Targeting Lipopolyplexes Using Bifunctional Peptides Incorporating

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Bioconjugate Chem. 2007, 18, 1800–1810

Targeting Lipopolyplexes Using Bifunctional Peptides Incorporating Hydrophobic Spacer Amino Acids: Synthesis, Transfection, and Biophysical Studies Michael A. Pilkington-Miksa,† Michele J. Writer,‡ Supti Sarkar,§ Qing-Hai Meng,§ Suzie E. Barker,‡ Parviz Ayazi Shamlou,¶,§ Helen C. Hailes,† Stephen L. Hart,‡ and Alethea B. Tabor*,† Departments of Chemistry, University College London, Christopher Ingold Laboratories, 20 Gordon Street, London WC1H 0AJ, Wolfson Centre for Gene Therapy of Childhood Disease, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, and Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, United Kingdom. Received March 16, 2007; Revised Manuscript Received July 26, 2007

We have developed efficient synthetic routes to two hydrophobic amino acids, suitably protected for solid-phase peptide synthesis, and have successfully synthesized peptides containing these or other hydrophobic amino acids as spacers between a Lys16 moiety and an integrin-targeting motif. These peptides have in turn been used to formulate a range of lipopolyplex vectors with Lipofectin and plasmid DNA. The transfection efficiencies of these vectors and their aggregation behavior in buffers and in serum have been studied. We have shown that vectors containing peptides incorporating long linkers that are entirely hydrophobic are less efficient transfection agents. However, linkers of equivalent length that are in part hydrophobic show improved transfection properties, which is probably due to the improved accessibility of the integrin-binding motif.

INTRODUCTION The development of gene delivery methods for the treatment of various diseases is still limited by the efficiency, reliability, and safety of the vectors used. Although viral vectors frequently exhibit excellent gene transfer efficiencies, recent concerns about their safety and immunogenicity (1–3) have led to renewed interest in the design and synthesis of nonviral gene delivery vectors (4–6). We are currently investigating the use of a ternary synthetic lipopolyplex vector, the LID vector, for gene delivery. The first generation of these targeted, self-assembling vector complexes (7) consisted of plasmid DNA, a lipid component, Lipofectin (a 1:1 mixture of the cationic lipid DOTMA with a co-lipid DOPE), and a cationic peptide (1) (comprising a K16 domain which binds to the plasmid DNA, a targeting domain (CRRETAWAC), which binds to R5β1 integrins found on cell surfaces, and a flexible spacer sequence (GA)). LID vectors displayed high transfection efficiency and low toxicity in Vitro and in ViVo, transfected nondividing cells efficiently, and were well-tolerated upon repeated administration with low immunogenicity in ViVo (7–9). Our working hypothesis for the transfection pathway is that the LID complexes bind to the R5β1 integrin and are then internalized by receptor-mediated endocytosis. One factor which might have a major effect on the efficiency of vector binding and internalization, therefore, would be the length and structure of the spacer sequence: longer spacers might position the targeting domain away from the vector surface and therefore render it more accessible. We also hypothesized that the * [email protected]. † Department of Chemistry, University College London. ‡ Wolfson Centre for Gene Therapy of Childhood Disease, UCL Institute of Child Health. § Department of Biochemical Engineering, University College London. ¶ Present address: Process Engineering Center, Eli Lilly and Company, Lilly Corporate Center, DC 3127, Indianapolis, Indiana 46285, USA.

condensed DNA was surrounded by a lipid coat and reasoned that hydrophobic spacers might possess the ability to “stack” within the lipid coating, thus simultaneously increasing the stability of the resulting lipopolyplex vector. We have therefore synthesized peptide 2, containing ll-aminoundecanoic acid (Aua) as a spacer, and peptide 3, containing 16-aminohexadecanoic acid (Ahd) as spacer. Furthermore, in an attempt to make the spacer amino acid closer in structure to the unsaturated tail of the lipid DOTMA, we have synthesised the novel amino acid 4 (Fmoc-Laa), and used it to prepare peptide 5. Finally, we have designed and used peptide 7, incorporating a spacer containing 6-aminohexanoic acid (Ahx) units. We reasoned that the sequence Ahx-Ser-Ahx would be suitably hydrophobic and act

10.1021/bc0700943 CCC: $37.00  2007 American Chemical Society Published on Web 10/05/2007

Targeting Lipopolyplexes Using Bifunctional Peptides

as a longer spacer, but would not be able to stack within a lipid bilayer so efficiently. In this paper, we report the synthesis of amino acids 4 and 6, and the synthesis of the peptides; we also describe the effect of these spacers on the transfection efficiencies and biophysical properties of lipopolyplex vectors formulated from peptides 2, 3, 5, and 7.

EXPERIMENTAL PROCEDURES General Procedures and Materials. All reagents used for synthesis were purchased from commercial suppliers, with the exception of 9-phenylxanthen-9-ol, which was prepared from xanthone and phenylmagnesium bromide following a literature procedure (10). Solvents were purified using standard techniques, with the exception of THF, which was distilled from sodium wire without the use of benzophenone for the synthesis of 13. NMR spectra were recorded on a Bruker AMX300 spectrometer; chemical shifts (δ) are reported in parts per million (ppm) using residual isotopic solvent as an internal reference. Coupling constants (J) are reported in hertz (Hz). Electrospray mass spectra were recorded on Micromass Quattro LC, Thermo Finnegan MAT 900XP, or VG ZAB 2SE instruments, and fast atom bombardment mass spectra on Thermo Finnegan MAT 900XP or VG ZAB 2SE instruments. IR spectra were recorded on a Shimadzu FT-IR 8700. Solid-phase peptide synthesis was carried out either manually on a Merrifield bubbler or automatically on a peptide synthesizer (MilliGen 9050Plus PepSynthesiser). All reagents used for either manual or automatic synthesis were purchased from commercial suppliers, with the exception of the Fmoc-protected amino acids 4 (Fmoc-Laa) and 6 (FmocAua). HIPERSOLV DMF of HPLC grade was used straight from the bottle, while dichloromethane and piperidine were freshly distilled over calcium hydride before use. All peptide syntheses were carried out under nitrogen. Preparative HPLC was carried out on a Waters 600E instrument with Waters 486 UV detector, using a Vydac 218TP reverse-phase HPLC column (25 × 250 mm). Analytical HPLC was carried out on a Varian ProStar 210 with Varian ProStar 320 UV detector, using a Vydac 218TP reverse-phase HPLC column (2.1 × 250 mm). Water/ acetonitrile gradients were used as described, with both water and acetonitrile containing 0.1% trifluoroacetic acid and monitoring at 215 nm. Synthesis of Fmoc-Laa 4. 6-Bromohexanoic Acid tertButyl Ester 8 (11). To 6-bromohexanoic acid (2.50 g, 12.8 mmol) in dry dichloromethane (50 mL) at 0 °C under nitrogen was added tert-butyl alcohol (5.5 mL, 66 mmol, 5 equiv) followed by dimethylaminopyridine (0.156 g, 1.28 mmol, 0.1 equiv). After 5 min, dicyclohexylcarbodiimide (2.90 g, 14.08 mmol, 1.1 equiv) was added to this solution at 0 °C. The solution was then left to warm to room temperature and stirred for 12 h. After 12 h, the reaction was filtered and then partitioned with water (50 mL). The organic fraction was dried over anhydrous MgSO4 and concentrated in Vacuo. The remaining residue was purified by normal-phase silica gel chromatography, eluting with hexane/ethyl acetate (90:10) to give 8 as a clear oil (2.51 g, 78%). 1H NMR (300 MHz, CDCl3) δ 1.45 (9H, s, C(CH3)3), 1.46 (2H, m, Br(CH2)2CH2), 1.62 (2H, quintet, J ) 7.1 Hz, Br(CH2)3CH2CH2), 1.88 (2H, quintet, J ) 7.1 Hz, BrCH2CH2(CH2)3), 2.23 (2H, t, J ) 7.3 Hz, CH2COOC(CH3)3), 3.41 (2H, t, J ) 6.8 Hz, BrCH2); 13C NMR (75 MHz, CDCl3) δ 24.6, 28.0, 28.5, 32.8, 33.8, 35.7, 80.5, 173.2; IR νmax/cm-1 (film) 2977, 2936, 1728; C10H19O2Br (FAB+) m/z 253 (M + H+ [81Br], 15%), 251 (M + H+ [79Br], 18%), 197 ([C6H12O2 81 Br]+, 34%), 195 ([C6H12O2 79Br]+, 36%), 57 ([C4H9]+, 100%). 6-Iodohexanoic Acid tert-Butyl Ester 9 (12). To 6-bromohexanoic acid tert-butyl ester 8 (2.30 g, 9.20 mmol) in dry

