[Methods and Principles in Medicinal Chemistry] Fragment-based Approaches in Drug Discovery (JAHNKE: FRAGMENT-BASED APPROACHES IN DRUG DISCOVERY O-BK) || Click Chemistry for Drug Discovery

Part 4: Emerging Technologies in Chemistry

Fragment-based Approaches in Drug Discovery. Edited by W. Jahnke and D. A. ErlansonCopyright � 2006 WILEY-VCH Verlag GmbH & Co. KGaA,WeinheimISBN: 3-527-31291-9

15Click Chemistry for Drug DiscoveryStefanie Röper and Hartmuth C. Kolb

15.1Introduction

Nature’s secondary metabolites (“natural products”) capture the imagination ofthe synthetic organic chemist, due to their complex structures and carbon frame-works, and their oftentimes interesting biological properties. Despite their pro-mise as lead structures for drug discovery, optimization efforts are usuallythwarted by complex and lengthy syntheses, which make analog generation diffi-cult. For this reason, combinatorial chemistry approaches have been developed torapidly scan the chemical universe for new molecules with interesting biologicalproperties that may serve as lead structures for the development of new therapeu-tics. Guida et al. estimated the size of the universe of “drug-like” compounds tobe on the order of 1063 molecules [1]. For this calculation, compounds were de-fined as being drug-like if they were stable in water, weighed no more than 500 Daand contained only H, C, N, O, P, S, F, Cl and Br. Obviously, only an infinitesimalportion of the potential medicinal chemistry universe has been explored to date;and it seems clear that a chemical toolkit containing only the most reliable andversatile processes is needed if one is to make any kind of impact. Click chemistry,introduced in 2001 by Sharpless, Finn and Kolb, was developed with these ideasin mind. It is a modular approach that employs a set of highly reliable reactionsfor assembling fragments to facilitate the discovery of new lead structures andtheir optimization [2]. It focuses exclusively on highly energetic, “spring-loaded”reactants and a pure, kinetically controlled outcome (Fig. 15.1). Click chemistry re-actions are wide in scope, stereoselective, insensitive to water and oxygen and theyutilize readily available starting materials to produce the desired products in highyields, while giving only inoffensive by-products. The reaction work-up is simple,requiring no purification by chromatography. Click chemistry not only simplifiescompound synthesis, but also makes it more reliable, which may result in fasterlead discovery and optimization. It does not replace conventional methods fordrug discovery but rather complements and extends them.

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Fragment-based Approaches in Drug Discovery. Edited by W. Jahnke and D. A. ErlansonCopyright � 2006 WILEY-VCH Verlag GmbH & Co. KGaA,WeinheimISBN: 3-527-31291-9

15.2Click Chemistry Reactions

Inspired by how Nature performs combinatorial chemistry, click chemistry fo-cuses on carbon–heteroatom bond forming reactions. Olefins and acetylenes pro-vide the carbon frameworks. Sample reactions include:� cycloaddition reactions, e.g. hetero-Diels–Alder [3] and 1,3-dipolar cycloaddition [4];� carbonyl chemistry, e.g. the formation of hydrazones, oxime ethers and hetero-

aromatic systems;� addition to carbon–carbon multiple bonds, such as epoxidation [5], aziridination

[6], dihydroxylation [7] and sulfenyl [8] and nitrosyl halide additions;� nucleophilic substitution on strained compounds and intermediates, such as

epoxides, aziridines and episulfonium and aziridinium ions [2].

From the beginning, water has played an important role as a reaction medium;and many click chemistry reactions proceed optimally in pure water, particularwhen the reactants are insoluble [9]. Scheme 15.1 illustrates the phenomenon ofrate acceleration that occurs under heterogeneous conditions (the reactants arestirred in aqueous suspension). Without water or water/alcohol mixtures, the reac-tions are slower and less selective, and are more dangerous on a large scale, be-cause of their highly exothermic nature. Water, owing to its large heat capacity,has a great advantage over other solvents on safety issues, but it also facilitatesproduct isolation.

314 15 Click Chemistry for Drug Discovery

Fig. 15.1Energetically highly favorable linking reactions, which are partof the click chemistry universe.

