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Click chemistry patents and their impact on drug discovery and chemical biology
First introduced by K Barry Sharpless in 2001, the term ‘click chemistry’ soon became a widely used description of chemical reactions that proceed rapidly, cleanly and in a manner that is often compatible with aqueous solutions. Click chemistry is frequently employed throughout the process of drug discovery, and greatly helps advance research programs in the pharmaceutical industry. It facilitates library synthesis to support medicinal chemistry optimization, helps identify the targets and off-targets of drug candidates, and can facilitate the determination of drug efficacy in clinical trials. In the last decade, a large number of patent applications covering the various types and utilities of click chemistry have been filed. In this review, we provide the first analysis of click chemistry applications.
Background In 2001, the concept of click chemistry was first introduced by Sharpless et al. to describe reactions defined by a set of certain criteria: “the reaction must be modular, wide in scope, give very high yields, generate only inoffensive by-products that can be removed by nonchromatographic methods, and be stereospecific (but not necessarily enantioselective). The required process characteristics include simple reaction conditions, readily available starting materials and reagents, the use of no solvents or a solvent that is benign (such as water) or easily removed, and simple product isolation” [1] . Since then, a number of chemical reactions have been developed that fulfill these criteria. The favorable features of click chemistry, especially its modular and high yielding nature, enable the rapid synthesis of compound libraries, therefore facilitating the process of lead optimization. In addition, many click reactions are biocompatible and bioorthogonal, making them exceptionally useful for diverse chemical biology applications, such as live cell imaging, bioconjugation and chemoproteomics, approaches that are fast becoming embedded within drug discovery [2–5] . There have been many review articles that summarize the development and utility of
10.4155/PPA.14.59 © 2015 Future Science Ltd
Hua Xu1 & Lyn H Jones*,1 Worldwide Medicinal Chemistry, Pfizer, 610 Main Street, Cambridge, MA 02139, USA *Author for correspondence: Tel: +1 617 674 7085; [email protected]
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click chemistry from different angles [6–8] . However, to our knowledge, thus far, there has not been a systematic review of the click chemistry patent landscape, although the number of patent applications disclosing various types and utilities of click reactions has been rapidly growing in the last decade. Here we attempt to fill this gap by providing an analysis of click chemistry patent applications in the drug discovery arena. 1,3-dipolar cycloaddition First described in late 19th century [9] , the 1,3-dipolar cycloaddition between an alkyne and organic azide was mechanistically investigated by Rolf Huisgen in the 1960s [10] . However, the reaction lacked selectivity, yielding both 1,4- and 1,5-disubstituted [1,2,3]-triazoles. It also required heating and a long time to proceed to completion. In 2002, it was reported that the copper (I) salt significantly accelerated the reaction (107–108 times faster than the uncatalyzed reaction), and only produced one of the two regioisomers, 1,4-disubstituted [1,2,3]-triazole at room temperature or with slight heating (Figure 1A) [11,12] . Having met the criteria of click chemistry mentioned earlier, the potential of the 1,3-dipolar cycloaddition is fully realized.
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Figure 1. Metal-catalyzed azide-alkyne cycloaddition chemistry. (A) copper-catalyzed azide-alkyne cycloadditions; (B) ruthenium-catalyzed azide-alkyne cycloaddition; and (C) examples of compounds generated using CuAAC. Compound 1: a branched hydroxamate HDAC inhibitor [13] ; compound 2: an antimicrobial conjugate formed from ciprofloxacin and neomycin B [14] . RT: Room temperature.
It not only accelerates the library synthesis of various [1–3]-triazoles that possess different biological activities, but also becomes one of the most reliable and popular tools to tackle some of the toughest problems in biology and chemistry. A ‘traditional’ 1,3-dipolar cycloaddition reaction requires the presence of a Cu(I) catalyst, which is usually produced in situ by a Cu(II) salt and a reducing agent, such as TCEP or sodium ascorbate. A patent application from The Scripps Research Institute disclosed a broad range of copper-catalyzed azide-alkyne cycloadditions (CuAAC) [15] , which are described as being useful for the development of novel small molecular drugs, bioactive nanomaterials, antibacterial and nonimmunogenic coatings for medical implants. Specifically the application disclosed a process of producing [1–3]-triazoles via the cycloaddition of a terminal alkyne with an azide using Cu(I) as the catalyst. The terminal alkyne could be prepared Key terms Click chemistry: Chemical reactions that proceed efficiently and stereospecifically and are often compatible with aqueous solutions. Copper-catalyzed azide-alkyne cycloaddition: Most commonly used 1,3-dipolar cycloaddition reaction between an azide and a terminal alkyne using copper(I) as the catalyst. Strain-promoted azide-alkyne or azide-nitrone cycloaddition (SPAAC or SPANC): Copper-free variants of the CuAAC, which activates the alkyne by introducing ring strain. Staudinger ligation: Ligation reaction between an azide and a phosphine containing an ester moiety, generating a stable amide linkage between the two reactants.
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from an amine containing molecule (e.g., condensation with propargyl methanesulfonate ester). In addition, The Scripps Research Institute filed a separate patent application which disclosed a variety of ligands and the copper complex comprising the claimed ligand and a Cu(I) or Cu(II) ion, which could both be used to promote CuAAC [16] . In 2005, it was reported by Zhang et al. that pentamethylcyclopentadienyl ruthenium chloride complexes, such as Cp*RuCl(PPh3)2, catalyze the regioselective cycloaddition reaction between organic azides and terminal alkynes, forming 1,5-disubstituted triazoles (Figure 1B) [17] . In contrast to CuAAC, this rutheniumcatalyzed azide-alkyne cycloaddition (RuAAC) was also found to be applicable to internal alkynes [17,18] . The Scripps Research Institute also filed a patent application disclosing the aforementioned synthesis of 1,5-disubstituted and 1,4,5-trisubstitued [1–3]-triazoles using the ruthenium-catalyzed cycloaddition of alkynes and azides [19] . Both CuAAC and RuAAC enable ready access to [1–3]-triazole regioisomers and have led to the discovery of novel compounds that exhibit desired biological activities [6] . For example, a branched hydroxamate HDAC inhibitor synthesized by CuAAC, as described in a patent application filed by Accendatech (1 in Figure 1C), demonstrated potent growth inhibition (GI50 less than 1 μM) against several tumor cell lines [13] . Technion R&D Foundation Ltd disclosed the conjugation of two antimicrobial agents, for instance, quinolones and aminoglycosides (compound 2, Figure 1C ), in order to improve activity and overcome bacterial resistance [14] . The CuAAC reaction has limited applications in living cell systems, owing to the cytotoxicity of copper (I).
