Bioconjugation with strained alkenes and alkynes.

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.

Monitoring protein-polymer conjugation by a fluorogenic Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition.

The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has recently proven to be a powerful synthetic tool in various fields of chemistry, including protein-polymer conjugation. In this article, we describe a fluorogenic CuAAC, which allows for efficient monitoring of protein-polymer conjugation. We show that profluorescent 3-azido coumarin-terminated polymers can be reacted with an alkyne-functionalized protein to produce a strongly fluorescent triazole-linked conjugate. Upon formation of the product, the evolution of fluorescence can accurately be determined, providing information about the course of the CuAAC. As a proof of concept, we synthesized several 3-azido coumarin terminated poly(ethylene glycol) (PEG) chains and investigated their conjugation with alkyne-functionalized bovine serum albumin (BSA) as a model protein. CuAAC conjugation was shown to be very efficient and proceeded rapidly. Conversion plots were constructed from measuring the fluorescence as function of reaction time. An additional benefit of the fluorogenic CuAAC is the in situ labeling of bioconjugates. We envision that the fluorogenic protein-polymer conjugation is not restricted to the reaction system reported in this work, but may also be ideal to screen for optimal reaction conditions of various other systems.

Controlled integration of polymers into viral capsids.

In this paper, we describe the controlled incorporation of two synthetic polymers with different structures in the cowpea chlorotic mottle virus (CCMV) capsid. Poly(ethylene glycol) (PEG) chains have been attached to the amine groups of lysine residues on the outer surface of the viral capsid. The functionalization of CCMV with PEG chains provoked a slow but irreversible dissociation of the virus into PEG-coat protein (CP) subunits, likely due to steric interference between the protein-protein subunits as a result of the presence of the PEG chains. This thermodynamic instability, however, can be overcome if a second polymer, such as polystyrene sulfonate (PSS), is present within the capsid. After complete disassembly of the PEG-CCMV conjugates and removal of the viral RNA, incubation of the PEG-functionalized coat proteins with PSS resulted in the formation of much more robust PSS-CCMV-PEG capsids with a diameter of 18 nm (T = 1 capsids). These are the first virus-like particles bearing synthetic organic polymers both inside and outside the viral capsid, opening a new route to the synthesis of biohybrid nanostructured materials based on viruses.