Shameer M. Kondengadan,Shubham Bansal,Ce Yang,Dongning Liu,Zach Fultz,Binghe Wang
Click chemistry and drug delivery: A bird's-eye view
Bioorthogonal chemistry,Bond-breaking reaction,PROTAC,Antibody–drug conjugates,Gasotransmitters
Click chemistry has been proven to be very useful in drug delivery. Due to the availability of a large number of click reactions with a various characteristics, selection of appropriate chemistry for a given application is often not a trivial task. This review is written for pharmaceutical researchers who are interested in click chemistry applications and yet may not be click chemistry experts. For this, the review gives an overview of available click reactions organized by application types. Further, the general understanding of click reactions being fast and high yielding sometimes overshadows the need to analyze reaction kinetics in assessing suitability of a given reaction for certain applications. For this, we highlight the need to analyze the relationship among reaction kinetics, concentration effects, and reaction time scales, knowing that lack of such analysis could easily lead to failures. Further, possible issues such as chemical stability with various click reagents are also discussed to aid experimental designs. Recent examples and extensive references are also provided to aid in-depth understanding of technical details. We hope this review will help those interested in using click chemistry in drug delivery to select the appropriate reactions/reagents and minimize the number of pitfalls.
Click chemistry and drug delivery: A bird's-eye view

Click chemistry has been proven to be very useful in drug delivery. Due to the availability of a large number of click reactions with a various characteristics, selection of appropriate chemistry for a given application is often not a trivial task. This review is written for pharmaceutical researchers who are interested in click chemistry applications and yet may not be click chemistry experts. For this, the review gives an overview of available click reactions organized by application types. Further, the general understanding of click reactions being fast and high yielding sometimes overshadows the need to analyze reaction kinetics in assessing suitability of a given reaction for certain applications. For this, we highlight the need to analyze the relationship among reaction kinetics, concentration effects, and reaction time scales, knowing that lack of such analysis could easily lead to failures. Further, possible issues such as chemical stability with various click reagents are also discussed to aid experimental designs. Recent examples and extensive references are also provided to aid in-depth understanding of technical details. We hope this review will help those interested in using click chemistry in drug delivery to select the appropriate reactions/reagents and minimize the number of pitfalls.

In the world of drug discovery, a major issue is the optimization of pharmaceutical properties including pharmacokinetics, metabolism, absorption, distribution, toxicity, and drug targeting. Developing new drug delivery approaches often allows for solutions to many of such issues. In recent years, approaches of click chemistry and bioorthogonal chemistry have been widely applied in drug delivery, broadly defined, with very exciting results. Along this line, there have been reviews addressing specific aspects of click chemistry in drug delivery including cell engineering and drug delivery; cell tracking and tissue engineering; polymers, dendrimers, and hydrogels for drug delivery; as well as other general topics8, 9, 10. This review is written for pharmaceutical researchers and is aimed at providing a birds-eye view of the various types of click chemistry that impacts drug delivery and highlighting some common issues that need to be carefully considered when using click chemistry for drug delivery applications. The review is structured in such a way that addresses specific needs of interested readers. First, we have a section that provides an overview of available click reactions for drug delivery applications. This section should allow readers to quickly achieve an understanding of the types of reactions needed for a given type of applications. Second, we are mindful that not all readers are aware of the intricate details as to the relationship among reaction kinetics, concentrations of the reaction partners, and reaction time scale. It is very important to note that the choice of reaction and associated reaction kinetics is a critical factor to consider; success or failure of a project could rest on the selection of not only the right type of reactions, but also the appropriate reaction kinetics. For this reason, we highlight key reaction kinetic parameters and calculated reaction half-lives at various concentrations of the reagents that one would expect to use. Such kinetic analysis will especially help those who do not deal with reaction kinetics on a routine basis. Additionally, experts will benefit from such analysis as well. Third, very rarely a click reagent is completely benign, stable, and inert towards biomolecules. Some reactivity issues of click reaction partners are discussed whenever relevant and applicable. Such analysis is important for readers to consider in experimental design. Fourth, we organize the contents based on types of applications such as direct conjugation, triggered drug release, and delivery of gaseous signaling molecules as a unique class of small molecules. Such an organization will help readers to readily focus on reactions that match the intended application. Fifth, we show recent examples in various types of applications for readers to gain in-depth understandings of relevant applications. By bringing major types of applications into one unifying theme of drug delivery, the review provides a birds-eye view of the field and covers a wide range of concepts useful to both novice and experts. With all these in mind, we begin by giving the basic characteristics of the commonly used click reactions in drug delivery and then highlight different types of applications.

Bioorthogonal reactions are generally fast and high-yielding reactions that do not interfere with the native biological processes,. Many click reactions are bioorthogonal and have been extensively used for different applications. Diverse types of reactions for various purposes have been reported over the years. In this section, we give an overview of click reactions in the context of applications in drug delivery. Further, we discuss certain characteristics important for drug delivery applications including water solubility and reaction kinetics. For various purposes, there are several types of reactions suitable for applications including strain-promoted azido-alkyne cycloaddition (SPAAC) (Table 1, Entry 1), Cu-catalyzed azido-alkyne cycloaddition (CuAAC) (Table 1, Entry 2), Staudinger ligation (Table 1, Entry 3), cycloaddition reactions between a tetrazine and a trans-alkene or a strained alkyne (Table 2, Entries 1, 2, 4, 8; Table 1, Entry 9), reactions between an isocyanopropyl group and tetrazine (Table 2, Entry 9), and Diels–Alder reactions between a cyclopentadienone and a strained alkyne (Table 2, Entry 5; Table 3, Entry 1). Each reaction type has its own characteristics that are suitable for certain types of drug delivery applications. Table 1, Table 2, Table 3 highlight key characteristics for easy reference in selecting appropriate reactions for an intended application. These reactions have a range of reaction rates. Some are slow and are largely limited to solution-phase conjugation-related work. For in-vivo applications, reaction kinetics need to be compatible with concentrations easily attainable in vivo. There are many reactions with second order rate constants in the range of 1 to 1 × 103 L/mol·s, which would allow for effective in-situ applications in the low to mid-micromolar concentration range. As a reference point for meaningful time-scale discussions, the first half-life at 5 μmol/L of each partner is 3 min for a reaction with a rate constant of 1 × 103 L/mol·s and about 56 h for a reaction with a rate constant of 1 L/mol·s.

