Maleimide-Functionalized Lipids that Anchor Polypeptides to Lipid

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Bioconjugate Chem. 2000, 11, 832−841

Maleimide-Functionalized Lipids that Anchor Polypeptides to Lipid Bilayers and Membranes John T. Elliott† and Glenn D. Prestwich* Department of Physiology and Biophysics, The University at Stony Brook, Stony Brook, New York, Department of Medicinal Chemistry, The University of Utah, Salt Lake City, Utah. Received March 27, 2000; Revised Manuscript Received June 13, 2000

Two maleimide-containing diacylglycerol derivatives were synthesized to permit the anchoring of short peptides and longer polypeptides to phospholipid bilayers and membranes. The maleimide was introduced at the site normally occupied by a phospholipid headgroup. The first lipid, the dipalmitoyl ester of 1-maleimido-2,3-propanediol, was developed as a membrane anchor for extracellular domains of transmembrane proteins. The second anchoring lipid, in which the 3-position contained a 6-aminohexanoate, was designed for convenient modification with amine-reactive reporter groups. Specifically, the NBD fluorophore, 7-nitrobenzo-2-oxa-1,3-diazole-aminohexanoic-N-hydroxysuccinimide ester, was attached to give an fluorescent anchoring reagent. Next, these reagents were applied to the anchoring of a C-terminally cysteamine-modified 8 kDa polypeptide that comprises the extracellular N-terminal domain of the human thrombin receptor, a transmembrane protease-activated receptor (PAR-1). Gel filtration and fluorescence analysis showed that the fluorescent lipopolypeptide spontaneously inserted into preformed phospholipid vesicles, but it did not insert into whole cell membranes. In contrast, the dipalmitoyl derivative could only be reconstituted into artificial membranes by mixing the lipopolypeptide and phospholipid before vesicle formation. These results suggest that biophysical interactions governing the lipopolypeptide insertion into artificial and cellular membranes may differ. The thiol-reactive lipidating reagents should be valuable materials for studying the structure and function of peptides and polypeptides at phospholipid bilayer surfaces.

INTRODUCTION

Lipid modification of soluble peptides and polypeptides confers amphipathic character to the molecules and typically changes their behavior in aqueous solutions. The amphiphiles have a tendency to aggregate, form micelles and vesicles, and associate with phospholipid bilayers. This property makes it possible to use acylated peptides and polypeptides to investigate biophysical phenomena that occur on cellular membranes. Versatile reagents for the synthesis of lipopeptides are thus an important requirement for the success of such studies. We describe herein two new thiol-reactive lipids and the attachment of these reagents to a thiol-functionalized recombinant 8-kDa polypeptide. Synthetic lipoconjugates can be used to evaluate the biophysics of protein-membrane interactions. Membrane binding properties of signal transduction and oncogenic proteins that are palmitoylated or myristoylated in vivo have been examined with lipopeptides that represent the native proteins (1-3). Naturally occurring lipopeptides that function as bacterial cell wall components (4), biosurfactants (5), or antibiotics (6) have been investigated using artificially synthesized analogues under in vitro and in vivo conditions. Lipid conjugates of peptides have also been used to study biologically relevant struc* To whom correspondence should be addressed at The University of Utah. Voice: (801) 585-9051; (801) 581-7063. Fax: (801) 585-9053; (801) 581-7087. E-mail: gprestwich@ deans.pharm.utah.edu. † The University at Stony Brook. Present address: National Institute of Standards and Technology, Biotechnology Division, 100 Bureau Drive, Gaithersburg, MD 20899.

tural conformations that are induced at phospholipid membrane surfaces (7). A lipid modification can significantly increase the lifetime of the membrane-bound complex, thereby improving the data obtained from a structural study. For example, the structural properties of peptide hormones CCK (8), gastri (9), and yeast R-factor (10) were studied in membrane-bound preparations using this approach. A variety of synthetic methods are available for preparing lipid conjugates of peptides and polypeptides. Acylations can be performed at reactive amino acid side chains (11, 12) or during peptide synthesis on solid-phase resins (13-16). In the former method, conjugation via a thiol group is convenient because thiol function is often unique in a primary sequence (i.e., Cys). In addition, molecular biology techniques allow site-specific incorporation of cysteine residues when the peptides or polypeptides are produced in cellular systems (17). The maleimide group is routinely used as a thiol acceptor, as it exhibits high selectivity and reactivity under essentially neutral aqueous conditions. Several maleimide-based lipidating reagents are commercially available. Two reagents are based on a heterobifunctional four- or sixcarbon linker conjugated to the phosphatidylethanolamine headgroup (Avanti Polar Lipids, Alabaster, AL). Another features a 3500 Da poly(ethylene glycol) extending from the phospholipid headgroup (Shearwater Polymers, Huntsville, AL), but the flexible tether introduces considerable uncertainty as to where coupled peptide would reside relative to the membrane surface. To study the extracellular polypeptide domains of transmembrane membrane proteins, we required a reagent that could anchor a sulfhydryl-containing peptide

10.1021/bc000022a CCC: $19.00 © 2000 American Chemical Society Published on Web 09/12/2000

Maleimide-Functionalized Lipids That Anchor Polypeptides

Figure 1. Structures of the maleimido-lipids. Lipid 2 contains a BOC protecting group on the primary acyl chain. Upon BOC removal, the amino-acyl chain can be modified with amine reactive reporter groups.

to a phospholipid bilayer. For our application, it was important that the conjugation site was confined to the region of a phospholipid bilayer normally occupied by the headgroups. Thus, two lipids that contained maleimide functional groups on a diacylglycerol-like backbone were prepared (Figure 1). The first lipid, Pam2Mal (1), was constructed with two palmitoyl chains to serve as a membrane anchor. The second lipid, BOC-AH-PamMal (2), was prepared with a palmitoyl and a BOC-protected1 6-aminohexanoyl chain. The protected amino-acyl chain was introduced so the lipid could ultimately be modified with amine reactive reporter groups (e.g., fluorophores) to allow facile quantitation and localization of the lipoconjugate (18). In this study, the lipid was derivatized to give the fluorescent lipidating reagent 7-nitrobenzo2-oxa-1,3-diazole-6-aminohexanoyl-AH-PamMal (NBDPamMal) (10). To evaluate the utility of the lipids, a soluble 8 kDa polypeptide representing the N-terminal extracellular domain of the G protein-coupled human thrombin receptor (PAR-1) (19) was selectively derivatized at a Cterminal cysteamine with Pam2Mal and NBD-PamMal. The lipopolypeptides were assessed for their ability to associate with artificial and cellular membranes by gel filtration and fluorometric assays. The results from this study demonstrate that these new thiol-reactive lipidating reagents will be valuable for preparing lipoconjugates that can be anchored into phospholipid membranes, and can be employed for biological structure and function studies. EXPERIMENTAL PROCEDURES

Materials and General Methods. NMR spectra were collected on a Varian Gemini 300 instrument. Highresolution FAB-MS was performed at The University of California, Riverside, mass spectrometry facility. MALDI1 Abbreviations: BOC, di-tert-butyl-dicarbonate; CBZ, carbobenzyloxy protecting group; CHRF-288, megakaryoblastic celltype; DCC, dicyclohexylcarbodiimide; DIC, diisopropylcarbodiimide; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPG, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol; DTT, dithiothreitol; Hex, hexane; NBD, 7-nitrobenzo-2-oxa-1,3-diazole; NBD-Cl, 4-chloro-NBD; NBD-X-NHS, NBD-6-aminohexanoicN-hydroxysuccinimide ester; NMM, N-methoxycarbonyl-maleimide; NTTR, extracellular N-terminal domain of the human thrombin receptor (residues Glu30-Ser99); NTTR-ML, NTTRdipalmitoyl lipid conjugate; NTTR-(NBD)ML, NTTR-fluorescent lipid conjugate; NTTR-SH, NTTR with a C-terminal sulfhydryl group; PAR-1, protease activated human thrombin receptor; PB, phosphate buffer; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; RP-HPLC, reversed-phase HPLC; S2, Drosophila melangaster cell-line; TEA, triethylamine; TX-100, Triton X-100.

