organic & biomolecular chemistry · click-chemistry is an approach based on cycloaddition...

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Organic &BiomolecularChemistrywww.rsc.org/obc Volume 10 | Number 47 | 21 December 2012 | Pages 9297–9508

PERSPECTIVE Jose M. Palomo Click reactions in protein chemistry: from the preparation of semisynthetic enzymes to new click enzymes

Organic &BiomolecularChemistry

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Cite this: Org. Biomol. Chem., 2012, 10, 9309

www.rsc.org/obc PERSPECTIVE

Click reactions in protein chemistry: from the preparation of semisyntheticenzymes to new click enzymes

Jose M. Palomo*

Received 19th July 2012, Accepted 11th September 2012DOI: 10.1039/c2ob26409a

Click-chemistry is an approach based on cycloaddition reactions which has been successfully used as achemical approach for complex organic molecules and which has recently starred in a boom in the worldof protein chemistry. The advantage of the use of this technique in protein chemistry is based on a veryhigh and efficient chemoselectivity, which usually requires simple or no purification and is extremelyrate-accelerated in aqueous media. The perspective discusses some of the most recent advances in theapplication of this reaction in selective enzyme surface modification for the creation of new semisyntheticenzymes (fluorescence labeled enzymes, peptide-enzyme conjugates, glycosylated enzymes), andinterestingly, the recent design and creation of “click” enzymes.

Introduction

The click chemistry concept, introduced by Barry Sharpless in2001, describes the chemistry of tailored reactions to generatecompounds quickly and easily by joining small units under verysimple reaction conditions.1 Several efficient reactions, whichare capable of producing a wide catalogue of functional syntheticmolecules and organic materials, have been grouped accordingly

under the term click reactions. Characteristics of modular clickreactions include mild, high-yielding, high-selective, reliable,and clean transformations of broad scope that usually requiresimple or no purification.

Molecular processes considered to fit all or most of these cri-teria include certain cycloaddition reactions, such as the Huisgen1,3-dipolar azide–alkyne2 and the Diels–Alder cycloaddition3

and other methods such as the Staudinger ligation4–6 or thethiol–ene reaction7,8 (Fig. 1).

This click concept has been rapidly applied to biomoleculesmodification, surface functionalization, polymer science, studies

Jose M. Palomo

Jose M. Palomo received hisPh.D. degree (summa cumlaude) in 2003 at the AutonomaUniversity of Madrid workingat the Biocatalysis Departmentin Institute of Catalysis (IPC,CSIC) in Madrid. For postdoc-toral research he joined theChemical Biology Departmentof Max Planck Institute in Dort-mund in 2004. In 2006, hebegan his appointment as anAssociate Research Scientist atthe Biocatalysis Department in

ICP-CSIC. Since 2009, he has been a Tenured Scientist (Associ-ate Professor) in ICP-CSIC. His current research interestsinclude design of semisynthetic enzymes, carbohydrate chemistrywith enzymes, asymmetric biotransformations and immobiliz-ation of biomolecules. Fig. 1 General scheme of click reactions.

Departamento de Biocatálisis. Instituto de Catálisis (CSIC). C/ MarieCurie 2. Cantoblanco. Campus UAM, 28049 Madrid, Spain.E-mail: [email protected]; Fax: +34-91 585 47 60

This journal is © The Royal Society of Chemistry 2012 Org. Biomol. Chem., 2012, 10, 9309–9318 | 9309

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http://dx.doi.org/10.1039/c2ob26409a

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of biological processes, etc.9–20 and so relies on easy andefficient coupling technologies.

The copper(I)-catalyzed 1,3-dipolar azide–alkyne cyclo-addition (CuAAC), the undisputed leader among click chemistryreactions, is a quantitative and highly chemoselective reactionbetween an azide group and a terminal alkyne group to form atriazole-linked product. This chemical reaction was described byRolf Huisgen as a 1,3-dipolar cycloaddition2 where the chemicalcoupling needed to use high temperature to form the triazolemolecule. Therefore, the discovery of the Cu(I) catalysis per-mitted the cycloaddition to proceed within minutes at roomtemperature, opening the door to a broad application, especiallyin biological systems. Sharpless and co-workers and Meldal andco-workers developed a Cu-catalyzed rendition of this reactionusing terminal alkynes as substrates, but the metal’s cytotoxicitylimits its use in living systems.21

Despite the demonstrated reliability of CuAAC for theefficient functionalization of biomacromolecules in vitro, thenegative effects of the Cu catalyst have greatly limited its use forin vivo applications. Thus, although CuAAC has been used tolabel bacterial22,23 and mammalian cells,24,25 the presence of Cuhas often been found to be detrimental to living cells.