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Click chemistry patents & their impact on drug discovery & chemical biology
Copper ions are readily chelated by native amino acids and can induce the formation of reactive oxygen species, resulting in damage to cells and tissues. Considerable efforts have therefore been made to eliminate the need for copper in the reaction. Ring strain was first exploited by Bertozzi and colleagues as an alternative means to activate alkynes and promote the cycloaddition with azides in living systems (strain-promoted azide-alkyne cycloaddition, SPAAC) [20] . Then, various cyclooctynes (mono or difluorinated, or fused with other aryl rings) were reported to have improved reaction rates with azides, which demonstrated the successful labeling of biomolecules in living cells and even animals [21–23] . In a 2006 patent application filed by the University of California, Bertozzi et al. disclosed a series of cycloalkynes and heterocycloalkynes (structures 3 and 4, Figure 2) to label azide-containing biomolecules [24] . The azide target substrates can either be generated in vitro and then introduced into the cells by a variety of well-established methods, (e.g., microinjection or liposome-mediated delivery), or produced in vivo using metabolic labeling [25] or genetic engineering [26] . Later, Stichting Katholiek Universiteit described the strain-promoted cycloaddition of cyclopropanefused cyclooctynes with azides (SPAAC) or nitrones (SPANC), (5–7, Figure 2) [27] . The methods to prepare the cyclooctynes by cyclopropanation of a cyclooctadiene followed by bromination and subsequent dehydrobromination reactions revealed the desired alkyne functionality. Fluorometric tags or pharmaceutical compounds could be ligated to the cyclooctynes via the functional group Q (e.g., –OH) for diagnostic and therapeutic applications. A patent application filed by the University of Georgia Research Foundation discloses the click reactions of cyclooctynes fused to two aryl groups (benzannulated systems) (general structure 8, Figure 2) [28] . Such compounds were also shown to be useful for cell surface modifications and the X group in 8 can provide an attachment site for bioconjugation (e.g., C=O, CHOH, CHNH2). The University of Melbourne has filed a patent application related to the preparation and use of
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bi-cyclo[6.1.0]-non-4-yne as a biomolecular tether, for example, the ligation of proteins to quantum dots for the use in single-cell, single-molecule imaging methods [29] . Various utilities of SPAAC and SPANC have also been described in several recently filed patent applications, including methods that improve RNA functionalization [30] , oligonucleotide ligation [31] , and the delivery of diagnostics such as 18F PET probes and therapeutic agents to specific cell and tissue types, or pathogens, using in vivo click chemistry approaches [32] . Targeted delivery in this way has considerable potential to limit the exposure of chemotherapeutic drugs to healthy cells, so reducing cytotoxicity (see below). The application of sequential SPAAC and CuAAC was reported by our group in the development of trifunctional scaffolds with applications in chemical biology and bioconjugation [33,34] . As a proof-of-principle, we showed that a variety of biomolecules, including peptides and sugars, and tags such as dyes and biotin, could be clicked onto a core template in a sequential, reliable and high yielding manner. Staudinger ligation The first traceless Staudinger ligation was reported by Bertozzi and colleagues in 2000 [35] . It is a modified version of the classical Staudinger reaction that introduces a methyl ester group to the triarylphosphine as an electrophilic trap so that a stable amide bond is formed between the two reactants (Figure 3) . The Staudinger ligation has proven to be selective and efficient, and these modified triarylphosphines can even selectively label azides installed on cell surface glycoconjugates [35] . Later, the Staudinger ligation was also applied to the visualization of glycans in live cells and animals [3,36] . Notably, a fluorogenic Staudinger phosphine probe based on a FRET-quenching mechanism was later developed and used to image sugars containing azide groups in live cells [37] . Koninklijke Philips Electronics discloses a kit comprising one targeting probe and either an imaging or therapeutic probe that relied on the Staudinger ligation [38]. Specifically, an azide group is incorporated into the target of interest, say a cell surface receptor, (H)8-n
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and then an imaging probe, which contains the desired triphenylphosphine derivative, undergoes a Staudinger ligation with the target to enable MRI, PET or SPECT imaging. Similarly, a therapeutic agent incorporating a triphenylphosine group can be targeted to the site of interest using the Staudinger ligation. A recent patent application filed by Siemens Medical Solutions disclosed a two-step method to generate a radiolabeled tracer for PET imaging [39] . The first synthetic step condenses a carboxylic acid with the hydroxyl group of a phosphine molecule to form a phosphine ester. Second, a Staudinger ligation between the phosphine ester and an azide bearing a PET radioisotope functionality produced the desired radiolabeled tracer. An issue regarding the utility of the Staudinger ligation is the susceptibility of the phosphine reagents to oxidation by oxygen which slows the reaction. Additionally, the intrinsic rate of the Staudinger ligation is relatively slow [40] , making it less appealing compared with other types of click reactions, particularly for in vivo labeling (see below). Inverse electron demand Diels–Alder click chemistry (tetrazine ligation) There have been continuous efforts to search for novel click reactions since the original CuAAC and SPAAC reports. For example, it was reported in 2008 that strained dienophiles, such as norbornene and trans-cyclooctene (TCO) react rapidly and efficiently with 1,2,4,5-tetrazines in an aqueous solution [41,42] . As a result of exceptionally high reaction rates, this type of inverse electron-demand Diels−Alder reaction (IEDDA) has been utilized in bioconjugation chemistry with a number of diverse applications, including targeted imaging in live cells and animals [42–44] . A small library of tetrazine derivatives were synthesized recently and shown to exhibit varying degrees of stability and reactivity with TCO (200 to 30000 M-1S-1), thus facilitating the design of appropriate click reacting pairs [45] . Devaraj and colleagues reported the Key term Inverse electron-demand Diels−Alder reaction: Cycloaddition reaction between an electron-rich dienophile and an electron-poor diene.
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in situ synthesis of alkenyl tetrazine derivatives, and showed they possess strong fluorescent turn-on properties (up to 400-fold enhancements) when reacted with cyclopropenes and trans-cyclooctenes [46] . Some coumarin tetrazines were also reported to exhibit 11,000fold fluorescence enhancement upon ligation with TCOc (a trans-cyclooctene derivative) [47] . Such highly fluorogenic probes assist in lowering the background fluorescence signals caused by nonspecific binding of the fluorophore to the cellular components and allow for ‘no-wash’ cell imaging. A patent application filed by the University of Delaware discloses a method of performing the ligation reaction between 1,2,4,5-tetrazines and dienophiles in an organic or aqueous medium (9–17, Figure 4) [48] . The method of preparing such trans-cyclooctenes and several novel 3-substituted cyclopropenes was also disclosed. The rapidity and bioorthogonality of the tetrazine-based Diels–Alder reaction makes it an excellent option for the synthesis of radiotracers and their subsequent use in PET imaging. A 18F-labeled cyclic RGD peptide (which was shown to achieve specific tumor uptake) was generated in 5 min with more than 90% purity using the tetrazine-TCO ligation [49] . Similarly, Reiner et al. described the rapid synthesis of an 18 F-labeled poly[ADP-ribose]polymerase 1 (PARP1) inhibitor, that generated high-quality PET images of cancer cells [50] . The same group filed a patent application which disclosed a PARP1 tracer and the use of the tracer to detect and image cancer cells [51] . In addition, in a patent application filed by University of Southern California and University of Delaware, the use of tetrazine-TCO ligation to selectively and efficiently synthesize a Diels–Alder adduct bearing a radionuclide for imaging in animals and humans was disclosed [52] . The abovementioned methods utilizing IEDDA will undoubtedly assist in the rapid synthesis of radiotracers to detect and quantify the engagement of pharmaceuticals with their molecular targets in live animals. Proof of target engagement is a key pillar for successful target validation [53] and clinical progression, resulting in reduced attrition rates in drug discovery [54] .
