Active Template Synthesis of Protein[n]Catenanes Using Engineered Peptide–Peptide Ligation Tools

The expansion of protein topological diversity requires new and efficient synthetic tools.Herein,we report the second and third generations of the SpyStapler-mediated SpyTag/BDTag ligation system for the efficient synthesis of 4-arm star proteins and the repurposing of the third generation as an active template to enable the synthesis of higher-order protein[n]catenanes(n=3,4,and 5).SpyStapler003 has a higher affinity to its cognate SpyTag and BDTag reactive pairs relative to the original SpyStapler.Hence,it can overcome much more profound steric hindrance in protein ligation and improve the efficiency of the resulting active template tool to facilitate the construction of radial protein[n]catenanes.Various proteins of interest,such as dihydrofolate reductase and the nanobody KN035,can be modularly incorporated into the[n]catenanes with intact activity.Combination of passive and active template strategies gives rise to linear protein[4]catenanes,which further expands the current topological diversity.Moreover,higher-order protein catenation not only leads to enhanced thermal stability and proteolytic resistance but also higher affinity of the nanobody via multivalent effects.Our study provides tools useful for bioconjugation and new topological protein scaffolds for the multivalent display of enzymes and antibodies.

Size-controlled synthesis and near-infrared photothermal response of Cp^(*)Rh-based metalla[2]catenanes and rectangular metallamacrocycles

A variety of research reports on novel supramolecular topologies have been published over the last years.However,it is still a great challenge to tap into the inner functional properties of these complexes.Herein,two tetranuclear metallamacrocycles 1 and 2 and four octonuclear[2]catenanes 3-6 were constructed successfully via a coordination-driven self-assembly strategy,by conscious design and use of the tetramethyl bidentate pyridine ligand L1,and the appropriate selection of six binuclear half-sandwich rhodium building units with different longitudinal dimensions.The complexes have been fully characterized by single crystal X-ray diffraction analysis and NMR spectroscopy.Furthermore,near-infrared photothermal studies of the obtained[2]catenanes reveal different photothermal response in solid and solution states,which may be attributed to a strong fluorescence quenching effect of the half-sandwich organometallic fragment and different conjugated effect of Cp^(*)Rh based building blocks in the interlocking structures.The photothermal conversion efficiencies of[2]catenanes 4-6 fall in the range 30.5%-16.5% respectively.This contribution aims to play a key role in the experimental development of Cp^(*)-based photothermal materials.

Stereoselective Self-Assembly of Chiral Metalla[2]Catenanes and Lemniscular Macrocycles

Designing and stereoselectively constructing chiral mechanically interlocked molecules(MIMs)is a goal that many chemists are pursuing due to their potential in the development of functional molecular machines involved in catalysis,sensing,and chiroptical switching.However,the synthesis of enantiopure supramolecular entities with crystallographic structures remains challenging.Herein,the stereoselective self-assembly of chiral metalla[2]catenanes interlocked by two D-shaped macrocycles was realized by combining long binuclear half-sandwich organometallic Cp*Rh^(III)(Cp*=η^(5)-pentamethylcyclopentadienyl)clip and ditopic monodentate ligand incorporating L-leucine residues.The resulting metalla[2]catenane displayed unique co-conformational mechanical helical chirality characteristics arising from the chirality transfer of the chiral ligand.Furthermore,decreasing the length of the binuclear Cp*Rh^(III)clip and adjusting the steric hindrance of the amino-acid side chains(L-alanine and L-phenylalanine)in the ligands resulted in two lemniscular macrocycles with left-handed(M)-twisted conformational chirality.To the best of our knowledge,the synthesized macrocycles are the first family of homochiral abnormal lemniscate-shaped organometallic macrocycles featuring figure-eight geometries.Metalla[2]catenanes and macrocycles with opposite chirality were also generated by employing corresponding D-amino acid ligands,as evidenced by single-crystal X-ray diffraction,nuclear magnetic resonance,mass,and circular dichroism spectroscopies.Our present study demonstrates that homochiral MIMs can be accessed readily using amino acid–containing ligands through rational molecular design.