In the context of drug delivery, click reactions have been used for direct conjugation in solution, in cell culture, and in animal-model studies. Such applications include protein modifications in solution or PROTAC (See Section 3.3 for highlighted examples) assembly in vivo. Bioconjugation involves linking two or more molecular partners: often a small molecule with a functionalized biomolecule. Such conjugation requires some or all of the following features: high efficiency and yield, short reaction time, high selectivity, and inertness toward functional groups of biomolecules. Over the last two decades, a series of bioorthogonal reactions have been reported for such bioconjugation applications. Among the most commonly used reactions are strain-promoted reactions of an alkyne with an azido group (Table 1, Entry 1) and Cu-catalyzed azido-alkyne [2 + 3]-cycloaddition (CuAAC) (Table 1, Entry 2). CuAAC is a modification of the classical Huisgen cycloaddition, which is the reaction between an azido group with a terminal or internal alkyne under somewhat harsh conditions (e.g., refluxing in toluene) to give a 1,2,3-triazole as a mixture of 1,4- and 1,5-regioisomers,, However, copper catalysis makes this reaction feasible under near physiological conditions, except for the issue of copper toxicity. Further, an appropriate ligand is often used to facilitate CuAAC reactions, which yield the 1,4-regioisomer of triazole as the major product (Table 1, Entry 2). The fast reaction kinetics (second-order rate constant of 4.6 × 105 L/mol·s) for this type of reactions means that conjugation chemistry can be carried out within a short duration (e.g., t1/2 = 2.1 s at 1 μmol/L), which is especially desirable if conjugation involves chemically or conformationally unstable biomolecules. As a result, this type of reactions has been widely used in a range of applications including modifications of fragile biomolecules such as peptides, nucleic acids, proteins, and virus-like particles. Still, the application of CuAAC is limited due to Cu toxicity. This issue has been overcome by using strained alkynes. Such strain-promoted reactions are still fast with examples of rate constants of about 2.9 L/mol·s (Table 1, Entry 1), which is sufficiently fast for labeling live cells. Subsequent to Bertozzi's initial work, a large number of strained alkynes have been reported for various applications with variable rate constants,23, 24, 25. Besides being used in [2 + 3] cycloaddition reactions with an alkyne, an azido group has been widely used in Staudinger ligation, which enables the formation of an amide bond between an azido nitrogen and a neighboring carboxyl group after phosphine-mediated reduction of the azido group,. Similar ligation reactions have been explored in multiple areas including biomolecular labeling, radiolabeling,, DNA labeling, and drug delivery. As an example, Staudinger ligation was used for bioluminescence imaging of glycans on live cells (Table 1, Entry 3), drug (doxorubicin) release, and targeted delivery by using polymeric micelles. However, there are certain limitations of the Staudinger ligation including oxidative degradation of the phosphine reagent, the sluggish reaction rate (e.g., second-order rate constant of 2.3 × 10−3 L/mol·s and thus t1/2 of ∼100 days at 50 μmol/L), and the often hydrophobic nature of these molecules. There are also reactions that allow for tight and reversible conjugation. Along this line, boronic acids are known for their ability to reversibly bind with compounds with Lewis bases such as hydroxyl and diol groups. Because of the boron open shell, it is also an excellent Lewis acid, facilitating reversible conjugation reactions of carbonyl groups (Table 1, Entries 5–8). Most of the examples in using the boronic acid group for drug delivery applications are based on its ability to facilitate carbonyl-amine/hydrazine conjugations via “iminium” formation or a similar reaction (Table 1, Entries 5–8). Some of these reactions are known to have very high affinity with Kd values between low μmol/L and low nmol/L. Such high affinities allow for conjugation using reaction partners at concentrations as low as nmol/L. The boronic acid group also tends to aid in water solubility, affording desirable physicochemical properties. Further, the boronic acid group shows a dual-role by promoting conjugation with carbonyl group and serving as a “handle” to allow for further coupling/conjugation (Table 1, Entry 4). Thus, boronic acid chemistry has led to the development of a unique class of bioorthogonal reactions,. These includes reactions with amines (Table 1, Entry 5), hydrazines (Table 1, Entry 5), oximes (Table 1, Entry 5), and α-amino-hydrazide (Table 1, Entry 6). Interestingly, there are significant differences in the Kd values for the association of 2-acetylphenylboronic acid with different functional groups including amine, hydrazine, and oximes. For instance, the dissociation constant (Kd value) for the association of 2-acetylphenylboronic acid has been reported to be 0.6 mmol/L for hydrazide, 0.07 μmol/L for phenylhydrazine, and 14 μmol/L for oximes. The boronic acid group also provides reversibility as iminoboronates have been reported as intermediates for N-terminal cysteine functionalization due to their reversible nature (Table 1, Entry 7). Recently, new chemistry with much-slowed dissociation kinetics than the iminoboronates has been developed (Table 1, Entry 8). In the iminoboronate chemistry described, the boronic acid is always kept ortho to the carbonyl group. Whereas, in one recent design, the boronic acid and carbonyl groups are placed on two different rings. In doing so, a second order reaction rate of 2.1 × 10−2 L/mol·s and Kd of 1.2 mmol/L have been reported for the reaction between lysine and boronic acid molecule RMR1 (Table 1, Entry 8). Collectively, all the reactions described above give a diverse set of options to choose from for a given application. The advancement in the field of labeling and drug delivery has led to development of variations of strain-promoted reactions for diverse applications. For instance, strained alkyne–tetrazine reactions have been reported with reaction rate constants in the range of 2.3–125 L/mol·s (Table 1, Entry 9),. These reactions are essentially strain-promoted inverse-electron demand Diels–Alder reactions (SPIEDAR) with 1,2,4,5-tetrazine. The fast reaction rate is important for various labeling applications at low micromolar concentrations. For example, the reaction half-life is 121 h with a second-order rate constant of 2.3 L/mol·s and 2 h with a rate constant of 125 L/mol·s at low micromolar concentrations (e.g., 1 μmol/L),. Similarly, strain-promoted alkyne-nitrone cycloaddition (SPANC) has been developed for labeling proteins (rate of 3.9 L/mol·s) using nitrone functionalized interleukin-8 as an example (Table 1, Entry 10). This reaction adds to the toolbox for site-specific conjugations of proteins, beyond the traditional methods such as modification of cysteine thiol or lysine amine. However, there are also some limitations such as hydrolytic stability issues with acyclic nitrones and rearrangement of the cycloadduct N-alkylated isoxazolines. To improve the reaction rate further, other activation methods have also been explored, including a photo-triggered click reaction between tetrazole and strained alkyne with a reported second order rate constant of 3.92 × 104 L/mol·s (Table 1, Entry 11), which gives a t1/2 of 25 s at 1 μmol/L. As an example, the reaction between a strained alkyne and a substituted tetrazole allowed for labeling of the glucagon receptor in live cells within 15 s, consistent with the calculated t1/2. Despite the ultrafast kinetics, the use of a photochemical reaction is limited to topical applications. Additional bioorthogonal reactions include [3 + 2] cycloaddition reactions of mesoionic compounds with strained alkynes with the second-order rate constant in the range of 0.006–29.9 L/mol·s. For example, a [3 + 2] cycloaddition reaction between phenyl sydnone and a strained alkyne has a second-order rate constant of 0.054 L/mol·s (Table 1, Entry 12). The examples in this section accommodate a diverse set of functional groups such as an azido group, strained alkyne, and the boronic acid group; provide fast and tunable reaction kinetics; and serve as very useful tools for direct conjugation for monitoring biological processes and protein labeling. These reactions are summarized in Table 1 with information on the salient features of each reaction together with rate constants or ranges of rate constants. Additionally, we have also provided the reaction half-lives at different initial concentrations. This is to provide some intuitive information on the time scale issue without the need for readers to do individual calculations from rate constants. We hope such information will be especially useful to those who are not familiar with chemical reaction kinetics. It should be noted that these half-life values are based on the condition that both reaction partners are at the same specified concentrations. However, for biolabeling work when the concentration of the molecules to be labeled is much lower than the labeling agent, then careful attention is needed to examine the concentration effect on labeling efficiency and time scale. Furthermore, by providing the half-life information, we would like to emphasize the need to consider therapeutically relevant concentrations of click partners when designing experiments. It is important to note that stability plays an important role in applications involving direct conjugation. For some of the reactions discussed above, there are stability issues with some functional groups including azido (prone to reduction and fragmentation), BCN (acid-catalyzed degradation), boronic acid (prone to oxidation and protodeboronation), and tetrazine (reduction). These stability issues present some problems for drug delivery applications. However, the specific compatibility matter will depend on the nature of the intended applications. It is important to note that these reactions have been widely studied in vitro and in vivo. Additionally, similar click reactions have contributed towards solving delivery problems associated with gaseous signaling molecules as discussed in Sections 2.3, 4.2, 5 (Table 2, Entries 5–6, and Table 3).

In recent years, there have been a wave of new reports of strategies that lead to bond-breaking reactions after the initial click reaction, resulting in the ready release of a payload and thus prodrug activation. These bond-breaking reactions range from allylic elimination, hydrolysis of an iminium group, lactonization, 1,4-elimination, 1,6-elimination via quinone methide chemistry, and extrusion. Table 2 shows representative examples that have been used in drug delivery applications. Among all, a widely used reactions is the one between tetrazine and trans-alkene, which is fast and can overcome the major problem of the sluggish reaction kinetics of Staudinger ligation (K2 = ∼10−3 L/mol·s) as discussed before. The tetrazine-trans-alkene reaction has fast reaction kinetics, and has been extensively exploited for applications in targeted delivery of drugs such as doxorubicin (Table 2, Entry 1). The reaction is compatible with various functional groups including alcohol, ester, carbonate, ether, and carboxylic acid (Table 2, Entry 1), making it an attractive approach for prodrug activation. In the majority of examples, the drug moiety is tethered to the trans-cyclooctene part, while tetrazine is used as an activator for drug release. However, there are also examples of using trans-cyclooctene as a trigger for the release of a payload drug tethered to the tetrazine part via a cleavable bond. Using a wide range of trans-cyclooctene analogs and modified tetrazines, reaction rate can be tuned to rate constants as high as ∼104 L/mol·s (Table 2, Entry 2). Further, many other permutations of strained alkene-tetrazine reactions have been reported, where a tetrazine reacts with various activated-alkenes such as benzonorbornadienes (Table 2, Entry 3) and vinyl ether (Table 2, Entry 7). Similar chemistry has been utilized to deliver a drug payload by lactonization where the click reaction acts as trigger (Table 2, Entry 8). In addition, thiocarbamate functionalized trans-cyclooctene has been used to deliver H2S, through the release of carbonyl sulfide as a precursor to H2S via carbonic anhydrase-catalyzed hydrolysis (Table 2, Entry 4). Apart from trans-cyclooctene and tetrazine, other masking groups have also been explored for these types of bioorthogonal activations. One such example is caging by a 3-isocyanopropyl group, which reacts with tetrazine to yield a 3-oxopropyl groups, followed by β-elimination of the 3-oxopropyl group to release the drug payload (Table 2, Entry 9). Dual drug release has also been explored with the reaction between 3-isocyanopropyl and tetrazine where one drug is caged by 3-isocyanopropyl and another caged by tetrazine. There are also click reactions for prodrug activation, which releases two payloads, first carbon monoxide (CO) (in-depth discussions in Sections 4, 5) followed by anticancer drug floxuridine (Table 2, Entries 5–6),. Further, the facile reaction between a strained alkene and a properly designed azido compound can be used to generate an amino group, leading to subsequent prodrug activation via quinone-methide chemistry (Table 2, Entry 10). Similarly, drug release can be achieved by the reaction of an arylazido group with acrolein, which enables prodrug activation by this endogenously produced trigger in cancer cells (Table 2, Entry 11).