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acetone (400 mL) under nitrogen was added sodium iodide (41 g, 275 mmol, 30 equiv). The reaction mixture was left to stir in darkness at room temperature for 6 days, after which time it was filtered. The separated solid was then washed with diethyl ether, which was added to the filtrate. The filtrate was concentrated in Vacuo and then redissolved in diethyl ether (250 mL). This solution was partitioned with 5% aqueous sodium thiosulfate (100 mL) and saturated aqueous sodium chloride (100 mL). The aqueous phase was back-extracted with diethyl ether (150 mL). The organic solutions were combined, dried over anhydrous MgSO4, and concentrated in Vacuo to give a yellow oil. This oil was purified by silica gel chromatography, eluting with hexane/ethyl acetate (90:10) to give 9 as a yellow oil (2.53 g, 93%). 1H NMR (300 MHz, CDCl3) δ 1.39–1.49 (2H, m, I(CH2)2CH2), 1.45 (9H, s, C(CH3)3), 1.62 (2H, quintet, J ) 7.2 Hz, I(CH2)3CH2), 1.84 (2H, quintet, J ) 7.2 Hz, ICH2CH2), 2.22 (2H, t, J ) 7.3 Hz, CH2COOC(CH3)3), 3.19 (2H, t, J ) 7.0 Hz, ICH2(CH2)4); 13C NMR (75 MHz, CDCl3) δ 6.9, 24.4, 28.5, 30.3, 33.6, 35.7, 80.5, 173.2; IR νmax/cm-1 (film) 2977, 2933, 1729; C10H19O2I (FAB+) m/z 299 (M + H+, 24%), 243 ([C6H12O2I]+, 55%), 57 ([C4H9]+, 100%). 8-Bromooctan-1-ol (13). To 1,8-octanediol (5.96 g, 40.0 mmol) in toluene (350 mL) was added hydrobromic acid (5.25 mL, 48% aq solution). The reaction mixture was heated to reflux for 24 h after which time a further quantity of hydrobromic acid was added (1.5 mL). The reaction mixture was again left to reflux for 24 h. The reaction mixture was then cooled to room temperature, and diethyl ether was added (250 mL). The aqueous hydrobromic acid layer was separated from the organic layer; the organic phase was partitioned with saturated aqueous NaHCO3 (100 mL), then 0.1 M phosphate buffer (pH 7, 100 mL). The aqueous phase was back-extracted with diethyl ether (100 mL); the organic solutions were combined, dried over anhydrous MgSO4, and concentrated in Vacuo to give a yellow oil (8.56 g). This oil was purified by silica gel chromatography, eluting with hexane/diethyl ether (50:50) to give 8-bromooctan1-ol as a pale yellow oil (8.29 g, 39.64 mmol, 99%). 1H NMR (300 MHz, CDCl3) δ 1.33–1.59 (10H, m, BrCH2CH2(CH2)5), 1.86 (2H, quintet, J ) 7.1 Hz, BrCH2CH2), 3.40 (2H, t, J ) 6.8 Hz, BrCH2(CH2)5), 3.63 (2H, t, J ) 6.6 Hz, CH2OH); 13C NMR (75 MHz, CDCl3) δ 26.0, 28.5, 29.1, 29.6, 33.1, 33.2, 34.3, 63.3; IR νmax/cm-1 (film) 3333, 2930, 2855; C8H15OBr (FAB+) m/z 211 (M + H+ [81Br], 40%), 209 (M + H+ [79Br], 42%), 69 ([C5H9]+, 100%), 55 ([C4H7]+, 82 %). HRMS (FAB, NOBA matrix): C8H16OBr calcd for [MH]+ 209.0541, found 209.0550. 1-Bromo-8-(9-phenyl-xanthen-9-yloxy)octane 10. To 8-bromooctan-1-ol (5.41 g, 25.9 mmol) and 9-phenylxanthen-9-ol (10) (7.10 g, 25.9 mmol) was added glacial acetic acid (75 mL). Once complete dissolution had occurred, the solution was concentrated in Vacuo. A further volume of glacial acetic acid (75 mL) was added, and again the resulting solution was concentrated in Vacuo. This was repeated a further five times. Once all the glacial acetic acid had been removed for the final time, the remaining oil was redissolved in hexane (250 mL). This solution was partitioned with saturated aqueous NaHCO3 and saturated aqueous NaCl, and the aqueous phase was backextracted with hexane (100 mL). The organic solutions were combined, dried over anhydrous MgSO4, and concentrated in Vacuo to give 10 as a yellow oil (12.04 g, 100%). 1H NMR (300 MHz, CDCl3) δ 1.20–1.42 (8H, m, BrCH2CH2(CH2)4), 1.51 (2H, m, CH2CH2OPx), 1.84 (2H, quintet, J ) 7.2 Hz, BrCH2CH2), 2.96 (2H, t, J ) 6.2 Hz, CH2CH2OPx), 3.40 (2H, t, J ) 6.8 Hz, BrCH2), 7.01–7.41 (13H, m, aromatic); 13C NMR (75 MHz, CDCl3) δ 26.5, 28.5, 29.1, 29.5, 30.2, 33.2, 34.3, 63.4, 75.5, 116.5, 123.7, 124.2, 126.8, 126.9, 128.1, 129.2, 130.0, 149.9, 151.7; IR νmax/cm-1 (film) 3033, 2932, 2855,

