to assay platform in vitro, the same preferences might also legitimately extrapolate to living Figure 5. Inhibition of bee PLA2 by thioether amide–PC. The log of increasing mole fractions of thioether amide–PC sonicated with 0.5 µM DBPC at Xd = 0.001/0.2% (w/w) in Triton X‑100 micelles is plotted versus initial velocities (n = 2) generated upon addition of 0.01 U well–1 of bee venom PLA2. Calculated Xi(50) = 0.004, corresponding to 2 µM thioether amide–PC. w w w. a c s c h e m i ca l biology.org

b

120 100 80 60 40 20 0 -20

DBPA DBPC DBPE DBPG

Relative Activity (%)


a

120 100 80 60 40 20 0 -20

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Probes

d

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DBPA DBPC DBPE DBPG



c

120 100 80 60 40 20 0 -20

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systems. Thus, these data validate the applicability of the much more easily utilized DBPL fluorogenic assay for further biological studies and inhibitor discovery. Probe Design Features. Our probe design provides the benefit of unmodified phospholipid head groups and ester linkages that should reflect authentic protein-ligand interactions. Binding models (33, 34 ) predict shallow insertion by sPLA2 into the membrane and place the PL acyl chains outside the phospho lipid-binding pocket beyond approximately the ninth carbon of the sn-2 chain and the fourth carbon of the sn-1 chain (35 ). This leads to the assumption that, for sPLA2, once a probe is properly inserted into liposomes/micelles, minor modifications to the chain termini compatible with a hydrophobic lipid environment will not be an important factor in enzyme selectivity. The correlation between our head group selectivity data and those of assays using different methods (Table 1) seems to support this assumption. However, the aromatic fluor and quencher groups at the chain termini of the DBPLs may disrupt the micelles sufficiently to result in attenuation of enzyme activity at higher mole fractions (36, 37 ) and could www.acschemicalbiolog y.o rg

DBPA DBPC DBPE DBPG

Probes



e

DBPA DBPC DBPE DBPG

120 100 80 60 40 20 0 -20

Figure 6. Head group selectivities of commercially available PLA2. a) 0.1 U well–1 of bovine pancreas, b) 20 ng well–1 of human Type V, (c) 0.2 U well–1 of S. violaceoruber, d) 0.6 U well–1 of LysoMaxS, e) 0.5 U well–1 of Naja mossambica venom, and f) 0.1 U well–1 of bee venom PLA2 were assayed against DBPA, DBPC, DBPE, and DBPG in Triton X‑100 (reduced) mixed micelles (Xd = 0.0001/0.02% (w/w) for all probes). Head group selectivities are reported as percentages of the largest slope obtained over a 3 min incubation of enzyme with DBPL mixed micelles.

DBPA DBPC DBPE DBPG

Probes

also affect subcellular localization of the probes (38 ). Additionally, the saturated acyl linkages connecting the glyceryl backbone to the Dabcyl and BODIPY groups are expected to resist cleavage by cPLA2, which insert more deeply into lipid bilayers and thereby maintain a preference for sn-2 arachidonic acid (34, 39 ). The DBPL probes have also been designed so that fluorescence is completely quenched until cleavage occurs. In this regard, they are like molecular beacons, often used to detect the hybridization of nucleic acids (40 ). Another paradigm for probe design uses a FRET pair in which the donor and acceptor both emit fluorescence. The acceptor quenches donor fluorescence while simultaneously emitting at another distinct wavelength. The advantage of a design that uses two fluorophores is that the degree of cleavage can be assessed by measuring emission ratios between the donor and acceptor. Such ratiometric probes have been used in single molecule detection studies (41 ) and as substrates for phosphodiesterases (42 ) and PLA2 (23 ), among other examples. However, bis-fluorophore probes suffer from higher background, lower sensitivity, and a requirement for more complex data analyVOL.1 NO. 2 • ACS CHEMICAL BIOLO GY

