Strain-promoted Azide-alkyne Click Research Articles

Site-Specific Conjugation of Bottlebrush Polymers to Therapeutic Protein via Bioorthogonal Chemistry.

Achieving efficient and site-specific conjugation of therapeutic protein to polymer is crucial to augment their applicability in the realms of biomedicine by improving their stability and enzymatic activity. In this study, we exploited tetrazine bioorthogonal chemistry to achieve the site-specific conjugation of bottlebrush polymers to urate oxidase (UOX), a therapeutic protein for gout treatment. An azido-functionalized zwitterionic bottlebrush polymer (N3-ZBP) using a "grafting-from" strategy involving RAFT and ATRP methods was synthesized, and a trans-cyclooctene (TCO) moiety was introduced at the polymer end through the strain-promoted azide-alkyne click (SPAAC) reaction. The subsequent coupling between TCO-incorporated bottlebrush polymer and tetrazine-labeled UOX using a fast and safe bioorthogonal reaction, inverse electron demand Diels-Alder (IEDDA), led to the formation of UOX-ZBP conjugates with a 52% yield. Importantly, the enzymatic activity of UOX remained unaffected following polymer conjugation, suggesting a minimal change in the folded structure of UOX. Moreover, UOX-ZBP conjugates exhibited enhanced proteolytic resistance and reduced antibody binding, compared to UOX-wild type. Overall, the present findings reveal an efficient and straightforward route for synthesizing protein-bottlebrush polymer conjugates without compromising the enzymatic activity while substantially reducing proteolytic degradation and antibody binding.

Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging.

A novel family of julolidine-containing fluorescent rhodols equipped with a wide variety of substituents was synthesized in a versatile two-step process. The prepared compounds were fully characterized and exhibited excellent fluorescence properties for microscopy imaging. The best candidate was conjugated to the therapeutic antibody trastuzumab through a copper-free strain-promoted azide-alkyne click reaction. The rhodol-labeled antibody was successfully applied for in vitro confocal and two-photon microscopy imaging of Her2+ cells.

Photoredox-Catalyzed Labeling of Hydroxyindoles with Chemoselectivity (PhotoCLIC) for Site-Specific Protein Bioconjugation.

We have developed a novel visible-light-catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site-specifically installed 5-hydroxytryptophan residue (5HTP) on full-length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light-emitting diodes (455/650 nm) for rapid site-specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure formed likely through a singlet oxygen-dependent modification of 5HTP. PhotoCLIC has a wide substrate scope and its compatibility with strain-promoted azide-alkyne click reaction, enables site-specific dual-labeling of a target protein.

Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging.

An asymmetric cyanine-type fluorescent dye was designed and synthesized via a versatile, multi-step process, aiming to conjugate with an Her2+ receptor specific antibody by an azide-alkyne click reaction. The aromaticity and the excitation and relaxation energetics of the fluorophore were characterized by computational methods. The synthesized dye exhibited excellent fluorescence properties for confocal microscopy, offering efficient applicability in in vitro imaging due to its merits such as a high molar absorption coefficient (36 816 M-1 cm-1), excellent brightness, optimal wavelength (627 nm), larger Stokes shift (26 nm) and appropriate photostability compared to cyanines. The conjugated cyanine-trastuzumab was constructed via an effective, metal-free, strain-promoted azide-alkyne click reaction leading to a regulated number of dyes being conjugated. This novel cyanine-labelled antibody was successfully applied for in vitro confocal imaging and flow cytometry of Her2+ tumor cells.

Homogeneity of antibody-drug conjugates critically impacts the therapeutic efficacy in brain tumors.

SUMMARYGlioblastoma multiforme (GBM) is the most aggressive and fatal disease of all brain tumor types. Most therapies rarely provide clinically meaningful outcomes in the treatment of GBM. Although antibody-drug conjugates (ADCs) are promising anticancer drugs, no ADCs have been clinically successful for GBM, primarily because of poor blood-brain barrier (BBB) penetration. Here, we report that ADC homogeneity and payload loading rate are critical parameters contributing to this discrepancy. Although both homogeneous and heterogeneous conjugates exhibit comparable in vitro potency and pharmacokinetic profiles, the former shows enhanced payload delivery to brain tumors. Our homogeneous ADCs provide improved antitumor effects and survival benefits in orthotopic brain tumor models. We also demonstrate that overly drug-loaded species in heterogeneous conjugates are particularly poor at crossing the BBB, leading to deteriorated overall brain tumor targeting. Our findings indicate the importance of homogeneous conjugation with optimal payload loading in generating effective ADCs for intractable brain tumors.