1802 Bioconjugate Chem., Vol. 18, No. 6, 2007

1603, 1574; C27H29O2Br (FAB+) m/z 465 (M + H+[79Br], 1%), 275 ([C19H15O2]+, 12%), 257 ([C19H13O]+, 100%), 197 ([C13H9O2]+, 63%), 181 ([C13H8O]+, 14%). Anal. Calcd for C27H29O2Br: C, 69.68; H, 6.28; Br, 17.16. Found: C, 69.72; H, 6.20; Br, 17.15. 1-Iodo-8-(9-phenylxanthen-9-yloxy)octane 11. To 1-bromo8-(9-phenyl-xanthenyloxy)octane 10 (12.04 g, 25.9 mmol) in dry acetone (500 mL) was added sodium iodide (155.1 g, 1.03 mol, 40 equiv). The reaction was left stirring under nitrogen, in darkness, and at room temperature for seven days. After seven days, the reaction mixture was concentrated in Vacuo. The remaining solid was dissolved in diethyl ether (400 mL) and saturated aqueous NaHCO3 (400 mL). The organic phase was then partitioned with 5% aqueous sodium thiosulfate (150 mL) and saturated aqueous NaCl (150 mL). The aqueous solutions were combined and back-extracted with diethyl ether (200 mL). The organic solutions were combined, dried over anhydrous MgSO4, and concentrated in Vacuo to give a dark yellow oil. This oil was purified by normal-phase silica gel chromatography, eluting with chloroform to give 11 as a pale yellow oil (12.9 g, 97%). 1H NMR (300 MHz, CDCl3) δ 1.22–1.41 (8H, m, ICH2CH2(CH2)4), 1.54 (2H, m, CH2CH2OPx), 1.82 (2H, m, ICH2CH2), 2.98 (2H, t, J ) 6.3 Hz, CH2OPx), 3.2 (2H, t, J ) 7.0 Hz, ICH2CH2), 7.01–7.41 (13H, m, aromatic); 13C NMR (75 MHz, CDCl3) δ 7.5, 26.5, 28.8, 29.5, 30.2, 30.8, 33.9, 63.4, 75.5, 116.5, 123.7, 124.2, 126.8, 126.9, 128.1, 129.2, 130.0, 149.9, 151.7; IR νmax/cm-1 (film) 3033, 2932, 2855, 1603, 1574; C27H29O2I (FAB+) m/z 535 (M + Na+, 3%), 513 (M + H+, 1%), 257 ([C19H13O]+, 100%), 197 ([C13H9O2]+, 47%); HRMS (FAB, NOBA matrix): C27H30O2I calcd for [MH]+513.1291, found 513.1270. 1-(9-Phenylxanthen-9-yloxy)-dec-9-yne 12. To lithium acetylide ethylenediamine (11.5 g, 125 mmol, 40 equiv) under nitrogen was added dry tetrahydrofuran (30 mL). This suspension was cooled to -78 °C, and hexamethylphosphoramide (6 mL) was added. A solution of 1-iodo-8-(9-phenyl-xanthen-9yloxy)octane 11 (12.8 g, 25.0 mmol) in THF (30 mL) was slowly added to the suspension at -78 °C. Once all the solution had been added to the suspension, the reaction was allowed to warm to room temperature and left stirring overnight. After 24 h, the reaction was concentrated in Vacuo. The remaining oil was dissolved in diethyl ether (350 mL). This solution was filtered and then partitioned with 5% aqueous sodium thiosulfate (150 mL) and saturated aqueous NaCl (150 mL). The aqueous solutions were combined and back-extracted with diethyl ether (100 mL). The organic solutions were combined, dried over anhydrous MgSO4, and concentrated in Vacuo to give a brown oil. This oil was purified by normal-phase silica gel chromatography, eluting with hexane/chloroform (50:50) with a few drops of triethylamine, to give 12 as a pale yellow oil (10.05 g, 98%). 1H NMR (300 MHz, CDCl3) δ 1.24–1.63 (12H, m, HCtCCH2(CH2)6), 1.95 (1H, t, J ) 2.6 Hz, HCtCCH2), 2.27 (2H, dt, J ) 7.0 Hz and 2.6 Hz, HCtCCH2), 2.98 (2H, t, J ) 6.3 Hz, CH2OPx), 7.01–7.41 (13H, m, aromatic); 13C NMR (75 MHz, CDCl3) δ 18.8, 26.6, 28.9, 29.1, 29.4, 29.6, 30.3, 63.4, 68.5, 75.5, 85.1, 116.5, 123.7, 124.3, 126.8, 126.9, 128.1, 129.2, 130.0, 149.9, 151.7; IR νmax/cm-1 (film) 3304, 3034, 2932, 2856, 2120, 1603, 1575; C29H30O2 (FAB+) m/z 433 (M + Na+, 2%), 411 (M + H+, 2%), 257 ([C19H13O]+, 100%); HRMS (FAB, NOBA matrix): C29H31O2 calcd for [MH]+ 411.2324, found 411.2300. 2-Methylprop-2-yl 16-(9-phenylxanthen-9-yloxy)hexadeca7-ynoate 13. To freshly purified 1-(9-phenyl-xanthen-9-yloxy)dec-9-yne 11 (2.05 g, 5.00 mmol) in dry THF (30 mL) under N2 was added HMPA (4 mL). This solution was cooled to -78 °C, and then n-butyl lithium (2.6 mL, 2.5 M, 6.5 mmol, 1.3 equiv) was slowly added. The solution was left to stir for 30

Pilkington-Miksa et al.

