Orthogonal Reactions Research Articles

Efficient Encapsulation of Self-Healing Agents in Polymer Nanocontainers Functionalized by Orthogonal Reactions

An orthogonal polymerization reaction between two monomers partitioned in the two liquid phases of a miniemulsion yielded nanocapsules with various functional groups (sulfonate, amine, carboxylic acid, and poly(ethylene glycol) (PEG). The formation of the nanocapsules could be realized in the presence of a self-healing agent in the liquid core. We present here the conditions for the successful preparation of functional polymer nanocapsules by free-radical polymerization as orthogonal reaction for self-healing materials. The stability of the nanocapsules was assessed by AFM measurements in the dried state as well as in water.

Metal-Chelating Polymers by Anionic Ring-Opening Polymerization and Their Use in Quantitative Mass Cytometry

Metal-chelating polymers (MCPs) are important reagents for multiplexed immunoassays based on mass cytometry. The role of the polymer is to carry multiple copies of individual metal isotopes, typically as lanthanide ions, and to provide a reactive functionality for convenient attachment to a monoclonal antibody (mAb). For this application, the optimum combination of chain length, backbone structure, end group, pendant groups, and synthesis strategy has yet to be determined. Here we describe the synthesis of a new type of MCP based on anionic ring-opening polymerization of an activated cyclopropane (the diallyl ester of 1,1-cyclopropane dicarboxylic acid) using a combination of 2-furanmethanethiol and a phosphazene base as the initiator. This reaction takes place with rigorous control over molecular weight, yielding a polymer with a narrow molecular weight distribution, reactive pendant groups for introducing a metal chelator, and a functional end group with orthogonal reactivity for attaching the polymer to the mAbs. Following the ring-opening polymerization, a two-step transformation introduced diethylenetriaminepentaacetic acid (DTPA) chelating groups on each pendant group. The polymers were characterized by NMR, size exclusion chromatography (SEC), and thermogravimetric analysis (TGA). The binding properties toward Gd(3+) as a prototypical lanthanide (Ln) ion were also studied by isothermal titration calorimetry (ITC). Attachment to a mAb involves a Diels-Alder reaction of the terminal furan with a bismaleimide, followed by a Michael addition of a thiol on the mAb, generated by mild reduction of a disulfide bond in the hinge region. Polymer samples with a number average degree of polymerization of 35, with a binding capacity of 49.5 ± 6 Ln(3+) ions per chain, were loaded with 10 different types of Ln ions and conjugated to 10 different mAbs. A suite of metal-tagged Abs was tested by mass cytometry in a 10-plex single cell analysis of human adult peripheral blood, allowing us to quantify the antibody binding capacity of 10 different cell surface antigens associated with specific cell types.

Synthesis of Bispecific Antibodies using Genetically Encoded Unnatural Amino Acids

Bispecific antibodies were constructed using genetically encoded unnatural amino acids with orthogonal chemical reactivity. A two-step process afforded homogeneous products in excellent yield. Using this approach, we synthesized an anti-HER2/anti-CD3 bispecific antibody, which efficiently cross-linked HER2+ cells and CD3+ cells. In vitro effector-cell mediated cytotoxicity was observed at picomolar concentrations.

Catalyst‐Free and Site‐Specific One‐Pot Dual‐Labeling of a Protein Directed by Two Genetically Incorporated Noncanonical Amino Acids

The genetic incorporation of one azide-containing and one keto-containing noncanonical amino acids into a protein at one amber and one ochre mutation sites respectively followed by their orthogonal reactions with one hydroxylamine-containing and one cyclooctyne-containing dyes allows highly efficient one-pot site-specific dual labeling of the protein in a catalyst-free fashion.

A General Regioselective Synthesis of 2,4-Diarylpyrimidines from 2- Thiouracil through Two Orthogonal Cross-Coupling Reactions

A novel efficient synthesis of 2,4-diarylpyrimidines was developed based on phosphonium-mediated Suzuki coupling of 2-(methylsulfanyl)pyrimidin-4(3<i>H</i>)-one (at position 4) followed by the Liebeskind–Srogl cross-coupling (at position 2) under microwave irradiation.

