Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

ABSTRACT

Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder ‘click’ reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by ¹²⁵I) and subsequent accumulation of an ¹¹¹In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of ¹¹¹In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using ¹¹¹In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.     

Preparation of diazenecarboxamide-carboplatin conjugates by click chemistry

An alternative, mild and highly efficient synthetic approach to platinum complexes with bioactive carrier ligands features a platinum-complex-tolerant copper(I)-catalyzed 1,3-dipolar cycloaddition. As demonstrated by the preparation of novel diazenecarboxamide-carboplatin conjugates, this approach is superior to other methodologies.     

Sequential and parallel dual labeling of nanoparticles using click chemistry

Bioorthogonal ‘click’ reactions have recently emerged as promising tools for chemistry and biological applications. By using a combination of two different ‘click’ reactions, ‘double-click’ strategies have been developed to attach multiple labels onto biomacromolecules. These strategies require multi-step modifications of the biomacromolecules that can lead to heterogeneity in the final conjugates. Herein, we report the synthesis and characterization of a set of three trifunctional linkers. The linkers having alkyne and cyclooctyne moieties that are capable of participating in sequential copper(I)-catalyzed and copper-free cycloaddition reactions with azides. We have also prepared a linker comprised of an alkyne and a 1,2,4,5-terazine moiety that allows for simultaneous cycloaddition reactions with azides and trans-cyclooctenes, respectively. These linkers can be attached to synthetic or biological macromolecules to create a platform capable of sequential or parallel ‘double-click’ labeling in biological systems. We show this potential using a generation 5 (G5) polyamidoamine (PAMAM) dendrimer in combination with the clickable linkers. The dendrimers were successfully modified with these linkers and we demonstrate both sequential and parallel ‘double-click’ labeling with fluorescent reporters. We anticipate that these linkers will have a variety of application including molecular imaging and monitoring of macromolecule interactions in biological systems.     

Direct and nondestructive verification of PNA immobilization using click chemistry

The fluorogenic 1,3-Huisgen dipolar cycloaddition reaction was used as part of a novel immobilization strategy of PNA capture probes on a microarray. By using this click chemistry, azidocoumarin-anchored PNA probes were immobilized on phenyl acetylene-modified glass slides with the simultaneous generation of the fluorescent triazolylcoumarin moiety. Since the emitting moieties are generated in the immobilization reaction itself, fluorescent signals can be used to directly monitor the integrity of immobilization in a nondestructive manner. By using this strategy, PNA microarrays were prepared and successfully employed to perform microarray-based diagnosis of selected mutations in the breast cancer susceptibility gene BRCA1.     

In situ synthesis of fluorescent membrane lipids (ceramides) using click chemistry

Ceramide analogues containing azide groups either in the polar head or in the hydrocarbon chains are non-fluorescent. When incorporated into phospholipid bilayers, they can react in situ with a non-fluorescent 1,8-naphthalimide using click chemistry giving rise to fluorescent ceramide derivatives emitting at ≈440 nm. When incorporated into giant unilamellar vesicles, two-photon excitation at 760 nm allows visualization of the ceramide-containing bilayers. This kind of method may be of general applicability in the study of model and cell membranes.     

Efficient synthesis of a fluorescent tripod detection system for pesticides by microwave-assisted click chemistry

A new tripod molecule containing an aromatic core bearing three peracetylated cyclodextrins was synthesized via a microwave-assisted Huisgen 1,3-dipolar cycloaddition and was studied by fluorescence spectroscopy. The photoluminescent properties of complexation phenomena with different pesticides were evaluated in acetonitrile. Fluorescence titrations have been performed to calculate binding constants, sensitivity factors, and limit of detection of the resulting complexes. 2D NMR experiments confirmed the inclusion of pesticide in the hydrophobic cavity of the macrocycle and validated the supramolecular association responsible for the quenching of the fluorescence.     