Alternatively, benign Cu-free AAC strategies, not requiringcytotoxic metals and additives, have also appeared. The Bertozzigroup has taken advantage of the inherent ring strain of cyclo-octynes as an effective way for lowering the activation barrier ofAAC and an alternative to the use of metal catalysts (Fig. 1b).26

This strain-promoted AAC variant, SPAAC, has been exploitedas a bioorthogonal strategy for the fluorogenic labeling of pro-teins and is currently used in many different chemicalapplications.27

Interestingly, the different reactivity of CuAAC and SPAACcan be used in applications where the two processes can beimplemented sequentially for orthogonal and dual-labellingpurposes.

Together with it, the Diels–Alder reaction is another quiteinteresting click reaction. It is a [4π + 2π] cycloaddition betweenan electron-rich 1,3-diene and an electron-deficient dienophileleading to the formation of a six-membered carbocycle (Fig. 1c).This is also a highly selective transformation and in water turnedout to be accelerated by a factor up to 104 when compared tothat in organic solvents.28

These advantages have permitted the employment of thisbioorthogonal methodology in peptide and protein chemistry andsurface functionalization.29–33

The azide-Staudinger ligation4 represents another excellentexample of click reaction. Hermann Staudinger5 described thereaction between an azide and a phosphine for the first time in1919. However, it was not until recently that the Bertozzi grouprecognized the potential of this reaction as a method for biocon-jugation and transformed it into the so-called Staudinger lig-ation.6 The bio-orthogonal character of both the azide and thephosphine functions has resulted in the Staudinger ligationfinding numerous applications in cell surface engineering, syn-thesis of peptides and proteins, probing post-translational modifi-cations, labelling of glass surfaces or fluorogenic molecules.34

Starting with an ortho-phosphine terephthalic acid derivative,aza-ylide is formed through a reaction with azide and undergoescyclization to give an oxazaphosphetane as an initial

intermediate. In aqueous media, spontaneous hydrolysis yieldsthe desired amide, into which the phosphine oxide by-product isincorporated (Fig. 1d).6,35,36

From a practical standpoint, they showed that basic and morenucleophilic phosphines react faster, however it should be notedthat the very basic aliphatic phosphines are more prone to oxi-dation limiting their use in biological systems.37

Highly efficient reactions of thiols with reactive carbon–carbon double bonds, or simply “enes”, discovered in the early1900s,38 can proceed in two different ways: a radical (termedthiol–ene reaction) or an anionic chain (termed thiol Michaeladdition), carrying many of the attributes of click reactions(Fig. 1e).

This exceptional versatility and its propensity for proceedingto quantitative conversions under even the mildest of conditionsmake thiol–ene chemistry amenable to application in bioorganicchemistry and biomedicine. The selectivity and high yieldsassociated with the thiol–ene reaction are a perfect marriage withthe requirements of biomaterial applications. Numerous accountsexist in the literature on the implementation of the thiol–ene reac-tion to functionalize biological materials and biological mo-lecules, and to incorporate biological molecules into othersynthetic materials and molecules.39–45

Herein, the most recent examples of the potential applicationof these click reactions on the preparation of new semisyntheticenzymes have been reviewed. The design and discovery of clickenzymes have also been emphasized in the present reviewarticle.

Click-chemistry in site-specific modification of enzymes

The introduction of small molecules such as bioorthogonal moi-eties is of special relevance for site-specific modification byclick reactions. The chemical modification of enzymes usingclick reactions has been specially focused on the introduction offluorescent label molecules in a different manner,46–49 or proteinpost-translational modification such as glycosylation or lipidationreactions.50–55

Fluorescent labeled proteins synthesis

The application of new chemical methods for labeling proteinshave garnered much attention in the fields of molecular imagingdue to their straightforward procedures, general applicability, andthe small size of chemical labels, at least relative to the geneti-cally-encoded protein-based labels, such as Green FluorescentProtein (GFP). The click reactions that can be combined withbiological techniques have been actively investigated.55

One protocol to create engineered fluorescent semisyntheticenzymes has been recently performed by genetically encodedincorporation of different clickable unnatural amino acids(UAAs).56 The introduction of p-azido- (1) and alkyne L-phenyl-alanine (2) generated different tyrosine synthetase (TS) ofMethanocaldococcus jannaschii variants. In a second approach,p-azido- 3 and alkyne L-phenylalanine 4 were successfully incor-porated on tyrosine synthetase of Methanosarcina mazei.

