Click Chemistry with dPEG® Products
Click chemistry is an essential, nearly indispensable tool for bioconjugation reactions. It provides a simple, straightforward route to the creation of numerous different structures and conjugated biomolecules. Since its discovery, the use of the click chemistry reaction has spread into many different fields, sub-fields, and disciplines of chemistry and biology. In this review, we will explore the types of click chemistry reactions, discuss some of their strengths and limitations, and review the various types of single molecular weight, discrete PEG (dPEG®) products for click chemistry applications that we at Quanta BioDesign make and sell internationally.
If you are unfamiliar with Quanta BioDesign's dPEG® products, please visit the following two pages for a thorough explanation of dPEG® technology and answers to frequently asked questions.
Frequently Asked Questions (about dPEG® products)
Introduction to Click Chemistry
In 2001, Hartmuth C. Kolb, M. G. Finn, and K. Barry Sharpless coined the term "click chemistry" to refer to a diverse set of reactions with a shared set of features that make the reactions appear to be " 'spring-loaded' for a single trajectory." Kolb, Finn, and Sharpless set forth a strict set of criteria that reactions had to meet to be called "click chemistry" reactions. Their paper identified the 1,3-dipolar cycloaddition reaction, which Rolf Huisgen analyzed and reported on in 1967 , as an exemplar of a click chemistry reaction. Although the 1,3-dipolar cycloaddition reaction frequently is named the "Huisgen 1,3-dipolar cycloaddition," the first reported reaction of this type dates back to 1893 and a report by A. Michael. Huisgen's 1,3-dipolar cycloaddition between an azide and alkyne proceeds at 100°C and yields a mixture of the 1,4- and 1,5-disubstituted triazoles.2 See Figure 1.
In 2002, Tornøe, Christensen, and Meldal and independently, Rostovtsev, Green, Fokin, and Sharpless related that copper(I) salts catalyze the rapid, regiospecific formation of 1,4-disubstituted, 1,2,3,-triazoles between azides and terminal alkynes. See Figure 2. This reaction is commonly abbreviated as CuAAC, for "copper(I)-catalyzed azide-alkyne cycloaddition." These two independent reports marked the beginning of the widespread scientific awareness of the power and specificity of click chemistry.
|What makes a chemical process "click chemistry?"|
|The criteria set out by Kolb, Finn, and Sharpless1 define the rules by which a reaction is or is not a click chemistry reaction. For a process to be "click chemistry," the reaction must:|
· be modular;
|The process should have the following characteristics:|
· simple reaction conditions;
|Characteristics of a "click chemistry" reaction product:|
· The reaction product is stable under physiological conditions.
In their 2001 paper,1 Kolb, Finn, and Sharpless also identified four classes of chemical transformations that they considered as click chemistry reactions. These four classes were as follows:
- cycloadditions of unsaturated species (such as 1,3-dipolar cycloadditions and Diels-Alder reactions);
- nucleophilic substitution chemistry (including, for example, ring-opening epoxide reactions);
- non-aldol carbonyl chemistry (examples include oxime and hydrazone bond formation); and
- additions to carbon-carbon multiple bonds (for instance, epoxidation, sulfenyl halide addition, and the Michael addition).
Since the seminal 2001 paper by Kolb, Finn, and Sharpless, and subsequent discovery in 2002 of the CuAAC, many more reactions have been discovered or recognized as "click chemistry." These reactions include ruthenium-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, strain-promoted alkyne-nitrone cycloaddition, photoclick chemistry, sulfur fluoride exchange, inverse electron-demand Diels-Alder, and more. We will discuss these different types in the sections below.
Types of Click Chemistry: Metal-Catalyzed Click Chemistry
Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)
The copper(I)-catalyzed cycloaddition reaction of terminal alkynes to organic azides to form 1,4-disubstituted 1,2,3-triazoles is one of the best known, most popular click chemistry reactions today. The reaction works well on solid supports5 as well as in solution.6 Although copper(I) salts catalyze the reaction, copper(II) salts do not affect the reaction. However, copper(II) salts combined with a reducing agent such as sodium ascorbate form copper(I) salts that can catalyze the reaction. Moreover, metal turnings of pure copper metal catalyze the formation of the 1,4-disubstituted, 1,2,3-triazoles. 5,6 The CuAAC works with a broad span of reagents across a wide range of temperatures (0° – 160°C), pH values (4 – 12), and functional groups. The reaction works best in aqueous media, including serum and whole blood. Side reactions are rare and easily managed.6,
The CuAAC works on terminal alkynes, but not internal ones.7 This limitation is mechanistic. The first step in the CuAAC reaction is the formation of a copper-acetylide complex. This complex then reacts with the three nitrogen atoms of the azide in a stepwise fashion, leading to the 1,2,3-triazole product.
Copper(I)-catalyzed nitrile oxide-azide cycloaddition (CuNOAC)
While studying the CuAAC, Himo et al. found a similar copper(I)-catalyzed reaction that is also a click chemistry reaction.7 Although not as well-known as the azide-alkyne cycloaddition, copper(I) also catalyzes the reaction of terminal alkynes with nitrile oxides to form isoxazoles. Consequently, this reaction is known as the copper(I)-catalyzed nitrile oxide-azide cycloaddition, which is abbreviated CuNOAC. See Figure 3. Limited investigations have been conducted on this reaction. See references , , and .
Ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC)
In 2005, Li Zhang et al. reported the discovery of a new type of click chemistry reaction catalyzed by ruthenium(II).,, At 80°C in benzene, in the presence of 5% Ru(OAc)2(PPh3)2, Zhang and colleagues found that benzyl azide and phenylacetylene reacted to form the 1,4-disubstituted, 1,2,3-triazole product. However, by switching to any one of four different ruthenium catalysts – Cp*RuCl(PPh3)2, [Cp*RuCl]4, CpRuCl(COD), or Cp*RuCl(NBD) – the azide-alkyne ligation formed the 1,5-disubstituted product with a terminal alkyne. See Figure 4.a.
Further study showed that the RuAAC reaction works with both terminal and internal alkynes. Ruthenium(II)-catalyzed cycloaddition reactions with internal alkynes form 1,4,5-trisubstituted-1,2,3-triazoles.12,14 Although Cu(I)-catalyzed methods for forming trisubstituted 1,2,3-triazoles exist, the RuAAC provided the first facile method to 1,4,5-trisubstituted-1,2,3-triazoles. See Figure 4.b.
Although the RuAAC is a "click chemistry" reaction like the CuAAC, there are key differences. First, the solvent must be aprotic. Examples of compatible solvents for the RuAAC include acetone, benzene, dichloromethane, dimethylformamide, and toluene. Protic solvents such as water, methanol, ethanol, and isopropanol, as well as hexanes, diethyl ether, and ethyl acetate, inhibit catalysis. Second, unlike CuAAC, which progresses at room temperature, the RuAAC requires heating to 50 – 80°C to proceed. Third, while the CuAAC accepts a wide variety of copper sources for reaction catalysis (Cu(I) salts, Cu(II) salts plus a reducing agent, and pure copper metal), the RuAAC is quite limited in catalysts that form the 1,5-isomer.
Other metal-catalyzed azide-alkyne cycloadditions
Metals other than copper(I) and ruthenium (II) catalyze azide-alkyne cycloadditions. In scientific literature, researchers have reported 1,3-dipolar azide-alkyne cycloadditions using indium, iridium,,, nickel, rhodium, and zinc. Also, a 2017 report demonstrated a [4+3] azide-alkyne cycloaddition using yttrium triflate.
Types of Click Chemistry: Bioorthogonal Click Chemistry
Bioorthogonality in bioconjugation
The toxicity of copper is one of the problems with the CuAAC and potentially with other metal-catalyzed azide-alkyne cycloadditions.,, Prolonged exposure of living cells to toxic metals such as Cu(I) damages or kills living cells. Attempts have been made to create non-toxic Cu(I) catalysts that permit live cells to be labeled using the CuAAC. Also, metal-catalyzed cycloaddition reactions must react in aqueous media. Thus, for example, the RuAAC cannot be used in aqueous media or with living cells because the metal is toxic, and the catalysts do not work in water. Therefore, a requirement for metal catalysis can limit the in vitro and in vivo utility of azide-alkyne cycloaddition reactions.
"Bioorthogonal chemistry" describes chemical reactions that occur inside of biological systems without the reactants or product interacting or interfering with the systems' natural biochemical processes.24,, The reactants and product must be stable in biological systems under physiological conditions. Ideally, the reaction should be highly specific and quite fast. This last consideration is essential when labeling low-abundance structures or molecules or reporting on physiological processes that occur in or on living cells.
One early example of bioorthogonal chemistry is the well-known, well-studied reaction between ketones and hydrazides or aminooxy compounds. This reaction forms a hydrazone (ketone plus hydrazide) or oxime (ketone plus aminooxy) bond. For more information on this reaction, please see our page titled, Oxime and Hydrazone Bioconjugation Reactions. Hydrazides and aminooxy compounds are absent in living systems, and ketones are uncommon. Lara K. Mahal, Kevin J. Yarema, and Carolyn R. Bertozzi took advantage of this reactivity to modify cell surface oligosaccharides and thereby change the cell surface recognition epitopes.
The Bertozzi group also modified the Staudinger reaction for use as a bioconjugation tool to create new structures in living cells. The Staudinger reaction occurs between a phosphine and an azide leading to the formation of an aza-ylide that hydrolyzes spontaneously in the presence of water to yield an amine and the analogous phosphine oxide. A modified phosphine containing an electrophilic trap (in the form of a methyl ester), stabilized the aza-ylide producing an amide bond rather than an amine. The Bertozzi group termed this a "Staudinger ligation." The initial Staudinger ligation work retained the phosphine in the bioconjugate product. However, later work resulted in the so-called "traceless" Staudinger ligation," in which the phosphine became a byproduct of the amide bond ligation.