Rational Design and Synthesis of Interlocked[2]Catenanes Fea-turing Half-Sandwich Cp^(*)Rh/Ir Units and Pyrene-Based Ligands

The construction of interlocked topologies by metal-cation-template methods has been extensively explored.However,construction of these species using template-free chemical self-assembly methods is hard to rationally achieve due to the inherent unpredictability of self-assembly processes.In this work,a strategy to construct interlocked[2]catenanes by coordination-driven template-free self-assembly is demonstrated,whereby combining rigid bidentate ligands with large conjugated-πarea units and building blocks with appropriate lengths facilitated construction of interlocked[2]catenane compounds.Two rigid bidentate ligands,L1 and L2,were chosen to self-assemble with three dinuclear building blocks B1,B2 and B3.Ultimately,the successful construction of three[2]catenanes demonstrated the validity of the strategy.Although[2]catenanes are the simplest of all interlocked topologies,this motif is the basis for constructing more intricate topologies.This successful strategy may therefore lead to construction of other,more intricate topologies in the future.

Self-assembly of Novel Hetero[3]rotaxane,[2]Rotaxanes and [2]Catenane

One linear template 13 and one cyclophane template 15, both incorporating two electron rich 1,4-dialkoxybenzene units and one diamide unit, have been synthesized. By utilizing donor-acceptor interaction and/or intermolecular hydrogen bonding assembling principles, one novel hetero[3]rotaxane 22·4Cl, possessing one neutral and one tetracationic ring components, has been synthesized from 13, through neutral [2]rotaxane 21 as intermediate. With 15 as template, tetracationic [2]catenane 23·4PF6 was assembled by using donor-acceptor interaction, but no neutral [2]rotaxane could be obtained under the typical conditions of hydrogen bonding assembling principle. The interlocked supramolecular compounds have been characterized and their spectral properties are investigated.

Synthesis and Characterization of Two [2]Catenanes Based on Phenylene-diacetylene Crown Ethers

Two charged donor-acceptor [2]catenanes were synthesized by the cyclization of the π-acceptor 1,1 ＇-[1, 4-phenylenebis-（methylene）]bis（4,4＇-bipyridinium）bis（hexafluorophosphate）（2） and bis（bromomethyl）benzene with the templates of the π-donor phenylene-diacetylene crown ethers la and lb in the yields of 31%, 35%, respectively. The mass spectra, 1H and 13C NMR spectra confirm that the phenylene-diacetylene crown ethers, with a nonaromatic ~r-system, successfully template the cyclization of CBPQT4＋. The X-ray crystallography analysis shows that the CBPQT4＋ ring encircles around the phenylene motif of the crown ethers, leaving the 1,3-butadiyne fragment out of the cavity of CBPQT4＋ ring. The mechanically interlocked structure of [2]catenanes was stabilized by the cooperative effects of π-stacking and hydrogen bonding interactions.

Construction of a hetero pseudo[2]rota[2]catenane

A novel ammonium template containing three ammonium sites was synthesized. Two ammoniums located on the linear component served as template for cucurbit[6]uril to form the CB-based pseudo[2]rotacane while another one located on the macrocyclic component played a role of template for clipping reaction. As a result of a ＇＇threading-followed-by-clipping＇＇ approach, a novel hetero pseudo[2]rota[2]catenane was successfully constructed.

Toward Selective Synthesis of Protein Olympiadanes via Orthogonal Active Templates in One Step

Unlike small molecules,the topological complexity of macromolecules remains largely unexplored due to the huge synthetic challenge.Herein,we report the development of orthogonal active templates for concise and selective synthesis of protein[n]heterocatenanes toward protein olympiadanes.An active template(AT-Snoop)was first developed based on the isopeptide-bond-forming RrgA domain with comparable efficiency and excellent orthogonality to the previously reported active template(AT-Spy)based on the CnaB2 domain.Their combination facilitated the selective synthesis of protein[n]catenanes from multiple components in one step and the resulting structures were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis,size exclusion chromatography,liquid chromatography-mass spectrometry,and proteolytic digestion experiments.The results offered a promising solution to tackling the daunting challenge of precision synthesis of protein olympiadane with five distinct ring components.Not only did the success provide new tools for proteintopology engin eering but alsospurred and fueled the future exploitation of topology-related functional benefits in protein science.

Rational Design and Cellular Synthesis of Proteins with Unconventional Chemical Topology

Chemical topology refers to the three-dimensional arrangement(i.e.,connectivity and spatial relationship)of a molecule's constituent atoms and bonds.The molecular mechanism for translation defines the linear configuration of all nascent proteins.Nontrivial protein topology arises only upon post-translational processing events and often imparts functional benefits such as enhanced stability,making topology a unique dimension for protein engineering.Utilizing the assembly-reaction synergy,our group has developed several methods for the effective and convenient cellular synthesis of a variety of topological proteins,such as lasso proteins,protein rotaxanes,and protein catenanes.The work opens the access to new protein classes and paves the road toward illustrating the topological effects on structure-function relationship of proteins,which lays solid foundation for exploring topological proteins’practical application.

