Copper -free cycloaddition

Squaraines 17b and 17c have terminal acetylene residues, which allowed to convert the squaraine dyes and tetralactam macrocycles into permanently interlocked rotaxane structures using copper-catalyzed and copper-free cycloaddition reactions with bulky stopper groups [58]. 

The presence of Cu ions leads to RNA damage and is toxic for in vivo systems. Copper-free click reactions are therefore highly desirable. Several groups established copper-free cycloaddition reactions on nucleic acids. Recent developments in the field are presented in the following sections in detail. In the same context, a modified Staudinger reaction has been employed for bioorthogonal labeling of nucleic acids for completeness, this method is therefore also referenced briefly below. 

Utility of Enol and Aryl Triflates. Enol and aryl triflates are extensively used for cross-coupling reactions, the formation of carbon-carbon, carbon-tin, carbon-nitrogen, carbon-sulfur, carbon-phosphorus, and carbon-halogen bonds, and reduction/ deoxygenation. In recent examples, they were used to form enamines or enamides or were eliminated to cyclooctynes for copper-free cycloadditions in biological systems. 

The group of Rutjes reported on the low temperature, copper(I)-free cycloaddition between an azide and an electron deficient and strained double bond (for example oxanor-bomadiene 58 in Scheme 10.17) at 25-37 °C to form a triazoline, followed by a spontaneous retro Diels-Alder reaction that expelled furan to produce a 1,4,5-substituted triazole (59) 50 rpjjg copper-free cycloaddition-retro Diels-Alder was anticipated to be compatible... 

Scheme 14 Copper-free cycloaddition reaction between a 3,3-difluorocyclooctyne derivative and an azide...

POC-click is formed by thermo-cross-linking the mixture of pre-POC-N (azide-containing POC prepolymer) and pre-POC-Al (alkyne-containing POC prepolymer) the process applies synchronous binary cross-link mechanism, esterification, and thermal click reaction, and the residual azide groups on the surface of POC-click film or scaffold paved the way of surface bioconjugation through strain-promoted alkyne-azide cycloaddition (SPAAC), another copper-free click reaction. 

For an example of such interaction in the TS of non-catalyzed alkyne/azide cycloaddition (click reaction), see Gold, B., Shevchenko, N. E., Bonus, N., Dudley, G. B., Alabugin, 1. V. (2012). Selective Transition State Stabihzation via Hyperconjugative and Conjugative Assistance Stereoelectronic Concept for Copper-Free Click Chemistry. The Journal of Organic Chemistry, 77(1), 75-89. 

Debets MF, van Berkel SS, Schoffelen S et al (2010) Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3 -F 2) cycloaddition. Chem Commun 46 97-99... 

Ning X, Guo J, Wolfert MA, Boons GJ (2008) Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast Huisgen cycloadditions. Angew Chem Int Ed 47 2253-2255... 

Likewise, Hirao et al. applied copper free chck chemistry for RNA functionalization via the 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbalde-hyde (Pa) unnatural base pair [152], For this, an azide-modified Pa nucleobase (N3-Pa) was incorporated as triphosphate in a T7 in vitro transcription reaction using Ds as corresponding nucleobase in the DNA template (Fig. 3b, 2). Post-transcriptional modification was achieved using fluorescent dibenzocyclooctyne (DIBO) derivatives in a strain-promoted azide-alkyne cycloaddition reaction. Transcription and efficient site-specific labeling of a 260mer RNA was demonstrated. 

An azide-modified nucleoside has also been used for copper-free DNA detection with fluorescent cyclooctynes via strain-promoted azide-alkyne cycloaddition. 5-(Azidomethyl)-2 -deoxyuridine (AmdU) is, in contrast to various other azide containing nucleosides, stable in solution and metabolically stable and could be incorporated in cellular DNA [35]. Recently, this method has been extended for the detection of cellular RNA by 5-azidopropyl-modified UTP analogs [168]. 

Shelbourne M, Chen X, Brown T, El-Sagheer AH (2011) Fast copper-free click DNA ligation by the ring-strain promoted alkyne-azide cycloaddition reaction. Chem Commun 47(22) 6257-6259. doi 10.1039/clccl0743g... 

Gutsmiedl K, Wirges CT, Ehmke V, Carell T (2009) Copper-free click modification of DNA via nitrile oxide-norbornene 1,3-dipolar cycloaddition. Org Lett ll(ll) 2405-2408. doi 10.1021/ O19005322... 

A drawback of the copper(l)-catalyzed variant of this reaction is the presence of the transition metal, which is potentially toxic for hving cells. Therefore, ring-strained cyclooctyne derivatives have been introduced as substrates for the azide-alkyne cycloaddition whose reaction with the azide does not require a transition metal catalyst (Figure 16). The driving force of the corresponding copper-free click reaction stems for the release of ring strain associated with the conversion of the cyclooctyne s triple bond into a double bond. Fluorine substituents make the system even more reactive. ... 

Copper-free strain-promoted azide-alkyne cycloaddition... 

Figure 14.5 Reaction scheme of copper-free strain-promoted azide-alkyne cycloaddition. The reaction occurs because of the interaction between functionahzed polymer with azide (blue) and the triple bond (red) of the reactive cyclooctyne, in absence of any catalysts.

Click chemistry is commonly used in organic synthesis due to its high yield and its rapid kinetic. Although Cu-catalyzed allqme-azide cycload-dition, also called CuAAC, is the most utilized one, " " the intrinsic toxicity of the copper catalyst and its associated removal difficulties limit its application in biology. For instance, to improve dendrimer s water solubility, PEG groups are often incorporated via this method but a recent study conducted by Week and coworkers has observed substantial copper contamination in them. Copper-free strain promoted allq ne-azide cycloaddition (SPAAC) has been more and more employed to avoid this drawback.Nevertheless this last method introduces rigid cyclooctatriazole units that can modify the dendrimer scaffold and thus its properties. 

Currently, copper-catalyzed and copper-free alkyne-azide cycloadditions are the most widely applied cycloaddition reactions for the modification of biomolecules. We developed an approach based on the combination of CuAAC or strain-promoted copper-free variants with enzymatic tailing (and optional enzymatic hgation), which allows the convenient postsynthetic introduction of an azide into existing unmodified RNA strands (chemically or enzymatically synthesized or even isolated from biological sources) at a desired position, and the subsequent functionalization with a wide variety of commercially available or self-synthesized functional alkynes [16c]. 

Scheme 16.21 Cyclooctyne derivatives for use in copper-free, strain-promoted azide-alkyne [3+2] cycloadditions designed by Bertozzi (81,83," and Boons j- The second-generation difluorinated derivative 84 is easier to synthesize than 83...

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