Hydrophobic Cluster Formation Is Necessary for Dibenzofuran-Based Amino Acids to Function as .beta.-Sheet Nucleators

Abstract

Three dibenzofuran-based amino acid residues, namely 4-(2-aminoethyl)-6-dibenzofuranpropanoic acid (1), 4-(aminomethyl)-6-dibenzofuranethanoic acid (2), and 4-amino-6-dibenzofuranmethanoic acid (3), were prepared in order to compare their physical properties so as to further understand residue l's ability to nucleate antiparallel 3-sheet formation within small peptides in aqueous solution. FT-IR, variable temperature NMR, and an X-ray crystallography study reveal that amide analogs of 1 and 2 can adopt intramolecularly hydrogen bonded conformations in nonpolar solvents, whereas amides composed of residue 3 cannot. Spectroscopic studies reveal that linear heptapeptides containing 1 are capable of adopting a dynamic antiparallel 3-sheet structure in aqueous solution, whereas residues 2 and 3 are incapable of nucleating a 3-sheet structure in an otherwise identical peptide sequence. The efficacy of residue 1 as a 3-sheet nucleator appears to result from a 15-membered ring intramolecularly hydrogen-bonded hydrophobic cluster conformation which serves as a partial 3-sheet template enabling neighboring residues to be added to the growing sheet with a favorable equilibrium constant. The hydrophobic cluster conformation in these heptapeptides is stabilized by (S-Sheet Formation via Dibenzofuran-Based Amino Acids acid sequence which will adopt a partial /8-sheet structure capable of nucleating a /8-sheet,* 2 **S.*'7 we set out to design an amino acid that will stabilize a partial /8-sheet structure and thus nucleate /3-sheet formation in aqueous solution.8The concept of introducing a conformationally rigid molecule into a peptide to stabilize a single conformation was established by Hirschmann, Veber, Freidinger, Nutt, and their colleagues at Merck.9 Since then and previous to our efforts, several elegant dipeptide and related 8-turn mimetics have been prepared either to control the conformation of peptides and/or as potential pharmaceuticals.10 The ability to design a molecular fragment which nucleates 8-sheet formation was first demonstrated by Kemp by employing the epindolidione nucleus. When the epindolidione ring system is incorporated into an a-amino acid sequence, it serves as an artificial 8-strand and mediates sheet formation via hydrogen bonding between the epindolidione skeleton and the covalently attached peptide chain.10'0'11 The goal of the work described here is to develop an amino acid that is a dipeptide mimetic of the i + 1 and i + 2 residues of a 8-turn in an effort to nucleate 8-sheet folding without significantly perturbing the resulting antiparallel 8-sheet structure. Protein

The nucleation of monomeric parallel β-sheet-like structures and their self-assembly in aqueous solution

“…9,26 Brie¯y, dibenzofuran was treated with secbutyllithium and TMEDA in diethyl ether at À78 C to aord 4,6-bis-lithiated dibenzofuran. Addition of iodine to the dianion aords 4,6-diiododibenzofuran in 83% yield.…”

“…The preparation of amide 4 uses the monoacid monoester derivative 5 reported previously (Scheme 3). 9 The acid functional group was transformed to the acid chloride 6 and treated with isobutyl amine yielding the monoamide monoester intermediate 7. The remaining ester group was hydrolyzed to aord acid 8, which was converted to the HOBT ester 9 with BOP.…”

“…26 This template is designed to function analogous to the 4-(2-aminoethyl)-6-dibenzofuranpropionic acid template which nucleates antiparallel b-sheet formation in aqueous solution employing a hydrogen-bonded hydrophobic cluster as discerned from NMR-based structural studies. 9,27,28 The desired perpendicular orientation of the dibenzofuran ring system in 1 relative to the plane of the parallel b-sheet should be stabilized by hydrophobic interactions between the dibenzofuran skeleton and the hydrophobic side chains of the aamino acid residues that¯ank 1, Figure 1. 9,27,28 This hydrogen-bonded hydrophobic cluster is expected to be stable under a variety of conditions and should facilitate parallel sheet folding once the solution conditions permit long range residue±residue interactions between strands.…”

“…57 Previous studies on simple amides show that the antiparallel dibenzofuran-based template strongly promotes a 15-membered ring intramolecular hydrogen-bonded conformation without detectable intermolecular amide± amide hydrogen bonding in a 1.5 mM CH 2 Cl 2 solution. 9,56 In the amides studied here, a 14-membered ring intramolecular amide±amide hydrogen bond was anticipated, the type required to initiate parallel b-sheet formation (Scheme 2). The IR spectra of a 1.5 mM solution of amide 3 in CH 2 Cl 2 reveals absorption bands at 3441 and 3336 cm…”

“…9,52,56 The temperature dependence of the chemical shift is less reliable than the FT-IR data, e.g. in cyclic systems and in rare acyclic systems where the enthalpies of the equilibrating conformations are similar, the temperature dependencies of the amide NH chemical shift show opposite trends to those discussed directly above.…”