The Staudinger ligation is a useful bioorthogonal reaction for bioconjugation. Indeed, the Staudinger ligation has low toxicity, high specificity, and minimal-to-no background labeling under a variety of conditions, making it ideal for biological labeling reactions.28 On the other hand, as Carolyn Bertozzi and others have noted, the Staudinger ligation has slow reaction kinetics.24, It is not "click chemistry." However, recent reports have suggested that engineering of the phosphine and the azide can significantly enhance the reaction's kinetics and atom economy, thus improving its utility in living systems.
Strain-promoted azide-alkyne cycloaddition (SPAAC)
Cyclooctyne is the smallest stable cycloalkyne. In 1953, Blomquist and Liu observed that cyclooctyne reacted spontaneously and explosively with neat phenyl azide, an observation that Wittig and Krebs confirmed in 1961. See Figure 5.
These reports led Carolyn Bertozzi, then at the University of California, Berkeley (now at Stanford University), to assess the utility of strained cyclooctyne derivatives for click chemistry. The resulting fruitful research has led to the discovery of numerous strained ring systems that promote click chemistry reactions without the need for a metal catalyst. These reactions collectively are called "strain-promoted azide-alkyne cycloaddition" reactions or SPAAC. See Figure 6. Numerous published reviews of the SPAAC research cover this topic in greater detail and depth than will be discussed here.
Two critical issues in SPAAC are hydrophobicity and reaction rates. Cyclooctyne compounds are lipophilic. Poor water solubility can hinder SPAAC reactions, particularly in vivo. Moreover, as noted by several publications, the highly hydrophobic cyclooctyne can bind to serum albumin in vivo, resulting in sequestration of the cyclooctyne from azides that have been introduced into cells or on cell surfaces. For example, the Bertozzi group found that in mice, DIFO (Figure 7) bound to mouse serum albumin, which prevented DIFO from reacting with azides in the mouse tissue. Thus, despite DIFO's fast ex vivo reaction kinetics with azides, in vivo, DIFO was less efficient than the phosphine catalysts used in the Staudinger ligation.
The scientific literature contains numerous reports of cyclooctyne derivatives with varying degrees of lipophilicity and reactivity. Alterations in electronic configuration or overall ring strain modulate the cyclooctyne's reactivity. Some of the most reactive cyclooctyne compounds are unstable, and their rapid decomposition makes them unsuitable for bioconjugation. See reference 39(c), infra. Substitutions on the cyclooctyne ring also affect lipophilicity.
In 2010, Debets et al. at Radboud University Nijmegen in The Netherlands developed dibenzoazacyclooctyne (DIBAC) seeking to combine the fast reaction kinetics of dibenzocyclooctyne (DIBC) with the increased hydrophilicity of aza-dimethoxycyclooctyne (DIMAC). See Figure 8.
DIBAC was combined with mPEG2000 to form DIBAC-mPEG2000 and then conjugated that compound to the enzyme Candida Antarctica Lipase B (CalB) that had been modified to contain azides (AHA-CalB). AHA-CalB contains five azidohomoalanaine residues DIBAC-mPEG2000, only one of which is exposed on the enzyme's surface. A direct comparison of DIBAC-mPEG2000 with DIBC-mPEG2000 under the same conditions showed that DIBAC-mPEG2000 reacted with AHA-CalB faster and more efficiently than DIBC-mPEG2000. Full conversion of AHA-CalB to mPEG2000-AHA-CalB occurred in three (3) hours with only five (5) equivalents of DIBAC-mPEG2000 relative to AHA-CalB. Furthermore, DIBC-mPEG2000 reacted only with the surface-exposed azide, while DIBAC-mPEG2000 conjugated to both the surface-exposed azide group and one of the buried azide groups.42 In scientific literature, DIBAC is also known as DBCO.
Conjugation of the terminal carboxylic acid group of DIBAC/DBCO to PEG creates a water-soluble product that can increase the water solubility and reduce the immunogenicity of the partner (i.e., azide-functionalized) molecules. By lessening the lipophilicity of DIBAC/DBCO through PEG conjugation, the range of bioconjugations in which DIBAC/DBCO can participate increases. Reports also suggest that DIBAC/DBCO has less affinity for serum albumin than DIFO and other lipophilic cyclooctyne compounds.
The choice of solvent affects the reaction kinetics of DIBAC/DBCO. In 2016, Derek Davis, Erin Price, and coworkers under the direction of Jennifer M. Heemstra of the Department of Chemistry at the University of Utah systematically explored the effects of buffer identity, ionic strength, and organic cosolvents on the reaction rate between commercially available DBCO-PEG4-acid and mPEG3-azide. (Note: the paper did not provide the sources of these two products, but Quanta BioDesign offers similar products, as discussed below.)
The researchers found that SPAAC was tolerant of a wide range of aqueous buffers, pH, and ionic strength. However, for buffers with a pH of 5.5 or below, the reaction rate of the SPAAC between DBCO-PEG4-acid and mPEG3-azide decreased slightly. Different organic cosolvents had different effects on the SPAAC reaction rate, and these effects varied by both the identity of the cosolvent and the amount of cosolvent used. Five cosolvents were tested – acetonitrile, dimethyl sulfoxide (DMSO), ethanol, methanol, and N-methyl-2-pyrrolidone (NMP) – at 10%, 40%, and 70% cosolvent ratios. As cosolvents, acetonitrile and methanol did not improve the reaction rates compared to aqueous buffer with no organic cosolvent, and indeed, they slowed the reaction rates with each increase in the amount of the organic cosolvent in aqueous buffer. With NMP, the reaction rate rose slightly from 10% to 40% cosolvent, but then fell sharply at 70% cosolvent. Ethanol and DMSO both increased the reaction rates of SPAAC compared to aqueous buffer.
Moreover, both ethanol and DMSO showed sharp increases in the reaction rate from 10% to 40% cosolvent, followed by a moderate (ethanol) or slight (DMSO) drop in the reaction rate at 70% cosolvent. Exploring the effects of DMSO further, the researchers found that 60% DMSO as an organic cosolvent gave the most substantial increase in the SPAAC reaction rate. The variation between the slowest and fastest reaction rates using organic cosolvents was nearly 4-fold.
A 2016 report by Dommerholt, Rutjes, and van Delft found similar effects on the SPAAC reaction rate for methanol and acetonitrile. In addition, the impact of THF-water mixtures on the reaction rate was studied, and THF was found to slow the reaction rate even more than methanol or acetonitrile. See reference 39(c), infra.
One potential problem with using cyclooctyne derivatives for SPAAC in biological systems is that the alkyne bond reacts with free thiols. The thiol-yne reaction is a well-known, well-studied type of Michael addition frequently used in polymer chemistry. Thiol-yne chemistry is a type of click chemistry. Studies have shown that cyclooctyne and its derivatives react with free thiols, and this finding has implications for SPAAC reactions in biological systems., Thiol-yne chemistry is potentially useful in biological systems, for example, in bridging protein disulfide bonds., However, free thiols that occur naturally in biological systems can interfere with SPAAC conjugations. One possible result of such interference is that control reactions in which no azide is present in the reaction mixture may still exhibit some degree of conjugation because of the free thiol groups.43
In 2012, van Geel and coworkers reported that DIBAC/DBCO, DIBO, and bicyclo[6.1.0]nonyne (BCN) (another strained cycloalkyne) reacted with free thiols in cell lysates. The free thiols came from cysteine residues on peptides and proteins in the lysate. However, the results showed that these strained cycloalkyne compounds reacted preferentially with azides. Moreover, incubation of the cell lysate with iodoacetamide (IAM) abolished the undesired thiol-cycloalkyne reaction by alkylating the free thiols in the lysate. IAM is compatible with SPAAC. Consequently, the specificity of the SPAAC reaction improved with IAM preincubation.
Despite some limitations and problems with SPAAC, this click chemistry reaction is enormously popular and highly useful. SPAAC applications include in vitro and in vivo bioimaging, glycoengineering,24,39(d),42(b) the formation of peptide- and protein-functionalized hydrogels, the development of radiopharmaceuticals,, and the development of antibody-drug conjugates (ADCs).,,
Strain-Promoted Alkyne-Nitrone Cycloaddition (SPANC)
Another type of copper-free click chemistry is strain-promoted alkyne-nitrone cycloaddition, known by the acronym SPANC. In SPANC reactions, a nitrone, rather than an azide, reacts with a cyclooctyne derivative via strain. The nitrone accepts multiple functional groups, allowing the simultaneous incorporation of various labels onto a biomolecule in a single reaction. Also, by endocyclic and acyclic nitrones react in SPANC. See Figure 10.
SPANC was first reported in 2010 by Ning et al. as a method to label N-terminal serines of peptides and proteins rapidly. Oxidation of the N-terminal serine's side-chain oxygen with sodium periodate formed the aldehyde with loss of formaldehyde. The nitrone was then created by treating the aldehyde with p-methoxybenzenethiol and N-methylhydroxylamine. Adding a strained cyclooctyne gave the isoxazoline product. The use of p-anisidine accelerated the reaction. See Figure 11. The researchers speculated that the acceleration by p-anisidine arose from a mechanism similar to the p-anisidine-catalyzed acceleration of oxime bond formation from aldehydes and hydroxylamines.