In using click reactions for drug delivery applications, the delivery of gaseous signaling molecules including carbon monoxide and sulfur dioxide represents unique challenges. Medical use of gaseous molecules is well-documented as discussed in detail in Sections 4, 5. Nitrous oxide (for anesthesia, also referred to as laughing gas), nitric oxide (as a vasodilator), and oxygen (as a supplement for patients with breathing problems) are some prominent examples. Recent years have seen increasing attention on developing therapeutics based on three endogenously generated gaseous molecules: carbon monoxide (CO), hydrogen sulfide (H2S), nitric oxide (NO). CO has been reported as a potential therapeutic agent with multiple pharmacological effects, including anti-inflammatory, organ protection, and circadian clock regulation81, 82, 83, 84. All these issues have been extensively discussed in various publications and are not the subject of this section. Instead, the focus is on using unique chemistry to construct delivery systems for these gaseous molecules. Along this line, there is a unique category of reactions, cheletropic reactions, which allow for extrusion of CO or SO2 from an otherwise stable molecule. In one case, cheletropic reactions of norbornadien-7-ones release CO under near physiological conditions (Table 3, Entries 1, 2). As a proof of concept, CO release from the organic prodrug has been demonstrated from the reaction of a strained alkyne and a tetrasubstituted cyclopentadienone, with a second-order rate constant of 0.6 L/mol·s. Moreover, similar chemistry has been used for the targeted delivery of CO into the mitochondria through enrichment-triggered release (Table 3, Entry 1). Further, the same kind of chemistry has been adapted for the dual release of anticancer drug floxuridine and CO (Table 2, Entry 5),. An alternative approach is to bring both the dienone and alkyne moieties into one molecule by taking on an intramolecular approach (Table 3, Entries 2, 4). While doing so, a proper balance between stability and release rate is critical. An ideal CO prodrug should be stable enough during preparation and storage but rapidly release CO under physiological conditions. In doing so, one can take advantage of the facilitation of Diels–Alder reactions in aqueous solution via hydrophobic forces. Similar chemistry has been used for the dual release of metronidazole and CO (Table 2, Entry 6). Further, to control the activation of the unimolecular prodrug, an esterase sensitive prodrug has been developed to release CO. Besides CO, SO2 is also being studied as a possible endogenous gaseous signaling molecule; it is produced in the body from the metabolism of sulfur-containing amino acids and hydrogen sulfide, and is considered as playing important roles in cardiovascular functions (discussed in depth in Section 5). Moreover, it has also been reported to have anti-hypotensive, anti-bacterial, and anti-oxidative activities. Similar cheletropic chemistry has been applied in preparing prodrugs for SO2 (Table 3, Entries 3, 4),. The chemistry discussed in this section demonstrates different ways of designing prodrugs for molecules with no functional handles for modification. The use of such unique cheletropic chemistry contributes significantly to the development of research tools and possibly therapeutics based on gaseous signaling molecules.

One of the most straightforward ways of applying click chemistry is direct conjugation of a moiety capable of selective binding or enrichment for targeted delivery of a payload. In this section, we describe examples of click chemistry applications for in-situ labeling, preparation of antibody‒drug conjugates (ADC), in-situ assembly of PROTAC molecules, and targeted delivery of radionuclides.

Biological processes involve coordinated signaling and metabolic pathways of numerous biomolecules and related metabolites. Real-time monitoring of alterations in these molecules provides an approach to understanding cellular processes. Earlier studies often use fluorescent proteins such as the Green Fluorescent Protein (GFP) for labeling. To extend similar studies beyond proteins to other biomacromolecules such as lipids and glycans, there is a need for other labeling techniques, where traceable probes are delivered to tag the intended biomolecules for identification and visualization. One approach is the incorporation of a functional group into a biomolecule for subsequent chemical ligation using bioorthogonal reactions of appropriate reaction rates. Due to the heterogeneity and complexity of the cellular environment and the often fragile nature of biomacromolecules, diverse chemical conjugation techniques have been developed to suit various needs. These reactions include carbonyl condensation, azido-alkyne click chemistry (Table 1, Entry 1), Staudinger ligation (Table 1, Entry 3), and inverse electron demand Diels–Alder reaction (IEDDA) (Table 1, Entry 9) among many others. Here, we present an overview of the applications of click chemistry for targeted delivery of probes for in-situ labeling with relevant examples. As discussed earlier, to overcome copper toxicity issues associated with CuAAC reactions (Table 1, Entry 2), activated alkynes have been developed for use in bioorthogonal reactions with an azido handle, mostly through the introduction of strain energy (Table 1, Entry 1)23, 24, 25. The feasibility of using such strained alkynes for biological applications has been demonstrated by targeted labeling of live cells. In this section, only a few examples are discussed with a focus on recent ones with unique and new characteristics. One example is the identification of specific protein glycoforms on cell surface via targeted delivery of a click chemistry partner to the glycoform of interest for subsequent covalent labeling and then analysis (Fig. 1). Specifically, two aptamers sgc8c21 and TD05.1 with known affinity towards the PTK-7 protein and the mu heavy chain of the mIgM, respectively, were used to direct one “partner” reagent to the intended glycan. In this case, Jurkat cells and Ramos cells were treated with peracetylated N-azidoacetylmannosamine (Ac4ManNAz), which was metabolically converted to N-azidoacetlyneuraminic acid (SiaNAz) and incorporated into the glycoproteins on the cell surface. Then, Jurkat cells were treated with a sgc8c21 aptamer–cyclooctyne conjugate, leading to selective binding to the PTK-7 protein, as confirmed by western blot analyses. This binding was for the delivery of a strained cyclooctyne moiety to the proximity of the azido group on the targeted glycan, leading to covalent labeling via click chemistry. Similarly, incubation of Ramos cells with a TD05.1 aptamer–cyclooctyne conjugate led to the labeling of the light (30 kDa) and heavy (60 kDa) chains of mIgM protein. Control experiments with mismatched aptamer-cyclooctyne conjugates did not lead to the same labeling. Further, control experiments by removing the azido-label also led to negative results. Such results demonstrate the power of click reaction for selective glycan labeling using covalent chemistry through targeted delivery of click partners. Direct conjugation has also been used for tagging an antibody recruiting molecule (ARM) on cells for addressing resistance issue in cancer; the same approach is also applicable for targeting bacterial and viral infection. A critical step in this approach is to use covalent linkage to prolong the duration for ARM to stay on the cell surface for optimal efficiency in recruiting and retaining antibodies for the subsequent cell killing. In one example, click chemistry was used to create a stable-covalent insertion of a hapten by exploiting the azido-alkyne click chemistry (Fig. 2). Specifically, the high-affinity folate receptor is overexpressed in tumor cells,. Hence folate conjugation with an azido sugar allowed its selective delivery into the intended tumor cells. Wherein, folate-PEG was conjugated with N-azidoacetylmannosamine (Ac3ManNAz) via a disulfide linker with the idea of enriching this conjugate inside the tumor cells by receptor-mediated endocytosis. The conjugate was designed to release the free azidosugar by the elevated level of glutathione (GSH) in cancer cells for subsequent metabolic conversion to sialic acid and incorporation into cell-surface glycans. Next, an antibody-recruiting hapten (l-rhamnose) was covalently linked to the cell surface by using click chemistry with dibicyclooctyne rhamnose (DBCO-Rha) (Table 1, Entry 1). The ability for such cells to recruit antibodies was confirmed by incubation with 20% human serum and human complements. Cell killing by antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) were observed. For example, CCK-8 assay indicated ∼50% cell death in FR+ KB cells (FR upregulated) under the experimental conditions while in KB cells, cell death was at 20%. In short, these results suggest the success of using click chemistry for labeling tumor cells with an antibody-recruiting molecule and subsequent activation of immune response leading to cytotoxicity. In another approach, boronic acid-enhanced carbonyl-amine/hydrazide conjugation chemistry was used for the site-specific conjugation of antibodies (Table 1, Entry 6). For example, 2-formyphenylboronic acid (2fPBA) functionalized antibody was conjugated with α-amino-hydrazide. Specifically, aryl carbonyls with a boronic acid moiety at the ortho position exhibit fast condensation with an amine or hydrazide in comparison to traditional carbonyl groups. The main promoter for this rapid kinetics is the additional stability of the condensation product, 2,3,1-benzodiazaborine (DAB) derivative (Fig. 3). Introduction of an α-amino group to the hydrazide produces a tricyclic structure, in which the amino group coordinates with the boron atom and forms an anionic tetracoordinate boron species. Besides this extra stabilization, the zwitterionic property of the DAB product of 2fPBA and α-amino-hydrazide helps to improve the solubility and plasma clearance of the conjugation products. The bioorthogonality of the chemistry was proven by labeling trastuzumab (Tras), a clinically relevant antibody with known affinity towards human epidermal growth factor receptor 2 (HER2) overexpressed in breast cancer. First, the light chain of trastuzumab was tagged with amine functionalized 2-fPBA via transglutaminase (TG) ligation, to get a 2fPBA tagged light chain (Tras-LC-2-fBA). For the functionalization of the heavy chain, Tras with a K334C mutation was conjugated with a maleimide linker (Tras-HC-2fBA). These modified antibodies along with their unmodified peers were reacted with Texas Red−α-amino-hydrazide (Tx Red−am-zide). The results from SDS PAGE and ESI-MS indicated specific labeling on 2fPBA functionalized antibodies. Moreover, BODIPY conjugates of the light and heavy chains (Tras-LC and Tras-HC) of trastuzumab was synthesized by a similar procedure. These BODIPY conjugates were tested with different cell lines; flowcytometry studies showed fluorescence increases in SK-BR-3 (HER+) but not in MB-231 (HER−) cells. Additionally, this method along with Tz-TCO reaction was used for multi-bioorthogonal labeling to generate site-specific conjugates (Fig. 3). Initially, Tras-LC was modified with TCO via transglutaminase-mediated ligation (1); then the heavy chain was deglycosylated with peptide−N-glycosidase F (PNGaseF) to 2, so that the Q295 residue becomes available for transglutaminase-mediated ligation with 2fPBa (3). This dual functionalized antibody LC−TCO/HC−2fPBA (4) was then reacted with BODIPY−Tz (5) and Tx Red−am-zide (6) in one pot. SDS-PAGE and ESI-MS results indicated site-specific conjugation within 30 min, wherein BODIPY−Tz was conjugated through the TCO of light chain and Tx Red−am-zide was conjugated to the 2fPBA of heavy chain. Briefly, this method provided a novel bioconjugation technique to generate a less hydrophobic conjugate than the conjugate prepared using SPAAC and Tz-TCO reactions. Such properties make this boronic acid-facilitated condensation method an attractive tool for ADCs, where increased hydrophobicity may cause antibody aggregation and rapid clearance. Moreover, its reliability for multiplex applications makes this approach attractive for the synthesis of dual-payload ADCs capable of targeting two antigens. Photoactivated tetrazole-BCN click chemistry (Table 1, Entry 11) has recently been reported for live cell labeling of membrane proteins. Specifically, the aim was rapid photo-labeling of the glucagon receptor (GCGR). In doing so, there was a need to design a click pair that can be initiated via photoactivation to optimize the reaction rate for spatio-temporal controls. Herein, a reaction between sterically shielded diphenyl tetrazole and BCN was used. The tetrazole was conjugated with N-sulfonylpyrrole at the ortho positions of the C-phenyl ring with the intention of favoring the click product over the competing nucleophilic addition product and helping to improve the water solubility (confirmed by HPLC results). Kinetic studies on different tetrazole-BCN pairs indicated 2-3-fold slower reaction rate for bulkier substitutions at N-sulfonylpyrrole. Nevertheless, the rate constant for the fastest BCN-tetrazole reaction was found to be 39,200 L/mol·s under physiological condition. Thus, the tetrazole-BCN reaction is faster than many other click reactions with BCN, but lags behind some tetrazine-BCN reactions (K2 = ∼105–106 L/mol·s),. Successful application of this tetrazole-BCN ligation method was demonstrated by live-cell imaging of a low-abundant glucagon receptor on the cell membrane using HEK 293T cells. Specifically, BCN was genetically encoded to K372 of the receptor, followed by treatment with Cy-5 modified triazole and photoirradiation at 302 nm UV for 15 s. Confocal microscopy studies indicated high fluorescence intensity in the Cy-5 channel, which was colocalized with the GFP channel. In contrast, the control experiment with H372BocK (protein with BCN in 372 is replaced by a Boc group) did not show any signals in the Cy-5 channel, supporting labeling specificity of this novel method. In short, this photo-tetrazole-BCN click reaction is faster than many other bioorthogonal reactions with BCN, allowing for live cell labeling of proteins within 15 s. Besides the methods discussed in the section, there are many other applications of direct conjugation using click chemistry and new methods are being added to the list,,,111, 112, 113, 114, 115. For instance, there is a novel cysteine-specific modification of proteins or bacteriophage using isonitrile-chloroxime ligation chemistry with a rate constant of 306 L/mol·s. There are extensive reviews as further comprehensive coverage,,,111, 112, 113, 114, 115.