Site-specific DNA-antibody conjugates for specific and sensitive immuno-PCR

Antibody conjugates are widely used as diagnostics and imaging reagents. However, many such conjugates suffer losses in sensitivity and specificity due to nonspecific labeling techniques. We have developed methodology to site-specifically conjugate oligonucleotides to antibodies containing a genetically encoded unnatural amino acid with orthogonal chemical reactivity. These oligobody molecules were used in immuno-PCR assays to detect Her2(+) cells with greater sensitivity and specificity than nonspecifically coupled fragments, and can detect extremely rare Her2(+) cells in a complex cellular environment. Such designed antibody-oligonucleotide conjugates should provide sensitive and specific reagents for diagnostics, as well as enable other unique applications based on oligobody building blocks.

Synthesis of Water-Soluble Scaffolds for Peptide Cyclization, Labeling, and Ligation

The synthesis and applications of water-soluble scaffolds that conformationally constrain side chain unprotected linear peptides containing two cysteines are described. These scaffolds contain a functionality with orthogonal reactivity to be used for labeling and ligation. This is illustrated by the chemical ligation of two dissimilar constrained peptides via oxime ligation or strain-promoted azide-alkyne cycloaddition in aqueous media.

Acrylamide-Based Copolymers Bearing Photoreleasable Thiols for Subsequent Thiol–Ene Functionalization

A new set of monomers is presented in order to incorporate thiols into radical polymers using a protecting chemistry/photocleavage route. The (co)polymerization kinetics of an o-nitrobenzyl thioether-containing acrylamide derivative are reported. The presence of the o-nitrobenzyl moiety is found to strongly affect the polymerization. Nevertheless, water-soluble copolymers with N,N-dimethylacrylamide (DMAAm) as a comonomer are obtained either by free radical polymerization (10 000 ≤ Mn ≤ 17 500 g mol–1; 1.5 ≤ PDI ≤ 1.8) or by reversible addition–fragmentation transfer (RAFT)-mediated controlled/living radical polymerization (2000 ≤ Mn ≤ 5700 g mol–1; 1.1 ≤ PDI ≤ 1.2). Deprotection under UV light (λ = 366 nm) at ambient temperature is followed by UV/vis monitoring of the protecting group release, which proceeds to completion between 40 min and 2 h within the studied range of concentration as demonstrated by 1H NMR spectroscopy. Thiol–maleimide addition is subsequently carried out and found to proceed with a nearly quantitative yield (ca. 90%) as measured by 1H NMR. Different block copolymers (9400 ≤ Mn ≤ 16 500 g mol–1; 1.3 ≤ PDI ≤ 1.4) with a PDMAAm water-soluble block, a polystyrene hydrophobic block, or a poly(N-isopropylacrylamide) thermosensitive block as the first segment and possessing the photoreleasable thiol moieties in the second block are subsequently synthesized by RAFT-mediated polymerization. We finally demonstrate the orthogonal sequential deprotection and reaction with benzyl maleimide of two different thiol species originating from the thiocarbonylthio functionality and the o-nitrobenzyl protected lateral groups, respectively.

Controlling the Assembly of Nanoparticle Mixtures With Two Orthogonal Polymer Complexation Reactions

Self-assembly from mixed dispersions of three sizes of monodisperse polystyrene nanoparticles, large (L), medium (M), and small (S), was controlled by coating each particle type with either a monofunctional or bifunctional polymer capable of participating in specific complexation reactions. The complexation reactions were (1) complexation between phenolic polymers and polyethylene glycol (PEG) containing polymers and (2) condensation of phenylboronic acid containing polymers with polyols. These complexation reactions function independently and can be "turned off" independently; phenylboronic acid complexation was reversed by lowering the pH, whereas the interactions of phenolic copolymers with PEG copolymers could be reversed by adding excess PEG homopolymer. The specificity and reversibility of the interactions was demonstrated by the formation of simple binary aggregates from mixtures. The bifunctional copolymers were poly(vinyl phenol-co-diallyldimethyl ammonium chloride), Ph-DADMAC, and poly(3-acrylamide phenylboronic acid-co-PEG methacrylate), PBA-PEG. The monofunctional polymer was polyvinylalcohol, PVA. Ph-DADMAC forms complexes with PBA-PEG (H-bonding) and with anionic surfaces or polymers (electrostatic/polyelectrolyte complexation). PBA-PEG complexes with Ph-DADMAC (H-bonding) and with PVA (boronate ester formation). PVA does not interact with Ph-DADMAC; therefore, PVA coated particles do not deposit onto Ph-DADMAC coated particles.