min after which time freshly purified iodohexanoic acid tertbutyl ester 9 (1.94 g, 6.5 mmol, 1.3 equiv) in THF (25 mL) was added dropwise. Once this was complete, the reaction was allowed to warm to room temperature and was left to stir for 24 h. The reaction was then concentrated in Vacuo to yield a brown gum, which was dissolved in diethyl ether (300 mL), and the resulting solution was filtered. The filtrate was partitioned with 5% aqueous sodium thiosulfate (150 mL) and saturated aqueous NaCl (150 mL). The aqueous solutions were combined and back-extracted with diethyl ether (150 mL). The organic solutions were combined, dried over anhydrous MgSO4, and concentrated in Vacuo to give a brown oil. This oil was purified by normal-phase silica gel chromatography, eluting with hexane/chloroform (40:60) with a few drops of triethylamine, to give 13 as a pale yellow oil (2.03 g, 70%). 1H NMR (300 MHz, CDCl3) δ 1.18–1.68 (18H, m, PxOCH2(CH2)6 CH2CtCCH2(CH2)3), 1.45 (9H, s, C(CH3)3), 2.13–2.16 (4H, m, PxO(CH2)7CH2CtCCH2), 2.22 (2H, t, J ) 7.4 Hz, CH2COOC(CH3)3), 2.96 (2H, t, J ) 6.3 Hz, PxOCH2), 6.99–7.39 (13H, m, H aromatic); 13C NMR (75 MHz, CDCl3) δ 19.0, 19.2, 25.1, 26.6, 28.5, 28.7, 29.2, 29.47, 29.55, 29.61, 30.1, 30.3, 35.9, 63.4, 75.4, 80.3, 80.31, 80.8, 116.5, 123.7, 124.3, 126.8, 126.8, 128.1, 129.2, 130.0, 149.9, 151.7, 173.5; IR νmax/cm-1 (film) 2931, 2856, 1733, 1603, 1575; C39H48O4 (FAB+) m/z 603 (M + Na+, 5%), 269 ([C16H29O3]+, 4%), 257 ([C19H13O]+, 100%). 2-Methylprop-2-yl 16-hydroxyhexadeca-7-ynoate 14. To 2-methylprop-2-yl 16-(9-phenylxanthen-9-yloxy)hexadeca-7-ynoate 13 (1.39 g, 2.39 mmol) in dichloromethane (50 mL), under nitrogen was added pyrrole (1.65 mL, 1.6 g, 23.9 mmol, 10 equiv) followed by dichloroacetic acid (1.25 mL, 1.95 g, 15.0 mmol). This solution was left to stir for 10 min at room temperature. An excess of saturated aqueous NaHCO3 was then added, and the reaction was stirred vigorously to ensure mixing of aqueous and organic phases. The organic and aqueous phases were separated, and the aqueous phase was partitioned with dichloromethane (100 mL). The organic solutions were combined, partitioned with saturated aqueous NaCl (50 mL), dried over anhydrous MgSO4, and concentrated in Vacuo to yield a dark brown oil. This oil was purified by normal-phase silica gel chromatography, using only 60 mm in height of silica and eluting with chloroform with a few drops of triethylamine, to give 14 as a clear oil (0.77 g, 95%). 1H NMR (300 MHz, CDCl3) δ 1.28–1.68 (18H, m, HOCH2(CH2)6CH2CtCCH2(CH2)3), 1.45 (9H, s, C(CH3)3), 2.15–2.18 (4H, m, HO(CH2)7CH2CtCCH2), 2.22 (2H, t, J ) 7.5 Hz, CH2COOC(CH3)3), 3.63 (2H, t, J ) 6.6 Hz, HOCH2); 13C NMR (75 MHz, CDCl3) δ 19.0, 19.01, 25.1, 26.1, 28.5, 28.7, 29.1, 29.2, 29.5, 29.7, 33.2, 35.9, 63.3, 80.3, 80.4, 80.7, 173.5; IR νmax/cm-1 (film) 3419 (broad), 2931, 2856, 1732; C20H36O3 (FAB+) m/z 325 (M + H+, 4%), 269 ([C16H29O3]+, 27%), 57 ([C4H9]+, 100%). (Z)-2-Methylprop-2-yl 16-hydroxyhexadeca-7-enoate 15. A suspension of Lindlar catalyst (500 mg) in hexane/methanol (90:10, 20 mL) was prepared under nitrogen. This suspension was connected to a hydrogenation apparatus and exposed to hydrogen at atmospheric pressure at room temperature until no further change in gas volume was detected. 2-Methylprop-2-yl 16-hydroxy-hexadeca-7-ynoate 14 (0.73 g, 2.26 mmol) in hexane (10 mL) was then added to this suspension, and the reaction was left to absorb approximately 58 cm3 of hydrogen, at room temperature. Once this had occurred, the reaction mixture was dissolved in dichloromethane (80 mL) and filtered. The filtrate was concentrated in Vacuo to yield 15 as a yellow oil (0.74 g, 100 %). 1H NMR (300 MHz, CDCl3) δ 1.23–1.68 (18H, m, HOCH2(CH2)6CH2CHdCHCH2(CH2)3), 1.45 (9H, s, C(CH3)3), 2.02 (4H, m, HO(CH2)7CH2CHdCHCH2), 2.22 (2H, t, J ) 7.5 Hz, CH2COOC(CH3)3), 3.65 (2H, t, J ) 6.6 Hz,


HOCH2), 5.37 (2H, m, CHdCH); 13C NMR (75 MHz, CDCl3) δ 25.4, 26.1, 27.5, 27.6, 28.5, 29.2, 29.55, 29.60, 29.8, 29.9, 30.1, 33.2, 36.0, 63.3, 80.3, 130.0, 130.4, 173.7; IR νmax/cm-1 (NaCl) 3383 (broad), 3004, 2927, 2855, 1732; C20H38O3 (FAB+) m/z 349 (M + Na+, 1%), 57 ([C4H9]+, 100 %); HRMS (FAB, NOBA matrix): C20H38O3Na calcd for [M + Na]+ 349.2719, found 349.2730. (Z)-2-Methylprop-2-yl-16-azidohexadeca-7-enoate 16. To (Z)2-methylprop-2-yl-16-hydroxyhexadeca-7-enoate 15 (1.57 g, 4.82 mmol) in dry dichloromethane/triethylamine (1:1, 40 mL) at 0 °C, under nitrogen, was slowly added methanesulfonyl chloride (0.45 mL, 0.66 g, 5.78 mmol, 1.2 equiv). Once addition was complete, the reaction was left to stir for 2 h at room temperature. An excess of saturated aqueous NaHCO3 was then added, and the reaction was stirred vigorously for 10 min. The reaction was then concentrated in Vacuo until only a small volume of aqueous solution remained. The remaining concentrate was partitioned with dichloromethane (2 × 100 mL). The organic phase was partitioned with saturated aqueous NaCl (75 mL), dried over anhydrous MgSO4, and concentrated in Vacuo to give the intermediate mesylate as an oil. The recovered oil was immediately dissolved in dry dimethylformamide (20 mL) and sodium azide (1.25 g, 19.3 mmol, 4 equiv) was added. This solution was left to stir under nitrogen at room temperature for five days. The solution was then concentrated in Vacuo to yield a thick oil, which was dissolved in diethyl ether (200 mL) and filtered. The filtrate was partitioned with water (70 mL) and saturated aqueous NaCl (70 mL). The aqueous solutions were combined and partitioned with diethyl ether (80 mL). The organic solutions were combined, dried over MgSO4, and concentrated in Vacuo to yield a pale brown oil. This oil was purified by normal-phase silica gel chromatography, eluting with chloroform, to give 16 as a yellow oil (1.52 g, 90%). 1H NMR (300 MHz, CDCl3) δ 1.21–1.41 (14H, m, N3CH2(CH2)6 CH2CHdCHCH2(CH2)3), 1.45 (9H, s, C(CH3)3), 1.58 (4H, m, N3CH2(CH2)6CH2CHdCHCH2(CH2)3-), 2.02 (4H, m, N3(CH2) 7CH2CHdCHCH2), 2.21 (2H, t, J ) 7.6 Hz, CH2COOC(CH3)3), 3.26(2H,t,J)6.9Hz,N3CH2),5.37(2H,m,N3CH2(CH2)7CHdCH); 13C NMR (75 MHz, CDCl3) δ 25.4, 27.1, 27.4, 27.6, 28.5, 29.1, 29.2, 29.5, 29.6, 29.75, 29.8, 30.1, 36.0, 51.9, 80.3, 130.0, 130.4, 173.6; IR νmax/cm-1 (film) 2929, 2855, 2096, 1732; C20H37O2N3 (FAB+) m/z 324 ([C20H38O2N]+, 9%), 57 ([C4H9]+, 100 %); HRMS (FAB, NOBA matrix): C20H37O2N3Na calcd for [M + Na]+ 374.2783, found 374.2760. (Z)-2-Methylprop-2-yl-16-aminohexadeca-7-enoate 17. To (Z)-2-methylprop-2-yl-16-azidohexadeca-7-enoate 16 (1.28 g, 3.65 mmol) in dry THF (40 mL), under nitrogen was added triphenylphosphine (1.43 g, 5.50 mmol, 1.5 equiv). The solution was left to stir at room temperature for 5 h, after which time water (10 mL) was added and the reaction was left to stir for a further 48 h. The reaction was then concentrated in Vacuo, redissolved in diethyl ether/hexane (1:1, 60 mL), and filtered. The filtrate was concentrated in Vacuo to yield an oil, which was purified by normal-phase silica gel chromatography, eluting with a gradient of chloroform and triethylamine (99:1 to 90:10), to give 17 as a pale yellow oil (1.11 g, 93%). 1H NMR (300 MHz, CDCl3) δ 1.24–1.41 (14H, m, H2NCH2(CH2)6 CH2CHdCHCH2(CH2)3), 1.45 (9H, s, C(CH3)3), 1.51–1.66 (4H, m, H2NCH2(CH2)6CH2CHdCHCH2(CH2)3), 2.02 (4H, m, H2N(CH2)7CH2CHdCHCH2), 2.21 (2H, t, J ) 7.5 Hz, CH2COOC(CH3)3), 2.69 (2H, t, J ) 6.9 Hz, H2NCH2(CH2)7), 5.37 (2H, m, H2NCH2(CH2)7CHdCH(CH2)5); 13C NMR (75 MHz, CDCl3) δ 25.4, 27.3, 27.4, 27.6, 28.50, 29.1, 29.6, 29.79, 29.85, 29.89, 30.0, 30.1, 36.0, 42.6, 80.2, 130.0, 130.4, 173.6; IR νmax/cm-1 (film) 3300, 3004, 2926, 2855, 1733; C20H39O2N (FAB+) m/z 326 (M + H+, 56%), 270 ([C16H32O2N]+, 87%),


