The depth of molecular recognition: voltage-sensitive blockage of synthetic multifunctional pores with refined architecture

Voltage-sensitive blockage by ADP, ATP and phytate (IP6) demonstrates that active-site contraction toward the middle of newly synthesized rigid-rod beta-barrels provides a general strategy to rationally create and modulate the voltage sensitivity (and to increase the efficiency) of molecular recognition by synthetic multifunctional pores.     

Rigid-rod beta-barrels as lipocalin models: probing confined space by carotenoid encapsulation

Herein, we describe the design, synthesis, structure, and function of synthetic, supramolecular beta-barrel models. Assembly of octi(p-phenylene)s with complementary -Lys-Leu-Lys-NH2 and Glu-Leu-Glu-NH2 side chains yielded water-soluble rigid-rod beta-barrels of precise length and with flexible diameter. A hydrophobic interior was evidenced by guest encapsulation. Host-guest complexes with planarized, monomeric beta-carotene within tetrameric rigid-rod beta-barrels, and disc micellar astaxanthin J-aggregates surrounded by about dodecameric rigid-rod "bicycle tires" were prepared from mixed micelles by dialytic detergent removal. The significance of these findings for future bioorganic chemistry in confined, intratoroidal space is discussed in comparison with pertinent biological examples.     

Thermodynamic and kinetic stability of synthetic multifunctional rigid-rod beta-barrel pores: evidence for supramolecular catalysis

The lessons learned from p-octiphenyl beta-barrel pores are applied to the rational design of synthetic multifunctional pore 1 that is unstable but inert, two characteristics proposed to be ideal for practical applications. Nonlinear dependence on monomer concentration provided direct evidence that pore 1 is tetrameric (n = 4.0), unstable, and "invisible," i.e., incompatible with structural studies by conventional methods. The long lifetime of high-conductance single pores in planar bilayers demonstrated that rigid-rod beta-barrel 1 is inert and large (d approximately 12 A). Multifunctionality of rigid-rod beta-barrel 1 was confirmed by adaptable blockage of pore host 1 with representative guests in planar (8-hydroxy-1,3,6-pyrenetrisulfonate, KD = 190 microM, n = 4.9) and spherical bilayers (poly-L-glutamate, KD < or = 105 nM, n = 1.0; adenosine triphosphate, KD = 240 microM, n = 2.0) and saturation kinetics for the esterolysis of a representative substrate (8-acetoxy-1,3,6-pyrenetrisulfonate, KM = 0.6 microM). The thermodynamic instability of rigid-rod beta-barrel 1 provided unprecedented access to experimental evidence for supramolecular catalysis (n = 3.7). Comparison of the obtained kcat = 0.03 min(-1) with the kcat approximately 0.18 min(-1) for stable analogues gave a global KD approximately 39 microM3 for supramolecular catalyst 1 with a monomer/barrel ratio approximately 20 under experimental conditions. The demonstrated "invisibility" of supramolecular multifunctionality identified molecular modeling as an attractive method to secure otherwise elusive insights into structure. The first molecular mechanics modeling (MacroModel, MMFF94) of multifunctional rigid-rod beta-barrel pore hosts 1 with internal 1,3,6-pyrenetrisulfonate guests is reported.     

Bioorganic chemistry à la baguette: studies on molecular recognition in biological systems using rigid-rod molecules

Initial studies using rigid-rod molecules or "baguettes" to address bioorganic topics of current scientific concern are reported. It is illustrated how transmembrane oligo(p-phenylene)s as representative model rods can be tuned to recognize lipid bilayer membranes either by their thickness or polarization. The construction of otherwise problematic hydrogen-bonded chains along transmembrane rods yields "proton wires," which act by a mechanism that is central in bioenergetics but poorly explored by means of synthetic models. Another example focuses on multivalent ligands assembling rigid-rod cell-surface receptors into transmembrane dynamic arene arrays. The potassium transport mediated by these ligand-receptor complexes provides experimental support for the potential biological importances of the controversial cation-pi mechanism. More complex supramolecular architecture is portrayed in the first artificial beta-barrels. It is shown how programmed assembly of toroidal rigid-rod supramolecules in detergent-free water permits control of diameter of the chemical nature of their interior. Reversed rigid-rod beta-barrels are assembled to function as self-assembled ionophores, ion channel models, and transmembrane nanopores. The potential of future intratoroidal chemistry is exemplified by encapsulation and planarization of beta-carotene in water and the construction of transmembrane B-DNA at the center of a second-sphere host-guest complex à al baguette.     