Bioorthogonal click chemistry to assay mu-opioid receptor palmitoylation using 15-hexadecynoic acid and immunoprecipitation

We have developed a modification of bioorthogonal click chemistry to assay the palmitoylation of cellular proteins. This assay uses 15-hexadecynoic acid (15-HDYA) as a chemical probe in combination with protein immunoprecipitation using magnetic beads in order to detect S-palmitoylation of proteins of interest. Here we demonstrate the utility of this approach for the mu-opioid receptor (MOR), a G-protein-coupled receptor (GPCR) responsible for mediating the analgesic and addictive properties of most clinically relevant opioid agonist drugs. This technique provides a rapid, non-isotopic, and efficient method to assay the palmitoylation status of a variety of cellular proteins, including most GPCRs.     

A nascent proteome study combining click chemistry with 2DE

To investigate the dynamic cellular response to a condition change, selective labeling of the nascent proteome is necessary. Here, we report a method combining click chemistry protein labeling with 2D DIGE. To test the relevance of the method, we compared nascent proteomes of actively growing bacterial cells with that of cells exposed to protein synthesis inhibitor, erythromycin. Cells were incubated with methionine analog, homopropargyl glycin, and their nascent proteome was selectively labeled with monosulfonated neutral Cy3 and Cy5 azides specially synthesized for this purpose. Following fluorescent labeling, the protein samples were mixed and subjected to standard 2D DIGE separation. The method allowed us to reveal a dramatic reduction of newly synthesized proteins upon erythromycin treatment, while the total proteome was not significantly affected. Additionally, several proteins, whose synthesis was resistant to erythromycin, were identified.     

Synthesis of a fluorescence-labeled K30 antigen repeating unit using click chemistry

An N-dansyl-labeled K30 antigen repeating unit, [4-[5-(N,N'-dimethylamino)naphthalene-1-sulfonamine]-1H-1,2,3-triazol-1-yl]hexyl beta-D-glucopyranosyluronate-(1-->3)-alpha-D-galactopyranosyl-alpha-D-mannopyranosyl-(1-->3)-beta-D-galactopyranoside, was synthesized using click chemistry, the copper(I)-catalyzed 1,3-dipolar cycloaddition reaction of an azide and an alkyne. The target compound could further facilitate the studies of interactions among K30 oligosaccharides and proteins.     

Synthesis and cytotoxic activity of novel acyclic nucleoside analogues with functionality in click chemistry

We describe synthesis of novel acyclic nucleoside analogues which are building blocks for CuAAC reaction and their activity against two types of human cancer cell lines (HeLa, KB). Three of chosen compounds show promising cytotoxic activity. Synthesis pathway starting from simple and easily accessible substrates employing DMT or TBDPS protective groups is described. Adenosine and thymidine analogues containing alkyne moiety and adenosine analogue containing azido group were synthesized. The obtained units showed ability of forming triazole motif under the CuAAC reaction conditions.     

Development of a bispecific monoclonal antibody against a gallium-67 chelate and the human melanoma-associated antigen p97 for potential use in pretargeted immunoscintigraphy

A bispecific monoclonal antibody (bsmAb) has been developed against the human melanoma-associated antigen p97 and an octahedral gallium chelate (Ga-HBED) using the hybrid hybridoma technology. As tetradomas were expected to produce a maximum of ten different molecular species of immunoglobulins, the bispecific antibody was purified from this mixture by consecutive protein A affinity and cation-exchange chromatographic techniques. Although it was established by sodium dodecyl sulphate/polyacrylamide gel electrophoresis that the heavy (H) and light (L) chains of the two parental immunoglobulins were mismatched in the bispecific antibody, results from cell enzyme-linked immunosorbent assay indicated significant dual specific binding to both the melanoma cells and⁶⁷Ga-HBED. Other in vitro techniques further confirmed that the bsmAb Bi 5-56-II-17 still retained about 30%–40% simultaneous binding capacity to both the antigens, as would have been expected in a bsmAb that has ideally matched H and L chains. Preliminary in vivo experiments using nude mice bearing the human melanoma xenografts showed that the bsmAb Bi 5-56-II-17 was able to target the radioactive gallium chelate to the tumours twice as efficiently compared to the monospecific, bivalent gallium chelate antibody.     

