Click Chemistry, A Powerful Tool For Pharmaceutical Sciences

Click Chemistry, a Powerful Tool for Pharmaceutical
Sciences
Christopher D. Hein,1 Xin-Ming Liu,1 and Dong Wang1,2,*
Author information Copyright and License information Disclaimer
The publisher's final edited version of this article is available at Pharm Res
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Abstract
Click chemistry refers to a group of reactions that are fast, simple to

use, easy to purify, versatile, regiospecific, and give high product
yields. While there are a number of reactions that fulfill the criteria,
the Huisgen 1,3-dipolar cycloaddition of azides and terminal alkynes
has emerged as the frontrunner. It has found applications in a wide
variety of research areas, including materials sciences, polymer
chemistry, and pharmaceutical sciences. In this manuscript, important
aspects of the Huisgen cycloaddition will be reviewed, along with
some of its many pharmaceutical applications. Bioconjugation,
nanoparticle surface modification, and pharmaceutical-related
polymer chemistry will all be covered. Limitations of the reaction will
also be discussed.
Keywords: click chemistry, polymer, bioconjugation, nanomedicine,

drug delivery
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1. Introduction
In the field of pharmaceutical science, researchers are constantly

seeking for new molecules and constructs that exhibit specific
properties. While one could easily come up with a structure design
that would fit the needs, the real struggle lies in its synthesis and
purification. If only molecular synthesis were as simple as building a
LEGO® castle or assembling an IKEA® desk. If only readily available
structure units could be easily linked together to form numerous
molecules of desire in just a few crisp steps. While it still remains a
far-reaching dream, the new concept of click chemistry seems to offer
a glimpse of hope.
Since being first introduced by Dr. Barry Sharpless’ group in 1999 at

the 217th American Chemical Society annual meeting, click chemistry
has become a very popular topic, as evidenced by a nearly exponential
growth in the amount of related publications. A literature search via
SciFinder Scholar®, performed on December 31st of 2007, revealed a
total of 788 publications containing the keywords “click chemistry” or
“click reaction”, which included journal articles, reviews, preprints,
abstracts, patents, and dissertations. As shown in Figure 1,
publications in this area have quickly increased over the past four
years.
Fig. 1
Number of publications containing the key words “click chemistry” or “click reaction”
from 1999-2007. The literature search was performed via SciFinder Scholar® on Dec.
31, 2007 and included journal articles, abstracts, preprints, dissertations, patents, and
reviews. The years 1999 and 2000 each contain one publication.
In his landmark review in 2001, Dr. Sharpless defined click chemistry

as a group of reactions that “...must be modular, wide in scope, give
very high yields, generate only inoffensive byproducts that can be
removed by nonchromatographic methods, and be stereospecific (but
not necessarily enantioselective). The required process characteristics
include simple reaction conditions (ideally, the process should be
insensitive to oxygen and water), readily available starting materials
and reagents, the use of no solvent or a solvent that is benign (such as
water) or easily removed, and simple product isolation. Purification, if
required, must be by nonchromatographic methods, such as
crystallization or distillation, and the product must be stable under
physiological conditions” (1). The identification of this group of
reactions was a direct result of Mother Nature’s strategy of achieving
astonishing biological diversity from a very limited number of
monomers (i.e. proteins from amino acids, nucleic acids from
nucleotides, etc.). Puzzled by the slowness and inefficiency of the
conventional drug discovery process, Dr. Sharpless proposed a new
tactic, one in where large combinatorial libraries could be easily
prepared by linking available building blocks via click reactions. The
rule of thumb for this approach was that “...all searches must be
restricted to molecules that are easy to make” (1).
Although this novel philosophy for drug discovery is very appealing,

the medicinal chemists in this field seem to be hesitant. As shown
in Figure 2, among all the publications identified through SciFinder
Scholar®, only 14% are drug discovery related. Interestingly,
applications of click reactions in polymer sciences are warmly
welcomed and considered as an immediate success. Biomedical
applications of click chemistry, especially in pharmaceutical sciences,
are also emerging as a field of great interest. One reason is in many
areas of research, such as drug delivery and nanomedicine, linker
chemistry plays a pivotal role. Biological therapeutics needs to be
tagged with probes for the convenience of detection and evaluation.
Nanoscale delivery vehicles need to be assembled to provide drug
transportation in vivo. Targeting moieties and drug(s) need to be
attached or loaded onto delivery systems. These are very tedious tasks
as there are many constraints on performing successful linker
chemistry. For instance, they are typically performed in water.
Furthermore, the reaction conditions are preferred to be mild so that
biologicals and other fragile structures present will not loss their
functions. Protections and deprotections also need to be considered to
avoid unwanted side reactions. Click chemistry, through its unique
features, easily satisfies all of these constraints. It has repeatedly
shown to serve the needs of the pharmaceutical community
exceedingly well, despite the fact that it was intended to serve an
entirely different purpose. In this review, important aspects of click
chemistry will be discussed, along with its emerging applications in
drug delivery and nanomedicine. We will also provide some of our
concerns and vision for its future development in pharmaceutical
sciences. Several reviews on click chemistry have already been
published (1-3). The readers may find them helpful in understanding
other aspects of click chemistry or in obtaining more background
information.
Fig. 2
Major classifications of the applications of click chemistry. Analysis was performed
based on a literature search via SciFinder Scholar® on Dec. 31, 2007. The search
included journal articles, abstracts, preprints, dissertations, patents, and reviews. The
field of drug discovery was considered separate from pharmaceutical and the category
“other” contains miscellaneous applications that cannot be grouped into the other three
categories, such as applications in materials sciences, certain reviews, and novel
methods for improved catalysts. Many applications are subjective in that they can fall
into more than one category (i.e. the synthesis of certain block copolymers could be
considered both pharmaceutical and polymer-related). In these cases, the applications
were categorized as pharmaceutical if and only if their corresponding abstracts
specifically mentioned that the involved compound(s) could play a role in a
pharmaceutical setting. It is intended for this chart to represent a general overview and
should not be taken literally.
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2. Classification of Click Reactions

As already implicated, click chemistry encompasses a group of
powerful linking reactions that are simple to perform, have high
yields, require no or minimal purification, and are versatile in joining
diverse structures without the prerequisite of protection steps. To
date, four major classifications of click reactions have been identified
(Figure 3).
Fig. 3
Major classifications of click chemistry reactions, along with corresponding examples.
Nu = nucleophile; EWG = electron withdrawing group.
 Cycloadditions - these primarily refer to 1,3-dipolar cycloadditions,

but also include hetero-Diels-Alder cycloadditions (2).
 Nucleophilic ring-openings - these refer to the openings of strained
heterocyclic electrophiles, such as aziridines, epoxides, cyclic sulfates,
aziridinium ions, episulfonium ions, etc. (2).
 Carbonyl chemistry of the non-aldol type- examples include the
formations of ureas, thioureas, hydrazones, oxime ethers, amides,
aromatic heterocycles, etc. (1). Carbonyl reactions of the aldol type
generally have low thermodynamic driving forces, hence they have
longer reaction times and give side products, and therefore cannot be
considered click reactions (1).
 Additions to carbon-carbon multiple bonds - examples include
epoxidations, aziridinations, dihydroxylations, sulfenyl halide
additions, nitrosyl halide additions, and certain Michael additions
(1,2).
Among the four major classifications, cycloadditions, particularly the

CuI-catalyzed Huisgen 1,3-dipolar cycloaddition (HDC) of azides and
terminal alkynes to form 1,2,3-triazoles (4), are the most widely used.
Based on the literature search mentioned earlier, nearly 100% of the
publications referred to this click reaction, which has found
applications across many diverse research areas. In the following
sections, the potential of this click reaction and its pharmaceutical
applications will be reviewed.
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3. CuI-Catalyzed Huisgen 1,3-dipolar Cycloaddition of Azides

and Terminal Alkynes
The CuI-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and

terminal alkynes to form 1,2,3-triazoles is the model example of a click
reaction (Figure 3). It fulfills all of the criteria of click chemistry
perfectly, no matter how subjective they may be, and is therefore
extremely reliable and easy to use. This reaction exclusively forms 1,4-
substituted products, making it regiospecific. It typically does not
require temperature elevation but can be performed over a wide
range of temperatures (0-160°C), in a variety of solvents (including
water), and over a wide range of pH values (5 through 12). It proceeds
as much as 107 times faster than the uncatalyzed version, and
purification essentially consists of product filtration (3,5-7).
Furthermore, it is unaffected by steric factors. “Variously substituted
primary, secondary, tertiary, and aromatic azides readily participate
in this transformation. Tolerance for variations in the acetylene
component is also excellent” (6). All of these characteristics make this
cycloaddition particularly popular among the other click reactions
described above.
Two additional reasons for the popularity of this cycloaddition are

azides and terminal alkynes are fairly easy to install and they are
extremely stable at standard conditions (2,8). They both can tolerate
oxygen, water, common organic synthesis conditions, biological
molecules, a large range of solvents and pH’s, and the reaction
conditions of living systems (reducing environment, hydrolysis, etc.)
(2,3,9). Even though the decomposition of aliphatic azides is
thermodynamically favored, a kinetic barrier exists that allows them
to be stable in the aforementioned conditions (3). They will essentially
remain “invisible” in solution until a dipolarophile, such as an alkyne,
comes into contact (3).
3.1 Mechanism of HDC raction
In general, cycloadditions proceed through a concerted mechanism.

However, experimental kinetic data (10) and molecular modeling (7)
performed on the HDC reaction seem to favor a stepwise reaction
pathway (3,5). It has been calculated that the activation barrier for a
catalyzed concerted HDC reaction is actually greater than that for an
uncatalyzed concerted reaction (27.8 kcal/mol vs. 26 kcal/mol in one
particular reaction using density functional theory calculations) (7).
Furthermore, a stepwise-catalyzed HDC reaction has an activation
barrier 11 kcal/mol lower than a concerted catalyzed reaction (3).
Based on experimental evidence (5,6) and the fact that CuI can readily
insert itself into terminal alkynes (Sonogashira coupling, 11), it is
envisioned that the first step of the reaction involves π complexation
of a CuI dimer to the alkyne (1 in Figure 4). Thereafter, deprotonation
of the terminal hydrogen occurs to form a Cu-acetylide (5). There are
actually several different kinds of Cu-acetylide complexes that can
form, depending on the reaction conditions utilized; 2 represents just
one possibility (7). The π complexation of CuI lowers the pKa of the
terminal alkyne by as much as 9.8 pH units, allowing deprotonation to
occur in an aqueous solvent without the addition of a base (3). If a
non-basic solvent such as acetonitrile was to be used then a base, such
as 2,6-lutidine or N,N’-diisopropylethylamine (DIPEA), would have to
be added (12).
Fig. 4
Proposed mechanism for the HDC reaction. Ligands are represented by “L” and
symbolize a wide variety of possible compounds, depending on the catalyst used. As an
example, if CuBr was used as the catalyst then the ligand would be bromide. Figure
adapted from reference 3 with permission from Wiley-VCH.
In the following step, N(1) displaces one of the ligands from the
second Cu in the Cu-acetylide complex to form 3. In turn, this
“activates” the azide for nucleophilic attack (5). Due to proximity and
electronic factors, N(3) can now easily attack C(4) of the alkyne,
leading to a metallocycle (not shown for simplicity). The metallocycle
then contracts when the lone pair of electrons of N(1) attacks C(5) to
form the respective triazole 4. Once 4 forms, the attached Cu dimer
immediately complexes to a second terminal alkyne. However, this
second alkyne cannot undergo a cycloaddition due to the unfavorable
structure of the complex, and it dissociates upon protonation to
reform 4. One final protonation releases the CuI catalyst from the
1,2,3-triazole product 5, to undergo a second catalytic cycle with
different substrates (3). Both of these protonations are most likely the
result of interactions with protonated external base and/or solvent,
but further studies are needed to conclusively confirm (3).
3.2 Catalysts
There are a number of methods to generate the active catalyst for the
HDC reaction. One of the most common techniques is to reduce
CuII salts, such as CuSO4·5H2O, in situ to form CuI salts. Sodium
ascorbate is typically used as the reducing agent in a 3- to 10-fold
excess (3), but other reducing agents, including hydrazine (13) and
tris(2-carboxyethyl)phosphine (TCEP) (9), have been used with
reasonable success. The advantages of this strategy are it is cheap, it
can be performed in water, and it does not require deoxygenated
atmosphere (3,14). Not only does an aqueous solvent remove the need
for a base, as previously explained, but it also eliminates the need for
protecting groups (O-H and N-H functional groups essentially remain
“invisible” in aqueous solutions) and it is environmentally safe (1).
The main disadvantage is the reducing agent might reduce Cu II down
to Cu0. This can generally be prevented, though, by using a proper
ratio of reducing agent to catalyst and/or adding a copper-stabilizing
agent, such as tris-(hydroxypropyltriazolylmethyl)amine (THPTA) (3).
A second way to create the catalyst is to directly add CuI salts. Many