Synthetic pores with sticky pi-clamps

In this report, we describe design, synthesis, evaluation and molecular dynamics simulations of synthetic multifunctional pores with pi-acidic naphthalenediimide clamps. Experimental evidence is provided for the formation of unstable but inert, heterogeneous and acid-insensitive dynamic tetrameric pores that are sensitive to base and ionic strength. Blockage experiments reveal that the introduction of aromatic electron donor-acceptor interactions provides access to the selective recognition of pi-basic intercalators within the pore. This breakthrough is important for the application of synthetic pores as multianalyte sensors.     

Substrate-independent transduction of chromophore-free organic and biomolecular transformations into color

The concept of synthetic multifunctional pores as substrate-independent optical signal transducers of chemical reactions is introduced with emphasis on the combination with substrate-specific signal generation in biomolecular transformations. Comparison with the general electrochemical transduction, known from conventional biosensors, and the general optical transduction of analyte-specific biomolecular recognition (rather than transformation), known from immunosensing, reveals the fundamental nature of the concept as well as an attractive complementarity to existing methods. Examples with transferases, hydrolases, lyases, and even an isomerase demonstrate that optical transduction with synthetic multifunctional pores is general far beyond the substrate-specific signal generators of electrochemical transduction, that is, the oxidoreductases, and absolutely unproblematic. In part very recent breakthroughs are used to highlight the remarkable promise of synthetic multifunctional pores as optical transducers of biomolecular transformation with regard to practical sensing and screening applications.     

Ligand-gated synthetic ion channels

Supramolecular pi-stack architecture is fundamental in DNA chemistry but absent in biological and synthetic ion channels and pores. Here, a novel rigid-rod pi-stack architecture is introduced to create synthetic ion channels with characteristics that are at the forefront of rational design, that is, ligand gating by a conformational change of the functional supramolecule. Namely, the intercalation of electron-rich aromatics is designed to untwist inactive electron-poor helical pi-stacks without internal space into open barrel-stave ion channels. Conductance experiments in planar lipid bilayers corroborate results from spherical bilayers and molecular modeling: Highly cooperative and highly selective ligand gating produces small, long-lived, weakly anion selective, ohmic ion channels. Structural studies conducted under conditions relevant for function provide experimental support for helix-barrel transition as origin of ligand gating. Control experiments demonstrate that minor structural changes leading to internal decrowding suffice to cleanly annihilate chiral self-organization and function.     

Synthetic multifunctional pores with external and internal active sites for ligand gating and noncompetitive blockage

Design, synthesis, and multifunctionality of p-octiphenyl beta-barrel pores with external LRL triads and internal HH dyads are described. Molecular recognition of anionic fullerenes > calixarenes > pyrenes by guanidinium arrays at the outer pore surface is shown to result in pore opening, whereas alpha-helix recognition within the topologically matching internal space is shown to result in noncompetitive pore blockage. This experimental evidence for multifunctionality is supported by comparison with pertinent control pores and blockers, by structural studies using FRET from p-octiphenyl donors in the pore to BODIPY acceptors in the bilayer, and by molecular mechanics simulations. Practical usefulness of ligand-gated synthetic multifunctional pores is exemplified with the continuous detection of chemical processes.     

Interface engineering of synthetic pores: towards hypersensitive biosensors

Hydrophilic anchoring is introduced as a promising strategy to constructively control the various interactions of synthetic pore sensors with the surrounding biphasic environment. Artificial rigid-rod beta barrels are selected as classical synthetic multifunctional pores and random-coil tetralysines are attached as hydrophilic anchors. The synthesis of this advanced pore is accomplished in 32 steps from commercially available starting materials. With regard to pore activity as such, the key impact of hydrophilic anchoring is a change from a Hill coefficient n<1 to n=4. This change confirms successful suppression of the competing self-assembly with precipitation from the aqueous phase as the origin of the accomplished increase in pore activity. The hydrophilic anchors do not interfere with the blockage of the synthetic pore sensors by anionic analytes. In the case of stoichiometric binding of blockers (K(D)=EC(50) of the pore; EC(50)=concentration needed to observe 50 % pore activity), however, the increase in pore activity achieved by hydrophilic anchoring results in improved pore blockage under high dilution conditions. Controls confirm that this increase does not occur with analytes that do not exhibit stoichiometric binding (K(D)>EC(50)). These results not only reveal stoichiometric binding as the expected origin of the sensitivity limit of synthetic pore sensors, they also provide promising solutions for this problem. The combination of hydrophilic anchoring with targeted pore formation emerges as a particularly promising strategy to further reduce effective pore concentrations. The scope and limitations of this approach are exemplified with pertinent analyte pairs that are essential for the sensing of sucrose, lactose, acetate, and glutamate with synthetic pores in samples from the supermarket.     