Bioconjugate Chem., Vol. 11, No. 6, 2000 833

MS was performed on a Bruker Protein-TOF system using a 3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid) matrix (CASM, University at Stony Brook, Stony Brook, New York). Uncorrected melting points were determined on a Fisher-Johns apparatus. The purity of the compounds was estimated by TLC, reversed-phase HPLC (RP-HPLC), mass spectrometry and NMR. NMethoxycarbonylmaleimide (NMM) was obtained from Sigma Chemical (St. Louis, MO). Chemical reactions were carried out in HPLC grade solvents and reagent grade solvents were used for silica gel chromatography (flash grade). Phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL). HPLC purifications were performed on an LKB/Bromma 2152 system and the eluate was monitored at 220 or 280 nm. BOC-6-aminohexanoic acid (20) and CBZ-(()-1-amino-2,3-propanediol (21) were prepared as previously described. TLC Stains. Lipid products were identified with 10% phosphomolybdic acid in ethanol (EtOH). Primary amines were detected with 5% ninhydrin in EtOH. Maleimides were identified as previously described (22). Fluorescent lipid products were visualized directly under longwavelength UV illumination (∼350 nm). CBZ-Amino-2,3-dipalmitoyl-propane (4). CBZ-Amino-propanediol 3 (0.45 g, 2.0 mmol) was mixed with palmitic acid (1.22 g, 4.8 mmol) and (dimethylamino)pyridine (DMAP, 0.49 g, 4.0 mmol) in CHCl3 (20 mL). Diisopropylcarbodiimide (DIC, 790 µL, 5.0 mmol) was added and the mixture was stirred for 16 h at room temperature. The solvent was removed by rotary evaporation and the residual oil was dissolved in methanol (MeOH) (ca. 50 mL) and stored at -20 °C for 1 h. Precipitated material was collected by filtration, redissolved in CHCl3 (1-2 mL) and recrystallized with MeOH. The product was collected by filtration and dried under vacuum. A white solid (0.81 g, 87% yield) was obtained: FAB-MS for C43H75NO6, MH+ at m/z 702.5679 (calcd 702.5673); mp 49-50 °C; 1H NMR (300 MHz, CDCl3) δ 0.83-0.87 (t, 6 H), 1.17-1.23 (m, 48 H), 1.54-1.60 (m, 4 H), 2.24-2.31 (m, 4 H), 3.36-3.48 (m, 2 H), 4.08-4.28 (m, 2 H), 4.96 (b, 1 H), 5.08 (s, 2 H), 7.24-7.34 (m, 5 H). The product was observed on TLC, Rf ) 0.57 in hexane (Hex):EtOAc (2:1). CBZ-Amino-3-(BOC-6-aminohexanoyl)-2-propanol (5). CBZ-Amino-propanediol 3 (1.2 g, 5.3 mmol) was mixed with BOC-6-aminohexanoic acid (1.17 g, 5.0 mmol) and DMAP (0.65 g, 5.3 mmol) in CHCl3 (30 mL). The solution was cooled to 0 °C and dicyclohexylcarbodiimide (DCC, 1.65 g, 8.0 mmol) was added. The major product was observed by TLC, Rf ) 0.51 in EtOAc:Hex (2:1). After 24 h, the urea byproduct was removed by filtration. The solvent was evaporated and the residue was dissolved in a small volume of CHCl3:EtOAc:Hex (3: 2:1). The mixture was purified on silica gel by eluting with EtOAc:Hex (2:1). A clear oil was obtained after solvent removal (1.1 g, 45% yield): FAB-MS for C22H34N2O7, MH+ at m/z 439.2465 (calcd 439.2444); 1H NMR (300 MHz, CDCl3) δ 1.29-1.35 (m, 2 H), 1.41-1.48 (m, 11 H), 1.58-1.65 (m, 2 H), 2.30-2.35 (m, 2 H), 3.07 (br t, 2 H), 3.16-3.47 (m, 2 H), 3.64 (br s, 1 H), 3.93 (m, 1 H), 4.04-4.14 (m, 1 H), 4.64 (br s, 1 H), 5.09 (s, 2 H), 5.43 (br s, 1 H), 7.29-7.33 (m, 5 H). CBZ-Amino-2-palmitoyl-3-(BOC-6-aminohexanoyl)propane (6). Secondary alcohol 5 (1.1 g, 2.5 mmol) was mixed with palmitic acid (0.77 g, 3.0 mmol) and DMAP (0.31 g, 2.5 mmol) in CHCl3 (25 mL). DCC (0.64 g, 3.13 mmol) was added and the solution was stirred for 16 h at room temperature. The mixture was cooled (-20 °C) and the urea byproduct was removed by filtration. The

834 Bioconjugate Chem., Vol. 11, No. 6, 2000

solvent was removed by rotary evaporation and the remaining residue was dissolved in small volume of CHCl3:EtOAc:Hex (2:1:1), purified on silica gel with EtOAc:Hex (1:1), and concentrated to give a clear oil (1.6 g, 95% yield): FAB-MS for C38H64N2O8, MNa+ at m/z 699.4590 (calcd 699.4560); 1H NMR (300 MHz, CDCl3) δ 0.82-0.87 (t, 3 H), 1.19-1.68 (m, 41 H), 2.20-2.30 (m, 4 H), 3.04-3.08 (t, 2 H), 3.37-3.44 (m, 2 H), 4.05-4.12 (m, 2 H), 4.23-4.28 (m, 1 H), 5.06 (m, 3 H), 7.27-7.35 (m, 5 H); TLC, Rf ) 0.52 in Hex:EtOAc (2:1). 1-Amino-2,3-dipalmitoyl-propane (7a). CBZ-protected lipid 4 (2.0 g, 2.8 mmol) was dissolved in warm absolute EtOH (100 mL) containing 5% Pd/C (ca. 500 mg). The solution was saturated with H2 (ca. 2 atm) and shaken for 6 h at room temperature. CHCl3 (100 mL) was added to the solution, the catalyst was removed by filtration through Celite, and the solvent was removed in vacuo to give a white solid. The product migrated slightly above the origin on TLC in Hex:EtOAc (2:1) and stained positive with ninhydrin. Pam2Mal (1). Amino lipid 7a (300 mg, 0.48 mmol) was mixed with NMM (147 mg, 0.94 mmol) and tetrabutylammonium hydrogen sulfate (150 mg, 0.44 mmol) in CHCl3 (4 mL) on ice. TEA (66 µL, 0.64 mmol) was slowly added over 5 min, and the solution was stirred for an additional 10 min. The solution was removed from the ice and saturated NaHCO3 (8 mL) was added to the organic layer while being vigorously stirred. The maleimido-lipid was observed in the organic layer by TLC, Rf ) 0.83 in Hex:EtOAc (2:1). After 3 h at room temperature, the phases were separated by low speed centrifugation and the reddish organic layer was concentrated in a rotary evaporator. The residue was dissolved in a small volume of CHCl3:Hex:EtOAc (3:2:1) and the maleimidolipid was purified on silica gel with a Hex:EtOAc (2:1) and concentrated to give a white crystalline product (211 mg, 69% yield): FAB-MS for C39H69NO6, MNa+ at m/z 670.5005 (calcd 670.5023); mp 62-63 °C; 1H NMR (300 MHz, CDCl3) δ 0.83-0.87 (m, 6 H), 1.16-1.23 (m, 48 H), 1.51-1.61 (m, 4 H), 2.21-2.33 (m, 4 H), 3.68-3.81 (m, 2 H), 4.04-4.25 (m, 2 H), 5.19-5.24 (m, 1 H), 6.69 (s, 2 H). The maleimide proton peaks were observed at 6.69 ppm. The product could be stored as a powder at -20 °C or as a 50 mM solution in CHCl3 at -80 °C for at least one year with less than 5% maleimide hydrolysis. BOC-AH-PamMal (2). Lipid 2 was prepared using the identical procedure described above for the CBZ removal and maleoylation of dipalmitoyl lipid 4. The 1-amino-2palmitoyl-3-(BOC-6-aminohexanoyl)-propane lipid (7b) was a clear oil. The maleoylated product (2) was observed by TLC (Rf ) 0.5), developed in Hex:EtOAc (2:1), and purified on silica gel with Hex:EtOAc (2:1). After solvent removal and overnight drying in vacuo, a clear oil was obtained. The oil crystallized into a white waxy solid after standing overnight at -20 °C (277 mg, 69% yield): FABMS for C34H58N2O8, MNa+ at m/z 645.4113 (calcd 645.4091); mp 37-39 °C; 1H NMR (300 MHz, CDCl3) δ 0.82-0.87 (t, 3 H), 1.22-1.67 (m, 41 H), 2.21-2.34 (m, 4 H), 3.07 (br t, 2 H), 3.66-3.81 (m, 2 H), 4.03-4.25 (m, 2 H), 5.18-5.25 (m, 1 H), 6.69 (s, 2 H). The product was stored at -20 °C for at least 5 months without detectable maleimide hydrolysis. Modified Route to NBD-6-aminohexanoic-NHS Ester (NBD-X-NHS, 9). NaHCO3 (0.63 g, 7.5 mmol) and 6-aminohexanoic acid (0.66 g, 5.0 mmol) were added to H2O (5 mL). NBD-Cl (0.5 mg, 2.5 mmol) dissolved in MeOH (20 mL) was added and the mixture was stirred at 60 °C for 30 min (23). The solution was cooled in ice, acidified to pH 2.0 with concentrated HCl, and the MeOH