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table 1. Comparison of head group selectivity data

unsaturated acyl chains and for which the DBPL probes could serve as fluorogenic PLA2 Phospholipid selectivity Assay system substrates. For example, serum LPA is S. violaceoruber PC >> PE > PA Triton X-100/chromagenica biosynthesized primarily by PLD cleavage S. violaceoruber PC > PG > PE > PA Triton X-100/DBPLs of LPLs that have been generated either Pig pancreas PG >> PC Triton X-100 and other detergents/pH statb directly by serum LCAT (lecithin–cholesterol Pig pancreas PG >> PE > PC Continuous fluorescence displacementc acyl transferase) or by cellular sPLA2 and Bovine pancreas PG >> PE > PC > PA Triton X-100/DBPLs PS–PLA1 (2 ). Direct cleavage of PA by sPLA2 Bee venom PG >> PC > PE Continuous fluorescence displacementc or mPA–PLA1 can also give serum LPA in a Bee venom PE ≥ PC Triton X-100/pH-statd mechanism that may be important for local Bee venom PC > PG ≥ PE > PA Triton X-100/DBPLs signaling events like wound healing and N. naja venom PC > PG >> PE Continuous fluorescence displacementc inflammation (2 ). LPA production is thought N. naja venom PC >> PE Triton X-100/pH-statd to be regulated in cells by initial PLD converN. mossambica venom PC > PG >> PE > PA Triton X-100/DBPLs sion to PA followed with cleavage by an Human group V PC > PG > PA > PE Mixed POPL vesicles/LC-ESIe sPLA2 or PA–PLA1 (4 ). Human group V PG > PC > PE Polymerized mixed liposomes/ Further examples include a human Group Pyrene‑labeled PLf III sPLA2 with homology to bee venom sPLA2 Human group V PG > PC > PE > PA Triton X-100/DBPLs that has an apparent preference for PG. Its unique tissue distribution relative to other aAdapted from ref 48. bAdapted from ref 49. cAdapted from ref 50. dAdapted from ref 51. sPLA2 suggests a specialized biological role eAdapted from ref 52. fAdapted from ref 53. (18 ). A lysosomal PLA2 from macrophages (17 ) having a demonstrated preference for sis. Incomplete quenching and inadequate spectral PG has been implicated in the biosynthesis of LBPA overlap can result in a high fluorescence background. (lysobisphosphatidic acid), an important component An extreme example of this occurred when DBPC was of vesicle structure (43 ). In addition, tafazzin, the gene compared with BBPC (bis-BODIPY-PC, Invitrogen) and responsible for Barth syndrome, has been shown to found to generate a vastly superior signal (21 ). encode a transacylase responsible for remodeling of PG The nonratiometric data obtained with the DBPL and cardiolipin (44 ). On the basis of sPLA2 hydrolysis substrates is qualitative in a cellular context: comparaexperiments, LPE or its N-acyl derivatives are proposed tive fluorescence intensity is used to monitor subcelluintermediates in the biosynthesis of N-acyl ethanollar enzyme activity. The straightforward synthetic route amines, endogenous ligands for cannabinoid and vanilto the DBPL analogues would readily allow substitution loid receptors having anti-inflammatory activity (10 ). of the BODIPY–dabcyl pair with a ratiometric fluoroAnother example of a signaling sPLA2 with a phore pair, should applications require such probes. demonstrated head group preference, a PC-preferring But, notwithstanding the quantitative limitations of the lysosomal PLA2, is selectively expressed in alveolar “dequenching” paradigm, a probe design in which fluor macrophages and may play a role in pulmonary surescence is completely quenched prior to enzyme cleavfactant catabolism (12 ). LPC generation by iPLA2 can age, and only one fluor (rather than two) is released stimulate the attraction of phagocytes to apoptotic cells following cleavage, offers an advantage in fluorescence (13 ). And, Group X sPLA2, which is uniquely located in or laser scanning confocal microscopy. When used peripheral and neuronal fibers, demonstrates neuritowith these visualization techniques, dequenching genic activity dependent on LPC production (14 ). The probes like the DBPL substrates offer the potential for literature contains descriptions of many other sPLA2 achieving excellent sensitivity and detailed detection and iPLA2 with uncertain or unknown head group of enzyme activity at subcellular locations without the selectivities. need for specialized equipment. Furthermore, cPLA2 enzymes hydrolyze a variety of Potential Probe Applications. An increasing body saturated phospholipids and can be important signal of work indicates important cell signaling roles for transducers. A cPLA2 of this kind is likely to play a role cPLA2, sPLA2, and iPLA2 that do not necessarily prefer in transducing stimulation of muscarinic receptors to 88