57 ([C4H9]+, 100%); HRMS (FAB, NOBA matrix): C20H40O2N calcd for [MH]+ 326.3059, found 326.3040. (Z)-2-Methylprop-2-yl-16-(9-fluorenylmethyloxycarbonylamido)hexadeca-7-enoate 18. To (Z)-2-methylprop-2-yl-16aminohexadeca-7-enoate 17 (0.46 g, 1.42 mmol) in dry THF (20 mL), under nitrogen at 0 °C was added N-methylmorpholine (0.32 mL, 0.286 g, 2.83 mmol, 2 equiv). This solution was left to stir for 10 min, and then fluorenylmethyl chloroformate (0.73 g, 2.83 mmol, 2 equiv) in dry THF (15 mL) was slowly added. Once addition was complete, the reaction was stirred for a further hour at 0 °C and then left to stir at room temperature overnight. After 24 h, the reaction was concentrated in Vacuo, redissolved in dichloromethane (150 mL), and filtered. The filtrate was partitioned with 5% aqueous potassium hydrogen sulfate (70 mL) and saturated aqueous NaCl (70 mL). The aqueous solutions were back-extracted with dichloromethane (100 mL). The organic solutions were combined, dried over anhydrous MgSO4, and concentrated in Vacuo to give a brown solid. This solid was purified by normal-phase silica gel chromatography, eluting with chloroform, to give 18 as a pale brown solid (0.69 g, 89%). 1H NMR (300 MHz, CDCl3) δ 1.20–1.43 (14H, m, FmocHNCH2(CH2)6CH2CHdCHCH2 (CH2)3), 1.45 (9H, s, C(CH3)3), 1.57–1.66 (4H, m, FmocHNCH2(CH2)6CH2CHdCHCH2(CH2)3), 2.03 (4H, m, FmocHN(CH2)7CH2CHdCHCH2(CH2)4), 2.21 (2H, t, J ) 7.5 Hz, CH2COOC(CH3)3), 3.17 (2H, m, J ) 6.1 Hz, FmocHNCH2), 4.21 (1H, t, J ) 6.7 Hz, NHCOOCH2CHC12H8), 4.40 (2H, d, J ) 6.7 Hz, NHCOOCH2C13H9), 4.98 (1H, bs, NH), 5.37 (2H, m, FmocHNCH2(CH2)7CHdCH), 7.31 (2H, t, J ) 7.4 Hz, Ar), 7.40 (2H, t, J ) 7.4 Hz, Ar), 7.61 (2H, d, J ) 7.4 Hz, Ar), 7.77 (2H, d, J ) 7.4 Hz, Ar); 13C NMR (75 MHz, CDCl3) δ 25.5, 27.2, 27.5, 27.6, 28.5 (C(CH3)3), 29.2, 29.6, 29.7, 29.8, 29.9, 30.0, 30.1, 36.0, 41.53, 47.8, 66.9, 80.2 (C(CH3)3), 120.3, 125.4, 127.4, 128.0, 130.0, 130.4, 141.7, 144.5, 156.9 (CdO), 173.6 (CdO); IR νmax/cm-1 (KBr disc): 3325 (st.), 3005 (wk.), 2926, 2854 (st.), 1730 (st.), 1689 (st.), 1542 (med.), 1266, 1152 (st.); C35H49O4N (FAB+) m/z 492 ([C31H42O4N]+, 7%), 270 ([C16H32O2N]+, 9.6%), 252 ([C16H30ON]+, 5.8%), 179 ([C14H11]+, 100%), 165 ([C13H9]+, 12%), 57 ([C4H9]+, 44.1%); HRMS (FAB, NOBA matrix): C35H49O4NNa calcd for [M + Na]+ 570.3559, found 570.3580. (Z) 16-(9-Fluorenylmethyloxycarbonylamido)-hexadeca-7enoic Acid 4. To (Z)-2-methylprop-2-yl 16-(9-fluorenylmethyloxycarbonylamido)hexadeca-7-enoate 18 (0.75 g, 1.37 mmol) under nitrogen at room temperature was added trifluoroacetic acid/water (95:5, 63 mL). To this solution was immediately added 1,3-propanedithiol (0.693 mL, 0.747 g, 6.9 mmol, 5 equiv). The reaction was left to stir for 1.5 h, after which it was concentrated in Vacuo to yield a pale brown solid. The solid was purified by normal-phase silica gel chromatography, eluting initially with chloroform (100 %), then gradually changing to chloroform/methanol/acetic acid (92:6:2), to give 4 as an offwhite solid (0.64 g, 95 %). 1H NMR (300 MHz, CDCl3) δ 1.27–1.65 (18H, m, FmocHNCH2(CH2)6CH2CHdCHCH2 (CH2)3CH2CO2H), 2.01 (4H, m, FmocHN(CH2)7CH2CHd CHCH2(CH2)4CO2H), 2.21 (2H, t, J ) 4.9 Hz, -CH2CO2H), 3.17 (2H, m, FmocHNCH2), 4.21 (1H, t, J ) 6.7 Hz, NHCOOCH2CHC12H8), 4.40 (2H, d, J ) 6.7 Hz, -NHCOOCH2C13H9), 4.88 (1H, bs, -NH-), 5.37 (2H, m, -CHdCH(CH2)5CO2H), 7.31 (2H, dt, J ) 7.5 Hz and 0.9 Hz), 7.40 (2H, t, J ) 7.4 Hz), 7.61 (2H, d, J ) 7.4 Hz), 7.77 (2H, d, J ) 7.5 Hz); 13C NMR (75 MHz, CDCl3) δ 25.1, 27.2, 27.4, 27.6, 29.1, 29.6, 29.68, 29.77, 29.83, 30.1, 30.4, 34.5, 41.5, 47.7, 66.9, 120.4, 125.4, 127.4, 128.1, 130.0, 130.1, 141.8, 144.4, 157.0, 179.7; IR νmax/cm-1 (KBr) 3325 (st.), 3005 (wk.), 2926, 2854 (st.), 2600–2400 (wk.), 1730 (st.), 1689 (st.), 1542 (med.), 1465 (wk.), 1266, 1152 (st.); C32H41O4N (FAB+) m/z 492 (M + H+, 3.2%),