The four different variants of TS were selectively activatedwith the respective alkyne or azide single-molecule dyes by the

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copper-catalyzed alkyne–azide click (CuAAC) reaction. Theenzyme modified with 4 was the best variant for the incorpor-ation of an alkyne modified Alexa Fluor® 488 (green) by clickchemistry. The advantages of using this distribution of theorthogonal groups in this case are that the alkyne moiety is lessprone to reduction compared to the azide (particularly importantduring long-term expression in living cultures).

This new fluorescent enzyme was subsequently modified bythe introduction of a second fluorescent label Alexa Fluor® 594(red) attached to equivalent cysteines using standard maleimidechemistry to study the Foerster resonance energy transfer(FRET) interaction (Fig. 2).

The preparation of these kinds of semisynthetic enzymes byclick chemistry can be used as an efficient and general strategyto make proteins accessible to single-molecule studies, with afocus on the specific demands necessary for high-resolutionsingle-molecule FRET (smFRET), as a precise structural biologytool.

A different method for the introduction of fluorescent mo-lecules on enzymes is based on the site-specific lysine modifi-cation via a kinetic control.57 The activation of a unique Lysfrom 10 available on the enzyme surface of an RNase was poss-ible by kinetic control using biotin-TEO-ethynyl-N-hydroxysuc-cinimide. This strategy permitted the introduction of an alkyneon the enzyme surface for click reaction and a biotin moleculefor efficient purification of the modified enzyme using an avidin-resin (Fig. 3). Thus, a purified alkyne activated enzyme wasspecifically labeled using an azide–coumarin derivative 5 inexcellent yield. This method represents an interesting andefficient method for the creation of pure fluorescent semisyn-thetic enzymes.

A third strategy for site-specific enzyme labeling via Cu cata-lyzed click chemistry was described by using the biotin–strepta-vidin system.58 This technique permitted the determination ofin vivo activity based labeling of lipases with in situ detection oflipolytic activities by on slide click chemistry and imaging byfluorescence microscopy. Cytosolic as well as organelle residentlipases are specifically labeled in intact living cells.

First, the introduction of the alkyne was performed directly onthe lipase serine active site by treatment of the enzymes withalkyne-p-nitrophenyl phosphonate molecule (6), an irreversible

inhibition for serine hydrolyses. The labeled cells were clickedwith biotin–azide by Cu catalysis and subsequently stained witha streptavidin–Atto532 conjugate (Fig. 4). This strategy serves asa starting point for many commercially available signal amplifi-cation systems.

A more sophisticated example was the development of a fluor-escent labeling method of cell surface proteins. The Ting Grouphas developed an entire research program optimizing a two-stepenzymatic/chemical labeling scheme used to tag and image avariety of amino acid recognition sequence (LAP) fusion pro-teins in multiple mammalian cell lines with diverse fluorophoresincluding Cy3, coumarin, fluorescein, rhodamine, biotin, AlexaFluor 568, AttO 647N and AttO 655 (Fig. 5).59 The method isbased on the incorporation of azide moieties (7) onto an engi-neered LplA acceptor peptide (LAP) catalyzed by mutated ver-sions of Escherichia coli enzyme lipoic acid ligase (LplA). Forexample, the W37I mutant of LplA catalyzes site-specific

Fig. 2 Engineered fluorescent semisynthetic enzymes by geneticallyencoded incorporation of different clickable unnatural amino acids.

Fig. 3 Introduction of reporters by click chemistry on site-specificlysine modified-enzymes.

Fig. 4 Two step labeling of lipases with in situ detection of lipolyticactivities via Cu catalyzed click chemistry using a streptavidin system.


ligation of 10-azidodecanoic acid to LAP in cells, in nearlyquantitative yields after 30 min.

