Facile Synthesis of Hydrocarbon-Stapled Peptides

Wei Qiu, Ling Sheng, Peng Zou, Ke Yang, Anita Hong and Xiaohe Tong
AnaSpec, Inc.

Introduction
Intracellular protein-protein interactions that govern many biological pathways are frequently mediated by a-helical structures of protein; however, the use of short protein fragments (peptides) leads to a loss of secondary structure, such as alpha helical structure. Short peptides also are easily degraded by proteolysis and have difficulty in intact cells penetration.¹ Verdine’s group^1-2 has shown that these problems could be overcome by a chemical modification of a-helical peptides they termed hydrocarbon stapling. They used (S)-a-(2’-pentenyl)alanine containing olefin-bearing tethers to generate an all hydrocarbon “staple” by ruthenium-catalyzed olefin metathesis. The (S)-a-(2’-pentenyl)alanine peptides were made to flank three (substitution positions l and l + 4) or six (l and l + 7) amino acids within the peptide, so that reactive olefinic residues would reside on the same face of the a-helix. The modified hydrocarbon-stapled peptides were helical, relatively protease-resistant, and cell-permeable peptides that bound with increased affinity for its target. Hydrocarbon stapling may provide a useful strategy in researching experimental and therapeutic modulation of protein-protein interactions as well as in in vivo pharmacokinetics studies.

Here we report a versatile synthesis method for hydrocarbon-stapled peptides. Asymmetric synthesis of (S)-Fmoc-a-(2’-pentenyl)-alanine was successfully accomplished via an Ala-Ni (II)-BPB-complex [3] in three steps with a 40% total yield. The 12-mer peptide containing two a- pentenyl-alanines on positions 4 and 8 was synthesized by Fmoc solid phase synthesis method. After olefin metathesis and cleavage, the peptide was purified by HPLC to obtain the hydrocarbon-stapled peptide.

Methods and Results
In contrast with Verdine’s method [2] for (S)-Fmoc-a-(2’-pentenyl)-alanine, we chose Ala-Ni (II)-BPB-complex method [3] for asymmetric synthesis. The Ala-Ni (II)-BPB-complex [4] was reacted with 5-bromo-1-pentene in acetone under basic conditions to give a mixture of a Ni(II) complex of Schiff base of (S)-BPB-(S)-trans-a-(2’-pentenyl)alanine [a-(S)-2] and Ni(II) complex of Schiff base of (S)-BPB-(R)-trans-a-(2’-pentenyl)-alanine [a-(R)-2] with ratio 6:1. After separation with silica gel column, diastereo-pure a-(S)-2 complexes were obtained at 44% yield.

a-(S)-2 complexes were decomposed with 3N HCl/MeOH to afford (S)-a-(2’-pentenyl)alanine (3) as well as a chiral ligand which was extracted with DCM. After work up, (S)-a-(2’-pentenyl)alanine (3) was protected with Fmoc-OSu to give the (S)-Fmoc-a-(2’-pentenyl)alanine (4) with yield 93% (two steps).

Peptide 1 was synthesized manually by Fmoc solid phase synthesis method using Rink amide MBHA resin. For normal amino acids, couplings were performed with fourfold excess of amino acids. Fmoc-amino acids were activated using the ratio of Fmoc-amino acid:HBTU:HOBt:DIEA, 1:1:1:2. For (S)-Fmoc-a-(2’-pentenyl)alanine , coupling was performed with twofold excess of amino acid which was activated with DIC:HOAt (1:1). For peptide olefin metathesis, the peptide resin with N-terminal protected by Fmoc group was treated with degassed 1, 2 dichloroethane containing Bis(tricyclohexyl-phosphine)-benzylidine ruthenium (IV) dichloride at room temperature for two hours and the reaction repeated once for completion. After de-Fmoc, the resin bound peptide was cleaved using standard protocols (95% TFA, 2.5% water, 2.5% TIS). The cleaved peptide was purified by RP-HPLC using 0.1% (v/v) TFA/water and 0.1% (v/v) TFA/acetonitrile. Chemical composition of the pure product was confirmed using MS. For fluorescently labeled Peptide 2, the N-terminal group of Peptide 1 was further derivatized with ß-Ala followed by FITC (DMF/DIEA) on the resin before the cleavage. The other cleavage, purification and confirmation steps were the same as above. Peptide 1 not only showed enhanced a-helicity and resistance to proteolysis, but also had antiviral activity (manuscript in preparation).

Conclusions
- Asymmetric synthesis of (S)-Fmoc-α-(2’-pentenyl)alanine was successfully prepared via an Ala-Ni (II)-BPB-complex with 40% total yield.
- Hydrocarbon-stapled peptides were synthesized.
- Peptide 1 not only showed enhanced α-helicity and resistance to proteolysis, but also had antiviral activity.

References
1. Walensky, LD. et al. Science 305, 1466 (2004).
2. Schafmeister, CE. et al. J. Am. Chem. Soc. 122, 5891 (2000).
3. Qiu, W. et al. Tetrahedron 56, 2577 (2000).
4. Belokon, YN. et al. Tetrahedron: Asymmetry, 9, 4249 (1998).

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