Биомолекулаларды қосу үшін "нақылыш химиясы" әдісі

Small molecule reactions allowing the joining of substrates of choice with specific biomolecules

In chemical synthesis, click chemistry is a class of simple, atom economy reactions commonly used for joining two molecular entities of choice. Click chemistry is not a single specific reaction, but describes a way of generating products that follow examples in nature, which also generates substances by joining small modular units. In many applications, click reactions join a biomolecule and a reporter molecule. Click chemistry is not limited to biological conditions: the concept of a "click" reaction has been used in chemoproteomic, pharmacological, biomimetic and molecular machinery applications. However, they have been made notably useful in the detection, localization and qualification of biomolecules. Click reactions occur in one pot, are not disturbed by water, generate minimal and inoffensive byproducts, and are "spring loaded"—characterized by a high thermodynamic driving force that drives it quickly and irreversibly to high yield of a single reaction product, with high reaction specificity (in some cases, with both regio and stereo specificity). These qualities make click reactions particularly suitable to the problem of isolating and targeting molecules in complex biological environments. In such environments, products accordingly need to be physiologically stable and any byproducts need to be non toxic (for in vivo systems). By developing specific and controllable bioorthogonal reactions, scientists have opened up the possibility of hitting particular targets in complex cell lysates. Recently, scientists have adapted click chemistry for use in live cells, for example using small molecule probes that find and attach to their targets by click reactions. Despite challenges of cell permeability, bioorthogonality, background labeling, and reaction efficiency, click reactions have already proven useful in a new generation of pulldown experiments (in which particular targets can be isolated using, for instance, reporter molecules which bind to a certain column), and fluorescence spectrometry (in which the fluorophore is attached to a target of interest and the target quantified or located). More recently, novel methods have been used to incorporate click reaction partners onto and into biomolecules, including the incorporation of unnatural amino acids containing reactive groups into proteins and the modification of nucleotides. These techniques represent a part of the field of chemical biology, in which click chemistry plays a fundamental role by intentionally and specifically coupling modular units to various ends. The term "click chemistry" was coined by K. Barry Sharpless' wife, Jan Dueser, in 1998, and was first fully described by Sharpless, Hartmuth C. Kolb, and M. G. Finn of The Scripps Research Institute in 2001. In 2022, the Nobel Prize in Chemistry was jointly awarded to Carolyn R. Bertozzi, Morten P. Meldal and K. Barry Sharpless, "for the development of click chemistry and bioorthogonal chemistry".

Click chemistry is a method for attaching a probe or substrate of interest to a specific biomolecule, a process called bioconjugation. The possibility of attaching fluorophores and other reporter molecules has made click chemistry a very powerful tool for identifying, locating, and characterizing both old and new biomolecules. One of the earliest and most important methods in bioconjugation was to express a reporter on the same open reading frame as a biomolecule of interest. Notably, green fluorescent protein (GFP) was first (and still is) expressed in this way at the N or C terminus of many proteins. However, this approach comes with several difficulties. For instance, GFP is a very large unit and can often affect the folding of the protein of interest. Moreover, by being expressed at either terminus, the GFP adduct can also affect the targeting and expression of the desired protein. Finally, using this method, GFP can only be attached to proteins, and not post translationally, leaving other important biomolecular classes (nucleic acids, lipids, carbohydrates, etc.) out of reach. To overcome these challenges, chemists have opted to proceed by identifying pairs of bioorthogonal reaction partners, thus allowing the use of small exogenous molecules as biomolecular probes. A fluorophore can be attached to one of these probes to give a fluorescence signal upon binding of the reporter molecule to the target—just as GFP fluoresces when it is expressed with the target. Now limitations emerge from the chemistry of the probe to its target. In order for this technique to be useful in biological systems, click chemistry must run at or near biological conditions, produce little and (ideally) non toxic byproducts, have (preferably) single and stable products at the same conditions, and proceed quickly to high yield in one pot. Existing reactions, such as Staudinger ligation and the Huisgen 1,3 dipolar cycloaddition, have been modified and optimized for such reaction conditions. Today, research in the field concerns not only understanding and developing new reactions and repurposing and re understanding known reactions, but also expanding methods used to incorporate reaction partners into living systems, engineering novel reaction partners, and developing applications for bioconjugation. Biotech company Shasqi is a company leveraging click chemistry in humans.

