Strain-promoted Alkyne-nitrone Cycloaddition Research Articles

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The back cover picture shows a novel bioorthogonal‐activated fluorescent turn‐on probe based on strain‐promoted alkyne‐nitrone cycloaddition (SPANC). The spiro rearrangement products originating from nitrone‐alkyne cycloadducts exhibited hundreds‐fold fluorescence enhancement, large Stokes shift and high fluorescence quantum yield. More importantly, this strategy was successfully applied for labeling proteins in vitro as well as mitochondria in living cells with high signal‐to‐noise ratio under catalyst‐free and no‐wash conditions. More details are given in the article by Tian et al. on page 209—214. image

A Bioorthogonal‐Activated Fluorescence Turn‐On Probe Based on Nitrone‐Modified 1,8‐Naphthalimide for Live‐Cell Imaging

Comprehensive SummaryA nitrone‐modified 1,8‐naphthalimide was designed as a novel bioorthogonal‐activated turn‐on probe based on strain‐promoted alkyne‐nitrone cycloaddition (SPANC). The bioorthogonal cycloadducts were subsequently transformed into fluorescent rearrangement products by photo‐acceleration, which exhibited significant fluorescence enhancement, large stokes shift, and high fluorescence quantum yield. DFT calculations were performed to elucidate the fluorescence OFF‐ON mechanism. This fluorogenic strategy was successfully applied to labeling of proteins and visualizing mitochondria in live cells in real time.

Highly Electron-Deficient Pyridinium-Nitrones for Rapid and Tunable Inverse-Electron-Demand Strain-Promoted Alkyne-Nitrone Cycloaddition.

Highly accelerated inverse-electron-demand strain-promoted alkyne-nitrone cycloaddition (IED SPANC) between a stable cyclooctyne (bicyclo[6.1.0]nonyne (BCN)) and nitrones delocalized into a Cα-pyridinium functionality is reported, with the most electron-deficient "pyridinium-nitrone" displaying among the most rapid cycloadditions to BCN that is currently reported. Density functional theory (DFT) and X-ray crystallography are explored to rationalize the effects of N- and Cα-substituent modifications at the nitrone on IED SPANC reaction kinetics and the overall rapid reactivity of pyridinium-delocalized nitrones.

Dual-Bioorthogonal Molecular Tool: "Click-to-Release" and "Double-Click" Reactivity on Small Molecules and Material Surfaces.

The development of reactive moieties that enable molecular control of bond-forming and bond-breaking reactions within complex media is highly important in materials and biomaterials research as it provides opportunities to carefully manipulate small molecules and material surfaces in a reliable manner. Despite recent advances in the realization of new ligation strategies and "click-and-release" systems, there has been little development of multifunctional moieties that feature a broad range of chemical capabilities. To address this challenge, we designed a molecular tool that can utilize four well-defined bioorthogonal chemistries interchangeably for the attachment, replacement, and release of molecules within a system: the Staudinger-Bertozzi ligation (SBL), perfluoroaryl azide Staudinger reaction (PFAA-SR), strain-promoted alkyne-azide cycloaddition (SPAAC), and strain-promoted alkyne-nitrone cycloaddition (SPANC). We demonstrate "click-to-release" and "double-click" reactivity on small molecules and gold nanoparticles (AuNPs) as a model material substrate. As a proof of concept for material derivatization, we employed 5 nm AuNPs-functionalized with a Rhodamine B derivative and biotin through the double-click strategy-and showed their potential as a pretargeted delivery nanocarrier. This multifunctional molecular tool enables the design and production of molecular and material systems with unique, modular, and tunable dynamic properties that can be altered under mild and bioorthogonal conditions.

Membrane-specific spin trap, 5-dodecylcarbamoyl-5-N-dodecylacetamide-1-pyroline-N-oxide (diC12PO): theoretical, bioorthogonal fluorescence imaging and EPR studies.