The triazole forming Huisgen [4] 1,3-dipolar cycloaddition of alkynes and azidesis the premier example of a click chemistry reaction. Despite the fact that the reac-tion is thermodynamically favored by 30–35 kcal mol–1, it possesses a very high ac-tivation barrier, which prevents it from taking place at room temperature. In itsclassic form, the reaction requires elevated temperature or pressure to achieve rea-sonable rates, yielding mixtures of regioisomers. Despite their high intrinsicenergy, azides and alkynes are surprisingly unreactive, which is very advanta-geous, since it provides for a high degree of stability towards aqueous media andbiological molecules. In 2002, the groups of Sharpless/Fokin [10] and Meldal [11]independently discovered a copper(I)-catalyzed, stepwise variant of the 1,3-dipolarcycloaddition between azides and terminal acetylenes, which not only proceedsunder mild conditions, but also is regiospecific for the 1,4-disubstituted 1,2,3-tri-azole (Scheme 15.2).

This new method has enjoyed rapid acceptance; and numerous applications fordrug discovery and material sciences have been developed, due to its reliability,tolerance to a wide variety of functional groups, regiospecificity and the readyavailability of starting materials. The 1,4-disubstituted 1,2,3-triazole unit of the re-action products is an interesting, if underappreciated, pharmacophore that isnearly impossible to oxidize, reduce or cleave. It has been demonstrated to activelyengage in interactions with proteins, due to its large dipole moment of ~5 Debye,and the ability of N-3 to function as a hydrogen bond acceptor [12]. These pharma-

31515.2 Click Chemistry Reactions

Scheme 15.1Water-based reactions.

Scheme 15.2The Cu(I)-catalyzed generation of 1,4-disubstituted 1,2,3-triazoles.

cophoric properties and the rigidity of 1,4-disubstituted triazoles are reminiscentof peptide bonds, suggesting this linking unit to be useful for developing new bio-logically active molecules.

15.3Click Chemistry in Drug Discovery

Since the discovery of the regiospecific copper(I)-catalyzed triazole process, nu-merous chemical biology applications of this linking reaction have been published[13]. Simple experimental procedures, complete conversion, high yields and speci-ficity for 1,4-disubstituted 1,2,3-triazoles make product purification easy and pro-vide excellent opportunities for combinatorial library synthesis and natural pro-duct modification. The inertness of azides and alkynes to biological systemsmakes this ligation process interesting for a wide range of biomolecular applica-tions, ranging from target-templated in situ chemistry to DNA and proteomics re-search, via bioconjugation reactions.

15.3.1Lead Discovery Libraries

The Kolb laboratory at Coelacanth Corporation [14] developed the click chemistryapproach to combinatorial chemistry. Over 170 000 individual compounds across75 different structural themes were made on a 25–50 mg scale using semi-auto-mated liquid phase synthesis approaches. Some of the “spring-loaded” buildingblocks that were used for library generation were epoxides and aziridines, whichprovided 1,2-difunctionalized compounds by nucleophilic ring opening [15], azidesand �-ketoesters, which were converted to triazoles via 1,3-dipolar cycloaddition[15], imidoesters for the preparation of 5-membered aromatic heterocycles [17] and3-aminoazetidines for the synthesis of non-aromatic heterocyclic libraries [18].Also, targeted amino acid-derived libraries were made, which led to the discovery ofpotent peroxisome proliferator-activated receptor � (PPAR-�) agonists [19].

Recent studies have shown that the copper-catalyzed triazole process is suitablefor the solid phase synthesis of combinatorial libraries. The group led by Gmeinerused this process for the preparation of a polystyrene based formyl indol methyltriazole (FIMT) resin for parallel solid phase synthesis of a series of BP-897-typearylcarboxamides (Scheme 15.3) [20]. A library of 42 compounds was generated ina five-step sequence that involved resin loading by reductive amination, followedby amide coupling, deprotection, palladium-catalyzed N-arylation and acidic clea-vage. The final products were screened for binding to monoaminergic G protein-coupled receptors without further purification. Five library members showed ex-cellent dopamine D3 receptor affinities; and carboxamide (Scheme 15.3; 1) dis-played a Ki value of 0.28 nM, which exceeds the binding properties of drug candi-date BP897.


