Copper-free click chemistry
Copper-free click chemistry is a bioorthogonal reaction first developed by Carolyn Bertozzi as an activated variant of an azide alkyne Huisgen cycloaddition, based on the work by Sharpless et. al. Unlike the CuAAC, Cu-free click chemistry has been modified to be bioorthogonal by eliminating a cytotoxic copper catalyst, allowing reaction to proceed quickly and without live cell toxicity. It was developed as a faster alternative to the Staudinger ligation, with the first generation producing rate constants over 63 times faster.
Although the reaction produces a regioisomeric mixture of triazoles, the lack of regioselectivity in the reaction is not a major concern for its applications in bioorthogonal chemistry. More regiospecific and less bioorthogonal requirements are best served by the traditional Huisgen cycloaddition, especially given the low yield and synthetic difficulty (compared to the addition of a terminal alkyne) of synthesizing a strained cyclooctyne.
The azide group is particularly bioorthogonal because it is extremely small (favorable for cell permeability and avoids side perturbation), metabolically stable, and does not exist in natural cells and thus has no competing biological side reactions. Although the alkyne is not as small, it still has the stability and orthogonality necessary for in vivo labeling.
The incredible bioorthogonality of the reaction has allowed the Cu-free click reaction to be applied within cultured cells, live zebrafish, and even mice.
Development of strained cyclooctynes
OCT was the first of all cyclooctynes developed for Cu-free click chemistry; it had only ring strain to drive the reaction forward, and the kinetics were barely improved over the Staudinger ligation. After OCT and MOFO (monofluorinated cyclooctyne), the difluorinated cyclooctyne (DIFO) was developed.
The substituted cyclooctyne is activated for a 1,3-dipolar cycloaddition by its ring strain and electron-withdrawing fluorine substituents, which allows the reaction to take place with kinetics comparable to the Cu-catalyzed Huisgen cycloaddition. Ring strain (~18 kcal/mol) arises from the deviation of the bond angles from the ideal 180 in order to form an eight-membered ring, the smallest of all cycloalkynes. The electron-withdrawing fluorine substituents were chosen due to synthetic ease and compatibility with living biological systems. Additionally, the group cannot produce cross-reacting Michael acceptors that could act as alkylating agents towards nucleophilic species within cells.
Computational studies in Gaussian, using gas phase B3LYP and SCS-MP2, by Houk have examined the reactivity of DIFO. Like most cyclooctynes, DIFO prefers the chair conformation in both the ground state and the minimum energy traction path, although boat transition states may also be involved. Gas phase regioselectivity is calculated to favor 1,5 addition over 1,4 addition by up to 2.9 kcal/mol in activation energy in the gas phase; solvation corrections give the same energy barriers for both regioisomers, explaining the regioisomeric mix that results from DIFO cycloadditions. While the 1,4 isomer is disfavored by its larger dipole moment (all electron-rich substituents on one side), solvation stabilizes it more strongly than the 1,5 isomer, eroding regioselectivity. Experimental studies by Bertozzi report a nearly 1:1 ratio of regioisomers, confirming the predicted lack of regioselectivity in the addition.
Furthermore, Houk’s examination of distortion energies finds that nearly all of the distortion energy (92%) arises from the distortion of the 1,3 dipole rather than the cyclooctyne, which has a pre-distorted ground state geometry which increases its reactivity. Fluorination decreases the distortion energy by allowing the transition state to be achieved with a lesser distortion of the 1,3-dipole during reaction, resulting in a larger dipole angle.
Based on reports by Boons et. al. that a fusion of a cyclooctyne to two aryl rings also increased reaction rate, the cyclooctyne reagents of the Bertozzi group proceeded through a series of fusions that sought to increase the ring strain even further. DIBO (dibenzocyclooxtyne) was developed as a precursor to BARAC (biarylazacyclooctynone), although calculations had predicted that a single fused aryl ring would be optimal. Attempts to make a difluorobenzocyclooctyne (DIFBO) were unsuccessful due to the instability of the compound.
Further adjustments to produce DIBAC/ADIBO were performed by Van Delft and others who discovered that adding distal ring strain and reducing sterics around the alkyne could further increase reactivity.
Problems with DIFO with in vivo mouse studies illustrate the difficulty of producing bioorthogonal reactions. Although DIFO was extremely reactive in the labeling of cells, it was a relatively poor reagent in mouse studies due to binding with albumin within the mice. In response, DIMAC (dimethoxyazacyclooctyne) was developed with greater pharmacokinetics and water solubility, although efforts in bioorthogonal labeling of mouse models is still in development.
- ^ Jeremy M. Baskin , Jennifer A. Prescher , Scott T. Laughlin , Nicholas J. Agard , Pamela V. Chang , Isaac A. Miller, Anderson Lo , Julian A. Codelli , and Carolyn R. Bertozzi (2007). "Copper-free click chemistry for dynamic in vivo imaging". Proceedings of the National Academy of Sciences 104 (43): 16793–16797. doi:10.1073/pnas.0707090104.
- ^ Ellen M. Sletten, Carolyn R. Bertozzi (2011). "From Mechanism to Mouse: A Tale of Two Bioorthogonal Reactions". Accounts of Chemical Research 44 (9): 666–676. doi:10.1021/ar200148z.
- ^ Franziska Schoenebeck, Daniel H. Ess, Gavin O. Jones and K. N. Houk (2009). "Reactivity and Regioselectivity in 1,3-Dipolar Cycloadditions of Azides to Strained Alkynes and Alkenes: A Computational Study". Journal of the American Chemical Society 131 (23): 8121–8133. doi:10.1021/ja9003624.
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