Elliott and Prestwich

was removed by rotary evaporation. Excess water (ca. 50 mL) was added and the suspension was homogeneously dispersed by bath sonication. A fine black powder was collected on a 0.45 µm cellulose acetate filter (Fisher Scientific), washed with water, and dried under vacuum. The total yield of crude NBD-6-aminohexanoic acid was 0.6 g (81% yield). Next, the crude NBD-6-aminohexanoic acid (250 mg, 0.85 mmol) and N-hydroxysuccinimide (120 mg, 1.02 mmol) were dissolved in acetone (10 mL); DIC (200 µL, 1.28 mmol) was added and the solution was stirred for 16 h at room temperature. The solvent was removed under reduced pressure and the residue was suspended in MeOH (8 mL) in a sonicator bath. The insoluble material was collected by centrifugation (6000g for 10 min), washed with MeOH (2 mL), and dried under vacuum. The total yield was 200 mg (57% yield) and the product was greater than 85% pure as determined by RPHPLC (0-80% CH3CN/0.1% TFA in 40 min, 1 mL/min, on a C8 semianalytical column) and NMR (data not shown). NBD-PamMal (10). The aminohexanoyl chain of lipid 2 (50 mg, 80 µmol) was quantitatively deprotected in CHCl3:TFA (1:1, 1 mL) for 90 min at room temperature. The solvent was evaporated under N2 and the residue was redissolved in a small amount of CHCl3, and reconcentrated in vacuo to eliminate residual TFA. Next, the deprotected AH-PamMal (8) was dissolved in 90%CHCl3/ 10%MeOH (1 mL), and then NBD-X-NHS 9 (31 mg, 80 µmol) and TEA (27 µL, 200 µmol) were added. The product NBD-PamMal was identified by TLC developed in EtOAc:Hex (2:1, Rf ) 0.09) or EtOAc (Rf ) 0.32). The sample was mixed for 60 min at room temperature, concentrated in vacuo to give an orange residue; this oil was dissolved in a small volume of CHCl3:EtOAc:Hex (2: 2:1) and purified on silica gel with EtOAc:Hex (2:1) to remove a major fluorescent byproduct, followed by elution with EtOAc. A highly fluorescent product was obtained as an orange oil (64.2 mg, 83% yield). FAB-MS for C41H61N6O10, MNa+ at m/z 821.4428 (calcd 821.4425); 1H NMR (300 MHz, CDCl3) δ 0.83-0.87 (t, 3 H), 1.16-1.84 (m, 38 H), 2.18-2.35 (m, 6 H), 3.21-3.28 (m, 2 H), 3.513.53 (m, 2 H), 3.72-3.75 (m, 2 H), 4.02-4.29 (m, 2 H), 5.22-5.25 (m, 1 H), 5.78 (m, 1 H), 6.14-6.17 (d, 1 H), 6.70 (s, 2 H), 8.45-8.48 (d,1 H). Less than 5% of maleimide hydrolysis was detected by 1H NMR. The product was stored at -80 °C as a 50 mM stock solution in CHCl3. Preparation of the Thrombin Receptor Tether Polypeptide (NTTR-SH). The complete details of the molecular biology and protein purification (24) will be published elsewhere. The method is briefly described here. The cDNA encoding the extracellular N-terminal region of the human thrombin receptor (PAR-1) (19) Glu30-Ser99, was amplified by PCR and subcloned into the pET-31b polypeptide expression system (Novagen, Milwaukee, WI). After expression, purification and preparative CNBr cleavage, the C-terminal homoserine lactone of the 66 amino acid polypeptide was derivatized with the free-base form of cystamine (25) in DMSO essentially as described (17). The disulfide containing polypeptide was purified by HPLC, reduced with dithiothreitol (DTT), and again purified by HPLC to generate the 8 kDa polypeptide with a C-terminal cysteamine (called NTTR-SH). The total yield was generally greater than 70% for a 25 mg scale reaction. Lipid-Polypeptide Conjugation. Either Pam2Mal 1 or NBD-PamMal 10 (3.0 µmol, 60 µL from 50 mM stock in CHCl3) was mixed with n-octyl-glucopyranoside (0.1 mmol, 100 µL from 1 M stock in CHCl3) in a glass test