15.3.2Natural Products Derivatives and the Search for New Antibiotics

Bacterial resistance to antibiotic drugs is a growing problem in antibacterial ther-apy; and new classes of anti-infectives are desperately needed [21]. Predictionssuggested that by 2005 nearly 40 % of all Streptococcus strains will be resistant toboth penicillin and macrolide antibiotics in the United States [22]. Of special con-cern is the drug resistance of Gram-positive pathogens such as Streptococcus pneu-moniae to penicillin, Staphylococcus aureus (MRSA) to methicillin and in particularEnterococcus faecium (VRE) to vancomycin. The biological activity of natural pro-ducts is often mediated by their carbohydrate epitopes, which influence the phar-macokinetic properties, targeting and mechanism of action in general. Conse-quently, diversification of the carbohydrate groups may lead to new therapeutics[23]. With the aid of the Cu(I)-catalyzed process, 15 triazole-linked monoglycosy-lated derivatives of vancomycin were synthesized (Fig. 15.2) [24]. Antibacterialscreens of this library revealed that acid derivative 2 (Fig. 15.2; 2) was twice as ac-tive as vancomycin against both E. faecium and S. aureus.

31715.3 Click Chemistry in Drug Discovery

Scheme 15.3Solid-phase supported synthesis of dopamine D3 receptor ligands.

Another approach for modifying natural products was described by Marriott etal., who synthesized a triazole derivative of Kabiramide C in the presence of a cataly-tic amount of Cu(I) and NEt3 (Fig. 15.2) [25]. Kabiramide C is a macrolide drug thattargets actin, a highly conserved and abundant protein, which plays an essentialrole in cytokinesis, cell mobility and vesicle transport. Each triazole derivative wasshown to tightly bind G-actin with the same stoichiometry as the natural product.

A new class of synthetic antibacterial agents against Gram-positive bacteria, con-taining the oxazolidinone pharmacophore, targets bacterial protein synthesis. How-ever, growing drug resistance has been observed with E. faecium and S. aureus. Addi-tionally, oxazolidinone antibiotics can cause severe hypertensive crisis, an undesiredside-effect caused by the inhibition of monoamine oxidase (MAO). In an effort to ad-dress these issues, researchers at AstraZeneca made analogues of lineozolide [26] byreplacing the acetamide functionality with a 1,2,3-triazole and the morpholine ringby a thiapyran sulfone [27]. Both the copper-catalyzed and thermal [3+2]cycloaddi-tion processes were employed to generate triazoles; and vinylsulfones were found togive 1,4-disubstituted triazoles with good selectivities (Scheme 15.4).

SAR studies revealed that 5-substitued triazoles were generally inactive or onlymarginally active. In contrast, many triazoles with a small substituent in the 4-po-sition showed good antibacterial activities and at the same time displayed signifi-cantly reduced inhibition of MAO.

Even though tyrocidine antibiotics are attractive therapeutic agents, they causelysis of human red blood cells. In an attempt to decrease this toxic side effect,Walsh et al. employed a chemoenzymatic approach to make carbohydrate-modi-fied cyclic peptide tyrocidine (tyc) antibiotics, which are nonribosomal peptides(NRP; Scheme 15.5) [28]. They generated a library of 247 glycopeptides by con-ducting an enzymatic macrocyclization, followed by Cu(I)-catalyzed triazole for-


Fig. 15.2Triazole derivatives of natural products.


Scheme 15.4Triazole/thiapyran analogs of the oxazolidinone antibiotic linezolid.

Scheme 15.5Chemoenzymatic approach to glycopeptides via copper-catalyzed triazole formation.

mation with azidoalkyl glycosides in 96-well plates. The excised thioesterase (TE)domain from the tyrocidine synthetase was used to catalyze the formation of thehead-to-tail cyclic peptide derivatives from linear, propargylglycine-containing,peptide N-acetyl cystamine (SNAC) thioesters. The cyclization was followed bycopper-catalyzed triazole formation, which proceeded cleanly, allowing the crudereaction mixtures to be screened in antibacterial and hemolytic assays. Screeninghits were validated by re-testing the purified compounds, leading to the identifica-tion of two glucopeptides that displayed a 6-fold better therapeutic index thanwild-type tyrocidine while maintaining its high antibacterial potency.

15.3.3Synthesis of Neoglycoconjugates

All cell surfaces carry oligosaccharides linked to proteins or lipids. These glyco-conjugates modulate a number of cellular recognition processes, for example theinflammatory response; and their importance for the generation of therapeuticagents has been increasingly recognized in recent years. However, there are someinherent disadvantages associated with this class of compounds that complicatedrug discovery efforts. The synthesis of N-and O-linked glycopeptides is difficult;and the products are susceptible to chemical and enzymatic hydrolysis [29, 30].Isosteric and metabolically stable replacements for the glycosidic linkage maysolve these issues. Triazoles have been investigated as linkers for stable glycopep-tide analogues (Scheme 15.6). They can be made by coupling azide-functionalizedglycosides with acetylenic amino acids or acetylenic glycosides with azide-contain-ing amino acids, using the Cu(I)-catalyzed process [31].