270 ([C16H32O2N]+, 10%), 252 ([C16H30ON]+, 5.3%), 179 ([C14H11]+, 100%), 165 ([C13H9]+, 12%); HRMS (FAB, NOBA matrix): C32H42O4N calcd for [MH]+ 492.3114, found 492.3100. Preparation of Liposomes. Lipopolyplex formulations were prepared by mixing the components in the following order: 50 µL of Lipofectin (InVitrogen Ltd., Paisley, UK: 30 µg/µL in OptiMEM); 70 µL of peptide (at between 0.029 mg/mL and 0.1 mg/mL in OptiMEM, to afford peptide charge ratios of either 4:1 or 1.5:1 (taking into account the TFA counterions associated with each peptide)); followed by 50 µL of the luciferase reporter plasmid pCILux (40 µg/mL in OptiMEM). Plasmid pCILux was prepared by subcloning a luciferase gene from pGL3 Control (Life Technologies, Paisley, UK) into the eukaryotic expression vector pCI (Promega, Southampton, UK). The complex was mixed by pipetting briefly before diluting in OptiMEM to a final volume of 1.57 mL. Transfection of Cells. The normal human airway epithelial cell line 1HAEo- (16) and the cystic fibrosis (CF) cell line 2CFSMEo- (17) (D. Gruenert, University of California, San Francisco) were maintained in Eagle’s Minimal Essential Medium (MEM) HEPES modification (Sigma, Poole, UK) containing 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 µg/mL streptomycin and 2 mM L-glutamine, as described previously (18). Neuro-2A cells (ATCC) were maintained in Dulbecco’s Modified Eagle Medium (DMEM)Glutamax-1 (Life Technologies, Paisley, UK) containing 10% fetal calf serum (FCS) (Sigma, Poole, UK), 100 IU/mL penicillin, 100 µg/mL streptomycin, 1% sodium pyruvate, and 1% nonessential amino acids (Life Technologies, Paisley, UK). AJ3.1 is a mouse embryonic fibroblast line derived from A/J embryos 13.5 days postcoitum (Barker et al., manuscript in preparation). The complete growth medium was removed from cells plated at 2 × 104 cells/well overnight in 96-well plates, and 200 µL of complex (0.25 µg of plasmid DNA) was added to each well, leaving minimal time between preparing the complex and adding to the cells. All transfections were carried out in 6 wells each. The cells were incubated with the complexes for 4 h before replacing with normal media for 24 to 48 h, after which reporter gene expression was analyzed by luciferase assay (Promega, Southampton, UK). Luciferase assays. Cells were washed twice with PBS before the addition of 100 µl of 1 × Reporter Lysis Buffer (Promega, Southampton, U. K.) to the cells for 20 min at 4 °C before freezing at -20 °C for at least 30 min followed by thawing at room temperature ((20 °C). Twenty µl of the lysate at room temperature was transferred to a white polystyrene 96-well plate (Porvair Sciences Ltd., Shepperton, U. K.) and the luciferase activity was measured using the Luciferase Assay System (Promega) and a Lucy-1 Luminometer (Anthos Ltd., Saltzburg, Austria). The amount of protein present in each transfection lysate was determined with the Bio-Rad protein assay reagent by the manufacturer’s instructions, adding 20 µl from the luciferase test to 200 µl of the reagent diluted 1 in 5 and incubating at room temperature for 10 min before comparing the OD590 to a range of BSA standards. Luciferase activity was expressed as Relative Light Units (RLU) per mg of protein (RLU/mg). DLS. The complexes were formulated as described above, but using the PSVβ plasmid (a 6.9 kilobase plasmid). All experiments were conducted at a weight ratio of lipid/peptide/ DNA 0.75:4:1. This resulted in a 4:1 charge ratio, when the TFA counterions associated with the peptide are taken into account. The DLS measurements were performed using a Malvern Zetasizer 3000 instrument (Malvern Instruments Ltd., UK) operating at a wavelength of 633 nm and a power output of 5 mW. All experiments were conducted at a constant

Pilkington-Miksa et al. Scheme 1a

a

(i) DCC, DMAP, tert-butyl alcohol, CH2Cl2, 78%; (ii) Nal, acetone (93%). Scheme 2a

a (i) HBr, toluene, rfx (99%); (ii) 9-phenylxanthen-9-ol, AcOH (100%); (iii) Nal, acetone (97%).

temperature of 25 °C and a scattering angle of 90°, and spectra were collected every 180 s for at least 30 min. For broad, irregularly shaped distributions, where the method of cumulants was inappropriate, the CONTIN “constrained regularization” program (19, 20) was used for data analysis.

RESULTS Synthesis of the Lipophilic Amino Acids Fmoc-Laa (4) and Fmoc-Aua (6). 6-Aminohexanoic acid is commonly used as a lipophilic spacer in a variety of applications (21) and has been shown to increase the serum stability of some bioconjugates in in ViVo applications (22). However, longer or more structurally complex ω-amino acids have not been extensively investigated as spacers or as conformational constraints. The flexible 11-aminoundecanoic acid (Aua) spacer was recently synthesized by the Campbell group as the Fmoc-protected Pfp ester and incorporated into analogues of atrial natriuretic factor (15). The only report of a longer, unsaturated linker being used in bioconjugate applications was the use of cis-alkenyl tethers, derived from alkynyl bis-carboxylic acids, as conformationally restricted cross-linked peptide inhibitors of HIV protease (23). In order to prepare peptides containing the long-chain ω-amino acids Aua and Laa using solid-phase peptide synthesis techniques, we had to first synthesize the Fmoc-protected amino acids, 4 and 6, themselves. For the cis-alkenyl amino acid Fmoc-Laa 4, the most efficient synthetic route appeared to be to prepare this from a suitable acetylenic ω-hydroxy acid by selective hydrogenation. This could in turn be synthesized from a protected ω-iodo alcohol, a protected ω-iodo acid, and acetylene. As the other functionality and protecting groups in 4 made it desirable that the protecting group on the ω-iodo acid should not need hydrogenation or base treatment for its removal, we elected to protect the acid as the tert-butyl ester. Initial attempts to esterify the available 8-bromohexanoic acid using perchloric acid and tert-butyl acetate following a procedure by Jost and Rudinger (24) were lowyielding and slow (>5 days), and the low boiling point of the resulting ester 8 made separation from tert-butyl acetate difficult. DCC-mediated coupling of tert-butyl alcohol (25) with the carboxylic acid gave greatly improved yields of the desired ester

Targeting Lipopolyplexes Using Bifunctional Peptides Scheme 3a

a

(i) LiCCH(CH2NH2)2, HMPA, THF (98%); (ii) n-BuLi, THF/ HMPA -78°C, then 9 (70%); (iii) 0.3 M Cl2CHCOOH, pyrrole, CH2Cl2 (95%); (iv) Pd/C, H2, MeOH/hexane (100%); (v) MsCl, Et3N, CH2Cl2; (vi) NaN3, DMF (90% over 2 steps); (vii) PPh3, H2O, THF (93%); (viii) Fmoc-Cl, NMM, THF (89%); (ix) TFA, 5% H2O, 1,3-propanedithiol (95%).

Bioconjugate Chem., Vol. 18, No. 6, 2007 1805 Scheme 5a

a (i) HOAt, HATU, 1:1 DMF/CH2Cl2, 4; (ii) DBU/piperidine/DMF 1:1:48; (iii) 16 cycles of Fmoc deprotection and coupling of FmocLys(Boc)-OH, Fmoc deprotection; (iv) TFA (85%), thioanisole (5%), phenol (5%), water (2.5%), and triethylsilane (2.5%); (v) H2O, 7 d.