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Click chemistry patents & their impact on drug discovery & chemical biology
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Thiol-ene addition The thiol-ene addition is an efficient and high yielding reaction that is also tolerant to aqueous solutions. The most popular example is the reaction between a thiol and maleimide, generating a thiol-succinimide linker (Figure 5A) . A common problem of the thiol-succinimide is its intrinsic instability. It is known that thiolsuccinimides are susceptible to retro-Michael addition and thereby subsequent thiol exchange process with albumin, cysteine or glutathione. A recent report described the rapid dissociation of a cytotoxic drug from a targeting mAb (a so-called antibody–drug conjugate, or ADC), that reduced the therapeutic index of the molecule in an animal model, as a result of the reversible nature of the thiol-maleimide reaction [55] . Nektar Therapeutics disclosed a method to solve the stability issue by hydrolyzing the thiol-maleimide adduct. This led to >95% of the succinimide group being in a ring opened form, and the resultant thiol-succinamic acid conjugate was found to be more A
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stable and did not undergo the thiol exchange [58] . In this patent application, they described using the method to generate polyethylene glycol (PEG) modified proteins, that often improve the pharmacokinetics and pharmacodynamics of the conjugated therapeutic, whilst reducing immunogenicity [59,60] . A series of linker compounds disclosed by Syschem Inc contain a double bond as the thiol acceptor (general chemical structures 18–20, Figure 5B), allowing conjugation of a drug payload molecule to a free thiol group within a cellular recognition ligand [56] . The payload molecules Z include, but are not limited to, cytotoxic agents, targeted chemotherapeutic agents, radionuclides and immunomodulating agents. Another development in this area involved the use of next generation maleimides, including bromomaleimides and aryloxymaleimides, to enable disulfide bridging and the dual functionalization of disulfide bonds [61–63] . This has led to the controlled assembly of near homogenous ADCs by loading the small molecule drug via the
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Figure 5. Thiol-ene addition. (A) Ligation reaction between thiol and maleimide. (B) Various linker compounds disclosed in the patent applications: 18–20 in patent US20130323169 [56] ; 21 in patent WO2011018613, where Y is an electrophilic leaving group [57] .
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Figure 6. Aldehyde/ketone ligation. (A) Ligation reaction between aldehyde/ketone and hydrazine/ alkoxyamine. (B) The Pictet–Spengler ligation.
interchain disulfide bonds of the antibody [64] . UCL Business PLC filed application patent on a conjugation method using the maleimides with good electrophilic leaving groups at 3- and/or 4-positions (general structure 21, Figure 5B) [57] . An acid-cleavable thiomaleamic acid linker recently developed by the same group [65] was found to be stable at physiological pH, but cleaved in the acidic environment of the lysosome, thereby enabling the design of an ADC cleavable linker technology [66] . Aldehyde/ketone ligation The carbonyl group in aldehydes and ketones can be selectively ligated with hydrazine and alkoxyamine nucleophiles even under physiological conditions to form hydrazones and oximes, respectively (Figure 6A) . These reactions have been exploited to label a variety of biomolecules, including proteins and glycans [25,67–69] . A recent development was the facile incorporation of aldehyde tags into proteins so enabling site-specific bioconjugation [70] . The method relies on the use of formylglycine generating enzyme (FGE) to recognize a natural amino acid tag sequence that then converts a cysteine residue into a unique amino acid bearing the aldehyde functionality [71] . A patent application disclosing the use of this method to produce a carrier protein–drug conjugate has been published [72] . In 2001, a patent application disclosing a method of generating aminodrug peptide conjugates by way of aldehyde/ketone ligation for targeted therapeutics published [73] . Specifically, the patent A
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Cyanobenzothiazole & thiol conjugation As the last step of biosynthesis of luciferin, the condensation reaction between 2-cyanobenzothiazole and 1,2-aminothiol is relatively mild (Figure 7A) . Rao’s group at Stanford first demonstrated that the reaction can occur in vivo under the control of pH, disulfide reduction and proteolytic cleavage. It is further shown that this reaction could be used to visualize protease activity in single live cells [78] . Promega discloses a series of cyanobenzothiazole derivatives (Figure 7B) that can be used to site-specifically install a reporter moiety (including a fluorophore, affinity moiety, or a photocrosslinking label) to a protein with an N-terminal cysteine residue for the purpose of detection and affinity isolation [79] . Promega filed another patent application on the ligation between cyanobenzothiazole and cysteine [80] .
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application describes a linker containing a ketone or aldehyde group, which is attached to the amino group of a cytotoxic drug, such as anthracyclin antibiotics, and then ligated with an O-alkylhydroxylamine derivative of a carrier peptide. The resulting conjugates showed high affinity to tumor tissues, thus improving the therapeutic index of the parent drug. The classical Pictet–Spengler reaction refers to the condensation reaction between a β-arylethylamine, for instance, tryptamine, and an aldehyde or ketone, followed by ring closure. It was first utilized for protein N-terminal labeling by Kodama et al. [74] , and was disclosed in a patent application as a general method for protein modification [75] . In order to improve the rate of reaction (10– 4 M-1 S-1 at pH 4–5), Bertozzi and colleagues developed a modified Pictet–Spengler reaction between N-alkoxytryptamine and aldehyde/ketone (Figure 6B). They demonstrated the site-specific chemical modification of glyoxal- and formylglycine-functionalized proteins, including an aldehyde-tagged variant of the therapeutic monoclonal antibody Herceptin [76] . Moreover, the reaction rate is 4–5-times higher than the classical Pictet–Spengler reaction. The labeling method was disclosed by the Regents of the University of California as useful for chemically modified biomolecules, including proteins, glycans, nucleic acids and cofactors [77] .