Copper-catalyzed triazole formation from carbohydrate-derived acetylenes andazides is considerably faster under microwave irradiation, allowing reaction timesto be reduced from several hours at room temperature to a few minutes. This al-lowed the preparation of a series of multivalent, triazole-linked neoglycoconju-gates with complete regiocontrol and in high yields, using organic soluble Cu(I)-complexes as catalysts [32]. Polyvalent mannosylated aromatic and heteroaromaticsystems were obtained in this way, in addition to a heptavalent manno-�-cyclodex-trin (Scheme 15.7).


Scheme 15.6Synthesis of triazole-linked glycopeptides.

Norris et al. reported the rapid, regiospecific synthesis of various glucosyl-1,4-disubstituted 1,2,3-triazoles in water, based on the CuSO4/ascorbate system [33].Sabesan et al. generated a combinatorial library of triazole-linked oligosaccharideanalogs [34] by using thermal [3+2]cycloaddition to generate a variety of mimics ofnatural and unnatural carbohydrates with a wide variety of substituents placed onthe carbohydrate groups.

15.3.4HIV Protease Inhibitors

Every year, particularly in sub-Saharan Africa, human immunodeficiency virustype 1 (HIV-1) causes the deaths of millions of people; and the number of indivi-duals who carry the virus continues to rise. HIV-protease [35] is responsible for


Scheme 15.7Multivalent neoglycoconjugates.

the final stage of virus maturation and its inhibitors are used together with reversetranscriptase inhibitors for “highly active anti-retroviral therapy” (HAART). Unfor-tunately, numerous drug-resistant and cross-resistant mutant HIV-1 proteaseshave evolved, making the development of new inhibitors necessary [36].

Cu(I)-catalyzed triazole formation was used to prepare a 100-member librarybased on hydroxyethylamine peptide isosteres (Scheme 15.8; 3–6) [37]. Two differ-ent azide cores (3 and 4), inspired by the existing drugs (aprenavir [38], nelfinavir[39], saquinavir [40]), were connected with acetylenes for library production. Directscreening of the aqueous reaction mixtures against HIV-protease and three mu-tants (G48V,V82F,V82A) led to the identification of four hits with inhibition con-stants in the 10 nM range, which were all derived from scaffold 3. In contrast,scaffold 4 failed to provide any hits.


Scheme 15.8Development of HIV-1 inhibitors through combinatorial triazoleformation, combined with high-throughput screening.

15.3.5Synthesis of Fucosyltranferase Inhibitor

Fucosyltranferases (Fuc-T) catalyze the final glycosylation step in the biosynthesisof the sialyl Lewis x (sLex ) and sialyl Lewis a (sLea ) epitopes of cell surface glyco-proteins and glycolipids. These fucosylated oligosaccharides mediate a variety ofcrucial cell–cell recognition processes such as fertilization, embryogenisis, lym-phocyte trafficking, immune response and cancer metastasis [41]. The trans-ferases catalyze the attachment of L-fucose to a specific hydroxyl group of sialyl N-acetyl-lactosamine, using guanosine diphosphate �-L-fucose (GDP-fucose) as thefucosyl donor. Selective fucosyltransferase inhibitors may block the generation ofthese fucosylated products and the pathology they trigger. However, due to lowsubstrate affinity and the absence of enzyme structure data, rational design of in-hibitors has been difficult [42].

Wong et al. discovered nanomolar inhibitors from a triazole library prepared bythe copper-catalyzed reaction of 85 azides with a GDP-derived alkyne in water[43]. High yields and the absence of protecting groups made it possible to directlyscreen the crude reaction mixtures (Scheme 15.9). This led to the identification ofthe biphenyl derivative 7 as a hit, which was purified and tested against a varietyof fucosyl and galactosyl tranferases and kinases, to discover that it was not onlyhighly selective for human �-1,3-fucosyltransferase VI but also very potent.


Scheme 15.9Synthesis and in situ screening of fucosyltransferase inhibitors.