The Bertozzi group further developed one of Huisgen's copper free click reactions to overcome the cytotoxicity of the CuAAC reaction. Instead of using Cu(I) to activate the alkyne, the alkyne is instead introduced in a strained (DIFO), in which the electron withdrawing, propargylic, gem fluorines act together with the ring strain to greatly destabilize the alkyne. This destabilization increases the reaction driving force, and the desire of the cycloalkyne to relieve its ring strain. This reaction proceeds as a concerted [3+2] cycloaddition to the triple bond in a cyclooctyne in the same mechanism as the Huisgen 1,3 dipolar cycloaddition. Substituents other than fluorines, such as benzene rings, are also allowed on the cyclooctyne. This reaction has been used successfully to probe for azides in living systems, even though the reaction rate is somewhat slower than that of the CuAAC. Moreover, because the synthesis of cyclooctynes often gives low yield, probe development for this reaction has not been as rapid as for other reactions. But cyclooctyne derivatives such as DIFO, dibenzylcyclooctyne (DIBO) and biarylazacyclooctynone (BARAC) have all been used successfully in the SPAAC reaction to probe for azides in living systems.

Diaryl strained cyclooctynes including dibenzylcyclooctyne (DIBO) have also been used to react with 1,3 nitrones in strain promoted alkyne nitrone cycloadditions (SPANC) to yield N alkylated isoxazolines. Because this reaction is metal free and proceeds with fast kinetics (k2 as fast as 60 1/Ms, faster than both the CuAAC or the SPAAC) SPANC can be used for live cell labeling. Moreover, substitution on both the carbon and nitrogen atoms of the nitrone dipole, and acyclic and endocyclic nitrones are all tolerated. This large allowance provides a lot of flexibility for nitrone handle or probe incorporation. However, the isoxazoline product is not as stable as the triazole product of the CuAAC and the SpAAC, and can undergo rearrangements at biological conditions. Regardless, this reaction is still very useful as it has notably fast reaction kinetics.

Strained alkenes also utilize strain relief as a driving force that allows for their participation in click reactions. Trans cycloalkenes (usually cyclooctenes) and other strained alkenes such as oxanorbornadiene react in click reactions with a number of partners including azides, tetrazines and tetrazoles. These reaction partners can interact specifically with the strained alkene, staying bioorthogonal to endogenous alkenes found in lipids, fatty acids, cofactors and other natural products.

Strained cyclooctenes and other activated alkenes react with tetrazines in an inverse electron demand Diels Alder followed by a retro [4+2] cycloaddition (see figure). Like the other reactions of the trans cyclooctene, ring strain release is a driving force for this reaction. Thus, three membered and four membered cycloalkenes, due to their high ring strain, make ideal alkene substrates.

The tetrazole alkene "photoclick" reaction is another dipolar addition that Huisgen first introduced in the late 1960s ChemBioChem 2007, 8, 1504. (68) Clovis, J. S.; Eckell, A.; Huisgen, R.; Sustmann, R. Chem. Ber. 1967, 100, 60.) Tetrazoles with amino or styryl groups that can be activated by UV light at 365 nm (365 does not damage cells) react quickly (so that the UV light does not have to be on for a long time, usually around 1–4 minutes) to make fluorogenic pyrazoline products. This reaction scheme is well suited for the purpose of labeling in live cells, because UV light at 365 nm damages cells minimally. Moreover, the reaction proceeds quickly, so that the UV light can be administered for short durations. Quantum yields for short wavelength UV light can be higher than 0.5. This allows tetrazoles to be used wavelength selectively in combination with another photoligation reaction, where at the short wavelength the tetrazole ligation reaction proceeds nearly exclusively and at longer wavelength another reaction (ligation via o quinodimethanes) proceeds exclusively. Finally, the non fluorogenic reactants give rise to a fluorogenic product, equipping the reaction with a built in spectrometry handle. Both tetrazoles and the alkene groups have been incorporated as protein handles as unnatural amino acids, but this benefit is not unique. Instead, the photoinducibility of the reaction makes it a prime candidate for spatiotemporal specificity in living systems. Challenges include the presence of endogenous alkenes, though usually cis (as in fatty acids) they can still react with the activated tetrazole.

The Scripps Research Institute has a portfolio of click chemistry patents. Licensees include Invitrogen, Allozyne, Aileron, Integrated Diagnostics, and the biotech company , a BASF spin off created to sell products made using click chemistry. Moreover, holds a worldwide exclusive license for the research and diagnostic market for the nucleic acid field. Fluorescent azides and alkynes are also produced by companies such as Cyandye.