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In this application, Promega describes assays to detect the activity of nonluciferase enzymes and determine their modulator activity by employing 2-cyano-6-hydroxy- or 2-cyano-6-amino-benzothiazole derivatives, which have a specific enzyme recognition motif for the desired nonluciferase enzyme coupled to the benzothiazole backbone via the 6-hydroxy or a 6-amino site. One purported advantage of these benzothiozole derivatives is that they would be more readily accepted by the nonluciferase enzymes as a substrate than luciferin, which contains a carboxylic acid group and is not tolerated by certain enzymes, such as CYP2D6. It is disclosed that these derivatives, after modification by the nonluciferase enzyme, can still react with D-cysteine, but do not give the light producing luciferin, resulting in a decrease in bioluminescence signals, compared with the ‘no enzyme’ control. It was shown that the bioluminogenic assay could be performed with recombinant proteins, microsomes, and even intact cells. More importantly, it was used to measure the effect of a given compound on the activity of UGT and CYP450, which are key metabolizing enzymes monitored for potential drug–drug interactions. In addition, this method could be adapted to a high-throughput format, making it more appealing for drug discovery. Other useful click chemistry Recently, Carroll and co-workers disclosed a series of novel covalent inhibitors and probes that irreversibly modify the activities of proteins via the reaction of a sulfenic acid- or sulfenamide-modified cysteine residue (Figure 8) [81] . The compounds disclosed contain a substituted aryl or heterocyclic core structure that promoted binding interactions with a specific protein, such as protein kinases and phosphatases, and a nucleophilic warhead that is capable of forming a covalent bond with a sulfenylated cysteine residue (e.g., 3-cyclohexanedione, barbituric acid, thiazinanonedioxide, etc). By targeting only the redox-sensitive cysteine residues, this strategy may help to reduce the unwanted irreversible modifications of off-target proteins. Additionally, the disclosed methods include using the probe compounds to determine the potency of an inhibitor against a specific protein containing a cysteine residue in the sulfenyl modified form. The reaction between N-hydroxysuccinimide and amine (e.g., ɛ-NH2 group of a Lys residue) is often employed in protein bioconjugation chemistry. A patent application filed by APIT Lab GmbH disclosed the utilization of the reaction to install at least one PEG moiety to a lysine residue in Lys/Arg oxidoreductase by applying succinimide as the linker group [82] . The resultant PEG modified Lys/Arg oxidoreductase
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Figure 8. Novel covalent inhibitors/probes targeting sulfenic acid- or sulfenamide-modified cysteine residue [81]. R1 is a sulfenic acid specific nucleophile, such as 3-cyclohexanedione, barbituric acid, and thiazinanonedioxide; R 2 is H, OH or alkoxy; X is H, halogen, OH or alkoxy; and Y is H, halogen, OH or alkoxy.
exhibits significantly improved activity and stability in plasma, and the anticancer efficacy in mouse is improved compared with the unmodified protein, which suffers short circulation time in plasma after administration, and thus lacks therapeutic efficacy. In addition, sulfur(VI) fluoride exchange has emerged as a new click reaction that can serve as a powerful tool in chemical biology, due to the balance of reactivity and aqueous stability of the sulfonyl fluoride functionality (R-SO2-F). Sulfonyl fluorides are usually quite unreactive, but react with nucleophilic amino acids when placed in an appropriate orientation within a protein binding site for example. In fact, sulfonyl fluoride reagents have been shown to label a variety of proteins by forming covalent bonds with Ser, Tyr or Lys residues, as summarized in a recent review article by KB Sharpless and colleagues [83] . As the reactivity is controllable, sulfonyl fluorides may also serve as warheads for covalent drugs. Furthermore, Jiang et al. reported the application of SuFEx to generate polymers containing sulfur(VI) -SO2- connectors via the condensation of fluoro-substituted and silyl-substituted monomers in the presence of a base catalyst [84] . The Scripps Research Institute filed a patent application and disclosed this polymerization method [85] (examples of the disclosed monomers are shown in Figure 9). This method may enable production of novel polymeric materials with more favorable properties. Conclusion & future perspective Copper-mediated azide-alkyne cycloadditions are the most popular click chemistry reactions, partly due to the ready accessibility of the reagents, its Key term Sulfur(VI) fluoride exchange (SuFEx): Facile and selective reaction between sulfur(VI) fluoride and a nucleophile.
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compatibility with biological systems and the bioorthogonality of the chemistry. However, with the recent discovery and development of other types of click reaction, and increasing access to reagents that enable exploratory chemistries, we expect to see even greater utilization of click chemistry in the future. We believe expansion of the click chemistry toolbox will be required to meet the ever increasing demands of biomolecule specific labeling, especially in complex cellular environments. In particular, imaging techniques are important in many aspects of drug discovery and chemical biology, and the number of tools will continue to grow rapidly in
this area. Additionally, we expect novel click reactions will be developed to facilitate the development of macromolecular therapy, which is a fast-growing business in the pharmaceutical industry. Financial & competing interests disclosure The authors are employees and shareholders of Pfizer. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Executive summary • First introduced by KB Sharpless, click chemistry refers to reactions that are ‘modular, wide in scope, give very high yields, generate only inoffensive by-products that can be removed by nonchromatographic methods, and be stereospecific (but not necessarily enantioselective).’ • Various types of chemical reactions have been adapted to meet the criteria of click chemistry. Most famously, with Cu(I) as the catalyst, the azide-alkyne cycloaddition is greatly accelerated and can be performed at room temperature in aqueous solutions. • Click chemistry has impacted drug discovery from early exploratory research through to clinical stages of development. It facilitates the synthesis of compound libraries, supports high throughput screening, helps to identify the targets of hits from phenotypic screens and determine target engagement by drug candidates. • The number of click chemistry patent applications has grown considerably in recent years, many of them focusing on the site-specific/selective modification of macromolecules. In one aspect, these facile modifications could improve the properties of therapeutic proteins or antibody–drug conjugates (e.g., ADME, efficacy, etc). In another aspect, the modified protein may be used for imaging purposes to confirm target engagement. • Patent applications disclosing kits and methods that provide screening assays for enzyme modulators may be useful in the identification of potential liabilities for compounds during development, such as drug–drug interactions.
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References
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Zhang L, Chen X, Xue P et al. Ruthenium-catalyzed cycloaddition of alkynes and organic azides. J. Am. Chem. Soc. 127(46), 15998–15999 (2005).
Papers of special note have been highlighted as: • of interest; •• of considerable interest 1
Kolb HC, Finn MG, Sharpless KB. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. Engl. 40(11), 2004–2021 (2001).
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First introduction and description of the concept of click chemistry.
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One of the first reports on copper-catalyzed azide-alkyne cycloaddition.
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Discloses an interesting method to address the stability issue by hydrolyzing the thiol-maleimide adduct.
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Schumacher FF, Nunes JP, Maruani A et al. Next generation maleimides enable the controlled assembly of antibody–drug conjugates via native disulfide bond bridging. Org. Biomol. Chem. 12(37), 7261–7269 (2014).