such compounds have been utilized over the past few years, including
CuBr, CuI, CuOTf·C6H6 (OTf = trifluoromethanesulfonate), [Cu(NCCH 3)4]
[PF6], etc. (6). This method does not require a reducing agent, but it
has to be done in a deoxygenated environment and in an organic
solvent (or a mixed solvent), meaning that protection groups will
probably be needed along with a base (14). It has been shown that
using excess amounts of both the bases 2,6-lutidine and DIPEA
produce the best results, causing the least amount of side products
(6,12). Still, CuI salts are not as reliable as the CuII procedure (6).
Oxidizing copper metal with an amine salt is another way to generate

the catalyst (7,14). There are a considerable number of disadvantages
with this strategy. Longer reaction times are needed, as well as larger
amounts of copper, it is more expensive, and requires a slightly acidic
environment to dissolve the metal, which could be damaging to any
acidic-sensitive functional groups present in the reactants (3). For
more information on this method or any of the others mentioned thus
far, refer to reference 3 for an exquisite review.
Recently, CuI-modified zeolites were reported as catalysts for the HDC

reaction (15). “Zeolites” refers to a family of aluminosilicate minerals,
occurring both naturally and synthetically, that are highly porous and
therefore have large surface areas (16-18). They are particularly
desired as catalysts because of their “...high concentration of active
acid sites, their high thermal/hydrothermal stability, and high size
selectivity” (18). In one particular HDC reaction, the zeolite USY,
modified with CuI, exhibited far better results than CuCl. Its product
yield was thirteen percent higher and its reaction time was roughly
three times as short (15). Pending more research, this approach could
prove to be very valuable.
Another research group has attempted to find a replacement for

copper as the catalyst. Knowing that other transition metals can insert
into alkynes (19,20), Golas et al. reacted propargyl ether with azide-
terminated polystyrene to make polymers using three different
catalyst: NiCl2, PtCl2, and PdCl2 (13). Although none of them displayed
catalytic activity as high or as fast as CuBr, they all produced
polymerization products. PtCl2 produced polymers with the highest
molecular weight, followed by PdCl2, followed by NiCl2.
In 2005, pentamethyl cyclopentadienyl ruthenium (II) complexes

(Cp*Ru), such as Cp*RuCl(PPh3)2, were discovered as novel catalysts
for click chemistry. Contrary to all previously mentioned catalysts,
Cp*Ru complexes afford only 1,5-substituted 1,2,3-triazoles (21).
Furthermore, they can work on both terminal and internal alkynes
alike (22). An example of such a reaction is shown in Figure 5. Due to
their recent discovery, though, limited information is available
detailing the role of ruthenium(II) complexes in click chemistry, as
evidenced by a search via SciFinder Scholar®.
Fig. 5
Using a Cp*Ru catalyst in the click reaction exclusively forms 1,5-substituted 1,2,3-
triazoles. These catalysts can also work on internal alkynes (not shown), contrary to all
other known catalysts.
Finally, studies have shown that catalysts are not always required for
the cycloaddition to proceed. By using electron deficient alkynes, the
reaction can proceed readily at ambient conditions (23). However,
electron deficient alkynes are very reactive toward nucleophiles and
can lead to side products (24), which has kept these types of reactions
estranged from the field of click chemistry. Other studies have shown
that the cycloaddition can occur rapidly if the alkyne is first
incorporated into an eight-member ring, forming a cyclooctyne (24-
26). Cyclooctynes are very unstable, due to a high degree of ring strain
(18 kcal/mol), which causes them to react readily with azides (24).
While this method leaves no side products and requires no cytotoxic
catalysts, it requires the prerequisite of connecting the alkyne of
interest to an eight-member ring. This can be a very challenging task
and is much more complicated than simply adding an acetylene
functional group, as in traditional click reactions. An additional
concern with the cyclooctyne method is it produces a racemic mixture
of regioisomers (Figure 6) (24). This stipulation is typically
inconsequential to pharmaceutical applications, but it defies the very
definition of click chemistry, which requires click reactions to be
regiospecific.
Fig. 6
I
Huisgen 1,3-dipolar cycloadditions of azides and alkynes without Cu catalyst. If an
alkyne is first incorporated into an eight-member ring then a 1,3-dipolar cycloaddition
of azides and alkynes can proceed rapidly without the aid of a catalyst. However, a
racemic mixture of regioisomers is obtained.
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4. Applications of Click Chemistry in Pharmaceutical Sciences
Since its debut in 1999, click chemistry has stimulated enormous

amount of interests in many different research fields, being utilized in
everything from microelectronics to virus labeling to treatments for
cancer. In the following sections, an in-depth look will be taken at
some of its applications pertaining to the field of pharmaceutical
sciences. The area of drug discovery will be excluded since multiple
review papers on this subject already exist (2,27-29).
4.1 Polymer Therapeutics and Click Chemistry
“‘Polymer therapeutics’ is an umbrella term used to describe

polymeric drugs, polymer-drug conjugates, polymer-protein
conjugates, polymeric micelles to which drug is covalently bound, and
multi-component polyplexes that are being developed as non-viral
vectors. All subclasses use specific water-soluble polymers, either as
the bioactive itself or as an inert functional part of a multifaceted
construct for improved drug, protein, or gene delivery” (30). While
polymer therapeutics have been used to treat numerous diseases, they
are particularly effective against those that have an enhanced
permeability and retention (EPR) effect (31), such as rheumatoid
arthritis (32) and solid tumors (33). Non-viral gene therapy
formulations based on synthetic polymers have been extensively
investigated and have shown better safety profiles than viral gene
vehicles, though their gene transfection efficacy is relatively low (34).
Conjugation of biocompatible polymers, such as poly(ethylene glycol)
(PEG), to therapeutic proteins and peptides has proved to be a very
effective strategy in reducing immunogenicity and increasing
bioavailability (35,36). Clearly, biocompatible polymers have played
vital roles in modern pharmaceutical sciences, especially in drug
delivery and formulations. Nevertheless, simpler and more efficient
polymer chemistry is needed to meet the ever-increasing demand for
highly diverse pharmaceutical properties.
As explained in previous sections, click chemistry was initially

developed as a drug discovery tool. However, its most successful
applications thus far have been in the field of polymer chemistry. As
seen in Figure 2, over 1/3 of all publications containing the keywords
“click chemistry” or “click reaction” are related to polymer synthesis
and/or modification. Generally, these publications can be classified
into five broad categories: block copolymer synthesis, linear
multifunctional copolymer synthesis, dendrimer synthesis, polymer
network synthesis, and polymer analogous modification. In addition to
significantly improving product yields, most of these click chemistry
applications drastically simplified the synthetic routes and
purification procedures. Therefore, it is the belief of the authors that
this new “tool in the box” may shift the paradigm of polymer synthesis
and lead to new strategies of polymer therapeutics development.
4.1.1 Synthesis of Block Copolymers with Click Reaction
Biocompatible amphiphilic block copolymers have many

pharmaceutical applications. They have been extensively used in the
formulation of various nanoparticulate structures, such as micelles,
nanospheres, nanocapsules, polymersomes, etc. (37-39). Typically,
block copolymers are synthesized via two routes: (A) sequential
addition of different monomers into polymerizations containing living
reaction centers (40,41). Living ionic polymerizations, atom transfer
free radical polymerizations (ATRP), reversible addition
fragmentation chain transfer (RAFT) polymerizations, ring-opening
polymerizations (ROP), or their combination have all been utilized to
obtain well-defined block copolymers of different components. (B)
Linking different linear polymer chains via their terminal
functionalities. While the latter method allows the combination of
polymer blocks that may not be compatible with the first, the lack of
efficient linker chemistry has made this route rarely used.
The emergence of click chemistry drastically changed the scientific

community’s views on block copolymer synthesis. Because of its
extremely high reaction efficiency and tolerance to a variety of
functional groups, click chemistry has become the hallmark of linker
chemistry. It is one of the most efficient ways to join two substances
together and has thus been used repeatedly to link well-defined
homopolymers to form block copolymers. Recently, Van Camp et al.
reported a synthetic strategy for diverse amphiphilic copolymer
structures by combination of ATRP and the HDC reaction. Using a
modular approach, polymers with alkyne functionalities as well as
polymers with azide functionalities [e.g. poly(1-ethoxyethyl acrylate)
and poly(acrylic acid)], were first synthesized via ATRP. They were
then subsequently “clicked” together to yield block copolymers (42).
Similarly, Opsteen et al. described the synthesis of polystyrene (PS),
poly(tert-butyl acrylate) (PtBA), poly(methyl acrylate) (PMA) block
copolymers using click chemistry (43). Using an initiator containing a
triisopropylsilyl (TIPS) protected acetylene, the three homopolymer
blocks were obtained via ATRP and the terminal bromides were then
converted to azides. Following TIPS deprotection, the heterotelechelic
homopolymers were joined together via HDC reactions. When RAFT
polymerization was employed to obtain the homopolymer blocks,
however, specially functionalized chain transfer agents had to be
synthesized to allow the introduction of terminal azides or acetylenes
(44,45). Additionally, this modular strategy of clicking different
homopolymer blocks together has also been exploited by numerous
other research teams (46-49). Among these works, one report focused
on the synthesis of well-defined block copolymers composed of a rigid
polypeptide sequence poly(g-benzyl-L-glutamate), or PBLG, and a
poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) block (46).
The azide and terminal alkyne functionalities were introduced into the
α-positions of both the PBLG and PDMAEMA precursors by using
appropriate α-ω-functionalized initiators. The PBLG block was
obtained by ring-opening polymerization of γ-benzyl-L-glutamate N-
carboxyanhydride (NCA), and the PDMAEMA blocks from ATRP of 2-
(dimethylamino)ethyl methacrylate. Although this approach of
incorporating a polypeptide block into a copolymer is truly innovative,
the need of column chromatography for purification violates
Sharpless’ definition of a click reaction. Hence, one could make the
argument that this copolymer application does not actually utilize
click chemistry.
Clearly, click chemistry has revitalized the second strategy of block
copolymer synthesis. Many monomers that cannot be used to produce
block copolymers via living polymerizations (due to extremely
disparate reactivities or solubility differences) can now be easily
incorporated through the second strategy. Quite literally, with click
chemistry, any two homopolymer blocks can be joined together to
form block copolymers. This opens the door for combinatorial block
copolymer synthesis, allowing diverse copolymers with very unique
properties to be synthesized quickly and easily, which could
potentially lead to great strides in the field of pharmaceutical sciences.
4.1.2 Click Chemistry and Polymeric Micelles
One of the main pharmaceutical applications of amphiphilic block

copolymers is the construction of polymeric micelles as delivery
vehicles for therapeutic, imaging, or diagnostic agents (50-52).
Although polymeric micelles are simple and effective delivery systems
that have been evaluated in clinical treatments for various cancers,
they still face several challenges such as stability and control of drug
release. To improve these problem areas, many different
methodologies have been explored (53,54). One of the most promising
methods was developed by Wooley’s group at Washington University.
As a novel method for stabilizing polymeric micelles, the group