Synthetic multifunctional pores: deletion and inversion of anion/cation selectivity using pM and pH

We report the characterization of multifunctional rigid-rod beta-barrel ion channels with either internal aspartates or arginine-histidine dyads by planar bilayer conductance experiments. Barrels with internal aspartates form cation selective, large, unstable and ohmic barrel-stave (rather than toroidal) pores; addition of magnesium cations nearly deletes cation selectivity and increases single-channel stability. Barrels with internal arginine-histidine dyads form cation selective (PK/Pc1 = 2.1), small and ohmic ion channels with superb stability (single-channel lifetime > 20 seconds). Addition of "protons" results in inversion of anion/cation selectivity (Pc1-/Pk+ = 3.8); addition of an anionic guest (HPTS) results in the blockage of anion selective but not cation selective channels. These results suggest that specific, internal counterion immobilization, here magnesium (but not sodium or potassium) cations by internal aspartates and inorganic phosphates by internal arginines (but not histidines), provides access to synthetic multifunctional pores with attractive properties.     

Synthetic ion channels and pores (2004-2005)

This critical review covers synthetic ion channels and pores created between January 2004 and December 2005 comprehensively. The discussion of a rich collection of structural motifs may particularly appeal to organic, biological, supramolecular and polymer chemists. Functions addressed include ion selectivity and molecular recognition, as well as responsiveness to light, heat, voltage and membrane composition. The practical applications involved concern certain topics in medicinal chemistry (antibiotics, drug delivery), catalysis and sensing. An introduction to principles and methods is provided for the non-specialist; some new sources of inspiration from fields beyond chemistry are highlighted.     

Sugar sensing with synthetic multifunctional pores

Recently, synthetic multifunctional pores have been identified as "universal" detectors of chemical reactions. In this report, we show that with the assistance of enzymes as variable co-sensors, synthetic multifunctional pores can serve as similar universal sensors of variable components in mixed analytes. Sugar sensing in soft drinks is used to exemplify this new concept. This is achieved using invertase and hexokinase as co-sensors and a new synthetic multifunctional pore capable of discriminating between ATP and ADP in an "on-off" manner as sensor. The on-off discrimination between ATP as good and ADP as poor pore blocker is shown to be reasonably tolerant of changing experimental conditions. These results identify universal sensing with synthetic multifunctional pores as a robust, sensitive, and noninvasive method with appreciable promise for practical applications.     

Molecular recognition: from solution science to nano/materials technology

In the 25 years since its Nobel Prize in chemistry, supramolecular chemistry based on molecular recognition has been paid much attention in scientific and technological fields. Nanotechnology and the related areas seek breakthrough methods of nanofabrication based on rational organization through assembly of constituent molecules. Advanced biochemistry, medical applications, and environmental and energy technologies also depend on the importance of specific interactions between molecules. In those current fields, molecular recognition is now being re-evaluated. In this review, we re-examine current trends in molecular recognition from the viewpoint of the surrounding media, that is (i) the solution phase for development of basic science and molecular design advances; (ii) at nano/materials interfaces for emerging technologies and applications. The first section of this review includes molecular recognition frontiers, receptor design based on combinatorial approaches, organic capsule receptors, metallo-capsule receptors, helical receptors, dendrimer receptors, and the future design of receptor architectures. The following section summarizes topics related to molecular recognition at interfaces including fundamentals of molecular recognition, sensing and detection, structure formation, molecular machines, molecular recognition involving polymers and related materials, and molecular recognition processes in nanostructured materials.     

核酸适配体及其在化学领域的相关应用

核酸适配体是一小段经体外筛选得到的寡核苷酸序列,能与相应的配体进行高亲和力和强特异性的结合,它的出现为化学生物学界和生物医学界提供了一种新的高效快速识别的研究平台,并在许多方面展示了良好的应用前景。本文从核算适配体的性质和体外筛选过程等方面出发,着重综述了核算适配体的化学修饰方法及其在分析化学和酶化学中应用的研究进展。     

Self-assembly of the beta2-microglobulin NHVTLSQ peptide using a coarse-grained protein model reveals a beta-barrel species

Although a wide variety of proteins can assemble into amyloid fibrils, the structure of the early oligomeric species on the aggregation pathways remains unknown at an atomic level of detail. In this paper we report, using molecular dynamics simulations with the OPEP coarse-grained force field, the free energy landscape of a tetramer and a heptamer of the beta2-microglobulin NHVTLSQ peptide. On the basis of a total of more than 17 ns trajectories started from various states, we find that both species are in equilibrium between amorphous and well-ordered aggregates with cross-beta-structure, a perpendicular bilayer beta-sheet, and, for the heptamer, six- or seven-stranded closed and open beta-barrels. Moreover, analysis of the heptamer trajectories shows that the perpendicular bilayer beta-sheet is one possible precursor of the beta-barrel, but that this barrel can also be formed from a twisted monolayer beta-sheet with successive addition of chains. Comparison with previous aggregation simulations and the fact that nature constructs transmembrane beta-sheet proteins with pores open the possibility that beta-barrels with small inner diameters may represent a common intermediate during the early steps of aggregation.     