Scheme 6a

Scheme 4a

a (i) TsOH, PhCH2OH, benzene (91%); (ii) Na2CO3 (aq), dioxane, Fmoc-Cl (95%); (iii) H2, Pd/C, THF (88%).

7 within 24 h; this was then converted to the iodide 9 with sodium iodide (12) in 93% yield (Scheme 1). We elected to protect the ω-iodo alcohol with the highly acidlabile 9-(9-phenyl)xanthenyl group (pixyl, Px), as this could be removed under very mild conditions without deprotection of the tert-butyl ester. Selective monopixylation of 1,8-octanediol followed by bromination proved to be unsuccessful as, although the monoprotected diol was effectively synthesized, the 9-phenylxanthyl protecting group proved to be unstable to the conditions required to convert the hydroxyl group to a bromo group (CBr4, PPh3 in CH2Cl2). 8-Bromooctan-1-ol was therefore synthesized first (Scheme 2) using hydrobromic acid (48%) (26). The hydroxy group was then protected with 9-phenylxanthen9-ol, using glacial acetic acid both as a proton source and for azeotroping off water formed in the reaction, following a procedure by Gaffney and Reese (27) to give 10. Treatment with sodium iodide afforded 11 in 97% yield. To form the required substituted acetylene, 11 was then reacted with lithium acetylide ethylenediamine complex (LAEDA), using a procedure adapted from DeJarlais and Emken (28),

a

(i) HOAt, HATU, 1:1 DMF/CH2Cl2, 4; (ii) DBU/piperidine/DMF 1:1:48; (iii) 16 cycles of Fmoc deprotection and coupling of FmocLys(Boc)-OH, Fmoc deprotection; (iv) TFA (85%), thioanisole (5%), phenol (5%), water (2.5%), and 1,2-ethanedithiol (2.5%); (v) H2O, 7 d.

to give 12 in excellent yield (Scheme 3). The organolithium derivative of 12 was then prepared by adding n-butyl lithium in THF and HMPA at -78 °C; this was followed by addition of 9 to give the desired disubstituted acetylene 13. Fortuitously, the tert-butyl groups of both 13 and 9 appeared to withstand the use of n-butyl lithium under the conditions of this reaction. The selective removal of the 9-(9-phenyl)xanthenyl group from 13 was achieved with 0.1 M dichloroacetic acid in dichloromethane using pyrrole as the scavenger (27), to give 14. A small amount (less than 5% by TLC analysis of the crude mixture) of the tert-butyl group was removed under these conditions. Catalytic hydrogenation at atmospheric pressure using



Scheme 7a

a (i) HOAt, HATU, 1:1 DMF/CH2Cl2, Fmoc-Aua 6; (ii) DBU/ piperidine/DMF 1:1:48; (iii) 16 cycles of Fmoc deprotection and coupling of Fmoc-Lys(Boc)-OH, Fmoc deprotection; (iv) TFA (85%), thioanisole (5%), phenol (5%), water (2.5%), and triethylsilane (2.5%); (v) H2O, 7 d.

Figure 2. (a) Transfection of 1HAEo- cells with LPD-3, LPD-5, and LPD-1 at different charge ratios. (b) Transfection of 1HAEo- cells with LPD-3 and LPD-5 at a 4:1 peptide ratio, in varying concentrations of serum.

Figure 1. (a) Transfection of 1HAEo- cells with LPD-1(4:1) and LPD7(4:1). (b) Transfection of CFSMEo- cells with LPD-1(4:1) and LPD7(4:1).

Lindlar catalyst in methanol and hexane gave the cis-hydroxy compound 15, which was then mesylated and converted to the azide 16. Staudinger reduction of 16, using a procedure originally developed for the synthesis of allylic amines (29), gave the amine 17 in 80% yield. A slight modification to the original procedure, adding PPh3 first and leaving to stir for 5 h before adding water, increased the yield to 93%. Fmoc protection to give 18 (30) was followed by removal of the tert-butyl

group (31) to give the desired lipophilic amino acid Fmoc-Laa 4 in 46% overall yield from 11. For the synthesis of peptide 2, it was necessary to prepare the Fmoc-derivative of commercially available 11-aminoundecanoic acid. Reaction of the poorly soluble 11-aminoundecanoic acid with fluorenylmethyl chloroformate in water/aqueous sodium hydrogen carbonate failed, and an inseparable mixture of the Fmoc-protected amino acid and the Fmoc-protected dipeptide and tripeptide were recovered. Boumrah et al. (15) circumvented the problem of solubility by preparing Fmoc-Aua from 11-(9-fluorenylmethyloxycarbonylamido)undecanoic acid benzyl ester; however, the initial benzyl ester formation was inconvenient because it required the use of dry hydrochloric acid gas. We therefore used a modification (14) of a procedure (32) for the preparation of amino acid benzyl esters using the less hazardous toluenesulfonic acid to prepare 11-aminoundecanoic acid benzyl ester p-toluenesulfonate 19. Subsequent treatment of 19 with fluorenylmethyl chloroformate in dioxane/ aqueous sodium bicarbonate gave 11-(9-fluorenyl-methyloxycarbonylamido)undecanoic acid benzyl ester 20 in 95% yield (Scheme 4). Benzyl deprotection of 20 to give 11-(9-fluorenylmethyloxycarbonylamido)undecanoic acid (Fmoc-Aua, 6) was accomplished by hydrogenation (15) in 88% yield. Thus, 6 was conveniently prepared in three steps in an overall yield of 76%. Peptide Synthesis. As the integrin-targeting C-terminus is the same for all of the peptides, this sequence was first synthesized on a large scale using automated solid-phase peptide synthesis to give the resin-bound protected peptide 21 (Scheme 5). Incorporation of the lipophilic amino acid Fmoc-Laa 4 using DIC and HOBt in DMF, whether manually or on the peptide synthesizer, was unsuccessful, probably because of the poor



Figure 4. Average aggregate sizes of lipopolyplexes at pH 7.4 as a function of time. Data refer to LPD-1 in H2O ((), LPD-1 in PBS (9), LPD-3 in H2O (4), LPD-3 in PBS ()), LPD-5 in H2O (*), LPD-5 in PBS (solid pentagon), LPD-5 in H2O + 150 mM NaCl (0). All experiments were conducted at a weight ratio of lipid/peptide/DNA 0.75:4:1 resulting in a +4 charge ratio.

