Synthetic Multifunctional Pores Research Articles

Chirality sensing with pores: Reactive signal amplifiers for otherwise undetectable small molecules

Recently, we have introduced a concept to determine enantiomeric excess (ee) with synthetic multifunctional pores (Tanaka and Matile, Chirality 2008;20:307-312). The reported approach is, however, limited to macromolecules and not applicable to small molecules. The problem is that the ability of synthetic pores to respond to chemical stimulation decreases with the size and the charge of the analyte. Here we demonstrate that this problem can be overcome with reactive signal amplifiers that covalently capture elusive analytes for sensitive recognition by the pore. For proof of principle, we use L-lactate and D-lactate as representative pair of enantiomers, L-lactate oxidase as stereospecific signal generator, and cascade blue hydrazide as reactive signal amplifier to capture the produced pyruvate. After stereospecific signal generation and reactive signal amplification, L-lactate was detectable quantitatively and without further optimization in the presence of at least 99% ee of D-lactate. Attempts to sense the traces of impurity in commercial samples of D-lactate gave values in the expected range (99.6% ee expected, 99.3 +/- 0.1% ee found).

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.

Direct Visualization of Glucose Phosphorylation with a Cationic Polythiophene

a-D-Glucose is the chief energy source in organisms and plays an important role in the metabolism of most mammalian tissues and cells. Glycolysis, the major route of glucose metabolism, is initiated by the catalytic phosphorylation to D-glucose-6-phosphate (G-6-P) by hexokinase (HK) in the presence of adenosine triphosphate (ATP) and Mg. Phosphorylation of glucose is important for both energy metabolism and biosynthesis in the cell. It accelerates glucose metabolism and prevents glucose from effusing from cells. Furthermore, the rates of ATP-dependent glucose phosphorylation can be used to identify new members of the hexokinases. Glucose phosphorylation is currently measured by coupling to the reduction of NADP+ using glucose-6-phosphate dehydrogenase as an auxiliary enzyme. The generated NADPH is then monitored by absorption spectroscopy and/or HPLC techniques. These methods share the drawback of including a coupled additional enzymatic reaction and are timeconsuming discontinuous assays. As a way to circumvent these limitations, a synthetic multifunctional pore, octakis(Gla-LeuArg-Trp-His-Val-NH2)-p-octiphenylene (SMP), has been used for developing fluorometric assays. Such methods require intricate design and the synthesis of SMPs. These considerations provide motivation for developing simpler and more convenient methods to detect and monitor glucose phosphorylation. Conjugated polymers have been used as the optical platform for chemical and biological detection. Water-soluble polythiophene derivatives display changes in their conformation and aggregation states upon forming interpolyelectrolyte complexes with DNA. Recently, a cationic conjugated polythiophene was used to successfully respond to the presence of ATP, via a combination of electrostatic and hydrophobic interactions, and to detect DNA cleavage. As demonstrated here, it is possible to use these properties for designing a simple, homogeneous, continuous and real-time protocol to assay glucose phosphorylation. The overall strategy is illustrated in Scheme 1. Cationic poly[3-(3′-N,N,N-trimethylamino-1′-propyloxy)-4-methyl-2,5thiophene hydrochloride] (CPT) was chosen as the optical probe. Upon mixing, electrostatic interactions bring together

Chirality sensing with synthetic pores

A concept to determine enantiomeric excess with synthetic multifunctional pores is introduced. To do so, the poor stereoselectivity of molecular recognition by stimuli-responsive pores is coupled with the stereospecificity of enzymes. With substrates as good and products as poor pore blockers, enzymatic conversion of one enantiomer is shown to readily reveal the concentration of the other one. Calculations suggest that high substrate/product discrimination by the synthetic pores may provide access to the accurate detection of the extreme enantiomeric excess that is of interest in chemistry, pharmacology, and medicine, but otherwise possibly problematic to detect. Validity of the introduced concept is experimentally confirmed with poly-L-glutamate and poly-D-glutamate as enantiomeric substrates with high blockage efficiency, L-glutamate and D-glutamate as enantiomeric products with poor blockage efficiency, subtilisin A as enzyme, and a classical rigid-rod beta-barrel as synthetic pore.

Synthetic pores with sticky π-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.

Adhesive π-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).

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.

Cover Picture: Molecular Recognition by Synthetic Multifunctional Pores in Practice: Are Structural Studies Really Helpful? (Adv. Funct. Mater. 2/2006)

AbstractStefan Matile and co‐workers describe their recent results on synthetic multifunctional pores on p. 169. In Nature, pores are used to transport materials across cellular membranes. Synthetic pores, such as the one shown on the cover, can be used for molecular recognition, leading to applications such as hosting, sensing, or catalysis. The authors argue that demonstration of function is not enough to confirm that synthetic pores work as designed: structural studies and molecular modeling provide complementary, yet vital, information.This account summarizes five years of research devoted to the development of the concept of synthetic multifunctional pores. The objective is to complement a comprehensive graphical summary of molecular recognition with a survey of structural studies on the same topic. The relevance of the latter for research focusing on creation and application of supramolecular functional materials is discussed briefly in a subjective manner.