tube. The solvent was evaporated with a stream of nitrogen, and the residue was dried under vacuum for 2 h. NTTR-SH (5 mg, 0.6 µmol) dissolved in degassed 50 mM phosphate buffer (PB), pH 7.0 (1 mL), was added to the detergent/lipid film. The tube was filled with argon and sealed with Parafilm, and the suspension was homogenized by sonication. After stirring for 2 h at room temperature, the sample was dehydrated in a Speed-Vac. CHCl3 (0.7 mL) was added and the excess lipid, and detergent was extracted with brief bath sonication. The lipopolypeptide was recovered with the addition of MeOH (0.7 mL) and centrifugation (14000g for 10 min). The pellet was air-dried and dissolved in 200 µL of DMSO. Water (1 mL) was added and any insoluble material was removed by centrifugation (14000g). The supernatant was injected onto a semipreparative C4 HPLC column (Vydac, 1.0 × 250 mm) and the lipoconjugate was purified with the gradient 20 to 100% B over 60 min at 2 mL/ min; mobile phase A, 0% CH3CN/0.052% TFA; mobile phase B, 90% CH3CN/0.06% TFA. Product fractions were concentrated in a Speed-Vac, dissolved in water and lyophilized to give polypeptide conjugated to Pam2Mal 1, designated NTTR-ML, and the fluorescent lipopolypeptide conjugate from NBD-PamMal 10, designated NTTR(NBD)ML. The identity and purity of the products were determined by analytical HPLC, MALDI-MS, UV spectroscopy and fluorometry. Both lipopolypeptides were freely soluble in water and MeOH at concentrations of at least 4 mg/mL. Reconstitution of Lipopolypeptides into Phospholipid Membranes. Reconstitution experiments were performed with approximately a 200:1 phospholipid to lipopolypeptide molar ratio unless otherwise indicated. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (7 mg, 10 µmol) was transferred into a small round-bottom flask and the solvent was removed under N2 and then overnight in vacuo. NTTR-ML (23 nmol, 500 µL of a 46 µM stock solution in MeOH) was added to the lipid and homogeneously dispersed by bath sonication. The MeOH was removed by rotary evaporation and the residue was placed under vacuum for 8 h. The lipid film was hydrated with 50 mM PB, pH 7.2 (1 mL), and the contents were transferred to a glass test tube. The tube was filled with argon, sealed with Parafilm, and bath-sonicated (Heat Systems, Farmingdale, NY) for 30 min at 37 °C. Large multi-lamellar vesicles were removed by filtration through a 0.45 µm PVDF membrane (Whatman) and the samples were stored at 37 °C until used (26). Lipid loss from filtration was determined by including 0.1 mol % NBDDMPE in the phospholipid sample and measuring the change in fluorescence after Triton X-100 (TX-100) (2% final concentration) was added to samples corresponding to before and after filtration. The lipid loss varied from 50% for DMPC to 10 for 20% (molar ratio) 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG)/1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicle samples. The lipopolypeptide loss from filtration was determined by the fluorescamine assay used in the gel filtration studies described below. The quantity of lipopolypeptide recovered after filtration corresponded to phospholipid recovery (data not shown). The fraction of the lipopolypeptide on the exterior of the vesicles was estimated by comparing the fluorescence intensity of reconstituted vesicle samples treated with fluorescamine before or after solubilizing with 2% TX-100 (27). Spontaneous insertion of NTTR-(NBD)ML into preformed vesicles was achieved by adding NTTR-(NBD)ML (10 nmol, 10 µL of 10 µM stock in H2O) directly to a


vesicle preparation (ca. 2 mM lipid) and mixing by gentle pipet action. The insertion process could be monitored fluorometrically. NTTR-(NBD)ML (10 nmol) was added to 25 mM PB, pH 7.2 (1 mL), in a fluorescence cuvette. The sample was excited at 475 nm and the emission was monitored at 530 nm. Lipid vesicles (DMPC, 200 µL, from 10 mM stock), prepared as described above without the addition of lipopeptide, were added to the cuvette and the increase in fluorescence was recorded. Similar experiments were also performed with 20% DMPG/DMPC (molar ratio), POPC, and 20% POPG/POPC (molar ratio) vesicles. Spontaneous insertion of NTTR-(NBD)ML into whole cells was also attempted. Human platelets (outdated, University Blood Bank, The University at Stony Brook), CHRF-288 (R. W. Johnson Pharmaceutical Research Institute) and Drosophila melangaster cell-lines (S2) (Invitrogen) were prepared by collecting the cells under low speed centrifugation and resuspending the cells in phosphate-buffered saline, pH 7.4, containing 5 mM EDTA. The final cell density was approximately 109 and 107 cells/mL for platelets and CHRF-288 or S2 cells, respectively. Cells (100 µL) were added to a NTTR(NBD)ML sample and changes in fluorescence intensity were monitored at 25 °C. After the fluorescence intensity change had come to equilibrium, POPC vesicles were added determine the quantity of lipopolypeptide that remained in solution and was able to interact with the artificial membranes. Gel Filtration Analysis of Reconstituted Lipopolypeptides. The extent of lipopolypeptide reconstitution into lipid vesicles or whole cells was determined by incubating the samples at 25 or 37 °C for up to 6 h, followed by separation on CL-6B Sepharose (Pharmacia, 1.5 × 28 cm) with 25 mM PB, pH 7.2. The gravity-driven flow rate was ∼0.6 mL/min and 5 min fractions were collected. The 10-nm separation between excitation and emission wavelengths was used to reduce the intensity of scattered light at the detector. Fractions containing vesicles or whole cells could be identified visually as cloudy suspensions or by measuring scattered light intensity (λex ) 400 nm; λem ) 410 nm) with a fluorometer. Fractions containing the nonfluorescent lipopolypeptide, NTTR-ML, were determined by a fluorescamine assay (28): TX-100 (2% final concentration) and TEA (0.1% final concentration) were mixed with an aliquot (1 mL) of each fraction. Fluorescamine (20 µL, 10 mM in dioxane) was added to each sample, and the sample was vortexed for 1 min. Relative concentrations of NTTR-ML in each fraction were determined fluorometrically (λex ) 395 nm; λem ) 475 nm). Samples containing NTTR(NBD)ML were determined by adding TX-100 (2% final concentration) to an aliquot (1-mL) of each fraction and measuring the fluorescence intensity of the NBD fluorophore (λex ) 475 nm; λem ) 535 nm). Control gel filtration experiments were performed with the NTTR polypeptide (unconjugated) preincubated with vesicles and with each lipopolypeptide in the absence of vesicles and whole cells. RESULTS

Design and Synthesis of the Maleimido-Lipids. Two new maleimide-based lipid reagents were synthesized to permit conjugation with thiol-containing polypeptides and other biomacromolecules (Figure 1). 1-Aminopropanediol was chosen as the glycerol-like backbone for lipid construction. Maleoylation could then be performed at the primary amine to position the conjugation site proximal to the headgroups in a phospholipid membrane



Figure 2. Lipid modification of the N-terminal extracellular domain of the human thrombin receptor. (A) The primary structure of the lipopolypeptide prepared from a maleimide lipid (1 or 10) and the 8 kDa polypeptide (NTTR) from the human thrombin receptor (Glu30-Ser99) is shown. The thrombin cleavage site between Arg41 and Ser42 is indicated by the scissors. The sulfhydryl group at the C-terminal of the thrombin receptor polypeptide (NTTR) is generated with cystamine as described in the text. The maleimide lipid is shown adjacent to a phospholipid to illustrate the expected location of the conjugation site with respect to a phospholipid bilayer. The orientation of the polypeptide does not imply any structural characteristics. R1 is palmitoyl or NBD-Xaminohexanoyl and R2 is palmitoyl. (B) HPLC chromatograms after NTTR-SH was coupled to Pam2Mal (1). The hydrophobic modification increased the retention time on a C-4 semipreparative column by 20 min. Similar results were obtained after NTTRSH was conjugated to NBD-PamMal (10). (C) MALDI-MS analysis of the NTTR-ML product. A single product with a mass at 8767.5 Da was observed (calcd 8762.9). The second mass peak is a doubly charged ion.