The reaction kinetics are fast — often much quicker than SPAAC — varying by the substitutions on the nitrone.39(b), However, a drawback to SPANC is that the starting materials and the resulting isoxazoline product can be unstable under certain conditions. For example, acyclic nitrones, but not endocyclic nitrones, rapidly hydrolyze in acidic and basic environments. Also, some isoxazoline products (such as those derived from the highly reactive cyclooctyne derivative known as BARAC) undergo acid-catalyzed rearrangement. Other SPANC-derived cycloadducts may thermally rearrange. As with SPAAC, DIBAC/DBCO has fast reaction kinetics in SPANC and forms products that are reasonably stable under physiological conditions.39(b),57,58
Inverse Electron-Demand Diels-Alder (IEDDA) Click Chemistry
In 2008, two different groups reported the reaction between a strained cycloalkene and a 1,2,4,5-tetrazine to give a 4+2 cycloaddition product., The release of ring strain drives the IEDDA reaction as it also does in SPAAC and SPANC. IEDDA has extremely rapid reaction kinetics. Moreover, it is both bioorthogonal and orthogonal to other types of click chemistry. IEDDA is useful for labeling live cells in vitro and in vivo.25,39(d),62 Moreover, scientific literature reports applications of IEDDA in the synthesis or development of pharmaceutical drugs and drug delivery systems,25,39 peptide synthesis, the development of ADCs, and the creation of pretargeted radiopharmaceutical therapeutics.
The use of light provides scientists with robust spatial and temporal control over chemical reactions, including synthesis and live-cell labeling. In the presence of light, tetrazoles and alkenes react to form fluorogenic pyrazoline products with the release of nitrogen. The light used for photoclick chemistry is in the range of 350 – 365 nanometers (nm), which lessens the damage to living cells and makes this technique suitable for labeling live cells. However, in the presence of a photoinitiator, many alkenes (as well as terminal alkynes) can react with thiols in a click-type reaction using 365 nm light. Thus, it may be necessary to remove or cap all free thiols before proceeding with a photoclick chemical reaction. The thiol-ene, thiol-yne, and photoclick-thiol-ene reactions are reviewed below. Figure 13 shows a general photoclick chemical reaction.
Numerous reviews and research papers on photoclick chemistry exist. Uses for photoclick chemistry include creating hydrogels and other crosslinked networks, labeling biomolecules (peptides, proteins, glycans, and nucleic acids) and live cells, and synthesizing antibody-drug conjugates.
Types of Click Chemistry: Other Types
There are several different types of click chemistry besides the ones listed above. Three notable types are Oxime/Hydrazone Bond Formation, Thiol-Maleimide Click Chemistry, and Thiol-Ene/Thiol-Yne Click Chemistry. Two of these three types have separate discussion pages at the links below:
Oxime and Hydrazone Bioconjugation Reactions
For information about oxime and hydrazone bond formation or thiol-maleimide click chemistry, please click the links above.
Thiol-Ene Click Chemistry
Theodore Posner discovered the reaction known as alkene hydrothiolation (now commonly known as thiol-ene chemistry or the thiol-ene click reaction) in 1905. This anti-Markovnikov addition reaction forms a thioether bond between an alkene and a sulfhydryl (thiol) group. Two types of thiol-ene click reactions are known. One category is a radical-mediated addition that is initiated by light, heat, or another radical initiator. A second category is a Michael addition catalyzed by a base or nucleophile. In both types of thiol-ene click chemistry, the addition product is anti-Markovnikov. A quick survey of relevant scientific literature shows that most reported thiol-ene chemical reactions are photoinitiated, radical-mediated reactions. See Figure 14.
Thiol-ene click chemistry meets nearly all of the criteria for "click chemistry" set forth by Kolb et al. 1, Some reaction products may not be entirely stable under physiological conditions. Some reactions may proceed slowly rather than rapidly. Still, these problems are not exclusive to thiol-ene click chemistry. For example, thiosuccinimide conjugates can undergo a retro-Michael addition over time in vivo resulting in "payload migration," and hydrazone compounds can hydrolyze in some types of environments found in vivo. However, most experts agree that the thiol-ene reaction is a type of click chemistry.
Thiol-ene click chemistry became popular among polymer scientists starting in the 1990s, and it has driven the creation of a large, diverse group of functional polymeric materials. However, thiol-ene click chemistry is increasingly important and popular for bioconjugation work. Examples of applications using thiol-ene chemistry in bioconjugation include the following:
- synthesis of cell-encapsulating PEG hydrogels for 3D cell culture and regenerative medicine applications;
- generation of photo-thiol-ene-crosslinked, biocompatible hybrid vesicles to control the dispersion of water-soluble dyes;
- creation of covalently immobilized DNA on silicon-based microarrays using thiol-ene chemistry; and
- modification of wool keratin with mPEG using a photo-initiated thiol-ene reaction.
As noted above, most thiol-ene bioconjugations that are reported in the scientific literature are radical-mediated, photoinitiated reactions. These reactions are compatible with water and oxygen, making them quite useful for biomolecules. However, oligonucleotides and some proteins are unstable in the presence of UV light., Thus, for these molecules, photoinitiated, radical-mediated thiol-ene click chemistry may be an unsuitable bioconjugation strategy. For these molecules, other types of radical initiators or a Michael addition reaction strategy may prove useful. Also, because free thiols and tetrazines both react with strained alkenes, researchers wanting to use IEDDA in bioconjugation should take care to oxidize, cap, or otherwise remove free thiols from reaction mixes before undertaking the IEDDA reaction.
Thiol-Yne Click Chemistry
In 1949, Bader et al. discovered that sulfhydryl (thiol) groups react with alkynes to form thiol-ether bonds. This reaction is known as the thiol-yne reaction or, more formally, alkyne hydrothiolation. Chemists mostly neglected this reaction until Fairbanks et al. popularized it in 2009. As with the thiol-ene click chemistry reaction, the thiol-yne reaction was initially widely adopted by polymer chemists. However, bioconjugation chemists have also found this reaction useful.
As with the thiol-ene reaction, the thiol-yne click chemistry is a radical-mediated reaction that is triggered by photo (UV) or thermal initiation. The alkyne reacts with a sulfhydryl to form an alkenyl sulfide. The alkene product can then react with another sulfhydryl, forming a second thioether bond.86 See Figure 15 for a schematic of the thiol-yne reaction.
In polymer and materials chemistry, the ability to join two sulfhydryl groups to the alkyne carbons permits the formation of complex networks.44,45,86 In bioconjugation, thiol-yne chemistry allows the disulfide bonds in peptides and proteins to be bridged with various functional groups.47 As with thiol-ene click chemistry, ultraviolet photoinitiation to trigger radical formation has limited the use of thiol-yne chemistry for oligonucleotides and proteins.83 However, photoinitiation is not an absolute requirement for thiol-yne chemistry as the reaction proceeds under various mild conditions.83,
Examples of thiol-yne chemistry that are relevant to this review include the following:
- Development of FRET-based fluorescent and luminescent probes;83
- Attachment of carbohydrates to peptides;,
- Preparation of micelles and the creation of functional lipid mimetics;
- Construction of dendrimers;
- Conjugation of multiple serum albumin molecules into a single unit;
- Peptide stapling;
- Functionalization of hydrogel surfaces for drug delivery; and
- Creation of a biocompatible antimicrobial peptide with improved performance characteristics.
Sulfhydryl groups also can react with cyclooctyne derivatives. Dadfar et al. showed that, sans photoinitiator, cyclooctyne derivatives conjugate spontaneously to a single sulfhydryl group leading to a thioether joined to a cyclooctene derivative. With a photoinitiator, the cyclooctyne derivative forms two thioether bonds, and the cyclooctyne becomes a cyclooctane. Earlier, Griebenow, Dilmaç, Greven, and Bräse demonstrated that disulfide bonds in peptides and antibody fragments (Fab) are bridged by cyclooctyne compounds. Similarly to the thiol-ene click chemistry reaction, researchers wanting to conduct SPAAC reactions with a cyclooctyne derivative may find it prudent to take steps to reduce or eliminate unwanted thiol-yne reactions. Alternatively, the thiol-yne click chemistry reaction may provide a useful method of obtaining a desirable bioconjugate without the use of an azide functional group that is not native to the proteome.
Quanta BioDesign's Click Chemistry Product Lines
Quanta BioDesign offers a range of click chemistry reagents that are useful for the types of click chemistry described above. These are discussed below.
Quanta BioDesign offers azido-dPEG®-acid, azido-dPEG®-NHS ester, and azido-dPEG®-TFP ester crosslinkers. These crosslinkers conjugate to primary amines, such as the ε amine of lysine, through the carboxyl functional group to form amide bonds. The carboxylic acid-functionalized crosslinkers couple to amines using a carbodiimide such as EDC to drive the reaction. The NHS- and TFP-esters spontaneously react with primary amines in aqueous buffer at pH > 7. In organic solvent, the NHS- and TFP-esters react with amines in the presence of organic bases such as DIPEA or TEA.
Quanta BioDesign offers several azido-dPEG®-amine crosslinkers ranging from n = 3 to n = 35. The amine functional group couples to carboxylic acids and their active esters to form an amide bond. The coupling chemistry is the same as for the carboxyl-based crosslinkers, above.