reported the synthesis of well-defined core crosslinked polymeric
micelles, utilizing multi-functional dendritic crosslinkers (55).
Amphiphilic diblock copolymers of poly(acrylic acid)-b-poly(styrene)
(PAA-b-PS) were first functionalized with terminal alkynes
throughout the hydrophobic polystyrene block segment to form
micelles that were click-readied for crosslinking. Azide functionalized
1st generation dendrimers were then “clicked” with the alkynyl groups
of the micelles to form core crosslinked micelles. It was estimated, on
average, that 3.4 polymer chains were conjugated to each dendrimer.
The remaining click-readied functionalities (both terminal alkynes
and azides) could be used as handles for further chemical
modifications, such as conjugating dyes.
In addition to crosslinking micelles, the same group was able to

introduce click-readied functionalities selectively throughout either
the core or shell of micelles (56). After obtaining a poly(acrylic acid)-
b-polystyrene copolymer micelle, it was modified so that poly(acrylic
acid) within the micelle shell displayed either azido or alkynyl
functional groups via amidation chemistry. Using amidation chemistry
once more, the unreacted acrylic acids were linked together via a
diamine linker, crosslinking the shell of the micelle in an intramicellar
fashion. Following similar chemistry, a shell-crosslinked micelle
containing click-readied functional groups in the polystyrene core was
also prepared. The availability and reactivity of the functional groups
in both of these crosslinked micelles towards click chemistry was
demonstrated by reactions with complementary click-functionalized
fluorescent dyes. Studies by analytical ultracentrifugation
sedimentation confirmed the covalent attachment of the fluorescent
tags in the core or shell regions of the crosslinked micelles.
While the strategies used by this research team are very appealing,
they are still in their early stages of development for pharmaceutical
applications. The polymers used in their studies are not
biocompatible. Therefore, it would be of immense importance to see if
the same chemistry is applicable to biocompatible block copolymers
such as polyester/polyethylene glycol block copolymer systems,
which are often used in drug delivery.
4.1.3 Click Chemistry and Linear Multifunctional Polymeric Delivery Systems
The most commonly used biocompatible water-soluble polymers for

drug delivery include N-(2-hydroxypropyl)methacrylamide (HPMA)
copolymers, polyglutamate, and PEG. Among them, HPMA copolymer
is the most extensively studied drug carrier, with more than half of all
polymeric drug conjugates in clinical evaluations based on this
polymer (57,58). Polyglutamate-drug conjugates, on the other hand,
are the most advanced in clinical evaluation with polyglutamate-
paclitaxel in phase III clinical trial (59,60). As for PEG, its main clinical
applications are PEGylations of protein therapeutics to alter their
pharmacokinetics and render them nonimmunogeneic (61). It has also
been used as a carrier for chemotherapeutic agents (62).
While the field of water-soluble polymeric drug conjugates has

gradually matured over the past few decades, challenges still remain
in many aspects of its development: (1) compared to the construction
of other drug delivery systems (e.g. liposome, micelles and
nanoparticles), the synthesis of polymer-drug conjugate is relatively
complex. For carriers based on vinyl polymers such as HPMA
copolymer, the introduction of a new drug to the conjugate often
involves multi-step synthesis of a new drug-containing vinyl monomer
and its subsequent copolymerization with HPMA and other co-
monomers. (2) Another issue is the nondegradable nature of vinyl
polymers and PEG. While a polymer carrier with high molecular
weight (MW) would render a long half-life for the conjugated drug in
circulation, the MW must be set lower than the threshold of the renal
glomerular filtration to avoid chronic accumulation of the polymer
carrier in vivo and its potential negative impact. (3) Due to the recent
advancement of living free radical polymerizations, the MW and
polydispersity index (PDI) of vinyl polymer - drug conjugates may be
properly controlled. Nevertheless, the distribution of functionality
along the polymer backbone still cannot be controlled. (5) Different
from vinyl polymers and polyglutamate, PEG has limited sites for
functionalization. Only the two chain termini can be used for drug
conjugation, which significantly lowers the drug loading capacity of
PEG as a carrier.
As one of the potential solutions to these challenges, a linear

multifunctional PEG design has been proposed as a polymeric drug
carrier (63). The concept is to join short telechelic PEGs with tri-
functional linkers. While two functionalities will be used to link the
PEGs, the third one will be used for drug conjugation. Different from
traditional PEG designs, it has multiple functionalities pendent to the
polymer backbone. Short peptide sequences can also be introduced
into the polymer main chain to allow biodegradation of the polymer
carrier into short PEG segments. The length of the short PEG
predetermines the distance between adjacent functionalities. With
this method vinyl monomer synthesis is no longer necessary. While
several approaches have been made to synthesize the linear
multifunctional PEG construct (63-66), further improvements are
desperately needed. In particular, the chemistry that joins the short
PEG segments should be much more efficient, and tolerant of pendent
functionality conjugation.
Recently, Liu et al. used click chemistry to synthesize a linear

multifunctional PEG as a novel drug delivery system (67). PEG
oligomer diol was first activated with phosgene and capped with
propargyl amine so that it was acetylene terminated. This was to serve
as the polymer building block. A diazide monomer, 2,2-
bis(azidomethyl)propane-1,3-diol, and various functional derivatives
were synthesized as the functionality building blocks. All of these
building blocks were dissolved in water and copolymerized in a step-
growth fashion via the HDC reaction. Due to the self-catalyzing feature
that has been observed in click reactions using 2,2-
bis(azidomethyl)propane-1,3-diol (5), this click copolymerization was
shown to be highly efficient. The strategy was used to synthesize a
prototype bone-targeting PEG conjugate that used alendronate (a
bisphosphonate) as the targeting moiety. The content of alendronate
could be easily adjusted since the copolymerization was performed in
water, a good solvent for bisphosphonates. Compared to traditional
synthetic polymeric delivery systems, the advantages of this approach
are that the entire synthetic procedure is modular, simple, free from
vinyl chemistry, and produces high yields; no special reaction
condition(s) or protection steps are needed; oligopeptide sequences
can be easily inserted between PEG and the acetylene functionality to
allow biodegradability of the delivery system. Furthermore, by adding
some monoacetylene-functionalized PEG to the system, Liu et al. have
been able to control the average molecular weight. More work needs
to be done, however, to improve the wide polydispersity of the
copolymer.
4.1.4. Other Polymer-Related Applications of Click Chemistry
Due to their concentrated surface functionality and narrow

polydispersity, dendrimers have been actively investigated as
potential drug delivery carriers (68,69). They are typically
synthesized via two strategies: by the divergent approach or by the
convergent approach. Without elaboration, both approaches are often
complicated, involving multiple reaction steps, and both require
extensive purification. Click chemistry can greatly simplify the
synthesis of dendrimers, making them more applicable and affordable.
As an example of a dendrimer used for drug delivery, Gopin et al.

reported the synthesis and evaluation of a single-triggered
disassemble dendrimer as a potential platform for a multi-prodrug
(70). “These unique structural dendrimers can release all of their tail
units through a self-immolative chain fragmentation that is initiated
by a single cleavage at the dendrimer’s core” (70). To allow enzymatic
activation of the second-generation of self-immolative dendrimers,
azide-monoterminated PEG was conjugated to the dendritic platform
via click chemistry, which prevented aggregate formation of the
hydrophobic drugs.
Polymer networks such as hydrogels are also often used in drug

delivery (71, 72). A few attempts have been made to use click
chemistry to synthesize polymer networks with high efficiency and
reduced defects (73-75). The problem with this approach, however, is
the difficulty of removing copper catalyst from the network.
As discussed above, polymer analogous reactions are not very efficient
due to the steric hindrance caused by the polymer backbone. There
are often unreacted pendent functional groups leftover. Because of its
high reaction yield and mild reaction conditions, people have tried to
introduce azides or acetylenes as novel pendent functionalities to the
polymer backbone so that it can be used for further click modifications
(76, 77). One concern, however, is that this strategy will make sense
only when the click functionalities are introduced by
copolymerization, not polymer analogous reaction.
4.2 Click Chemistry and Nanoparticular Delivery Systems
Nanoparticular delivery systems are the carriers ranging in size from

10 to 1000 nm. Over the past few decades, delivery systems such as
quantum dots, magnetic nanoparticles, gold nanoparticles, micelles,
and liposomes have all been extensively investigated for imaging and
drug/gene delivery applications (78, 79). Due to the small size, they
are able to penetrate through fenestrated vasculature, allowing
efficient drug accumulation at target sites. Moreover, drug targeting
by nanoparticular delivery systems offers several important
advantages: they reduce drug dose, minimize side-effects, protect
drugs against degradation, and enhance drug stability (80).
Surface modifications of these carriers can have significant impacts on

their physical-chemical properties and therapeutic efficacy. They can
alter nanoparticle zeta potentials, hydrophobicities, and targeting
capabilities. These modifications can be performed using a variety of
techniques, including physical adsorption, electrostatic binding,
specific recognition, and covalent coupling, each of which has its own
advantages and disadvantages (79). In this part of the review, the
application of click chemistry towards the modification of
nanoparticular carrier surfaces will be examined.
4.2.1 Gold and Magnetic Nanoparticles

The use of gold nanoparticles in pharmaceutical research has become
a well-established practice, as evidenced by the ever-growing plethora
of scientific literature. Due to their unique physical properties (81),
they have been used to label antibodies (82), target polynucleotides
(83), in cancer cell diagnostics (84), in drug delivery systems (85), etc.
Many of these methods, however, rely on the non-covalent
attachments of biological molecules, which have important drawbacks
(86). For instance, non-covalent interactions are much weaker than
covalent bonds, meaning that it is much easier to break apart the
nanoparticle-biological molecule conjugate. For long-term
conjugation, covalent bonds must be used. One study performed by
Brennan et al. showed that bioconjugations to gold nanoparticles via
covalent bonds could occur easily and rapidly with click chemistry
(86).
Gold nanoparticles were first prepared and functionalized with azide

groups at the surface. A lipase enzyme (a 30 kDa globular recombinant
protein) was also modified to express a single terminal alkyne and
was allowed to react with the nanoparticles. Gel electrophoresis
verified that several lipases had covalently attached to each
nanoparticle, without any nonspecific binding, and a lipase assay
showed that they still retained their enzymatic activity. Following a
similar procedure, Fleming et al. were able to conjugate several
different alkynyl derivatives, including ferrocene, aniline, and PEG, to
gold nanoparticles via the click reaction (87).
Magnetic nanoparticles are of great interest in pharmaceutical

sciences because of their good biocompatibility, injectability, and
highly specific accumulation in target tissues under a local magnetic
field (88). Several methods have been investigated to functionalize
magnetic nanoparticles (89), but Lin et al. were one of the first to take
advantage of click chemistry. They successfully attached several
different organic molecules to magnetic particles via the HDC reaction
(90). These molecules included Tn antigen, flag peptide, biotin, 2,4-
dinitrophenol (DNP), and maltose binding protein (MBP), each of
which had been functionalized to contain a terminal alkyne. The
magnetic nanoparticles, on the other hand, were azide functionalized.
Fluorescent tests showed that each of the bioconjugations was a
success and that there was no nonspecific binding of the 1,2,3-triazole
or unreacted azide. Furthermore, it was observed that the azide linker
length of the magnetic nanoparticles drastically affects how well MBP,
and most likely other proteins, can bind. As linker length increased, so
did binding efficiency, which was attributed to steric hindrance effects
between the bulky nanoparticle and MBP.
Deferent from Lin et al., White et al. chose ligands containing either a
phosphonic acid or a carboxylic acid group at one terminus to bind
strongly to the surface of a γ-Fe2O3 nanoparticle, and either an azide or
acetylene group at the other terminus to provide a chemical handle for
modification (91). Both benzyl azide and 5-chloropentyne were then
successfully conjugated through click chemistry. To demonstrate the
versatility of the strategy, an acetylene terminated polymeric ligand
was also attached to the nanoparticles.
4.2.2 Liposomes
Since the pioneering work of Bangham and co-workers in the late