(see Figure 2A). The dipalmitoyl derivative Pam2Mal (1) was constructed to ensure that the conjugated polypeptide would remain anchored into a phospholipid bilayer. Binding studies have shown that molecules acylated with two long hydrocarbon chains can exhibit long lifetimes in a membrane-bound complex (t1/2 exceeding several hours) (29). The BOC-protected AH-PamMal reagent (2) was constructed to serve as a phospholipid membrane anchor that could be conveniently modified with amine reactive reporter groups. BOC was chosen as the amino acyl protecting group since it could be removed under acidic conditions that would not alter the maleimide group (22, 30). After CBZ protection, the diol was acylated in the presence of DMAP and a carbodiimide (Scheme 1). Use of N,N-diisopropylcarbodiimide during the formation of the dipalmitoyl derivative allowed simultaneous crystallization of 1 and extraction of the urea byproduct with MeOH, thereby eliminating a chromatographic step. Sequential acylation of CBZ-1-amino-propanediol with BOC-6-aminohexanoic acid at the primary alcohol followed by palmitic acid at the secondary alcohol took advantage of the differential reactivity between the primary and secondary alcohols (31). Quantitative hydrogenolysis of the CBZ protecting group unmasked the primary amine used for maleoylation. Maleoylation of Lipids. NMM maleoylates primary amines under mild aqueous conditions (22). Maleimide cyclization is dependent on bicarbonate ion; thus, the

Scheme 1. Assembly of Lipid Acyl Chains

reactants must be partially soluble in aqueous NaHCO3. Since the amino lipids 7a and 7b were completely insoluble in aqueous solutions, maleoylation was accomplished in a two-phase system containing a phase transfer catalyst (Scheme 2). An amide-imide intermediate (22) was initially formed in the CHCl3 phase and the

Maleimide-Functionalized Lipids That Anchor Polypeptides Scheme 2. Maleoylation of Lipids

addition of NaHCO3 with vigorous mixing resulted in cyclization to the maleimide. Maleimido-lipids 1 and 2 were recovered in greater than 65% yield with this procedure. The overall reaction rate was approximately 6-fold lower than that reported for the totally aqueous system (22). The biphasic system described here should be useful for preparation of other lipid, drug, or fluorophore-conjugating reagents that are not compatible with standard maleimide-forming procedures (32-34). Amino Acyl Deprotection and Fluorescent Modification of Lipid 2. BOC protected AH-PamMal (2) was designed to improve the experimental versatility of the conjugating reagent. This lipid was found to be very stable upon storage, thus it can be prepared on a semipreparative scale and utilized as required with different amine reactive reporter groups. We chose to modify the lipid with NBD-X-NHS ester, since the derivatization produces a molecule similar to wellcharacterized fluorescent phospholipids (35) and phosphoinositides (31, 36, 37). In addition, NBD is an environmentally sensitive fluorophore that exhibits a large increase in quantum yield in the presence of hydrophobic environments (38). BOC-AH-PamMal (2) was quantitatively deprotected with TFA; no hydrolysis of the maleimide group was observed. Maleimide hydrolysis was undetectable when the deprotected lipid was incubated for 1 h in an organic solvent system (90% CHCl3/10% MeOH with 5 mol equiv of TEA) commonly used for amine acylation with activated reagents. Modification with NBD-X-NHS ester using this solvent system afforded NBD-PamMal (10) in 80% yield (Scheme 3). Most activated amine-reactive reporter groups can be used to acylate the amino-acyl lipid under similar conditions. Direct reaction of AHPamMal (8) with NBD-Cl resulted in a number of side products, and the corresponding NBD-lipid could only be purified in a 1% final yield (data not shown). The synthesis of NBD-6-aminohexanoic-NHS ester (9) presented here offers an economical way to prepare large quantities of this reagent. It does not require chromatography and results in a 50-60% overall yield of amine reactive product. The major impurity (less than 15%) is a nonreactive, nonfluorescent hydrolyzed product of NBD-Cl that does not interfere with coupling reactions. This protocol should be applicable to large scale preparations of fluorescent bioconjugates. Lipid Conjugation to the NTTR Polypeptide. Generation of the thrombin receptor extracellular domain, NTTR, with a C-terminal sulfhydryl group provided the opportunity to conjugate the polypeptide to the maleimido-lipids. Detergent was required to solubilize the lipid and polypeptide, but only n-octyl-glucopyranoside, but not SDS or TX-100, resulted in homogeneous suspensions. The lipopolypeptides were easily purified by HPLC on a C4 column (Figure 2B). MALDI-MS analysis of

Bioconjugate Chem., Vol. 11, No. 6, 2000 837 Scheme 3. Deprotection and Fluorescent Modification of the Amino-Acyl Chain of Lipid 2

NTTR-ML indicated the formation of a single lipid conjugate with the correct mass (Figure 2C). The observed mass was 8767.5 Da (calcd 8762.9 Da). The fluorescent NTTR-(NBD)ML lipopolypeptide did not generate reproducible MALDI-MS values, but the HPLC chromatogram indicated that only a single new product was created (tR ) 29 min) after the reaction. The conjugation yields were >70% (4 mg) as judged by the preparative HPLC chromatogram (Figure 2B). Lipopolypeptide formation was not observed when the maleimidolipids were incubated with the NTTR polypeptide that did not contain a free sulfhydryl group. We attempted to form lipoconjugates by adding NTTRSH directly to DMPC vesicles containing 2% maleimide lipids. Conjugates could not be detected by HPLC or MALDI-MS suggesting the maleimide group was not available in a sufficiently reactive environment for coupling to occur. Reconstitution of the Lipopolypeptides into Phospholipid Vesicles. Analysis of the gel filtration chromatograms after application of each lipopolypeptide in the absence of phospholipid vesicles revealed that they elute before the unconjugated 8 kDa NTTR polypeptide (Figure 3A). This suggested that a majority of the lipopolypeptide (>90%) is aggregated in a micellar form at the concentrations (ca. 50-100 nM) used in these experiments. It also indicated that the CMC value for the lipopolypeptides is lower than 50 nM. Analysis of the micelles on a 50 kDa limit size-exclusion gel showed the lipopolypeptides eluted one fraction after the void volume, thereby suggesting the micelles were composed of between 6 and 9 monomers (data not shown). Both lipoconjugates were tested for the ability to spontaneously insert into phospholipid vesicles. This method of lipopolypeptide incorporation is beneficial in many biochemical applications since it can be performed under gentle in vivo conditions and results in lipoconjugates on the exterior of the membrane surface. Spontaneous insertion of NTTR-ML into DMPC or POPC vesicles after 10 h at 37 °C or 1 h at 50 °C could not be observed using gel filtration analysis (data not shown). The elution profiles of these preparations were identical to NTTRML in the absence of vesicles indicating the lipopolypeptide remained in micellar form (Figure 3A). Greater than 90% of NTTR-ML did reconstitute into vesicles when the phospholipids and lipopolypeptide were premixed before



Figure 3. Gel filtration (CL-6B Sepharose) and fluorometric analysis of lipopolypeptide association with phospholipid membranes. (A) Co-mixture of NTTR-ML with DMPC before vesicle formation resulted in greater than 90% incorporation of the lipopolypeptide in vesicles found in the void volume (fractions 6 and 7) (9). The NTTR polypeptide (unconjugated) does not associate with phospholipid membranes ([). NTTR-ML in the absence of lipid membranes eluted between the vesicle and the free polypeptide fractions indicating the lipopolypeptide is in micellar form (×). A similar chromatogram was observed when NTTR-ML was incubated with preformed DMPC or POPC vesicles (37 °C at 5 h) indicating NTTR-ML does not spontaneously insert into artificial membranes. NTTR-ML concentrations were measured with a fluorescamine assay. (B) NTTR-(NBD)ML was added to DMPC or POPC vesicles and incubated for 5 min before gel filtration analysis. NBD fluorescence was totally associated with the vesicle fractions indicating NTTR-(NBD)ML spontaneously inserted into the artificial membranes (×). The fluorescent lipopolypeptide in the absence of vesicles eluted with a profile similar to NTTR-ML indicating NTTR-(NBD)ML exists in micellar form (9). Whole cells (results from Drosophila S2 cells are shown in the figure) were incubated with NTTR-(NBD)ML before subjecting the cells to gel filtration analysis ([). Less than 1% of the total fluorescence was recovered in the void volume indicating the lipopolypeptide did not incorporate into whole cell membranes. Similar results were observed with human platelets and CHRF-288 cells. The chromatograms suggested NTTR-(NBD)ML remained in a micellar structure. (C) Spontaneous insertion of NTTR-(NBD)ML into POPC vesicles could be monitored fluorometrically. In micellar form, the NBD fluorophore was quenched [(×) before 2 min]. A 20-fold increase in fluorescence intensity was observed upon the addition of POPC vesicles (×). Addition of human platelet (9), CHRF-288 (2) or Drosophila S2 (b) whole cells induced less than 15% change in fluorescence. Gel filtration results indicated that the increase was not due to spontaneous insertion into the cell membranes. After ca. 25 min, the addition of POPC vesicles to the cell mixtures resulted in the expected fluorescence increase indicating the lipopolypeptide remained in solution (in micellar form) and preferentially interacted with the artificial membranes.

