Imaging the sialome during zebrafish development with copper-free click chemistry.
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ABSTRACT:
SUBMITTER: Dehnert KW
PROVIDER: S-EPMC3385855 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Dynamic imaging of proteins in live cells is routinely performed by using genetically encoded reporters, an approach that cannot be extended to other classes of biomolecules such as glycans and lipids. Here, we report a Cu-free variant of click chemistry that can label these biomolecules rapidly and selectively in living systems, overcoming the intrinsic toxicity of the canonical Cu-catalyzed reaction. The critical reagent, a substituted cyclooctyne, possesses ring strain and electron-withdrawing fluorine substituents that together promote the [3 + 2] dipolar cycloaddition with azides installed metabolically into biomolecules. This Cu-free click reaction possesses comparable kinetics to the Cu-catalyzed reaction and proceeds within minutes on live cells with no apparent toxicity. With this technique, we studied the dynamics of glycan trafficking and identified a population of sialoglycoconjugates with unexpectedly rapid internalization kinetics.
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Project description:Chemical reactions that enable selective biomolecule labeling in living organisms offer a means to probe biological processes in vivo. Very few reactions possess the requisite bioorthogonality, and, among these, only the Staudinger ligation between azides and triarylphosphines has been employed for direct covalent modification of biomolecules with probes in the mouse, an important model organism for studies of human disease. Here we explore an alternative bioorthogonal reaction, the 1,3-dipolar cycloaddition of azides and cyclooctynes, also known as "Cu-free click chemistry," for labeling biomolecules in live mice. Mice were administered peracetylated N-azidoacetylmannosamine (Ac(4)ManNAz) to metabolically label cell-surface sialic acids with azides. After subsequent injection with cyclooctyne reagents, glycoconjugate labeling was observed on isolated splenocytes and in a variety of tissues including the intestines, heart, and liver, with no apparent toxicity. The cyclooctynes tested displayed various labeling efficiencies that likely reflect the combined influence of intrinsic reactivity and bioavailability. These studies establish Cu-free click chemistry as a bioorthogonal reaction that can be executed in the physiologically relevant context of a mouse.
| S-EPMC2836626 | biostudies-literature
Project description:The 1,3-dipolar cycloaddition of cyclooctynes with azides, also called "copper-free click chemistry", is a bioorthogonal reaction with widespread applications in biological discovery. The kinetics of this reaction are of paramount importance for studies of dynamic processes, particularly in living subjects. Here we performed a systematic analysis of the effects of strain and electronics on the reactivity of cyclooctynes with azides through both experimental measurements and computational studies using a density functional theory (DFT) distortion/interaction transition state model. In particular, we focused on biarylazacyclooctynone (BARAC) because it reacts with azides faster than any other reported cyclooctyne and its modular synthesis facilitated rapid access to analogues. We found that substituents on BARAC's aryl rings can alter the calculated transition state interaction energy of the cycloaddition through electronic effects or the calculated distortion energy through steric effects. Experimental data confirmed that electronic perturbation of BARAC's aryl rings has a modest effect on reaction rate, whereas steric hindrance in the transition state can significantly retard the reaction. Drawing on these results, we analyzed the relationship between alkyne bond angles, which we determined using X-ray crystallography, and reactivity, quantified by experimental second-order rate constants, for a range of cyclooctynes. Our results suggest a correlation between decreased alkyne bond angle and increased cyclooctyne reactivity. Finally, we obtained structural and computational data that revealed the relationship between the conformation of BARAC's central lactam and compound reactivity. Collectively, these results indicate that the distortion/interaction model combined with bond angle analysis will enable predictions of cyclooctyne reactivity and the rational design of new reagents for copper-free click chemistry.
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Project description:Attachment of DOTA to a novel monofluoro-cyclooctyne facilitates bioconjugation to an azide-modified peptide via Cu-free click chemistry. The resulting conjugate was radiolabeled with (111)In to afford a potential targeted molecular imaging agent with high specific activity and an excellent radiochemical purity.
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Project description:The 1,3-dipolar cycloaddition of azides and activated alkynes has been used for site-selective labeling of biomolecules in vitro and in vivo. While copper catalysis has been widely employed to activate terminal alkynes for [3 + 2] cycloaddition, this method, often termed "click chemistry", is currently incompatible with living systems because of the toxicity of the metal. We recently reported a difluorinated cyclooctyne (DIFO) reagent that rapidly reacts with azides in living cells without the need for copper catalysis. Here we report a novel class of DIFO reagents for copper-free click chemistry that are considerably more synthetically tractable. The new analogues maintained the same elevated rates of [3 + 2] cycloaddition as the parent compound and were used for imaging glycans on live cells. These second-generation DIFO reagents should expand the use of copper-free click chemistry in the hands of biologists.
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Project description:We describe the use of a strain-promoted copper-free click reaction in the post-self-assembly functionalization of organoplatinum(II) metallacycles. The coordination-driven self-assembly of a 120° cyclooctyne-tethered dipyridyl donor with 60° and 120° di-Pt(II) acceptors forms molecular rhomboids and hexagons bearing cyclooctynes. These species undergo post-self-assembly [3+2] Huisgen cycloaddition with a variety of azides to give functionalized ensembles under mild conditions.
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Project description:A method of DNA monolayer formation has been developed using copper-free click chemistry that yields enhanced surface homogeneity and enables variation in the amount of DNA assembled; extremely low-density DNA monolayers, with as little as 5% of the monolayer being DNA, have been formed. These DNA-modified electrodes (DMEs) were characterized visually, with AFM, and electrochemically, and were found to facilitate DNA-mediated reduction of a distally bound redox probe. These low-density monolayers were found to be more homogeneous than traditional thiol-modified DNA monolayers, with greater helix accessibility through an increased surface area-to-volume ratio. Protein binding efficiency of the transcriptional activator TATA-binding protein (TBP) was also investigated on these surfaces and compared to that on DNA monolayers formed with standard thiol-modified DNA. Our low-density monolayers were found to be extremely sensitive to TBP binding, with a signal decrease in excess of 75% for 150 nM protein. This protein was detectable at 4 nM, on the order of its dissociation constant, with our low-density monolayers. The improved DNA helix accessibility and sensitivity of our low-density DNA monolayers to TBP binding reflects the general utility of this method of DNA monolayer formation for DNA-based electrochemical sensor development.
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Project description:Bifunctional, fluorinated cyclooctynes were used for the in situ "click" crosslinking of azide-terminated photodegradable star polymers, yielding photodegradable polymeric model networks with well-defined structures and tunable gelation times.
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