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modulation of calcium channel activity (45 ) in neurons. In a related example, which also demonstrates the use of DBPC in a cell-based enzyme assay, SCG (superior cervical ganglion) neurons from mice expressing cPLA2 and deficient in sPLA2 were labeled with DBPC and found to exhibit a distinct increase in gross PLA2 activity following stimulation with the muscarinic agonist Oxo-M (oxotremorine-M, Figure 7). In conclusion, DBPA, DBPC, DBPE, and DBPG are useful new substrates for analysis of PLA activity in vitro and in living cells and tissues. The fluorogenic assay allows expeditious detection of enzyme activity in real time with a continuous readout. The assay is amenable to high-throughput screening for PLA2 isozyme inhibitors and for examination of the PL head group selectivity of virtually all PLA2 isozymes. The utility of DBPC in cell-based enzyme assays has been shown here as well as previously (21 ). Reports of its use in whole cell monitoring of PLA in studies investigating a putative PLA2 involved in ovarian cancer progression (46 ), and for another PLA2 involved in neuronal L-channel inhibition (Liu, L., Zhao, R., Bai, Y., Stanish, L. F., Evans, J. E.,

METHODS

NMR and MS data can be found online in the Supporting Information. 6-(p-Methyl Red)aminohexanoic Acid (1). The sodium salt of p-methyl red (Acros) was converted to its acid form by treatment with 2 M HCl, followed by lyophilization. The acid form (6.8 g, 25 mmol) was then combined with NHS (N-hydroxysuccinimide, 4.4 g, 38 mmol) and EDCI (N-(3-dimethylaminopropyl)-N´-ethyl carbodiimide hydrochloride, 7.3 g, 38 mmol) in DMF (60 mL). After stirring overnight, the reaction was concentrated under vacuum and washed (H2O) to give the NHS–ester of p-methyl red. The NHS–ester (226 mg, 0.62 mmol) was further reacted with 6-aminocaproic acid (123 mg, 0.94 mmol) in a 2:1 solution of DMF/H2O containing 7% DMAP. After 24 h, the reaction was acidified with 3 M HCl, then extracted with 10% MeOH in CH2Cl2. Solvents were removed in vacuo, and the product was precipitated from EtOH/H2O as a deep red solid (200 mg, 77% yield, two steps). 1-O-(6-(p-Methyl Red)aminohexanoyl)-2-O-(6-(p-methyl red)aminohexanoyl)-sn-glyceryl Phosphatidylcholine (2). A solution of 6-(p-methyl red)aminohexanoic acid (40 mg, 0.11 mmol) and carbonyldiimidazole (25 mg, 0.16 mmol) in anhydrous DMSO (0.5 mL) was stirred under Ar for 30 min. This thick reaction mixture was transferred via syringe in 1 mL DMSO to a flask containing 4.5 mg (0.02 mmol) sn-glycero-3-phosphocholine (Bachem) and DBU (21 µL, 0.14 mmol) stirring in DMSO (0.25 mL) at 40 °C. After 24 h, the reaction was concentrated under vacuum and purified on SiO2 (Flash Chromatography ASTM 230–400 Silica Gel) by elution with 65:25:4 CH2Cl2/ MeOH/H2O to give a red solid (7 mg, 40% yield). 1-O-(6-(p-Methyl Red)aminohexanoyl)-sn-glyceryl Phosphatidylcholine (3). Compound 2 (100 mg) was dissolved in MeOH (300 µL) and then treated with 1 mg (ca. 1400 U) PLA2

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Figure 7. Fluorescence of DBPC in a cell-based assay of stimulation of an adult mouse superior cervical ganglion neuron expressing cPLA2 but deficient in sPLA2. PLA2 activation is shown at 0 min (a, baseline PLA2 activity), and after 6 min exposure to 10 µM Oxo-M (b). Images provided by Ann R. Rittenhouse, Rubing Zhao, Yen Bai, and Michael J. Sanderson; used with permission.