hydration and sonication (Figure 3A). Approximately, 66% ((10%) of the lipopolypeptide was accessible on the outer surface of the vesicles as determined by the fluorescamine assay described in the Experimental Procedures. The unconjugated NTTR polypeptide did not associate with phospholipid vesicles after incubation for several hours at 37 °C (Figure 3A). It eluted approximately four fractions after the lipid derivative indicating that it exists as a smaller molecular weight entity (e.g., monomer) in solution. NTTR-(NBD)ML did spontaneously insert into preformed vesicles as indicated by complete recovery of the lipopolypeptide in the void volume from the gel filtration column (Figure 3B). Similar results were obtained with 20% DMPG/DMPC and POPC vesicles. Since the quantum yield of the NBD fluorophore is environmentally sensitive, we were able to monitor lipopolypeptide insertion fluorometrically. The fluorescence intensity of NTTR(NBD)ML reached maximum value (ca. 15-fold increase) within 2 min after vesicle addition suggesting the lipopolypeptide was completely inserted within this period (Figure 3C). This was confirmed by gel filtration, which showed complete lipopolypeptide association with vesicles after only a 5 min incubation (Figure 3B). The increase

in quantum yield is likely a result of two environmental changes in the vicinity of the fluorophore. First, the NBD fluorophore becomes positioned into a hydrophobic phospholipid membrane environment. Second, the intermolecular quenching that exists when the lipopolypeptide is in a micellar state is eliminated upon phospholipid membrane association. Spontaneous insertion of NTTR-(NBD)ML or NTTRML into whole cell membranes after 6 h (28 °C for S2 cells, and 37 °C for platelets and CHRF-288 cells) was not observed by gel filtration analysis. Figure 3B shows the results from gel filtration analysis of NTTR-(NBD)ML incubated with the S2 cells at 28 °C for 6 h. Less than 1% of the NBD fluorescence was recovered in the void volume where the S2 cells were observed by light scattering measurements. Similar results were obtained after gel filtration analysis of NTTR-(NBD)ML preincubated with platelets and CHRF-288 cells (data not shown). The ability of the fluorescent lipopolypeptide to associate with whole cells was also examined fluorometrically. After a 25 min incubation of the fluorescent lipopolypeptide with platelets, CHRF-288 or S2 cells, the fluorescence intensity increased less than 15% when compared to the increase observed upon phospholipid


vesicle addition (Figure 3A). This result suggested that the addition of whole cells could promote an environmental change around the NBD fluorophore, but gel filtration analysis indicated that high-affinity membrane association did not occur. The reasons for the small fluorescent changes are unknown, but the fact that NTTR-(NBD)ML did not spontaneously insert into all three cell-types demonstrated that the behavior was not a cell-specific phenomena. Once the NBD fluorescence reached equilibrium after cell addition (ca. 25 min), the addition of POPC vesicles resulted in an immediate increase in fluorescence to levels that were obtained with only the addition of POPC vesicles (Figure 3C). This suggested that the fluorescent lipopolypeptide remained in solution (in micellar form) and preferentially inserted into the artificial vesicles. DISCUSSION

Two maleimide-based lipidating reagents were prepared to anchor sulfhydryl-containing peptides and polypeptides into phospholipid membranes. The lipopolypeptide conjugates prepared from each of the lipids were shown to form soluble micellar structures in aqueous solutions. This behavior is consistent with that of lipid-based amphiphiles. Interestingly, the ability of the lipid conjugates to interact with phospholipid membranes appeared to be dependent on the chemical details of the lipid modification and the type of biological membrane. To further understand this phenomenon, it is worth summarizing the properties that govern the interaction between lipid-based amphiphiles and phospholipid membranes. Two models have been proposed to characterize the interaction between amphiphiles (e.g., lipopeptides) and biological membranes. The first model depicts amphiphile insertion through a monomer intermediate. In this model, the probability of spontaneous insertion is dependent upon the equilibrium between micelle and monomer formation (i.e., CMC value), the rate of monomer escape from the micelle and the rate of monomer insertion into the acceptor membrane (39, 40). The steady-state concentration of free monomer is not necessarily important since studies have shown that spontaneous insertion can occur at amphiphile concentrations that are above their CMC values (39, 41). The dipalmitoyl-based lipopolypeptide, NTTR-ML, appears to be above its CMC value as demonstrated by the gel filtration data. However, the chromatogram also suggests that up to 10% of the conjugate may exist in smaller aggregates or in monomeric form (Figure 3A). This lipopolypeptide did not insert into artificial membranes at room temperature, after 10 h at 37 °C, or 1 h at 50 °C, indicating that it does not exist in a form that allows interaction between the conjugate lipid chains and the membrane surface. In terms of the monomer intermediate model, this suggests that monomer escape and insertion rates were not significantly affected by the increases in temperature. The fluorescent derivative, NTTR-(NBD)ML, did spontaneously insert into POPC, DMPC, and 20% DMPG/ DMPC vesicles at room temperature. This indicates that the surface charge and acyl chain composition of the acceptor membrane did not have a significant impact on the NTTR-(NBD)ML insertion process. This lipopolypeptide appears to be above the CMC value at experimental concentrations, although the tailing chromatogram suggests that some portion of NTTR-(NBD)ML may exist as smaller aggregates or monomers (Figure 3B). The only difference between the dipalmitoyl lipid derivative and the fluorescent lipid derivative is the