After that, a copper free-click reaction was performed by treat-ment of the introduced alkyl azide 8 with five different cyclo-octyne structures (Fig. 5a). From all of them, fluorophore conju-gates to aza-dibenzocyclooctyne (ADIBO) gave the highest andmost specific derivatization of azide-conjugated LAP in cells.The hydrophobicity of ADIBO was detrimental by targeting ofhydrophobic fluorophores such as AttO 647N obtaining a betterconjugation yield using the less hydrophobic monofluorinatedcyclooctyne (MOFO) (Fig. 5c). Therefore, this methodologyshould provide general access to biochemical and imagingstudies of cell surface proteins, using small fluorophores intro-duced via a short peptide tag.

An alternative to the previous examples is the combination ofboth click reactions: CuAAC and SPAAC for dual labeling ofbiomolecules.60,61 In this sense, an alkyne-protein was obtainedby the introduction of the maleimide linker 9 through the freesulfhydryl group of bovine serum albumin (BSA) (Fig. 6). Theincorporation of another kind of alkyne-pentynoic acid succini-mide ester 10 was subsequently introduced by modification ofamino groups in the protein (numerous alkyne sites) for thecopper-mediated click reaction.

A copper free click reaction was firstly performed by additionof azide 11 (strongly fluorescent) selectively producing themono-fluorescent labeled BSA. After purification, this mono-labeled BSA was treated with the second azide 12 (fluorescentonly after the click chemistry) in the presence of CuI (Fig. 7). Asexpected, the intensity of both emission bands increased as thesecond click reaction proceeded and indicated the presence ofFRET.

Another click-reaction with excellent characteristics forenzyme labeling has been the Diels–Alder cycloaddition.

Indeed, lipases, useful enzymes in organic synthesis, have beenspecifically modified for fluorescent labeling by this strategy.62

Candida antarctica (fraction B) (CAL-B) lipase was firstmodified by the introduction of a trans–trans hexadiene group.A 7-nitrobenzofurazan (NBD)-labeled maleimide 13 was selec-tively incorporated on the modified diene-enzyme by cyclo-addition reaction at pH 6 and 25 °C in almost quantitative yield(Fig. 8). This methodology was also applied for specific fluor-escent labeled Rab proteins for bioconjugation studies.32

The modifications discussed above were all at a single tag.Nature, in contrast, frequently installs multiple and distinctmodifications on proteins.

Therefore, an efficient system for enzyme modification will beone approach with tandem bioorthogonal ligations. The firstreport of a tandem bioorthogonal ligation in complex biologicalsamples involved a Staudinger–Bertozzi ligation and a Diels–Alder cycloaddition procedure, which utilizes mutually ortho-gonal reagents but suffers from the need to mask free thiolgroups prior to the ligation step to avoid nonspecific labeling.63

Fig. 6 Site-specific modification of enzymes by introducing two differ-ent alkyne groups for click reactions.

Fig. 7 CuAAC and SPAAC for dual labeling of biomolecules.

Fig. 5 Site-specific labeling of LAP fusion proteins with fluorophores.(a) General scheme of the click reaction; (b) protein labeling with AlexaFluor 568; (c) intracellular protein labeling using the indicated cyclo-octyne-fluorophore conjugate MOFO-ATTO 647N or MOFO-ATTO655.Reprinted with permission from ref. 59. Copyright 2012 AmericanChemical Society.


More recently, it was reported that a copper-free azide–cyclooctyne cycloaddition can be used concurrently with aninverse-electron-demand Diels–Alder reaction between tetrazineand trans-cyclooctene for the simultaneous labeling of twodifferent receptors on cell surfaces, provided that the properreagents are carefully selected so that cross reactivity isminimized.64

Nevertheless, the Overkleeft group has recently developed atriple ligation strategy employing the tetrazine ligation, Staudin-ger–Bertozzi ligation, and copper(I)-catalyzed Huisgen [2 + 3]cycloaddition for the selective and simultaneous labeling ofthree different enzymatic activities in a single experiment(Fig. 9).65 They have demonstrated that the reagents used for thetetrazine ligation are orthogonal to those used in Staudinger–Bertozzi ligation and copper(I)-catalyzed click reactions.Although tetrazines are not directly compatible with copper-cata-lyzed click chemistry, this problem can easily be overcome by asimple washing step between the two ligation reactions. Biotinor Bodipy azide, tetrazines or alkyne derivatives, biotin–

phosphine were used for these strategies to site-specific labeling.The triple ligation strategy can be extended for the study of otherbiological processes such as post-translational modifications,allowing the selective monitoring of multiple targets at the sametime. Moreover, the possibility to perform both tetrazine andStaudinger–Bertozzi ligation inside living cells66 should alsoenable tandem labeling procedures in vivo.

