Effects of condensing agent and nuclease on the extent of ejection from phage lambda

We have recently demonstrated, that DNA ejection from bacteriophage lambda can be partially or completely suppressed in vitro by external osmotic pressure. This suggests that DNA ejection from phage is driven by an internal mechanical force consisting of DNA bending and DNA-DNA electrostatic repulsion energies. In the present work we investigate the extent to which DNA ejection is incomplete at zero osmotic external pressure when phage is opened with its receptor in vitro. The DNA fragment remaining in the capsid and the tail that is no longer bent or compressed -and hence for which there is no internal driving force for ejection- is shown not to be ejected. We also demonstrate that DNA can be "pulled" out from the capsid by DNase I acting as a DNA binding protein or spermine acting as a DNA condensing agent. In particular, cryo electron microscopy and gel electrophoresis experiments show the following: (i) DNA ejection from bacteriophage lambda incubated in vitro with its receptor is incomplete at zero external osmotic force, with several persistence lengths of DNA remaining inside the phage capsid, if no nuclease (DNase I) or DNA condensing agent (spermine) is present in the host solution; (ii) in the presence of both DNase I and spermine in the host solution, 60% (approximately 29 kbp) of wild-type lambda DNA (48.5 kbp) remains unejected inside the phage capsid, in the form of an unconstrained toroidal condensate; (iii) with DNase I added, but no spermine, the ejection is complete; (iv) with spermine, but without DNase I added, all the DNA is again ejected, and organized as a toroidal condensate outside.     

Cooperatively Enhanced Receptors for Biomimetic Molecular Recognition

The concept of preorganization suggests that organizing a receptor around its guest during binding is detrimental, because the cost of conformational change is assumed to be paid out of the binding energy. Although this concept has historically guided the synthesis of a great many synthetic hosts, in recent years, chemists have begun to synthesize receptors that resemble proteins in their cooperative conformational changes. Such changes could enhance the host–guest interactions, in particular if the binding of the guest triggers previously unengaged noncovalent interactions within the host. These hosts, referred to as cooperatively enhanced receptors, corroborate with their biological counterparts to support the approach of creating high‐affinity receptors through the combined strategies of cooperativity and preorganization. Solvents, often the invisible participants of any solution‐based supramolecular process, should be properly considered in the design of synthetic receptors, whether preorganized or cooperatively enhanced.     

Aryl Crown Ethers Based on d-Glucose Scaffold – Highly Selective Receptors of Amino Acid Ester Hydrochlorides

Amino acids are important biomolecules with a broad scope of applications in chemical and biological sciences. Their functions and properties depend on their absolute configuration. Therefore, methods for chiral recognition and separation of amino acids are highly sought after. For the purposes of diagnostic and medicinal applications chiral recognition of amino acids in water is particularly relevant. However synthetic receptors for enantioselective binding of amino acids in aqueous media are rare. Recently we reported a d -glucose-based crown ether for chiral recognition of amino acid esters in water. We achieved enantioselectivities towards amino acids with hydrophobic sidechains which were among the highest ever reported for a small molecule receptor. The binding affinities were however moderate. Herein we disclose analogs of that receptor, containing aryl functionalities in the crown ether fragment. The new receptors show considerably improved binding affinities for amino acid ester hydrochlorides in water, while retaining high enantio- or chemoselectivities.     

Solid phase synthesis and opioid receptor binding properties of presumable dermorphin precursor derivatives.

Presumable dermorphin precursor peptide derivatives comprised of 35 amino acids and their fragments, which are based on the amino acid sequence determined by recombinant deoxyribonucleic acid (DNA) techniques, were synthesized by the solid phase method. A 35-residue peptide amide containing L-Ala2-dermorphin sequence at the N-terminus (1) as well as its D-Ala2 isomer (2) and the C-terminal 20-residue peptide amide were found to be unexpectedly stable against aminopeptidase M digestion and in rat brain membrane fractions mixture, suggesting that the C-terminal Glu-rich moiety of 1 and 2 serves to protect from enzymatic breakdown. In the opioid receptor binding assay, 2 showed 40 and 25-fold higher affinities than 1 for mu and delta-receptors, respectively. The N-terminal 15-residue peptide fragment of 2 showed greatly increased affinities for both receptors, being one half of those of dermorphin, whereas that of 1 showed low affinities. Opioid receptor binding properties of these synthetic peptides may be useful in investigation of the processing to dermorphin.     

