Strain-promoted Azide-alkyne Click Chemistry Research Articles

Self-immolative Polymer Hydrogels viaIn SituGelation.

Hydrogels are of interest for a wide range of applications. The ability to control when the hydrogel degrades can provide beneficial properties such as controlled degradation in the environment or the stimulated release of drugs or cells. Self-immolative polymers are a class of degradable polymers that undergo complete end-to-end depolymerization upon the application of a stimulus. They have been explored for hydrogel development, but the ability to prepare and selectively degrade self-immolative hydrogels under neutral aqueous conditions has so far been limited. We describe here the preparation of water-soluble polyglyoxyamides with cross-linkable pendent azides and their cross-linking to form hydrogels with 4-arm poly(ethylene glycol)s having unstrained and strained alkynes using copper-assisted and strain-promoted azide-alkyne click chemistry respectively. The influence of pendent azide density and solution polymer content on the resulting hydrogels was evaluated. A polyglyoxylamide with a 70:30 ratio of pendent hydroxyl:azide successfully provided hydrogels with compressive moduli ranging from 1.3 - 6.3 kPa under copper-free conditions at 10 - 20% (w/w) of polymer in phosphate-buffered saline. Selective depolymerization and degradation of the hydrogels upon irradiation with light was demonstrated, resulting in reductions in the compressive moduli and the release of depolymerization products that were detected by NMR spectroscopy.

Surface modification for site-directed covalent attachment of molecules via strain-promoted azide-alkyne click-chemistry reaction and photolithography

Copper-free “click” chemistry on appropriately modified surfaces and photolithography were explored for site-directed immobilization of biomolecules. The surfaces were modified either with self-assembled monolayers of silanes or an epoxy resin and then used as they were or further modified through adsorption of rabbit gamma-globulins to increase their reactive amine-content. All surfaces were then reacted with a succinimidyl ester cyclooctyne derivative and the efficiency of each modification approach was determined through click reaction with an azide derivative of fluorescein. It was found that the highest fluorescence signal was provided by the surface that had been modified with the epoxy resin and then coated with rabbit gamma-globulins. Surfaces prepared following the different modification procedures have been analyzed with ToF-SIMS to gain insight into the chemical changes of the surfaces after each step of the modification procedure. Analysis of intensities of characteristic ions signals confirmed the successful outcome of click reaction following the proposed surface modification approach. In addition, the ability to define through photolithography areas for site-directed immobilization of biomolecules onto surfaces modified with the succinimidyl ester cyclooctyne derivative was demonstrated through the creation of two molecules patterns.

Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs.

Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of bi- and multivalent nanobody constructs with oriented assembly by Cu-free strain promoted azide-alkyne click chemistry (SPAAC). We used sortase A for ligation of click chemistry functional groups site-specifically to the C-terminus of nanobodies before creating C-to-C-terminal nanobody fusions and 4-arm polyethylene glycol (PEG) tetrameric nanobody constructs. We demonstrated the viability of this approach by generating constructs with the SARS-CoV-2 neutralizing nanobody Ty1. We compared the ability of the different constructs to neutralize SARS-CoV-2 pseudotyped virus and infectious virus in neutralization assays. The generated dimers neutralized the virus similarly to a nanobody-Fc fusion variant, while a 4-arm PEG based tetrameric Ty1 construct dramatically enhanced neutralization of SARS-CoV-2, with an IC50 in the low picomolar range.

A microfluidic approach for sequential assembly of siRNA polyplexes with a defined structure-activity relationship