Antibody‒drug conjugates (ADC) take advantage of the high selectivity of antigen–antibody interactions for targeted delivery of cytotoxic drugs to tumor cells. As an indication of great success, 9 ADCs have been approved by the US Food and Drug Administration (FDA) and nearly 100 are in clinical trials. The ADC molecules work by relying on binding-initiated internalization and subsequent release of the cargo molecule. As such, there are certain features required for the linker/conjugation chemistry. If the conjugation is too labile, then the attached drug may release prematurely. If it is too stable, then it presents a challenge to the timely release at the desired location. Further, the conjugation chemistry needs to be compatible with the often-fragile nature of antibodies. Traditionally, the most commonly used method is tethering through a lysine amino or a cysteine sulfhydryl group of the antibody. However, the chemistry needed for such modifications often results in heterogeneous products and instability that affect the therapeutic efficacy of ADCs. The synthesis of ADCs requires special chemical ligation tools so that the functional characteristics of both elements are preserved. Considering the mild conditions of click chemistry, it is an ideal tool for modification of antibodies without compromising their binding affinity towards cell surface antigens. For site-specific conjugation of an antibody with a drug molecule to obtain homogenous, stable, and potent ADCs, non-canonical amino acids have been used for expression into an antibody. For example, an azido-modified amino acid was inserted into an antibody. This allowed specific click conjugation with an alkyne tethered to a drug molecule. As a proof of concept, the 4D5 antibody against HER/neu (a well-known target in tumor) was modified with auristatin F (AF) or a pyrrolobenzodiazepine (PBD) on four different sites: two positions (H274, H359) in the heavy chain and two positions in the light chain (L70, L81). These four sites were selected because they are distant from the binding site and hinge region but are part of the Fc receptor domain. The in-vitro cytotoxicity of the conjugates was assessed against HER2-expressing tumor cells. In comparison with free AF, 4D5-AF demonstrated 1000-fold higher toxicity towards cells with high HER2 expressions. In contrast, irrespective of HER2 expression, free AF showed similar toxicity towards all the cell lines. In addition, cytotoxicity results from different 4D5-AF analogues indicated that higher drug loading alone was not enough for the antitumor effect; site of conjugation also mattered. Importantly, the equal activity of conjugates using either CuAAC (Table 1, Entry 2) and SPAAC (Table 1, Entry 1) chemistry indicated that Cu toxicity was not an issue for the conjugation reaction and did not affect the integrity of the protein or binding affinity of ADCs. Antitumor activity was studied in a mouse model by using different conjugates, 4D5-PBD, a-hIL6-PBD, HER2-malPBD (generated by conventional thiol-maleimide conjugation) and 4D5-AF. Among these studies, only mice treated with 4D5-PBD and HER2-malPBD showed tumor regression. Control experiments where the mice were treated with a-hIL6-PBD or vehicle did not show signs of antitumor activity. Surprisingly, 4D5-AF did not re-capitulate the same effect in vivo, consistent with previous comparative studies of PBD dimers and tubulin inhibitor ADCs. In short, the use of click chemistry allowed for careful control of conjugation site, which is important for potency. Copper toxicity of CuAAC conjugation was not an issue and did not compromise potency of ADCs. Further, click chemistry has also been extensively used for triggered release of drug payload from ADCs. These are discussed in the section on triggered drug release and targeting (Section 4.1).