15.3.6Glycoarrays

Carbohydrate arrays (glycoarrays) are used for the high-throughput analysis of su-gar–receptor interactions [44, 45] and for studying cell–cell recognition processesmediated by carbohydrates. Wong et al. created a covalent array by reacting azide-bearing saccharides with an alkyne that was bound to microtiter plates via a disul-fide linker (Scheme 15.10) [46]. The disulfide bond survives a range of biologicalassay conditions and can readily be cleaved with reducing agents, allowing charac-terization and quantitative analysis by mass spectrometry. The utility of thismethod was demonstrated by performing two binding assays with Lotus tetragono-lobus lectin (LTL), which recognizes �-l-fuctose [47], and Erythrina cristagalli lectin(EC) [48], which recognizes galactose.

A carbohydrate array was employed for screening a compound library of GDPderivatives for Fuc-T inhibitors (Scheme 15.11) [49]. N-acetyllactosamine (LacNac)was displayed on the surface of a microtiter plate by Cu(I)-catalyzed triazole for-mation with an immobilized lipid alkyne. The compound library was mixed withFuc-T and GDP-fucose and then incubated in the LacNAc-containing microtiterplate wells. The transfer of fucose to LacNAc was quantified with a lectin from Tet-ragonolobus pupureas (TP), conjugated to a peroxidase, leading to the identificationof four compounds with nanomolar inhibition constants. Three of these hits con-tained a biphenyl substituent. Remarkably, these hits had not been identified in apreceding fluorescence-based assay [50].

This method can be used for high-throughput screening of large compoundlibraries at low cost.


Scheme 15.10Glycoarray synthesis based on the copper-catalyzed triazole process.

15.4In Situ Click Chemistry

The groups of Sharpless and Kolb developed a new strategy for drug discovery,which relies on the irreversible, target-guided synthesis (TGS) of high infinity in-hibitors from small click chemistry fragments (Fig. 15.3) [51–54]. The latter are as-sembled within the biological target’s binding pockets through selective bindingand irreversible interconnection to generate potent inhibitors that engage in mul-tiple interactions with the target. Since this target-templated, fragment-based ap-proach requires relatively few reagents (which are sampled by the protein in thou-sands of different ways), it promises to be more efficient than traditional combina-torial chemistry, which involves the synthesis, purification and screening of thou-sands of library compounds. Follow-up tests are performed only with the target-generated hits to measure their binding affinities and specificities. These hits areusually very potent, due to the multivalent nature of the interactions with the pro-tein.

15.4.1Discovery of Highly Potent AChE by In Situ Click Chemistry

The target-guided click chemistry approach was first tested with acetylcholine es-terase (AChE) (Fig. 15.4). The enzyme plays a key role in neurotransmitter hydro-lysis in the central and peripheral nervous system [55, 56]. It has two separatebinding sites on either end of a narrow gorge [57]. For fragment assembly by the

32515.4 In Situ Click Chemistry

Scheme 15.11Identification of fucosyl transferase inhibitors using a glycoarray.

enzyme, the thermal 1,3-dipolar cycloaddition reaction between azides and acety-lenes [4] was employed, since the reaction is extremely slow at room temperatureand the reactants do not react with the protein. The fragments were designed tobind to either the peripheral anionic site or the active center. They carried azideand acetylene groups, respectively, via flexible spacers. From a total of 52 possiblereagent combinations, the enzyme selected only four pairs, leading to the forma-tion of four reaction products in a highly regioselective fashion. These compoundsturned out to be more potent than any other non-covalent organic AChE inhibitor,the most potent one having a dissociation constant Kd of 99 fM in the case of eelAChE (Fig. 15.4, left).

Additional experiments demonstrated that this fragment-based approach is suit-able for the discovery of novel AChE inhibitors from reagents that were not pre-viously known to interact with the enzyme’s peripheral site. Thus, a total of24 acetylene reagents were incubated in sets of up to ten compounds with AChEand the active site ligand, tacrine azide (Fig. 15.4, right) [58]. The triazole pro-ducts, formed by the enzyme, were identified by HPLC-MS analysis of the crudereaction mixtures. The enzyme selected only the two phenyltetrahydroisoquino-line building blocks that were in the reagent library and combined them with the


Fig. 15.3In situ click chemistry.

Fig. 15.4In situ click chemistry with acetylcholine esterase (AChE).

tacrine azide within the active center gorge, to form multivalent inhibitors that si-multaneously associate with the active and peripheral binding sites. The winningcombination involved reagents that have relatively weak binding affinities forAChE (7.8–34 µM), demonstrating that this target-guided strategy is successfuleven with micromolar binders. The new inhibitors are not only more “drug-like”than the previous phenylphenanthridinium-derived compounds, due to the lackof permanent positive charge and aniline groups and fewer fused aromatic rings,but they are also three times as potent. With dissociation constants as low as33 fM, these compounds are the most potent non-covalent AChE inhibitorsknown.