1950s (92), liposomes have stimulated ongoing interest in
pharmaceutical sciences. Several liposomal formulations have been
approved by the US Food and Drug Administration (FDA) for clinical
use, especially for the treatment of cancers, over the past decade (93).
However, this particular drug carrier still have not attained its full
potential; there are still some aspects that need optimization. Their
surfaces have to be modified to avoid uptake by the phagocytic cells of
the reticular endothelial system (RES), drug release mechanisms need
to be improved so that the drug is completely and exclusively
deposited at the disease target site, and more efficient strategies for
conjugating targeting moieties (such as peptides and antibodies) to
liposomal surfaces are required. Although great strides have been
taken to resolve each of these issues (94-96), the latter still remains a
problem. Many strategies are highly dependent upon chemical
reactions that are not well-controlled, which often lead to unwanted
products. One of the most popular methods involves the reaction
between functionalized carboxylic acid groups and primary amines to
form amide bonds (97). This method does not require prior ligand
modification, thus reduces the risk of losing ligand bioactivity, but
there is often a lack of specificity, resulting in an uncontrolled number
of covalent bonds between the liposome and the targeting ligands. A
much more efficient conjugation method relies on the reaction of thiol
and maleimide functional groups to produce stable thioether bonds
(98). However, in many cases, native thiol functional groups are either
absent or present in insufficient amounts. Recently, hydrazone
linkages have been studied for conjugation, which involves a mild
oxidation of hydroxyl groups to aldehydes (99). It is obvious that this
oxidation reaction can cause the ligand to loss its activity.
As already mentioned in previous sections, the HDC reaction is the

epitome of linker chemistry and has proven to be a highly useful
conjugation method. Despite this, few papers can be found relating
click chemistry to liposomal conjugations. Two of these articles will
now be discussed.
In a recent report Schuber et al. developed a novel strategy for

conjugating mannose ligands to the surfaces of preformed liposomes
using the click reaction (100). The resulting mannosylated liposomes
can serve as vehicles to target specific cells, such as human dendritic
cells (101). In this strategy, an alkyne terminated lipid anchor was
first incorporated into liposomes. Then an unprotected mannosyl
derivative was functionalized with an azide group and conjugated to
the surface of the liposome in one single step. The group found that
catalytic quantities of the ascorbate/CuSO4 system, however, were not
enough to drive the click reaction to completion. A stabilizing agent
that protects the Cu(I) oxidation state from degradation pathways had
to be added (for more information on the role of Cu(I)-stabilizing
agents in click chemistry refer to reference 3), which was found to
greatly accelerate the reaction with high yields and reasonable
reaction times (1 hr). This conjugation reaction did not damage the
integrity of the bilayers and did not significantly change the particle
size. Residual copper in the conjugated liposome was not measurable
(<0.03 μg/mL), meaning that the oxidation of unsaturated
phospholipids by copper ions is avoided (102).
Cavalli et al. investigated the facile in-situ surface modification of

liposomes using the click reaction (103). Firstly, a mixture of alkyne-
terminated 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE),
1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), and a lissamine
rhodamine derivative of DOPE (DOPE-LR) were sonicated together to
prepare unilamellar liposomes bearing terminal alkyne groups at their
surface. Then an azide-functionalized nitro-benzoxadiazole (NBD)
derivative, N3-Lys(NBD)-NH2, and CuBr were added to the vesicle
solution. Using a method based on fluorescence resonance energy
transfer (FRET), the reaction was followed in time, showing that the
chemical modifications truly did occur at the surfaces of the
liposomes. The reaction was completed within 4 hr, with no dramatic
size changes of the liposomes. These two studies clearly indicate the
speed and versatility of the HDC reaction, allowing both
carbohydrates and biologicals to be linked to liposome surfaces,
regardless of the other functional groups present.
4.3 Bioconjugation
The use of bioconjugation and biopharmaceuticals are quickly

becoming common practice. As of May 1st, 2007, an estimated 30
biopharmaceuticals are currently in clinical trials (104). This
popularity stems from the fact that bioconjugation reactions can serve
many purposes and satisfy many functional requirements. They are
frequently employed to increase aqueous solubility, reduce
immunogenicity, increase circulation time, and improve stability. They
can also be used for radiolabeling and to attach fluorescent tags.
Excluding drug discovery and polymer chemistry, the most common

pharmaceutical applications of click chemistry are bioconjugation
reactions. One reason for this is 1,2,3-triazoles make ideal linkers.
They are extremely water soluble, making in vivo administration much
easier. In fact, there electronic properties are very similar to amide
bonds, but they are not subject to the same hydrolysis reactions (3).
They are also stable in typical biological conditions, which tend to be
aqueous and mildly reducing in nature (2). Another advantageous
property is they are extremely rigid, ensuring that the two linked
substances are not interacting with each other (3). If the linker were
flexible then the substances could easily aggregate and/or react with
each other. Furthermore, side reactions at unintended sites of the
substances are a major problem in bioconjugation reactions. The fact
that azides and alkynes are relatively unreactive towards most
functional groups further makes this click reaction sought after; it
ensures that bioconjugation will only occur at the desired location(s).
Finally, as previously mentioned, this reaction tolerates many
different types of azides and alkynes. Primary, secondary, tertiary, and
aromatic azides will all undergo click chemistry; the steric bulkiness
of the attached group seems to be insignificant. This is important since
many biological molecules, such as globular proteins, are sterically
bulky. Following now is an overview of some bioconjugation studies
that have been performed via click chemistry.
4.3.1 Radiolabeling
Radiolabeling has become a powerful tool in the pharmaceutical

scientist’s arsenal. By connecting a radionuclide to a drug of interest,
one can track its in vivo distribution (105), what specific receptors it
attaches to (106), its metabolic pathway (105), etc. All that is required
is a radionuclide that has desirable decay characteristics and an
instrument that can detect the amount of radiation present. One
particular radionuclide that is commonly used is technetium-99m,
or 99mTc. This radioisotope has an ideal half-life of six hours and emits
readily detectable 140 keV gamma rays (107). It currently has several
applications in the medical field, ranging from identifying
angiogenesis via attachment to monoclonal antibodies to evaluating
ventricular function by conjugation to red blood cells (108,109).
A major obstacle with 99mTc, however, is it can be difficult to attach to

organic molecules. One of the most common methods used is to take
an organometallic precursor, such as the tricarbonyl molecule
[99mTc(OH2)3(CO)3]+ (110), and complex it to a tridentate ligand that is
already attached to the molecule of interest (107). This ligand is
typically based on the amino acids cysteine, lysine, and especially
histidine (111). The problems associated with this method, though,
are numerous. The synthesis of such ligands requires multiple steps,
which can be quite challenging, they are not always easily
incorporated into the molecules of interest, and they can cross react
with other functional groups present (107,111).
To avoid these problems, Mindt et al. turned to click chemistry (111).

Realizing that 1,2,3-triazoles share similar electronic properties to the
imidazoles in histidines and that they too might be able to complex
with organometallic precursors, the HDC reaction was employed. The
facts that triazoles can be made in one easy step and azides can be
easily incorporated into organic molecules made this strategy further
attractive. By reacting various alkynes and azides, allowing each to
complex with either [99mTc(OH2)3(CO)3]+ or [ReBr3(CO)3]2-, and
determining the complexation efficiencies, EC50 values as high as 2 to 3
× 10-7 M were obtained. These numbers are almost as high as that for
NЄ-methyl histidine (∼1 × 10-7 M), proving that 1,2,3-triazoles can
chelate organometallic precursors just as well as histidine. To prove
the versatility of the reaction, biomolecules from every major
classification were used (a peptide [bombesin], a nucleic acid
[thymidine], a carbohydrate, and a phospholipid) as click reaction
substrates and subsequently radiolabeled. The bombesin triazole
derivative, in particular, showed in vivo stability and receptor affinity
similar to that of a bombesin histidine derivative. This further
suggests that triazoles could be a beneficial alternative to histidine
when it comes to complexation with radionuclides.
4.3.2 Tagging E. coli
The proteins expressed on the surfaces of microbial cells can be used

and manipulated for various applications. They have proved quite
useful in a broad range of fields, including protein engineering,
screening for antibody fragments, whole cell catalysts, adsorbents for
bioremediation, etc. (112,113). A major limitation, however, is there
are only 20 naturally-occurring amino acids. If one could expand the
set of amino acids expressed or specifically replace certain amino
acids with synthetic derivatives then surface proteins could be further
manipulated and the number of potential applications would be
increased exponentially. In this direction, some research groups have
already demonstrated remarkable success (113).
Capitalizing on the high reliability of the HDC reaction, Link and Tirrell
incorporated an azide-containing synthetic amino acid into the
surface proteins of E. coli (113). The amino acid they chose was
azidohomoalanine (AHA). It had been shown previously that AHA can
replace methionine residues in proteins (114) and the outer
membrane protein C (OmpC) of E. coli contains three such residues
(115). A mutant E. coli containing nine methionine residues per OmpC
was also created for comparison. Once AHA had been incorporated
into OmpC for both strains, click reactions using a biotinylated alkyne
reagent were performed. Both cells were successfully biotinylated, but
only the mutant E. coli would bind with the large molecule avidin,
presumably due to less steric crowding surrounding the additional
methionine residues.
4.3.3 Tagging CPMV
When wanting to test the bioconjugation abilities of one or more
compounds, Cowpea mosaic virus (CPMV) is frequently used as the
scaffold. It can be made inexpensively on the gram scale and it is easy
to separate from small unreacted reagents. Furthermore, it contains
60 identical copies of an asymmetric two-protein unit, each of which
contains one cysteine and one lysine (9,116). These 60 cysteines and
lysines can be manipulated and attached to various functional groups
and biomolecules, as evidenced by countless studies (117-119).
In one study, conducted by Wang et al., fluorescein dye derivatives

were attached to both azide-functionalized and alkyne-functionalized
CPMV using click chemistry (116). Not only were all of the reactions
successful, but also their product yield was as high as 100%. Two
years later, in 2005, they were able to attach three more hemicyanine
dyes to CPMV using the same reaction (9). Purification can be simply
performed by dialysis or ultrafiltration.
4.3.4 Labeling DNA
Oligonucleotides represent a very important class of biomolecules.

Although they have been “shunned” by the pharmaceutical community
in the past due to lack of knowledge, they have recently found many
applications and are considered to be the most important tools to
many areas of research (120). They have been used for gene therapy
(121), as antisense agents to treat diseases like leukemia (122), as
molecular probes (123), etc. Adding different functional groups
further increases their versatility, especially when one considers that
functionalization can be introduced at either the 3′-end, 5′-end, or an
internal position. The newly added functional groups can serve as
handles for bioconjugation with a wide variety of biological molecules.
Current methods for DNA bioconjugation, however, are inefficient.
The procedure must be able to tolerate aqueous conditions, give high
yields, and the resulting linkage must be stable in biological conditions
(124). This is the perfect situation for click chemistry.
Utilizing the HDC reaction, Seo et al. were able to tag a fluorophore to
the 5′-end of single-stranded DNA (124). The oligonucleotide was
modified, through several reactions, to display a terminal alkyne at its
5′-end and the fluorophore contained an azido functional group. A
91% product yield was obtained, but no catalyst was used in the
reaction, leading to a mixture of 1,4-substituted and 1,5-substituted
1,2,3-triazole products. If a copper catalyst were used then it is highly
likely that only 1,4-substituted triazoles would be produced and that
the reaction would proceed much faster. Seela et al. took DNA labeling
one step further and synthesized nucleosides that each contained a
single terminal alkyne on their aromatic nucleobase (125). Modified
deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), and
deoxythymidine (dT) were all included. Using solid phase synthesis,
several oligonucleotides were subsequently synthesized, which either
contained one modified nucleoside, two, or none at all (to serve as
controls). The properties of the oligonucleotides and their duplexes
were not significantly affected by the modified nucleosides, as
evidenced by the similar melting temperatures when compared to the
controls. Reporter molecules containing azido functional groups were
then conjugated to the modified bases through click chemistry. Upon
conjugation the reporter molecules began to fluoresce, indicating that
the oligonucleotides had been successfully labeled. If made
commercially available, Seela’s modified nucleosides could make
oligonucleotide labeling trivial.
4.3.5 Polysaccharides
Polysaccharides are another major class of macromolecules that have