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Develops next generation maleimides that allow generation of antibody–drug conjugate with controlled drug-to-antibody ratios via native disulfide bond bridging.
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Click chemistry patents and their impact on drug discovery and chemical biology
First introduced by K Barry Sharpless in 2001, the term ‘click chemistry’ soon became a widely used description of chemical reactions that proceed rapidly, cleanly and in a manner that is often compatible with aqueous solutions. Click chemistry is frequently employed throughout the process of drug discovery, and greatly helps advance research programs in the pharmaceutical industry. It facilitates library synthesis to support medicinal chemistry optimization, helps identify the targets and off-targets of drug candidates, and can facilitate the determination of drug efficacy in clinical trials. In the last decade, a large number of patent applications covering the various types and utilities of click chemistry have been filed. In this review, we provide the first analysis of click chemistry applications.
Background In 2001, the concept of click chemistry was first introduced by Sharpless et al. to describe reactions defined by a set of certain criteria: “the reaction must be modular, wide in scope, give very high yields, generate only inoffensive by-products that can be removed by nonchromatographic methods, and be stereospecific (but not necessarily enantioselective). The required process characteristics include simple reaction conditions, readily available starting materials and reagents, the use of no solvents or a solvent that is benign (such as water) or easily removed, and simple product isolation” [1] . Since then, a number of chemical reactions have been developed that fulfill these criteria. The favorable features of click chemistry, especially its modular and high yielding nature, enable the rapid synthesis of compound libraries, therefore facilitating the process of lead optimization. In addition, many click reactions are biocompatible and bioorthogonal, making them exceptionally useful for diverse chemical biology applications, such as live cell imaging, bioconjugation and chemoproteomics, approaches that are fast becoming embedded within drug discovery [2–5] . There have been many review articles that summarize the development and utility of
10.4155/PPA.14.59 © 2015 Future Science Ltd
Hua Xu1 & Lyn H Jones*,1 Worldwide Medicinal Chemistry, Pfizer, 610 Main Street, Cambridge, MA 02139, USA *Author for correspondence: Tel: +1 617 674 7085; [email protected]
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click chemistry from different angles [6–8] . However, to our knowledge, thus far, there has not been a systematic review of the click chemistry patent landscape, although the number of patent applications disclosing various types and utilities of click reactions has been rapidly growing in the last decade. Here we attempt to fill this gap by providing an analysis of click chemistry patent applications in the drug discovery arena. 1,3-dipolar cycloaddition First described in late 19th century [9] , the 1,3-dipolar cycloaddition between an alkyne and organic azide was mechanistically investigated by Rolf Huisgen in the 1960s [10] . However, the reaction lacked selectivity, yielding both 1,4- and 1,5-disubstituted [1,2,3]-triazoles. It also required heating and a long time to proceed to completion. In 2002, it was reported that the copper (I) salt significantly accelerated the reaction (107–108 times faster than the uncatalyzed reaction), and only produced one of the two regioisomers, 1,4-disubstituted [1,2,3]-triazole at room temperature or with slight heating (Figure 1A) [11,12] . Having met the criteria of click chemistry mentioned earlier, the potential of the 1,3-dipolar cycloaddition is fully realized.
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CuSO4 Sodium ascorbate RT
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Figure 1. Metal-catalyzed azide-alkyne cycloaddition chemistry. (A) copper-catalyzed azide-alkyne cycloadditions; (B) ruthenium-catalyzed azide-alkyne cycloaddition; and (C) examples of compounds generated using CuAAC. Compound 1: a branched hydroxamate HDAC inhibitor [13] ; compound 2: an antimicrobial conjugate formed from ciprofloxacin and neomycin B [14] . RT: Room temperature.
It not only accelerates the library synthesis of various [1–3]-triazoles that possess different biological activities, but also becomes one of the most reliable and popular tools to tackle some of the toughest problems in biology and chemistry. A ‘traditional’ 1,3-dipolar cycloaddition reaction requires the presence of a Cu(I) catalyst, which is usually produced in situ by a Cu(II) salt and a reducing agent, such as TCEP or sodium ascorbate. A patent application from The Scripps Research Institute disclosed a broad range of copper-catalyzed azide-alkyne cycloadditions (CuAAC) [15] , which are described as being useful for the development of novel small molecular drugs, bioactive nanomaterials, antibacterial and nonimmunogenic coatings for medical implants. Specifically the application disclosed a process of producing [1–3]-triazoles via the cycloaddition of a terminal alkyne with an azide using Cu(I) as the catalyst. The terminal alkyne could be prepared Key terms Click chemistry: Chemical reactions that proceed efficiently and stereospecifically and are often compatible with aqueous solutions. Copper-catalyzed azide-alkyne cycloaddition: Most commonly used 1,3-dipolar cycloaddition reaction between an azide and a terminal alkyne using copper(I) as the catalyst. Strain-promoted azide-alkyne or azide-nitrone cycloaddition (SPAAC or SPANC): Copper-free variants of the CuAAC, which activates the alkyne by introducing ring strain. Staudinger ligation: Ligation reaction between an azide and a phosphine containing an ester moiety, generating a stable amide linkage between the two reactants.
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from an amine containing molecule (e.g., condensation with propargyl methanesulfonate ester). In addition, The Scripps Research Institute filed a separate patent application which disclosed a variety of ligands and the copper complex comprising the claimed ligand and a Cu(I) or Cu(II) ion, which could both be used to promote CuAAC [16] . In 2005, it was reported by Zhang et al. that pentamethylcyclopentadienyl ruthenium chloride complexes, such as Cp*RuCl(PPh3)2, catalyze the regioselective cycloaddition reaction between organic azides and terminal alkynes, forming 1,5-disubstituted triazoles (Figure 1B) [17] . In contrast to CuAAC, this rutheniumcatalyzed azide-alkyne cycloaddition (RuAAC) was also found to be applicable to internal alkynes [17,18] . The Scripps Research Institute also filed a patent application disclosing the aforementioned synthesis of 1,5-disubstituted and 1,4,5-trisubstitued [1–3]-triazoles using the ruthenium-catalyzed cycloaddition of alkynes and azides [19] . Both CuAAC and RuAAC enable ready access to [1–3]-triazole regioisomers and have led to the discovery of novel compounds that exhibit desired biological activities [6] . For example, a branched hydroxamate HDAC inhibitor synthesized by CuAAC, as described in a patent application filed by Accendatech (1 in Figure 1C), demonstrated potent growth inhibition (GI50 less than 1 μM) against several tumor cell lines [13] . Technion R&D Foundation Ltd disclosed the conjugation of two antimicrobial agents, for instance, quinolones and aminoglycosides (compound 2, Figure 1C ), in order to improve activity and overcome bacterial resistance [14] . The CuAAC reaction has limited applications in living cell systems, owing to the cytotoxicity of copper (I).