Azido-alcohol click chemistry products
Quanta BioDesign's azido-dPEG®-alcohol products can function as crosslinkers or as surface modification agents. After reacting the azide group with an alkyne, the alcohol end of the molecule can be left unmodified to provide additional water solubility to the conjugate. Alternatively, the OH group can be modified with N,N-disuccinimidyl carbonate, to create an amine-reactive end group for crosslinking. Other modifications to the OH group are possible to open up different crosslinking options.
m-dPEG®-azide and m-dPEG®-DBCO products
We designed our m-dPEG®-azide and m-dPEG®-DBCO products to modify surfaces. CuAAC, RuAAC, and SPAAC all work with the azide-terminated products on alkyne or cycloalkyne surfaces. Conversely, the DBCO-functionalized product is useful for SPAAC and SPANC reactions with azide-functionalized surfaces. The terminal methyl group maintains a neutral charge on the molecule. These products can be useful for constructing dendrimers, coating surfaces, or adding hydrophobic volume and immunological "stealth" properties to alkyne-functionalized biomolecules. It is important to note that the DBCO-functionalized products have somewhat diminished water solubility due to the presence of the hydrophobic DBCO moiety.
Terminal alkyne products for click chemistry
Quanta BioDesign offers two terminal alkyne products for click chemistry. These products can be used with azide partners for copper- and ruthenium-catalyzed azide-alkyne cycloadditions and with thiol partners in thiol-yne click chemistry reactions.
Product number 10510, propargyl amine, is a three-carbon alkyne with a terminal amine. The amine forms amide bonds with carboxylic acids and their active esters. This product can be used to form an enormous number of different compounds by reacting the amine with our acid- and active ester-functionalized products. The terminal alkyne then can react with azides or sulfhydryls under suitable click chemistry conditions.
Similarly, product number 10511, propargyl-dPEG®1-NHS ester, is a short, amine-reactive linker with a terminal alkyne. The NHS ester reacts with any of the primary amines offered by Quanta BioDesign. Thus, our customers can design click-chemistry-enabled dPEG® compounds for their specific applications.
The maleimide functional group is unstable in the presence of azide. The spontaneous reaction between maleimide and azide results in nearly complete degradation within hours. Thiol-ene or thiol-yne click chemistry is one way to avoid this problem. Some companies sell kits that allow users to synthesize the crosslinker in situ. However, Quanta BioDesign offers a unique solution to this problem.
The bromoacetyl group reacts with free thiols to form thioether linkages. (Please see our page titled Bromoacetyl Reaction Chemistry for additional information on this topic.) Also, the bromoacetyl group is stable in the presence of azide. Thus, our line of Bromoacetamido-dPEG®n-azide crosslinking products offers a unique solution to the problem of maleimide-azide instability. The azide reacts with alkynes as described above, while the bromoacetyl group, which is linked via an amide bond to a dPEG® spacer, selectively reacts with sulfhydryl groups at pH 7.5 and above. This reaction is much slower than the maleimide-thiol reaction, but it works well and avoids the stability issues of maleimide-azide compounds.
Biotinylation with click chemistry products
Quanta BioDesign offers a selection of dPEG® products functionalized with biotin and either azide or DBCO. Click here to view these click-dPEG® biotinylation products. These products combine the highly useful benefits of the biotin-(strept)avidin reaction with the powerful benefits of click chemistry. We also have a page discussing biotinylation with dPEG® products.
DBCO-dPEG® products for click chemistry
PEG chelates metal ions effectively. Thus, Quanta BioDesign has avoided creating dPEG® products with terminal alkynes for azide-alkyne cycloaddition applications. Instead, our efforts focus on developing DBCO-functionalized dPEG® compounds that facilitate SPAAC and SPANC reactions and can be used in thiol-yne click chemistry, if desired.
The DBCO-dPEG®-maleimide crosslinkers provide a simple means of crosslinking azide-functionalized molecules or surfaces with sulfhydryls. Because of the instability of maleimides in the presence of azides, it may be advisable to react the maleimide end of the crosslinker first, followed by the DBCO end.
DBCO-dPEG®-TFP ester crosslinkers
DBCO-dPEG®-TFP ester crosslinkers permit crosslinking between azide-functionalized moieties (surfaces, small molecules, or biomolecules, for example) and primary amines. The TFP ester is less likely to hydrolyze and more reactive with amines than NHS esters. For more information on TFP esters, please click here.
The Bromoacetamido-dPEG®-amido-DBCO crosslinkers enable crosslinking between a sulfhydryl and an azide. The DBCO participates in SPAAC and SPANC reactions, and the bromoacetyl group reacts selectively with free thiols at pH >7.5. For more information about the bromoacetyl-thiol reactivity, please see our page on Bromoacetyl Reaction Chemistry.
Dye-labeled dPEG®-DBCO products
Quanta BioDesign offers two dyes containing a dPEG® spacer and a DBCO functional group for SPAAC and SPANC reactions. The two dyes are PN11812, DBCO-dPEG®12-carboxyfluorescein, and PN11813, DBCO-dPEG®12-meso-TP-IR775.
Listed in our catalog under Phospholipid-dPEG® Derivatives, PN11383, DBCO-dPEG®24-amido-dPEG®24-DSPE contains a DBCO-functionalized dPEG® spacer attached to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine. In liposomes and micelles, this product permits SPAAC conjugation to an azide-functionalized moiety such as a targeting peptide.
Other DBCO Products
Other DBCO-functionalized dPEG® products offered by Quanta BioDesign include a homobifunctional DBCO-dPEG® crosslinker, and a homotetrafunctional DBCO-dPEG® crosslinker.
Quanta BioDesign's Thiol- and Protected Thiol-functionalized Products
We do not list our thiol-functionalized and protected thiol-functionalized dPEG® products with our click chemistry reagents. The reason for this is simple: most of our customers purchase thiol and protected thiol products to thiolate molecules or coat metal surfaces (especially gold). Nevertheless, our m-dPEG®x-thiol and Thiol-dPEG®x-acid reagent categories also are usable in thiol-ene and thiol-yne click chemistry reactions.
"Click chemistry" is a general term that covers a multitude of different types of reactions. Quanta BioDesign, Ltd. offers a wide range of dPEG® linkers and spacers that are functionalized with functional groups suitable for most of these different reactivities. Also, we offer customers reagents that facilitate the design of additional click chemistry reagents, thus broadening the range of compounds that are useful for the various types of click chemistry reactions. For a complete list of all our dPEG® products, please see our online catalog, online flip catalog, or downloadable (PDF) catalog.
If you like this review of click chemistry reactions and bioconjugation, please share it on social media! Also, we are on LinkedIn, Facebook, and Twitter. We would be delighted for you to follow us on any or all of those social media sites.
 Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angewandte Chemie International Edition 2001, 40(11), 2004–2021. https://doi.org/10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5.
 Huisgen, R.; Szeimies, G.; Möbius, L. 1.3-Dipolare Cycloadditionen, XXXII. Kinetik der Additionen organischer Azide an CC-Mehrfachbindungen. Chemische Berichte 1967, 100(8), 2494–2507. https://doi.org/10.1002/cber.19671000806.
 Huisgen, R. Kinetics and Reaction Mechanisms: Selected Examples from the Experience of Forty Years. Pure and Applied Chemistry 1989, 61(4), 613–628. https://doi.org/10.1351/pac198961040613.
 Michael, A. Ueber Die Einwirkung von Diazobenzolimid Auf Acetylendicarbonsäuremethylester. Journal für Praktische Chemie 1893, 48 (1), 94–95. https://doi.org/10.1002/prac.18930480114.
 Tornøe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides. J. Org. Chem. 2002, 67(9), 3057–3064. https://doi.org/10.1021/jo011148j.
 Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. Angewandte Chemie International Edition 2002, 41(14), 2596–2599. https://doi.org/10.1002/1521-3773(20020715)41:14<2596::AID-ANIE2596>3.0.CO;2-4.
 Himo, F.; Lovell, T.; Hilgraf, R.; Rostovtsev, V. V.; Noodleman, L.; Sharpless, K. B.; Fokin, V. V. Copper(I)-Catalyzed Synthesis of Azoles. DFT Study Predicts Unprecedented Reactivity and Intermediates. J. Am. Chem. Soc. 2005, 127(1), 210–216. https://doi.org/10.1021/ja0471525.
 Rodionov, V. O.; Fokin, V. V.; Finn, M. G. Mechanism of the Ligand-Free CuI-Catalyzed Azide–Alkyne Cycloaddition Reaction. Angew. Chem. Int. Ed. 2005, 44(15), 2210–2215. https://doi.org/10.1002/anie.200461496.
 Gregory F. Morehouse. Mechanistic Investigations into the Sulfonamide Metathesis and Copper(I)-Catalyzed Nitrile Oxide-Alkyne Cyclization Reactions; the Synthesis of Chemogenetic and Proteomic Tools and Ligands for Novel Imaging Agents, and Theoretical Calculations on Carbenes with Organometallic Ligands - ProQuest. Thesis, Northeastern University, Boston, MA, 2009.
 Tang, S.; Gong, T.; Fu, Y. Mechanistic Study of Copper-Catalyzed Intramolecular Ortho-C-H Activation/Carbon-Nitrogen and Carbon-Oxygen Cyclizations. Sci. China Chem. 2013, 56 (5), 619–632. https://doi.org/10.1007/s11426-012-4795-3.
 James S. Oakdale. Homogeneous Catalysis: From Ruthenium(II)-Catalyzed Azide Alkyne Cycloadditions (Part I) to Super-Base Mediated Sulfur(VI) Fluoride Exchange Reactions (Part II) - ProQuest. Doctoral Dissertation, The Scripps Research Institute, La Jolla, CA, 2015.
 Zhang, L.; Chen, X.; Xue, P.; Sun, H. H. Y.; Williams, I. D.; Sharpless, K. B.; Fokin, V. V.; Jia, G. Ruthenium-Catalyzed Cycloaddition of Alkynes and Organic Azides. J. Am. Chem. Soc. 2005, 127(46), 15998–15999. https://doi.org/10.1021/ja054114s.