Recent work in the Kolb and Fokin laboratories has shown that the target-guided lead discovery method also works with targets other than acetylcholine es-terase, e.g. other enzymes such as carbonic anhydrase II and HIV protease [59],and non-enzymes, such as transthyretin.

Carbonic anhydrases are Zinc–enzymes that catalyze the interconversion ofHCO3

– and CO2. They are involved in key biological processes related to respira-tion and the transport of CO2/HCO3

–, bone resorption, calcification and tumori-genicity [60]. Systemically and topically administered carbonic anhydrase inhibi-tors have long been used to control the elevated intraocular pressure associatedwith glaucoma [61, 62]. Most inhibitors are aromatic or heteroaromatic systemsthat carry a sulfonamide functional group [63–65], which coordinates the Zn2+ ionin the active site. Based on this information, Kolb et al. designed 4-ethynylbenze-nesulfonamide as an anchor molecule for the target-guided formation of carbonicanhydrase inhibitors (Fig. 15.5) [53]. The benzenesulfonamide anchor moleculewas incubated with each of 24 azide reagents in the presence of bovine carbonicanhydrase II for 40 h. Analysis of all 24 reaction mixtures by liquid chromatogra-phy with mass spectrometry/selected ion mode detection (LC/MS-SIM) revealed

32715.4 In Situ Click Chemistry

Fig. 15.5In situ click chemistry with Carbonic anhydrase-II.

that 11 mixtures had formed triazoles inside the enzyme. Control experiments va-lidated these hits: (a) no product was observed in the absence of protein, (b) noproduct was generated with bovine serum albumin as a result of non-specific pro-tein binding and (c) no product was observed when the reaction was performed inthe presence of a low-nanomolar carbonic anhydrase inhibitor. These results de-monstrate that carbonic anhydrase is able to assemble triazoles from azide andacetylene building blocks and that the reaction occurred inside or near its activesite. The most potent hit compound is 185 times more active (Kd = 0.2 nM) thanthe acetylenic scaffold (Kd = 37 nM).

15.5Bioconjugation Through Click Chemistry

15.5.1Tagging of Live Organisms and Proteins

Click chemistry was recently employed to selectively tag biomolecules in livingcells or organisms to monitor their involvement in biological process and/or de-tect their biodistribution and metabolism [66, 67].

The azido group is a very useful chemical handle for bioconjugation, since it islargely inert in biological systems and readily undergoes highly selective reactions,such as the Staudinger ligation with phosphines [68] as well as [3+2]cycloadditionwith strained [69] and terminal alkynes [70]. The Cu(I)-catalyzed reaction betweenazides and acetylenes enables the selective tagging of virus particles [71], nucleicacids [72], enzymes [73], cells [74] and proteins from complex tissue lysates [75].

The utility of the Cu(I)-catalyzed triazole formation for this purpose was estab-lished by labeling intact cowpea mosaic virus particles (CPMV) [76] with fluores-cein (Scheme 15.12). CMPV is composed of 60 identical copies of a two sub-unitprotein assembled around a single-stranded RNA. Through traditional bioconju-gation techniques, a total of 60 azides were attached per virus particle, setting thestage for Cu(I)-catalyzed tagging with alkynefluorescein. Finn et al. developed spe-


Scheme 15.12Labeling of cowpea mosaic virus particles with fluorescein.

cial conditions for the triazole formation: CuSO4 was reduced in situ with tris(2-carboxyethyl)phosphine (TCEP) and a Cu(I)-stabilizing ligand, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), was used to stabilize Cu(I) in water andprotect the virus from Cu-triazole-induced disassembly [71].

Tirrel and Link replaced methione residues in the outer membrane protein C(OmpC) of Escherichia coli cells by azidohomoalanine (Fig. 15.6; 8) and subsequentlyreacted the azide groups with a biotinylated alkyne reagent, using the TBTA/copper-catalyzed triazole process to tag E. coli with biotin (Scheme 15.13) [74].

Further studies revealed that highly pure (99.999%) CuBr allowed cell surfacelabeling to be enhanced by a factor of 20. This led to the discovery that even azi-doalanine (Fig. 15.6; 9), which was previously not thought to be a good replace-ment for methionine in protein synthesis, was incorporated in cell surface pro-teins. Also, azidonorvaline 10 and azidonorleucine 11 were detected [77].