Proteolysis targeting chimera (PROTAC) is an approach for degrading proteins of interest by combining the power of proteosomal degradation through polyubiquitination with ligand-based targeting. The design takes a heterobifunctional approach by tethering a protein ligand to an E3-ligase binder. Successful binding of a PROTAC molecule with its target protein recruits an E3-ligase, which promotes ubiquitination and subsequent proteasomal degradation of the protein. However, the bifunctional nature of a PROTAC molecule often means high molecular weight and increased polar surface area (800–1000 Da, ∼200 Å2 respectively), reduced solubility, decreased cell permeability, and poor pharmacokinetics. One approach to address the high-molecular weight issue is to examine the possibility of delivering the individual functional moieties separately and then allow for in-situ assembly. In doing so, click reactions offer unique benefits. In one specific example, BRD4 ligand JQ1 and E3-ligase recruiter thalidomide were conjugated to TCO and tetrazine partners respectively. Incubation of these partners in cell culture (HeLa) led to click assembly of proteolysis targeting chimera (CLIP-TAC) 10, which was shown to lead to degradation of BRD4, presumably through ubiquitination of BRD4 and its subsequent proteosomal degradation (Fig. 4). In addition, control experiments with extracellularly synthesized CLIPTACs did not degrade the targeted protein, underscoring the advantage of delivering individual partners of the final PROTAC molecule for improved membrane permeability. Of course, there is the possibility that the two click partners may assemble outside the cell leading to reduced intracellular concentration of the PROTAC molecule. Fine tuning of the reaction kinetics may allow for optimization of the chance of intracellular assembly. Moreover, click-synthesized PROTACs have been successfully used for the degradation of drug resistance and gain-of-function (GOF) mutants of the estrogen receptor α (ERα). ERα is overexpressed in ∼70% breast cancer cases and an important target in tumor therapy. Existing ERα-targeting therapies such as ER modulators (tamoxifen) and selective ER degraders (fulvestrant) are often ineffective due to drug resistance and evolution of GOF mutants. Development of PROTACs that degrades all mutant forms of ERα is an attractive approach. Herein, a ERα inhibitor against WT and GOF mutants of ERα were click-conjugated with two different VHL-based E3-ligase binders to give PROTACs 11 and 12 (Fig. 5), with ERα DC50 (concentration for 50% degradation of target protein) values of 6 and 37 nmol/L respectively in a degradation assay in MCF-7 cells. A competitive experiment with VHL-298, a standard E3 binder, resulted in a significant decrease in activity due to competitive inhibition of PROTAC binding with E3 ligase. Such results are consistent with the PROTACs activity being dependent on VHL-E3 interaction. Moreover, cell viability studies of compounds 11 and 12 in multiple cell lines showed selective cytotoxicity in ER positive cells, indicating minimal off-target effects. In contrast with the in-vitro effect, compound 12 shows 2-fold higher activity (TGI ∼85% at 10 mg/kg) than that of compound 11. This decreased activity of compound 11 was found to be primarily due to its reduced tumor accumulation and rapid plasma clearance.

Radioimmuno-conjugation has been used in targeted delivery of radioisotopes for imaging applications. An important goal of this approach is mostly for minimizing radiation exposure to healthy tissues. To a great extent, this can be achieved by pre-targeted imaging, wherein an antibody with specific affinity towards a biomarker is conjugated in-situ with a short-lived radio nuclei carrier component. PET-imaging of pancreatic tumor by a short-lived 18F nuclide (t1/2 = 109.8 min) is a good example for this approach. Herein, Tetrazine-TCO click chemistry (Table 2, Entry 1) was adapted for the pre-targeted imaging of pancreatic adenocarcinoma. Specifically, tumor-bearing mice were injected with 5BI-TCO, a pancreatic tumor-targeting antibody conjugate, which helps TCO accumulation in tumor cells. After 72 h, Tz-PEG11-Al[18F]-NOTA was injected to allow for the conjugation reaction between TCO and tetrazine (Tz). Time-dependent radioactivity accumulation at tumor sites was observed with the peak level being at the 4 h point. Conversely, the radioactivity in the blood decreased over time. These results support the idea that click reaction-mediated accumulation of radionuclei at the intended site. In agreement with this, PET imaging results showed tumoral uptake within 1 h with the ratio of tumor to background radioactivity levels increasing in a time-dependent fashion. This approach was shown to be 60 times more effective in tumor imaging when compared with using a pre-assembled antibody conjugate with a long lived radioligand. Another important advancement is the introduction of theranostic molecules into a carrier component to facilitates diagnosis, along with monitored drug delivery. Feasibility studies of image-guided pretargeted drug delivery were performed on mice with HER2+ breast cancer through treatment with mertansine (DM1) as a model drug. Here, pretargeting component and therapeutic carrier component were conjugated with two different carrier proteins, trastuzumab (Tras) and human serum albumin (HSA) respectively. The treatment group was injected with TCO tagged pretargeting component Tras-TCO-[89Zr-DFO] and tetrazine tagged therapeutic carrier component HSA-DM1-Tz [99mTc-HyNic]. Tumor uptake of both components was confirmed by PET-CT and SPECT-CT imaging respectively. The delivery component undergoes multiple Tz-TCO click reactions with the HER2-bound pretargeting component and release the drug molecule. Antitumor activity was analyzed by tumor growth reduction. Tumor volume in treatment group decreased to 9% of the original size in 26 days with minimal toxicity, while tumor in the control group treated with delivery vehicle alone increased by 78% within 11 days. Clearly, incorporation of a theranostic molecule brings additional advantages. Imaging results in the pretargeting step helps the diagnostic application, while imaging the drug delivery step provides real time data on drug distribution and tumor accumulation.

Much of the prior chemistry in prodrug development relies on balancing the need for stability of the linker or masking moiety and ready conversion to the original drug under appropriate conditions. Recently, prodrug activation using click reactions has emerged as a powerful approach, affording both stability and controlled release of the parent drug in the living system. Along this line, several reactions have been reported for this type of applications including cycloaddition-initiated allylic elimination (Table 2, Entry 1), Diels–Alder reaction-initiated extrusion (Table 3, Entries 1–4), lactonization/lactamization initiated by extrusion or cycloaddition (Table 2, Entries 5–6), and cycloaddition (isocyano)-initiated β-elimination (Table 2, Entry 9). This section highlights some interesting recent applications of these reactions.

As discussed in previous section (Section 3.2), ADCs take advantage of the targeting ability of antibodies to selectively deliver a drug molecular to the intended site/organ. As with all drug-vector conjugates, activation or release of the parent drug is a critical step. Current clinically used ADCs rely on intracellular biological activation mechanisms, which afford limited control. Therefore, there have been efforts to develop approaches allowing for extracellular activation of ADCs. In using a click-activated allylic elimination (Table 2, Entry 1), Robillard and co-workers described one such approach wherein an IEDDA reaction is followed by a self-immolative release of doxorubicin from the ADC. As shown in (Fig. 6), an antibody was conjugated with doxorubicin via a cleavable TCO linker that was designed to be activated by an IEDDA reaction with tetrazine. The cycloaddition reaction between TCO and tetrazine would yield an 1,4-dihdropyridazine intermediate, leading to an elimination reaction to release CO2 and doxorubicin. In one example, TAG72, a non-internalizing receptor present in solid tumor, was selected as the molecular target for homing purposes. To target this receptor, anti-TAG72 mAb (CC49) was conjugated with doxorubicin by a TCO linker to give compound 13. In-vitro doxorubicin release of a few analogs was evaluated by HPLC at various time points. The reaction rates of tetrazine 15 and tetrazine 16 with the drug conjugate 13 were found to be 100- and 10-fold slower than that of tetrazine 14, but the doxorubicin release yields with 15 and 16 (55% and 79% yield, respectively) were much higher than that of 14 (7% yield) in PBS, serum, and cell culture. Anti-tumor experiments were performed with compound 13 in mice bearing colon carcinoma xenografts. No significant tumor blocking was observed upon intravenous (iv) administration of 15 and significant but incomplete blocking was observed upon intraperitoneal (ip) administration of 15, suggesting fast clearance being the reason for insufficient drug accumulation at the tumor site. Significant tumor blocking was observed upon both iv and ip administration of 16. To increase the on-tumor reaction time and to improve the reaction yields, compounds 17 and 18 were designed as slow-clearing dextran carriers for tetrazines. Complete tumor blocking was achieved without any off-target toxicity after iv administration of 17 and 18. RP-HPLC analysis of the extracts from tumor tissues revealed doxorubicin release yields of 44% and 37% after administration of 17 and 18, respectively. In short, these results demonstrate the utility of using click chemistry to trigger release of drug payload from ADCs extracellularly. In using click-activated β-elimination as a way to release a drug molecule (Table 2, Entry 9), Franzini and colleagues designed an approach to use an isocyanopropyl moiety, which can be activated via reaction with tetrazine, leading to a β-elimination reaction as the drug activation step. As shown in (Fig. 7), by design an isocyanopropyl group can undergo a cycloaddition reaction with tetrazine followed by formation of a pyrazole-imine intermediate 26. Hydrolysis of this intermediate gives an aldehyde 27 and 4-aminopyrazole 28. An appropriate leaving group (part of the payload) at the β-position can then undergo β-elimination to achieve payload release and formation of acrolein. To demonstrate utility, 1,8-naphthalimide was modified with an isocyanopropyl group to study the unmasking reaction by monitoring the anticipated ratiometric changes in absorbance and fluorescence spectra. Incubation of these masked dyes with tetrazine in PBS at 37 °C led to complete consumption of isocyanopropyl naphthalimide and release the parent fluorophore as analyzed by HPLC. Near-quantitative release of the dye molecule was achieved upon reaction of tetrazine with the isocyanopropyl masked dye. When phenols or amines were used as the leaving group, it was found that the release was much slower, which is in agreement with the anticipated chemistry because of their poor leaving group ability. However, fluorophore release in diluted human serum reached completion in a few minutes, which was attributed to albumin catalyzed β-elimination reaction. As demonstration of utility in delivering therapeutics, isocyanopropyl prodrugs of doxorubicin, mitomycin C, mercaptopurine, and SN-38 were synthesized. High yields of drug release were observed by HPLC (SN-38, 91%; doxorubicin, 91%; mercaptopurine, 94%; mitomycin C, 79%) upon incubation of the prodrugs with tetrazine in PBS. Cytotoxicity studies demonstrated the release of the active parent drug. In contrast, the prodrugs alone showed little toxicity. Further experiments were performed in zebrafish models implanted with tetrazine-modified beads for in-vivo release of a fluorophore and mexiletine. Thus, fish implanted with tetrazine-modified polystyrene beads exhibited significantly higher fluorescence staining when exposed to isocyanopropyl resorufin than controls without the modified beads. Incubation with an isocyanopropyl-mexiletine conjugate caused dose-dependent decrease in heart rate in fish with implanted tetrazine modified beads. The effects were similar to that of the parent drug, mexiletine. These experiments demonstrated the feasibility of using the reaction between tetrazine and an isocyanopropyl group for controlled drug release in vivo. Since conventional chemotherapy for cancer still relies on the use of cytotoxic agents such as doxorubicin, cisplatin and paclitaxel, the development of prodrugs with controllable release profiles is highly desirable to complement existing drug delivery approaches. Further studies with 3-isocynopropyl derivatives are likely to lead to very promising results. A very unique approach in using click-activated prodrugs is to take advantage of a Diels–Alder reaction-initiated extrusion reaction. This has been extensively used in the design of prodrugs of two endogenous gaseous signaling molecules: carbon monoxide (CO), and sulfur dioxide (SO2),. Section 5 discusses in detail the merit of using these two gaseous molecules for developing therapeutics. Herein, the principles of using extrusion reactions for designing their prodrugs are discussed. Several reactions have been used for this purpose (e.g., Table 3, Entry 2). Specifically, the organic CO prodrug in (Fig. 8), has an alkyne unit positioned for an intramolecular cycloaddition reaction with the cyclopentadienone moiety, leading to the formation of substituted norbornadien-7-one. Subsequent extrusion reaction leads to CO release. With the introduction of a naphthalene group in the dienone moiety (30), the by-product after CO release (31) is highly fluorescent, allowing for real-time tracking of CO release and a “click, release, and fluoresce” approach. The ability to track drug release is very valuable for the interrogation of prodrug activation and effects. It should be noted that the intramolecular Diels–Alder reaction is designed for activation in aqueous solution through hydrophobicity-enhanced reaction rate. This unique property allows for synthesis and purification of the prodrug in organic solvents and CO release in aqueous environments. Additional unique applications of click reactions for preparation of prodrugs of gaseous signaling molecules are described in Section 5.