Figure 3. Transfection properties of LPD-2 and LPD-1 at 1:1 and 4:1 ratios, in (a) 1HAEo-, (b) N2A, and (c) AJ3.1 cells.

solubility of 4 in DMF. Manual coupling of 4 to 21, using HATU and HOAt (33, 34) in 1:1 CH2Cl2/DMF, however, proved successful, and the coupling was confirmed by the Kaiser test and by MS analysis of a cleaved and deprotected sample. As the Laa residue is very hydrophobic, a mixture of DBU and piperidine (35) was used for the removal of the Fmoc group. The peptide synthesis was then completed using automated methods to give the resin-bound peptide 22. Cleavage of 22 from the resin and deprotection, using 85% TFA and thioanisole, phenol, and triethylsilane as scavengers, proved problematic. After purification and MS analysis, it was discovered that reduction of the double bond of Laa had taken place to give peptide 23, containing the saturated Ahd residue. This was surprising, as, although there are instances where double bonds can be reduced with trifluoroacetic acid and an alkylsilane at room temperature, there are very few examples (36) of it occurring with nonconjugated alkenes. Aerial oxidiation to form the disulfide bond was then attempted using 0.01 M aqueous ammonium bicarbonate (37) or DMSO (38). However, both of these methods presented problems in the final HPLC purification

of the peptide, as the excess salts or DMSO led to poor retention of the peptide on the column and hence a very low recovery. The disulfide bond formation was therefore carried out in water at high dilution over 7 days to give complete formation of peptide 3. With these observations taken into account, intermediate 21 was then used to prepare peptide 5. Two Laa residues were incorporated manually using HATU and HOAt, with the Fmoc groups being removed with a mixture of DBU and piperidine. The peptide was completed via automated synthesis and cleaved and deprotected using 85% TFA and thioanisole, phenol, and ethanethiol as scavengers; no reduction of either of the double bonds was detected by MS analysis. Aerial oxidation in water was again carried out to yield peptide 5 (Scheme 6). A similar strategy was used to prepare peptide 2, using Fmoc-Aua 6 (Scheme 7). Transfection. A series of lipopolyplexes were formulated, using the previously described procedure (7) from Lipofectin, plasmid DNA, and peptides 1, 2, 3, 5, and 7, respectively, to give lipopolyplexes LPD-1 to LPD-7. Additionally, each lipopolyplex was formulated at three different peptide/DNA (P/ N) charge ratios: 0.5:1; 1:1; and 4:1. We initially investigated the complex LPD-7, with the AhxSer-Ahx spacer. This performed slightly better in transfection than the original LID complex, LPD-1, in a range of cell lines (Figure 1). Encouraged by this success, we tested the complexes containing lipid-like spacers, LPD-3 and LPD-5, in 1HAEocells (Figure 2). However, the results were disappointing: virtually no transfection was observed with these complexes (Figure 2a) and the complexes showed no stability when placed in even low amounts of serum (Figure 2b). Peptide 2 contains the 11-aminoundecanoic acid spacer, which is shorter than the other spacer regions but more hydrophobic than the spacer in peptide 7. The LPD-2 complexes formulated from this peptide had moderate transfection properties (Figure 3) which were highly dependent on the cell type. In N2A cells, transfection exceeded the original vector (LPD-1), whereas in 1HAEo-cells, the transfection was not as good, and in AJ3.1 cells, the transfection was comparable. Stability Measurements. In order to assess whether the poor transfection results obtained with the lipophilic peptides were



Figure 5. Relationship between zeta potential and particle size. The line represents the Monte Carlo simulation data and the circles the experimental data.

related to aggregation of vector particles, we investigated the aggregation properties of the lipopolyplex complexes containing peptides 3 and 5. The lipopolyplexes LPD-1, LPD-3, and LPD-5 were formulated from Lipofectin, plasmid DNA, and the relevant peptide as described previously (7) at 4:1 charge ratios, and the size of the resulting particles measured in water, NaCl solutions, and at different concentrations of phosphate buffered saline (PBS) to mimic physiological conditions. In order to determine the size of the complex, dynamic light scattering (DLS) measurements were made (39) and the hydrodynamic radius or diameter of the particles can be calculated (40, 41). In Figure 4, the changes in size distribution over time of these lipopolyplexes are shown. In water, very little aggregation takes place for any of the complexes; however, when increasing amounts of NaCl are added, the rate of aggregation increases. In PBS, a physiologically relevant buffer, both the original complex and the complexes containing lipophilic spacers aggregate dramatically over time. It is also notable that the starting size of each of the complexes is rather different, with LPD-3 being the most compact.

DISCUSSION We have developed efficient routes to two hydrophobic amino acids, suitably protected for solid-phase peptide synthesis. The novel amino acid 4 has been designed to imitate the unsaturated chain of the lipid DOTMA, and we have also developed an improved route to the previously described 6. We have then successfully synthesized peptides containing these or other hydrophobic amino acids as spacers, for use in nonviral gene delivery studies of targeted ternary lipopolyplex vectors. Several factors influence the transfection efficiencies of targeted lipopolyplex vectors (6). For the present study, three factors could be important. It is probable that a long spacer unit between the DNA-condensation and integrin-targeting moieties of the peptide could lead to better interactions between the vector and the cell surface receptor. We also initially hypothesized that, if these spacer units could be designed to stack within a lipid bilayer, the stability of the vector, and hence its transfection efficiency (39), could be enhanced. However, the overall hydrophobicity of the spacer unit will also be important in determining the stability of the resulting vector, and hence the transfection efficiency, as vectors which dissociate rapidly or aggregate in buffer or serum solutions will have greatly reduced transfection efficiencies. We have previously carried out a theoretical study (42) to model the dynamics of vector aggregation. The vectors LPD-1 and LPD-3 were studied, using Monte Carlo simulation

techniques to model the dynamics of vector aggregation. It was clearly demonstrated, both experimentally and in the simulation, that LPD-3 aggregated much more rapidly than LPD-1. It was shown that the levels of aggregation of vector particles under Brownian motion could be predicted on the basis of the zeta potential of the vector particles. Vector buffer systems with a zeta potential less than 20 mV exhibit significant aggregation (Figure 5). These results are in agreement with the poor transfection efficiencies of LPD-3 (Figure 2) and also with the rapid aggregation of LPD-3 in PBS (Figure 4). The hydrophobicity of the Ahd linker clearly outweighs any beneficial effect of interaction with the hydrophobic portion of either DOTMA or DOPE, although it appears to lead to a structure that is initially more compact than LPD-1 (Figure 4). Moreover, when two Laa amino acids are used as lipid-like linkers, in peptide 5, the resulting complex (LPD-5) demonstrates equally poor transfection efficiencies (Figure 2) and serum (Figure 2) and buffer (Figure 4) stability. These results suggest three possible alternative hypotheses: either packing of lipid-like linkers within a lipid bilayer is not possible; or it does not result in a more stable vector particle; or ternary lipopolyplex vectors of this type do not have an ordered lipid layer. It is notable that in other, unrelated biological systems where hydrophobic spacers have been employed (43) the results have not been as good as with hydrophilic, PEG-based spacers. In comparison, peptide 7 has a linker sequence of approximately the same length as peptide 5. However, the transfection efficiency of LPD-7 is much better than that of LPD-5, and also an improvement on the original vector, LPD-1 (Figures 1 and 2). LPD-7 is also more efficient than LPD-2, with a shorter but equally hydrophobic linker (Figures 1 and 3). We assume that peptide 7 has a spacer unit which is sufficiently long and inflexible to position the integrin targeting motif at the exterior of the vector, rendering it more accessible to cell surface receptors, and that it is the length, rather than the hydrophobicity, of the spacer which is important for efficient transfection. Further biophysical studies into the macromolecular organization and stability properties of ternary targeted lipopolyplex vectors are underway.

ACKNOWLEDGMENT The EPSRC are thanked for DTA studentships to M.P.M. and S.S. M.W. was supported by a grant from the BBSRC, and S.B. by a studentship from the MRC. The authors thank Dieter Gruenert, California Pacific Medical Center Research Institute, San Francisco, CA, for kindly providing the cell lines CFSMEoand 1HAEo-.


Supporting Information Available: Experimental details of the synthesis of Fmoc-Aua 6, and of the synthesis of all peptides described. This material is available free of charge via the Internet at http://pubs.acs.org.

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