Introduction
The intracellular protein-protein interactions that govern many
biological pathways are frequently mediated by α-helix structure of
protein. Theoretically, helical peptides also can interfere with or stabilize
protein-protein interactions, but native helical peptides have major
shortcomings as experimental or therapeutic agents because of low
potency, instability, and inefficient delivery to cells. Verdine’s group
[1-2] has shown that these problems could be overcome by a chemical
modification of α-helical peptides they termed hydrocarbon stapling.
They used (S)-α-(2’-pentenyl)alanine containing olefin-bearing tethers
to generate an all-hydrocarbon “staple” by ruthenium-catalyzed olefin
metathesis. The (S)-α-(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 α-helix. The modified hydrocarbon-stapled peptides are
helical, relatively protease-resistant, and cell-permeable peptides that
bind with increased affinity for its target, and may provide a useful
strategy for experimental and therapeutic modulation of protein-protein
interactions in many signaling pathways.
Here we report a versatile synthesis method for hydrocarbon-stapled
peptides. Asymmetric synthesis of (S)-Fmoc-α-(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 α-
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.
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, L.D., et al., (2004) Science 305, 1466-1470.
2. Schafmeister, C.E., et al., (2000) J. Am. Chem. Soc. 122, 5891-5892.
3. Qiu, W., et al., (2000) Tetrahedron 56, 2577-2582.
4. Belokon, Y.N., et al., (1998) Tetrahedron: Asymmetry, 9, 4249-4252.
Sequence of Peptide 1. XXFZDLLZYYGX
Sequence of Peptide 2. FITC-(βA)XXFZDLLZYYGX
Z = (S)-α-(2’-pentenyl)alanine
Figure 2. Strategy for hydrocarbon-stapled peptide with enhanced α helix structure.
was 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 β-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 α-helicity and resistance to proteolysis,
but also had antiviral activity (manuscript in preparation).
Results
In contrast with Verdine’s method [2] for (S)-Fmoc-α-(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-α-(2’-pentenyl)alaninealanine [α-(S)-2] and Ni(II) complex of Schiff base of (S)-BPB-(R)-trans-α-(2’-pentenyl)-
alanine [α-(R)-2] with ratio 6:1. After separation with silica gel column, diastereopure
α-(S)-2 complexes were obtained at 44% yield.
Figure 1. Synthesis of (S)-Fmoc-α-(2’-pentenyl)alanine
The α-(S)-2 complexes were decomposed with 3N HCl/MeOH to afford (S)-
α-(2’-pentenyl)alanine (3) as well as a chiral ligand which was extracted with
DCM. After work up, (S)-α-(2’-pentenyl)alanine (3) was protected with Fmoc-
OSu to give the (S)-Fmoc-α-(2’-pentenyl)alanine (4) with 93% yield (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-α-
(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
Figure 2. Strategy for hydrocarbon-stapled peptide with enhanced α helix structure.
was 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 β-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 α-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, L.D., et al., (2004) Science 305, 1466-1470.
2. Schafmeister, C.E., et al., (2000) J. Am. Chem. Soc. 122, 5891-5892.
3. Qiu, W., et al., (2000) Tetrahedron 56, 2577-2582.
4. Belokon, Y.N., et al., (1998) Tetrahedron: Asymmetry, 9, 4249-4252.