One of the challenges in targeted delivery using conjugates with an antibody or a receptor ligand is the regeneration of the parent drug at the desired site. As discussed earlier, doing so requires a careful balance of stability and reversibility of the linker chemistry. One way to tackle this problem is to use the facile nature of click reactions to initiate enrichment-triggered release by taking advantage of the concentration-dependent nature of a bimolecular reaction. In doing so, Wang's lab developed the first enrichment-triggered drug release strategy based on controls by reaction kinetics using specially designed tetrazine and cyclooctyne click partners (Table 1, Entry 9). By tethering to the same targeting moiety, both click partners can be enriched at the same site. Therefore, the local concentrations for these two components would increase dramatically and the rate of this bimolecular reaction would significantly accelerate to the point that enables meaningful payload release at the target site. In contrast, due to the low concentrations in the systemic circulation, these two components would not react or react very slowly, and thus no meaningful drug release is expected. To establish proof of concept, mitochondria-targeted delivery of doxorubicin was tested for enrichment-triggered drug release. For this, a special tetrazine-cyclooctyne pair was designed to allow for drug release through cycloaddition-initiated lactonization. As shown in (Fig. 9), this tetrazine-cyclooctyne pair with a triphenylphosphonium (TPP) moiety was used for mitochondria-targeting with enrichment on the order of hundreds of folds. HeLa cells were treated with TPP-tethered click partners 32, 33 or negative controls without the TPP moiety, respectively. Cytotoxicity studies showed that the EC50 of the click partners tethered with the TPP moiety was about 2 μmol/L and that of its non-TPP conjugated counterparts were more than 10 μmol/L. Such results demonstrated that conjugation with the TPP moiety enhanced toxicity of doxorubicin prodrug towards HeLa cells as compared to non-TPP conjugated partners, presumably due to enrichment-triggered release of doxorubicin in mitochondria by the former. The same concept was also studied using carbon monoxide as an example, which is discussed in Section 5.1. In summary, enrichment-triggered drug release using click chemistry can be used to diminish general toxicity and side effects of conventional chemotherapies.

The aforementioned examples showcased click reaction-triggered drug release with one payload per prodrug. To advance a strategy for combinational therapy, the development of “one prodrug, two payloads” approach is highly desirable. For example, CO has been shown to present synergistic or additive effects when used in combination with anti-inflammatory, anti-tumor, or anti-bacterial agents. Therefore, it is highly desirable to develop a drug delivery system for the co-delivery of CO and another payload for potential combinational therapy. In 2018, Wang and coworkers reported a cascade prodrug for CO and a antibacterial payload by using an intramolecular Diels–Alder reaction followed by a lactonization reaction (Table 2, Entry 6). As shown in (Fig. 10), the initial intramolecular Diels–Alder reaction leads to the release of CO and the formation of an intermediate 36, which has a nucleophile (a hydroxyl group) positioned in a 1,5-relationship with an ester group, leading to the release of an alcohol after lactonization. Further, the side product 37 following drug release is a blue fluorophore that can be used for tracking the release of drug payload in vitro. As proof of concept, metronidazole was used as the drug payload; and co-delivery of CO and metronidazole led to much enhanced antibacterial effect against Helicobacter pylori with MIC90 of 0.31 μg/mL, as compared to 2.5 μg/mL for metronidazole alone, suggesting the sensitization effect of CO towards H. pylori. However, it should be noted that H. pylori is special in that urease is a critical enzyme for its ability to control pH and this enzyme has nickel at its active site. Nickel is known to have high affinity for carbon monoxide. Therefore, one would not expect the same type of synergistic or additive effects in bacteria without a nickel enzyme. However, similar drug delivery strategies should be applicable to other “one prodrug, two payloads” applications. In another example (Table 2, Entry 5), Wang and colleagues developed a bimolecular strategy for co-delivery of CO and another drug payload for combinational therapy by taking advantage of the Diels–Alder reaction between cyclooctyne and cyclopentadienone. As shown in (Fig. 11), a hydroxyl group is introduced to the propargyl position of a cyclooctyne and a drug payload is appended to a cyclopentadienone moiety via an ester bond. The prodrug is designed to undergo a click reaction between cyclooctyne and cyclopentadienone for CO release followed by lactonization to release the appended payload. A proof-of-concept study was conducted with floxuridine as the drug payload. Using HPLC to monitor the reaction, it was found that 50% of floxuridine was released within about 4 h upon co-incubation of 38 (100 μmol/L) and 39 (5 mmol/L) in 30% of DMSO/PBS. The in-vitro release of floxuridine was also validated in MB-231 cells by tracking a fluorescent reporter. However, no augmentation effect was observed in the cytotoxicity of floxuridine from this prodrug pair at 100 μmol/L. In retrospect, with the typical reaction rate constant being about 0.17 L/mol·s, the half-life is expected to be 16 h. At this slow-release rate, the lack of augmentation effect is not surprising. Future efforts will need to take the reaction rate issue into consideration. Nevertheless, the principle exhibited in such a drug delivery system of having two payloads in one prodrug could serve as a very powerful approach for developing combination therapies. In an example that delivers two fluorophores (Table 2, Entry 9), Franzini and colleagues used both a 3-isocyanopropyl moiety and a tetrazylmethyl group as an unmasking group, leading to cycloaddition and then hydrolysis for “click and release”. The rationale behind the design was similar to the example discussed earlier (Fig. 7); conversion of isonitrile 42 by tetrazine 43 into 4-aminopyrazole, which spontaneously eliminates the tethered leaving groups, 44 and 45 respectively. As shown in (Fig. 12), fluorescein and resorufin were tethered with tetrazylmethyl and 3-isocyanopropyl groups, respectively. By reacting tetrazylmethyl fluorescein with 3-isocyanopropyl resorufin in zebrafish embryos, the caging molecules were shown to undergo a single dissociative bioorthogonal reaction to liberate both fluorophores. As expected, double fluorescence turn-on was simultaneously observed by fluorescence microscopy. This idea of dual release by a single bioorthogonal reaction presents yet another approach to developing prodrugs for combination therapy. Relative to other prodrug strategies, the click-triggered drug release approaches described above have several advantages including fast and somewhat controllable release, stability of the linker during drug circulation, and ability to target the drug molecules. However, many challenges remain in this field of using click chemistry in prodrug activation. For example, with the two-component prodrug activation approach, the control of the pharmacokinetics of the individual components needs careful optimization. Nevertheless, we hope this section will stimulate more interests in this promising field.

