To test this prediction, we synthesized an analog of -peptide 27 containing (O-allyl)-3-L-Ser at positions 3 and 6 (2(3-6), Number 1), and subjected it to on-resin ring-closing metathesis using bis(tricyclohexylphosphine)benzylidene ruthenium (IV) dichloride34 to generate 2(3-6)s.45 The circular dichroism (CD) spectra of 2, 2(3-6) and 2(3-6)s were identical (Number S1), indicating that this 21-atom diether bridge is accommodated between positions 3 and 6. others have reported that insertion of a hydrocarbon bridge between the and positions of an Chelix also raises cell uptake. Here we describe a series of Cpeptides comprising diether and hydrocarbon bridges and compare them on the basis of cell uptake and localization, affinities for hDM2, and 14-helix structure. Our results spotlight the relative Bergenin (Cuscutin) merits of cationic patch and hydrophobic bridge strategies for improving Cpeptide uptake and determine a surprising correlation between uptake effectiveness and hDM2 affinity. -peptides1-4 possess several features that are desired in peptidomimetics;5,6 they are easily synthesized, fold into helices1-3,7 in physiologic buffers,8 and resist proteolysis.9 They also bind to proteins such as hDM2,10-14 hDMX,10 gp41,15,16 as well as others,17-19 and inhibit their interactions with -helical ligands. -peptides are not usually cell permeable, however, and this feature limits their power as research tools and potential therapeutics. Appending an Arg8 sequence to a -peptide can improve uptake20,21 but adds substantial mass. We reported that embedding a small cationic patch within a PPII,22 -23 or -peptide11 helix improves uptake without the addition of significant mass.24,25 Similarly, Verdine, Walensky, and others26-33 reported that insertion of a hydrocarbon bridge (a staple) between the and positions of an -helix34 increases uptake.26,29,32,34-38 Here we describe a variety of -peptides containing diether- and hydrocarbon bridges and compare them on the basis of cell uptake and localization, affinity for hDM2, and 14-helix structure. Our results spotlight the relative merits of cationic patch and hydrophobic bridge strategies for improving -peptide uptake and determine an unprecedented correlation between uptake effectiveness and hDM2 affinity and positions of a 14-helix. To test this prediction, we synthesized an analog of -peptide 27 comprising (O-allyl)-3-L-Ser at positions 3 and 6 (2(3-6), Number 1), and subjected it to on-resin ring-closing metathesis using bis(tricyclohexylphosphine)benzylidene ruthenium (IV) Bergenin (Cuscutin) dichloride34 to generate 2(3-6)s.45 The circular dichroism (CD) spectra of 2, 2(3-6) and 2(3-6)s were identical (Number S1), indicating that this 21-atom diether bridge is accommodated between positions 3 and 6. Bergenin (Cuscutin) Intro of the diether bridge did not significantly increase or decrease the degree of 14-helix structure as judged by CD. Open in a separate window Number 1 Helical online representation of -peptides analyzed herein. 3-homoamino acids are recognized from the single-letter code utilized for the related Camino acid. Orn represents ornithine. Z represents 3-(S)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid. In order to evaluate the relative uptake of bridged -peptides in the context of a functional molecule of varied sequence, we synthesized a series of variants of 53-12,10 an inhibitor of p53-hDM2 complexation (Number 1). These variants Bergenin (Cuscutin) contained either (O-allyl)-3-L-Ser (to generate a diether bridge) or (and positions 2 and 5 (25.O-s and 25.C-s, respectively) or 4 and 7 Bergenin (Cuscutin) (47.O-s and 47.C-s, respectively). According to the CD spectra (Number 2), all bridged -peptides assumed a 14-helical structure and were modestly more helical than unbridged analogs (Number S2). Open in a separate window Number 2 CD analysis of -peptides comprising hydrocarbon or diether bridges between residues (A) 2 and 5 or (B) 4 and 7. Fluorescence polarization (FP) analysis of hDM2 binding by -peptides comprising (C) hydrocarbon or (D) diether bridges. Like a prelude to evaluating cell uptake and localization, we used a direct fluorescence polarization assay to compare hydrocarbon and diether bridged -peptides on the basis of affinity for hDM21-188 (Number 2B). -peptides comprising KRT4 a diether or hydrocarbon bridge between positions 4 and 7 bound hDM21-188 2-collapse better (analysis suggests that the lower hDM21-188 affinity of -peptides 25.C-s and 25.O-s results from steric hindrance between the hydrocarbon bridge and the hDM2 surface that is absent in the complex with peptides 47.C-s and 47.O-s (Number 3, compare A and B). Open in a.