Sanderson, M. J., Bonventre, J. V., and Rittenhouse, A. R., unpublished results) will be described in detail in other publications. Further collaborative demonstrations of the utility of DBPLs bearing other head groups as substrates for a variety of animal, plant, and bacterial PLA enzymes in vitro and in cells are in progress and will be reported in due course.

(Naja mossambica mossambica, Sigma) in 600 µL of 0.1 M sodium borate buffer containing 0.1 M CaCl2 and adjusted to pH 7.8. This solution was allowed to stir at 40 °C. Over 48 h, another 1 mg of PLA2 was added in a total of 1.2 mL of 1:2 MeOH/buffer. After 72 h, the solvents were removed under a stream of Ar and compound 3 (56 mg, 90% yield) was collected as a red solid from a small SiO2 column with 60:35:7 CH2Cl2/MeOH/H2O. 1-O-(6-(p-Methyl Red)aminohexanoyl)-2-O-(12-Boc-amino dodecanoyl)-sn-glyceryl Phosphatidylcholine (4). To 65 mg (0.21 mmol) of 12-(Boc-amino)dodecanoic acid (Fluka) and TPSNT (23, 47 ) (76 mg, 0.20 mmol) stirring in distilled CH2Cl2 (2 mL) was added 30 µL (0.38 mmol) of 1-methylimidazole. The solution was stirred for 30 min and then added to a stirred solution of 3 (40 mg, 0.06 mmol) in 2 mL of CH2Cl2. After 72 h, the solvent was evaporated and product 4 (50 mg, 85% yield), a red solid, was isolated from SiO2 with a step gradient of 1:9 MeOH/CH2Cl2; 65:25:4 CH2Cl2/MeOH/H2O; then, 60:35:7 CH2Cl2/MeOH/H2O. 1-O-(6-(p-Methyl Red)aminohexanoyl)-2-O-(12-(5-BODIPYpentanoyl)aminododecanoyl)-sn-glyceryl Phosphatidylcholine (DBPC). Compound 4 (10 mg, 0.01 mmol) was dissolved in a solution of 1:2 TFA/CHCl3 (1.5 mL, total). After stirring for 1 h, the solvent was removed in vacuo and the crude amine was used without purification in the next step. Freshly made 1 M TEAB buffer (600 µL, pH 8.5) was added to the crude amine (8.9 mg, 0.01 mmol), followed by a solution of 5 mg (0.01 mmol) of C5–BODIPY(FL), SE (Molecular Probes) in 400 µL of DMF. The reaction was stirred for 24 h, at which time the solvents were removed with a stream of Ar. DBPC (9.5 mg, 78% yield) was isolated from a short SiO2 column as a red solid, using a step gradient of 30% MeOH in CH2Cl2; then, 65:25:4 CH2Cl2/MeOH/H2O. VOL.1 NO. 2 • ACS CHEMICAL BIOLO GY