Semisynthetic tailor-made peptide modified proteins

The site-specific introduction of peptide sequences on proteinshas represented a tremendous advance in biological studies viathe creation of tailor-made semisynthetic enzymes with specificpost-translational modifications.67,68

The preparation of tailor-made GTPases (Ras, Rab), enzymeswith a critical role in tumour processes,69 has been one of themost successful examples. A practical chemical tailor-madepeptide synthesis on the solid phase has been developed duringrecent years by the Waldmann Group, focused on the preparationof complex lipidated GTPase peptides.32,33,70,71

Particularly, the use of the sulfonamide linker in peptides syn-thesis permitted the preparation of a small library of differenttailor-made peptides in high yields and with a simple purificationstep.33,71 Peptides synthesized by this strategy were applied forthe semisynthesis of Ras proteins by thiol–ene conjugation forprotein membrane location72 and transporting studies.69 Differentlong peptides and proteins were synthesized by the Diels–Alderreaction between azide, alkyne tailor-made peptides or modifiedprotein.73

Recently, the combination of expressed protein ligation (EPL)and click chemistry was developed on the preparation of semi-synthetic Rab GTPases (Fig. 10).74 The azide–Rab protein wasgenerated by the introduction of an azide–cysteine by EPL onthe protein. Different tailor-made alkyne containing peptides14–16 were synthesized, including the geranylgeranyl moiety.Therefore quantitative click chemistry was obtained yieldingfolded and functional geranylgeranylated Rab GTPases(Fig. 10). This ligation approach could be a fascinating way forhigh yield production of lipidated proteins for biologicalprocesses.

Semisynthetic glycosylated enzymes

The precise study of the biological function of glycoproteins iscomplicated by the fact that they are produced as mixtures ofdifferent glycoforms, protein isoforms that possess the same

Fig. 8 Preparation of NBD-labeled lipases by Diels–Aldercycloaddition.

Fig. 9 Schematic overview of the triple ligation strategy for simul-taneous labeling of multiple enzymatic activities involving tetrazine–norbornene cycloaddition, Staudinger–Bertozzi ligation, and copper(I)-catalyzed click reaction. The stars correspond to different fluorescentlabel groups.

Fig. 10 Synthesis of fluorescent labeled semisynthetic Rab protein.


protein backbone, but differ in both the nature of the glycan andthe glycosylation site. Therefore, the development of strategies tocreate tailor-made semisynthetic glycosylated proteins has beenpublished in the last few years for example by the Davis groupwho developed the use of alkyne–azide reaction or Staudingerligation to site-specific incorporation of sugars into differentproteins.75

The strategies were concentrated on the genetical incorpor-ation of the orthogonal group. For example, the introduction ofazidohomoalanine (Aha) can be incorporated into proteins as amethionine surrogate; reassigning the Met triplet codon to anazide tag. N-Acetylglucosamine (GlcNAc)-modified proteinswere prepared by this method introducing the alkyne–sugar 17and used to probe the substrate tolerance of endoglycosidase A(Fig. 11a).75b A second approach consisted of introducing thealkyne group on the protein, specifically the homopropargylgly-cine (Hpg) tag incorporated as a methionine surrogate (reassign-ing the Met codon to an alkyne tag) to synthesize 18F-labeledglycoproteins by the introduction of fluorinated sugars 18(Fig. 11b). This experiment was completed in just over 2 h,demonstrating that radiolabeled glycoproteins can be synthesizedin a time frame compatible with the short half-life of 18F.75c

Considering the rate accelerated by photoinduced click reac-tions, Wittrock et al. have recently used the photoinduced thiol–ene coupling to couple a glycopeptide antigen 19 on a proteincore such as BSA (which serves as a carrier for the antigens)(Fig. 11c).76

Using tailor-made thiolated glycopeptides it was possible tointroduce eight molecules of glycopeptides on the allyl-

functionalized BSA core. This precise control afforded by theclick reaction could be one of the most promising aspects inregards to creating future vaccines based on this approach.

Designing enzymes with “click-activity”

Enzymes have been honed over millions of years of evolution tohave unique specificities for catalyzing chemical transformationsin the crowded environment of a cell. Over the last few decades,chemists have begun to take advantage of the catalytic propertiesof enzymes to improve the speed and efficiency of organic reac-tions. Researchers have used both rational design and directedevolution methods to improve the catalytic activity, stability andselectivity of native enzymes.

