Bioorthogonal Click Reaction Research Articles

Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications.

Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2β subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2α/CK2β interface, a bioorthogonal click reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated into CK2α. Performing the SPAAC click reaction (Strain-Promoted Azide-Alkyne Cycloaddition) by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) led to a specifically labeled human protein kinase CK2α. This site-specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermore a dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate αS1-casein towards CK2α. This labeling strategy was also applied to CK2β subunit on Escherichia coli, indicating the site-specific modifications of proteins on the bacterial cell surface when displayed by Autodisplay.

Shape-Encoded Chitosan-Polyacrylamide Hybrid Hydrogel Microparticles with Controlled Macroporous Structures via Replica Molding for Programmable Biomacromolecular Conjugation.

Polymeric hydrogel microparticle-based suspension arrays with shape-based encoding offer powerful alternatives to planar and bead-based arrays toward high throughput biosensing and medical diagnostics. We report a simple and robust micromolding technique for polyacrylamide- (PAAm-) based biopolymeric-synthetic hybrid microparticles with controlled 2D shapes containing a potent aminopolysaccharide chitosan as an efficient conjugation handle uniformly incorporated in PAAm matrix. A postfabrication conjugation approach utilizing amine-reactive chemistries on the chitosan shows stable incorporation and retained chemical reactivity of chitosan, readily tunable macroporous structures via simple addition of low content long-chain PEG porogens for improved conjugation capacity and kinetics, and one-pot biomacromolecular assembly via bioorthogonal click reactions with minimal nonspecific binding. We believe that the integrated fabrication-conjugation approach reported here could offer promising routes to programmable manufacture of hydrogel microparticle-based biomacromolecular conjugation and biofunctionalization platforms for a large range of applications.

Bioorthogonal two-component drug delivery in HER2(+) breast cancer mouse models.

The HER2 receptor is overexpressed in approximately 20% of breast cancers and is associated with tumorigenesis, metastasis, and a poor prognosis. Trastuzumab is a first-line targeted drug used against HER2(+) breast cancers; however, at least 50% of HER2(+) tumors develop resistance to trastuzumab. To treat these patients, trastuzumab-based antibody-drug conjugates (ACDs) have been developed and are currently used in the clinic. Despite their high efficacy, the long circulation half-life and non-specific binding of cytotoxic ADCs can result in systemic toxicity. In addition, standard ADCs do not provide an image-guided mode of administration. Here, we have developed a two-component, two-step, pre-targeting drug delivery system integrated with image guidance to circumvent these issues. In this strategy, HER2 receptors are pre-labeled with a functionalized trastuzumab antibody followed by the delivery of drug-loaded nanocarriers. Both components are cross-linked by multiple bioorthogonal click reactions in situ on the surface of the target cell and internalized as nanoclusters. We have explored the efficacy of this delivery strategy in HER2(+) human breast cancer models. Our therapeutic study confirms the high therapeutic efficacy of the new delivery system, with no significant toxicity.

Functionalised type-I collagen as a hydrogel building block for bio-orthogonal tissue engineering applications.

In this study, we derivatized type I collagen without altering its triple helical conformation to allow for facile hydrogel formation via the Michael addition of thiols to methacrylates without the addition of other crosslinking agents. This method provides the flexibility needed for the fabrication of injectable hydrogels or pre-fabricated implantable scaffolds, using the same components by tuning the modulus from Pa to kPa. Enzymatic degradability of the hydrogels can also be easily fine-tuned by variation of the ratio and the type of the cross-linking component. The structural morphology reveals a lamellar structure mimicking native collagen fibrils. The versatility of this material is demonstrated by its use as a pre-fabricated substrate for culturing human corneal epithelial cells and as an injectable hydrogel for 3-D encapsulation of cardiac progenitor cells.