received limited attention. Carbohydrates make up 5 to 10% of plasma
membrane mass, in the form of glycoconjugates, and mediate a variety
of events, such as cell-cell recognition, metastasis, fertilization, and
immunological response (126,127). Alterations to cell surface
oligosaccharides have been linked to a number of different diseases,
including cancer and tuberculosis (127). Despite these pivotal
biological functions, however, carbohydrates are rarely found in
pharmaceutics. Their synthesis typically involves multiple reaction
steps and many stereoisomers, making it challenging to even the most
skilled chemists. Furthermore, their moderate affinity towards target
receptors and enzymes and poor pharmacological properties make
them seldom used as targeting moieties/lead compounds (2). Physical
modifications of the carbohydrates can improve both of these issues,
but chemical handles are restricted to alcohol functional groups and
the steric bulkiness of polysaccharides often results in low product
yields.
Due to the easy introduction of azide functional groups and high

reliability of the HDC reaction towards the bulkiest of reactants,
Liebert et al. used click chemistry to modify the surface of cellulose
(128). It was thought that the 1,2,3-triazoles themselves would
interact with biological targets through hydrogen bonding and dipole
interactions, increasing the overall affinity of cellulose towards its
desired target. The team first introduced azide functional groups into
cellulose through two simple steps: tosylation of the secondary
alcohol groups followed by azidation with sodium azide. The overall
percent yield ranged from 60% to 99%. Three different terminal
alkyne-containing low molecular weight compounds
(methylpropiolate, 2-ethynylaniline, and 3-ethynylthiophene) were
then mixed with three different strands of azide-celluloses in separate
reactions, using a copper(II) salt catalyst and sodium ascorbate. All
reactions were successful with product yields ranging from 75% to
98%, depending on the reaction conditions utilized.
In that same year, Hafrén et al. modified cellulose with click chemistry
but took a slightly different approach (129). In one single step,
terminal alkynes were introduced through the secondary alcohols of
cellulose using 5-hexynoic acid in a neat reaction. 3-Azidocoumarin
and copper catalyst were then added. Upon reaction, the coumarin-
cellulose compound began to fluoresce a bright blue color, indicating
that the reaction was successful, but no product yield was calculated.
The work of the Liebert and Hafren research teams clearly show that
polysaccharides can be modified via click chemistry. Both azide and
terminal alkyne functional groups can be easily introduced and the
HDC reaction still occurs in high yields despite steric hindrance.
Go to:
5. The Pitfalls of Click Chemistry
Up until now, this review may have given the impression that the
Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of terminal alkynes
and azides is an “invincible” reaction, always giving high yields no
matter what the situation. However, the HDC reaction has a number of
limitations, each of which will be discussed. Firstly, like with any
cycloaddition, if the diene (the azide in the case) is too electron
deficient then it will not undergo the reaction. The energy of its
ground state configuration is far too low for it to interact with a
dienophile (the terminal alkyne). Likewise, the dienophile cannot be
too electron rich. These situations are highly unlikely to occur, though,
and require functional groups that are not commonly seen in
biological systems or administered drugs.
A more common problem is alkyne homocoupling. This occurs when

an alkyne reacts with a second alkyne instead of the azide. As shown
in Figure 7, there are several alkyne homocoupling side reactions that
can occur, three of which include Glaser (130), Straus (131), and
Eglinton couplings (132,133). Some of these require a CuI catalyst
(Glaser and Straus), while others require CuII (Eglinton) (133). Some
need the presence of oxygen to react (Glaser) while others can
continue in inert atmosphere (Straus) (133). Most of these reactions,
though, can be minimized by using a sterically bulky base (3). It has
been shown that the smaller the base, the more it stabilizes the
reactive intermediates of the homocoupling reactions (not shown),
and the lower the percent yield of the cycloaddition (3,134,135). The
small bases tetramethylethylenediamine (TMEDA), pyridine, and
triethylamine are frequently the culprits when these side reactions
occur (133,134).
Fig. 7
Three types of alkyne homocouplings that can lower the percent yield of the HDC
reaction: Glaser (top), Straus (middle) and Eglinton (bottom).
A less common problem is CuI saturation. In order for the click

reaction to take place, the CuI-acetylide complex intermediate has to
have physical contact with the azide (see section 3.1 for the
mechanism). If the complex is closely surrounded by terminal alkynes,
however, then there is a chance that the alkynes will chelate with the
complex, thereby “saturating” it (3). This effectively prevents any
azide functional groups from reaching the complex and performing
displacement. CuI saturation is rare, though, as it requires a dienophile
that contains multiple terminal alkynes that can coordinate to a single
location. One notable example came through the work of Zhao et
al. (136). A substrate containing four terminal alkynes in close
proximity was unable to undergo an HDC reaction. However, when the
alkynes were replaced with azide functional groups, the substrate
readily reacted.
The stability of some azides may also be a limitation. It is known that if

the ratio of nitrogen atoms to carbon atoms in an organic molecule
exceeds, or is equal to, one then the molecule should be considered
explosive and very dangerous. As an example, methyl azide often
decomposes explosively and heavy-metal azides are frequently used
as detonators (8). All that is required is a slight input of external
energy, such as heat or pressure (8). Fortunately, this is generally not
a major issue for pharmaceutical research, which tends to focus on
larger molecules with high carbon contents.
The click reaction also has challenges pertaining specifically to the
field of pharmaceutical sciences. One of the most obvious
disadvantages is it requires a copper catalyst. Although the human
body requires copper in order to function, excessive intake can lead to
drastic consequences. Some associated side effects include hepatitis,
neurological disorders, kidney diseases, and Alzheimer’s disease
(137). The reason for this toxicity stems from the fact that copper can
easily accept and donate single electrons to change oxidation states,
thereby allowing it to catalyze toxic reactions, such as the in
vivo reduction of hydrogen peroxide to form hydroxyl free radicals
(138). Therefore, in order for the click reaction to find in
vivo applications, the copper catalyst must be completely removed.
This may not always be an easy task, but a few research groups have
demonstrated some success. As an example, in the click-PEG delivery
system developed by Liu et al. (67), about 98% of the copper was
effectively removed by incubation with ethylenediaminetetraacetic
acid (EDTA), followed by dialysis (unpublished data). However, this
approach can only be applied to large molecular weight structures.
Another group, Veinot et al., has used oxide-capped metallic iron
nanoparticles (139) as Cu sequesters. After performing a click
reaction with phenyl azide and benzyl acetylene, the crude product
was incubated with the nanoparticles twice and filtered twice.
Analysis revealed that the copper concentration had been significantly
reduced from 2026 ppm to 4.6 ppm.
Another challenge involves biocompatibility of 1,2,3-triazoles. Despite

the fact that they were first identified over a century ago, not much is
known about their biological pathways. The individual toxicities of
some 1,2,3-triazole-containing compounds have been extensively
scrutinized, but no generalities have been established. As evidenced
by a literature search via SciFinder Scholar on November 12th, 2007
there are no reports reviewing the in vivo metabolism of 1,2,3-
triazoles. This is very surprising since many drug candidates have
been synthesized via click chemistry and 1,2,3-triazoles have long
been used as bioisosteric replacements of amide functional groups. In
fact, the very intent of click chemistry is for drug discovery.
The final limitation discussed in this paper is, at the present, many
“click-readied” building units and “click products” are not yet
commercially available. This means it is the responsibility of the
pharmaceutical scientist to synthesize his/her own compounds or to
collaborate with others who can. Even though click reactions are some
of the easiest and high-yielding reactions known, this task can seem
daunting to even the most experienced pharmaceutical scientist,
whose typical background is not in organic synthesis. Fortunately,
click chemistry is still a relatively new idea. As time progresses and
this methodology gains increased acceptance, more and more
products may be made available on the market.
Go to:
6. Conclusions
Despite the fact it debuted less than ten years ago, click chemistry is
quickly revolutionizing the way scientists view their research.
Although all click reactions are easy to use, give high reaction yields,
do not require long reaction times, are regiospecific, and are easy to
purify, the CuI-catalyzed Huisgen 1,3-dipolar cycloaddition of azides
and terminal alkynes has emerged as the most popular click reaction
by far. It has found numerous applications across a wide variety of
disciplines, including materials research, polymer chemistry, and the
pharmaceutical sciences. It is particularly sought after by the
pharmaceutical community for its tolerance of typical biological
conditions, tolerance of most functional groups, and for the high
aqueous solubility of 1,2,3-triazoles. Specifically, the HDC reaction has
been used to synthesize novel polymeric delivery systems,
dendrimers, to cross-link micelles, and to modify the surfaces of
various nanoparticular delivery systems. The rigidity of 1,2,3-triazoles
also makes the reaction ideal for bioconjugations. Oligonucleotides,
proteins, polysaccharides, viruses, and bacteria such as E. coli have all
been successfully conjugated to various substances using this
approach.
The HDC reaction, however, is not perfect. There are a few important
limitations that need to be considered. The most significant is it
requires a copper catalyst. High level of copper in the body can lead to
serious, even deadly, consequences. It is also important to realize that
systemic studies need to be conducted to confirm the biocompatibility
of 1,2,3-triazoles. Other less serious problems include CuI saturation
and alkyne homocoupling.
Though the HDC reaction has several limitations, it is still one of the
most versatile and beneficial chemistry tools for pharmaceutical
applications. We believe that as the industry recognizes the
importance and large potential of this reaction and provides all of the
basic building blocks, it will continue to grow in popularity and
contribute to many major advances inpharmaceutical sciences and
other research fields. Hopefully as time progresses, other click
reactions will also gain momentum and add additional items to our
chemistry “tool box.”
Go to:
Acknowledgements
We acknowledge financial supports from NIH Grant AR053325 and
the College of Pharmacy, University of Nebraska Medical Center
(teaching assistantship to CDH).
Go to:
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Abstract
Click chemistry involves highly efficient organic reactions of two or more highly
functionalized chemical entities under eco-benign conditions for the synthesis of different
heterocycles. Several organic reactions such as nucleophilic ring-opening reactions, cyclo-
additions, nucleophilic addition reactions, thiol-ene reactions, Diels Alder reactions, etc. are
included in click reactions. These reactions have very important features i.e. high functional
group tolerance, formation of a single product, high atom economy, high yielding, no need
for column purification, etc. It also possesses several applications in drug discovery,
supramolecular chemistry, material science, nanotechnology, etc. Being highly significant
and valuable, we have elaborated on several aspects of click reactions in organic synthesis in
this chapter. Recent advancements in the field of organic synthesis using click chemistry
approach have been deliberated by citing last five years articles.
Keywords
 click chemistry
 organic synthesis
 eco-benign synthesis
 selectivity
 atom economy
 cyclo-addition
Author Information
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1. Introduction
Presently, researchers are paying considerable attention to devise eco-friendly approaches for
organic transformations. There has been a significant hike in interest among the scientists for
more environmentally acceptable processes in the chemical industries. Synthetic chemistry
has led us to the development of more potent structural analogs of natural products. The high
therapeutic efficiency, bioavailability, and pharmacological characteristics of synthetic
molecules have increased their use in medicinal chemistry as compared to natural products.
Pharmaceutical chemistry encompasses the design, synthesis, and evaluation of compounds.
In designing drugs, there is an upsurge demand for eco-benign pathways to accomplish the
green aspects of chemistry. Novel green pathways play a vital role in the synthetic chemistry
field by eradication of harmful solvents and chemicals or suitable handling of waste
materials. The quest for new and proficient approaches for the synthesis of numerous
biologically active scaffolds has made click chemistry a promising approach in chemistry.
Click chemistry is a fruitful approach for the fabrication of molecules.
Huisgen and co-workers demonstrated a click reaction, Cu(I)-catalyzed azide–alkyne

cycloaddition (CuAAC). The advanced use of this reaction and click chemistry was
introduced by K. Barry Sharpless in 2001. The term click chemistry not only refers to the
reaction which occurs fast but also to those that involve twelve principles of green
chemistry i.e. selective, easier product isolation, mild reaction condition, high yield, good
atom economy, avoid toxic catalyst and solvent, and so on. They encompass reactions that
are high yielding, fast, modular, and wide in scope. They are practical and tolerant for a
variety of functional groups. Finally, the product isolation is expected to be effortless due to
lack of by-products. These vast characteristics make click chemistry a powerful tool that
paves a path in several fields of research viz. designing of drugs and lead structures
[1, 2, 3, 4]. Therefore the synergy between these disciplines has given rise to an area of
intense research activity. The click chemistry has been such an engrossing topic of research
that a lot of review articles have been published so far which explained its applicability in
various fields of chemistry like manufacturing and alteration of metal–organic frameworks
[5], making devices in bio-sensing system for responsive copper identification [6], designing
bio-adhesives for hastening wound closure [7], in virus-related research [8], generation of
biosensors [9], proteomics analysis [10], in strategy for indirect 18F-labeling [11], in vivo bio-
imaging [12], to identify the interaction of curcumin with protein [13], synthesis of polymers
and material science [14], for surface modification [15], and so on. In this chapter, the state-
of-art modernization with a particular focus on click chemistry assisted synthesis and their
uses in various fields of science have been discussed. An attempt has been done to prepare an
outline of the importance of click chemistry and its foremost requirement in the research area.
It is predicted that this methodical study will pave the way for future opportunities in this
direction and design of safer, economical, and eco-friendly pathways.
A DVERTISEMENT
2. Green aspects of click reactions

