[PubMed] [Google Scholar](b) Kussie PH, Gorina S, Marechal V, Elenbaas B, Moreau J, Levine AJ, Pavletich NP. their cellular uptake. Among them, selective cross-linking of peptide side-chains (also referred to as peptide stapling) via selective chemical reactions, e.g., disulfide relationship formation,3 olefin mix metathesis,4 lactam formation,5 and 1,3-dipolar cycloaddition reactions,6 offers emerged to be probably one of the most encouraging methods.7 Recently, we reported the discovery of a distance-matching cross-linker, 6,6-bisbromomethyl-[3,3]bipyridine (Bpy), that reacts with a pair of cysteines located at em i /em , em i /em +7 positions of a short helical peptide, resulting in a significant enhancement in cell permeability.8 While it is known that attachment of the positively charged organizations to peptides generally prospects to enhanced cellular uptake, most studies in the literature have focused on either direct fusion of polyarginine-like sequences9 such as TAT sequence or substitution of arginines at strategically selected noncontinuous sites within the peptides.10 Herein, we report the 1st synthesis of polyamine-conjugated, stapled peptides and the characterization of the effect of polyamine conjugation on peptide bioactivity, helicity, and cell permeability. Polyamines such as spermine are positively charged in the physiological pH, and have been used through conjugation as an intracellular delivery aid for the lipophilic small-molecule medicines11 as well as the negatively charged siRNA.12 However, the effect of polyamine conjugation to synthetic peptides on cellular transport of peptides has not been reported.13 According to helical dipole magic size,14 placement of the positive-charged capping organizations in the carboxyl termini of short helical peptides prospects to increased helicity due to the favourable charge-dipole relationships.15 To probe whether this model is operative in the case of short stapled (-)-DHMEQ peptides, we conjugated spermine in the C-terminus of a dicysteine-containing peptide inhibitor of p53CMdm2 interaction (sequence = LTFCHYWAQLCS), which was used previously like a model in our peptide stapling studies.8 Plan 1 shows a representative synthesis of the spermine-conjugated, stapled peptide 4 in seven actions based on a solid phase procedure. Briefly, spermine was first immobilized onto solid support by treating 2-chlorotrityl chloride resin with extra amount of spermine in dichloromethane for 30 min. After considerable washing, selective safety of the secondary amines with Boc was achieved by 1st masking the terminal main amine with 2-acetyldimedone16 to form the em N /em -[1,(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) derivative followed by treatment with di- em tert /em -butyl dicarbonate. The Dde-protecting group was selectively eliminated by treating the resin with 2% hydrazine (-)-DHMEQ in DMF for 15 min. With the spermine-functionalized resin in hand, standard Fmoc-based solid phase peptide synthesis was performed and the linear cysteine-containing peptide was released from your resin after treatment having a TFA-containing cleavage cocktail. After HPLC purification, the fully deprotected linear peptide was incubated with 1.5 equiv of Bpy inside a mixed acetonitrile/30 mM NH4HCO3 solution at pH 8.5. HPLC analysis of the crude product indicated the stapling reaction of the spermine-conjugated peptide proceeded with greater than 95% conversion after 1.5 hours (Figure S1). By using this route, we also (-)-DHMEQ prepared spermine-conjugated, D,L-dicysteine-stapled peptide 8 because the D,L-dicysteine-stapled peptides have shown higher inhibitory activity towards Mdm2 in our earlier work.8 Open in a separate window Scheme 1 Representative synthesis of a spermine-conjugated stapled peptide 4 on solid support. To gauge how spermine conjugation affects stapled peptide’s biological activity, we identified the inhibitory activities of the spermine-conjugated cross-linked peptides with ELISA and compared them to additional stapled peptides reported previously8 (Number S2 and Table 1). Interestingly, whereas substitution of two residues at solvent-exposed part of the helix with arginines (His5Arg and Gln9Arg) led to addition of +2 charge accompanied by considerable drop (6~20-collapse) in inhibitory activity8 (compare 3 to 2; 7 to TSPAN32 6), spermine conjugation resulted in addition of +3 charge and significantly smaller reduction in inhibitory activity (compare 4 to 2; 8.