Architecture of Platonic and Archimedean polyhedral links

A new methodology for understanding the construction of polyhedral links has been developed on the basis of the Platonic and Archimedean solids by using our method of the ‘three-cross-curve and dou- ble-twist-line covering’. There are five classes of polyhedral links that can be explored: the tetrahedral and truncated tetrahedral links; the hexahedral and truncated hexahedral links; the dodecahedral and truncated dodecahedral links; the truncated octahedral and icosahedral links. Our results show that the tetrahedral and truncated tetrahedral links have T symmetry; the hexahedral and truncated hexahedral links, as well as the truncated octahedral links, O symmetry; the dodecahedral and truncated dodeca- hedral links, as well as the truncated icosahedral links, I symmetry, respectively. This study provides further insight into the molecular design, as well as theoretical characterization, of the DNA and protein catenanes.

Topology: a unique dimension in protein engineering

Controlling protein topology has been a long standing challenge to go beyond their linear configuration defined by the translation mechanism of cellular machinery. In this mini-review, we focus on the topological diversity in proteins and review the major categories of protein topologies known to date, including branched/star proteins, circular proteins, lasso proteins, knotted proteins, and protein catenanes. The discovery of these topologically complex natural proteins and their synthetic pathways, the rational design and recombinant synthesis of artificial topological proteins and their biophysical studies, are summarized and discussed with regard to their general features and broad implications. The complexity of protein topology is recognized and the routes to diverse protein topologies are illustrated. We believe that topology engineering is an important way to modify protein properties without altemating their native sequences and shall bring in valuable dynamic features central to the creation of artificial protein machinery.

Adaptive Self-Assembly and Induced-Fit Interconversions between Molecular Borromean Rings,Russian Dolls and Ring-in-Ring Complexes

Herein,we report an artificially assembled interlocked system that features adaptive self-assembly and induced-fit transformation properties in the presence of appropriate guest molecules.By careful choice of solvent and guests,a molecular Borromean rings complex based on organometallic half-sandwich units could be transformed in a stepwise manner to a ring-in-ring complex and a monomeric rectangle,leading to a conversion cycle between these structures.This adaptive self-assembly method was further applied to the construction of a Russian dolls complex,forming another conversion cycle composed of a molecular Borromean rings complex,a Russian dolls complex and a monomeric rectangle.A comprehensive map showing all of the structural transformations between these metallarectangles and interlocked species is drawn up,representing a recoverable and recyclable transformation mechanism.

Artificial molecular machines that can perform work

An artificial molecular machine consists of molecule or substituent components jointed together in a specific way so that their mutual displacements could be initiated using appropriate outside stimuli. Such an ability of performing mechanical motions by consuming external energy has endowed these tiny machines with vast fascinating potential applications in areas such as actuators, manipulating atoms/molecules, drug delivery, molecular electronic devices, etc. To date, although vast kinds of molecular machine archetypes have been synthesized in facile ways, they are inclined to be defined as switches but not true machines in most cases because no useful work has been done during a working cycle. More efforts need to be devoted on the utilization and amplification of the nanoscale mechanical motions among synthetic molecular machines to accomplish useful tasks. Here we highlight some of the recent advances relating to molecular machines that can perform work on different length scales, ranging from microscopic levels to macroscopic ones.

Mechanical Properties of Interlocked-ring Polymers: A Molecular Dynamics Simulation Study

Interlocked-ring polymers,also known as polycatenanes,possess an interesting molecular architecture.These polymers are composed of many interlocked rings in a linear chain.The topological constrain between neighboring rings distinguishes the interlockedring polymer from its linear counterpart.Here we present extensive molecular dynamic simulations on the interlocked-ring polymers and analyze the static properties of the polymer.By applying external forces to the polymer,we also study the force-extension curves of the polymer,which provides rich information about the mechanical properties of the interlockedring polymers.

Catenated Cages Mediated by Enthalpic Reaction Intermediates

Catenated cages are generally considered thermodynamically more stable than their constituent monomeric cages.However,the catenation mechanism is yet to be elucidated;it would require systematic investigation into the structural effects of the building blocks,their enthalpic and entropic contributions,and the effect of solvents.By inspecting these factors,we rationalized some design principles for the efficient construction of catenated cages.