Photodynamic therapy via FRET following bioorthogonal click reaction in cancer cells

Longer wavelength light (650-800nm) is desired to treat large tumors in photodynamic therapy (PDT). However, shorter wavelength light is needed in PDT for thin tumors, not to cause undesirable local side effects. We proposed a strategy for stepwise optical imaging and PDT using a bioorthogonal click chemistry and fluorescence resonance energy transfer (FRET). We prepared azidyl rhodamine (Rh-N3, clickable FD) and cyclooctynyl phthalocyanine [Pc-(DIBAC), clickable PS], with which, here, we demonstrate that the non-catalytic click chemistry is rapid and efficient in cancer cells and FRET from a fluorescence dye (FD) to a photosensitizer (PS) is sufficient to generate enough singlet oxygen killing cancer cells by using shorter wavelength light.

Biocompatible Supramolecular Catalytic One-Dimensional Nanofibers for Efficient Labeling of Live Cells.

Understanding complex cellular functions requires study and tracking of biomolecules such as proteins, glycans, and lipids in their natural environment. Herein, we report the first supramolecular nanocatalyst for bioorthogonal click reaction to label live cells. This biocompatible and biodegradable nanocatalyst was formed by self-assembled peptide nanofibers complexed with copper ions. The supramolecular nanocatalyst enhanced azide-alkyne cycloaddition reaction rate under physiological conditions and was shown to be useful for efficient bioorthogonal labeling of live cells.

Controlling Localization and Excretion of Nanoparticles by Click Modification of the Surface Chemical Structures inside Living Cells.

Surface modification of silica nanoparticles (SiNPs) inside living cells was conducted by using the copper-free bioorthogonal click reaction to evaluate the effects of surface chemical structures on dynamic behavior and excretion of SiNPs. The results demonstrated the spatial translocation of intracellular SiNPs to the plasma membrane upon surface modification in situ with phospholipids. Moreover, the extent and kinetics of excretion of the SiNPs from inside the cell were enhanced by modification.

Versatile click alginate hydrogels crosslinked via tetrazine–norbornene chemistry

Alginate hydrogels are well-characterized, biologically inert materials that are used in many biomedical applications for the delivery of drugs, proteins, and cells. Unfortunately, canonical covalently crosslinked alginate hydrogels are formed using chemical strategies that can be biologically harmful due to their lack of chemoselectivity. In this work we introduce tetrazine and norbornene groups to alginate polymer chains and subsequently form covalently crosslinked click alginate hydrogels capable of encapsulating cells without damaging them. The rapid, bioorthogonal, and specific click reaction is irreversible and allows for easy incorporation of cells with high post-encapsulation viability. The swelling and mechanical properties of the click alginate hydrogel can be tuned via the total polymer concentration and the stoichiometric ratio of the complementary click functional groups. The click alginate hydrogel can be modified after gelation to display cell adhesion peptides for 2D cell culture using thiol-ene chemistry. Furthermore, click alginate hydrogels are minimally inflammatory, maintain structural integrity over several months, and reject cell infiltration when injected subcutaneously in mice. Click alginate hydrogels combine the numerous benefits of alginate hydrogels with powerful bioorthogonal click chemistry for use in tissue engineering applications involving the stable encapsulation or delivery of cells or bioactive molecules.

Quantum Chemical Calculations and Experimental Validation of the Photoclick Reaction for Fluorescent Labeling of the 5' cap of Eukaryotic mRNAs.

Bioorthogonal click reactions are powerful tools to specifically label biomolecules in living cells. Considerable progress has been made in site-specific labeling of proteins and glycans in complex biological systems, but equivalent methods for mRNAs are rare. We present a chemo-enzymatic approach to label the 5’ cap of eukaryotic mRNAs using a bioorthogonal photoclick reaction. Herein, the N7-methylated guanosine of the 5’ cap is enzymatically equipped with an allyl group using a variant of the trimethylguanosine synthase 2 from Giardia lamblia (GlaTgs2). To elucidate whether the resulting N2-modified 5’ cap is a suitable dipolarophile for photoclick reactions, we used Kohn–Sham density functional theory (KS-DFT) and calculated the HOMO and LUMO energies of this molecule and nitrile imines. Our in silico studies suggested that combining enzymatic allylation of the cap with subsequent labeling in a photoclick reaction was feasible. This could be experimentally validated. Our approach generates a turn-on fluorophore site-specifically at the 5’ cap and therefore presents an important step towards labeling of eukaryotic mRNAs in a bioorthogonal manner.