Membranous organelles are major endogenous sources of reactive oxygen and nitrogen species. When present at high levels, these species can cause macromolecular damage and disease. To better detect and scavenge free radical forms of the reactive species at their sources, we investigated whether nitrone spin traps could be selectively targeted to intracellular membranes using a bioorthogonal imaging approach. Electron paramagnetic resonance imaging demonstrated that the novel cyclic nitrone 5-dodecylcarbamoyl-5-N-dodecylacetamide-1-pyroline-N-oxide (diC12PO) could be used to target the nitrone moiety to liposomes composed of phosphatidyl choline. To test localization with authentic membranes in living cells, fluorophores were introduced via strain-promoted alkyne-nitrone cycloaddition (SPANC). Two fluorophore-conjugated alkynes were investigated: hexynamide-fluoresceine (HYA-FL) and dibenzylcyclooctyne-PEG4-5/6-sulforhodamine B (DBCO-Rhod). Computational and mass spectrometry experiments confirmed the cycloadduct formation of DBCO-Rhod (but not HYA-FL) with diC12PO in cell-free solution. Confocal microscopy of bovine aortic endothelial cells treated sequentially with diC12PO and DBCO-Rhod demonstrated clear localization of fluorescence with intracellular membranes. These results indicate that targeting of nitrone spin traps to cellular membranes is feasible, and that a bioorthogonal approach can aid the interrogation of their intracellular compartmentalization properties.

"Shine & Click" Photo-Induced Interfacial Unmasking of Strained Alkynes on Small Water-Soluble Gold Nanoparticles.

In this study, we report the design, synthesis, and characterization of small 3 nm water soluble gold nanoparticles (AuNPs) that feature cyclopropenone-masked strained alkyne moieties capable of undergoing interfacial strain-promoted cycloaddition (i-SPAAC) with azides after exposure to UV-A light. A strained alkyne precursor was incorporated onto AuNPs by direct ligand exchange of a thiol-modified cyclopropenone-masked dibenzocyclooctyne (photoDIBO) ligand. These photoDIBO-AuNPs were characterized by 1 H NMR, IR, and UV/Vis spectroscopy, as well as transmission electron microscopy (TEM) and thermogravimetric analysis (TGA), and the extent of modification was quantified. Upon irradiation with UV-A light, photoDIBO-AuNPs underwent efficient and quantitative regeneration of the parent strained alkyne by photochemical decarbonylation to afford DIBO-derivatized AuNPs. DIBO-AuNPs were found to react cleanly and rapidly (k=5.3×10-2  m-1  s-1 ) by an interfacial strain-promoted alkyne-azide cycloadditon (i-SPAAC) with benzyl azide, which served as a simple model system. Furthermore, DIBO-AuNPs were reacted with various azides and a nitrone (interfacial strain-promoted alkyne-nitrone cycloaddition, i-SPANC) to showcase the generality of this approach for the facile modification of AuNP surfaces and their properties. The cyclopropenone-based photo-triggered click chemistry at the interface of water-soluble AuNPs offers exciting opportunities for the atom-by-atom control and assembly of functional materials for applications in materials and biomaterials science as well as in chemical biology.

Structural Manipulation of Ruthenium(II) Polypyridine Nitrone Complexes to Generate Phosphorogenic Bioorthogonal Reagents for Selective Cellular Labeling

We report a new class of ruthenium(II) polypyridine complexes functionalized with a nitrone group as phosphorogenic bioorthogonal probes. These complexes were very weakly emissive owing to rapid C=N isomerization of the nitrone moiety, but exhibited significant emission enhancement upon strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction with bicyclo[6.1.0]nonyne (BCN)-modified substrates. The modification of nitrone with a dicationic ruthenium(II) polypyridine unit at the α-C-position and a phenyl ring at the N-position led to remarkably accelerated reaction kinetics, which are substantially greater (up to ≈278 fold) than those of other acyclic nitrone-BCN systems. Interestingly, the complexes achieved specific cell membrane/cytosol staining upon specific labeling of an exogenous substrate, BCN-modified decane (BCN-C10), in live cells. Importantly, the in situ generation of the more lipophilic isoxazoline adduct in the cytoplasm resulted in increased cytotoxicity, highlighting a novel approach to apply the SPANC labeling technique in drug activation.