According to Sir John Cornforth, a Noble Prize laureate in chemistry in 1975, ideal reaction
has been defined as “The ideal chemical process is that which a one-armed operator can
perform by pouring the reactants into a bath-tub and collecting the pure product from the
drain hole” [16]. Click reactions are designed in such a way that it involves all the twelve
principles of green chemistry. Click chemistry includes synthetic methods that are designed
to maximize the inclusion of all resources used in the process into the final product. Due to
involvement of addition and rearrangement reactions, they have high atom economy. The
products are designed with maximum efficacy and minimum cytotoxicity [17]. The green
aspects have been depicted in Figure 1.
Figure 1.
Green aspects of click chemistry.
A DVERTISEMENT
3. Role of click reactions in synthetic chemistry

Click chemistry includes a cluster of powerful linking chemical reactions that are easy to
perform, have high yields, require no or minimal purification, and are flexible in the
unification of different structures without the prerequisite of protection steps. Molecular
diversity, modularity, and efficiency are essential in synthetic organic chemistry and expected
to be involved in the preparation of several complexes and multi-purpose compounds. In
general “Click Chemistry” is a class of biocompatible reactions, to link desired substrates
with particular biomolecules. Natural products are produced by joining tiny modular
units via biosynthesis as well as photosynthesis [18]. Click chemistry provides a route for the
synthesis of several heterocyclic scaffolds, amino acids, triazole-fused heterocycles, peptides,
and chromophores [19, 20].
3.1 Classification of click reaction
There is no specific classification of click reactions. The chief requisite for “Click
Chemistry” is well met by reactions that take place in nature and their mimic in the laboratory
is the closest and most desirable to the mind and spirit of most synthetic organic chemists.
Usually, four main classifications of click reactions have been identified [21, 22].
 Cycloadditions: These refers to 1,3-dipolar cycloadditions reactions and hetero-

Diels-Alder cycloadditions.
 Nucleophilic ring-opening reactions: This classification belongs to the opening of
strained heterocyclic electrophiles, such as epoxides, aziridines, aziridinium ions,
cyclic sulfates episulfonium ions, etc.
 Nucleophilic addition reaction: It includes the reaction of carbonyl groups like
formation of hydrazones, urease, thiourease, oxime ethers, aromatic heterocycles,
amides, etc.
 Additions to carbon–carbon multiple bonds: It involves epoxidation,
dihydroxylation, aziridination, nitrosyl halide addition, sulfenyl halide addition, and
certain Michael additions.
Presently, click chemistry inspired synthesis has become the most fascinating approach.
Several multi-component reactions have been designed in an eco-friendly manner like aldol
condensation followed by Michael addition, Ugi reaction/aldol reaction, Ugi
reaction/Huisgen reaction, Ugi Reaction/Diels-Alder reaction, Ugi reaction/Heck reactions,
Michael addition/Mannich reaction, etc. [23]
The most famous click reaction is the classical reaction between an azide and an alkyne. Both
the substrates do not react under physiological conditions and go through a cycloaddition
reaction only at a particular temperature. The uncatalyzed reaction is usually slow and not
regio-selective. On the other hand, it was found that the use of electron-deficient terminal
alkynes can cause 1,4-regioselectivity to a great extent. These factors limit the use of
uncatalyzed Huisgen cycloaddition as an efficient conjugation pathway [24].
3.2 Metal-catalyzed approach for the click synthesis
3.2.1 Synthesis of triazole derivatives
Metals have been used to catalyze several click reactions. The mechanism of metal catalyzed
azide alkyne click reaction involves formation of π-alkynyl complex with metal followed by
complexation of azide by metal of the π-coordinated triple bond. After cyclization,
metallacycle is formed followed by the reductive elimination to afford the relevant 1,2,3-
triazole. Several metal like Cu, Ru, Ag, Au, etc. have been employed to accomplish click
reactions [25, 26, 27, 28]. This section has been divided in two subsections:
3.2.1.1 Metal-catalyzed synthesis of triazole derivatives
Transition metals have been used to catalyze several organic reactions as they provide large
surface area and they have vacant d-orbitals due to which they can show variable oxidation
state that help in generation of intermediate for organic synthesis [29, 30]. The common
process for the click reaction is the transition metal catalyzed synthesis of 1,2,3-triazoles. 1,3-
dipolar cyclo-addition of an azide and an alkyne catalyzed by Cu is the most extensively used
click-chemistry pathway due to its high selectivity and simplicity [31]. In 2014, Guo and co-
workers synthesized β-cyclodextrin derivatives (1) using mono-6-azidocyclodextrin and
aromatic aldehydes by CuI-catalyzed azide–alkyne cyclo-addition. The mono, di, and tri
derivatives were synthesized upto 75% yield under mild reaction conditions [32] (Figure 2).
Figure 2.
Synthesis of β-cyclodextrin derivatives using click chemistry approach.
Later on, Kumar et al. [33] designed a library of new nucleosides (2 and 3) having 1,2,3-
triazole scaffold at the 2″-position of the sugar nucleus. It was synthesized by 2″-azidouridine
using the copper (I)-catalyzed Huisgen–Sharpless–Meldal 1,3-dipolar cyclo-addition reaction
(Figure 3). The reaction gave 52–82% yield and 1,4-disubstituted 1,2,3-triazoles were
obtained.
Figure 3.
Synthesis of library of traizole substituted nucleosides.
Tale and co-workers also synthesized 1,2,3-triazoles in excellent yields using (1-(4-
methoxybenzyl)-1-H-1,2,3-triazol-4-yl)methanol (MBHTM) ligand (1.1 mol%) and
CuSO4 (1 mol%) as a catalyst and sodium ascorbate (5 mol%) in DMSO:H2O(1:3) as a
solvent [34]. Shamla and co-workers synthesized coumarin substituted triazole derivatives (4)
in good yields using 4-bromomethylcoumarins, terminal alkynes, and sodium azide in the
presence of triethylamine and CuI as a catalyst [35] (Figure 4).
Figure 4.
Synthesis of coumarin substituted triazole derivatives.

Yarlagadda et al. synthesized N-((l-benzyl-slH-l,2,3-triazol-5-yl) methyl)-4-(6-methoxy
benzo[d]thiazol-2-yl)-2-nitrobenzamide derivatives (5) and examined their anti-microbial
activity. Among these compounds, compounds 5a, 5 h, 5i possessed promising activity in
comparison to standard drug ciprofloxacin and miconazole (Figure 5) [36].
Figure 5.
Synthesis of N-((l-benzyl-lH-l,2,3-triazol-5-yl) methyl)-4-(6-methoxy benzo[d]thiazol-2-yl)-

2-nitrobenzamide derivatives.
Anand et al. designed Cu(I) catalyzed regio-selective synthesis of iso-indoline-1,3-dione

linked 1,4 coumarinyl 1,2,3-triazoles (6) and Ru (II) catalyzed pathway of 1,5 coumarinyl
1,2,3-triazoles in high yields with no need for further purification (Figure 6) [37].
Figure 6.
Synthesis of iso-indoline-1,3-dione linked 1,4 coumarinyl 1,2,3-triazoles derivatives.
Anandhan et al. prepared a series of triazole-based macrocyclic amides (7) via click

chemistry using CuSO4 (5 mol%), sodium ascorbate (10 mol %) in the presence of H2O/THF
(1:1), RT. The synthesized compounds showed good anti-inflammatory activity although at
low concentration (50 μg/mL) in comparison to the reference drug prednisolone (Figure 7)
[38].
Figure 7.
Triazole based macrocyclic amides.
Li and co-workers designed triazole derivatives (8 and 9) by click chemistry using

CuSO4∙5H2O (0.1 g) and ascorbic acid (0.1 g) in tBuOH/H2O as a solvent and investigated
their applications to synthesize self-assembled membrane against copper corrosion. As per
the investigation results, it was found that 2-(1-tosyl-1H-1,2,3-triazol-4-yl)-ethanol (TTE) (8)
and 2-(1-tosyl-1H-1,2,3-triazol-4-yl)-propan-2-ol (TTP) (9) coating on film can sturdily
decrease the corrosion caused by copper in 3 wt.% NaCl solution and the inhibition
effectiveness of TTP and TTE were 93.1% and 89.4%, respectively (Figure 8) [39].
Figure 8.
Derivatives of triazole.
Savanur and co-workers developed facile click chemistry inspired synthesis of triazole ring
fused coumarin and quinolinone derivatives using CuSO4 (10 mol%), sodium ascorbate
(10 mol%), H2O:PEG, RT followed by K2CO3/DMF at 50–60 °C and examined their anti-
microbial activity. Among the synthesized compounds, compounds 10j,
11 g and 12f displayed good anti-bacterial activities. Derivatives 10e and 10j were found
highly active against yeast strains. Compound 11f was highly active against filamentous
strain A. niger and yeast fungi [40] (Figure 9).
Figure 9.
Triazole ring fused coumarin and quinolinone derivatives.
Yarovaya et al. [41] fabricated a conjugate of cytisine with camphor having triazole ring
using click chemistry pathway by employing CuSO4∙5H2O, sodium ascorbate, t-BuOH/H2O.
The designed molecules were examined for in vitro antiviral activity against
A/PuertoRico/8/34 influenza virus (H1N1). The compound (13) has highest inhibition
activity with IC50 = 8 ± 1 μmol (Figure 10).
Figure 10.
Cytisine conjugated triazole derivative.
Khanapurmath et al. synthesized various derivatives of triazole by click chemistry approach