Schultz and co-workers reported a site-specific, fast and irreversible method forprotein bioconjugation [73]. The incorporation of genetically encoded azide- or al-kyne-containing unnatural amino acids into proteins of Saccharomyces cerevisiae,followed by coupling with azide- or alkyne-dyes (Fig. 15.7, 12), allowed gel-fluores-cence scanning of the modified protein. This methodology for genetically encod-ing unnatural amino acids in prokaryotic and eukaryotic organisms was used inconjunction with phage display to synthesize polypeptide libraries with unnaturalamino acid building blocks [78]. For this study, a pIII fusion streptavidin-binding

32915.5 Bioconjugation Through Click Chemistry

Fig. 15.6Methionine, azidoalanine, azidohomoalanine azidonorvaline, azidonorleucine.

Scheme 15.13Tagging E. coli with biotin.

peptide was expressed in E. coli strain TTS (type III secretion) in the presence ofunnatural amino acids. The resulting phages displayed the unnatural amino acid-carrying peptides. In order to characterize this phage display system, the resultingphages were tagged with an alkyne dye, using the Tirrell conditions for the tria-zole-forming process [74]. SDS-PAGE and Western blot analysis showed that theunnatural amino acid was specifically introduced into the pIII coat protein of theunnatural phage. This technology enables the site-specific and irreversible attach-ment of a single PEG molecule to a protein, making possible the production ofwell defined and homogenous therapeutic PEGylated proteins.

15.5.2Activity-based Protein Profiling

The field of proteomics is engaged in the development of methods for analyz-ing protein expression and function. Conventional methods measure proteinvariations based on abundance, whereas activity-based protein profiling (ABPP)[75], a chemical proteomics method, utilizes active site-directed probes to deter-mine the functional state of enzymes in complex proteomes (e.g. to distinguishan active enzyme from its inactive precursors and inhibitor-bound forms). TheABPP probes contain two main elements: a reactive group (RG), which bindsto and covalently labels the active site, and a reporter tag (e.g. biotin and or a


Fig. 15.7Genetically encoded incorporation of unnatural amino acidsfor site-specific tagging of proteins.

fluorophore), which enables the rapid detection and isolation of the labeled en-zyme [66].

Sorensen et al. synthesized a series of rhodamine biotin-tagged compoundsusing the Cu(I)-catalyzed triazole process to investigate the FR182877 mechanismof action (Scheme 15.14), [78]. Incubation of the tagged derivatives with mouseproteome in vitro led to the identification of carboxylesterase-1 (CE-1) as a specifictarget of the natural product. The authors examined enzyme activity with a serinehydrolase-directed probe fluorophosphonate rhodamine and determined thatFr182877 is a selective inhibitor of CE-1 with an IC50 of 34 nM.

Despite the successful application of ABPP for several enzyme classes, the cur-rently used bulky, cell wall impermeable reporter tags require cell homogenizationprior to analysis, making measurement in living cells impossible. Cravatt et al. de-veloped a “tag-free” version of ABPP, in which the reporter group is attached tothe activity-based probe after covalent labeling of the protein targets(Scheme 15.15) [75]. Bioorthogonal coupling partners, azides and alkynes, wereemployed to join the probe and tag with the copper-catalyzed triazole process.With this method, Cravatt et al. detected glutathione S-transferase (GSTO 1–1),enoyl COA hydratase-1 (ECH-1) and ALDH-1 at endogenously expressed levels inviable cells. For these studies, an azide-bearing (tag-free) phenylsulfonate ester(PS-N3) probe was incubated with intact cells and then ligated with a rhodaminealkyne reporter group after cell homogenization. The results were consistent withtraditional methods.


Scheme 15.14Rhodamine biotin-tagged compounds for ABPP of carboxylesterase-1.

This click chemistry-based approach to ABPP allowed, for the first time, the de-termination of protein expression levels in living animals. ECH-1 was observed inthe heart tissue of mice by injecting PS-N3 into the living animal and staining thehomogenized tissue of the sacrificed animal with rhodamine alkyne.

The drawback of this methodology is a higher degree of background labeling ascompared to conventional ABPP, due to low-level non-specific labeling of abun-dant proteins of the proteome.

Further studies demonstrated that swapping the reaction groups (i. e. PS-al-kyne/RhN3 instead of PS-N3/Rh-alkyne) reduced the extent of background label-ing and enabled the visualization of lower-abundance enzyme activity [80]. How-ever, the PS-N3/Rh-alkyne combination reacts four times faster, making it the sys-tem of choice for fast analysis.