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Click chemistry patents & their impact on drug discovery & chemical biology
Copper ions are readily chelated by native amino acids and can induce the formation of reactive oxygen species, resulting in damage to cells and tissues. Considerable efforts have therefore been made to eliminate the need for copper in the reaction. Ring strain was first exploited by Bertozzi and colleagues as an alternative means to activate alkynes and promote the cycloaddition with azides in living systems (strain-promoted azide-alkyne cycloaddition, SPAAC) [20] . Then, various cyclooctynes (mono or difluorinated, or fused with other aryl rings) were reported to have improved reaction rates with azides, which demonstrated the successful labeling of biomolecules in living cells and even animals [21–23] . In a 2006 patent application filed by the University of California, Bertozzi et al. disclosed a series of cycloalkynes and heterocycloalkynes (structures 3 and 4, Figure 2) to label azide-containing biomolecules [24] . The azide target substrates can either be generated in vitro and then introduced into the cells by a variety of well-established methods, (e.g., microinjection or liposome-mediated delivery), or produced in vivo using metabolic labeling [25] or genetic engineering [26] . Later, Stichting Katholiek Universiteit described the strain-promoted cycloaddition of cyclopropanefused cyclooctynes with azides (SPAAC) or nitrones (SPANC), (5–7, Figure 2) [27] . The methods to prepare the cyclooctynes by cyclopropanation of a cyclooctadiene followed by bromination and subsequent dehydrobromination reactions revealed the desired alkyne functionality. Fluorometric tags or pharmaceutical compounds could be ligated to the cyclooctynes via the functional group Q (e.g., –OH) for diagnostic and therapeutic applications. A patent application filed by the University of Georgia Research Foundation discloses the click reactions of cyclooctynes fused to two aryl groups (benzannulated systems) (general structure 8, Figure 2) [28] . Such compounds were also shown to be useful for cell surface modifications and the X group in 8 can provide an attachment site for bioconjugation (e.g., C=O, CHOH, CHNH2). The University of Melbourne has filed a patent application related to the preparation and use of
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bi-cyclo[6.1.0]-non-4-yne as a biomolecular tether, for example, the ligation of proteins to quantum dots for the use in single-cell, single-molecule imaging methods [29] . Various utilities of SPAAC and SPANC have also been described in several recently filed patent applications, including methods that improve RNA functionalization [30] , oligonucleotide ligation [31] , and the delivery of diagnostics such as 18F PET probes and therapeutic agents to specific cell and tissue types, or pathogens, using in vivo click chemistry approaches [32] . Targeted delivery in this way has considerable potential to limit the exposure of chemotherapeutic drugs to healthy cells, so reducing cytotoxicity (see below). The application of sequential SPAAC and CuAAC was reported by our group in the development of trifunctional scaffolds with applications in chemical biology and bioconjugation [33,34] . As a proof-of-principle, we showed that a variety of biomolecules, including peptides and sugars, and tags such as dyes and biotin, could be clicked onto a core template in a sequential, reliable and high yielding manner. Staudinger ligation The first traceless Staudinger ligation was reported by Bertozzi and colleagues in 2000 [35] . It is a modified version of the classical Staudinger reaction that introduces a methyl ester group to the triarylphosphine as an electrophilic trap so that a stable amide bond is formed between the two reactants (Figure 3) . The Staudinger ligation has proven to be selective and efficient, and these modified triarylphosphines can even selectively label azides installed on cell surface glycoconjugates [35] . Later, the Staudinger ligation was also applied to the visualization of glycans in live cells and animals [3,36] . Notably, a fluorogenic Staudinger phosphine probe based on a FRET-quenching mechanism was later developed and used to image sugars containing azide groups in live cells [37] . Koninklijke Philips Electronics discloses a kit comprising one targeting probe and either an imaging or therapeutic probe that relied on the Staudinger ligation [38]. Specifically, an azide group is incorporated into the target of interest, say a cell surface receptor, (H)8-n
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Figure 2. Examples of cycloalkynes disclosed in the patent literature [24,27–28].
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Figure 3. Staudinger ligation.
and then an imaging probe, which contains the desired triphenylphosphine derivative, undergoes a Staudinger ligation with the target to enable MRI, PET or SPECT imaging. Similarly, a therapeutic agent incorporating a triphenylphosine group can be targeted to the site of interest using the Staudinger ligation. A recent patent application filed by Siemens Medical Solutions disclosed a two-step method to generate a radiolabeled tracer for PET imaging [39] . The first synthetic step condenses a carboxylic acid with the hydroxyl group of a phosphine molecule to form a phosphine ester. Second, a Staudinger ligation between the phosphine ester and an azide bearing a PET radioisotope functionality produced the desired radiolabeled tracer. An issue regarding the utility of the Staudinger ligation is the susceptibility of the phosphine reagents to oxidation by oxygen which slows the reaction. Additionally, the intrinsic rate of the Staudinger ligation is relatively slow [40] , making it less appealing compared with other types of click reactions, particularly for in vivo labeling (see below). Inverse electron demand Diels–Alder click chemistry (tetrazine ligation) There have been continuous efforts to search for novel click reactions since the original CuAAC and SPAAC reports. For example, it was reported in 2008 that strained dienophiles, such as norbornene and trans-cyclooctene (TCO) react rapidly and efficiently with 1,2,4,5-tetrazines in an aqueous solution [41,42] . As a result of exceptionally high reaction rates, this type of inverse electron-demand Diels−Alder reaction (IEDDA) has been utilized in bioconjugation chemistry with a number of diverse applications, including targeted imaging in live cells and animals [42–44] . A small library of tetrazine derivatives were synthesized recently and shown to exhibit varying degrees of stability and reactivity with TCO (200 to 30000 M-1S-1), thus facilitating the design of appropriate click reacting pairs [45] . Devaraj and colleagues reported the Key term Inverse electron-demand Diels−Alder reaction: Cycloaddition reaction between an electron-rich dienophile and an electron-poor diene.