However, not all organic reactions a chemist might want touse are found in nature. Indeed, the Diels–Alder cycloadditionreaction – a click reaction – one of the most famous reactions inorganic chemistry has been proposed as a key transformation inthe biosynthesis of many cyclohexene-containing secondarymetabolites.77,78 However, only four purified enzymes have thusfar been implicated in biotransformations that are consistent witha Diels–Alder reaction. These enzymes are namely solanapyronesynthase,79 LovB,80 macrophomate synthase,81 and riboflavinsynthase.82 Although the stereochemical outcomes of these reac-tions indicate that the product formation could be enzyme-guided in each case, these enzymes typically demonstrate morethan one catalytic activity, leaving their specific influence on thecycloaddition step uncertain.

Therefore the development of strategies to design “clickenzymes” represents a challenge in chemistry and biology,promising a greener and more productive future.

Until now, several strategies have gained the possibility toobtain a “Diels–Alderase” enzyme such as chemical biosyntheticstudies,83,84 discovery of Diels–Alder activity on different pro-teins,85,86 computational design87,88 or the creation of hybrid cat-alysts combining enzymes and organometallic complexes.89–94

Discovered click-enzymes

The explosive development of whole-genome sequencing andthe isolation of entire gene clusters encoding the proteins thatcarry out biosynthetic pathways make such speculation about thefact that a Diels–Alder reaction takes place in the course offorming a final product irresistible.

The Liu group has established in the biosynthesis of spinosynA that SpnF, an apparent S-adenosyl-l-methionine (SAM)-depen-dent methyltransferase according to primary sequence compari-sons, in fact accelerates the rate of spontaneous Diels–Alderconversion of pentene 20 (Fig. 12) to the tricyclic product 23 bya factor of approximately 500.83,84 Both the ene and diene com-ponents of this reaction are similarly polarized by vinylogous(conjugated) carbonyl groups, as indicated by the dipole arrowsin Fig. 12. Two different transition states (21, 22) have been pro-posed for the adduct formation. The rate of the Diels–Alder reac-tion is acutely sensitive to energy perturbations of the interactingmolecular orbitals. However, it was not possible to account forthe 500-fold catalytic effect and computational analyses areawaited, that can evaluate these alternatives and possibly others,

Fig. 11 Preparation of glycoproteins by click reactions. (a) N-Acetyl-glucosamine incorporation by Cu catalyzed click chemistry; (b) 18F flu-orescent labeled incorporation on proteins by Cu catalyzed clickchemistry; (c) glycopeptide incorporation by thiol–ene reaction.


and these will be further refined by an X-ray structure, ideallywith a substrate or an unreactive but accurate analogue bound.

Non-enzymatic proteins with cycloaddition activity

Another approach to obtain click enzymes has been based on thediscovery of proteins with Diels–Alder catalytic capacity.85,86

Currently, there are not many proteins that express this type ofactivity. Recently, hen egg white lysozyme was able to catalyze athree-component aza-Diels–Alder reaction between the aromaticaldehyde, aromatic amine, and 2-cyclohexen-1-one (Fig. 13).85

The enzyme catalyzed the reaction with a wide range of sub-strates and reaction conditions resulting in moderate to goodyields (up to 98%) and good stereoselectivity towards the endoadduct (endo/exo 90/10). The key step is the formation of theenol which is catalyzed by lysozyme. In this step, Glu 35 proto-nates the carbonyl functional group of cyclohexenone, whileAsp 52 acts as a nucleophile to attack the acidic proton. Next,the enol cyclize with the in situ generated imine via a Mannich–Michael process to give the final products with the aid of theprotein. This result opens the possibility of application of thisprotein for other interesting organic reactions.

A second protein with a Diels–Alderase activity is the mucin,a heavily glycosylated protein.86 The Gozin group demonstratedthat ovine submaxillary mucin and porcine gastric mucin acceler-ate the rate of carbon–carbon bond-forming Diels–Alder reactionbetween N-propylmaleimide and anthracene up to 200 times therates of the reference processes, which were performed in waterand chloroform, respectively.