Bridging cells of three colors with two bio-orthogonal click reactions.

Cell-cell interactions play a crucial role in the development and function of multicellular organisms. To study cell-cell interactions in vitro, it is a big challenge for researchers to artificially build up cell junctions to bridge different types of cells for this purpose. Herein, by employing two orthogonal click reactions, we rationally designed four click reagents Mal-CBT, Mal-Cys, Mal-Alkyne, and Mal-N3 and successfully applied them to bridge cells of three colors. Orthogonality between these two click reactions was validated in solution and characterized with HPLC and ESI-MS analyses. After modifications of fluorescent protein-expressing prokaryotic Escherichia coli (E. coli) cells (or eukaryotic HEK 293T cells) of three colors with the reagents Mal-Cys, Mal-CBT and Mal-Alkyne, or Mal-N3 , the cells were sequentially bridged. The HEK 293T cells showed a higher efficiency of cell bridging than the E. coli cells. Finally, using optical tweezers, we quantitatively measured the bridging probability between Mal-Cys-modified and Mal-CBT-modified HEK 293 cells, as well as the rupture force between two bridged cells. We found that the CBT-Cys click reaction markedly improved the efficiency of cell bridging and the rupture force between two bridged cells was measured to be 153.8 pN at a force-loading rate of 49 pN s-1. Our results demonstrate that it is possible to use two (or n) orthogonal click reactions to bridge three (or n + 1) types of cells. Taking the biological importance of cell junctions into consideration, we anticipate that our method of bridging three types of cells with two bio-orthogonal click reactions will be a useful tool for biologists to study cell-cell interactions with more convenience and efficiency.

An injectable and fast-degradable poly(ethylene glycol) hydrogel fabricated via bioorthogonal strain-promoted azide-alkyne cycloaddition click chemistry.

Biocompatible and degradable injectable materials prepared via bioorthogonal reactions are highly promising for biomedical applications because they can be formed in situ and administered in a minimally invasive way. In this work, a PEG-based injectable hydrogel was fabricated via a copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry. Azide and cyclooctyne moieties on the PEG backbones underwent a rapid click reaction to trigger the formation of the hydrogel within several minutes. Resulting from the introduction of ester groups into the cross-linked network, the hydrogel presented pH-dependent hydrolysis and biological fast degradability. Good biocompatibility of the hydrogel was verified by in vitro cytotoxicity assay and in vivo studies. The hydrogel formed in situ after subcutaneously injecting the gel precursors into Kungming (KM) mice. The implanted hydrogel caused a mild inflammatory response in vivo, and the surrounding tissues fully recovered a week after the injection. The injectable and fast-degradable hydrogel fabricated by the bioorthogonal click reaction may be useful as biomaterials such as embolic agents for interventional therapy.

Metabolic Glyco-Engineering in Eukaryotic Cells and Selected Applications.

By metabolic glyco-engineering cellular glycoconjugates are modified through the incorporation of synthetic monosaccharides which are usually analogues of naturally present sugars. In order to get incorporated, the monosaccharides need to enter the cytoplasm and to be substrates for the enzymes necessary for their transformation into activated sugars, most often nucleotide sugars. These have to be substrates for glycosyltransferases which finally catalyze their incorporation into glycans. Such pathways are difficult to reconstitute in vitro and therefore new monosaccharide analogues have to be tested in tissue culture for their suitability in metabolic glyco-engineering. For this, glycosylation mutants are the most appropriate since they are unable to synthesize specific glycans but through the introduction of the monosaccharide analogues they may express some glycans at the cell surface with the unnatural sugar incorporated. The presence of those glycans can be easily and quantitatively detected by lectin binding or by chemical methods identifying specific sugars. Monosaccharide analogues can also block the pathways leading to sugar incorporation, thus inhibiting the synthesis of glycan structures which is also easily detectable at the cell surface by lectin labeling. The most useful and most frequently employed application of metabolic glyco-engineering is the introduction of reactive groups which can undergo bio-orthogonal click reactions for the efficient labeling of glycans at the surface of live cells.