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in situ synthesis of alkenyl tetrazine derivatives, and showed they possess strong fluorescent turn-on properties (up to 400-fold enhancements) when reacted with cyclopropenes and trans-cyclooctenes [46] . Some coumarin tetrazines were also reported to exhibit 11,000fold fluorescence enhancement upon ligation with TCOc (a trans-cyclooctene derivative) [47] . Such highly fluorogenic probes assist in lowering the background fluorescence signals caused by nonspecific binding of the fluorophore to the cellular components and allow for ‘no-wash’ cell imaging. A patent application filed by the University of Delaware discloses a method of performing the ligation reaction between 1,2,4,5-tetrazines and dienophiles in an organic or aqueous medium (9–17, Figure 4) [48] . The method of preparing such trans-cyclooctenes and several novel 3-substituted cyclopropenes was also disclosed. The rapidity and bioorthogonality of the tetrazine-based Diels–Alder reaction makes it an excellent option for the synthesis of radiotracers and their subsequent use in PET imaging. A 18F-labeled cyclic RGD peptide (which was shown to achieve specific tumor uptake) was generated in 5 min with more than 90% purity using the tetrazine-TCO ligation [49] . Similarly, Reiner et al. described the rapid synthesis of an 18 F-labeled poly[ADP-ribose]polymerase 1 (PARP1) inhibitor, that generated high-quality PET images of cancer cells [50] . The same group filed a patent application which disclosed a PARP1 tracer and the use of the tracer to detect and image cancer cells [51] . In addition, in a patent application filed by University of Southern California and University of Delaware, the use of tetrazine-TCO ligation to selectively and efficiently synthesize a Diels–Alder adduct bearing a radionuclide for imaging in animals and humans was disclosed [52] . The abovementioned methods utilizing IEDDA will undoubtedly assist in the rapid synthesis of radiotracers to detect and quantify the engagement of pharmaceuticals with their molecular targets in live animals. Proof of target engagement is a key pillar for successful target validation [53] and clinical progression, resulting in reduced attrition rates in drug discovery [54] .
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Figure 4. Examples of dienophiles and tetrazines disclosed in patent US8236949 [48].
Thiol-ene addition The thiol-ene addition is an efficient and high yielding reaction that is also tolerant to aqueous solutions. The most popular example is the reaction between a thiol and maleimide, generating a thiol-succinimide linker (Figure 5A) . A common problem of the thiol-succinimide is its intrinsic instability. It is known that thiolsuccinimides are susceptible to retro-Michael addition and thereby subsequent thiol exchange process with albumin, cysteine or glutathione. A recent report described the rapid dissociation of a cytotoxic drug from a targeting mAb (a so-called antibody–drug conjugate, or ADC), that reduced the therapeutic index of the molecule in an animal model, as a result of the reversible nature of the thiol-maleimide reaction [55] . Nektar Therapeutics disclosed a method to solve the stability issue by hydrolyzing the thiol-maleimide adduct. This led to >95% of the succinimide group being in a ring opened form, and the resultant thiol-succinamic acid conjugate was found to be more A
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stable and did not undergo the thiol exchange [58] . In this patent application, they described using the method to generate polyethylene glycol (PEG) modified proteins, that often improve the pharmacokinetics and pharmacodynamics of the conjugated therapeutic, whilst reducing immunogenicity [59,60] . A series of linker compounds disclosed by Syschem Inc contain a double bond as the thiol acceptor (general chemical structures 18–20, Figure 5B), allowing conjugation of a drug payload molecule to a free thiol group within a cellular recognition ligand [56] . The payload molecules Z include, but are not limited to, cytotoxic agents, targeted chemotherapeutic agents, radionuclides and immunomodulating agents. Another development in this area involved the use of next generation maleimides, including bromomaleimides and aryloxymaleimides, to enable disulfide bridging and the dual functionalization of disulfide bonds [61–63] . This has led to the controlled assembly of near homogenous ADCs by loading the small molecule drug via the
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Figure 5. Thiol-ene addition. (A) Ligation reaction between thiol and maleimide. (B) Various linker compounds disclosed in the patent applications: 18–20 in patent US20130323169 [56] ; 21 in patent WO2011018613, where Y is an electrophilic leaving group [57] .
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Figure 6. Aldehyde/ketone ligation. (A) Ligation reaction between aldehyde/ketone and hydrazine/ alkoxyamine. (B) The Pictet–Spengler ligation.
interchain disulfide bonds of the antibody [64] . UCL Business PLC filed application patent on a conjugation method using the maleimides with good electrophilic leaving groups at 3- and/or 4-positions (general structure 21, Figure 5B) [57] . An acid-cleavable thiomaleamic acid linker recently developed by the same group [65] was found to be stable at physiological pH, but cleaved in the acidic environment of the lysosome, thereby enabling the design of an ADC cleavable linker technology [66] . Aldehyde/ketone ligation The carbonyl group in aldehydes and ketones can be selectively ligated with hydrazine and alkoxyamine nucleophiles even under physiological conditions to form hydrazones and oximes, respectively (Figure 6A) . These reactions have been exploited to label a variety of biomolecules, including proteins and glycans [25,67–69] . A recent development was the facile incorporation of aldehyde tags into proteins so enabling site-specific bioconjugation [70] . The method relies on the use of formylglycine generating enzyme (FGE) to recognize a natural amino acid tag sequence that then converts a cysteine residue into a unique amino acid bearing the aldehyde functionality [71] . A patent application disclosing the use of this method to produce a carrier protein–drug conjugate has been published [72] . In 2001, a patent application disclosing a method of generating aminodrug peptide conjugates by way of aldehyde/ketone ligation for targeted therapeutics published [73] . Specifically, the patent A
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Cyanobenzothiazole & thiol conjugation As the last step of biosynthesis of luciferin, the condensation reaction between 2-cyanobenzothiazole and 1,2-aminothiol is relatively mild (Figure 7A) . Rao’s group at Stanford first demonstrated that the reaction can occur in vivo under the control of pH, disulfide reduction and proteolytic cleavage. It is further shown that this reaction could be used to visualize protease activity in single live cells [78] . Promega discloses a series of cyanobenzothiazole derivatives (Figure 7B) that can be used to site-specifically install a reporter moiety (including a fluorophore, affinity moiety, or a photocrosslinking label) to a protein with an N-terminal cysteine residue for the purpose of detection and affinity isolation [79] . Promega filed another patent application on the ligation between cyanobenzothiazole and cysteine [80] .
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application describes a linker containing a ketone or aldehyde group, which is attached to the amino group of a cytotoxic drug, such as anthracyclin antibiotics, and then ligated with an O-alkylhydroxylamine derivative of a carrier peptide. The resulting conjugates showed high affinity to tumor tissues, thus improving the therapeutic index of the parent drug. The classical Pictet–Spengler reaction refers to the condensation reaction between a β-arylethylamine, for instance, tryptamine, and an aldehyde or ketone, followed by ring closure. It was first utilized for protein N-terminal labeling by Kodama et al. [74] , and was disclosed in a patent application as a general method for protein modification [75] . In order to improve the rate of reaction (10– 4 M-1 S-1 at pH 4–5), Bertozzi and colleagues developed a modified Pictet–Spengler reaction between N-alkoxytryptamine and aldehyde/ketone (Figure 6B). They demonstrated the site-specific chemical modification of glyoxal- and formylglycine-functionalized proteins, including an aldehyde-tagged variant of the therapeutic monoclonal antibody Herceptin [76] . Moreover, the reaction rate is 4–5-times higher than the classical Pictet–Spengler reaction. The labeling method was disclosed by the Regents of the University of California as useful for chemically modified biomolecules, including proteins, glycans, nucleic acids and cofactors [77] .
