SPAAC

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. 

Fig. 6 Depiction showing the differences in bond angles between linear alkyne and strained cyclooctyne used in SPAAC...

Strain-promoted azide-alkyne cycloaddition (SPAAC)... 

The successful preparation of cycloalkynes also opened up the possibility to explore their unique chemical reactivity. In fact, the transient existence of the cycloalkyne species could initially only be indirectly corroborated by fast in situ trapping of the smaller-sized rings (seven carbons and below) before decomposition [31]. While not strictly applicable to cyclooctyne, which is the smallest cyclic alkyne that can be isolated and stored in pure form, Blomquist already noted that nevertheless careful exclusion of air was requisite to avoid rapid decomposition. More importantly, he was also the first to observe that cyclooctyne reacts explosively when treated with phenyl azide, forming a viscous liquid product [8]. This remark is in fact the first historic administration of a process that has now become known as strain-promoted azide-alkyne cycloaddition (SPAAC). 

From Fig. 5, it also becomes apparent that while active development of cyclooctynes took place in the years 2008-2010, the intensity in the field has more or less subsided in the past years. One possible reason for this observation may be found in the fact that further boosting of the reactivity of cyclooctyne for azide is typically penalized by loss in stability, as earlier mentioned for BARAC, DIFBO, and TMTH. Another explanation lies in the current commercial availability of the cyclooctynes DIBO, DIB AC, and BCN, the three of which have dominated the field of SPAAC in the past years. 

While significant effort has been devoted over the years to the development of more reactive cyclooctynes, as delineated above, only scant investigations so far have focused on the increase of SPAAC rates by modulation of the complementary component, i.e., the azide. In fact, the vast majority of reported applications of SPAAC are based on reaction with simple aliphatic azides. As a logical consequence, reaction rate constants are also nearly always determined with an aliphatic azide (typically benzyl azide), but seldom with an aryl azide. One possible reason that aromatic azides are generally avoided for SPAAC may lie in a report... 

The main determinant of the quality of any given cyclooctyne for SPAAC reaction is its reaction rate constant with azide (ahphatic or aromatic). Throughout the years, a large number of reaction rate constants have been experimentally determined for different cycloalkynes and azides, mainly by four analytical techniques (1) NMR, (2) UV spectroscopy, (3) IR spectroscopy, and (4) fluorescence. 

The most commonly apphed method to determine a SPAAC reaction rate constant is by NMR [1]. To this end, a cyclooctyne and an azide are mixed in a deuterated solvent and formation of product is quantified by integration of diagnostic peaks of the formed triazole product. Given the fact that the triazole ring itself is fully substituted, other diagnostic protons in the product with a unique, non-... 

The most sensitive method for determination of reaction rate constants of cyclooctynes is by means of a reaction with a fluorogenic azide substrate [35]. By definition, a fluorogenic SPAAC process involves a reaction between a non-fluorescent alkyne and azide, allowing the ligation of two biomolecules to afford a highly fluorescent triazole product. Besides that, compounds that become fluorescent upon reaction with a chemical reporter and without the need of copper have... 

Strain-promoted azide-alkyne cycloaddition (SPAAC), since its inception in 2004, has firmly established itself as a powerful click chemistry tool. The commercial access of starting materials, its ease of operation, the nowadays practical reaction... 

Debets MF, Prins IS, Merckx D et al (2014) Synthesis of DIBAC analogues with excellent SPAAC rate constants. Org Biomol Chem 12 5031-5037... 

Grost C, Berg T (2015) PYRROC the first functionalized cycloalkyne that facilitates isomer-free generation of organic molecules by SPAAC. Org Biomol Chem 13 3866-3870... 

Scheme 3 Summary of photo-triggered cycloaddition reactions useful in biological applications, a Reactions between aUcenes and photochemically generated 1,3-dipoles or 1,3-ienes. b SPAAC between photoprotected cyclooctynes and azides...

Fig. 1 Cycloaddition reactions employed in nucleic acid labeling with reporter groups (green star). A Cu -mediated azide-alkyne cycloaddition (CuAAC) of a terminal alkyne with an azide. B Strain-promoted azide-alkyne cycloaddition (SPAAC) of an azide with a cyclooctyne derivative. C Staudinger ligation of an azide with a phosphine derivative (not a cycloaddition reaction, see below). D Norbornene cycloaddition of a nitrile oxide as 1,3-dipole and a norbornene as dipolarophile. E Inverse electron-demand Diels- Alder cycloaddition reaction between a strained double bond (norbornene) and a tetrazine derivative. F Photo-cUck reaction of a push-pull-substituted diaiyltetrazole with an activated double bond (maleimide)...

SPAAC has further been employed for RNA labehng with 2 -azido-modified nucleotides incorporated at the 3 -end of RNA ohgonucleotides by an enzymatic approach with poly(A) polymerase [7]. Ligation of these 3 -azido modified RNAs employing a splinted ligation approach allows the preparation of internally azido-modified RNAs [7]. Besides this, azido-modified capped RNA was successfully labeled via SPAAC [14]. 

In summary, SPAAC reactions with dibenzocyclooctyne-modified nucleic acids have proven to be valuable tools for a catalyst-free bioorthogonal chck reaction with biologically inert, non-natural azides. One drawback is the rather slow reaction with second order rate constants of approximately 0.05 s [50, 58]. Wagenknecht... 

Rentmeister et al. developed a two-step approach to site-specifically label the 5 -cap of eukaryotic mRNAs via CuAAC [15] and SPAAC [14] click chemistry. For this, a variant of trimethylguanosine synthase 2 from Giardia latnblia is employed. 

For RNA, artificial self-cleaving hammerhead ribozymes with a triazole linkage at the active site are reported [26]. Interestingly, triazole backbone modifications in RNA by CuAAC and SPAAC chemical hgation of 3 -azide and 5 -cyclooctyne oligonucleotides can further be reverse transcribed into DNA while one nucleotide is omitted at the linkage [95]. 

DNA nanopatterns on surfaces have been immobilized using click chemistry [177], and branched, Y-shaped DNA molecules can be prepared from tripropar-gylated oligonucleotides [178] by CuAAC click reactions, which are useful building blocks for higher DNA nanostructures. Moreover, SPAAC click chemistry in combination with orthogonal photochemical fixation has been used to synthesize oligomeric DNA scaffolds from cyclic DNA nanostructures which are stable towards denaturation and allow facile purification [179]. 

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