 Oakdale, James S.; Fokin, Valery V. Preparation of 1,5-Disubstituted 1,2,3-Triazoles via Ruthenium-Catalyzed Azide Alkyne Cycloaddition. Org. Synth. 2013, 90, 96–104. https://doi.org/10.15227/orgsyn.090.0096.
 Johansson, J. R.; Beke-Somfai, T.; Said Stålsmeden, A.; Kann, N. Ruthenium-Catalyzed Azide Alkyne Cycloaddition Reaction: Scope, Mechanism, and Applications. Chem. Rev. 2016, 116(23), 14726–14768. https://doi.org/10.1021/acs.chemrev.6b00466.
 (a) Krasiński, A.; Fokin, V. V.; Sharpless, K. B. Direct Synthesis of 1,5-Disubstituted-4-Magnesio-1,2,3-Triazoles, Revisited. Org. Lett. 2004, 6(8), 1237–1240. https://doi.org/10.1021/ol0499203. (b) Spiteri, C.; Moses, J. E. Copper-Catalyzed Azide–Alkyne Cycloaddition: Regioselective Synthesis of 1,4,5-Trisubstituted 1,2,3-Triazoles. Angew. Chem. Int. Ed. 2010, 49(1), 31–33. https://doi.org/10.1002/anie.200905322. (c) Smith, C. D.; Greaney, M. F. Zinc Mediated Azide–Alkyne Ligation to 1,5- and 1,4,5-Substituted 1,2,3-Triazoles. Org. Lett. 2013, 15(18), 4826–4829. https://doi.org/10.1021/ol402225d. (d) Yuan, H.; Zhang, L.; Liu, Z.; Liu, Y.; Wang, J.; Li, W. A NHC-Catalyzed 1,3-Dipolar Cycloaddition Reaction of Allyl Ketones with Azides: Direct Access to 1,4,5-Trisubstituted 1,2,3-Triazoles. Org. Biomol. Chem. 2017, 15(20), 4286–4290. https://doi.org/10.1039/C7OB00906B.
 Yanai, H.; Taguchi, T. Indium(III) Triflate Catalyzed Tandem Azidation/1,3-Dipolar Cycloaddition Reaction of ω,ω-Dialkoxyalkyne Derivatives with Trimethylsilyl Azide. Tetrahedron Letters 2005, 46(50), 8639–8643. https://doi.org/10.1016/j.tetlet.2005.10.053.
 Rasolofonjatovo, E.; Theeramunkong, S.; Bouriaud, A.; Kolodych, S.; Chaumontet, M.; Taran, F. Iridium-Catalyzed Cycloaddition of Azides and 1-Bromoalkynes at Room Temperature. Org. Lett. 2013, 15(18), 4698–4701. https://doi.org/10.1021/ol402008u.
 Ding, S.; Jia, G.; Sun, J. Iridium-Catalyzed Intermolecular Azide–Alkyne Cycloaddition of Internal Thioalkynes under Mild Conditions. Angewandte Chemie International Edition 2014, 53(7), 1877–1880. https://doi.org/10.1002/anie.201309855.
 Luo, Q.; Jia, G.; Sun, J.; Lin, Z. Theoretical Studies on the Regioselectivity of Iridium-Catalyzed 1,3-Dipolar Azide–Alkyne Cycloaddition Reactions. J. Org. Chem. 2014, 79(24), 11970–11980. https://doi.org/10.1021/jo5018348.
 Kim, W. G.; Kang, M. E.; Lee, J. B.; Jeon, M. H.; Lee, S.; Lee, J.; Choi, B.; Cal, P. M. S. D.; Kang, S.; Kee, J.-M.; et al. Nickel-Catalyzed Azide–Alkyne Cycloaddition To Access 1,5-Disubstituted 1,2,3-Triazoles in Air and Water. J. Am. Chem. Soc. 2017, 139(35), 12121–12124. https://doi.org/10.1021/jacs.7b06338.
 Liao, Y.; Lu, Q.; Chen, G.; Yu, Y.; Li, C.; Huang, X. Rhodium-Catalyzed Azide–Alkyne Cycloaddition of Internal Ynamides: Regioselective Assembly of 5-Amino-Triazoles under Mild Conditions. ACS Catal. 2017, 7(11), 7529–7534. https://doi.org/10.1021/acscatal.7b02558.
 Smith, C. D.; Greaney, M. F. Zinc Mediated Azide–Alkyne Ligation to 1,5- and 1,4,5-Substituted 1,2,3-Triazoles. Org. Lett. 2013, 15(18), 4826–4829. https://doi.org/10.1021/ol402225d.
 Han, Y.-P.; Song, X.-R.; Qiu, Y.-F.; Zhang, H.-R.; Li, L.-H.; Jin, D.-P.; Sun, X.-Q.; Liu, X.-Y.; Liang, Y.-M. Lewis Acid Catalyzed [4 + 3] Cycloaddition of Propargylic Alcohols with Azides. Org. Lett. 2016, 18(5), 940–943. https://doi.org/10.1021/acs.orglett.5b03657.
 Sletten, E. M.; Bertozzi, C. R. From Mechanism to Mouse: A Tale of Two Bioorthogonal Reactions. Acc. Chem. Res. 2011, 44(9), 666–676. https://doi.org/10.1021/ar200148z.
 Kim, E.; Koo, H. Biomedical Applications of Copper-Free Click Chemistry: In Vitro, In Vivo, and Ex Vivo. Chem. Sci. 2019, 10(34), 7835–7851. https://doi.org/10.1039/C9SC03368H.
 Ostrovskis, P.; M. R. Volla, C.; Turks, M.; Markovic, D. Application of Metal Free Click Chemistry in Biological Studies. Curr. Org. Chem. 2013, 17(6), 610–640. https://www.ingentaconnect.com/content/ben/coc/2013/00000017/00000006/art00006.
 (a) Uttamapinant, C.; Tangpeerachaikul, A.; Grecian, S.; Clarke, S.; Singh, U.; Slade, P.; Gee, K. R.; Ting, A. Y. Fast, Cell-Compatible Click Chemistry with Copper-Chelating Azides for Biomolecular Labeling. Angew. Chem. Int. Ed. 2012, 51(24), 5852–5856. https://doi.org/10.1002/anie.201108181. (b) Hong, V.; Steinmetz, N. F.; Manchester, M.; Finn, M. G. Labeling Live Cells by Copper-Catalyzed Alkyne−Azide Click Chemistry. Bioconjugate Chem. 2010, 21(10), 1912–1916. https://doi.org/10.1021/bc100272z.
 Patterson, D. M.; Nazarova, L. A.; Prescher, J. A. Finding the Right (Bioorthogonal) Chemistry. ACS Chem. Biol. 2014, 9(3), 592–605. https://doi.org/10.1021/cb400828a.
 For those readers who are fascinated by the etymology of words, the following may be of interest. The prefix "bio" is Greek for "life," and the word "orthogonal" comes from the ancient Greek word orthogonios, meaning "right-angled." Thus, a bioorthogonal chemical reaction is one that is unrelated to processes that occur normally in living systems.
 McKay, C. S.; Finn, M. G. Click Chemistry in Complex Mixtures: Bioorthogonal Bioconjugation. Chemistry & Biology 2014, 21(9), 1075–1101. https://doi.org/10.1016/j.chembiol.2014.09.002.
 Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R. Engineering Chemical Reactivity on Cell Surfaces Through Oligosaccharide Biosynthesis. Science 1997, 276(5315), 1125–1128. https://doi.org/10.1126/science.276.5315.1125.
 Staudinger, H.; Meyer, J. Über Neue Organische Phosphorverbindungen III. Phosphinmethylenderivate Und Phosphinimine. Helvetica Chimica Acta 1919, 2(1), 635–646. https://doi.org/10.1002/hlca.19190020164.
 Saxon, E.; Bertozzi, C. R. Cell Surface Engineering by a Modified Staudinger Reaction. Science 2000, 287(5460), 2007–2010. https://doi.org/10.1126/science.287.5460.2007.
 (a) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. A "Traceless" Staudinger Ligation for the Chemoselective Synthesis of Amide Bonds. Org. Lett. 2000, 2(14), 2141–2143. https://doi.org/10.1021/ol006054v. (b) Sowa, S.; Mühlberg, M.; Pietrusiewicz, K. M.; Hackenberger, C. P. R. Traceless Staudinger Acetylation of Azides in Aqueous Buffers. Bioorganic & Medicinal Chemistry 2013, 21(12), 3465–3472. https://doi.org/10.1016/j.bmc.2013.02.045. (c) van Berkel, S. S.; van Eldijk, M. B.; van Hest, J. C. M. Staudinger Ligation as a Method for Bioconjugation. Angew. Chem. Int. Ed. 2011, 50 (38), 8806–8827. https://doi.org/10.1002/anie.201008102. (d) Schilling, C. I.; Jung, N.; Biskup, M.; Schepers, U.; Bräse, S. Bioconjugation via Azide–Staudinger Ligation: An Overview. Chem. Soc. Rev. 2011, 40(9), 4840–4871. https://doi.org/10.1039/C0CS00123F. (e) Bednarek, C.; Wehl, I.; Jung, N.; Schepers, U.; Bräse, S. The Staudinger Ligation. Chem. Rev. 2020, 120(10), 4301–4354. https://doi.org/10.1021/acs.chemrev.9b00665.