Metal- and Strain-Free Bioorthogonal Cycloaddition of o-Diones with Furan-2(3H)-one as Anionic Cycloaddend.

The development of new bioorthogonal reactions with mutual orthogonality to classic bioorthogonal reactions such as the strain-promoted azide-alkyne click reaction and the inverse-electron-demand Diels-Alder reaction is of great importance in providing chemical tools for multiplex labelling of live cells. Here we report the first anionic cycloaddend-promoted bioorthogonal cycloaddition reaction between phenanthrene-9,10-dione and furan-2(3H)-one derivatives, where the high polarity of water is exploited to stabilize the highly electron-rich anionic cycloaddend. The reaction is metal- and strain-free, which proceeds rapidly in aqueous solution and on live cells with a second-order rate constant up to 119 M-1 s-1 . The combined utilization of this reaction together with the two other widely used bioorthogonal reactions allows for mutually orthogonal labelling of three types of proteins or three groups of living cells in one batch without cross-talking. Such results highlight the great potential for multiplex labelling of different biomolecules in live cells.

Surfaces with Dual Functionality through Specific Coimmobilization of Self-Assembled Polymeric Nanostructures.

Coimmobilization of functional, nanosized assemblies broadens the possibility to engineer dually functionalized active surfaces with a nanostructured texture. Surfaces decorated with different nanoassemblies, such as micelles, polymersomes, or nanoparticles are in high demand for various applications ranging from catalysis, biosensing up to antimicrobial surfaces. Here, we present a combination of bio-orthogonal and catalyst-free strain-promoted azide-alkyne click (SPAAC) and thiol-ene reactions to simultaneously coimmobilize various nanoassemblies; we selected polymersome-polymersome and polymersome-micelle assemblies. For the first time, the immobilization method using SPAAC reaction was studied in detail to attach soft, polymeric assemblies on a solid support. Together, the SPAAC and thiol-ene reactions successfully coimmobilized two unique self-assembled structures on the surfaces. Additionally, poly(dimethylsiloxane) (PDMS)-based polymersomes were used as "ink" for direct immobilization from a PDMS-based microstamp onto a surface creating locally defined patterns. Combining immobilization reactions has the advantage to attach any kind of nanoassembly pairs, resulting in surfaces with "desired" interfacial properties. Different nanoassemblies that encapsulate multiple active compounds coimmobilized on a surface will pave the way for the development of multifunctional surfaces with controlled properties and efficiency.

Facile metabolic glycan labeling strategy for exosome tracking

BackgroundExosomes are nano-sized vesicles derived from the fusion of multivesicular bodies with the surrounding plasma membrane. Exosomes have various diagnostic and therapeutic potentials in cancer and other diseases, thus tracking exosomes is an important issue. MethodsHere, we report a facile exosome labeling strategy using a natural metabolic incorporation of an azido-sugar into the glycan, and a strain-promoted azide-alkyne click reaction. In culture, tetra-acetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz) was spontaneously incorporated into glycans within the cells and later redistributed onto their exosomes. These azido-containing exosomes were then labeled with azadibenzylcyclooctyne (ADIBO)-fluorescent dyes by a bioorthogonal click reaction. ResultsCellular uptake and the in vivo tracking of fluorescent labeled exosomes were evaluated in various cells and tumor bearing mice. Highly metastatic cancer-derived exosomes showed an increased self-homing in vitro and selective organ distribution in vivo. ConclusionOur metabolic exosome labeling strategy could be a promising tool in studying the biology and distribution of exosomes, and optimizing exosome based therapeutic approaches. General significantA facile and effective exosome labeling strategy was introduced by presenting azido moiety on the surface of exosome through metabolic glycan synthesis, and then conjugating a strain-promoted fluorescent dye.