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1-O-(6-(p-Methyl Red)aminohexanoyl)-2-O-(12-(5-BODIPYpentanoyl)-aminododecanoyl)-sn-glyceryl Phosphatidic Acid (DBPA). To a solution of 2 mg (0.002 mmol) of 4 in 300 µL of CHCl3 was added 140 U of peanut PLD (Sigma) dissolved in 700 µL of 0.1 M sodium acetate buffer (pH 5.6) that contained 0.1 M CaCl2. The reaction was stirred vigorously at 35–40 °C for 2 days, at which time the solvents were removed by vacuum. The crude PLD product was purified as a red solid from SiO2 with 20% MeOH in CH2Cl2. The PLD product (ca. 4 mg) was dissolved in a 1:5 solution of TFA/CH2Cl2 (1.2 mL, total). After stirring for 2 h, the solvents were removed, first under a stream of nitrogen and then in vacuo. The product (2 mg, 0.003 mmol) was then dissolved in 1 M TEAB buffer (600 µL), and 1.3 mg (0.003 mmol) of C5–BODIPY(FL), SE was added in 400 µL of DMF. The reaction was stirred for 6 h, and solvents were removed in vacuo. The product was isolated from SiO2 with a step gradient elution of 1:9 MeOH/CH2Cl2; then, 65:25:4 CH2Cl2/MeOH/H2O + 1% TEA. DBPA was further purified by passage through Sephadex LH‑20 in 1:9 MeOH/CH2Cl2. It was then passed through DOWEX 50WX8-200 (sodium form) to give DBPA as a red, solid sodium salt (1.6 mg, 25% yield, 3 steps). 1-O-(6-(p-Methyl Red)aminohexanoyl)-2-O-(12-Boc-amino dodecanoyl)-sn-glyceryl Phosphatidylglycerol (5). Compound 4 (5 mg, 5.4 µmol) was dissolved in 1.3 mL of CHCl3. An excess (~250 µL, total) of glycerol, 97 mg of Amberlite IRC-50 beads, and 1000 U of Streptomyces sp. PLD(P) (Genzyme) were then added. After stirring for several days at 40–45 °C, no reaction was observed. Approximately 250 µL of buffer (0.2 M NaOAc and 0.08 M CaCl2, pH 5.6) and 25 U of additional PLD were then added. After stirring overnight, the reaction had proceeded almost to completion. The DBPG precursor 5 was purified on SiO2 using 25% MeOH/CH2Cl2 to give a red solid. 1-O-(6-(p-Methyl Red)aminohexanoyl)-2-O-(12-(5-BODIPYpentanoyl)aminododecanoyl)-sn-glyceryl Phosphatidylglycerol (DBPG). Compound 5 (2 mg, 2.2 µmol) was dissolved in a 50% solution of TFA in CH2Cl2. After stirring for 2.5 h at room temperature, the solvents were removed under a stream of Ar. After 12 h under vacuum, the residue was dissolved in DMF (200 µL) containing C5–BODIPY(FL), SE (1.2 mg, 2.9 µmol). To this solution was added 400 µL of 1 M TEAB buffer (pH 8.4), and the reaction was stirred for 4 h. Solvents were removed with a stream of argon and then in vacuo. DBPG (1.7 mg, 68% yield, two steps), a red solid, was isolated from SiO2 using 10% MeOH/CH2Cl2; 20% MeOH/CH2Cl2; then, 70:20:2 CH2Cl2/MeOH/H2O. 1-O-(6-(p-Methyl Red)aminohexanoyl)-2-O-(12-Boc-amino dodecanoyl)-sn-glyceryl Phosphatidylethanolamine (6). Amberlite IRC-50 beads (50 mg) and CHCl3 (500 µL) were added to a vial containing dried 4 (6 mg, 6.5 µmol). Ethanolamine (10 µL, 164 µmol), then 200 U of Streptomyces sp. PLD(P) (Genzyme) in 400 µL of buffer (0.2 M NaOAc and 0.08 M CaCl2, pH 5.6) was added. The reaction was stirred at 40–45 °C. After 24 h, another 200 U of PLD was added, and then 200 U were added again after another 24 h. After stirring overnight, the solvents were stripped with a stream of nitrogen and DBPE precursor 6 was collected as a red solid from a SiO2 column with 65:25:4 CH2Cl2/MeOH/H2O. 1-O-(6-(p-Methyl Red)aminohexanoyl)-2-O-(12-Boc-amino dodecanoyl)-sn-glyceryl Phosphatidylethanolamino-Fmoc Carbamate (7). To compound 6 (1.6 mg, 1.8 µmol) was added Fmoc-Cl (0.6 mg, 2.4 µmol) in 0.9 mL of dioxane, followed by 0.5 mL of a saturated solution of NaHCO3. After stirring for 24 h, the reaction was diluted with EtOAc and washed with water and brine. The solvents were removed, and the red, solid product 7 (1.5 mg, 78% yield) was purified on SiO2 with a step gradient of 20% MeOH/CH2Cl2, then 65:25:4 CH2Cl2/MeOH/H2O. 1-O-(6-(p-Methyl Red)aminohexanoyl)-2-O-(12(5-BODIPY-pentanoyl)aminododecanoyl)-sn-glyceryl