Exploring the relation between amplification and binding in dynamic combinatorial libraries of macrocyclic synthetic receptors in water

Herein we describe an extensive study of the response of a set of closely related dynamic combinatorial libraries (DCLs) of macrocyclic receptors to the introduction of a focused range of guest molecules. We have determined the amplification of two sets of diastereomeric receptors induced by a series of neutral and cationic guests, including biologically relevant compounds such as acetylcholine and morphine. The host–guest binding affinities were investigated using isothermal titration calorimetry. The resulting dataset enabled a detailed analysis of the relationship between the amplification of selected receptors and host–guest Gibbs binding energies, giving insight into the factors affecting the design, simulation and interpretation of DCL experiments. In particular, two questions were addressed: Is amplification by a given guest selective for the best receptor? And does the best guest induce the largest amplification of a given receptor? Our experimental results and computer simulations showed that the relative levels of amplification of hosts by a guest are well‐correlated with their relative affinities, and simulations have confirmed previous observations that amplification can be selective for the best receptor when only modest amounts of guest are used. In contrast, the correlation between guest binding and the extent of amplification of a given receptor across a wide range of guests tends to be poorer, because every guest has its own unique set of affinities for competing receptors in the DCL. This implies that the results of screening a DCL for selective receptors by comparing the response of the mixture to two different guests should be interpreted with caution. DCLs are complex mixtures in which all compounds are connected through a set of equilibria. Obtaining quantitative information about all host–guest binding constants from such systems will require the explicit and simultaneous consideration of all of the main equilibria within a DCL.     

Total synthesis of pinnatoxins A and G and revision of the mode of action of pinnatoxin A

Pinnatoxins belong to an emerging class of potent marine toxins of the cyclic imine group. Detailed studies of their biological effects have been impeded by unavailability of the complex natural product from natural sources. This work describes the development of a robust, scalable synthetic sequence relying on a convergent strategy that delivered a sufficient amount of the toxin for detailed biological studies and its commercialization for use by other research groups and regulatory agencies. A central transformation in the synthesis is the highly diastereoselective Ireland-Claisen rearrangement of a complex α,α-disubstituted allylic ester based on a unique mode for stereoselective enolization through a chirality match between the substrate and the lithium amide base. With synthetic pinnatoxin A, a detailed study has been performed that provides conclusive evidence for its mode of action as a potent inhibitor of nicotinic acetylcholine receptors selective for the human neuronal α7 subtype. The comprehensive electrophysiological, biochemical, and computational studies support the view that the spiroimine subunit of pinnatoxins is critical for blocking nicotinic acetylcholine receptor subtypes, as evidenced by analyzing the effect of a synthetic analogue of pinnatoxin A containing an open form of the imine ring. Our studies have paved the way for the production of certified standards to be used for mass-spectrometric determination of these toxins in marine matrices and for the development of tests to detect these toxins in contaminated shellfish.     

Synthetic receptors for the differentiation of phosphorylated peptides with nanomolar affinities

Artificial ditopic receptors for the differentiation of phosphorylated peptides varying in i+3 amino acid side chains were synthesized, and their binding affinities and selectivities were determined. The synthetic receptors show the highest binding affinities to phosphorylated peptides under physiological conditions (HEPES, pH 7.5, 154 mM NaCl) reported thus far for artificial systems. The tight and selective binding was achieved by high cooperativity of the two binding moieties in the receptor molecules. All receptors interact with phosphorylated serine by bis(ZnII-cyclen) complex coordination and a second binding site recognizing a carboxylate or imidazole amino acid side chain functionality.     

Tuning the specificity of a synthetic receptor using a selected nucleic acid receptor

Because of their relative simplicity, synthetic receptors often lack the selectivity observed for biopolymer receptors, such as aptamers. However, aptamer recognition of ligands is limited by the chemistries inherent in the four canonical nucleotides. Here, we report the design and selection of a ternary complex in which the specificity of a bis-boronic acid synthetic host (1) that binds to various carboxylic acids is tuned by a surrounding aptamer. Although, the synthetic receptor alone has higher selectivity for citrate over DL-tartrate, the formation of the aptamer:receptor complex reversed the organic host selectivity to preferentially bind tartrate. The RNA conformation changed upon the introduction of the synthetic host, consistent with an induced-fit mechanism for binding.     