Dual Strain-Promoted Alkyne–Nitrone Cycloadditions for Simultaneous Labeling of Bacterial Peptidoglycans

Bioorthogonal chemistry has been applied to study a multitude of biological processes in complex environments through incorporation and detection of small functional groups. However, few reactions are known to be compatible with each other to allow for studies of more than one biomolecule simultaneously. Here we describe a dual labeling method wherein two stereoelectronically contrasting nitrone tags are incorporated into bacteria peptidoglycan and detected via strain-promoted alkyne-nitrone cycloaddition (SPANC) simultaneously. Furthermore, we show orthogonality with the azide functionality broadening the potential for simultaneous biomolecular target labeling in less accommodating metabolic pathways. We also demonstrate the simultaneous labeling of two different food-associated bacteria, L. innocua (a model for the food-born pathogen L. monocytogenes) and L. lactis (a fermentation bacterium). The ability to monitor multiple processes and even multiple organisms concurrently through nitrone/nitrone or nitrone/azide incorporation strengthens the current bioorthogonal toolbox and gives rise to robust duplex labeling of organisms to potentiate the studies of rapid biological phenomena.

Bioorthogonal labelling of living bacteria using unnatural amino acids containing nitrones and a nitrone derivative of vancomycin.

Unnatural D-amino acids bearing endocyclic nitrones were developed for live-cell labelling of the bacterial peptidoglycan layer. Metabolic incorporation of D-Lys and D-Ala derivatives bearing different endocyclic nitrones was observed in E. coli, L. innocua, and L. lactis. The incorporated nitrones of these bacteria then rapidly underwent strain-promoted alkyne-nitrone cycloaddition (SPANC) reactions affording chemically modified bacteria.

Kinetics studies of rapid strain-promoted [3+2] cycloadditions of nitrones with bicyclo[6.1.0]nonyne

Strain-promoted alkyne−nitrone cycloaddition (SPANC) reactions represent a bioorthogonal labeling strategy that is both very rapid and at the same time efficient and selective. Nitrones provide increased reaction rates as well as greater susceptibility toward stereoelectronic modification when compared with organic azides. We find that strain-promoted cycloadditions of cyclic nitrones with bicyclo[6.1.0]nonyne react with second-order rate constants as large as 1.49 L mol−1 s−1 at 25 °C. These reactions display rate constants that are up to 37-fold greater than those of the analogous reactions of benzyl azide with bicyclo[6.1.0]nonyne. We observed that reactions of nitrones with bicyclo[6.1.0]nonyne showed a stronger dependence on substituent effect for the reaction, as evidenced by a larger Hammett ρ value, than that for biaryl-aza-cyclooctanone. We demonstrate the ability to stereoelectronically tune the reactivity of nitrones towards different cyclooctynes in SPANC reactions. This ability to introduce selectivity into different SPANC reactions through substituent provides the opportunity to perform multiple SPANC reactions in one reaction vessel and opens up potential applications in multiplex labeling.

N-terminal dual protein functionalization by strain-promoted alkyne–nitrone cycloaddition

Strain-promoted alkyne-nitrone cycloadditon (SPANC) was optimized as a versatile strategy for dual functionalization of peptides and proteins. The usefulness of the dual labeling protocol is first exemplified by the simultaneous introduction of a chloroquine and a stearyl moiety, two endosomal escape-improving functional groups, into the cell-penetrating peptide hLF (human lactoferrin). Additionally, we demonstrate that dual labeling of proteins is feasible by combining metal-free and copper-catalyzed click chemistry. First, SPANC is applied to enhanced green fluorescent protein to introduce both biotin and a terminal alkyne. The terminal acetylene then serves as a convenient anchor point for the CuAAC reaction with azido-containing fluorescein, thereby demonstrating the potential of combined SPANC and CuAAC for the straightforward, dual functionalization of proteins.

