Click Chemistry describes the biocompatible reactions that are high yielding, that are very simple to perform, have wide scope, that need no chromatography to remove their byproducts and that can be conducted in easily removable solvents. The synthesis of first triazole was reported by Arthur Michael in 1893. In the 20th century, Huisgen discovered the catalyst of copper, 1,3-dipolar cycloaddition which required the temperature of 100 degree Celsius. This reaction of copper is a highly versatile reaction which does not require any specific reaction conditions and can easily be performed with various solvents, wide temperature range and pH, with different reducing agents and can use different copper sources.
Mechanism of the Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC)
CuAAC is a click chemistry reaction that takes place between an aliphatic azide and a terminal alkyne in the presence of copper and leads to the formation of 1,4-disubstituted [1,2,3]-triazoles. The reaction takes place rapidly in the presence of Cu(I), and the product obtained from cycloaddition is chemically stable and inert towards redox reactions, has aromatic character and has the capacity to accept hydrogen bond. Although, the use of copper is very important for this cycloaddition reaction, the reaction can also be pushed further towards its desired product by the introduction of reducing agents or ligand molecules and regulating the temperature and solvent by microwave irradiation or by heating in oil bath.
Many copper sources can be used for this reaction like Copper Iodide, Copper sulfide and Copper Bromide. CuI and N,N-diisopropylethylamine was used by Meldal to form a copper-acetylene complex by pre-activating the Cu at 25 degree Celsius in N,N-dimethylformamide to form 1,4-disubstituted [1,2,3]-triazoles structures. General reaction for CuAAC reaction leading to the formation of triazole ring is shown in the figure below. In this reaction, it was also reported that if base is removed from the mixture, the reaction yield is not influenced significantly and base-free CuAAC is reported oftenly i.
Figure 1 General reaction for CuAAC reaction producing a triazole ring
Cu(I) catalyzed Azide-Alkyne cycloaddition is shown below in figure 2. In this reaction, the resin's choice, absence or presence of base, solvent and copper catalyst are the main variables ii.
Figure 2 Cu(I) catalyzed Azide-Alkyne cycloaddition
The mechanism of the CuAAC reaction in the figure 3 below shows that two Cu(I) ions have been involved in this reaction which are equivalent and can scramble. At the beginning of the reaction, Cu-alkyne π complex is formed which then leads to the formation of the copper acetylide after the alkyne proton is deprontonized. The acetylenic proton becomes more acidic due to the coordination of copper with the alkyne which increases its acidity by up to 9.7 pH units. This allows the deprontonation of the alkyne proton to occur even in aqueous media without the presence of any base. The nucleophilic attack on the alkyne's internal carbon by the terminal nitrogen of the azide group makes the complex to cyclize and produce a metallacycle. After that, the metallacycle takes the ring contraction because of transannular interaction between the C==Cu bond and the nitrogen of the azide that has lone pair of electrons. This step leads to the formation of the Cu triazolide which then undergoes prontonation to generate 1,4-desubstituted triazole .
Figure 3 Cu(I)-Catalyzed Azide−Alkyne Cycloaddition Mechanism
Various methods can be used for the formation of Azides for this CuAAC-SP reaction, like diazo-transfer or substitution . SN-type substitution is done with the highly nucleophilic azide ions to produce aliphatic azides. The most commonly used azide source is the Sodium azide. Diazo-transfer reagent is used for the synthesis of organic azides from primary amines in the diazo-transfer reaction. The use of click chemistry for Sp synthesis of 1,4-disubstituted [1,2,3]-triazoles structures has been used to replace the amide bonds with these triazoles to form peptidotriazoles, cyclic peptides, peptide mimics and amino acid modifications ii.
Mechanism of the Ruthenium-Catalyzed Azide-Alkyne Cycloaddition (RuAAC)
In the RuAAC click reaction, the regioselectivity and the catalytic activity are dependent on the ligand environment of the Ru center. Ruthenium-Catalyzed Azide–Alkyne Cycloaddition (RuAAC) Click Reactions has the preference of the formation of 1,5-disubstituted 1,2,3 -triazoles as compared to the Cu-catalyzed reactions that forms 1,4-disubstituted triazoles. The cycloaddition of azides to terminal alkynes is catalyzed with the help of pentamethylcyclopentadienyl ruthenium chloride [Cp*RuCl] complexes that leads to the formation of 1,5-disubstituted 1,2,3 -triazoles. Internal alkynes can also be used with RuAAC which provides fully substituted 1,2,3-triazoles that is similar to CuAAC .
Figure 4
A six-membered ruthenacycle structure is formed by the oxidative coupling of the alkine and the azide in the ruthenium-catalyzed azide-alkyne cycloaddition in which the carbon of the alkyne which is more electronegative fuse with the electrophilic nitrogen of the azide to form the first hydrogen-nitrogen bond. The triazole product is then formed by the reductive elimination. This step of reductive elimination is rate-determining step as suggested by DTF calculations iv.
Figure 5 Ruthenium-catalyzed azide-alkyne cycloaddition mechanism
Mechanism of Strain-Promoted Azide–Alkyne Cycloaddition (SPAAC) Reactions
Azide-alyne cycloaddition reactions can also be used for biological applications like cell surfaces' functionalization and modification of viruses and proteins. But, as the metal salts are harmful to living cells, the transition-metal-catalyzed reaction's use is not advisable for these applications. To overcome the cytotoxicity of the Copper catalyzed cycloaddition reactions, a copper-free click reaction was developed by the Bertozzi group. In this reaction, alkyne is destabilized by introducing it in a strained DIFO (difluorooctyne) in which the ring strain acts together with the electron-withdrawing, gem-fluorine and propargylic. Cu(I) is not used in this reaction for the alkyne activation . Once the alkyne is destabilized, the reactions' driving force is increased and the cycloalkyne thus relieves its ring strain. The ring strain in the cyclooctyne molecules can be increased by the introduction of the benzyl groups.
Figure 6 SPAAC click reaction's example iii
The SPAAC reaction works on the same mechanism as the 1,3-dipolar cycloaddition reaction by Huisgen but proceed as a [3+2] cycloaddition. The reaction rate of SPAAC is relatively less than that of CuAAC , but the reaction is very useful in living systems. The probe development of the SPAAC reaction is not as fast as other reactions because of low yield of cyclooctynes. Thus, in living systems, the azides are probed successfully with the help of DIFO(difluorooctyne), DIBO(dibenzylcyclooctyne) and BARAC(biarylazacyclooctynone) in the SPAAC reaction.
Click chemistry in Polypeptides
As drug candidates, peptides have many limitations due to their aqueous solubility, chemical stability, H-bond formation, metabolic stability and lipophilicity. Many strategies have already been adopted for increasing the stability of these drug candidate peptides like terminal protection, backbone modifications, introducing non-natural amino acids and cyclization etc. As a bioisostere of peptides' amide bond, 1,2,3-triazoles have gained much importance. The chemical and physical properties of 1,2,3-triazole is found to be similar to that of the amide bond. The triazole's overall dipolar moment (∼5 D) is larger than amide bond's (∼4 D) dipolar moment and thus, the properties of peptide mimicry are much improved as the acceptor and donor properties of the hydrogen bond of the 1,4-disubstituted 1,2,3-triazoles are much more than that of the amide bond ii. Moreover, with the help of hydrogen bonds, the triazole ring can align itself with other amide groups, and thus, can also align one amide group to other amide group in secondary structure of peptide.
The amide bonds' in peptides have been replaced via click chemistry with 1,4-disubstituted 1,2,3-triazoles' SP synthesis to form cyclic peptides, peptide mimics, amino acids modification and peptidotriazoles. In resins, the amide bonds' replacement with 1,4-disubstituted 1,2,3-triazoles results in peptidomimetics' analog formation. Triazoles have been applied in bioconjugation, peptide ligation and radiotracer conjugation also which thereby promotes the PEGYlated peptides, glycopeptides and peptides nucleic acids formation ii.
Click chemistry is also used for tagging biomolecules in peptides modification using single scaffold conjugation in which the resin-bound peptidotriazoles constructs are synthesized via CuAAC. When the peptides are labeled with radioactive molecules, it is easier to visualize the target of interest via fluorescent imaging or positron emission tomography i.
Implication of using a natural and non-natural linkage
Click chemistry can be used in the double strand formation with natural DNA. A Triazole-Linked Analogue of Deoxyribonucleic Acid (TLDNA) can be designed with a copper-catalyzed version of the Huisgen [3+2] cycloaddition reaction as it is high yielding, reaction conditions needed are simple and inoffensive byproducts are generated. A stable double strand is formed with the complementary strand of natural DNA following a elongated chain reaction using copper-catalyzed version of the Huisgen [3+2] cycloaddition reaction that results in giving artificial oligonucleotide. In TLDNA, the natural DNA's phosphodiester link is replaced by the triazole ring. The flexibility of the oligonucleotide chains is maintained by keeping the methylene bridge at psuedo position and hence the oligonucleotides' six-bond periodicity is also maintained .
Triozoles linkage can be non-natural also, where two or three components can be clicked together via trioazole linkages. For example, under mild hydrochloric conditions, Cu(I)-Catalyzed Azide−Alkyne Cycloaddition reaction can be cobined successfully with Silver(I)-Catalyzed trimethylsilyl −Alkyne deprotection reaction. Peptide ligation is successful with the help of these reactions which synthesize the bistriazole products in high yield after a simple chemical reaction. This click-click chemoselective ligation reactions have very wide scope as silylated alkynes, polyfunctionalized azides, and alkynes are preapared with ease. Thus, under mild conditions complex structures are accessed directly from simple building blocks using combinatorial chemistry .
Click chemistry has improved the polypeptides in terms of its structure and synthesis.
In peptide-based polymers, it is clear that their mechanical, structural, and biological properties are derived from sequential repetition on their backbone (like glue, antifreeze proteins, elastin, and collogens etc.), click chemistry proves very beneficial in creating high-molecular polypeptides. The analogs of the polypeptides can be created with the click chemistry and these analogs retain the enzyme inhibitory activity which demonstrates the effectiveness of 1,4-disubstituted [1,2,3]-triazoles as a trans peptide bond isostere .
The development of peptide drugs is accelerated by the development of click chemistry reactions such as native chemical ligation and solid-phase peptide synthesis. The natural peptides cannot be used as successful drug candidates as they are unstable and are subjected to proteolysis by various proteases. With the help of click reactions, the unstable bonds of the peptides are replaced with non-natural stable structures and which maintains the biological activity also, so, these are useful for improving the peptides' drugability i. Huisgen cycloaddition reaction can be utilized to synthesize multivalent dendrimeric peptides which has high yield ranging from 46 to 96 percent and which enhances the biological activity also significantly. Thus, these peptides are very useful in the preparation of vaccines synthetically, in the treatment and diagnosis of infections and protein mimics also viii.
The structural rigidity and stability of peptides is increased by head-to-tail cyclization as cyclopeptides are more resistant proteolytically. Ring closing copper-catalyzed reactions can be used to prepare head-to-tail cyclic peptides which gives a high yield of 79 percent. Using click chemistry both non-protected and protected peptides can be cyclized efficiently viii.
Implication of Click Chemistry on Biological Targets
The click cycloaddition reaction has has so much impact on the chemical transformations that leads to the production of libraries of molecules and new materials that can be applied in biological systems. Chemical modification is done in pharmaceuticals and biological tools to benefit nucleic acids and their oligomers. In Oligonucleotides, this modification chemically is very beneficial to mitigate their native structure's problematic features like polyanionic backbone and nucleus-mediated cleavage. Chemical modification can also be done to modify nucleic acid molecule's novel functionalities which can be applied for other biological uses like DNA microarrays synthesis in the field of molecular diagnostics, short-interfering RNAs, antisense oligonucleotides, and molecular probes .
For the regulation of gene expressions, and for use as anti-tumor and anti-viral therapies, derivatives of nucleic acids are continuously being modified, designed and synthesized. To overcome genes that are creating problems in diseased cells or to overcome viral infections, potential therapeutic agents that have been researched are ASOs, ANAs ( Antiviral nucleoside analogues), and siRNAs. These drugs are commercially available and has benefitted a lot many people. Some other active biologically nucleic acid derivatives have emerged with the help of click chemistry like unique aptamers, synthetic ribozymes, and triplex forming oligonucleotides ix.
Click chemistry is proving very successful in the modification of three main areas within nucleic acids which are the phosphodiester internucleotide linkage, the nitrogenous base, and the ribose sugar. Using a simple synthetic procedure in click chemistry, researchers have got an opportunity to target the backbone by negating the effects coming from problematic backbone that are imposed on derivatives of nucleic acids. Thus, click chemistry helps a lot in reducing the toxicity of nucleic acids and also in increasing the nucleic acid based therapies' bioavailability and all this is done while maintaining the potency of the nucleic acids.