chemical structure of the primary acyl chain. This indicates the acyl moieties of the lipopolypeptide influences the interaction with artificial membranes. The mechanism by which the fluorescent lipopolypeptide promotes spontaneous insertion is unclear. However, the reduced hydrophobicity of the NBD-labeled acyl chain could influence the monomer escape and insertion rates. It has been shown that an NBD group on a phospholipid acyl chain localizes to the polar headgroup environment, thereby altering the structure of the associated hydrocarbon chain (35). The fluorescent lipopolypeptide did not insert into whole cell membranes. This suggests that a property of whole cell membranes that is not exhibited in the artificial membrane systems significantly lowers the fluorescent lipopolypeptide insertion rate. The second model that has been used to describe an amphiphile insertion process depicts insertion as dependent on a direct interaction between the amphiphile micelle and the phospholipid membrane (9, 42). In this model, collisions between the fluorescent NTTR-(NBD)ML micelles and the artificial phospholipid vesicles would be responsible for inducing conditions that allow insertion. The same conditions are apparently not achieved when the fluorescent lipopolypeptide micelles collides with whole cell membranes. Two main differences between the artificial and cellular membrane systems exist. First, the sizes of the membrane structures differ. The sonicated vesicles are small in diameter (20-200 nm) (43) and the cells used in this experiments are between 2 and 10 µm in diameter. The second difference is that whole cells have a surface richly adorned with glycoproteins and integral proteins. The protein component of the cellular membranes may have prevented NTTR-(NBD)ML association with the cellular surface, but previous reports have shown that various lipidated peptides (7, 44, 45) and proteins (46-48) are capable of spontaneous insertion into whole cell membranes. The size of the molecular headgroups, the method of lipid attachment, and the amino acid sequences vary in these studies, thus it is difficult to identify specific factors that prevent the NTTR lipid conjugates from incorporating into cellular membranes. It is possible that the chemical structure of the maleimido-lipids and the specific amino acid sequence of the NTTR polypeptide inhibit insertion into whole cells. Studies of phospholipid transfer from vesicles to whole cell membranes indicate that the transfer rate is dependent on the chemical structure of the acyl chains, backbone, and headgroups of the lipids (29, 49). The lipid reagents described here were conjugated to an 8 kDa polypeptide (NTTR) from the N-terminal of the human thrombin receptor (protease activated receptor1, PAR-1) (19). These model compounds were prepared to test the ability of the lipids to anchor the polypeptide into phospholipid membranes. The lipoconjugates were also prepared as probes that would be useful for examining structural and biochemical properties of the human thrombin receptor activation mechanism. The G-protein coupled PAR-1 receptor is a seven transmembrane receptor that exhibits an extended extracellular N-terminus. The receptor is activated when thrombin proteolytically cleaves the N-terminus, thereby generating the tethered ligand for the receptor. The NTTR polypeptide is identical to the N-terminal portion of the receptor prior to the first transmembrane segment and includes the thrombin cleavage site (Figure 2A). The C-terminal lipid modifications used in this study were proposed to mimic the first transmembrane segment of the receptor. Structural features exhibited by the polypeptide while it is anchored into a phospholipid


membrane were considered to be more closely representative of those that may be present in the native receptor (24). The NTTR lipid conjugates were also postulated to be useful as biochemical probes for investigating the rate of thrombin receptor cleavage on whole cellular membranes. Specific membrane-associated proteins have been implicated in altering the catalytic activity of thrombin on cellular membrane (50). A polypeptide representing the thrombin receptor N-terminus that could be anchored into cellular membranes would aid in characterizing the in vivo elements that influence the rate of thrombin receptor cleavage on a cellular membrane surface. Unfortunately, these studies were prevented by the inability of the NTTR lipopolypeptide to insert into whole cell membranes. In summary, a general and convenient method for the lipidation of thiol-containing biological molecules was presented. The maleimide-based lipoconjugating reagents Pam2Mal (1) and NBD-PamMal (10) are selective for sulfhydryl groups, which can be introduced into proteins, peptides, nucleic acids, or carbohydrates by either chemical or genetic methods. The extracellular N-terminal domain of the human thrombin receptor was derivatized with each lipid as a model system. The lipidated products were formed in high yield and could be reconstituted into artificial phospholipid membranes, although the reconstitution protocols were different for the two conjugates. The lipopolypeptides could not be reconstituted into whole cell membranes for reasons that are not clear. The conjugating reagents will aid in the preparation of acylated peptides and polypeptides for investigating structural and biochemical phenomena that occur at membrane surfaces. ACKNOWLEDGMENT

We would like to thank Dr. B. S. Coller (The University at Stony Brook, Stony Brook, NY, USB, and Mt. Sinai Medical Center, New York, NY) and Dr. B. E. Maryanoff (R.W. Johnson Pharmaceutical Research Institute, Spring House, PA, RWJPRI) for financial and scientific support. Dr. Q.-M. Gu and Dr. J. F. Marecek (USB) provided valuable insights during development of the synthetic methods. The PAR-1 cDNA was gift from Dr. W. Bahou (USB). Dr. C. Derian (RWJPRI) supplied the CHRF-288 cells and C. Coburn (USB) supplied the Drosophila S2 cells. LITERATURE CITED (1) Sankaram, M. B. (1994) Membrane interaction of small N-myristoylated peptides: implications for membrane anchoring and protein-protein association. Biophys. J. 67, 105112. (2) Silvius, J. R., and l’Heureux, F. (1994) Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers. Biochemistry 33, 3014-3022. (3) Buser, C. A., Sigal, C. T., Resh, M. D., and McLaughlin, S. (1994) Membrane binding of myristylated peptides corresponding to the NH2 terminus of Src. Biochemistry 33, 13093-13101. (4) Muhlradt, P. F., Kiess, M., Meyer, H., Sussmuth, R., and Jung, G. (1997) Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentration. J. Exp. Med. 185, 1951-1958. (5) Maget-Dana, R., and Ptak, M. (1995) Interactions of surfactin with membrane models. Biophys. J. 68, 1937-1943. (6) Stachelhaus, T., Schneider, A., and Marahiel, M. A. (1995) Rational design of peptide antibiotics by targeted replacement of bacterial and fungal domains. Science 269, 69-72.

Elliott and Prestwich (7) Moroder, L., Romano, R., Guba, W., Mierke, D. F., Kessler, H., Delporte, C., Winand, J., and Christophe, J. (1993) New evidence for a membrane-bound pathway in hormone receptor binding. Biochemistry 32, 13551-13559. (8) Romano, R., Bayerl, T. M., and Moroder, L. (1993) Lipophilic derivatization and its effect on the interaction of cholecystokinin (CCK) nonapeptide with phospholipids. Biochim. Biophys. Acta 1151, 111-119. (9) Romano, R., Musiol, H. J., Weyher, E., Dufresne, M., and Moroder, L. (1992) Peptide hormone-membrane interactions: The aggregational and conformational state of lipogastrin derivatives and their receptor binding affinity. Biopolymers 32, 1545-1558. (10) Jelicks, L. A., Broido, M. S., Becker, J. M., and Naider, F. R. (1989) Interaction of the Saccharomyces cerevisiae alphafactor with phospholipid vesicles as revealed by proton and phosphorus NMR. Biochemistry 28, 4233-4240. (11) Shahinian, S., and Silvius, J. R. (1995) A novel strategy affords high-yield coupling of antibody Fab’ fragments to liposomes. Biochim. Biophys. Acta 1239, 157-167. (12) Pecheur, E. I., Hoekstra, D., Sainte-Marie, J., Maurin, L., Bienvenue, A., and Philippot, J. R. (1997) Membrane anchorage brings about fusogenic properties in a short synthetic peptide. Biochemistry 36, 3773-3781. (13) de Bont, H. B. A., van Boom, J. H., and Liskamp, R. M. J. (1992) A convenient synthesis of a lipopeptide containing a diacylglycerol moiety: preparation of a potential inhibitor of protein kinase C. Recl. Trav. Chim. Pays-Bas 111, 222-226. (14) Kung, V. T., and Redemann, C. T. (1986) Synthesis of carboxyacyl derivatives of phosphatidylethanolamine and use as an efficient method for conjugation of protein to liposomes. Biochim. Biophys. Acta 862, 435-439. (15) Winger, T. M., Ludovice, P. J., and Chaikof, E. L. (1996) Lipopeptide conjugates: biomolecular building blocks for receptor activating membrane-mimetic structures. Biomaterials 17, 437-441. (16) Yu, Y., Berndt, P., Tirrell, M., and Fields, G. B. (1996) Selfassembling amphiphiles for construction of protein molecular architecture. J. Am. Chem. Soc. 118, 12515-12520. (17) Kuliopulos, A., and Walsh, C. T. (1994) Production, purification and cleavage of tandem repeats of recombinant peptides. J. Am. Chem. Soc. 116, 4599-4607. (18) Thompson, N. L., Brian, A. A., and McConnell, H. M. (1984) Covalent linkage of a synthetic peptide to a fluorescent phospholipid and its incorporation into supported phospholipid monolayers. Biochim. Biophys. Acta 772, 10-19. (19) Vu, T. H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64, 1057-1068. (20) Lee, B. H., Miller, M. J., Prody, C. A., and Neilands, J. B. (1985) Artificial siderophores. 1. Synthesis and microbial iron transport capabilities. J. Med. Chem. 28, 317-323. (21) Canonica, L., Nali, M., and Rindone, B. (1986) New potential immunoenhancing compounds. 1. Syntheses of 1-amino-1-deoxyphosphatidylcholine derivatives. Gazz. Chim. Ital. 116, 19-23. (22) Keller, O., and Rudinger, J. (1975) Preparation and some properties of maleimido acids and maleoyl derivatives of peptides. Helv. Chim. Acta 58, 531-541. (23) Ahnoff, M., Grundevlk, I., Arfwidsson, A., Fonsellus, J., and Persson, B. (1981) Derivatization with 4-chloro-7-nitrobenzofurazan for liquid chromatographic determination of hydroxyproline in collagen hydrolysate. Anal. Chem. 53, 485489. (24) Elliott, J. T. PhD Dissertation, The University at Stony Brook, Stony Brook, NY, 1999. (25) Nathan, A. H., and Bogert, M. T. (1941) The synthesis of pyrimidine and purine derivatives of cystamine and of a new type of thiazolidinopyrimidine. J. Am. Chem. Soc. 63, 23612366. (26) Signor, G., Mammi, S., Peggion, E., Ringsdorf, H., and Wagenknecht, A. (1994) Interaction of bombolitin III with phospholipid monolayers and liposomes and effect on the activity of phospholipase A2. Biochemistry 33, 6659-6670.