Comparison of Strain-Promoted Alkyne-Azide Cycloaddition <newline/>With Established Methods for Conjugation of <newline/>Biomolecules to Magnetic Nanoparticles

The preservation of the bioreactivity of antibodies and proteins by immobilization on the surface of magnetic nanoparticles is essential for particle targeting applications in diagnosis and therapy. Here we compare the conjugation of a model antibody and of streptavidin to the surface of biocompatible 100 nm magnetic starch nanoparticles by strain-promoted alkyne-azide cycloaddition (SPAAC) with the established carbodiimide and maleimide chemistry. Under our reaction conditions the bioreactivity of the immobilized antibody was about 28% for the random amide bond formation using carbodiimide chemistry, the bioreactivity increased to about 61% for bioorthogonal SPAAC and to about 90% for maleimide conjugation. The same order was found for the biotin binding capacity of streptavidin, that was conjugated to the magnetic nanoparticles with the same methods. The described analytical methods are a platform for further studies with improved bioorthogonal conjugation reactions, e.g. the strain-promoted alkyne-nitrone cycloaddition (SPANC).

Site‐Specific Conjugation of ScFvs Antibodies to Nanoparticles by Bioorthogonal Strain‐Promoted Alkyne–Nitrone Cycloaddition

Particularly suitable: An N-terminal serine mutant of anti-HER2 scFv antibody was conjugated to polymer-coated magnetofluorescent nanoparticles by strain-promoted alkyne-nitrone cycloaddition. The resulting nanoparticles (see scheme) proved effective in targeting and labeling HER2-positive breast cancer cells.

Site-Specific Conjugation of ScFvs Antibodies to Nanoparticles by Bioorthogonal Strain-Promoted Alkyne-Nitrone Cycloaddition

Eine N-terminale Serinmutante des Anti-HER2-Antikörpers scFv wurde durch die Titelreaktion an polymerbeschichtete magnetofluoreszierende Nanopartikel konjugiert (siehe Schema), die sich anschließend als sehr effektiv für die Erkennung und Markierung HER2-positiver Brustkrebszellen erwiesen.