Diels–Alderase computational design

The power of computer modelling for the de novo design of astereospecific artificial Diels–Alderase has been recently demon-strated by the Baker group.87,88

Computational algorithms were used to generate stable proteinscaffolds for backbone geometries that allowed the substrates tointeract productively. From a set of 1019 possible active siteconfigurations, approximately 106 were matched to stableprotein scaffolds. Further constraints led to the identification of84 enzyme candidates and after production, expression andpurification, the Diels–Alderase activity was evaluated. Twopotential candidates were selected although with moderate

Diels–Alderase activity (Fig. 14a). The application of site-directed mutagenesis permitted one to yield artificial enzymescapable of stereoselectively catalyzing a Diels–Alder reactionwith acceptable values.

However, the resulting activity is still low, with rate enhance-ments over the non-catalysed reaction reaching up to 105 in onlythe most successful examples. In this way, the Baker group hasbeen developing the Foldit program, a specific game for back-bone remodelling of the structure of a computationally designedbimolecular Diels–Alderase, which has been recently applied toenhance the activity.88 Several iterations of design and character-ization generated a 24-residue helix–turn–helix motif, includinga 13-residue insertion, that increased enzyme activity >18-fold.X-ray crystallography showed that the large insertion adopts ahelix–turn–helix structure positioned as in the Foldit model(Fig. 14b).

Artificial metalloenzymes

Artificial metalloenzymes have emerged as a promising approachto merge the attractive properties of homogeneous catalysis and

Fig. 14 De novo design of an enantioselective artificial Diels–Alder-ase. (a) Scheme of adduct formation. (b) A. Crystal structure of startingdesign Diels–Alderase (yellow). The modeled transition state of theDiels–Alder reaction is depicted in cyan. B. Structure of starting Diels–Alderase overlaid on CE6 player predicted structure (purple). C. Crystalstructure of CE6 (light blue) overlaid with player predicted structure ofCE6 (purple). D. The player-designed side chains in CE6 that form theinterface between helix 1 (purple) and the transition state model (cyan)are found in the same rotameric conformation as in the crystal structureof CE6 (light blue). Reprinted with permission from Macmillan Publish-ers Ltd: Nature Biotechnology ref. 88, copyright 2012.

Fig. 12 Proposed mechanism of the Diels–Alder reaction by Spnf inthe biosynthesis of spinosyn A.

Fig. 13 Lysozyme catalyzed Diels–Alder reaction.


biocatalysis. The activity and selectivity, including enantioselec-tivity, of natural metalloenzymes are due to the second coordi-nation sphere interactions provided by the protein. Artificialmetalloenzymes aim at harnessing second coordination sphereinteractions to create transition metal complexes that displayenzyme-like activities and selectivities.

These are hybrid catalysts in which a catalytically active tran-sition metal complex is incorporated into a host biomacromole-cule, typically a protein95 or DNA.96,97

The motivations for the creation of artificial metalloenzymesare both practical and fundamental. It is predicted that artificialmetalloenzymes can combine the best of both worlds, that is,broad catalytic scope, a hallmark of homogeneous catalysis, andhigh activity and selectivity under mild conditions, which typi-cally characterize enzymatic catalysis. During the last threedecades different types of synthetic metalloenzymes have beenprepared, including those based on anchoring appropriateligands such as diphosphines, phthalocyanines, or dipyridyl moi-eties covalently or noncovalently onto proteins.98

However, in the last few years three different systems havebeen mainly run for the creation of active and selective hybridmetalloenzymes. One of them has been developed by the Wardgroup, which, inspired by a visionary report by Wilsonand Whitesides in 1978, uses the potential of biotin–avidin tech-nology in creating artificial metalloenzymes. Owing to theremarkable affinity of biotin for either avidin or streptavidin,covalent linking of a biotin anchor to a catalyst precursor ensuresthat, upon stoichiometric addition of (strept)avidin, the metalmoiety is quantitatively incorporated within the hostprotein.84,90,95,99

A second approach for the creation of biohybrids, based onthe use of DNA as a template, has been introduced by theFeringa and Roelfes group.97 An active site is created in or nearthe DNA groove by binding of a transition metal complex toDNA. The second chiral coordination sphere provided by theDNA directs the reaction towards one of the enantiomers of theproduct, resulting in an enantiomeric excess.

A third approach is focused on the application of directedevolution of hybrid catalysts.100 The Reetz group introduced thisconcept with the idea of tuning the selectivity and rate of syn-thetic transition metal complexes which are anchored to a givenprotein host. As a conventional directed evolution, libraries ofmutant proteins are generated. A protein is first chosen as a hostfor attaching a synthetic transition metal catalyst, preferably inan appropriate cavity which can subsequently function as a typeof binding pocket. The protein should be characterized bythermal and oxidative robustness, and it should be accessible inabundant amounts by an efficient expression system.