using CuSO4 and ascorbic acid in H2O:DMF solvent and assessed them
against Mycobacterium tuberculosis H37Rv. 6-Methyluracil and theophylline mono-triazole
compounds 14(a-d) and bis-triazole compounds, 15(a-e) showed reasonable inhibition of M.
tuberculosis H37Rv, with MIC values in the range of 55.62–115.62 μM. Benzimidazolone
bis-triazole derivatives 16(a-n) inhibited M. tuberculosis H37Rv with MIC 2.35–18.34 μM
(Figure 11) [42].
Figure 11.
Methyluracil and theophylline mono-triazole compounds and Bis-triazole compounds.
3.2.1.2 Metal Nano-particle based triazole synthesis
Green synthesis is the fundamental requirement of present synthetic protocol and use of
nanoparticles (Nps) is one of the key tackle for organic transformations. NPs are microscopic
particles with dimension between 1–100 nm. These are used as catalysts because they provide
large surface area, high catalytic activity, nontoxic, heterogeneous nature, etc. In lieu of this,
Chetia et al. designed copper Nps (nano particles) supported over hydrotalcite and used these
Nps (15 mg) to catalyze 1,3 dipolar cycloaddition reaction to form 1,4 disubstituted-1,2,3-
triazoles (17) (Figure 12) at room temperature using ethylene glycol as a solvent. The catalyst
is heterogeneous, easily recyclable, and further reusable [43]. In this context, Poshala and co-
workers developed copper Nps (0.1 mmol) using rongalite as a reducing agent and examined
their catalytic efficiency in synthesizing triazole (18) derivatives in the presence of β-
cyclodextrin (0.02 mmol) [44].
Figure 12.
Triazole derivatives.
Chavan et al. [45] designed a click chemistry assisted MCR strategy for the synthesis of
spirochromenocarbazole tethered 1,2,3-triazoles (19) using CuINps supported over cellulose
(7 mol%) as a catalyst in the presence of DMF:Water (1:2) (Figure 13). The synthesized
compounds were screened for anti-cancer activity against MCF-7, HeLa, MDA-MB-231, A-
549, PANC-1 and THP-1. Compounds 19i and 19j were observed to be the most potent
against MCF-7 with IC50 = 2.13 μM and 4.80 μM respectively. Compound 19 k was the most
potent one against MDA-MB-231 with (IC50 = 3.78 μM). All the products were found to be
safe against the human umbilical vein endothelial cells (HUVECs).
Figure 13.
Spirochromenocarbazole tethered 1,2,3-triazole derivatives.
Elavarasan et al. prepared nano rod shaped triazine functional hierarchical mesoporous
organic polymers (HMOP) containing Cu metal. This catalyst was used to synthesize triazole
derivatives (20) via stirring at 80 °C in the presence of water as a solvent [46]. In the same
year 2019, Gholampour et al. synthesized a library of 1,4-naphthoquinone-1,2,3-triazole
hybrids (21) using CuSO4 (0.15 mmol) and sodium ascorbate (.05 mmol) catalyzed click
chemistry approach from 2-(prop-2-ynylamino)naphthalene-1,4-dione and different
azidomethyl-benzene analogs. The anti-cancer activity of synthesized compounds was
anticipated against three cancer cell lines (MCF-7, HT-29 and MOLT-4) by MTT assay. The
compound 21f possessed the highest activity [47]. In continuation to this, magnetic
CuFe2O4/g-C3N4 hybrids were synthesized and their catalytic activity was examined in the
synthesis of triazole derivatives (22 and 23) using terminal alkyne, azide, epoxide or
haloarene (Figure 14) [48].
Figure 14.
Structures of different triazole derivatives.
Pourmohammad et al. synthesized (CuI@[PMMA-CO-MI]) (0.02 g) nano catalyst and

employed them in the synthesis of triazole derivatives using terminal alkynes, α-haloketones
or alkyl halides and sodium azide in H2O at RT to give 1,4-disubstitued 1,2,3-triazoles (24)
(Figure 15). The catalyst being heterogenous and regioselective gave high yields in short
reaction times [49].
Figure 15.
Triazole derivatives.
Thanh et al. [50] designed a hybrid structure of chromene and triazole by applying click
chemistry approach for the synthesis of 1H-1,2,3-triazole-tethered 4H-chromene-D-glucose
analogs (25) using Cu@MOF-5 (2 mol%) as a catalyst to afford 80–97.8% yields. The copper
supported over metal organic frame work was found better catalyst in comparison to
conventional catalysts viz. CuSO4.5H2O-sodium ascorbate, CuI, Cu Nps, CuIM2(IM is
imidazole) as it afforded high yields of desired products in less reaction time and in the
presence of ethanol whereas other required long reaction time and non green solvent. All the
derivatives were assessed for in vitro anti-microbial activity with MIC values in the range of
1.56–6.25 μM (Figure 15).
3.2.2 Synthesis of other organic molecules
Click chemistry has been used to synthesize biologically active hybrids of several synthetic
organic molecules. In lieu of this, Sharova et al. [51] demonstrated click chemistry inspired
phosphorylation of anabasine, camphor, and cytisine using Cu assisted 1,3-diploar
cycloaddition reaction. Later on, Touj et al. [52] synthesized copper N-heterocyclic carbene
(Cu-NHC) complexes using benzimidazolium salt as a catalyst. These complexes were
further used for the synthesis of triazole derivatives (26) (Figure 16).
Figure 16.
Synthesis of triazole derivatives.
The reaction involved mild reaction conditions, water as a green solvent with low catalyst
loading, no need of further purification which made the protocol eco-friendly. Bernard et al.
[53] investigated a cost effective, and convenient click chemistry inspired synthesis of
cyclooctyne (27) and trans-cyclooctene (28) using inexpensive Cu powder as a catalyst
(Figure 17).
Figure 17.
Synthesis of cyclooctyne and trans-cyclooctene.
Qui et al. [54] synthesized parthenolide–thiazolidinedione (29) hybrids using click chemistry-

mediated coupling. The compounds were screened for anti-proliferative activity against
prostate (PC3), breast (MDA-MB-231), and human erythroleukemia cell line (HEL) by MTT
assay. The compound (29f) having 3,5-dimethoxyphenyl group exhibited the highest
inhibitory effect against HEL (IC50 = 2.99 ± 0.22 μM), MDAMB-231 (IC50 = 2.07 ± 0.19 μM),
and PC3 (IC50 = 3.09 ± 0.20 μM) (Figure 18).
Figure 18.
Synthesis of parthenolide–thiazolidinedione and 3’-O-1,2,3-triazolyl-guanosine-5′-o-

monophosphate derivatives.
Senthilvelan et al. [55] designed synthesis of 3’-O-1,2,3-triazolyl-guanosine-5′-o-

monophosphate (30) from in situ generation of azide from the resultant bromide followed by
copper and β-cyclodextrin catalyzed cyclo-addition with 3′-O-propargyl guanosine
monophosphate in aqueous media. The designed pathway has high regioselectivity and gave
good yield of products (Figure 18).
3.3 Metal-free approach for click reaction

Several new metal-free click chemistry assisted syntheses of heterocyclic scaffolds have been
designed up to date. These pathways involve a variety of functional group tolerance in the
substrate of cyclo-addition reaction. These synthetic pathways can be achieved under mild
conditions and give high yields of desired products using organo-catalyst [56, 57]. In 2010,
Fokin and co-authors developed the first transition metal-free synthesis of 1,5-diaryl-1,2,3-
triazoles (31) employing azide-alkyne cyclo-addition [58] (Figure 19Method 1). In this
reaction, tetraalkylammonium hydroxide was used as the catalyst that provided moderate to
high yield of products. Later on, in 2013, Ramachary and co-workers [59] achieved a region-
selective synthesis of N-arylbenzotriazoles at room temperature using a cyclic enone and an
arylazide under pyrrolidine catalysis at room temperature. Additional aromatization by DDQ
gave fused heterocyclic scaffolds (32) (Figure 19Method 2). In the subsequent year, a one-
pot tandem, Knoevenagel/azide-alkyne cycloaddition reaction between indole, aromatic
aldehydes or pyrazole and phenylazide was fruitfully accomplished in the presence of
piperidinium acetate in methanol to furnish the desired triazole derivatives (33) (Figure
19Method 3) [60].
Figure 19.
Metal-free synthetic route of triazole based heterocycles.
In 2018, Han et al. [61] developed a metal-free and solvent-free approach for the synthesis of
4-trifluroacetyl 1,2,3-triazoles in good yields with great selectivity. The synthesized
compounds were examined for the anti-cancer activity and compound 34b possessed superior
activity as compared to others against HePG2 (0.0267 μmol/mL) (Figure 20).
Figure 20.
4-Trifluroacetyl 1,2,3-triazole derivatives.
In the same year, Tan et al. [62] used thiol-ene click chemistry for the controlled
functionalization of poly vinylidene fluoride in the presence of a base. The mechanism of the
reaction suggests that it involves addition reaction followed by both Markonikov and anti-
Markonikov mechanism and furnishes the same product. Later on in 2019, Moore and co-
workers [63] designed a novel methodology for the synthesis of ionic liquids which were
based on fluoroalkynyl imidazolium using thio-ene/yne click chemistry. The pathway has
high conversion efficiency and high yields with no need for further purification.
3.4 Visible light assisted click chemistry
Visible-light-assisted organic transformations have received a huge response in chemical

synthesis in order to design environmentally friendly approaches. The synthesis using
economical, easily available visible-light sources have become vanguard in the synthetic
chemistry as a prevailing approach for the activation of small molecules to furnish the desired
products [64, 65, 66].
Burykina et al. [67] synthesized different kinds of vinyl sulphides (35) in high yields with
good selectivity using thiol-yne click reaction using visible light. The designed pathway is
transition-metal-free and gave Markonvikov-type product through a radical photo-redox
pathway (Figure 21Method 1).
Figure 21.
Visible light assisted synthesis of vinyl sulphide.
Recently, Wu et al. [68] synthesized triazole analogs (36) through photo-redox electron-

transfer mechanism. The authors inspected the reaction of benzyl azide with phenylacetylene
using diverse photo-catalysts under ambient reaction conditions like room temperature (RT),
air, and visible light irradiation. The catalyst (piq)2Ir(acac) or TPPT-Cl catalyzed the
formation of triazole derivatives. The designed pathway is high region-selective, high
yielding, having a high atom economy, and using solar catalysis (Figure 21Method 2).
3.5 Ultrasound assisted click chemistry
Ultrasound assisted reactions are milder and faster. The mechanism of ultrasound is based on
an acoustic cavitation phenomenon. This technology hastens the reaction in both
heterogeneous and homogeneous media, due to amplified energy intake. It shortens the
reaction time and augments the competence of the system by triggering the catalyst surface
area and removing deposited impurities [69, 70]. A decades ago, Cintas et al. [71] depicted
the synthesis of 1,4- disubstituted 1,2,3-triazole analogs using Cu under ultrasound irradiation
exclusive of a ligand. Later on, a heterogeneous catalytic system, Cu(II) doped clay was used
at RT with ultrasonic irradiations [72]. The use of heterogeneous catalyst evaded needless
complexity due to copper (I) salt redox protocol that involved the presence of ligands and
protecting agents. The reaction is eco-friendly, easy to prepare, and recoverable. One-pot
synthesis of 1,4-disubstituted-1,2,3-triazoles was successfully achieved using a benzyl or
alkyl halide, sodium azide, and a terminal alkyne under these conditions [73]. The formation
of triazole starting from a TMS protected alkynylglycoside was also demonstrated under
ultrasound conditions with in situ deprotection of TMS group [74]. In 2020, Kritchenkov et
al. synthesized triazole chitin derivatives and used them in the synthesis of Pd(II) complexes.
Initially, ultrasound-assisted interaction of chitin with 1-azido-3-chloropropan-2-ol gave
azido chitin and was further converted to triazole derivatives (37) that were used as ligands
for the complex formation (Figure 22) [75].
Figure 22.
Synthesis of ultrasound assisted 1-azido-3-chloropropan-2-ol azido chitin derivatives.
3.6 Microwave-assisted click chemistry
The use of microwave irradiation in cyclo-addition reactions for click chemistry has also
been comprehensively deliberated. It allows efficient internal heat transfer and therefore
decreases the reaction time as well as enhances the reaction rate with high yield [76, 77]. The
increased temperature can be used over short periods thus avoiding decomposition or
polymerization. Ashok and co-workers demonstrated the synthesis of 1,2,3-triazole analogs
using microwave irradiations in 8–10 min and examined their antimicrobial activity [78]
(Figure 23Method 1). This method has also been applied in the preparation of 1,2,3-triazole
analogs of nucleosides [79] (Figure 23Method 2). In general, those reactions which require
prolonged conventional heating are accomplished in just 10–15 min using microwave
irradiation. A chronological one pot Ru catalyzed cycloaddition was also designed from
primary aryl or aliphatic bromides (Figure 23Method 3) [80].
Figure 23.
Microwave assisted synthesis of triazole based scaffolds.