 (a) Lin, F. L.; Hoyt, H. M.; van Halbeek, H.; Bergman, R. G.; Bertozzi, C. R. Mechanistic Investigation of the Staudinger Ligation. J. Am. Chem. Soc. 2005, 127(8), 2686–2695. https://doi.org/10.1021/ja044461m. (b) Soellner, M. B.; Nilsson, B. L.; Raines, R. T. Reaction Mechanism and Kinetics of the Traceless Staudinger Ligation. J. Am. Chem. Soc. 2006, 128(27), 8820–8828. https://doi.org/10.1021/ja060484k.
 Sowa, S.; Mühlberg, M.; Pietrusiewicz, K. M.; Hackenberger, C. P. R. Traceless Staudinger Acetylation of Azides in Aqueous Buffers. Bioorganic & Medicinal Chemistry 2013, 21(12), 3465–3472. https://doi.org/10.1016/j.bmc.2013.02.045.
 Xie, Y.; Cheng, L.; Gao, Y.; Cai, X.; Yang, X.; Yi, L.; Xi, Z. Tetrafluorination of Aromatic Azide Yields a Highly Efficient Staudinger Reaction: Kinetics and Biolabeling. Chemistry – An Asian Journal 2018, 13(14), 1791–1796. https://doi.org/10.1002/asia.201800503.
 (a) Blomquist, A. T.; Liu, L. H. Many-Membered Carbon Rings. VII. Cycloöctyne. J. Am. Chem. Soc. 1953, 75(9), 2153–2154. https://doi.org/10.1021/ja01105a039. (b) Wittig, G.; Krebs, A. Zur Existenz niedergliedriger Cycloalkine, I. Chemische Berichte 1961, 94(12), 3260–3275. https://doi.org/10.1002/cber.19610941213.
 (a) See references 24 and 30, supra. (b) Debets, M. F.; van Berkel, S. S.; Dommerholt, J.; Dirks, A. (Ton) J.; Rutjes, F. P. J. T.; van Delft, F. L. Bioconjugation with Strained Alkenes and Alkynes. Acc. Chem. Res. 2011, 44(9), 805–815. https://doi.org/10.1021/ar200059z. (c) Dommerholt, J.; Rutjes, F. P. J. T.; van Delft, F. L. Strain-Promoted 1,3-Dipolar Cycloaddition of Cycloalkynes and Organic Azides. Top Curr Chem (Z) 2016, 374(2), 16. https://doi.org/10.1007/s41061-016-0016-4. (d) Meghani, N. M.; Amin, H. H.; Lee, B.-J. Mechanistic Applications of Click Chemistry for Pharmaceutical Drug Discovery and Drug Delivery. Drug Discovery Today 2017, 22(11), 1604–1619. https://doi.org/10.1016/j.drudis.2017.07.007. (e) Row, R. D.; Prescher, J. A. Constructing New Bioorthogonal Reagents and Reactions. Acc. Chem. Res. 2018, 51(5), 1073–1081. https://doi.org/10.1021/acs.accounts.7b00606. (f) Devaraj, N. K. The Future of Bioorthogonal Chemistry. ACS Cent. Sci. 2018, 4(8), 952–959. https://doi.org/10.1021/acscentsci.8b00251.
 Chang, P. V.; Prescher, J. A.; Sletten, E. M.; Baskin, J. M.; Miller, I. A.; Agard, N. J.; Lo, A.; Bertozzi, C. R. Copper-Free Click Chemistry in Living Animals. PNAS 2010, 107(5), 1821–1826. https://doi.org/10.1073/pnas.0911116107. See also, reference 24 at pages 673-674 describing this problem.
 (a) Gordon, C. G.; Mackey, J. L.; Jewett, J. C.; Sletten, E. M.; Houk, K. N.; Bertozzi, C. R. Reactivity of Biarylazacyclooctynones in Copper-Free Click Chemistry. J. Am. Chem. Soc. 2012, 134(22), 9199–9208. https://doi.org/10.1021/ja3000936. (b) However the following reference found that only electronic effects could be correlated accurately to experimental data: Garcia‐Hartjes, J.; Dommerholt, J.; Wennekes, T.; Delft, F. L. van; Zuilhof, H. Electronic Effects versus Distortion Energies During Strain-Promoted Alkyne-Azide Cycloadditions: A Theoretical Tool to Predict Reaction Kinetics. European Journal of Organic Chemistry 2013, 2013(18), 3712–3720. https://doi.org/10.1002/ejoc.201201627.
 (a) Debets, M. F.; Berkel, S. S. van; Schoffelen, S.; Rutjes, F. P. J. T.; Hest, J. C. M. van; Delft, F. L. van. Aza-Dibenzocyclooctynes for Fast and Efficient Enzyme PEGylation via Copper-Free (3+2) Cycloaddition. Chem. Commun. 2010, 46(1), 97–99. https://doi.org/10.1039/B917797C.
(b) Ning, et al. reported the synthesis of DIBC in Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G.-J. Visualizing Metabolically Labeled Glycoconjugates of Living Cells by Copper-Free and Fast Huisgen Cycloadditions. Angewandte Chemie International Edition 2008, 47(12), 2253–2255. https://doi.org/10.1002/anie.200705456.
(c) Sletten and Bertozzi reported the synthesis of DIMAC in Sletten, E. M.; Bertozzi, C. R. A Hydrophilic Azacyclooctyne for Cu-Free Click Chemistry. Org. Lett. 2008, 10(14), 3097–3099. https://doi.org/10.1021/ol801141k.
 Davis, D. L.; Price, E. K.; Aderibigbe, S. O.; Larkin, M. X.-H.; Barlow, E. D.; Chen, R.; Ford, L. C.; Gray, Z. T.; Gren, S. H.; Jin, Y.; et al. Effect of Buffer Conditions and Organic Cosolvents on the Rate of Strain-Promoted Azide–Alkyne Cycloaddition. J. Org. Chem. 2016, 81(15), 6816–6819. https://doi.org/10.1021/acs.joc.6b01112.
 Hoyle, C. E.; Lowe, A. B.; Bowman, C. N. Thiol-Click Chemistry: A Multifaceted Toolbox for Small Molecule and Polymer Synthesis. Chem. Soc. Rev. 2010, 39(4), 1355–1387. https://doi.org/10.1039/B901979K.
 Fairbanks, B. D.; Sims, E. A.; Anseth, K. S.; Bowman, C. N. Reaction Rates and Mechanisms for Radical, Photoinitated Addition of Thiols to Alkynes, and Implications for Thiol−Yne Photopolymerizations and Click Reactions. Macromolecules 2010, 43(9), 4113–4119. https://doi.org/10.1021/ma1002968.
 Conte, M. L.; Staderini, S.; Marra, A.; Sanchez-Navarro, M.; Davis, B. G.; Dondoni, A. Multi-Molecule Reaction of Serum Albumin Can Occur through Thiol-Yne Coupling. Chem. Commun. 2011, 47(39), 11086–11088. https://doi.org/10.1039/C1CC14402B.
 Griebenow, N.; Dilmaç, A. M.; Greven, S.; Bräse, S. Site-Specific Conjugation of Peptides and Proteins via Rebridging of Disulfide Bonds Using the Thiol–Yne Coupling Reaction. Bioconjugate Chem. 2016, 27(4), 911–917. https://doi.org/10.1021/acs.bioconjchem.5b00682.
 Zhang, C.; Dai, P.; Vinogradov, A. A.; Gates, Z. P.; Pentelute, B. L. Site-Selective Cysteine–Cyclooctyne Conjugation. Angew. Chem. Int. Ed. 2018, 57(22), 6459–6463. https://doi.org/10.1002/anie.201800860.
 van Geel, R.; Pruijn, G. J. M.; van Delft, F. L.; Boelens, W. C. Preventing Thiol-Yne Addition Improves the Specificity of Strain-Promoted Azide–Alkyne Cycloaddition. Bioconjugate Chem. 2012, 23(3), 392–398. https://doi.org/10.1021/bc200365k.
 Kenry; Liu, B. Bio-Orthogonal Click Chemistry for In Vivo Bioimaging. TRECHEM 2019, 1(8), 763–778. https://doi.org/10.1016/j.trechm.2019.08.003.
 Pickens, C. J.; Johnson, S. N.; Pressnall, M. M.; Leon, M. A.; Berkland, C. J. Practical Considerations, Challenges, and Limitations of Bioconjugation via Azide–Alkyne Cycloaddition. Bioconjugate Chem. 2018, 29(3), 686–701. https://doi.org/10.1021/acs.bioconjchem.7b00633.
 Zeng, D.; Zeglis, B. M.; Lewis, J. S.; Anderson, C. J. The Growing Impact of Bioorthogonal Click Chemistry on the Development of Radiopharmaceuticals. J Nucl Med 2013, 54(6), 829–832. https://doi.org/10.2967/jnumed.112.115550.
 Mushtaq, S.; Yun, S.-J.; Jeon, J. Recent Advances in Bioorthogonal Click Chemistry for Efficient Synthesis of Radiotracers and Radiopharmaceuticals. Molecules 2019, 24(19), 3567. https://doi.org/10.3390/molecules24193567.
 Jain, N.; Smith, S. W.; Ghone, S.; Tomczuk, B. Current ADC Linker Chemistry. Pharm Res 2015, 32(11), 3526–3540. https://doi.org/10.1007/s11095-015-1657-7.
 Yao, H.; Jiang, F.; Lu, A.; Zhang, G. Methods to Design and Synthesize Antibody-Drug Conjugates (ADCs). International Journal of Molecular Sciences 2016, 17(2), 194. https://doi.org/10.3390/ijms17020194.
 Zhou, Q. Site-Specific Antibody Conjugation for ADC and Beyond. Biomedicines 2017, 5(4), 64. https://doi.org/10.3390/biomedicines5040064.