Therapeutic nucleic acids provide versatile treatment options for hereditary or acquired diseases. Ionic complexes with basic polymers are frequently used to facilitate nucleic acid’s transport to intracellular target sites. Usually, these polyplexes are prepared manually by mixing two components: polyanionic nucleic acids and polycations. However, parameters such as internal structure, size, polydispersity and surface charge of the complexes sensitively affect pharmaceutical efficiency. Hence a controlled assembly is of paramount importance in order to ensure high product quality. In the current study, we present a microfluidic platform for controlled, sequential formulation of polyplexes. We use oligo-amidoamines (termed “oligomers”) with precise molecular weight and defined structure due to their solid phase supported synthesis. The assembly of the polyplexes was performed in a microfluidic chip in two steps employing a design of two successive Y junctions: first, siRNA and core oligomers were assembled into core polyplexes. These core oligomers possess compacting, stabilizing, and endosomal escape mediating motifs. Second, new functional motifs were mixed to the core particles and integrated into the core polyplex. The iterative assembly formed multi-component polyplexes in a highly controlled manner and enabled us to investigate structure-function relationships. We chose nanoparticle shielding polyethylene glycol (PEG) and cell targeting folic acid (termed “PEG-ligands”) as functional components. The PEG-ligands were coupled to lipid anchor oligomers via strain promoted azide—alkyne click chemistry. The lipid anchors feature four cholanic acids for inserting various PEG-ligands into the core polyplex by non-covalent hydrophobic interactions. These core—lipid anchor—PEG-ligand polyplexes containing folate as cell binding ligand were used to determine the optimal PEG-ligand length for transfecting folate receptor-expressing KB cells in vitro. We found that polyplexes with 20 mol % PEG-ligands (relative to ncore oligomer) showed optimal siRNA mediated gene knock-down when containing defined PEG domains of in sum 24 and 36 ethylene oxide repetitions, 12 EOs each from the lipid anchor and 12 or 24 EOs from the PEG-ligand, respectively. These results confirm that transfection efficiency depends on the linker length and stoichiometry and are consistent with previous findings using core—PEG-ligand polyplexes formed by click modification of azide-containing core polyplexes with aforementioned PEG-ligands. Hence, successive microfluidic assembly might be a potentially powerful route to create defined multi-component polyplexes with reduced batch-to-batch variability.

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).

Covalent immobilisation of magnetic nanoparticles on surfaces via strain-promoted azide–alkyne click chemistry

We report a new family of clickable cyclooctynyl magnetic nanoparticles suitable for bioorthogonal click chemistry applications.

Pretargeted PET Imaging Using a Site-Specifically Labeled Immunoconjugate.

In recent years, both site-specific bioconjugation techniques and bioorthogonal pretargeting strategies have emerged as exciting technologies with the potential to improve the safety and efficacy of antibody-based nuclear imaging. In the work at hand, we have combined these two approaches to create a pretargeted PET imaging strategy based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between a (64)Cu-labeled tetrazine radioligand ((64)Cu-Tz-SarAr) and a site-specifically modified huA33-trans-cyclooctene immunoconjugate ((ss)huA33-PEG12-TCO). A bioconjugation strategy that harnesses enzymatic transformations and strain-promoted azide-alkyne click chemistry was used to site-specifically append PEGylated TCO moieties to the heavy chain glycans of the colorectal cancer-targeting huA33 antibody. Preclinical in vivo validation studies were performed in athymic nude mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts. To this end, mice were administered (ss)huA33-PEG12-TCO via tail vein injection and-following accumulation intervals of 24 or 48 h-(64)Cu-Tz-SarAr. PET imaging and biodistribution studies reveal that this strategy clearly delineates tumor tissue as early as 1 h post-injection (6.7 ± 1.7%ID/g at 1 h p.i.), producing images with excellent contrast and high tumor-to-background activity concentration ratios (tumor:muscle = 21.5 ± 5.6 at 24 h p.i.). Furthermore, dosimetric calculations illustrate that this pretargeting approach produces only a fraction of the overall effective dose (0.0214 mSv/MBq; 0.079 rem/mCi) of directly labeled radioimmunoconjugates. Ultimately, this method effectively facilitates the high contrast pretargeted PET imaging of colorectal carcinoma using a site-specifically modified immunoconjugate.

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.

Membranotropic Peptide-Functionalized Poly(lactide)-graft-poly(ethylene glycol) Brush Copolymers for Intracellular Delivery

A biodegradable delivery scaffold based on poly(lactide)-graft-poly(ethylene glycol) (PLA-g-PEG) is introduced and tested in vitro. The use of a functional ring-opening polymerization initiator (containing a masked aldehyde) on an azido PEG-lactide monomer combines two known methodologies to afford functionalized PLA. The resulting copolymer features two compatible functional groups and decreased hydrophobicity due to the PEG. The functional groups are capable of performing high-yielding orthogonal postpolymerization reactions, namely strain-promoted azide–alkyne click chemistry and reductive amination. PLA-g-PEG was functionalized with a fluorescent dye (7-nitrobenzoxadiazole, NBD) and a cell internalization peptide, gH625. The resulting delivery vehicle was investigated for cell uptake with HeLa cells, showing that the gH625 conjugation exhibited enhanced cellular uptake and localization in close proximity to the nuclei. The presented methodology is a new approach toward targeted delivery.