On the Solution Structure of PHB: Preparation and NMR Analysis of Isotopically Labeled Oligo[(R)-3-hydroxybutanoic Acids] (OHBs)

Abstract

Dedicated to Professor Edgar Heilbronner with best wishes on the occasion of his 80th birthdayWhile the chain conformation of poly-and oligo[(R)-3-hydroxybutanoate] (PHB, OHB) is known to be 2 1 -and 3 1 -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 13 C-labeled and non-labeled residues (1) and a 20-mer containing an O-13 CH( 13 CH 2 D)-13 CHD Si -13 CO residue in position 9 (from the O-terminus) and a fully 13 Clabeled residue in position 12 (2), both with (t-Bu)Ph 2 Si protection at the O-and Bn protection at the Cterminus. The labeled (R)-3-hydroxybutanoic acid building blocks were prepared by Noyori hydrogenation of the ethyl ester of fully 13 C-labeled acetoacetic acid, and the D-atoms were incorporated by D 2 /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)H 2 protons, and to determination of the conformationally averaged dihedral angles f 2 and f 3 (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 2 1 helix is not the predominant secondary structure; the structure of the HB chain is averaged, even at ±308. 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.

Transmembrane pores formed by synthetic p -octiphenyl β-barrels with internal carboxylate clusters: Regulation of ion transport by pH and Mg 2+ - complexed 8-aminonaphthalene-1,3,6-trisulfonate

“…The development of design strategies for the construction of easily variable active sites at the inner surface of transmembrane pores is the first step toward such organic chemistry within confined anisotropic space. In sharp contrast to breathtaking progress being made with biotechnologically modified natural pores (1), reliable and general design strategies for synthetic ion channels and pores with easily variable internal active sites do not exist (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Reasonably straightforward covalent synthesis of barrel-like macromolecules with the required Ϸ3.5-nm height and variably functionalizable interior of Ն1.0 nm diameter is not (yet) possible.…”

“…Reasonably straightforward covalent synthesis of barrel-like macromolecules with the required Ϸ3.5-nm height and variably functionalizable interior of Ն1.0 nm diameter is not (yet) possible. The more straightforward noncovalent syntheses (16)(17)(18)(19)(20) applied to barrellike supramolecules with ion channel activity is, with one possible exception (15), limited by apparent difficulties to position functional groups at internal concave surfaces (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). However, we have recently found that this trend toward ''peripheral crowding'' in supramolecular synthesis (16)(17)(18)(19)(20) can be bypassed in artificial ␤-barrels with p-octiphenyl ''staves'' and have used their rationally designed hydrophobic, ionophoric, and cationic interior to encapsulate guests with complementary characteristics in water and bilayer membranes (21,22).…”

The Role of Short-Chain Conjugated Poly-(R)-3-Hydroxybutyrate (cPHB) in Protein Folding

“…Moreover, polyesters such as PHB, in contrast to polyamides and polypeptides, have highly flexible backbone structures, since they lack the stabilizing element of internal hydrogen bonds and the rigidity of a peptide bond. The extremely high flexibility of the PHB backbone at physiological temperatures has been amply demonstrated by Seebach et al , using circular dichroism and fluorescence (FRET) measurements [31], nuclear magnetic resonance (NMR) spectroscopy [32,33] and molecular dynamics simulations [34]. However, it is important to note that the glass temperature of PHB is ~10 °C, so that the PHB backbone becomes increasingly more rigid as temperatures are lowered below the physiological range.…”

A Molecular-Dynamics Simulation Study of the Conformational Preferences of Oligo(3-hydroxyalkanoic acids) in Chloroform Solution

“…± investigations on the structure of PHB in solution were undertaken. Although there are hints for the presence of folded secondary structures on the very short time scale of UV/ VIS spectroscopy (provided by FRET (Fluorescence Resonance Energy Transfer) [7] and CD [8] [9] measurements), no predominant secondary structure has been discovered on the longer NMR time scale 4 ). The conclusion was that PHB possesses a highly flexible backbone.…”