Traditionally, the structure and properties of natural products have been determined by total synthesis and comparison with authentic samples. We have now applied this procedure to the first nonproteinaceous ion channel, isolated from bacterial plasma membranes, and consisting of a complex of poly(3-hydroxybutyrate) and calcium polyphosphate. To this end, we have now synthesized the 128-mer of hydroxybutanoic acid and prepared a complex with inorganic calcium polyphosphate (average 65-mer), which was incorporated into a planar lipid bilayer of synthetic phospholipids. We herewith present data that demonstrate unambiguously that the completely synthetic complex forms channels that are indistinguishable in their voltage-dependent conductance, in their selectivity for divalent cations, and in their blocking behavior (by La 3؉ ) from channels isolated from Escherichia coli. The implications of our finding for prebiotic chemistry, biochemistry, and biology are discussed.Synthetic and naturally occurring ion channels are amphiphilic structures with an outer coat of nonpolar residues and a lining of polar and charged residues (1-7). In this report, we demonstrate the formation of synthetic nonproteinaceous ion channels from two structurally distinct polymers that share the above attributes in a cooperative fashion. The polymers in question, poly(3-hydroxybutyrate) (PHB) and inorganic polyphosphate (polyP), are ubiquitous constituents of biological cells (8)(9)(10)(11)(12). PHB is an amphiphilic homopolymer of (R)-3-hydroxybutanoic acid, which in bacteria is synthesized from acetyl-CoA in three steps: dimerization to form acetoacetyl-CoA, reduction by NADPH to form 3-hydroxybutyrylCoA, and polymerization to form PHB (13,14). Under growth-limiting conditions, some bacteria produce highmolecular-mass PHB (60,000 to Ͼ1,000,000 Da) in amounts of up to 90% of the cell dry weight. PolyP, a polyanion composed of phosphate residues linked by anhydride bonds, is formed by repetitive phosphoryl transfer from ATP or other high-energy phosphates (15-17).Both PHB and polyP have molecular characteristics that are consistent with a role in ion conduction. PHB has salt-solvating properties that derive from the recurrence of electrondonating ester carbonyl oxygens at regular and frequent intervals along its flexible backbone (18,19). The solvating abilities of PHB are illustrated by studies that demonstrate its capacity to transport cations across methylene chloride layers in U-tubes (20), form ion-conducting complexes with lithium perchlorate (21), or large-conductance nonselective ion channels in planar lipid bilayers (22). PolyP has a flexible backbone and high density of monovalent negative charges that create a large capacity for ion exchange and a stronger affinity for multivalent over monovalent cations (23).Plasma membrane vesicles of Escherichia coli contain complexes of PHB and polyP that function as calcium-selective channels in planar lipid bilayers (24). In vivo, such channels may control the influx of calcium to ma...
Monodisperse Linear and Cyclic Oligo[(R)-3-hydroxybutanoates~ Conitaining up to 128 Monomeric UnitsUsing benzyl ester/(leri-buty1)diphenylsilyl ether protection, (COCl)'/pyridine esterification conditions, and a fragment-coupling strategy (with H,/Pd-C debenzylation and H F .pyridine desilylation), linear oligomers of (R)-3-hydroxybutanoic acid (3-HB) containing up to 128 3-HB building blocks (mol. weight > 11 000 Da) are assembled (Schemes I , 2,5, and 6 ) . In contrast to the previously employed protecting-group combination, and due to the low-temperature esterifying conditions, this procedure leads to monodisperse oligomers: all steps occur without loss of single 3-HB units. The product oligomers with two, one, and no terminal protecting groups (mostly prepared in multi-gram am.ounts) are characterized by all standard spectroscopic methods, especially by mass spectroscopy (Figs. 2 and 3 ) , by their optical activity, and by elemental analyses. Cyclization of the oligo[(R)-3-hydroxybutanoic acids] with up to 32 3-HB units, using thiopyridine activation and CuBr, for the ring closure, produces oligolides consisting of up to 128 ring atoms (Scheme 7). Mixed oligolides containing 3-HB and (R)-3-hydroxypentanoic units are prepared from the corresponding linear trimers, using Yamuguchi's method for the ring closure (Scheme 8 and Fig. 4 (X-ray crystal structures of two folded conformers)). Comparisons of melting points (Table I ) , of [a]:& values (Tables 2 and 3), of 'H-NMR coupling constants (Table 3 ) , and of molecular volume/hydroxyalkanoate unit (Table 4 ) of linear and cyclic oligomer derivatives and of the high-molecularweight polymer show that the monodisperse oligomers appear to be surprisingly good models for the polymer. Besides this insight, our synthesis is supplying the samples to further test the role of P(3-HB) (ca. 140 units) as a component of complexes forming channels through cell-wall phospholipid bilayers.