Protein Modification by Strain‐Promoted Alkyne–Nitrone Cycloaddition

Schnell und zielsicher: Für die metallfreie 1,3-dipolare Cycloaddition von Dibenzocyclooctinen mit Nitronen wurden Geschwindigkeitskonstanten bis 39 M−1 s−1 ermittelt, sodass diese Reaktion bis zu 300-mal schneller abläuft als ähnliche Reaktionen mit Aziden. Die Strategie ist nützlich für die ortsspezifische N-terminale Modifizierung von Peptiden und Proteinen. The bioorthogonal chemical reporter strategy is emerging as a versatile method for the labeling of biomolecules, such as nucleic acids, lipids, carbohydrates, and proteins.1 In this approach, an abiotic chemical functionality (reporter) is incorporated into a target biomolecule and can then react with a complementary bioorthogonal functional group linked to one of a diverse set of probes. The azide functional group, which is the most commonly employed reporter, can react in a Staudinger ligation with modified phosphines,2 in a copper(I)-catalyzed cycloaddition with terminal alkynes (CuAAC),3 or in a strain-promoted alkyne–azide cycloaddition (SPAAC).4 The last type of reaction5 is attractive because it does not require a cytotoxic metal catalyst and therefore provides unique opportunities for the labeling of cell-surface glycans4b, 6 and proteins7 of living cells, the decoration of polymeric nanostructures,8 the labeling of lipids,9 proteomics,10 and tissue reengineering.11 The first generation of cyclooctynes suffered from relatively slow reaction rates; however, it has been found that the rate of strain-promoted cycloaddition can be increased by appending electron-withdrawing groups adjacent to the triple bond. For example, reactions of difluorinated cyclooctynes, such as 1 (Figure 1), with azides proceed approximately 60 times faster than the corresponding reactions of unsubstituted derivatives.5a We have found that derivatives of the 4-dibenzocyclooctynol 2 react fast with azido-containing saccharides and amino acids and can be employed for the visualization of metabolically labeled glycans of living cells.12 Attractive features of dibenzocyclooctynols include easy synthetic access, nontoxicity, and the straightforward attachment of a variety of probes. Recently, we introduced the more polar azacyclooctyne 3,13 which exhibits a higher rate of reaction. Despite these advances, there is an urgent need for new and faster bioorthogonal reactions for labeling at low concentration.1b Ring-strained cyclooctynes for bioorthogonal cycloaddition reactions with azides. We report herein a novel bioorthogonal reaction pair based on strain-promoted alkyne–nitrone cycloaddition (SPANC) to give N-alkylated isoxazolines with exceptionally fast reaction kinetics. The new methodology was used in a one-pot three-step protocol for the site-specific modification of peptides and proteins. Nitrones 4 a–f were readily prepared by the condensation of appropriate aldehydes with N-methylhydroxylamine. Cycloaddition reactions of 4 a–f with cyclooctynol 2 in a mixture of acetonitrile and water gave the corresponding stable14 isoxazolines, in most cases in high yield (Table 1). We measured the rate constants of the cycloaddition reactions by 1H NMR or UV spectroscopy at 25 °C and found that the substituents on the nitrone greatly influenced the reaction kinetics. For example, the replacement of an N-methyl with a phenyl group (to give 4 c) led to a faster reaction,15 whereas nitrone 4 d, derived from a ketone, exhibited reaction kinetics that were too slow for accurate determination of the rate constant. Exceptionally high reaction rates were measured for the cycloaddition of 2 with α-carboxynitrones 4 e and 4 f. These reactions proceeded 18 and 32 times as fast, respectively, as the cycloaddition of 2 with benzyl azide (0.12 M−1 s−1).16 Also, we found that a high water content increased the reaction rate constants (e.g. 12.8 M−1 s−1 for a derivative of 2 in acetonitrile/water (1:9); see the Supporting Information).17 Finally, we determined a rate constant for the cycloaddition of azacyclooctyne 3 with 4 f. As expected,13 a further enhancement of the reaction rate (39 M−1 s−1) was observed when 3 was used in place of the carbon analogue 2. 