Molecular ferries: membrane carriers that promote phospholipid flip-flop and chloride transport

The facilitated transport of ionic or polar solutes through biological membranes is an essential process for cellular life, and a major technical goal of the pharmaceutical industry. Synthetic receptors with affinities for anions are shown to act as molecular ferries and facilitate the movement of chloride ions and salts across vesicle and cell membranes. A process that competes with chloride transport is phospholipid translocation or flip-flop. This has led to the development of synthetic scramblases that can alter the transmembrane distribution of phospholipids and induce biological responses such as membrane enzyme activation. The facilitated translocation of phospholipids with multiply-charged head groups, like phosphatidylserine, is a difficult supramolecular challenge that requires a complementary, multitopic receptor with appropriate amphiphilicity.     

Mapping Polyamide–DNA Interactions in Human Cells Reveals a New Design Strategy for Effective Targeting of Genomic Sites

Targeting the genome with sequence‐specific synthetic molecules is a major goal at the interface of chemistry, biology, and personalized medicine. Pyrrole/imidazole‐based polyamides can be rationally designed to target specific DNA sequences with exquisite precision in vitro; yet, the biological outcomes are often difficult to interpret using current models of binding energetics. To directly identify the binding sites of polyamides across the genome, we designed, synthesized, and tested polyamide derivatives that enabled covalent crosslinking and localization of polyamide–DNA interaction sites in live human cells. Bioinformatic analysis of the data reveals that clustered binding sites, spanning a broad range of affinities, best predict occupancy in cells. In contrast to the prevailing paradigm of targeting single high‐affinity sites, our results point to a new design principle to deploy polyamides and perhaps other synthetic molecules to effectively target desired genomic sites in vivo.     

Triphenylene: A versatile molecular receptor

Since the late seventies, the search for new molecular receptors has been constant in perfecting the affinity and selectivity of recognition in different media. At present, a renewed interest in (host:guest) chemistry focuses on the molecular detection of specific targets such as biological, pollutant, toxic or explosive species. This review of triphenylene-based receptors outlines their recent contribution to molecular recognition. Two main structural approaches were investigated to transform a simple triphenylene moiety into a host for neutral aromatic compounds or cations, by tailoring multivalent molecules provided with or without a flatten cavity. The properties of different receptors are presented along with the latest synthetic methods to prepare high-value triphenylenes and the perspectives in the field of sensing. In addition, the role of functionalized triphenylenes in extended (host:guest) systems is illustrated by the main examples of discotic liquid crystals and porous coordination polymers involving this polyaromatic compound.     

Ranking Peptide Binders by Affinity with AlphaFold**

AlphaFold has revolutionized structural biology by predicting highly accurate structures of proteins and their complexes with peptides and other proteins. However, for protein-peptide systems, we are also interested in identifying the highest affinity binder among a set of candidate peptides. We present a novel competitive binding assay using AlphaFold to predict structures of the receptor in the presence of two peptides. For systems in which the individual structures of the peptides are well predicted, the assay captures the higher affinity binder in the bound state, and the other peptide in the unbound form with statistical significance. We test the application on six protein receptors for which we have experimental binding affinities to several peptides. We find that the assay is best suited for identifying medium to strong peptide binders that adopt stable secondary structures upon binding.     

Logic‐Gate‐Actuated DNA‐Controlled Receptor Assembly for the Programmable Modulation of Cellular Signal Transduction

Programming cells to sense multiple inputs and activate cellular signal transduction cascades is of great interest. Although this goal has been achieved through the engineering of genetic circuits using synthetic biology tools, a nongenetic and generic approach remains highly demanded. Herein, we present an aptamer‐controlled logic receptor assembly for modulating cellular signal transduction. Aptamers were engineered as “robotic arms” to capture target receptors (c‐Met and CD71) and a DNA logic assembly functioned as a computer processor to handle multiple inputs. As a result, the DNA assembly brings c‐Met and CD71 into close proximity, thus interfering with the ligand–receptor interactions of c‐Met and inhibiting its functions. Using this principle, a set of logic gates was created that respond to DNA strands or light irradiation, modulating the c‐Met/HGF signal pathways. This simple modular design provides a robust chemical tool for modulating cellular signal transduction.     

Dramatic Enhancement of Binding Affinities Between Foldamer-Based Receptors and Anions by Intra-Receptor π-Stacking

As a synthetic model for intra-protein interactions that reinforce binding affinities between proteins and ligands, the energetic interplay of binding and folding was investigated using foldamer-based receptors capable of adopting helical structures. The receptors were designed to have identical hydrogen-bonding sites for anion binding but different aryl appendages that simply provide additional π-stacking within the helical backbones without direct interactions with the bound anions. In particular, the presence of electron-deficient aryl appendages led to dramatic enhancements in the association constant between the receptor and chloride or nitrate ions, by up to three orders of magnitude. Extended stacking within the receptor contributes to the stabilization of the entire folding structure of complexes, thereby enhancing binding affinities.     