Combined oxypalladation/C-H functionalization: palladium(II)-catalyzed intramolecular oxidative oxyarylation of hydroxyalkenes.

Combined oxypalladation/C-H functionalization: palladium(II)-catalyzed intramolecular oxidative oxyarylation of hydroxyalkenes.

Synthesis of multifunctional polymer brush surfaces via sequential and orthogonal thiol-click reactions

Fabrication of multifunctional surfaces with complexity approaching that found in nature requires the application of a modular approach to surface engineering. We describe a versatile post-polymerization modification strategy to synthesize multifunctional polymer brush surfaces via combination of surface-initiated photopolymerization (SIP) and orthogonal thiol-click reactions. Specifically, we demonstrate two routes to multifunctional brush surfaces: in the first approach, alkyne-functionalized homopolymer brushes are modified with multiple thiolsvia a statistical, radical-mediated thiol-yne co-click reaction; and in the second approach, statistical copolymer brushes carrying two distinctly-addressable reactive moieties are sequentially modified via orthogonal base-catalyzed thiol-X (where X represents an isocyanate, epoxy, or α-bromoester) and radical-mediated thiol-yne reactions. In both cases, we show that surface properties, in the form of wettability, can be easily tuned over a wide range by judicious choice of brush composition and thiol functionality.

Click–click chemistry on a peptidic scaffold for easy access to tetrameric DNA structures

The use of a peptidic template and orthogonal oxime ligation and CuAAC reaction allows a stepwise approach for the design of antiparallel tetrameric DNA structure. As a proof of concept template assisted synthesis of i-motif DNA was achieved. As anticipated, a dramatic improvement in the stability of such a tetrameric structure was observed. More interestingly the formation of the i-motif was still observed at neutral pH, which could be attributed to the spatial proximity of the basic centers.

Utilizing click chemistry to design functional interfaces through post-polymerization modification

Post-polymerization modification is a simple and effective method to add complex functionality to a polymeric interface. A wide variety of click reactions have been utilized as a means of post-polymerization functionalization on surface-bound polymers to tune interfacial properties, such as friction, wettability, and adhesion. Patterning surfaces with spatial control of chemical functionality has also been obtained through orthogonal or sequential click reactions. This review highlights the progress in post-polymerization modification of polymers covalently attached to a surface, focusing on the design of functional interfaces in terms of the various reactive functional groups.

Design of polymeric nanoparticles for biomedical delivery applications.

Polymeric nanoparticles-based therapeutics show great promise in the treatment of a wide range of diseases, due to the flexibility in which their structures can be modified, with intricate definition over their compositions, structures and properties. Advances in polymerization chemistries and the application of reactive, efficient and orthogonal chemical modification reactions have enabled the engineering of multifunctional polymeric nanoparticles with precise control over the architectures of the individual polymer components, to direct their assembly and subsequent transformations into nanoparticles of selective overall shapes, sizes, internal morphologies, external surface charges and functionalizations. In addition, incorporation of certain functionalities can modulate the responsiveness of these nanostructures to specific stimuli through the use of remote activation. Furthermore, they can be equipped with smart components to allow their delivery beyond certain biological barriers, such as skin, mucus, blood, extracellular matrix, cellular and subcellular organelles. This tutorial review highlights the importance of well-defined chemistries, with detailed ties to specific biological hurdles and opportunities, in the design of nanostructures for various biomedical delivery applications.

The dynamics and spectroscopic fingerprint of hydroxyl radical generation through water dimer ionization: ab initio molecular dynamic simulation study

Water decomposition process was investigated by ab initio molecular dynamic simulations using a model of (H(2)O)(2)(+) clusters. The proton transfer (PT) process from the cationic H-donor water to the H-acceptor water for the formation of (HO˙)·H(3)O(+) was predicted as about 90 fs on average calculated at CCSD level of theory. The valence-electron transfer (VET) process through the formation of hemibond interaction between neutral and cationic water, (H(2)O)(2)(+), was also identified in several collected trajectories. Both PT and VET processes were found to propagate along two orthogonal reaction coordinates, the former was through an intermolecular hydrogen bond and the latter required oxygen-oxygen hemibonding. Significant difference of the theoretical electronic transitions along the VET trajectories was also observed in comparison with the non-VET cases, being calculated at SAC-CI level. The strong absorption features of hemibonding (H(2)O)(2)(+) may introduce an interesting consideration for experimental design to monitor the water decomposition process.