Site-Specifically Labeled Antibody-Drug Conjugate for Simultaneous Therapy and ImmunoPET.

The conjugation of antibodies with cytotoxic drugs can alter their in vivo pharmacokinetics. As a result, the careful assessment of the in vivo behavior, and specifically the tumor-targeting properties, of antibody-drug conjugates represents a crucial step in their development. In order to facilitate this process, we have created a methodology that facilitates the dual labeling of an antibody with both a toxin and a radionuclide for positron emission tomography (PET). To minimize the impact of these modifications, this chemoenzymatic approach leverages strain-promoted azide-alkyne click chemistry to graft both cargoes to the heavy chain glycans of the immuoglobulin's Fc domain. As a proof-of-concept, a HER2-targeting trastuzumab immunoconjugate was created bearing both a monomethyl auristatin E (MMAE) toxin as well as the long-lived positron-emitting radiometal 89Zr ( t1/2 ≈ 3.3 days). Both the tumor targeting and therapeutic efficacy of the 89Zr-trastuzumab-MMAE immunoconjugate were validated in vivo using a murine model of HER2-expressing breast cancer. The site-specifically dual-labeled construct enabled the clear visualization of tumor tissue via PET imaging, producing tumoral uptake of ∼70%ID/g. Furthermore, a longitudinal therapy study revealed that the immunoconjugate exerts significant antitumor activity, leading to a >90% reduction in tumor volume over the course of 20 days.

Toward Personalized Peptide-Based Cancer Nanovaccines: A Facile and Versatile Synthetic Approach.

Personalized cancer vaccines (PCVs) are receiving attention as an avenue for cancer immunotherapy. PCVs employ immunogenic peptide epitopes capable of stimulating the immune system to destroy cancer cells with great specificity. Challenges associated with effective delivery of these peptides include poor solubility of hydrophobic sequences, rapid clearance, and poor immunogenicity, among others. The incorporation of peptides into nanoparticles has the potential to overcome these challenges, but the broad range of functionalities found in amino acids presents a challenge to conjugation due to possible interferences and lack of reaction specificity. Herein, a facile and versatile approach to generating nanosized PCVs under mild nonstringent conditions is reported. Following a simple two-step semibatch synthetic approach, amphiphilic hyperbranched polymer-peptide conjugates were prepared by the conjugation of melanoma antigen peptides, either TRP2 (hydrophobic) or MUT30 (hydrophilic), to an alkyne functionalized core via strain-promoted azide-alkyne click chemistry. Self-assembly of the amphiphiles gave spherical nanovaccines (by transmission electron microscopy) with sizes in the range of 10-30 nm (by dynamic light scattering). Fluorescently labeled nanovaccines were prepared to investigate the cellular uptake by antigen presenting cells (dendritic cells), and uptake was confirmed by flow cytometry and microscopy. The TRP2 nanovaccine was taken up the most followed by MUT30 nanoparticles and, finally, nanoparticles without peptide. The nanovaccines showed good biocompatibility against B16-F10 cells, yet the TRP2 peptide showed signs of toxicity, possibly due to its hydrophobicity. A test for immunogenicity revealed that the nanovaccines were poorly immunogenic, implying the need for an adjuvant when administered in vivo. Treatment of mice with melanoma tumors showed that in combination with adjuvant, CpG, groups with the peptide nanovaccines slowed tumor growth and improved survival (up to 24 days, TRP2) compared to the untreated group (14 days).

Efficient Homodifunctional Bimolecular Ring-Closure Method for Cyclic Polymers by Combining RAFT and Self-Accelerating Click Reaction.

An efficient metal-free homodifunctional bimolecular ring-closure method is developed for the formation of cyclic polymers by combining reversible addition-fragmentation chain transfer (RAFT) polymerization and self-accelerating click reaction. In this approach, α,ω-homodifunctional linear polymers with azide terminals are prepared by RAFT polymerization and postmodification of polymer chain end groups. By virtue of sym-dibenzo-1,5-cyclooctadiene-3,7-diyne (DBA) as small linkers, well-defined cyclic polymers are then prepared using the self-accelerating double strain-promoted azide-alkyne click (DSPAAC) reaction to ring-close the azide end-functionalized homodifunctional linear polymer precursors. Due to the self-accelerating property of DSPAAC ring-closing reaction, this novel method eliminates the requirement of equimolar amounts of telechelic polymers and small linkers in traditional bimolecular ring-closure methods. It facilitates this method to efficiently and conveniently produce varied pure cyclic polymers by employing an excess molar amount of DBA small linkers.