The coupling of 6 with the desilylated branches 7 and 10 finally gave the dendrimers of the first (green circle in formula 11/12) and of the second generation (ll), respectively. The selective hydrogenolysis of the terminal benzyl groups of the dendrimers was achieved by catalytic transfer hydrogenation[61 and gave the polyanionic dendrimers of the first and second generation (12a).The dendritic compounds were obtained in To study the biodegradability['' of the dendrimers, we tested their stability in the presence of various hydrolases. ' 0 x o t + n depicts the degradation of three dendritic compounds 6a, 6b, and llb with a PHB-depolymerase.[81 The linear tetrameric HB 2b was used as the standard substrate for the depolymerase. The protected dendrimers with dimeric HB-elongation units were not degraded by the depolymerase. whereas the free acids were moderately good substrates for this enzyme.['] This observation is surprising as the simple dimeric H B 2a is not a substrate for the depolymerase. 'H N M R spectroscopic investigations of the degradation products of the deprotected, first-generation dendrimer with dimeric HB-elongation units (green circle in formula 11/12) revealed free HB, the triester of 1.3,Sbenzenetricarboxylic acid with HB, and also compound 13.['01 The enzymatic hydrolysis of the deprotected, first-generation dendrimer, which has dimeric HB-elongation units, was followed by ti-
Repetitive treatment of the biopolymer P(3-HB) (molecular weight > lo5 Dalton, storuge OJ s-P(3-HB)), with lithium hexamethyl disilazanid (LHMDS) at -70" in THF leads to a mixture of oligomers with increasingly sharp distribution around a 15-, 30-, and 45mer. Discrete fragments are also isolated when P(3-HB) is heated under reflux (89O) in neat Et,N. Linear oligo(3-HB) derivatives (3-7) containing up to 96 3-HB units are synthesized using an exponential segment-coupling strategy. These oligomers are used to calibrate size-exclusion chromatography columns for the analysis of oligo(3-HB) samples from the different sources. The linear oligo-(3-HB) derivatives also served as a model with respect to the physical properties ofhigh molecular weight P(3-HB) and were investigated as such by transmission electron microscopy (TEM) and by small-and wide-angle X-ray scattering (SAXS and WAXS). The thicknesses of the lamellar crystallites (long periods) formed by the Rmer, 16mer, and 32mer, are ca.26, 52, and 53 A, respectively, indicating that the 32mer molecules are folded once, very tightly, into a 'hair-pin'-type conformation. High-molecular-weight P(3-HB). which was crystallized in a similar way, also has a lamellar crystallite thickness of ca. [50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65] A. Thus, the treatment of P(3-HB) with LHMDS at low temperature causes etching of the amorphous regions, an effect well known in polymer science for studying the regularity of chain folding. The ca. 50-A packing within the tight folds of P(3-HB) is discussed in view of its possible function in ion transport through cell membranes.Polyhydroxybutyrate (P(3-HB)) is the prototype of a class of biopolymers [I] [2] called polyhydroxyalkanoates (PHA; for review articles, see . They are used by microorganisms as a means to store energy and reductase equivalents and as a carbon source (high-molecular-weight s-P(3-HB), > lo5 Dalton). A copolymer of P(3-HB) and P(3-HV) (V = valerate) is produced by fermentation and sold as a biodegradable and biocompatible plastic material under the trade name BZOPOL [6]. Low-molecular-weight P(3-HB) (100-200 units) was found in the membranes of prokaryotic and eukaryotic cells. It forms complexes, for instance, with calcium polyphosphate and albumin (c-P(3-HB)) [7]. The structure and function of c-P(3-HB) are greatly in debate, concerning suggestions including stabilizing proteins to a Ca2+ polyphosphate (PPi), and acting as a DNA channel through lipid membranes [7]. We have embarked on an investigation to obtain information on the structure and properties of oligo(3-HB) derivatives [3].
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