Bioconjugation with Strained Alkenes and Alkynes

Abstract

The structural complexity of molecules isolated from biological sources has always served as an inspiration for organic chemists. Since the first synthesis of a natural product, urea, chemists have been challenged to prepare exact copies of natural structures in the laboratory. As a result, a broad repertoire of synthetic transformations has been developed over the years. It is now feasible to synthesize organic molecules of enormous complexity, and also molecules with less structural complexity but prodigious societal impact, such as nylon, TNT, polystyrene, statins, estradiol, XTC, and many more. Unfortunately, only a few chemical transformations are so mild and precise that they can be used to selectively modify biochemical structures, such as proteins or nucleic acids; these are the so-called bioconjugation strategies. Even more challenging is to apply a chemical reaction on or in living cells or whole organisms; these are the so-called bioorthogonal reactions. These fields of research are of particular importance because they not only pose a worthy challenge for chemists but also offer unprecedented possibilities for studying biological systems, especially in areas in which traditional biochemistry and molecular biology tools fall short. Recent years have seen tremendous growth in the chemical biology toolbox. In particular, a rapidly increasing number of bioorthogonal reactions has been developed based on chemistry involving strained alkenes or strained alkynes. Such strained unsaturated systems have the unique ability to undergo (3 + 2) and (4 + 2) cycloadditions with a diverse set of complementary reaction partners. Accordingly, chemistry centered around strain-promoted cycloadditions has been exploited to precisely modify biopolymers, ranging from nucleic acids to proteins to glycans. In this Account, we describe progress in bioconjugation centered around cycloadditions of these strained unsaturated systems. Being among the first to recognize the utility of strain-promoted cycloadditions between alkenes and dipoles, we highlight our report in 2007 of the reaction of oxanobornadienes with azides, which occurs through a sequential cycloaddition and retro Diels-Alder reaction. We further consider the subsequent refinement of this reaction as a valuable tool in chemical biology. We also examine the development of the reaction of cyclooctyne, the smallest isolable cyclic alkyne, with a range of substrates. Owing to severe deformation of the triple bond from ideal linear geometry, the cyclooctynes show high reactivity toward dienes, 1,3-dipoles, and other molecular systems. In the search for bioorthogonal reactions, cycloadditions of cyclic alkenes and alkynes have now established themselves as powerful tools in reagent-free bioconjugations.

Highly accelerated inverse electron-demand cycloaddition of electron-deficient azides with aliphatic cyclooctynes

“…Similar to earlier reported data 10,15 , an approximately three-to fourfold higher reactivity was observed for DIBAC versus BCN (k BCN :k DIBAC ). It must be noted, however, that the absolute rate constants as measured by this protocol are about twofold lower than those reported earlier in other solvent systems 1 . One sensible explanation lies in the fact that 1,3-dipolar cycloadditions generally proceed faster in a more polar environment (for example, 50% aqueous CH 3 CN) 23 , as for example was also demonstrated by us for strain-promoted nitrone cycloadditions 24 19,20 , reaction with BCN unexpectedly proceeded 2.9 Â faster in comparison with benzyl azide (A, entry 1).…”

“…R ecent years have seen a tremendous interest in a class of chemistry known as strain-promoted cycloadditions 1 . The high mutual reactivity of reaction components, yet (relative) inertness for a large repertoire of other molecular functionalities, explains the fast-growing popularity of strainpromoted cycloadditions for application in material sciences 2 , surface functionalization 3 , bioconjugation 4 , chemical biology and even in vivo use (known as bioorthogonal chemistry) 5,6 .…”

“…The major focus in the field of strain-promoted cycloadditionsgenerally defined 1 as a bimolecular reaction of a strained system (typically an unsaturated carbocycle) with a complementary component (typically a 1,3-dipole or a diene)-has centred around cyclooctyne, the smallest stable cycloalkyne. Its reactivity with 1,3-dipoles, 1,3-dienes, carbenes and other functional groups has been reported throughout the years 1 , and inspired Bertozzi et al 7 to develop strain-promoted alkyne-azide cycloaddition (SPAAC) as a copper-free version of the popular 'click reaction' for application in cellular assays and bioconjugation.…”

“…Its reactivity with 1,3-dipoles, 1,3-dienes, carbenes and other functional groups has been reported throughout the years 1 , and inspired Bertozzi et al 7 to develop strain-promoted alkyne-azide cycloaddition (SPAAC) as a copper-free version of the popular 'click reaction' for application in cellular assays and bioconjugation. Subsequently, a whole family of cyclooctynes has been developed ( Fig.…”

Synthesis of Heterobifunctional Protein Fusions Using Copper‐Free Click Chemistry and the Aldehyde Tag

“…[23][24][25][26] The cyclooctyne reagent can be tuned for fast kinetics and the reaction proceeds selectively under a wide range of conditions. [27][28][29][30][31] However, harnessing these qualities for heterobifunctional protein conjugate synthesis first requires a practical route for the site-specific introduction of the necessary reactive partners.…”