Synthesis of Peptide-Spermine
Peptides and Peptide Conjugates
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[00099] Peptides were synthesized using automated solid phase peptide synthesis as described, e.g., in Stewart et al. (1984) Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, Ill. Peptides were synthesized using a polyamide-Kieselguhr composite resin and a Milligen 9050 peptide synthesizer (Milligen/Biosearch, Burlington, Mass.). Coupling cycles were performed according to the manufacturer's recommendations. 9-Fluorenyl-methyloxy-carbonyl (Fmoc) amino acid was activated as a pentafluorophenyl ester (--OPfp ester); Peptides were deblocked using; (1) 20% piperidine in N,N-dimethylformamide (DMF) for alpha-amino groups; Peptides were cleaved from the resin and deprotected using 95% trifluoroacetic acid (TFA), (2) Reagent R [TFA (90%), thioanisol (5%), ethylene dithiole (3%) and anisole (2%)], Reagent B [TFA (88%), phenol (5%), triisopropylsilane (2%) and water (5%)] or Reagent T [TFA (95%), triisopropylsilane (5%); Deprotection agent being chosen as is understood in the art based on the protecting groups used and the type of amino acid residues in the peptide; Crude peptides were precipitated and washed with ether. Peptides were purified by high pressure liquid chromatography on a Vydac C-18 reverse-phase column using a Waters HPLC system. The mobile phase consisted of a gradient from 0.01% TFA in 95% water/acetonitrile to 0.01% TFA in 25% water/acetonitrile. Peptides were characterized by HPLC, amino acid analysis and mass spectrometry (ES or MALDI-TOF). Exemplary peptide sequences useful in this invention are listed in Tables 1-3.
[00100] It was found that certain peptides that were only partially deblocked, i.e., at least one amino acid protecting group had not been removed, showed significant enhancement of transfection. Appropriate choice of deprotection agent allows selective synthesis of peptides which retain a desired protecting group. For example, deprotection with Reagent T does not remove the Mtr protecting group on arginines allowing the synthesis of partially deblocked peptides with Mtr groups remaining on R residues.
Synthesis of Polyamine Conjugated Peptides
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[00101] Peptides can be modified with polyamines, such as spermine, using an automated peptide synthesizer. Either the Fmoc or Boc chemistries can be used. For example, spermine can be attached to the N-terminus of a peptide as illustrated in Scheme I. 5-Carboxy spermine and Boc-protected 5-carboxy spermine can be synthesized as described in Behr, J-P. et al. (1989) Proc. Natl. Acad. Sci., 86:6982-6986. Fmoc-carboxy spermine can be synthesized by treating carboxy spermine with 9-fluorenylmethyl chloroformate. Fmoc-carboxy spermine or the pentafluorophenyl ester can be used in the synthesizer to obtain spermine-modified peptides. More than one polyamine can be attached in this manner to a given peptide using an appropriate combination of protecting groups.
N.sup.I,N.sup.II,N.sup.III,N.sup.IV -tetra(9-fluorenylmethoxycarbonyl)-5-carboxyspermine (Fmoc-carboxyspermine)
[00102] 5-Carboxy spermine (11.0 g) was dissolved in 100 ml water. The solution was chilled on ice and diluted with 200 ml dioxane and flushed with argon. A solution of 50 g of 9-fluorenylmethyl chloroformate (Fmoc-Cl) in 200 ml of dioxane was slowly added to the chilled carboxy spermine. The reaction mixture was stirred under argon at 4° C. for an hour and at room temperature overnight under argon. The reaction was monitored by TLC (silica Gel, CHCl.sub.3 /MeOH::9/1). The reaction mixture was poured into 1.5 L ice cold water (1.5 L) and extracted with ethyl acetate (2 L). The organic layer was separated and sequentially extracted with 400 ml of 1 N HCl (2×) and 300 ml saturated NaCl. The organic layer was dried over Na.sub.2 SO.sub.4 and concentrated on a rotary evaporator. The resultant gummy material was subjected to flash chromatography (silica, CHCl.sub.3 /MeOH::95/5) to yield 25.5 g of the desired material as a white fluffy solid. ##STR2##
Fmoc-carboxy-spermine-OPfp ester
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[00103] A solution of pentafluorophenol (4.2 g) in 3 ml of dioxane solution, followed by 2 ml of dioxane, was added to a solution of Fmoc-carboxy-spermine (8.0 g) in 10 ml of dioxane. The resulting mixture was chilled in an ice water bath and flushed with argon. Freshly distilled dicyclohexylcarbodiimide (DCC, 1.6 g) in 4 ml of dioxane was added to the chilled reaction mixture. The mixture was stirred at 4° C. for about an hour and at room temperature overnight under argon. Precipitated dicyclohexyl urea (DCU) was filtered out of the reaction mixture, which was then concentrated to dryness on a rotary evaporator. The residue was dried under high vacuum overnight. The ester was obtained in quantitative yield as a slightly yellowish gum, which was used without further purification in the peptide synthesizer.