 Ning, X.; Temming, R. P.; Dommerholt, J.; Guo, J.; Ania, D. B.; Debets, M. F.; Wolfert, M. A.; Boons, G.-J.; van Delft, F. L. Protein Modification by Strain-Promoted Alkyne–Nitrone Cycloaddition. Angewandte Chemie International Edition 2010, 49(17), 3065–3068. https://doi.org/10.1002/anie.201000408.
 (a) McKay, C. S.; Chigrinova, M.; Blake, J. A.; Pezacki, J. P. Kinetics Studies of Rapid Strain-Promoted [3 + 2]-Cycloadditions of Nitrones with Biaryl-Aza-Cyclooctynone. Org. Biomol. Chem. 2012, 10(15), 3066–3070. https://doi.org/10.1039/C2OB07165G. (b) McKay, C. S.; Blake, J. A.; Cheng, J.; Danielson, D. C.; Pezacki, J. P. Strain-Promoted Cycloadditions of Cyclic Nitrones with Cyclooctynes for Labeling Human Cancer Cells. Chem. Commun. 2011, 47(36), 10040–10042. https://doi.org/10.1039/C1CC13808A.
 (a) MacKenzie, D. A.; Pezacki, J. P. Kinetics Studies of Rapid Strain-Promoted [3+2] Cycloadditions of Nitrones with Bicyclo[6.1.0]Nonyne. Can. J. Chem. 2014, 92(4), 337–340. https://doi.org/10.1139/cjc-2013-0577.
 MacKenzie, D. A.; Sherratt, A. R.; Chigrinova, M.; Cheung, L. L.; Pezacki, J. P. Strain-Promoted Cycloadditions Involving Nitrones and Alkynes—Rapid Tunable Reactions for Bioorthogonal Labeling. Current Opinion in Chemical Biology 2014, 21, 81–88. https://doi.org/10.1016/j.cbpa.2014.05.023.
 Blackman, M. L.; Royzen, M.; Fox, J. M. Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels−Alder Reactivity. J. Am. Chem. Soc. 2008, 130(41), 13518–13519. https://doi.org/10.1021/ja8053805.
 Devaraj, N. K.; Weissleder, R.; Hilderbrand, S. A. Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging. Bioconjugate Chem. 2008, 19(12), 2297–2299. https://doi.org/10.1021/bc8004446.
 Knall, A.-C.; Slugovc, C. Inverse Electron Demand Diels–Alder (IEDDA)-Initiated Conjugation: A (High) Potential Click Chemistry Scheme. Chem. Soc. Rev. 2013, 42(12), 5131–5142. https://doi.org/10.1039/C3CS60049A.
 Pagel, M. Inverse Electron Demand Diels–Alder (IEDDA) Reactions in Peptide Chemistry. Journal of Peptide Science 2019, 25(1), e3141. https://doi.org/10.1002/psc.3141.
 Oller‐Salvia, B.; Kym, G.; Chin, J. W. Rapid and Efficient Generation of Stable Antibody–Drug Conjugates via an Encoded Cyclopropene and an Inverse-Electron-Demand Diels–Alder Reaction. Angewandte Chemie International Edition 2018, 57(11), 2831–2834. https://doi.org/10.1002/anie.201712370.
 Xu, L.; Raabe, M.; Zegota, M. M.; Nogueira, J. C. F.; Chudasama, V.; Kuan, S. L.; Weil, T. Site-Selective Protein Modification via Disulfide Rebridging for Fast Tetrazine/Trans-Cyclooctene Bioconjugation. Org. Biomol. Chem. 2020, 18(6), 1140–1147. https://doi.org/10.1039/C9OB02687H.
 Verhoeven, M.; Seimbille, Y.; Dalm, S. U. Therapeutic Applications of Pretargeting. Pharmaceutics 2019, 11(9), 434. https://doi.org/10.3390/pharmaceutics11090434.
 Aubert, S.; Bezagu, M.; Spivey, A. C.; Arseniyadis, S. Spatial and Temporal Control of Chemical Processes. Nature Reviews Chemistry 2019, 3(12), 706–722. https://doi.org/10.1038/s41570-019-0139-6.
 The following is a non-comprehensive list of reviews of photoclick chemistry: (a) Lim, R. K. V.; Lin, Q. Photoinducible Bioorthogonal Chemistry: A Spatiotemporally Controllable Tool to Visualize and Perturb Proteins in Live Cells. Acc. Chem. Res. 2011, 44(9), 828–839. https://doi.org/10.1021/ar200021p. (b) Ramil, C. P.; Lin, Q. Photoclick Chemistry: A Fluorogenic Light-Triggered in Vivo Ligation Reaction. Current Opinion in Chemical Biology 2014, 21, 89–95. https://doi.org/10.1016/j.cbpa.2014.05.024. (c) Herner, A.; Lin, Q. Photo-Triggered Click Chemistry for Biological Applications. In Cycloadditions in Bioorthogonal Chemistry; Vrabel, M., Carell, T., Eds.; Topics in Current Chemistry Collections; Springer International Publishing: Cham, 2016; pp 77–107. https://doi.org/10.1007/978-3-319-29686-9_5. (d) Kaur, G.; Singh, G.; Singh, J. Photochemical Tuning of Materials: A Click Chemistry Perspective. Materials Today Chemistry 2018, 8, 56–84. https://doi.org/10.1016/j.mtchem.2018.03.002.
 (a) Yao, H.; Wang, J.; Mi, S. Photo Processing for Biomedical Hydrogels Design and Functionality: A Review. Polymers 2018, 10(1), 11. https://doi.org/10.3390/polym10010011. (b) Pereira, R. F.; Barrias, C. C.; Bártolo, P. J.; Granja, P. L. Cell-Instructive Pectin Hydrogels Crosslinked via Thiol-Norbornene Photo-Click Chemistry for Skin Tissue Engineering. Acta Biomaterialia 2018, 66, 282–293. https://doi.org/10.1016/j.actbio.2017.11.016. (c) Yu, F.; Cao, X.; Li, Y.; Chen, X. Diels–Alder Click-Based Hydrogels for Direct Spatiotemporal Postpatterning via Photoclick Chemistry. ACS Macro Lett. 2015, 4(3), 289–292. https://doi.org/10.1021/mz5007427. (d) Lee, S.; Park, Y. H.; Ki, C. S. Fabrication of PEG–Carboxymethylcellulose Hydrogel by Thiol-Norbornene Photo-Click Chemistry. International Journal of Biological Macromolecules 2016, 83, 1–8. https://doi.org/10.1016/j.ijbiomac.2015.11.050. (e) Díaz-Betancor, Z.; Bañuls, M.-J.; Maquieira, Á. Photoclick Chemistry to Create Dextran-Based Nucleic Acid Microarrays. Anal Bioanal Chem 2019, 411(25), 6745–6754. https://doi.org/10.1007/s00216-019-02050-3.
 See references 69(a) and 69(b); see also Wang, J.; Zhang, W.; Song, W.; Wang, Y.; Yu, Z.; Li, J.; Wu, M.; Wang, L.; Zang, J.; Lin, Q. A Biosynthetic Route to Photoclick Chemistry on Proteins. J. Am. Chem. Soc. 2010, 132 (42), 14812–14818. https://doi.org/10.1021/ja104350y.
 (a) Nainar, S.; Kubota, M.; McNitt, C.; Tran, C.; Popik, V. V.; Spitale, R. C. Temporal Labeling of Nascent RNA Using Photoclick Chemistry in Live Cells. J. Am. Chem. Soc. 2017, 139(24), 8090–8093. https://doi.org/10.1021/jacs.7b03121. (b) Li, J.; Kong, H.; Huang, L.; Cheng, B.; Qin, K.; Zheng, M.; Yan, Z.; Zhang, Y. Visible Light-Initiated Bioorthogonal Photoclick Cycloaddition. J. Am. Chem. Soc. 2018, 140(44), 14542–14546. https://doi.org/10.1021/jacs.8b08175.
 Holland, J. P.; Gut, M.; Klingler, S.; Fay, R.; Guillou, A. Photochemical Reactions in the Synthesis of Protein–Drug Conjugates. Chemistry – A European Journal 2020, 26(1), 33–48. https://doi.org/10.1002/chem.201904059.
 Posner, T. Beiträge Zur Kenntniss Der Ungesättigten Verbindungen. II. Ueber Die Addition von Mercaptanen an Ungesättigte Kohlenwasserstoffe. Berichte der deutschen chemischen Gesellschaft 1905, 38(1), 646–657. https://doi.org/10.1002/cber.190503801106.
 Hoyle, C. E.; Bowman, C. N. Thiol–Ene Click Chemistry. Angewandte Chemie International Edition 2010, 49(9), 1540–1573. https://doi.org/10.1002/anie.200903924.
 (a) Lowe, A. B. Thiol-Ene "Click" Reactions and Recent Applications in Polymer and Materials Synthesis. Polym. Chem. 2010, 1(1), 17–36. https://doi.org/10.1039/B9PY00216B. (b) Lowe, A. B. Thiol–Ene "Click" Reactions and Recent Applications in Polymer and Materials Synthesis: A First Update. Polym. Chem. 2014, 5(17), 4820–4870. https://doi.org/10.1039/C4PY00339J.
 Stenzel, M. H. Bioconjugation Using Thiols: Old Chemistry Rediscovered to Connect Polymers with Nature’s Building Blocks. ACS Macro Lett. 2013, 2(1), 14–18. https://doi.org/10.1021/mz3005814.
 Lin, C.-C.; Raza, A.; Shih, H. PEG Hydrogels Formed by Thiol-Ene Photo-Click Chemistry and Their Effect on the Formation and Recovery of Insulin-Secreting Cell Spheroids. Biomaterials 2011, 32(36), 9685–9695. https://doi.org/10.1016/j.biomaterials.2011.08.083.