Molecular Recognition by Synthetic Multifunctional Pores in Practice: Are Structural Studies Really Helpful?

AbstractThis account summarizes five years of research devoted to the development of the concept of synthetic multifunctional pores. The objective is to complement a comprehensive graphical summary of molecular recognition with a survey of structural studies on the same topic. The relevance of the latter for research focusing on creation and application of supramolecular functional materials is discussed briefly in a subjective manner.

Synthetic multifunctional pores that open and close in response to chemical stimulation

Studies on synthetic multifunctional pores with external and internal active sites for ligand gating and noncompetitive blockage are presented, with emphasis on the contribution of external ligands to the characteristics of pore. A comparison between different synthetic multifunctional pores reveals that the location of functional groups in rigid-rod β-barrel pores is precisely reflected in the function: molecular recognition at the outer barrel surface results in pore opening, while molecular recognition at the inner barrel surface results in pore closing. Negligible nonspecific leakage, disappearance of pH gating, inhibition of intervesicular pore transfer, and maybe also the flickering of currents of single open pores characterize external ligands as adhesive cushions that liberate the pore from lateral pressure exerted by the surrounding membrane. Refined molecular models show good agreement with pore design and experimental facts with regard to function.

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.

Contributions of Lipid Bilayer Hosts to Structure and Activity of Multifunctional Supramolecular Guests

The question of whether or not the surrounding lipid bilayer host contributes to structure and activity of included functional guests is a general topic of current scientific concern. We report that synthetic multifunctional pores are of use to address this elusive question, because the detection of their catalytic activity is membrane independent. According to their salt-rate profiles, unstable multifunctional supramolecules with permanent internal charges show highest membrane sensitivity, and the dependence of membrane sensitivity on the acidity of internal cations exceeds that on supramolecule stability. These results can, with all appropriate caution, be interpreted as indications for the existence of long-range EMP-ICR interactions (EMP: external membrane pressure, ICR: internal charge repulsion) between membrane hosts and functional guests that can, for instance, prevent the 'explosion' and promote the 'implosion' of over- and undercharged transmembrane barrel-stave supramolecules, respectively.

Rigid‐Rod Molecules in Biomembrane Models: From Hydrogen‐Bonded Chains to Synthetic Multifunctional Pores

AbstractFor Abstract see ChemInform Abstract in Full Text.

Rigid-Rod Molecules in Biomembrane Models: From Hydrogen-Bonded Chains to Synthetic Multifunctional Pores

Synthetic ion channels and pores formed by rigid-rod molecules are summarized. This includes work on hydrogen-bonded chains installed along membrane-spanning rigid-rod scaffolds to transport protons. As a second topic, programmed assembly of p-septiphenyls with terminal iminodiacetate-copper complexes for potassium transport by cation-pi interactions is described. The third topic concerns rigid push-pull rods as fluorescent alpha-helix mimics to probe the importance of dipole-potential interactions for voltage gating, both on the functional and the structural level. Topic number four deals with p-octiphenyl staves as key scaffolds for the synthesis of rigid-rod beta-barrel pores. The description of internal and external design strategies for these rigid-rod beta-barrels covers a rich collection of pH-, pM-, voltage-, ligand-, and enzyme-gated synthetic multifunctional pores that can act as hosts, sensors, and catalysts. As far as practical applications are concerned, the possibility to detect chemical reactions with synthetic multifunctional pores appears most attractive. Recent molecular mechanics simulations are presented as a valuable approach to insights on the elusive suprastructures of multifunctional pores made from rigid rods.

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.

Blocker efflux through blocked pores

AbstractThe objective of this paper is to clarify a frequent confusion that occurs with synthetic multifunctional pores: does molecular recognition by a synthetic pore exclude molecular translocation through the same pore? Evidence that this is not the case is provided with a set of competitive experiments using a rigid‐rod β‐barrel with internal histidine–arginine dyads as synthetic multifunctional pores (SMPs) as well as 8‐hydroxypyrene‐1,3,6‐trisulfonate (HPTS) as a fluorescent and 1,3,6,8‐pyrenetetrasulfonate (PTS) as a non‐fluorescent blocker. Direct evidence for the efflux of HPTS blockers through blocked SMPs is obtained by quenching of released HPTS with an externally added quencher. Blockage of blocker efflux through blocked pores was demonstrated by adding PTS as the opposing external blocker (KD = 3.3 μM). A Hill coefficient of n = 1.5 may indicate that binding of more than one PTS blocker is necessary to inhibit the efflux of HPTS blockers. Supported by structural information on blockage and selectivity from biological potassium channels, blocker efflux through blocked pores is discussed as being implicated in selectivity. Copyright © 2004 John Wiley &amp; Sons, Ltd.

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.

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.

Toward Active-Site Compression in Rigid-Rod ?-Barrels

Applying the Woodhull equation to organic chemistry, a concept to maximize remote control of chemical processes that take place within synthetic multifunctional pores is described.

Automation highlights from literature

Automation highlights from literature