Well-defined cyclopropenone-masked dibenzocyclooctyne functionalized polymers from atom transfer radical polymerization

Two functional atom transfer radical polymerization (ATRP) initiators (I-2 and I-3) were developed bearing a cyclopropenone-masked dibenzocyclooctyne group. ATRP was then explored on three main kinds of monomers for radical polymerization including acrylates, styrenics, and methacrylates based on these novel initiators. By a standard ATRP protocol, the polymerization behavior demonstrated the living characteristics for all three cases and the corresponding well-defined cyclopropenone-masked dibenzocyclooctyne end or middle functionalized polymers were produced conveniently. Since UV irradiation of the cyclopropenone-masked dibenzocyclooctynes could quantitatively release the dibenzocyclooctynes widely used in strain promoted azide-alkyne cycloaddition bioorthogonal click reaction, these novel ATRP initiators and the resultant well-defined polymers should play an important role in the preparation of topological polymers and bio-synthetic polymer conjugates and many other related fields.

Abstract 4572: Bioorthogonal, two-component drug delivery in HER2(+) breast cancer mouse models

Abstract The HER2 receptors are overexpressed in ∼25% of breast cancer patients and characterized by poor internalization capability. The humanized monoclonal antibody, trastuzumab (Tz), is used as a first-line treatment for HER2(+) breast cancers. However, approximately 50% of patients with HER2(+) disease do not benefit from trastuzumab or the disease becomes refractory to the drug. We have demonstrated a target-specific intracellular delivery of therapeutics using new two-step/two-component drug delivery system driven by click chemistry that results in the in situ complexation and rapid internalization. Two delivery strategies based on azide (Az)/dibenzylcyclooctyne (DBCO), (Click-1) and trans-cyclooctene (TCO)/tertrazine (Tt), (Click-2) bioorthogonal click reactions were tested in human HER2(+) breast cancer mouse models. Trastuzumab was functionalized with Az or TCO groups and labeled with NIR CF-680 dye, to prepare Tz(Az/TCO)a(CF-680)b as the pretargeting component. Albumin substituted with paclitaxel (Px), functionalized with DBCO or Tt, and labeled with NIR CF-750 dye to prepare Alb(DBCO/Tt)x(Px)y(CF-750)z was used as the second delivery component. BT-474 HER2(+) breast cancer models were grown in athymic nude mice. Five groups of mice (a) untreated/PBS (b) first-component without Az, control for Click-1 (c) treated by Click-1 (d) first-component without TCO, control for Click-2, and (e) treated by Click-2 were used for the study. On day 0 mice were imaged with in vivo Xenogen optical imaging system and injected with the first pretargeting component followed by the second drug delivery component after 6h. Similarly, the second dose was administered on day 7. Mice were imaged at days 0 and 7 after administration of therapy to confirm the accumulation of components in tumors. Tumor volumes were measured every forth day up to day 20. After 21 days mice were sacrificed and tumors were extracted and analyzed by H&E staining for possible necrosis. Accumulation of the pretargeting and delivery components were observed in mice treated under Click-1 and Click-2 conditions compared to the corresponding controls and untreated mice. However, the accumulation was significantly higher in Click-2 group in comparison to all other groups. In addition, both pretargeting and delivery components were retained in the tumor for much longer time in Click-2 group than in other groups. We observed a tumor shrinking and significant decrease of tumor growth rate in Click-2 group. The results were confirmed by H&E immunohistochemistry analysis showing significant necrotic area in the Click-2 treated groups. Citation Format: Sudath Hapuarachchige, Wenlian Zhu, Yoshinori Kato, Dmitri Artemov. Bioorthogonal, two-component drug delivery in HER2(+) breast cancer mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4572. doi:10.1158/1538-7445.AM2014-4572

