Editing Synthesis of Peptide-Spermine (section)

== <br>Synthesis of Spermine-Modified Peptides<br>  ==

<br> [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.<br> 

<br> [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.<br> [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.<br> 



[[Image:Peptide_conjugated_to_spermine_SMPB.jpg]]

<br> [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. <br> 



[[Image:Peptides_for_transfection.jpg]]



[[Category:Transfection]][[Category:Paul]]