Towards copper-free nanocapsules obtained by orthogonal interfacial “click” polymerization in miniemulsion

A facile method to produce nanocapsules by copper-free interfacial "click"-polymerization as orthogonal reaction for the encapsulation of functional molecules is successfully performed using stable miniemulsion droplets. Difunctional azides and alkynes have been used for polymerization around the miniemulsion droplets, leading to the formation of nanocapsules. The results were compared with copper-catalyzed systems.

Pd(ii)-catalyzed cycloisomerisation of γ-alkynoic acids and one-pot tandem cycloisomerisation/CuAAC reactions in water

Cycloisomerisation reactions of γ-alkynoic acids into cyclic enol-lactones can be conveniently performed in pure water as a solvent and under aerobic conditions by using a novel iminophosphorane–Pd(II) complex trans-[PdCl2{μ2-N,S-(PTA)NP(S)(OEt)2}]2 as a catalyst. It is important to note that the catalytic system could be recycled up to 10 consecutive runs. In addition and for the first time, a one-pot tandem orthogonal reaction involving the fast cycloisomerisation of γ-alkynoic acids, followed by an intermolecular atom economical process, i.e. the 1,3-dipolar cycloaddition of azides with terminal alkynes (CuAAC), is reported. This new tandem cycloisomerisation/click reaction proceeds also in water, at room temperature and under aerobic conditions, giving rise to unprecedented bicyclic triazol-enol-lactones under the principles of the so called “Green Chemistry”.

Cytocompatible click-based hydrogels with dynamically tunable properties through orthogonal photoconjugation and photocleavage reactions.

To provide insight as to how cells receive information from their external surroundings, synthetic hydrogels have emerged as systems for assaying cell function in well-defined microenvironments where single cues can be introduced and subsequent effects individually elucidated. However, as the field seeks to answer more complex biological questions, advanced material systems are needed that allow dynamic alteration of the 3D cellular environment with orthogonal reactions that enable multiple levels of control of biochemical and biomechanical signals. Here, we sought to synthesize one such 3D culture system using cytocompatible and wavelength-specific photochemical reactions to create hydrogels that allow orthogonal and dynamic control of the material properties through independent spatiotemporally-regulated photocleavage of crosslinks and photoconjugation of pendant functionalities. Results demonstrate the versatile nature of the chemistry to create programmable niches to study and direct cell function by modifying the local hydrogel environment.

Ortho-Bromo(propa-1,2-dien-1-yl)arenes: Substrates for Domino Reactions

o-Bromo(propa-1,2-dien-1-yl)arenes exhibit novel and orthogonal reactivity under Pd catalysis in the presence of secondary amines to form enamines (concerted Pd insertion, intramolecular carbopalladation, and terminative Buchwald-Hartwig coupling) and of amides to form indoles (addition, Buchwald-Hartwig cyclization, and loss of the acetyl group). The substrates for these reactions can be accessed in a reliable and highly selective two-step process from 2-bromoaryl bromides.

Innate C-H trifluoromethylation of heterocycles

Direct methods for the trifluoromethylation of heteroaromatic systems are in extremely high demand in nearly every sector of chemical industry. Here we report the discovery of a general procedure using a benchtop stable trifluoromethyl radical source that functions broadly on a variety of electron deficient and rich heteroaromatic systems and demonstrates high functional group tolerance. This C-H trifluoromethylation protocol is operationally simple (avoids gaseous CF(3)I), scalable, proceeds at ambient temperature, can be used directly on unprotected molecules, and is demonstrated to proceed at the innately reactive positions of the substrate. The unique and orthogonal reactivity of the trifluoromethyl radical relative to aryl radicals has also been investigated on both a complex natural product and a pharmaceutical agent. Finally, preliminary data suggest that the regioselectivity of C-H trifluoromethylation can be fine-tuned simply by judicious solvent choice.