Using Unnatural Amino Acids to Probe the Interaction between Tarantula Toxins and Voltage Sensing Domains in KV Channels

Peptide toxins folded into an inhibitor cystine knot (ICK) are found in the venoms of a variety of organisms (spider, scorpion, snakes, etc.), and many of these target the voltage-sensing domains in voltage-activated ion channels to modulate their gating properties. Our previous studies suggest that tarantula toxins partition into the lipid membrane and bind to the S3b-S4 paddle motif within the voltage-sensing domains to allosterically inhibit Kv channels. Alanine scanning mutagenesis studies have identified candidate residues in both toxin and channel that may contribute to forming the protein-protein interface when in complex. To definitively identify protein interaction surfaces between toxin and Kv channel, we employed unnatural amino acids and Strain - Promoted Azide Alkyne Click (SPAAC) chemistry to carry out site-specific crosslinking. Azidohomoalanine (AHA), an analog of methionine, was incorporated in Kv channels expressed in Xenopus laevis oocytes through endogenous methionyl t-RNA synthetase by supplementing excess of AHA in the medium. Simultaneously, guangxitoxin-1E (GxTx-1E) was engineered with spinster cysteine residues to incorporate an azide-reactive dibenzocyclooctyne (DBCO) group using cysteine-maleimide conjugation method. Currently, we are assaying for toxin-channel crosslinks in a functional manner by analyzing the recovery of macroscopic ionic currents and kinetics of toxin dissociation using two-electrode voltage clamp (TEVC) technique.

Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides.

BackgroundCarbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques.ResultsArabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, l-fucose, and l-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions.ConclusionsOur results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands the molecular toolbox for direct glycan imaging in plants, from three to eight glycan analogs, which enables more extensive future studies of spatiotemporal glycan dynamics in a wide variety of plant tissues and species. We also show, for the first time in metabolic labeling and imaging of plant glycans, the potential of two copper-free click chemistry methods that are bio-orthogonal and lead to more uniform labeling. These improved labeling methods can be generalized and extended to already existing and future click chemistry-enabled monosaccharide analogs in Arabidopsis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0907-0) contains supplementary material, which is available to authorized users.

Multisite clickable modification of proteins using lipoic acid ligase.

Approaches that allow bioorthogonal and, in turn, site-specific chemical modification of proteins present considerable opportunities for modulating protein activity and stability. However, the development of such approaches that enable site-selective modification of proteins at multiple positions, including internal sites within a protein, has remained elusive. To overcome this void, we have developed an enzymatic approach for multisite clickable modification based on the incorporation of azide moieties in proteins using lipoic acid ligase (LplA). The ligation of azide moieties to the model protein, green fluorescent protein (GFP), at the N-terminus and two internal sites using lipoic acid ligase was shown to proceed efficiently with near-complete conversion. Modification of the ligated azide groups with poly(ethylene glycol) (PEG), α-d-mannopyranoside, and palmitic acid resulted in highly homogeneous populations of protein-polymer, protein-sugar, and protein-fatty acid conjugates. The homogeneity of the conjugates was confirmed by mass spectrometry (MALDI-TOF) and SDS-PAGE electrophoresis. In the case of PEG attachment, which involved the use of strain-promoted azide-alkyne click chemistry, the conjugation reaction resulted in highly homogeneous PEG-GFP conjugates in less than 30 min. As further demonstration of the utility of this approach, ligated GFP was also covalently immobilized on alkyne-terminated self-assembled monolayers. These results underscore the potential of this approach for, among other applications, site-specific multipoint protein PEGylation, glycosylation, fatty acid modification, and protein immobilization.

Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest—of either synthetic or biological origin—is modified at its 3′-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide–alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2′-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2′-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules.