CLICK CHEMISTRY

Presented by;
Hamid Ali
M. Phil (1st)
Seminar
Contents
1. Introduction .......................................................................................2
2. Click Chemistry .................................................................................3
3. Types of Click Reactions ..................................................................3
3.1 Huisgen 1, 3-Dipolar Cycloaddition Reaction ............................................ 4
3.2. Copper Catalysed Azide-Alkyne Cycloaddition ........................................ 5
3.3. Copper-Free Click Chemistry ..................................................................... 7
4. Applications of Click Chemistry .......................................................8
4.1. Anti-HIV Agent Discovery......................................................................... 8
4.2 Enzyme Inhibitor Synthesis ......................................................................... 9
4.3 Bioconjugation ........................................................................................... 10
4.4 Drug Delivery ............................................................................................ 11
5. Advantages of click chemistry ........................................................12
6. Conclusion .......................................................................................12
 CLICK CHEMISTRY
1. Introduction
It is always a challenging and complex process in formation of molecules and materials.
Not only do we have to figure out how to put the different parts of compounds together to form
real bonds, but we also need to be able to master all the intricacies of joining atoms in the right
way to create the right products. Over the years, many creative chemist introduced many
methods to efficiently build molecules of different types and worked out principles on how to
control the outcomes. This development has enabled us to make anything that we can imagine
or find in nature. However, all of these developed methods require strict consideration.

In 1999 one of the most creative chemist K. Berry Sharpless introduced first time the term
“click chemistry” at the 217th American Chemical Society annual meeting. In 2001, he and his
co-workers Hartmuth C. Kolb and M.G. Finn, summarised in his landmark article as a concept
for conducting organic reactions, which was based upon the premise that organic synthesis
should focus attention on highly selective, simple orthogonal reactions that give heteroatom-
linked molecular systems with high efficiency under mild reaction conditions.

The concept of click chemistry was in part motivated by natural processes, examination of the
molecules created by nature reveals that nature prefer carbon-heteroatom bond over carbon-
carbon bond; for example, nuclei acids, proteins and polysaccharides are condensation polymer
of subunits linked through carbon-heteroatom bonds. The strategy of making large molecules
with simples building blocks are described perfectly by nature with remarkable modularity and
diversity. After realizing this, Sharpless and his co-workers proposed the reinvigoration of
establishment, well-tested reactions, with the goal to accelerate the discovery of substances
with useful properties.

The “click” in click chemistry was meant to convey the type of convenience and satisfaction
one is afforded by snapping objects together with a luggage strap connector. It does not matter
what the pieces are; if the two ends of the buckle can reach each other, the linkage is made.
This powerful idea can enable impactful chemical entities to be created by anyone in many
fields including materials science, surface science, analytical chemistry, chemical biology, and
drug development.
2. Click Chemistry
In general, the click chemistry is defined as;

1. Give very high chemical yields of desired products

2. Combination of readily available simple building blocks
3. Generate almost no by-products
4. Simple product isolation by non-chromatographic methods
5. Reaction proceeds in water, or a solvent that is benign or easily removed
6. They should be stereospecific (but not necessarily enantioselective) and be highly
selective for a single product

The logic behind click chemistry is simple:

 New molecular properties are needed everywhere.

 Such properties can emerge from the joining of small molecular building blocks.
 Scientists and engineers not trained in synthetic chemistry often lack the skills and
equipment needed to perform such connecting operations reliably.
 Chemical methods exist, and more can be developed, that make molecular connections
easily.

3. Types of Click Reactions

A range of different reaction that fulfil many of the criteria of click chemistry also listed
by Sharpless and his co-workers:

1. Cycloaddition reaction involving unsaturated structures, such as hetero–Diels-Alder

reactions and 1,3-dipolar cycloaddition
2. Addition to carbon–carbon multiple bonds; particularly oxidation reactions, such as
epoxidation, dihydroxylation, aziridination, and nitrosyl and sulfenyl halide additions,
but also certain Michael addition reactions
3. Non-aldol carbonyl chemistry, such as formation of ureas and amides
4. Nucleophilic ring opening of strained heterocycles, such as epoxides and aziridines
 Figure 1. Summary of most popular click reactions

Amongst all the reactions which achieve "click status", the Huisgen 1,3-dipolar cycloaddition
of alkynes and azides to yield 1,2,3-triazoles is undoubtedly the premier example of a click
reaction and have been widely explored among others due to its efficiency, versatility and
inertness toward other functional groups.

3.1 Huisgen 1, 3-Dipolar Cycloaddition Reaction

Rolf Huisgen, is a German chemist his major achievements was the development of the
1,3-dipolar cycloaddition reaction, also known as the Huisgen cycloaddition or Huisgen
reaction. Although the reaction was highly exothermic with low reaction rate and yield, it had
a great impact on further research. The requirement of high temperature and pressures for this
reaction were the biggest constraint for its application in living systems. One of the greater
drawback of Huisgen 1,3-dipolar cycloaddition reaction usually results in an approximately 1
: 1 mixture of 1,4- and 1,5-triazole stereoisomers as shown in figure.
Figure 2: Thermally induced 1,3-dipolar cycloaddition between azides and terminal alkynes
typically lead to mixtures of 1,4- and 1,5-disubstituted 1,2,3-triazoles

3.2. Copper Catalysed Azide-Alkyne Cycloaddition

The situation changed dramatically when the catalysed reaction was discovered. In
2001, while working on the identification of efficient and mild methods to introduce the 1,2,3-
triazole pharmacophore in peptides, Morten Meldal and Christian W. Tornøe discovered that
Cuᴵ substantially catalyses the cycloaddition reaction between azides and terminal alkynes. A
first study of this copper-catalysed azide-alkyne cycloaddition (CuAAC) was presented at a
conference, where they described that high yields (80–95%) could be obtained by allowing a
terminal alkyne (attached to a solid support) to react with different alkyl and aryl azides in the
presence of a Cuᴵ salt at room temperature, producing the 1,4-disubstituted 1,2,3-triazoles.
They could also show that the chemistry was compatible with standard Fmoc
(fluorenylmethyloxycarbonyl) chemistry and solid phase peptide synthesis. A more detailed
account was published a few months later, in which the copper-catalysis process was described
in more detail.
 Figure 3: Copper-catalysed reactions from a) Meldal and co-workers, and b) Sharpless and
co-workers
Independent of, and in parallel with, the work of Meldal and Tornøe, Sharpless, together with
Valery V. Fokin and coworkers, could also identify Cuᴵ as a catalyst for the 1,3-cycloaddition
reaction. In this case, it was found that Cuᴵᴵ salts could be used in combination with a reducing
agent, such as ascorbate, to yield Cuᴵ In situ, an approach that improved the reaction with
respect to cost and catalyst purity. Again, the copper-catalysed reaction proceeded in high
yields with a variety of starting materials, essentially only producing the 1,4-disubstituted
1,2,3-triazole product (Figure 2b).

The active copper-species (Cuᴵ) turned out to be a remarkable catalyst that accelerates the
cycloaddition reaction up to 10⁷ times while primarily producing the 1,4-disubstituted isomer.
The catalysed process is also very robust and exhibits a broad scope with respect to starting
materials. Thus, the triazole formation is nearly independent of the substituents of both reaction
partners, and the outcome of the CuAAC reaction is relatively insensitive to steric or electronic
effects of the substituents. Moreover, the functional group interference is low, and various
substituted primary, secondary, tertiary, and aromatic azides readily participate in the
transformation. The tolerance for variations in the acetylene component is also excellent. The
process is experimentally straightforward and the triazoles can often be obtained in a procedure
which generally involves little more than stirring the reagents and filtering off the pure
products. Typically, the reaction proceeds to completion within hours at ambient temperature
in a variety of solvents, including alcohols and water without any organic co-solvent. Although
most experiments are performed at close to neutral pH, the catalysis s seems to proceed well at
a wide range of pH-values.

One reason for the immediate success of CuAAC is the selective formation of 1,4-disubstituted
triazoles. Importantly, the triazole species is essentially inert to many reactive conditions, e.g.
oxidation, reduction, and hydrolysis.

3.3. Copper-Free Click Chemistry

The immense success of the CuAAC reaction rapidly triggered the interest in using this
transformation in biological systems. Apart from the applications in synthesizing drug
molecules with a triazole linkage, azide–alkyne cycloaddition reactions have also been used
for various biological applications such as site-specific protein/viruses modifications and
functionalization of cell surfaces. Use of transition-metal-catalyzed reactions for such
applications is not advisable as metal salts could be detrimental to living cells. Copper salts are
known to degrade oligonucleotide strands, and copper is cytotoxic at higher concentrations.
This has placed an importance on the search for click reactions that can be carried out without
the use of metal catalysts. The potential toxicity of Cuᴵ can be overcome by improving the rate
of the Huisgen alkyne-azide cycloaddition without using copper as a catalyst.

In 2004, Carolyn R. Bertozzi and coworkers documented an approach using a family of strained
alkynes ‘cyclooctynes’ to allow strain-promoted cycloaddition of alkynes and azide (SPAAC).
The mechanism of SPAAC is based on the introduction of ring strain into the alkyne molecule,
which is then released in a transition state during the reaction. The angle strain of the triple
bond, along with the ring strain present in cyclooctyne, contributes to a significantly faster
reaction rate. In the ligation reaction, the intermediate aza-ylide undergoes an intramolecular
reaction with an ester, forming an amide bond through a series of studies, these authors
demonstrated that the reaction rate could be tuned by the substituent on the cyclooctyne group.
Being an alkyne and azide cycloaddition reaction, it can lead to a mixture of stereoisomers.
Several other related systems have been investigated by using dibenzocyclooctynes or
oxanorbornadienes. Nevertheless, this version of the strain-promoted cycloaddition ligation
reaction has been successfully used to label glycoproteins both in vitro and in vivo on cellular
surfaces, without noticeable cytotoxicity.
 Figure 4: Strain-promoted azide-alkyne cycloaddition (SPAAC).

4. Applications of Click Chemistry

This reaction system affords desired products in almost 100% yield with no need of
purification, such as recrystallization or column chromatography. Thus, this methodology is an
ecofriendly reaction. Moreover, the combination of various alkynes and azides allows it to
rapidly construct large compound libraries, and 1,2,3-triazole itself exhibits various kinds of
biological activities, such as anti-allergenic or anti-bacterial activities. In addition, the reaction
proceeds even in water, and thus, click chemistry has been widely used in many research fields
as below.

4.1. Anti-HIV Agent Discovery

Whiting and Sharpless et al. have reported the synthesis of a series of 1,4-disubstituted-
1,2,3-triazoles as potential candidates for HIV protease inhibitors in a combination of azide-
containing fragments with a diverse array of functionalized alkyne containing building blocks
by using click chemistry. After further optimization, it was revealed that 1 has the highest
activity, exhibiting 8 nM of Ki value.
4.2 Enzyme Inhibitor Synthesis
Enzymes are drug targets for many diseases, including cancer, Alzheimer’s disease,
diabetes, TB, and many currently incurable diseases and, thus, could have a significant role in
curing such diseases. With an increasing number of patients with such incurable diseases, lick
chemistry is impacting the synthesis and development of compounds in a faster and more
efficient manner, providing novel approaches for the screening of compound libraries. The
highly modular and efficient reaction properties of click chemistry have enabled synthetic
chemists to build libraries of different classes of compound through fragment-based enzyme
inhibitor development. We show only representative structures for each enzyme inhibitor and
discuss only the key, most recently studies published. Protein kinases (PKs) are a family of
enzymes involved in controlling the function of other proteins through the phosphorylation of
hydroxyl groups of serine and threonine amino acid

Figure 6: Examples of chemical structures of various inhibitors synthesized by click

chemistry: (a,b) protein tyrosine phosphatase inhibitors; (c) protein kinase inhibitor; (d)
transferase inhibitors; (e) serine hydrolase inhibitor; (f) cysteine protease inhibitors; (g) aspartic
protease inhibitors; (h) matrix metalloproteinase inhibitors; (i) glycosidase inhibitor; (j,k)
VEGFR2 kinase inhibitors; (l) triazolyl glycolipid for MRSA (PBP2a inhibitors).
The advent of click chemistry has had a profound influence on almost every branch of chemical
science and it has become a ubiquitous chemical tool with applications in nearly all areas of
modern chemistry from chemical to material science. An overview of the recent advancements
of click chemistry in different fields have been given below.

4.3 Bioconjugation
Bioconjugation encompasses a broad arena of science at the interface between
chemistry and molecular biology, involving the formation of covalent links between synthetic
labels and biomolecular frameworks. It involves biocompatible reactions joining substrates to
biomolecules in a selective, fast and high-yielding manner. Covalently linking two molecular
entities is a challenge for molecular biologists and chemists while studying the biological
systems, for instance, attaching a small molecular probe (fluorescent dye, radical probe, affinity
tag, etc.) onto a biopolymer or linking a complex carbohydrate with a peptide. Because of the
diverse functional reactivity and structural complexity, one has to find out selective ligation
reactions, allowing the coupling of two mutually and uniquely reactive functional groups,
usually in an aqueous environment under physiological conditions. These functional groups
should be selective for each other as well as capable to tolerate other functionalities, hence
circumventing the need to use protecting groups and ideally allowing applications of the
molecules in the complex environment of a living cell.

Reactions employing azide moiety as a functional group are mostly employed in

bioconjugation and they are known to offer following advantages; (i) the azide moiety is absent
in almost all natural existing compounds and (ii) despite a high intrinsic reactivity, azides allow
selective ligation with a very limited set of reaction partners.

The Huisgen 1,3-dipolar cycloaddition of azides and acetylenes to give 1,2,3-triazoles was
identified as an interesting candidate after Sharpless et al. Realization of achieving higher
functional group compatibility by employing electrocyclic reactions instead of encountering
limitations imposed by electrophile-nucleophile reactions. The groups of Sharpless and Finn
employed the azide-alkyne coupling in the parallel synthesis of a highly active inhibitor of the
enzyme acetylcholinesterase. Sharpless and Finn choose a strategy that involved a biological
target that should literally guide the inhibitor synthesis by serving as a template for the
assembly of building blocks instead of using a conventional approach in which a diverse set of
chemical compounds is first synthesized and subsequently screened against the biological
target in question.
 Figure 7: Template-guided synthesis of the highly active inhibitor azide-alkyne coupling

4.4 Drug Delivery

Click chemistry is of tremendous help to meet the demand of modern-day chemistry
research, mainly drug discovery. It uses pairs of functional groups that rapidly and selectively
react (click reaction) with each other in eco-friendly, mild, aqueous conditions as well as in
organic solvents. The selection of each click reaction is based on its selectivity, reactivity,
biocompatibility, and stability. Click chemistry has greatly facilitated the overall drug
discovery process by providing easy access to the synthesis of building blocks for new
molecular entities (NMEs). Although it has by no means replaced existing methods for drug
discovery, it has complemented and extended them by aiding lead discovery and optimization.

A metal-free “click” conjugation protocol using strain-promoted click chemistry was explored
as a facile and fast tool for the functionalization of microbubbles with drug encapsulated
nanoparticles, siRNA encapsulated micelles and protein molecules. This microbubble–
therapeutic “click” conjugation established in the current study did not require any catalyst or
initiator, did not involve toxic agents, had ultra-fast reaction speed, and was versatile for the
ligation of different anticancer or therapeutic agents to the microbubbles. These advantages
made it a favourable protocol for use in various biomedical research and clinical applications
such as therapeutic delivery for inflammation, tumour treatment, thrombosis and angiogenesis
in different organs and real-time imaging.
5. Advantages of click chemistry
1. The mixture owns only stable compounds.
2. The reaction owns a high yield.
3. To form a desired product in a simple and quantitative way.
4. Energetically highly favourable linking reaction.
5. The purification can be done on large scale.
6. The linkage is chemoselective.
7. Click reaction must be of wide scope, giving consistently high yields with a variety of
starting materials.
8. It must be easy to perform, be insensitive to oxygen or water and use only readily
available reagents.
9. Reaction work-up and product isolation must be simple, without requiring
chromatographic purification.

6. Conclusion
Click chemistry has become one of the most powerful strategies in materials and
biomedical sciences due to the efficiency, selectivity and tolerance of this class of reactions to
a variety of solvents and functional groups. By far the most widely utilized of these efficient
transformation reactions is the CuI-catalysed azide–alkyne cycloaddition. This reaction has
been creatively employed to facilitate the preparation of versatile molecules to be employed
in different areas of chemistry including bioconjugation, drug delivery and polymer chemistry.
Click chemistry has enabled the researchers to explore complex materials while simplifying
their preparation methods. Additional reactions that can provide the benefits of click chemistry
have been increasingly investigated, which are helpful in expanding the range of available
functional groups that can participate in highly efficient chemical transformations. Overall, the
results from research to date suggest that click chemistry has emerged as a valuable tool in
biomedical fields as well as in material chemistry.