Iron(II) Organizes a Synthetic Peptide into Three-Helix Bundles

Journal of the American Chemical Society

Volumn 113, Issue 4, 1991, Pages 1470-1471

  Lieberman, Marya ^a   Sasaki, Tomikazu ^a  

^a UNIVERSITY OF WASHINGTON   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

EID: 85007885646     PISSN: 00027863     EISSN: 15205126     Source Type: Journal    
DOI: 10.1021/ja00004a090     Document Type: Article

References (41)

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19. Peptide 1 (Figure 1) was synthesized6b in a protected form on p-methylbenzhydrylamine (MBHA) resin. This peptide was designed to form an amphiphilic α-helix,2a and Sasaki and Kaiser2b showed that it forms a four-α-helix bundle when attached to a porphyrin template. While peptide 1 was bound to the resin, its N-terminus was covalently coupled with 2,2′-bipyridine-4,4′-dicarboxylic acid. 2,2′-Bipyridine-4,4′-dicarboxyiic acid was prepared in 37% yield from 4,4′-dimethyl-2,2′-bipyridine (Aldrich) by oxidation with KMnO4.6c The diacid (0.20 mmol) was dissolved in 3 mL of 5% diisopropylethylamine (DIEA) in CH2Cl2 in a flask equipped with a stir bar and drying tube. N-Hydroxybenzotriazole (HOBt) (0.50 mmol) and diisopropylcarbodiimide (0.50 mmol) were added to form the HOBt diester; after 45 min of stirring, the solution cleared and turned faint yellow. MBHA resin-(aa)15-BOC (0.10 mmol) was deprotected with trifluoroacetic acid (TFA) and washed with CH2Cl2. The HOBt diester solution was added, and the mixture was shaken for 8 h. The resin was washed with CH2Cl2 and 33% EtOH/CH2Cl2. A negative Kaiser ninhydrin test6d for free NH2 indicated that coupling was complete. Cleavage from the resin according to a published procedure6e using trimethylsilyl triflate in TFA as a deprotecting reagent in the presence of thioanisole and m-cresol at 0 °C for 2 h yielded crude peptide 2, which was purified7 (yield 19%) by gel permeation chromatography on a 1.2 cm × 20 cm Sephadex G-15 column, eluting with 2% NH4HCO3, followed by preparative reverse-phase HPLC (Vydak C4 prep column, 20%–80% CH3CN in water with 0.1% TFA over 20 min; 2 elutes at 54% CH3CN). As a control compound, we also synthesized the benzamide-modified peptide 4 (Figure 1 ), using the same synthetic methodology.7
20. Stewart, J. M.; Young, J. D. Solid Phase Peptide Synthesis, Pierce Chemical Company: Rockford, IL 1984.
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24. For 2: amino acid analysis (expected values in parentheses) Glu/Gln 8.68 (8), Ala 2.00 (2), Leu 4.75 (5); FAB mass spectrum 2003, (M + Na)+ = 2003, ΔM = 0; UV-vis (water) λmax = 289 nm, ϵ = 11 170 M−1 cm−1. For 4: Glu/Gln 8.52 (8), Ala 2.00 (2), Leu 5.35 (5); time-of-flight MS (252Cf fission fragment) 1880.7, (M + Na)+ = 1881, ΔM = −0.3; UV-vis λmax = 260 nm.
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27. Loss of bipyridine from [Fe(bipy)3]2+ is disfavored (Keq = 10−9.55) and accompanied by a transition from low spin (S = 0) to high spin (5 = 2). See: Irving, H.; Mellor, D. H. J. Chem. Soc. 1962, 5237. FeII(pepy)3 is diamagnetic on the basis of its 1H NMR spectrum.
28. Peaks observed (ppm in Diphosphate): 9.00 (br s), 8.93 (br s), 7.75 (br s), 7,70 (br s), 7.64 (m), 7.56 (m). The mer isomer has C1 symmetry and should theoretically give 18 peaks in this region, unless fast exchange is occurring. For a spectrum of mixture of fac and mer isomers, see: Cook, M. J.; Lewis, A. P.; McAuliffe, G. S. G.; Thomson, A. J. Inorg. Chim. Acta 1982, L25–L28.
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31. The CD spectrum of pepy did not change in the presence of 1 × 10−3 M ethylenediaminetetraacetic acid (EDTA).
32. The observed fac preference of the metal center and the high α-helicity (85%) strongly suggest that the most stable folded state of FeII(pepy)3 would be a symmetrical three-helix bundle.
33. On the basis of its CD spectrum, the preferred chirality of the metal center of FeII(pepy)3 can be tentatively assigned to the Λ-configuration as shown in Scheme I. Λ-(−)-[Fe(bipy)3]2+ exhibits a negative peak at 285 nm ([θ] ≤ −4.95 × 105 deg cm2 dmol−1) and a positive peak at 300 nm ([θ] ≥ 1.34 × 106 deg cm2 dmol−1) in its CD spectrum.4b−d
34. Milder, S. J.; Gold, J. S.; Kliger, D. S. J. Am. Chem. Soc. 1986, 108, 8295.
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38. Regan, L.; DeGrado, W. F. Science 1988, 241, 976.
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40. Cohen, C.; Parry, D. A. D. Proteins. Struct., Funct., Genet. 1990, 7, 1. Also see refs 2 and 3a-d.
41. Ho and DeGrado reported that a 33-residue peptide with two amphiphilic helical regions designed to dimerize to a four-helix bundle trimerized by forming two three-helix bundles. Ho, S. P.; DeGrado, W. F. J. Am. Chem. Soc. 1987, 109, 6751.