15.5.3Labeling of DNA

The Ju laboratory has fluorescently labeled single-stranded DNA (ssDNA), usingconcerted thermal 1,3-dipolar cycloaddition [72]. Thus, DNA was azido-labeledby reacting succinimidyl 5-azido-valerate with an amino linker-modified oligonu-cleotide (M13–40 universal forward sequencing primer); and its cycloadditionwith 6-carboxy-fluorescein (FAM) was carried out thermally in aqueous media(Scheme 15.16a). The resulting FAM-labeled ssDNA was successfully used in theSanger dideoxy chain termination reaction, using PCR-amplified DNA as a tem-plate to produce DNA sequencing products terminated by biotinylated dideoxya-denine triphosphate (ddATP-biotin). These fragments were analyzed by capillaryarray electrophoresis and the results matched exactly the sequence of the DNAtemplate. An electron-withdrawing group was attached to the alkyne to optimizethe cycloaddition reaction for DNA analysis and immobilization [81]. This allowedcatalyst-free 1,3-dipolar cycloaddition to proceed smoothly at room temperature inaqueous solution, conditions that are compatible with DNA modification undercell-biological conditions.


Scheme 15.15“Tag-free” ABPP.

The Cu(I) process has been employed to immobilize DNA on a solid supportfor fast sequencing (Scheme 15.16b) [82]. Thus, an alkyne-modified glass surfacewas coupled with azido-labeled DNA, using the very mild and selective Cu(I) tria-zole process. In order to determine the functionality, stability and accessibility ofthe immobilized DNA, polymerase extension reactions were successfully carriedout with three unique photocleavable (PC) fluorescent nucleotides. In a sequenceinvolving alternate incorporation of PC fluorescent nucleotides, fluorophore detec-tion and photolytic removal of the fluorophore, a seven-nucleotide sequence inthe DNA template was correctly identified.

15.5.4Artificial Receptors

In polar solvents, cyclodextrins are able to bind hydrophobic molecules withintheir cavities, making them useful as enzyme mimetics [83] and molecular recep-tors [84]. Due to the large number of hydroxyl groups, selective modification of cy-clodextrins is challenging. Most approaches to create functionalized cyclodextrinshave very low yields and often lead to complex mixtures of anomeric compoundsor to systems with different ring sizes. Gin et al. demonstrated that Cu(I)-cata-lyzed triazole formation can be used for making functionalized cyclodextrin mi-mics (Scheme 15.17; 14, 15), starting from azido acetylene-bearing trisaccharides(Scheme 15.17; 13) [85]. In the presence of Cu(I), a �-cyclodextrin analog was ob-tained in excellent yield, whereas the uncatalyzed reaction led to multiple pro-


Scheme 15.16(a) DNA labeling, (b) immobilization of DNA on a glass chip.

ducts. The deprotected macrocycle 15 was shown to bind 8-anilino-1-naphthalene-sulfonate with similar affinity as regular �-cyclodextrin. This new strategy allowedthe synthesis of functionalized cyclodextrin mimics that would have been difficultto make using traditional means.

15.6Conclusion

In summary, click chemistry has proven to be a powerful tool in biomedical re-search, ranging from combinatorial chemistry and target-templated in situ chem-istry for lead discovery, to bioconjugation strategies for proteomics and DNA re-search. A number of click chemistry reactions have been developed; and heteroge-neous, water-based reactions as well as the formation of triazoles from azides andacetylenes deserve special recognition. Despite their high energy content, azidesand acetylenes are stable across a broad range of organic reaction conditions andtheir irreversible combination to triazoles is highly exothermic, albeit slow. Thefull potential of this ligation reaction was unleashed with the discovery of Cu(I)catalysis, which allows the reaction to proceed rapidly with near-quantitativeyields. Since no protecting groups are used, the crude triazole products can besubjected to biological screens without purification. This triazole-forming process,and click chemistry in general, promises to accelerate both lead finding and leadoptimization, due to its great scope, modular design and reliance on extremelyshort sequences of near-perfect reactions.


Scheme 15.17�-Cyclodextrin analogs by cyclodimerization of trisaccharide via [3+2]-cycloaddition.

335References

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[Methods and Principles in Medicinal Chemistry] Fragment-based Approaches in Drug Discovery (JAHNKE: FRAGMENT-BASED APPROACHES IN DRUG DISCOVERY O-BK) || Click Chemistry for Drug Discovery