Synthesis of Spermine-Modified Peptides
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[00104] Spermine-modified peptides were synthesized using Fmoc chemistry on a Milligen 9050 synthesizer using the protocol suggested by the manufacturer. Peptides were synthesized conventionally and carboxyspermine was attached at the N-terminus of the synthesized peptide using Fmoc-carboxyspermine-OPfp ester as the last amino acid to be added on the synthesizer, as illustrated in Scheme 1. Deprotection reagents used were selected as discussed above. The peptide-spermine conjugates were stored frozen until use. Table 4 lists several examples of spermine-conjugated peptides that were synthesized using FMOC-carboxy-spermine. Modified peptides were analyzed and purified using HPLC on a Vydac C-18 column. Modified peptides were characterized by HPLC, amino acid analysis and mass spectrometry (ES or MALDI-TOF). This method can be employed or readily adapted in view of well-known techniques for synthesis of polyamine-peptide conjugates.
[00105] Peptide-spermine conjugates can also be prepared using a heterobifunctional cross-linking agent sulfo-SMPB (Pierce Chemical Co., Rockford, Ill.) as illustrated in Scheme II. See: S. S. Wong "Heterobifunctional Cross-linkers" in Chemistry of Protein Conjugation and Cross-linking CRC Press p.147-194. Briefly, 100 mg/mL sulfo-SMPB in DMF is diluted to 20 mg/mL using 50 mM sodium phosphate buffer (pH 7.5). Spermine (50 mg/mL in 50 mM sodium phosphate buffer) was then added to the sulfo-SMPB solution at a 3:1 molar ratio. After 1 hour at room temperature, the reaction mixture is fractionated (LH-20 column) using the sodium phosphate buffer. The first major peak (spermine-MPB) was collected. Spermine-MPB is mixed at a 1:1.5 to 1:2 ratio with a synthetic (or naturally-occurring) peptide with terminal cysteine (HS-), either in pure powder form or in acetonitrile/water solution. Excess peptide is separated on a LH-20 column eluted with water. The peptide-spermine conjugate is stored frozen until use. The reaction of spermine-MPB with HS-peptide should be performed under appropriate reducing conditions to avoid peptide dimer formation. This method can be employed or readily adapted in view of well-known techniques for synthesis of polyamine -peptide conjugates.
[00106] Peptide dimers of peptides containing cysteine residues can be formed, if desired, by oxidative coupling to form a disulfide bond between two peptides. Concatemers and Mixed concatemers of this invention can be prepared by automated peptide synthesis and if desired the concatemers, mixed concatemers and peptide oligomers (dimers, etc.) can be conjugated to nucleic acid-binding groups by methods described herein.
[00107] Scheme 3 illustrates appropriately protected polyamine species that can be used in automated peptide synthesizers to introduce polyamines at the carboxy terminus of peptides (e.g., Structure II, for carboxyspermine conjugation). The scheme illustrates a synthesis of the carboxyspermine derivative of Structure II, which can be readily generalized for the synthesis of any analogous polyamine derivatives. The compound of Structure II, activated by removal of the pentafluorophenol group, can be conjugated to the solid support to provide a carboxy spermine group at a peptide carboxy terminus. Standard automated peptide synthesis is performed using the support of Structure I. The fully protected carboxy spermine of Structure II can itself be employed as a reagent for addition of the carboxy spermine group at any position along a synthetic peptide chain. These methods can be readily and routinely adapted for conjugation of any polyamine.
Peptide-Lipid Conjugates
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[00108] Peptide-lipid conjugates are prepared as follows: A lipid with a reactive head group, for example a group such as 1,2-dioleoyl-S,N-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (N--PDP--PE, Avanti Polar Lipids, Inc. Alabaster, Ala.) is reacted with a cysteine-peptide (e.g., cysteine at either end of the functional peptide). Reaction can be performed at RT and followed by measuring the released chromophore. See Scheme 4. For example, N--PDP--PE is dissolved in methanol to a concentration of 0.2 M (about 200 mg/mL). Cys-peptides, including VSVG-Cys, (e.g., KFTIVFC, SEQ ID NO: 11); RGD-Cys (e.g., GRGDSPC, SEQ ID NO: 8); or Cys-NLS (e.g., CGWGPKKKRKVG, SEQ ID NO: 12), are dissolved in an appropriate solvent, e.g., DMF, to a concentration of 100 mg/mL and mixed with the N--PDP--PE solution at a molar ratio of 1.5-2:1. Peptide-lipid conjugates can be purified by HPLC using a Vydac protein and peptide C18 reverse phase column with an acetonitrile/water/TFA and methanol solvent system. The conjugate can be characterized by UV and MS analysis.
Useful Peptides
[edit]Sp-NLSNLS the peptide (NLSNLS): GGYGPKKKRKVGGGGYGPKKKRKVGG
Transfection Protocol
[edit]Dilute L2K in 25 μL of serum-free medium ("OPTIMEM-I" or D-MEM).
Dilute DNA in 25 μL of serum-free medium ("OPTIMEM-I" or D-MEM).
Add Sp-NLSNLS (1-4 μg, as a 1 mg/ml solution in water) into the DNA solution, and incubated at RT for 15 min to allow pre-complexation.
Mix DNA-Sp-NLSNLS solution together with diluted "LIPOFECTAMINE"
ncubated at RT for 30 min
Add to cells