Thus, recently two stereoselective Cu-hybrid enzymes bydifferent approaches have been described for Diels–Alder cyclo-addition catalysis.91,92,97b One strategy used a DNA-based cata-lysis to produce both enantiomers of the adduct product 26 fromdienophile 24 and diene 25 (Fig. 15).97b Different organometal-lic catalysts using different ligands for the Cu(II) ion (Cu–L1–L10) were used. The enantiomeric preference in the Diels–Alderreaction was related to the denticity of the ligand and the result-ing structure of the substrate bound copper complex has beenshown to be competitive with conventional homogeneous cata-lysts in several reactions, further optimization of the Cu-terpy

type catalysts, i.e. to enhance their catalytic activity, is requiredfor synthetic applications.

In a different way, directed evolved hybrid catalysts were suc-cessfully synthesized to catalyze the Diels–Alder reaction byintroducing a Cu2+ binding site on coordinating amino acids atgeometrically appropriate positions in a thermostable synthasefrom Thermotoga maritime as host protein.91 The metal wasspecifically coordinated to the designed Asp/His/His binding siteshowing notable enantioselectivity, enhanced endo-selectivity,and somewhat higher reaction rate of the model Diels–Aldercycloaddition between azachalcone 27 and cyclopentadiene 28for formation of cycloadduct 29 (Fig. 16). These two examplesillustrate the potential of both strategies to get highly selective“click” enzymes.

Conclusions

This perspective focuses on the most recent advances in thedesign and creation of new proteins. The development ofspecific and bioorthogonal schemes as click reactions constitutesideal methodologies for selective modification of proteins. Thisrepresents an alternative to other technologies in the preparationof tailor-made fluorescent proteins, or semisynthetic enzymeswith post-translational changes included for the study of aspecific protein function in different biological processes(cancer, Alzheimer, etc.).

Fig. 16 Engineered Cu-metalloenzymes catalyzing Diels–Alderreaction.

Fig. 15 DNA-based catalytic asymmetric reactions and Cu(II) com-plexes of bi- and terpyridine ligands.


The progress of these click strategies, however, is one of thefuture lines in protein chemistry, for example in the design ofenzymes with improved catalytic efficiency or a broad substrateactivity scope.

The design and discovery of enzymes with unknown syntheticcatalytic ability is still more interesting. This technology rep-resents a new way and one of the main priorities for the chem-istry and biology in the new era, promising a greener and moreproductive future. In this article, the advances in cycloadditionreactions catalyzed by enzymes have been emphasized, speciallybased on a computational approach (acquiring a “Hit” with syn-thetic activity from a huge amount of protein) or hybrid systems(combination of enzymes and organometallic complexes).

Abbreviations

Alexa Fluor® 488 4-carboxy-2-(3,6-diamino-4,5-disulfonatox-anthenium-9-yl)benzoate

Alexa Fluor® 594 [6-(2-carboxy-5-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}phenyl)-1,2,2,10,10,11-hexamethyl-8-(sulfomethyl)-10,11-dihydro-2H-pyrano[3,2-g:5,6-g′]diquinolin-1-ium-4-yl]methanesulfonate

Alexa Fluor 568 6-(2-carboxy-5-{[(2,5-dioxopyrrolidin-1-yl)-oxy]carbonyl}phenyl)-2,2,10,10-tetra-methyl-8-(sulfomethyl)-10,11-dihydro-2H-pyrano[3,2-g:5,6-g′]diquinolin-1-ium-4-yl]-methanesulfonate

Cy3 2-[(E,3Z)-3-(1,2-[(E,3Z)-3-(1,3-dihydroin-dol-2ylidene)prop-1-enyl]-3H-indol-1-ium,dihydroindol-2-ylidene)prop-1-enyl]-3H-indol-1-ium

Bodipy 4,4-difluoro-4-bora-3a,4a-diaza-s-indaceneDansyl 5-(dimethylamino)naphthalene-1-sulfonyl

Acknowledgements

This work has been sponsored by the Spanish National ResearchCouncil (CSIC). I would like to express my sincere gratitude toTanya Shew for the English proofreading of this manuscript.

Notes and references

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