Probing gorge dimensions of cholinesterases by freeze-frame click chemistry

Freeze-frame click chemistry is a proven approach for design in situ of high affinity ligands from bioorthogonal, reactive building blocks and macromolecular template targets. We recently described in situ design of femtomolar reversible inhibitors of fish and mammalian acetylcholinesterases (EC 3.1.1.7; AChEs) using several different libraries of acetylene and azide building blocks. Active center gorge geometries of those AChEs are rather similar and identical triazole inhibitors were detected in situ when incubating the same building block libraries in different AChEs. Drosophila melanogaster AChE crystal structure and other insect AChE homology models differ more in their overall 3D structure than other members of the cholinesterase family. The portion of the gorge proximal to the catalytic triad and choline binding site has a approximately 50% reduction in volume, and the gorge entrance at the peripheral anionic site (PAS) is more constricted than in the fish and mammalian AChEs. In this communication we describe rationale for using purified recombinant Drosophila AChE as a template for in situ reaction of tacrine and propidium based libraries of acetylene and azide building blocks. The structures of resulting triazole inhibitors synthesized in situ are expected to differ appreciably from the fish and mammalian AChEs. While the latter AChEs exclusively promote synthesis of syn-substituted triazoles, the best Drosophila AChE triazole inhibitors were always anti-substituted. The anti-regioisomer triazoles were by about one order of magnitude better inhibitors of Drosophila than mammalian and fish AChEs. Moreover, the preferred site of acetylene+azide reaction in insect AChE and the resulting triazole ring formation shifts from near the base of the gorge to closer to its rim due to substantial differences of the gorge geometry in Drosophila AChE. Thus, in addition to synthesizing high affinity, lead inhibitors in situ, freeze-frame, click chemistry has capacity to generate species-specific AChE ligands that conform to the determinants in the gorge.     

Synthesis of a C3-symmetric (1-->6)-N-acetyl-beta-D-glucosamine octadecasaccharide using click chemistry

A C3-symmetric (1-->6)-N-acetyl-beta-D-glucosamine octadecasaccharide was convergently synthesized on the basis of a copper(I)-catalyzed 1,3-dipolar cycloaddition reaction of azide and alkyne. The target octadecasaccharide showed good antitumor activity against H22 in the preliminary mice tests.     

Rapid access to new bioconjugates of betulonic acid via click chemistry

Plant-derived pentacyclic triterpenoids of lupane and oleanane families provide a versatile structural platform for the discovery of new biologically active compounds. A number of semisynthetic derivatives of these molecules, possess high medical efficiency including antiviral (HIV-1), anticancer and immunomodulating activity. Even small structural changes in these triterpenoid derivatives were reported to lead to significant changes in their activity, making a convincing case for a systematic study of structure-activity relationships in this class of compounds.Our earlier work opened synthetic access to alkynes derived from the betulonic scaffold and enabled the development of a new family of biohybrids using Click Chemistry (CC). The computer-aided prediction of several types of biological activity were performed with program PASS (Prediction Activity Spectra of Substances. Experimental studies based on mouse models verified the SAR predictions obtained by the PASS program. The observed correlation between the anti-inflammatory and antioxidant activity indicates substantial contribution of the latter in the mechanism of anti-inflammatory effect of the triazole derivatives of betulonic acid.     

Probing replacement of pyrophosphate via click chemistry; synthesis of UDP-sugar analogues as potential glycosyl transferase inhibitors

A series of potential UDP-sugar mimics were readily synthesised by copper(I) catalysed modified Huisgen cycloaddition of the corresponding α-propargyl glycosides with 5-azido uridine in aqueous solution. None of the compounds accessed displayed significant inhibitory activity at concentrations of up to 4.5 mM in an assay against bovine milk β-1,4-galactosyltransferase.     

