On the solution structure of PHB: Preparation and NMR analysis of isotopically labeled oligo[(R)-3-hydroxybutanoic acids] (OHBs)

While the chain conformation of poly- and oligo[(R)-3-hydroxybutanoate] (PHB, OHB) is known to be 2₁-and 3₁-helical in stretched fibers and in the crystalline state, respectively (Fig. 2), the structure in solution is unknown. To be able to determine the NMR-solution structure, specifically labeled linear oligomers have been prepared: a 16-mer consisting of alternating pairs of fully ¹³C-labeled and non-labeled residues (1) and a 20-mer containing an O-¹³CH(¹³CH₂D)-¹³CHD ^Si-¹³CO residue in position 9 (from the O-terminus) and a fully ¹³C-labeled residue in position 12 (2), both with (ι-Bu)Ph₂Si protection at the O- and Bn protection at the C-terminus. The labeled (R)-3-hydroxybutanoic acid building blocks were prepared by Noyori hydrogenation of the ethyl ester of fully ¹³C-labeled acetoacetic acid, and the D-atoms were incorporated by D₂/Pd-C reduction of a previously reported dibromo-1,3-dioxinone 8 (Scheme 1). The oligomers were obtained by a series of fragment couplings (Schemes 2 and 3). 600-MHz NMR COSY, HSQC, ROESY, and cross-correlated relaxation measurements (Figs. 4-6, 9, and 12, and Tables 1-3) at different temperatures and interpretations thereof led to assignments of all resonances, including those from the diastereotopic C(2)H₂ protons, and to determination of the conformationally averaged dihedral angles φ₂ and φ₃ (Figs. 2, 7, and 8) in the chain of the oligoester. The conclusions are: all but five or six terminal residues adopt the same conformation; the 2₁ helix is not the predominant secondary structure; the structure of the HB chain is averaged, even at -30°. Our investigation confirms the high flexibility of the polyester chain, a property that has been deduced previously from biological studies of PHB in membranes, in ion channels, and as appendage of proteins.