Rapidly polymerizing injectable click hydrogel therapy to delay bone growth in a murine re-synostosis model

Craniosynostosis is the premature fusion of cranial sutures, which can result in progressive cranial deformations, increased intracranial pressure, and restricted brain growth. Most cases of craniosynostosis require surgical reconstruction of the cranial vault with the goal of increasing the intracranial volume and correcting the craniofacial deformities. However, patients often experience rapid post-operative bone regrowth, known as re-synostosis, which necessitates additional surgical intervention. Bone morphogenetic protein (BMP) inhibitors have tremendous potential to treat re-synostosis, but the realization of a clinically viable inhibitor-based therapeutic requires the development of a delivery vehicle that can localize the release to the site of administration. Here, we present an in situ rapidly crosslinking injectable hydrogel that has the properties necessary to encapsulate co-administered proteins and demonstrate that the delivery of rmGremlin1 via our hydrogel system delays bone regrowth in a weanling mouse model of re-synostosis. Our hydrogel is composed of two mutually reactive poly(ethylene glycol) macromolecules, which when mixed crosslink via a bio-orthogonal Cu free click reaction. Hydrogels containing Gremlin caused a dose dependent inhibition of bone regrowth. In addition to craniofacial applications, our injectable click hydrogel has the potential to provide customizable protein, small molecule, and cell delivery to any site accessible via needle or catheter.

Trans-cyclooctene tag with improved properties for tumor pretargeting with the diels-alder reaction.

Radioimmunotherapy (RIT) of solid tumors is hampered by low tumor-to-nontumor (T/NT) ratios of the radiolabeled monoclonal antibodies resulting in low tumor doses in patients. Pretargeting technologies can improve the effectiveness of RIT in cancer therapy by increasing this ratio. We showed that a pretargeting strategy employing in vivo chemistry in combination with clearing agents, proceeds efficiently in tumor-bearing mice resulting in high T/NT ratios. A dosimetry study indicated that the chemical pretargeting technology, which centered on the bioorthogonal Diels-Alder click reaction between a radiolabeled tetrazine probe and a trans-cyclooctene-oxymethylbenzamide-tagged CC49 antibody (CC49-TCO(1)), can match the performance of clinically validated high-affinity biological pretargeting approaches in mice ( Rossin J Nucl Med. 2013 , 54 , 1989 - 1995 ). Nevertheless, the increased protein surface hydrophobicity of CC49-TCO(1) led to a relatively rapid blood clearance and concomitant reduced tumor uptake compared to native CC49 antibody. Here, we present the in vivo evaluation of a TCO-oxymethylacetamide-tagged CC49 antibody (CC49-TCO(2)), which is highly reactive toward tetrazines and less hydrophobic than CC49-TCO(1). CC49-TCO(2) was administered to healthy mice to determine its blood clearance and the in vivo stability of the TCO. Next, pretargeting biodistribution and SPECT studies with CC49-TCO(2), tetrazine-functionalized clearing agent, and radiolabeled tetrazine were carried out in nude mice bearing colon carcinoma xenografts (LS174T). CC49-TCO(2) had an increased circulation half-life, a 1.5-fold higher tumor uptake, and a 2.6-fold improved in vivo TCO stability compared to the more hydrophobic TCO-benzamide-CC49. As a consequence, and despite the 2-fold lower reactivity of CC49-TCO(2) toward tetrazines compared with CC49-TCO(1), administration of radiolabeled tetrazine afforded a significantly increased tumor accumulation and improved T/NT ratios in mice pretargeted with CC49-TCO(2). In conclusion, the TCO-acetamide derivative represents a large improvement in in vivo Diels-Alder pretargeting, possibly enabling application in larger animals and eventually humans.