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Figure 7. Benzothiozole and thiol ligation. (A) Ligation reaction between 2-cyano-6-hydroxy benzothiazole and cysteine [78] . (B) Benzothiolzole derivatives described in patent application US20090263843 [79] .
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In this application, Promega describes assays to detect the activity of nonluciferase enzymes and determine their modulator activity by employing 2-cyano-6-hydroxy- or 2-cyano-6-amino-benzothiazole derivatives, which have a specific enzyme recognition motif for the desired nonluciferase enzyme coupled to the benzothiazole backbone via the 6-hydroxy or a 6-amino site. One purported advantage of these benzothiozole derivatives is that they would be more readily accepted by the nonluciferase enzymes as a substrate than luciferin, which contains a carboxylic acid group and is not tolerated by certain enzymes, such as CYP2D6. It is disclosed that these derivatives, after modification by the nonluciferase enzyme, can still react with D-cysteine, but do not give the light producing luciferin, resulting in a decrease in bioluminescence signals, compared with the ‘no enzyme’ control. It was shown that the bioluminogenic assay could be performed with recombinant proteins, microsomes, and even intact cells. More importantly, it was used to measure the effect of a given compound on the activity of UGT and CYP450, which are key metabolizing enzymes monitored for potential drug–drug interactions. In addition, this method could be adapted to a high-throughput format, making it more appealing for drug discovery. Other useful click chemistry Recently, Carroll and co-workers disclosed a series of novel covalent inhibitors and probes that irreversibly modify the activities of proteins via the reaction of a sulfenic acid- or sulfenamide-modified cysteine residue (Figure 8) [81] . The compounds disclosed contain a substituted aryl or heterocyclic core structure that promoted binding interactions with a specific protein, such as protein kinases and phosphatases, and a nucleophilic warhead that is capable of forming a covalent bond with a sulfenylated cysteine residue (e.g., 3-cyclohexanedione, barbituric acid, thiazinanonedioxide, etc). By targeting only the redox-sensitive cysteine residues, this strategy may help to reduce the unwanted irreversible modifications of off-target proteins. Additionally, the disclosed methods include using the probe compounds to determine the potency of an inhibitor against a specific protein containing a cysteine residue in the sulfenyl modified form. The reaction between N-hydroxysuccinimide and amine (e.g., ɛ-NH2 group of a Lys residue) is often employed in protein bioconjugation chemistry. A patent application filed by APIT Lab GmbH disclosed the utilization of the reaction to install at least one PEG moiety to a lysine residue in Lys/Arg oxidoreductase by applying succinimide as the linker group [82] . The resultant PEG modified Lys/Arg oxidoreductase
future science group
Patent Review
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Figure 8. Novel covalent inhibitors/probes targeting sulfenic acid- or sulfenamide-modified cysteine residue [81]. R1 is a sulfenic acid specific nucleophile, such as 3-cyclohexanedione, barbituric acid, and thiazinanonedioxide; R 2 is H, OH or alkoxy; X is H, halogen, OH or alkoxy; and Y is H, halogen, OH or alkoxy.
exhibits significantly improved activity and stability in plasma, and the anticancer efficacy in mouse is improved compared with the unmodified protein, which suffers short circulation time in plasma after administration, and thus lacks therapeutic efficacy. In addition, sulfur(VI) fluoride exchange has emerged as a new click reaction that can serve as a powerful tool in chemical biology, due to the balance of reactivity and aqueous stability of the sulfonyl fluoride functionality (R-SO2-F). Sulfonyl fluorides are usually quite unreactive, but react with nucleophilic amino acids when placed in an appropriate orientation within a protein binding site for example. In fact, sulfonyl fluoride reagents have been shown to label a variety of proteins by forming covalent bonds with Ser, Tyr or Lys residues, as summarized in a recent review article by KB Sharpless and colleagues [83] . As the reactivity is controllable, sulfonyl fluorides may also serve as warheads for covalent drugs. Furthermore, Jiang et al. reported the application of SuFEx to generate polymers containing sulfur(VI) -SO2- connectors via the condensation of fluoro-substituted and silyl-substituted monomers in the presence of a base catalyst [84] . The Scripps Research Institute filed a patent application and disclosed this polymerization method [85] (examples of the disclosed monomers are shown in Figure 9). This method may enable production of novel polymeric materials with more favorable properties. Conclusion & future perspective Copper-mediated azide-alkyne cycloadditions are the most popular click chemistry reactions, partly due to the ready accessibility of the reagents, its Key term Sulfur(VI) fluoride exchange (SuFEx): Facile and selective reaction between sulfur(VI) fluoride and a nucleophile.
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Figure 9. Examples of monomers disclosed in the patent application WO2014089078 [85] .
compatibility with biological systems and the bioorthogonality of the chemistry. However, with the recent discovery and development of other types of click reaction, and increasing access to reagents that enable exploratory chemistries, we expect to see even greater utilization of click chemistry in the future. We believe expansion of the click chemistry toolbox will be required to meet the ever increasing demands of biomolecule specific labeling, especially in complex cellular environments. In particular, imaging techniques are important in many aspects of drug discovery and chemical biology, and the number of tools will continue to grow rapidly in
this area. Additionally, we expect novel click reactions will be developed to facilitate the development of macromolecular therapy, which is a fast-growing business in the pharmaceutical industry. Financial & competing interests disclosure The authors are employees and shareholders of Pfizer. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Executive summary • First introduced by KB Sharpless, click chemistry refers to reactions that are ‘modular, wide in scope, give very high yields, generate only inoffensive by-products that can be removed by nonchromatographic methods, and be stereospecific (but not necessarily enantioselective).’ • Various types of chemical reactions have been adapted to meet the criteria of click chemistry. Most famously, with Cu(I) as the catalyst, the azide-alkyne cycloaddition is greatly accelerated and can be performed at room temperature in aqueous solutions. • Click chemistry has impacted drug discovery from early exploratory research through to clinical stages of development. It facilitates the synthesis of compound libraries, supports high throughput screening, helps to identify the targets of hits from phenotypic screens and determine target engagement by drug candidates. • The number of click chemistry patent applications has grown considerably in recent years, many of them focusing on the site-specific/selective modification of macromolecules. In one aspect, these facile modifications could improve the properties of therapeutic proteins or antibody–drug conjugates (e.g., ADME, efficacy, etc). In another aspect, the modified protein may be used for imaging purposes to confirm target engagement. • Patent applications disclosing kits and methods that provide screening assays for enzyme modulators may be useful in the identification of potential liabilities for compounds during development, such as drug–drug interactions.
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One of the first reports on copper-catalyzed azide-alkyne cycloaddition.
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One of the first reports on copper-catalyzed azide-alkyne cycloaddition.
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Develops next generation maleimides that allow generation of antibody–drug conjugate with controlled drug-to-antibody ratios via native disulfide bond bridging.
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