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Phosphatidylethanolamino-Fmoc Carbamate (8). Compound 7 was dissolved in 1 mL of CH2Cl2 and stirred with 0.2 mL of TFA for 1 h at room temperature. Following removal of solvents by vacuum, the crude amine (3.1 mg, 3.1 µmol) was dissolved in 0.7 mL of 1 M TEAB buffer, followed by addition of 0.5 mL of DMF, then C5–BODIPY(FL), SE (1.4 mg, 3.4 µmol). The solution was concentrated by about half, after several minutes, with a stream of nitrogen, and another 0.5 mL of DMF was added to improve solubility. After stirring overnight, the reaction was stripped of solvent with nitrogen, and DBPE precursor 8 was purified on SiO2 using a step gradient of 10% MeOH/CH2Cl2, 20% MeOH/CH2Cl2, and 65:25:4 CH2Cl2/MeOH/H2O to give a red solid. 1-O-(6-(p-Methyl Red)aminohexanoyl)-2-O-(12(5-BODIPY-pentanoyl)aminododecanoyl)-sn-glyceryl Phosphatidylethanolamine (DBPE). A 5% solution of piperidine in DMF was added to a vial containing a dried film of 8 (3.3 mg, 2.5 µmol). The solution was allowed to stir for 15 min and then evaporated under a stream of nitrogen. The product, DBPE, was collected from a short SiO2 column as a red solid using 10% MeOH/CH2Cl2, then 65:25:4 CH2Cl2/MeOH/H2O. Fluorogenic Assays Using DBPC and DBPA with PLA2. DBPC or DBPA was combined with either PC or Triton X-100 (reduced) at the relative concentrations given in the text. Carrier solvents (usually chloroform, or 65:25:4 chloroform/methanol/water) were evaporated under a stream of nitrogen and then in vacuo before buffer (0.1 M Tris, 0.1 M CaCl2, and 0.05% NaN3, pH 8.9) was added. The resulting suspension was sonicated for 30 s on a probe sonicator set at output = 3 and duty cycle = 50%, and then for an additional 2 min at the same sonicator settings with ice-bath cooling. Aliquots (0–100 µL) of the resulting micelle/vesicle solution were diluted to 100 µL total volume with buffer in a 96-well plate. Enzyme in buffer was added to initiate hydrolysis of the probe following a separate thermal preequilibration (2 min) at 37 °C of the enzyme and micelle/vesicle solutions. Fluorescence readings were recorded (λex = 500 nm; λem = 530 nm) every 15 s for 5 min on a SpectraMax GeminiXS fluorescent plate reader. Plots of initial velocity (RFU s–1) versus probe concentration were plotted and fit to the MichaelisMenten equation using GraphPad Prism (GraphPad Software, Inc.) and were shown at various mole fractions of DBPA and DBPC in Triton mixed micelles (Figure 4). Thioether Amide–PC Inhibitor Assay. To a 2 µg dried film of DBPC was added 2.2 mL of 0.04% (w/v) Triton X-100 (reduced) that had been prepared in 0.1 M Tris buffer, pH 8.8, containing 0.1 M CaCl2 and 0.05% NaN3. Using a probe sonicator set to power output = 3 and duty cycle = 50%, we sonicated this suspension for 30 s at room temperature, then in an ice bath for 2 min. The resulting mixed micelle stock solution was 0.8 µM in DBPC, amounting to 0.25% (w/w) of the Triton micelles. Thioether amide–PC (Cayman Chemical) was aliquoted into test tubes from 1 ng µL–1 and 10 ng µL–1 stock solutions to give 0, 15, 46, 77, 152, 463, and 772 ng of inhibitor in 100 µL total of 0.04% (w/v) Triton X-100 (reduced) buffer. A total of 250 µL of the DBPC mixed micelle stock solution was added to each test tube of inhibitor. Each test tube was then sonicated as above for 30 min at room temperature and placed on ice for 2 min. When an approximate MW of 631 for Triton X-100 (reduced) was assumed, this gave thioether amide–PC/DBPC/Triton mixed micelle solutions of mole fraction XTEPC = 0.0001–0.005 and a final concentration of DBPC in Triton of 0.18% (w/w), or 0.6 µM relative to solution. From each test tube, 100 µL of solution was placed in a black 96-well plate in duplicate. Separate wells containing 0.0013 U µL–1 bee venom PLA2 were also prepared. After the plate was incubated at 37 °C for 2 min, 10 µL of bee venom PLA2 solution was added to each well of DBPC/thioether amide–PC solution. Readings were recorded at 15 s intervals on a fluorescent plate reader during 5 min (λex = 500 nm;