Adhesive pi-clamping within synthetic multifunctional pores

We report the design, synthesis, and evaluation of synthetic multifunctional pores with adhesive, that is, electron-deficient naphthalenediimide (NDI) pi-clamps at their inner surface. We find that, in lipid bilayer membranes, comparable synthetic pores with and without pi-clamps have similar, nanomolar activity. Functional relevance of adhesive pi-clamping within synthetic pores is demonstrated by means of an innovative in situ blocker screening method. The obtained line of experimental evidence includes (a) different blockage efficiency with and without pi-clamps (quantified as clamping factors), (b) increasing clamping factors with increasing blocker charge (supportive ion pairing), and, most importantly, (c) increasing clamping factors with increasing aromatic electron donor-acceptor interactions. The availability of advanced synthetic multifunctional pores with refined active sites is important for practical applications in domains such as drug discovery (enzyme inhibitor screening) and diagnostics (multianalyte sensing).     

Synthetic catalytic pores

Catalytic activity of a synthetic multifunctional pore is studied in large unilamellar vesicles under conditions where substrate and synthetic catalytic pore (SCP) approach the membrane either from the same side (cis catalysis) or from opposite sides (trans catalysis). A synthetic supramolecular rigid-rod beta-barrel with excellent ion channel characteristics is identified as SCP using 8-acetoxypyrene-1,3,6-trisulfonate (AcPTS) as model substrate. The key finding is that application of supportive membrane potentials increases the initial velocity of AcPTS esterolysis (v0). This results in an increase of Vmax beyond experimental error (+30%), whereas KM increases less significantly. Long-range electrostatic steering by the membrane potential, possibly guiding substrates into the transmembrane catalyst and, more importantly, accelerating product release (foff = 1.3) is discussed as one possible explanation of this global reduction of catalyst saturation. Control experiments show, inter alia, that similarly strong changes do not occur with opposing membrane potentials.     

分子识别指导下的有机分子设计、合成和组装——世纪交替时代的有机化学

在21世纪即将来临之际,有机化学将面临生命科学、环境科学和材料科学越来越多的挑战。本文回顾了在分子识别指导下的有机分子的设计、合成和组装这个新领域的诞生和发展,认为这个领域将成为新世纪有机化学发展的一个重要方向。它的发展和应用不仅使得有机化学可能较好地面对新挑战,同时能推动有机合成化学自身的发展。     

Synthesis, reactions, and applications of fluorine-containing multifunctional carbon compounds

The chemistry of compounds containing a carbon atom bearing three or four different labile functional groups has received little attention. These compounds should be of considerable significance in theoretical and synthetic organic chemistry. Among the compounds with multifunctional structures, those having both carbonyl and halogen groups in addition to other heteroatom groups seem especially valuable from a synthetic viewpoint. Their potential use as probes in pure and applied synthetic chemistry has not been exploited, presumably because of structural instability and a paucity of synthetic approaches. Keeping this background in mind, we focused on the synthesis of a new class of multifunctional carbon compounds in which ester carbonyl, halogen, and other heteroatom-derived functional groups are directly attached to the central carbon atom. Fluorine was chosen as the halogen because of the inherent stability of the CF bond and because of the fundamental chemical and biological interest in fluorine-containing compounds. The synthesis, reactions, and some applications of various fluorine-containing multifunctional carbon compounds are described.     

Synthetic functional pi-stack architecture in lipid bilayers

Neglected until recently, pi-stack architecture is rapidly emerging as a powerful strategy to create function in lipid bilayer membranes. Recent reports describe supramolecular rosettes acting as hosts of intercalating guests, to assemble in bilayer membranes and, in the case of stacked guanosine and folate quartets, to form ion channels. The introduction of rigid-rod pi-stack architecture allowed us to address one of the great challenges in the field, i.e. ligand gating. Inspiring pi-stack chemistry from related fields, covering rainbow coloration, conductivity, as well as the critical dependence of charge mobilities on the precision of supramolecular organization is summarized to zoom in on arguably the most promising application of functional pi-stack architecture in lipid bilayers, that is the creation of multifunctional photosystems.