There are several gaseous molecules that are endogenously produced and are believed to act as key mediators in cellular signal transduction within the body. This list includes nitric oxide (NO),, carbon monoxide (CO),, hydrogen sulfide (H2S),, and possibly sulfur dioxide (SO2). They have potential and proven therapeutic effects with a large number of indications depending on the molecule in question. There are many publications including a dedicated journal (Nitric Oxide) and books on the pharmacological effects of these gaseous signaling molecules,,143, 144, 145, 146, 147; therefore, detailed discussions of these gaseous molecules are not duplicated here. In this section, we focus on drug delivery issues, which are unique. On one hand, these four molecules share the common feature of being gaseous when in the pure form, being soluble (to different degrees) in aqueous solution, and being volatile even when dissolved, which complicates pharmacokinetic studies. Further, NO, CO, and H2S have high affinity to the same hemoproteins, though they may bind to proteins of different oxidation states. On the other hand, they have different chemical properties with NO being short-lived, sulfide being readily inter-convertible among different redox states, SO2 existing in different forms, and CO being relatively inert, largely eliminated through exhalation, and hard to derivatize for prodrug preparation. For their unique properties, direct delivery of these gaseous molecules is only applicable under limited conditions. Thus, donors/prodrugs of these gaseous molecules offer unique advantages for their controlled delivery. However, the preparation of prodrugs for gaseous molecules also presents unique challenges, some of which can be addressed by using bioorthogonal/click chemistry. In the next four sub-sections, we discuss prodrugs of these four gaseous molecules that rely on click chemistry for activation. For a comprehensive understanding of prodrugs for these gaseous molecules in general, there are excellent recent comprehensive reviews151, 152, 153. The gaseous molecules described above exhibit some distinct traits: rapid diffusion, fast permeation through cellular barriers and/or rapid excretion through exhalation (CO).

CO is produced endogenously through the breakdown of heme by the enzyme heme oxygenases (HO-1, -2) with HO-1 being inducible under stress. At appropriate doses, CO offers a broad range of therapeutic benefits. There have been earlier efforts in preparing metal-based CO donors (known as CO-releasing molecules, CORMs) by taking advantage of the unique affinity of CO for certain transition metals154, 155, 156. However, developing organic CO prodrugs represents a unique challenge for two reasons: the need for breaking a carbon-carbon bond in prodrug activation and the lack of a typical functional-group “handle” in CO for prodrug derivatization. Among all the theoretical possibilities, a bond-breaking cheletropic reaction of norbornadiene-7-ones offers a biocompatible way to break a carbon-carbon bond for CO release under physiological conditions (Fig. 13). One critical issue to address is the fact that norbornadiene-7-ones spontaneously release CO under near-physiological conditions. This means the need to prepare pro-prodrugs as an approach. As a result, the key is the design of an approach, which would lead to norbornadiene-7-ones. Along the theme of this review, both inter-molecular (Table 3, Entry 1), and intra-molecular (Table 3, Entry 2), Diels–Alder reactions have been used as a way for prodrug activation for CO release. Such reactions to various degrees rely on hydrophobic forces to accelerate the Diels–Alder reaction and CO release in aqueous solutions. It should be mentioned that there is also an elimination approach for the preparation of norbornadiene-7-ones,157, 158, 159, 160 and organic CO prodrugs can also be made via other decarbonylation approaches under near physiological conditions. In the interest of staying focused on the theme of click chemistry and drug delivery, detailed aspects of these CO prodrugs are not discussed. The subject of organic CO prodrugs has been comprehensively reviewed,. Using the general approach of cheletropic reactions, prodrugs of esterase-sensitive,, ROS-sensitive, and pH-sensitive natures have been designed. These prodrugs have also been studied for pharmacological efficacy in protecting organs in animal models of liver injury,, kidney injury,, gastric injury, systemic inflammation, and colitis as well as for pharmacokinetic behaviors,.

Sulfide has been found to possess a broad range of pharmacological and therapeutic functions,. While hydrogen sulfide can be administered through inorganic sulfide salts (NaSH and Na2S), this method generates a spike of sulfide concentration of short duration, lacks concentration control, and does not allow for a sustained concentration for an extended period of time. Over the years, there have been extensive efforts in developing sulfide donors of various types and different oxidations states,168, 169, 170, 171. In the interest of staying focused on the theme of this review, this section only presents an example that uses click chemistry as a way to activate the sulfide prodrug. Along this line, carbonyl sulfide (COS)-releasing molecules provide a promising way to offer sustained release of sulfide through a click reaction followed by the catalytic action of carbonic anhydrase (CA). An inverse-electron demand Diels–Alder reaction between trans-cyclooctene and tetrazine has been used as a way to activate the release of COS, which then subsequently releases H2S (Table 2, Entry 4). Specifically, the click reaction between these two compounds forms a dihydropyridazine that undergoes chemical decomposition to form a cyclooctylpyridazine product while also releasing COS and BnNH2. By masking hydrogen sulfide as a caged carbonyl, H2S release can be controlled by a click trigger, offering the chance of controllable and possibly targeted release.

Sulfur dioxide is emerging as a possible new member of endogenous gaseous signaling molecule. There currently exists two non-gaseous ways to administer SO2 for research: SO2 donors and salt(s). The salt form relies on its equilibrium with the hydrated SO2 (SO2.H2O) in an aqueous solution for delivery of SO2, which can be hard to control,. The development of prodrugs with controllable release of SO2 offers an attractive approach to developing tools for research applications. Along this line, there are multiple approaches by many,,174, 175, 176, 177, 178, 179. This section only focuses on those approaches that rely on click chemistry for SO2 release. Using a cheletropic reaction similar to that of CO prodrugs, prodrugs were designed for controlled release of SO2 (Table 3, Entry 3). Alterations of the substituents on the thiophene dioxide allowed for a 150-fold range in reactivity with release rate constants ranging from 0.01 to 1.50 L/mol·s in MeOH at room temperature. The rate constant is fast enough to complete the reaction in 3 h at mid-micromolar concentrations of each partner. Further work needs to be done to extend the tunability of the reactions as well as expanding the potential areas for targeting for directed delivery of the SO2 prodrugs. The problems of bimolecular prodrug systems using a click reaction have been stated previously and the pursuit for a unimolecular system for release of SO2 led to some interesting results (Table 3, Entry 4). It has been reported that compounds that rely largely on hydrophobic effects to initiate the intra-molecular Diels–Alder reaction undergo quick cycloaddition followed by a cheletropic reaction to release SO2 under near-physiological conditions with tunable release rates (t1/2: between 7 min to nearly 10 days by varying the substitution). However, this method lacks the aspect triggered release in response to an external stimulus, which limits its potential. Further work is needed to allow for controlled release.

Over the last few decades, the field of NO donors has been very active180, 181, 182, 183, 184, 185, 186. Moreover, high concentrations of NO released by NO-donors can produce anticancer effects by nitrating DNA, proteins, and lipids. Because of the short-lived nature of NO, local delivery is often needed for optimal therapeutic efficiency. Along this line, bioorthogonal click chemistry has been used for NO release from a prodrug. Diazeniumdiolates are well-researched and reliable NO donors. Pro-prodrugs of diazeniumdiolates can be prepared by protecting the oxygen with a linker, which is sensitive to glutathione S-transferase, DT-diaphorase or lysyl oxidase (LOX), leading to NO release. In one example, multiple diazeniumdiolates were inserted into a single molecule by using Cu-catalyzed azide alkyne click chemistry. Such kind of chemistry allowed the synthesis of enzyme/hydrolysis-triggered NO prodrugs that can release up to 8 molecules of NO per prodrug molecule. The integrity of the diazeniumdiolate groups remained throughout the click reactions. However, since these triggering enzymes are widely distributed, off-target NO release is a critical concern. Such kind of limitation was overcome by using tetrazine-triggered click chemistry of diazeniumdiolates. Briefly, protection of the oxygen with an isocyanopropyl group allows for a stable precursor 52, which undergoes 4 + 1 cycloaddition with tetrazine (53) to form a cyclic adduct (Table 2, Entry 9) and then a pyrazole-imine intermediate (55) after cycloreversion and tautomerization. Hydrolysis of this imine intermediate produces an aldehyde (57), which is prone to β-elimination, leading to the release of the original diazeniumdiolate and then NO (Fig. 14). This class of O2-3-isocyanopropyl derived diazeniumdiolates have been shown to be stable under physiological conditions but the co-release of acrolein, a highly reactive compound, needs to be further examined for possible undesirable side effects. Continued research into forming hybrid NO-donors through click chemistry remains an exciting pathway with many potential applications. Overall, gaseous signaling molecules present exciting possibilities for the development of therapeutic agents. In doing so, controlled delivery is a critical issue for these molecules. Click chemistry has opened new venues for bioorthogonal release, sometimes with the possibility of triggered release.

Click chemistry has been widely used in materials chemistry,. In this section, we only highlight work related to click chemistry used in preparing materials for drug delivery applications. For other applications, there are excellent reviews,,. This section includes two major types of click chemistry applications in materials chemistry. The first one is to use click chemistry for endowing special properties in materials for drug delivery such as hydrogels, peptides, and nanoparticles. The second one is to use certain materials for targeted drug delivery.