 Yu, B.; Jiang, X.; Qin, N.; Yin, J. Thiol–Ene Photocrosslinked Hybrid Vesicles from Co-Assembly of POSS and Poly(Ether Amine) (PEA). Chem. Commun. 2011, 47(44), 12110–12112. https://doi.org/10.1039/C1CC15252A.
 Escorihuela, J.; Bañuls, M.-J.; Grijalvo, S.; Eritja, R.; Puchades, R.; Maquieira, Á. Direct Covalent Attachment of DNA Microarrays by Rapid Thiol–Ene "Click" Chemistry. Bioconjugate Chem. 2014, 25(3), 618–627. https://doi.org/10.1021/bc500033d.
 Ye, X.; Yuan, J.; Jiang, Z.; Wang, S.; Wang, P.; Wang, Q.; Cui, L. Thiol-Ene Photoclick Reaction: An Eco-Friendly and Facile Approach for Preparation of MPEG-g-Keratin Biomaterial. Engineering in Life Sciences 2020, 20(1–2), 17–25. https://doi.org/10.1002/elsc.201900105.
 (a) Dondoni, A. The Emergence of Thiol–Ene Coupling as a Click Process for Materials and Bioorganic Chemistry. Angewandte Chemie International Edition 2008, 47(47), 8995–8997. https://doi.org/10.1002/anie.200802516. (b) Tramutola, A.; Falcucci, S.; Brocco, U.; Triani, F.; Lanzillotta, C.; Donati, M.; Panetta, C.; Luzi, F.; Iavarone, F.; Vincenzoni, F.; Castagnola, M.; Perluigi, M.; Di Domenico, F.; De Marco, F. Protein Oxidative Damage in UV-Related Skin Cancer and Dysplastic Lesions Contributes to Neoplastic Promotion and Progression. Cancers 2020, 12(1), 110. https://doi.org/10.3390/cancers12010110. (c) Roy, S. Impact of UV Radiation on Genome Stability and Human Health. In Ultraviolet Light in Human Health, Diseases and Environment; Ahmad, S. I., Ed.; Advances in Experimental Medicine and Biology; Springer International Publishing: Cham, 2017; pp 207–219. https://doi.org/10.1007/978-3-319-56017-5_17. (d) Du, C.; Barnett, G.; Borwankar, A.; Lewandowski, A.; Singh, N.; Ghose, S.; Borys, M.; Li, Z. J. Protection of Therapeutic Antibodies from Visible Light Induced Degradation: Use Safe Light in Manufacturing and Storage. European Journal of Pharmaceutics and Biopharmaceutics 2018, 127, 37–43. https://doi.org/10.1016/j.ejpb.2018.02.007. (e) Neves-Petersen, M. T.; Petersen, S.; Gajula, G. P. UV Light Effects on Proteins: From Photochemistry to Nanomedicine. Molecular Photochemistry - Various Aspects 2012. https://doi.org/10.5772/37947. (f) Kerwin, B. A.; Remmele, R. L. Protect from Light: Photodegradation and Protein Biologics. Journal of Pharmaceutical Sciences 2007, 96 (6), 1468–1479. https://doi.org/10.1002/jps.20815. (g) Hussain, R.; Longo, E.; Siligardi, G. UV-Denaturation Assay to Assess Protein Photostability and Ligand-Binding Interactions Using the High Photon Flux of Diamond B23 Beamline for SRCD. Molecules 2018, 23 (8), 1906. https://doi.org/10.3390/molecules23081906.
 Gunnoo, S. B.; Madder, A. Chemical Protein Modification through Cysteine. ChemBioChem 2016, 17(7), 529–553. https://doi.org/10.1002/cbic.201500667.
 Bader, H.; Cross, L. C.; Heilbron, I.; Jones, E. R. H. 132. Researches on Acetylenic Compounds. Part XVIII. The Addition of Thiolacetic Acid to Acetylenic Hydrocarbons. The Conversion of Monosubstituted Acetylenes into Aldehydes and 1 :2-Dithiols. J. Chem. Soc. 1949, 619–623. https://doi.org/10.1039/JR9490000619.
 Yet, see, Ichinose, Y.; Wakamatsu, K.; Nozaki, K.; Birbaum, J.-L.; Oshima, K.; Utimoto, K. Et3B Induced Radical Addition of Thiols to Acetylenes. Chem. Lett. 1987, 16(8), 1647–1650. https://doi.org/10.1246/cl.1987.1647.
 Fairbanks, B. D.; Scott, T. F.; Kloxin, C. J.; Anseth, K. S.; Bowman, C. N. Thiol−Yne Photopolymerizations: Novel Mechanism, Kinetics, and Step-Growth Formation of Highly Cross-Linked Networks. Macromolecules 2009, 42(1), 211–217. https://doi.org/10.1021/ma801903w.
 Shiu, H.-Y.; Chan, T.-C.; Ho, C.-M.; Liu, Y.; Wong, M.-K.; Che, C.-M. Electron-Deficient Alkynes as Cleavable Reagents for the Modification of Cysteine-Containing Peptides in Aqueous Medium. Chemistry – A European Journal 2009, 15(15), 3839–3850. https://doi.org/10.1002/chem.200800669.
 Monesi, A. Sulfanyl Radical Addition to Alkynes: Revisiting an Old Reaction to Enter the Novel Realms of Green Chemistry, Bioconjugation, and Material Chemistry. Tesi di dottorato, alma, 2012. Monesi, Alessandro (2012) Sulfanyl Radical Addition to Alkynes: Revisiting an Old Reaction to Enter the Novel Realms of Green Chemistry, Bioconjugation, and Material Chemistry, [Dissertation thesis], Alma Mater Studiorum Università di Bologna. Dottorato di ricerca in Scienze chimiche , 24 Ciclo. DOI 10.6092/unibo/amsdottorato/4555.
 Minozzi, M.; Monesi, A.; Nanni, D.; Spagnolo, P.; Marchetti, N.; Massi, A. An Insight into the Radical Thiol/Yne Coupling: The Emergence of Arylalkyne-Tagged Sugars for the Direct Photoinduced Glycosylation of Cysteine-Containing Peptides. J. Org. Chem. 2011, 76(2), 450–459. https://doi.org/10.1021/jo101906j.
 Kumar, J.; Bousquet, A.; Stenzel, M. H. Thiol-Alkyne Chemistry for the Preparation of Micelles with Glycopolymer Corona: Dendritic Surfaces versus Linear Glycopolymer in Their Ability to Bind to Lectins. Macromolecular Rapid Communications 2011, 32(20), 1620–1626. https://doi.org/10.1002/marc.201100331.
 Naik, S. S.; Chan, J. W.; Comer, C.; Hoyle, C. E.; Savin, D. A. Thiol–Yne 'Click' Chemistry as a Route to Functional Lipid Mimetics. Polym. Chem. 2011, 2(2), 303–305. https://doi.org/10.1039/C0PY00231C.
 Yao, B.; Hu, T.; Zhang, H.; Li, J.; Sun, J. Z.; Qin, A.; Tang, B. Z. Multi-Functional Hyperbranched Poly(Vinylene Sulfide)s Constructed via Spontaneous Thiol–Yne Click Polymerization. Macromolecules 2015, 48(21), 7782–7791. https://doi.org/10.1021/acs.macromol.5b01868.
 Conte, M. L.; Staderini, S.; Marra, A.; Sanchez-Navarro, M.; Davis, B. G.; Dondoni, A. Multi-Molecule Reaction of Serum Albumin Can Occur through Thiol-Yne Coupling. Chem. Commun. 2011, 47(39), 11086–11088. https://doi.org/10.1039/C1CC14402B.
 Tian, Y.; Li, J.; Zhao, H.; Zeng, X.; Wang, D.; Liu, Q.; Niu, X.; Huang, X.; Xu, N.; Li, Z. Stapling of Unprotected Helical Peptides via Photo-Induced Intramolecular Thiol–Yne Hydrothiolation. Chem. Sci. 2016, 7(5), 3325–3330. https://doi.org/10.1039/C6SC00106H.
 Hu, X.; Tan, H.; Wang, X.; Chen, P. Surface Functionalization of Hydrogel by Thiol-Yne Click Chemistry for Drug Delivery. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2016, 489, 297–304. https://doi.org/10.1016/j.colsurfa.2015.11.007.
 Pranantyo, D.; Xu, L. Q.; Kang, E.-T.; Mya, M. K.; Chan-Park, M. B. Conjugation of Polyphosphoester and Antimicrobial Peptide for Enhanced Bactericidal Activity and Biocompatibility. Biomacromolecules 2016, 17(12), 4037–4044. https://doi.org/10.1021/acs.biomac.6b01452.
 Dadfar, S. M. M.; Sekula‐Neuner, S.; Bog, U.; Trouillet, V.; Hirtz, M. Site-Specific Surface Functionalization via Microchannel Cantilever Spotting (ΜCS): Comparison between Azide–Alkyne and Thiol–Alkyne Click Chemistry Reactions. Small 2018, 14(21), 1800131. https://doi.org/10.1002/smll.201800131. Figure 1 of this paper excellently summarizes the reaction conditions under which these reactions occur.
 Griebenow, N.; Dilmaç, A. M.; Greven, S.; Bräse, S. Site-Specific Conjugation of Peptides and Proteins via Rebridging of Disulfide Bonds Using the Thiol–Yne Coupling Reaction. Bioconjugate Chem. 2016, 27(4), 911–917. https://doi.org/10.1021/acs.bioconjchem.5b00682.