Maleimide-Functionalized Lipids That Anchor Polypeptides (27) Roy, M. T., Gallardo, M., and Estelrich, J. (1997) Bilayer distribution of phosphatidylserine and phosphatidylethanolamine in lipid vesicles. Bioconjugate Chem. 8, 941-945. (28) Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., and Weigele, M. (1972) Fluorescamine: a reagent for assay of amino acids, peptides, proteins and primary amines in the picomole range. Science 178, 871-872. (29) Shahinian, S., and Silvius, J. R. (1995) Doubly lipidmodified protein sequence motifs exhibit long-lived anchorage to lipid bilayer membranes. Biochemistry 34, 3813-3822. (30) Moroder, L., Nyfeler, R., Gemeiner, M., Kalbacher, H., and Wu¨nsch, E. (1983) Immunoassays of peptide hormones and their chemical aspects. Biopolymers 22, 481-486. (31) Chen, J., Profit, A. A., and Prestwich, G. D. (1996) Synthesis of photoactivatable 1,2-O-diacyl-sn-glycerol derivatives of 1-L-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PtdInsP2) and 3,4,5-trisphosphate (PtdInsP3). J. Org. Chem. 61, 6305-6312. (32) Kalgutkar, A. S., Crews, B. C., and Marnett, L. J. (1996) Design, synthesis and biochemical evaluation of N-substituted maleimides as inhibitors of prostaglandin endoperoxide synthases. J. Med. Chem. 39, 1692-1703. (33) Nielsen, O., and Buchardt, O. (1991) Facile synthesis of reagents containing a terminal maleimido ligand linked to an active ester. Synthesis 819-821. (34) Tsou, K., Barrnett, R. J., and Seligman, A. M. (1955) Preparation of some N-(1-naphthyl)-maleimides as sulfhydryl group reagents. J. Am. Chem. Soc. 77, 4613-4616. (35) Abrams, F. S., and London, E. (1993) Extension of the parallax analysis of membrane penetration depth to the polar region of model membranes: use of fluorescence quenching by a spin-label attached to the phospholipid polar headgroup. Biochemistry 32, 10826-10831. (36) Prestwich, G. D. (1996) Touching All the Bases: Inositol polyphosphate and phosphoinositide affinity probes from glucose. Acc. Chem. Res. 29, 503-513. (37) Prestwich, G. D., Chaudhary, A., Chen, J., Feng, L., Mehrotra, B., and Peng, J., (1998) Probing Phosphoinositide Polyphosphate Binding to Proteins. In Phosphoinositides (K. Bruzik, Ed.) Vol. 818, pp 24-47, American Chemical Society, Washington, DC, 1998. (38) Chattopadhyay, A., and London, E. (1988) Spectroscopic and ionization properties of N-(7-nitrobenz-2-oxa-1,3-diazol4-yl)-labeled lipids in model membranes. Biochim. Biophys. Acta 938, 24-34. (39) Kanda, S., Inoue, K., Nojima, S., Utsumi, H., and Wiegandt, H. (1982) Incorporation of a ganglioside and a spinlabeled ganglioside analogue into cell and liposomal membranes. J. Biochem. 91, 2095-2098.

Bioconjugate Chem., Vol. 11, No. 6, 2000 841 (40) Roseman, M. A., and Thompson, T. E. (1980) Mechanism of the spontaneous transfer of phospholipids between bilayers. Biochemistry 19, 439-444. (41) Zalipsky, S., Mullah, N., Harding, J. A., Gittelman, J., Guo, L., and Defrees, S. A. (1997) Poly(ethylene glycol)-grafted liposomes with oligopeptide or oligosaccharide ligands appended to the termini of the polymer chains. Bioconjugate Chem. 8, 111-118. (42) Kremer, J. M., Kops-Werkhoven, M. M., Pathmamanoharan, C., Gijzeman, O. L., and Wiersema, P. H. (1977) Phase diagrams and the kinetics of phospholipid exchange for vesicles of different composition and radius. Biochim. Biophys. Acta 471, 177-188. (43) Woodle, M. C., and Papahadjopoulos, D. (1989) Liposome preparation and size characterization. Methods Enzymol. 171, 193-217. (44) Schroeder, H., Leventis, R., Shahinian, S., Walton, P. A., and Silvius, J. R. (1996) Lipid-modified, cysteinyl-containing peptides of diverse structures are efficiently S-acylated at the plasma membrane of mammalian cells. J. Cell Biol. 134, 647660. (45) Metzger, J. W., Sawyer, W. H., Wille, B., Biesert, L., Bessler, W. G., and Jung, G. (1993) Interaction of immunologically active lipopeptides with membranes. Biochim. Biophys. Acta 1149, 29-39. (46) James, G., and Olson, E. N. (1990) Fatty acylated proteins as components of intracellular signaling pathways. Biochemistry 29, 2623-2634. (47) Kooyman, D. L., Byrne, G. W., McClellan, S., Nielsen, D., Tone, M., Waldmann, H., Coffman, T. M., McCurry, K. R., Platt, J. L., and Logan, J. S. (1995) In vivo transfer of GPIlinked complement restriction factors from erythrocytes to the endothelium. Science 269, 89-92. (48) Medof, M. E., Nagarajan, S., and Tykocinski, M. L. (1996) Cell-surface engineering with GPI-anchored proteins. FASEB J. 10, 574-586. (49) Homan, R., and Pownall, H. J. (1988) Transbilayer diffusion of phospholipids: dependence on headgroup structure and acyl chain length. Biochim. Biophys. Acta 938, 155-166. (50) Brass, L. F., Pizarro, S., Ahuja, M., Belmonte, E., Blanchard, N., Stadel, J. M., and Hoxie, J. A. (1994) Changes in the structure and function of the human thrombin receptor during receptor activation, internalization, and recycling. J. Biol. Chem. 269, 2943-2952.

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Maleimide-Functionalized Lipids that Anchor Polypeptides to Lipid

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