λem = 530 nm). Initial velocities were calculated from the slopes generated within the first 75 s of enzyme reaction. The log(XTEPC) was plotted against the initial velocities and fit to a sigmoidal curve for one-site competitive binding using GraphPad Prism (Figure 5). Head Group Assay. Samples of DBPA, DBPC, DBPE, and DBPG were dried into separate vials and carefully weighed five times to get average masses. Stock solutions were generated by diluting the samples with 65:25:4 CH2Cl2/MeOH/H2O. On the basis of the calculated average concentration of each sample, 5 µg aliquots of each stock solution were withdrawn. To increase precision and decrease variability between experiments, the UV absorbance at 510 nm of each 5 µg aliquot was measured and compared to the absorbance at 510 nm of its mother solution. Aliquot concentrations were then adjusted as necessary to bring each absorbance to its expected value. The 5 µg aliquots were dried down to a film. To each resulting film was added a 0.76% (w/v) solution of Triton X-100 (reduced) in Tris buffer (0.1 M, containing 0.1 M CaCl2 and 0.05% NaN3, pH 8.8). These were sonicated as above to generate 1.2 µM solutions of each probe as mixed micelles in Triton (0.02% (w/w) probe). Each solution was added to the wells of a black 96-well plate in duplicate. The wells were incubated and inoculated with 0.1 U well–1 (70 ng well–1) bee venom (Sigma), 0.6 U well–1 LysoMaxS (Genencor), 0.5 U well–1 (260 ng well–1) N. mossambica venom (Sigma), 0.1 U well–1 (5 µg well–1) bovine pancreas (Sigma), 0.02 U well-1 S. violaceoruber (Sigma), or 20 ng well–1 human Type V PLA2 (Cayman Chemical) at 37 °C, and the fluorescence was measured as above. Slopes were obtained from 0–150 s, averaged, and compared (Figure 6). Cell Assay. Images (Figure 7) were recorded from enzymatically dissociated SCG neurons from cPLA2 +/+ mice. Dissociated neurons from 8–16 week-old SCG were plated at a density of one-half ganglion per one 35 mm poly-l-lysine-coated glass bottom petri dish (no. 1.5, MatTek Corp.) and incubated at 37 °C in a 5% CO2 environment in MEM. A 1 µg µL–1 stock solution of DBPC in chloroform was prepared, divided into 12 µL samples, dried with nitrogen, and stored at –80 °C. Aliquots were mixed with 44 µL of PS in chloroform and redried under nitrogen. The DBPC-PS mixture was rehydrated with 1 mL of PBS, sonicated for 15 min on ice to generate liposomes, and used within a few hours of preparation. Cells cultured in Matek dishes were preincubated in HBSS (Hank’s balanced salt solution) for 30 min. The DBPC-labeled liposomes were then added to the cell culture, yielding a final DBPC concentration of 0.01 µg µL–1. Cells were incubated for 40 min at 37 °C, washed three times with HBSS to remove any adherent liposomes, and then viewed on a custombuilt, video-rate confocal microscope with a 40× objective lens. An excitation wavelength of 488 nm was used, and emission spectra were collected with long pass filters (OGSIS) at 515 nm. After recording time-zero images (Figure 7a), cells were stimulated with Oxo-M (oxotremorine-M) or treated with HBSS for unstimulated control images (not shown). For each time point, images were collected at room temperature at 30 frames/s for 1 s using Video Savant (IO industries, Inc.) and directly written to a PC. Each set of 30 images was averaged to create an image for time intervals ranging from 1 to 9 min and analyzed using NIH ImageJ133. The image generated after 6 min stimulation is shown in Figure 7b. Acknowledgment: We gratefully acknowledge Rubing Zhao, Yen Bai, Michael J. Sanderson, and Ann R. Rittenhouse for the cell images and associated experimental details. We thank the Center for Cell Signaling, a member of the Utah Centers of Excellence Program (1997-2002) and the NIH (Grants HL070231 and NS29632) for financial support of this research. Supporting Information Available: This material is available free of charge via the Internet.

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Fluorogenic Phospholipids as Head Group-Selective Reporters of

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