A DVERTISEMENT
4. Click chemistry in polymer synthesis

In the past two decades, various polymers have been introduced through ionene synthesis,
click chemistry, and Michael addition via polycondensation and polyaddition process. Click
chemistry reactions are known as reliable, powerful, high-yielding, and selective for the
synthesis of novel and combinatorial compounds via Diels Alder cyclo-additions, copper-
catalyzed azide-alkyne cycloadditions (CuAAC), and azide nitrile cycloadditions process
[81, 82]. In 2013, Pasini reviewed the utility of click reaction for the efficient synthesis of
macrocyclic structures like polymers, bio-conjugates, and dendrimers in different contexts
[83]. Recently, Arslan and Tasdelen systematically reviewed the applications of click
chemistry in polymer design and synthesis, and studies based on their architecture like block,
cyclic, star, hyperbranched, and graftbrush comb polymers [84].
In 2018, Acik and co-authors demonstrated a simple copper (I)-catalyzed azide-alkyne cyclo-
addition “click” reaction for the synthesis of polypropylene-graft-poly(L-lactide) copolymers
(PP-g-PLAs) using different feeding ratio of alkyne end-functionalized poly(L-lactide) azide
and side-chain functionalized polypropylene in the presence of CuBr/PMDETA and CuAAC
[85]. This polymer exhibited special characteristics like good thermal property, wettability
and biodegradability.
Öztürk and companions introduced efficient click chemistry inspired synthesis of an

amphiphilic copolymer (41) from the reaction of propargyl-PEG and terminally azidepoly(ε-
caprolactone) in CHCl3 at ambient temperature (Figure 24) [86]. This method displayed a
synergistic arrangement of hydrophilic PEG and crystalline PCL to furnish novel materials
with good applicability.
Figure 24.
Poly(CL-co-EG)star-type amphiphilic coploymer.
Yang et al. synthesized poly(3-hexylthiophene)-multiwalled carbon nanotube (P3HT-

MWCNT) hybrid materials from in-situ click chemistry using Cu(I) / DBU catalytic system
[87]. This novel hybrid also termed as organic–inorganic donor-acceptor material displayed
special characteristics such as better thermal stability, higher melting point of 243.2 °C, good
solubility, and optical properties.
Wang et al. reported a novel and efficient method for the synthesis of amphiphilic star-like
rod-coil block copolymer poly(acrylic acid)-blockpoly(3-hexylthiophene) through the
combined effect of atom transfer radical polymerization, quasi-living Grignard metathesis
method, and thiol–ene click reaction to furnish narrow molecular weight distribution and
well-defined molecular structures [88].
Agrihari et al. introduced CuAAC catalyzed synthesis of p-tert-butylcalix[4]arene linked

benzotriazolyl dendrimers using CuSO4.5H2O and NaN3 to prepare N-1, N-2 type 6 fold
compounds in good yields [89]. The synthesized compounds were evaluated for in
vitro and in vivo anti-bacterial studies against a range of microbes and demonstrated good
biological potential. Chen and his companions devised superhydrophobic cotton fabric from
mercaptan and vinyl trimethoxysilane using ultraviolet irradiation via thiol-ene click
chemistry [90]. This fabric possesses special characteristics like economic, highly resistant
towards acids, acetone, UV light, water, and other liquids.
Henning et al. utilized copper-catalyzed azide/alkyne cycloaddition reaction for the efficient
synthesis of triazole-based photo-initiators (42) for the two-photon polymerization process
(Figure 25) [91]. Here Me-Mono and Ph-Mono initiators displayed higher tolerability and
sensitivity in microfabrication areas.
Figure 25.
Synthesis of triazole-based photo-initiators.
A novel, facile and efficient synthesis of 3- and 4-arm star-shaped poly(2-methyl-N-

aziridine)s (43, 44) from ring opening reaction of N-sulfonyl aziridines in the presence of
trimethylsilylazide and PMDETA (N,N,N′,N″,N″-pentamethyldiethylenetriamine) through
click reaction with CuBr and alkyne was demonstrated by Luo et al. (Figure 26) [92].
Figure 26.
3-and 4-arm star-shaped poly(2-methyl-N-aziridine)s.
Cai et al. presented a one-step click chemistry process for the synthesis of high performance
graphene oxide/ styrene-butadiene rubber (GO/SBR) composites using pentaerythritoltetra(3-
mercapto propionate) [93]. Experiments and molecular simulation results concluded that
these composites displayed upgraded gas permeability, thermal conductivity, dynamic, and
static mechanical performances.
Tian and companions demonstrated the synthesis of the functionalized poly(1-butene)

(45)via sequential thiol-ene click reaction and ring-opening polymerization using poly(1,3-
butadiene) as a substrate (Figure 27) [94]. Here C═C bond was further functionalized from
thiol-ene reaction using hydroxyl-containing thiol compounds.
Figure 27.
Functionalized poly(1-butene) synthesis.
Zhang et al. devised synthesis of thiol-maleimide ‘click’ chemistry based β-cyclodextrin

polymers in an aqueous medium without generating by-products [95]. The structure of
products was affected by temperature range i.e. higher temperature gave higher molecular
weighted and compact structures. The obtained polymers showed better dissolution
performance and drug complex-forming capacity as compared to the parental structure.
Gao et al. reported thiol-ene click reactions in polysulfide oligomers and acrylate monomers
to prepare processable and self-healable thermosets and elastomers (46)via different
pathways like photo-initiator, redox-initiator system and base mediated catalytic approaches
(Figure 28) [96]. Reprocessable and self-healable properties depend upon polymer structure
and their synthetic methodology, therefore, DBU based catalytic synthesis displayed better
activity as compared to other processes, due to their catalytic efficiency for disulfide bond
exchanges.
Figure 28.
Process able and self-healable polymer synthesis.
Zhu et al. introduced facile click chemistry assisted poly(2,2,6,6-tetramethylpiperidin-1-oxyl-

4-yl methacrylate) graphene oxide composite (PTMA-GO) (47) assisted reaction in ambient
conditions and utilized them as cathode materials (Figure 29) [97]. After completing 300
charge–discharge cycles,the specific capacity was found to be 2.3 times higher for this
composite as compared to the PTMA electrode.
Figure 29.
PTMA-GO polymer synthesis.
Shen et al. devised synthesis of superhydrophilic and superoleophobic PEGylated PAN

membrane (48) from poly (ethylene glycol) methyl ether methacrylate (PEGMA)
monomers via thiol-ene click chemistry (Figure 30) [98]. After this fabrication, pore size of
the membrane was reduced and displayed low flux. The fabricated membrane exhibited
excellent separation (99%) of various oil–water emulsion and fouling-resistant ability.
Figure 30.
PEGylated PAN membranes.
A DVERTISEMENT
5. Conclusion
Synthetic organic chemistry includes the synthesis of biologically active molecules and
designing of potent scaffolds. Click chemistry is one of the toolboxes for chemistry, biology,
nano, and material sciences. It has vivid applications in the synthesis of organic molecules,
polymers, nanoparticles, biosensors, and many more. The concept of click chemistry fulfills
the green aspects of a reaction. In this chapter, we have deliberated an incredible flurry of
activities in the field of click chemistry inspired synthesis. This study highlights the current
advancements in the synthesis of heterocyclic and other cyclic structures using click
reactions. The insertion of a triazole ring with the help of click reaction increases the
biological activity of the synthesized compounds. Different pathways with metal or metal-
free conditions using conventional or non-conventional reaction methods have also been
demonstrated in this chapter.
A DVERTISEMENT
Acknowledgments
The authors are grateful to the Department of Chemistry, Mohan Lal Sukhadia University,
Udaipur (Raj.), India for providing necessary library facilities for carrying out the work. A.
Sethiya is thankful to UGC-MANF (201819MANF-2018-2019-RAJ-91971) for providing
Senior Research Fellowship to carry out this work. N. Sahiba is very much grateful to CSIR-
Delhi (file no. 09/172(0088)2018-EMR-I), New Delhi for providing Senior Research
Fellowship as a financial support.
A DVERTISEMENT
Conflict of interest
The authors declare no conflict of interest.
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Sections
Author information
 1.Introduction
 2.Green aspects of click reactions
 3.Role of click reactions in synthetic chemistry
 4.Click chemistry in polymer synthesis
 5.Conclusion
 Acknowledgments
 Conflict of interest
References
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Click Chemistry, A Powerful Tool For Pharmaceutical Sciences

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Click Chemistry, a Powerful Tool for Pharmaceutical

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Click chemistry refers to a group of reactions that are fast, simple to

Keywords: click chemistry, polymer, bioconjugation, nanomedicine,

In the field of pharmaceutical science, researchers are constantly

Since being first introduced by Dr. Barry Sharpless’ group in 1999 at

In his landmark review in 2001, Dr. Sharpless defined click chemistry

Although this novel philosophy for drug discovery is very appealing,

2. Classification of Click Reactions

 Cycloadditions - these primarily refer to 1,3-dipolar cycloadditions,

Among the four major classifications, cycloadditions, particularly the

3. CuI-Catalyzed Huisgen 1,3-dipolar Cycloaddition of Azides

The CuI-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and

Two additional reasons for the popularity of this cycloaddition are

3.1 Mechanism of HDC raction

In general, cycloadditions proceed through a concerted mechanism.

A second way to create the catalyst is to directly add CuI salts. Many

Oxidizing copper metal with an amine salt is another way to generate

Recently, CuI-modified zeolites were reported as catalysts for the HDC

Another research group has attempted to find a replacement for

In 2005, pentamethyl cyclopentadienyl ruthenium (II) complexes

4. Applications of Click Chemistry in Pharmaceutical Sciences

Since its debut in 1999, click chemistry has stimulated enormous

4.1 Polymer Therapeutics and Click Chemistry

“‘Polymer therapeutics’ is an umbrella term used to describe

As explained in previous sections, click chemistry was initially

4.1.1 Synthesis of Block Copolymers with Click Reaction

Biocompatible amphiphilic block copolymers have many

The emergence of click chemistry drastically changed the scientific

One of the main pharmaceutical applications of amphiphilic block

As a novel method for stabilizing polymeric micelles, the group

In addition to crosslinking micelles, the same group was able to

The most commonly used biocompatible water-soluble polymers for

While the field of water-soluble polymeric drug conjugates has

As one of the potential solutions to these challenges, a linear

Recently, Liu et al. used click chemistry to synthesize a linear

Due to their concentrated surface functionality and narrow

As an example of a dendrimer used for drug delivery, Gopin et al.

Polymer networks such as hydrogels are also often used in drug

4.2 Click Chemistry and Nanoparticular Delivery Systems

Nanoparticular delivery systems are the carriers ranging in size from

Surface modifications of these carriers can have significant impacts on

4.2.1 Gold and Magnetic Nanoparticles

Gold nanoparticles were first prepared and functionalized with azide

Magnetic nanoparticles are of great interest in pharmaceutical

Since the pioneering work of Bangham and co-workers in the late

As already mentioned in previous sections, the HDC reaction is the

In a recent report Schuber et al. developed a novel strategy for

Cavalli et al. investigated the facile in-situ surface modification of

The use of bioconjugation and biopharmaceuticals are quickly

Excluding drug discovery and polymer chemistry, the most common

Radiolabeling has become a powerful tool in the pharmaceutical

A major obstacle with 99mTc, however, is it can be difficult to attach to

To avoid these problems, Mindt et al. turned to click chemistry (111).

The proteins expressed on the surfaces of microbial cells can be used

In one study, conducted by Wang et al., fluorescein dye derivatives

Oligonucleotides represent a very important class of biomolecules.

Polysaccharides are another major class of macromolecules that have

Due to the easy introduction of azide functional groups and high

5. The Pitfalls of Click Chemistry

A more common problem is alkyne homocoupling. This occurs when

A less common problem is CuI saturation. In order for the click

The stability of some azides may also be a limitation. It is known that if