Estrogen-derived steroidal metal complexes: agents for cellular delivery of metal centers to estrogen receptor-positive cells

Targeted cellular delivery of drugs to specific tissues is an important goal in biomedical chemistry. Achieving this requires harnessing and applying molecular-level recognition events prevalent in (or specific to) the desired tissue type. Tissues rich in estrogen receptors (ERs), which include many types of breast cancer, accumulate molecules that have high binding affinities for these receptors. Therefore, molecules that (i) bind to the ER, (ii) have favorable cellular transport properties, and (iii) contain a second functionality (such as a center that may be used for diagnostic imaging or medical therapy) are exciting synthetic targets in the field of drug delivery. To this end, we have prepared a range of metallo-estrogens based on 17alpha-ethynylestradiol and examined their binding to the ER both as isolated receptor and in whole cell assays (ER positive MCF-7 cells). Estrogens functionalized with metal binding units are prepared by palladium-catalyzed cross-coupling reactions and a wide range of metal centers introduced readily. All the compounds prepared and tested exhibit effective binding to the estrogen receptor and are delivered across the cell membrane into MCF-7 cells. In the whole cell assays, despite their monocationic nature, the palladium and platinum complexes prepared exhibit similar (and even enhanced) receptor binding affinities compared to their corresponding neutral free ligands. It is unprecedented for a higher ER binding affinity to be observed for a cationic complex than for its metal-free ligand.     

Dramatic Enhancement of Binding Affinities Between Foldamer‐Based Receptors and Anions by Intra‐Receptor π‐Stacking

As a synthetic model for intra‐protein interactions that reinforce binding affinities between proteins and ligands, the energetic interplay of binding and folding was investigated using foldamer‐based receptors capable of adopting helical structures. The receptors were designed to have identical hydrogen‐bonding sites for anion binding but different aryl appendages that simply provide additional π‐stacking within the helical backbones without direct interactions with the bound anions. In particular, the presence of electron‐deficient aryl appendages led to dramatic enhancements in the association constant between the receptor and chloride or nitrate ions, by up to three orders of magnitude. Extended stacking within the receptor contributes to the stabilization of the entire folding structure of complexes, thereby enhancing binding affinities.     

Screening of 5-HT1A receptor antagonists using molecularly imprinted polymers

Molecular imprinting produces network polymers with recognition sites for imprint molecules. The high binding affinity and selectivity in conjunction with the polymers' physical robustness positions molecular imprinted polymers (MIPs) as candidates for use as preliminary screens in drug discovery. As such, MIPs can serve as crude mimics of native receptors. In an effort to evaluate the relationship between MIPs and native receptors, imprinted polymers for WAY-100635, an antagonist of the serotonin (5-HT) receptor subtype 5-HT1A were prepared. The resulting MIP P(WAY) was evaluated as an affinity matrix in the screening of serotonin receptor antagonists with known affinities for the native receptor. Rough correlations in affinity between the synthetic P(WAY) and native receptor 5-HT1A were found. These findings provide some support for the analogy between MIPs and native receptors and their possible use as surrogates.     

Proteo-dendrimers designed for complementary recognition of cytochrome c: dendrimer architecture toward nanoscale protein complexation

"Proteo-dendrimers" in which polyanionic hepta(glutamic acids), fluorescent zinc porphyrinate cores, hydrophilic polyether surfaces, and nonpeptide hydrophobic dendrons are combined, were developed as a new series of synthetic receptors for protein recognition. They have polyanionic "patch" structures on their surfaces and undergo complementary electrostatic interactions with a positively charged cytochrome c patch, as observed in biological protein-protein recognition systems. Stability constants of the resulting supramolecular complexes were determined in phosphate buffer (pH 7) by monitoring the fluorescence quenching of the zinc porphyrinates. These proteo-dendrimer receptors exhibited higher affinities with cytochrome c proteins in aqueous solutions than with biological cytochrome b5. Furthermore, they effectively blocked complexation of biological cytochrome b5 with cytochrome c, indicating that the proteo-dendrimers and cytochrome b5 similarly occupy the polycationic patch of cytochrome c.