Synthesis and bioevaluation of radioiodinated nitroimidazole hypoxia imaging agents by one-pot click reaction

Eight radioiodinated 2-nitroimidazole derivatives for use as hypoxia imaging agents were synthesized by one-pot click reaction using four azides, two alkynes, and [¹³¹I]iodide ions and evaluated by hypoxic cellular uptake and biodistribution experiments. The results suggested that radiotracers with suitable partition coefficients (log P: −0.2–1.2) were more likely to have higher hypoxic cellular uptake. Among these eight molecules, [¹³¹I]15 ([¹³¹I]-(5-iodo-1-(2-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)ethyl)-4-((2-nitro-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazole)) had a suitable log P (0.05 ± 0.03) and contained two 2-nitroimidazole groups. The hypoxic/aerobic cellular uptake ratio of [¹³¹I]15 was 4.4 ± 0.5, and the tumor/blood (T/B) and tumor/muscle (T/M) ratios were 2.03 ± 0.45 and 6.82 ± 1.70, respectively. These results suggested that [¹³¹I]15 was a potential hypoxia imaging agent.     

Synthesis of camphecene derivatives using click chemistry methodology and study of their antiviral activity

A series of seventeen tetrazole derivatives of 1,7,7-trimethyl-[2.2.1]bicycloheptane were synthesized using click chemistry methodology and characterized by spectral data. Studies of cytotoxicity and in vitro antiviral activity against influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cells of the compounds obtained were performed. The structure-activity relationship analysis suggests that to possess virus-inhibiting activity, the compounds of this group should bear oxygen atom with a short linker (C2-C4), either as a hydroxyl group (18, 19, 29), keto-group (21) or as a part of a heterocycle (24). These compounds demonstrated low cytotoxicity along with high anti-viral activity.     

Discovery of novel free fatty acid receptor 1 agonists bearing triazole core via click chemistry

The free fatty acid receptor 1 (FFA1/GPR40) is a novel antidiabetic target based on particular mechanism in enhancing glucose-stimulated insulin secretion. Most of reported FFA1 agonists, however, have been suffered from relatively high lipophilicity and molecular weight. Aiming to develop potent agonists with improved physicochemical property, 25 compounds containing triazole scaffold and various carboxylic acid fragments were synthesized via the click chemistry. Among them, the optimal lead compound 26 with relatively low lipophicity (Log D_7.4 = 1.95) and molecular weight (Mw = 391.78) exhibited a considerable FFA1 agonistic activity (36.15%). In addition, compound 26 revealed a significant improvement in the glucose tolerance with a 21.4% and 14.2% reduction of glucose AUC_0–2h in normal ICR mice and type 2 diabetic C57BL/6 mice, respectively. All of these results demonstrated that compound 26 was considered to be a promising lead compound suitable for further optimization.     

Synthesis of xylan-graft-poly(l-lactide) copolymers via click chemistry and their thermal properties

Di-O-(6-azidohexanoyl)-xylan-graft-poly(l-lactide)s (XylC6N₃-g-PLLAs) were prepared by grafting propargyl-terminated poly(l-lactide) onto di-O-(6-azidohexanoyl)-xylan (XylC6N₃) via click chemistry. Di-O-(6-azidohexanoyl)-xylan (XylC6N₃) was prepared via two steps from xylan extracted from eucalyptus kraft pulp with aqueous sodium hydroxide solution. Propargyl-terminated poly(l-lactide)s (PLLA) with three different molecular weights were synthesized via ring-opening polymerization of l-lactide using propargyl alcohol as initiator and tin (II) octanoate (Sn(Oct)₂) as catalyst. XylC6N₃ and propargyl-terminated PLLAs were treated with N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA) and copper(I) bromide, and the graft copolymers XylC6N₃-g-PLLAs were obtained. DSC measurements revealed that the glass transition temperatures (T_g) of the copolymers decreased compared to that of XylC6N₃, suggesting that the grafted PLLA side-chains act as an internal plasticizer for xylan. TGA measurements revealed that XylC6N₃-g-PLLAs had higher decomposition temperatures than those of XylC6N₃ or PLLA, and that the decomposition temperatures of the copolymers increased with decrease in the number of PLLA side-chains grafted to the xylan main-chain.