Hydrogels are polymeric materials that experience significant property changes upon exposure to water. They are often used in drug delivery for controlled release, among other applications,. For fine-tuning their properties, often chemical modifications are needed. However, the limited availability of reactions that can be carried out under near-physiological conditions with proper controls, high yields, and no or few by-products is an issue. In this regard, click chemistry has the kind of features that can address most of these problems. Click chemistry has been used for preparing hydrogels of different sizes and chemical natures including proteins, polysaccharides (alginate) and polynucleotides. Click chemistry has also been used to make hydrogels for 3D printing. Below we describe some examples. Hyaluronic acid has excellent water solubility and is often used for hydrogel preparation via crosslinking. There are usually two general approaches. First, a specific chemical modification is carried out through cross-linking chemistry, but this modification process usually introduces some reactive molecules with cytotoxicity. The other is through thiolation, but this process may introduce disulfide bonds, affecting protein structures and functions. Nimmo et al. designed a method for synthesizing hyaluronic acid-based hydrogels (60) through cross linking using a Diels–Alder reaction. Herein, a Diels–Alder reaction between the furan moiety of 58 and maleimide moiety of 59 was used for such cross linking (Fig. 15). The product 60 was tested for cellular adhesion and viability because of the importance of these two properties in the application of such materials. Specifically, MDA-MB-231 cells (expressing CD44, a receptor for HA) were seeded on the hydrogel. Then cellular adhesion and viability on the hydrogel were assessed by the morphology of the cells. It was found that after 14 days, most of the cells were still attached and continued to spread on the hydrogel. Using a cell viability assay, it was found that the hydrogel allowed a high level of cell survival (>98%), demonstrating the cytocompatibility of the hydrogel. Such results indicate that the hydrogel materials allow for cellular attachment and proliferation, which are important properties for in-vivo applications. Another example is alginate, a hydrophilic polysaccharide. Calcium alginate hydrogels are widely used because of their low toxicity, low cost, and good biocompatibility. For drug delivery applications, further modification of alginate typically needs to rely on the derivatization of the carboxylate group. At the same time, the carboxylic group is also required for crosslinking with calcium to achieve certain mechanical strength for the hydrogel. Moody et al. overcame the above shortcomings by optimizing the cross-linking process using a click reaction between an azido group and BCN (Table 1, Entry 1), leading to alginate hydrogels that are suitable for in-vivo drug delivery. Specifically, multi-armed cyclooctyne (e.g., tetra-BCN) was cross-linked with azide-tagged alginates. Here, the alginate was tagged with excess of azido group so that ∼150 azido group per strand remain for capturing DBCO (dibenzyl cyclooctyne)-linked drugs (Fig. 16). The gel was injected into targeted tissues in the mice. Then the therapeutic conjugated with DBCO was administered through iv. Through blood circulation, the DBCO–drug conjugate was trapped in target tissues by alginate via click reaction. Biocompatibility studies with CD1 mice indicated only slight inflammation after four weeks. In-vivo fluorescence imaging was used to explore the retention of gels at intramuscular sites by injecting gels labeled by Cy7 fluorophore. The results showed that the gel retained at the intended muscle site for over two weeks, with little migration from the injection site. In this example, click chemistry is used for both cross linking and drug capturing, which makes it possible to do repeated dosing for targeted delivery purposes. In a different type of application, Royzen and co-workers described the use of locally deposited polymer with tetrazine modification for activation of prodrugs through click chemistry (Fig. 17). Specifically, a tetrazine-modified sodium hyaluronate-based biopolymer (61) was injected at the tumor site; this was followed by infusion of TCO-modified cytotoxic prodrug doxorubicin (62). The design is for the prodrug to be captured locally by the biopolymer at the desired site through a Diels–Alder reaction between tetrazine and TCO (Table 2, Entry 1), followed by release of doxorubicin locally. It is important to note that injection of the tetrazine-modified biopolymer (61) allowed for about 50% retention after 2 weeks, leading to sustained ability for click activation of TCO-modified doxorubicin (62) in vivo. For cytotoxicity studies, the TCO-modified DOX (62) was found to be 83-times less cytotoxic to murine colorectal MC38 cells than free doxorubicin, indicating effective masking of doxorubicin's activity upon prodrug derivatization with TCO. Tumor-bearing mice (immunocompetent C57BL/6 mice with syngeneic MC38 flank tumors) were used to evaluate this click-based drug delivery platform. Compared to 31 days survival of control treatment group (saline), dox-prodrug treatment significantly reduced MC38 tumor growth and prolonged overall survival to 47 days. All these demonstrate the success of the drug delivery strategy as described.

Natural killer (NK) cell-based immunotherapy has been widely studied for the treatment of different forms of cancer206, 207, 208. Cytotoxicity of NK cells are mainly modulated through the production of different cytokines such as interleukin-2 (IL-2) and interferon γ (INF-γ) among others. Such kind of immune response are often inhibited at tumor sites by the Smad3/transforming growth factor β (TGF-β) signaling pathway, resulting in poor effects by the antitumor agents. This situation can be overcome by the co-delivery of anticancer drug and of TGF-β/Smad3 inhibitor. In one specific example, pH responsive self-assembly of nanoparticle was used for targeted delivery of doxorubicin and SIS3 (TGF-β/Smad3 inhibitor) to tumor (Fig. 18). Two different types of micelles were coated with PEG500 through an acid-sensitive imine linker with the desire that the imine bond would break at tumor sites with a lower pH (pH = 6.5–7.0). Bond cleavage would result in the removal of PEG coating and expose the DBCO and azido groups for the click reaction, leading to cluster formation of these two types of micelles. As such, the materials would accumulate in tumor cells through enhanced permeation and retention (EPR) effects. Briefly, PEG500 coated micelles were modified with two different click partners DBCO and azido group, followed by loading with DOX and SIS3 respectively. Click conjugation of these two types of micelles resulted in cluster formation and this fusion process caused an increase in size (changed from 83 to 300 nm). Confocal laser scanning microscopy (CLSM) studies showed that the micelle-clusters resulting from the click reaction have higher cellular uptake than the DBCO micelles or azido micelles. B16F10 tumor-bearing C57 mice were used to do the in-vivo studies. Compared with other controls, tumor growth rate of the M-DN@DOX/SIS3 group was the slowest. In addition, tumor paraffin sections also showed the most obvious tumor cell apoptosis in the M-DN@DOX/SIS3 group. Meanwhile, M-DN@DOX/SIS3 was shown to increase the production of NK cells by around 2 folds. These results led to enhanced immune response and significant tumor inhibition in animal models. Protein therapy is a promising treatment method for cancer,, but delivering proteins to specific cells has always been a challenge. An alternative is to use metal organic framework (MOFs) such as zeolite imidazole framework (ZIF), composed of Zn2+ and an imidazole moiety (Fig. 19). ZIF-8 is a pH sensitive nanoparticle where the imidazole moiety is protonated at the endosomal pH, breaking the metal ligand conjugation and leading to disassembly. As a result, the cargo molecule is released. For the purpose of targeted delivery, a tumor-specific antibody can be conjugated with ZIF-8 nanoparticles. Along this line, RNase A and Cas9/sgRNA was successfully delivered into tumor cells over-expressing the epidermal growth factor receptor (EGFR). Briefly, norbornene modified imidazole (65) was doped with protein encapsulated ZIF-8 nanoparticles (66), which were then click-conjugated with tetrazine-modified cetuximab ((67), EGFR targeting antibody). Application of such nanoparticles for the delivery of functional proteins was demonstrated by using a model protein RNase A. The Cet@POI@ZIF-8 nanoparticles (68) (<100 nm) were characterized by using SEM, TEM, and Zeta potential measurements. In-vitro studies using EGFR-rich A431 cells showed more than 50% inhibition at 60 μg/mL. In contrast, control experiments using HeLa cells (EGFR−) at the same dose did not show significant toxicity. Additionally, the effect of these nanoparticles (68) was also examined in mice bearing A431 tumor via tail vein injection. After 10 days, tumor growth in nanoparticle-injected group (10 mg/kg) was only one-third the volume of the control group. In short, the results suggest success in using such nanoparticles to deliver functional proteins to EGFR-overexpressing tumor cells. Results from cell-culture and animal-model studies demonstrate the potential of this delivery platform for delivering protein-based therapies.

In this review we have provided an overview of commonly used click reactions with characteristic traits and highlighted different types of very successful applications by using recent examples. While the future is very bright for click chemistry in drug delivery, it is very important to understand the limitations of the various available click reactions. For this, we have discussed different issues with various click reagents including stability and reactivity. Further, we have highlighted the issue of reaction kinetics, which are sometimes overlooked in experimental designs. Just because click reactions are generally considered fast does not necessarily mean that a given reaction is efficient at certain low concentrations. There is a critical need to match reaction kinetics with reagent concentration, reaction time, and the concentration of the reaction partner. We hope the reaction overview, the in-depth examples, the discussions of various potential issues including reaction kinetics and reagent stability will help minimize the number of pitfalls for researchers in applying click chemistry in drug delivery. We also hope that this review will stimulate more research in drug delivery using click chemistry.