4 R1 R2 R3 k[b] [M−1 s−1] k′ Yield [%] a H Ph Me 1.3×10−2 1 95 b H CH2CH2Ph Me 3.2×10−2 3 80 c H Ph Ph >0.2[c] >17 89 d Me CH2CH2CO2Et Me <1×10−3 <0.1 33 e H CO2Et Me 3.9 330 92 f H C(O)NHBn Me 2.2 180 93 Next, the challenge was to find a strategy for the incorporation of nitrones into biomolecules. We first focused our attention on metabolic labeling with monosaccharide derivatives bearing a nitrone moiety.19 Unfortunately, the incubation of Jurkat cells in the presence of nitrones 6–9 (10, 20, 50, and 100 μM; Figure 2), followed by labeling with dibenzocyclooctyne–biotin and staining with an avidin–fluorescein isothiocyanate (FITC) conjugate, led to no detectable fluorescence labeling of the cells.12 Presumably, either the biosynthetic glycosylation machinery does not accept nitrone modifications, or nitrones undergo intracellular hydrolysis in acidic compartments. Nitrone derivatives of D-mannosamine (compounds 6 and 7), sialic acid (compound 8), and D-galactose (compound 9) for metabolic cell-surface labeling. Fortunately, SPANC could be employed for efficient peptide and protein modification by implementing a one-pot three-step procedure. Thus, the N-terminal serine residue of model peptide 10 was oxidized20 with sodium periodate (1.1 equiv) to rapidly generate aldehyde 11, which was first treated with p-methoxybenzenethiol (6.6 equiv, 30 min), and then with N-methylhydroxylamine (2.2 equiv), p-anisidine (5 equiv), and 2 (2.2 equiv) to give the desired isoxazoline 13 via nitrone 12 (Scheme 1). We found that treatment with p-MeOC6H4SH was essential to avoid the conversion of N-methylhydroxylamine into nitrosomethane dimer ((MeNO)2) by oxidation with iodate (IO3−) formed in the previous step.21 Furthermore, the rate of nitrone formation was greatly enhanced by the addition of p-anisidine, probably by a similar mechanism to that described for the formation of oximes from aldehydes and hydroxylamines.22 One-pot N-terminal conjugation of a hexapeptide by SPANC: a) 1. NaIO4, NH4OAc buffer, pH 6.8, room temperature, 1 h; 2. p-MeOC6H4SH, room temperature, 1 h; then p-MeOC6H4NH2, MeHNOH⋅HCl, room temperature, 20 min; b) 2, room temperature, 1 h. To examine whether the one-pot three-step protocol was suitable for protein modification, we selected the chemokine interleukin-8 (IL-8),23 as this prototypical protein has an N-terminal serine residue and a relatively low molecular weight (72 amino acids, MW=8382 Da), which facilitates direct analysis of chemical modification by mass spectrometry. Current labeling methods of IL-8, for example, for the installment of a radiolabel for scintigraphic imaging of infections,24 are based on random reactions of side-chain lysine amino groups with no control over the number of reactions that take place or the sites of reaction. Thus, IL-8 in NH4OAc buffer (2 mM, pH 6.9) was subjected to oxidation with NaIO4 (1.1 equiv, 1 h), followed by treatment with p-MeOC6H4SH (6.6 equiv, 2 h), then N-methylhydroxylamine (10 equiv) and p-anisidine (10 equiv), and finally cyclooctynol 2 (25 equiv, 21 mM). After 24 h, mass spectrometric analysis showed the presence of a single protein with a mass corresponding to the isoxazoline conjugate 15 (MW=8599 Da; Scheme 2). The one-pot three-step SPANC protocol was also successfully employed to PEGylate25, 26 IL-8 by using the PEG2000-modified dibenzocyclooctyne 16 (PEG=poly(ethylene glycol)). Quantitative formation of PEG-modified IL-8 17 was observed by HPLC analysis (Figure 3). HPLC traces of IL-8 (14) and crude PEGylated IL-8, 17. One-pot N-terminal functionalization of IL-8 by SPANC: a) 1. NaIO4, NH4OAc buffer, pH 6.9, room temperature, 1 h; 2. p-MeOC6H4SH, room temperature, 2 h; b) p-MeOC6H4NH2, MeNHOH⋅HCl, room temperature, 20 min; c) cyclooctynol 2 or PEG–cyclooctyne 16, room temperature, 20 h. We have shown that 1,3-dipolar cycloadditions of cyclooctynes with nitrones that contain ester or amide α substituents exhibit much faster kinetics than similar reactions with azides.27 The new methodology was successfully employed for the site-specific modification of a peptide and a protein by implementing a one-pot three-step protocol. Besides serine or threonine oxidation, a variety of methods have been described for the installment of carbonyl groups in proteins,28 and it is to be expected that SPANC is compatible with these approaches. Furthermore, metal-free click reactions have found entry into materials science.11 SPANC will provide an additional tool for the preparation of increasingly complex materials by simple and flexible chemical manipulations. Finally, we anticipate that SPANC will offer an attractive alternative to the well-established oxime ligation29 because the synthesis of nitrones is simple,19 the isoxazoline products are stable,14 and the combination of a functionalized nitrone (R3 is a functional group, Table 1) with a cyclooctyne conjugate (such as 16) will make it possible to introduce two different functionalities in a single process. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Protein modification by strain-promoted alkyne-nitrone cycloaddition.

The bioorthogonal chemical reporter strategy is emerging as a versatile method for the labeling of biomolecules, such as nucleic acids, lipids, carbohydrates, and proteins.1 In this approach, an abiotic chemical functionality (reporter) is incorporated into a target biomolecule and can then react with a complementary bioorthogonal functional group linked to one of a diverse set of probes.