Reprogramming Nonribosomal Peptide Synthetases for “Clickable” Amino Acids

AbstractNonribosomal peptide synthetases (NRPSs) are multifunctional enzymes that produce a wide array of bioactive peptides. Here we show that a single tryptophan‐to‐serine mutation in phenylalanine‐specific NRPS adenylation domains enables the efficient activation of non‐natural aromatic amino acids functionalized with azide and alkyne groups. The resulting 105‐fold switch in substrate specificity was achieved without appreciable loss of catalytic efficiency. Moreover, the effective communication of the modified A domains with downstream modules in dipeptide synthetases permitted incorporation of O‐propargyl‐L‐tyrosine into diketopiperazines both in vitro and in vivo, even in the presence of competing phenylalanine. Because azides and alkynes readily undergo bioorthogonal click reactions, reprogramming NRPSs to accept non‐natural amino acids that contain these groups provides a potentially powerful means of isolating, labeling, and modifying biologically active peptides.

Reprogramming nonribosomal peptide synthetases for "clickable" amino acids.

Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes that produce a wide array of bioactive peptides. Here we show that a single tryptophan-to-serine mutation in phenylalanine-specific NRPS adenylation domains enables the efficient activation of non-natural aromatic amino acids functionalized with azide and alkyne groups. The resulting 10(5)-fold switch in substrate specificity was achieved without appreciable loss of catalytic efficiency. Moreover, the effective communication of the modified A domains with downstream modules in dipeptide synthetases permitted incorporation of O-propargyl-L-tyrosine into diketopiperazines both in vitro and in vivo, even in the presence of competing phenylalanine. Because azides and alkynes readily undergo bioorthogonal click reactions, reprogramming NRPSs to accept non-natural amino acids that contain these groups provides a potentially powerful means of isolating, labeling, and modifying biologically active peptides.

Bioorthogonal, two-component delivery systems based on antibody and drug-loaded nanocarriers for enhanced internalization of nanotherapeutics

Nanocarriers play an important role in targeted cancer chemotherapy. The optimal nanocarrier delivery system should provide efficient and highly specific recognition of the target cells and rapid internalization of the therapeutic cargo to reduce systemic toxicity as well as to increase the cytotoxicity to cancer cells. To this end, we developed a two-step, two-component targeted delivery system based on antibody and drug-loaded nanocarrier that uses bioorthogonal click reactions for specific internalization of nanotherapeutics. The pretargeting component, anti-HER2 humanized monoclonal antibody, trastuzumab, functionalized with azide groups labels cancer cells that overexpress HER2 surface receptors. The drug carrier component, dibenzylcyclooctyne substituted albumin conjugated with paclitaxel, reacts specifically with the pretargeting component. These two components form cross-linked clusters on the cell surface, which facilitates the internalization of the complex. This strategy demonstrated substantial cellular internalization of clusters consisted of HER2 receptors, modified trastuzumab and paclitaxel-loaded albumin nanocarriers, and subsequent significant cytotoxicity in HER2-positive BT-474 breast cancer cells. Our results show high efficacy of this strategy for targeted nanotherapeutics. We foresee to broaden the applications of this strategy using agents such as radionuclides, toxins, and interfering RNA.

Facile and efficient Lewis acid catalyzed synthesis of an asymmetric tetrazine useful for bio-orthogonal click chemistry applications.

Bio-orthogonal tetrazine click reactions have recently attracted significant interest for applications spanning biological imaging, cancer targeting, and biomaterials science. Here, we report a simple and efficient two-step scheme for the synthesis of an asymmetric tetrazine molecule containing a carboxylic acid handle for subsequent macromolecular conjugation. Yields as high as 75% were achieved using as little as 0.005 equivalents of nickel triflate catalyst, which is a significant improvement over previous methodologies.