Click reactions in protein chemistry - Instituto de Catálisis y ...

Organic &BiomolecularChemistrywww.rsc.org/obc Volume 10 | Number 47 | 21 December 2012 | Pages 9297–9508ISSN 1477-0520PERSPECTIVEJose M. PalomoClick reactions in protein chemistry: from the preparation of semisyntheticenzymes to new click enzymes

Organic &BiomolecularChemistryCite this: Org. Biomol. Chem., 2012, 10, 9309www.rsc.org/obcDynamic Article LinksPERSPECTIVEClick reactions in protein chemistry: from the preparation of semisyntheticenzymes to new click enzymesJose M. Palomo*Received 19th July 2012, Accepted 11th September 2012DOI: 10.1039/c2ob26409aClick-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.IntroductionThe 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 accordinglyDepartamento de Biocatálisis. Instituto de Catálisis (CSIC). C/ MarieCurie 2. Cantoblanco. Campus UAM, 28049 Madrid, Spain.E-mail: josempalomo@icp.csic.es; Fax: +34-91 585 47 60under 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 criteriainclude certain cycloaddition reactions, such as the Huisgen1,3-dipolar azide–alkyne 2 and the Diels–Alder cycloaddition 3and other methods such as the Staudinger ligation 4–6 or thethiol–ene reaction 7,8 (Fig. 1).This click concept has been rapidly applied to biomoleculesmodification, surface functionalization, polymer science, studiesJose 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 postdoctoralresearch he joined theChemical Biology Departmentof Max Planck Institute in Dortmundin 2004. In 2006, hebegan his appointment as anAssociate Research Scientist atJose M. Palomo the Biocatalysis Department inICP-CSIC. Since 2009, he has been a Tenured Scientist (AssociateProfessor) in ICP-CSIC. His current research interestsinclude design of semisynthetic enzymes, carbohydrate chemistrywith enzymes, asymmetric biotransformations and immobilizationof biomolecules. Fig. 1 General scheme of click reactions.This journal is © The Royal Society of Chemistry 2012 Org. Biomol. Chem., 2012, 10, 9309–9318 | 9309

of biological processes, etc. 9–20 and so relies on easy andefficient coupling technologies.The copper(I)-catalyzed 1,3-dipolar azide–alkyne cycloaddition(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 cycloaddition 2 where the chemicalcoupling needed to use high temperature to form the triazolemolecule. Therefore, the discovery of the Cu(I) catalysis permittedthe 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. 21Despite 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 bacterial 22,23 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 cyclooctynesas an effective way for lowering the activation barrier ofAAC and an alternative to the use of metal catalysts (Fig. 1b). 26This strain-promoted AAC variant, SPAAC, has been exploitedas a bioorthogonal strategy for the fluorogenic labeling of proteinsand is currently used in many different chemicalapplications. 27Interestingly, 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 10 4 when compared tothat in organic solvents. 28These advantages have permitted the employment of thisbioorthogonal methodology in peptide and protein chemistry andsurface functionalization. 29–33The azide-Staudinger ligation 4 represents another excellentexample of click reaction. Hermann Staudinger 5 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 bioconjugationand transformed it into the so-called Staudinger ligation.6 The bio-orthogonal character of both the azide and thephosphine functions has resulted in the Staudinger ligationfinding numerous applications in cell surface engineering, synthesisof peptides and proteins, probing post-translational modifications,labelling of glass surfaces or fluorogenic molecules. 34Starting with an ortho-phosphine terephthalic acid derivative,aza-ylide is formed through a reaction with azide and undergoescyclization to give an oxazaphosphetane as an initialintermediate. In aqueous media, spontaneous hydrolysis yieldsthe desired amide, into which the phosphine oxide by-product isincorporated (Fig. 1d). 6,35,36From 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 oxidationlimiting their use in biological systems. 37Highly 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 reactionto functionalize biological materials and biological molecules,and to incorporate biological molecules into othersynthetic materials and molecules. 39–45Herein, 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 enzymesThe introduction of small molecules such as bioorthogonal moietiesis 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. 50–55Fluorescent labeled proteins synthesisThe 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 genetically-encodedprotein-based labels, such as Green FluorescentProtein (GFP). The click reactions that can be combined withbiological techniques have been actively investigated. 55One 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-phenylalanine(2) generated different tyrosine synthetase (TS) ofMethanocaldococcus jannaschii variants. In a second approach,p-azido- 3 and alkyne L-phenylalanine 4 were successfully incorporatedon tyrosine synthetase of Methanosarcina mazei.The four different variants of TS were selectively activatedwith the respective alkyne or azide single-molecule dyes by the9310 | Org. Biomol. Chem., 2012, 10, 9309–9318 This journal is © The Royal Society of Chemistry 2012

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.copper-catalyzed alkyne–azide click (CuAAC) reaction. Theenzyme modified with 4 was the best variant for the incorporationof 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 moleculeson enzymes is based on the site-specific lysinemodificationvia a kinetic control. 57 The activation of a unique Lysfrom 10 available on the enzyme surface of an RNase was possibleby kinetic control using biotin-TEO-ethynyl-N-hydroxysuccinimide.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 avidinresin(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 semisyntheticenzymes.A third strategy for site-specific enzyme labeling via Cu catalyzedclick chemistry was described by using the biotin–streptavidinsystem. 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 irreversibleFig. 4 Two step labeling of lipases with in situ detection of lipolyticactivities via Cu catalyzed click chemistry using a streptavidin system.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 amplificationsystems.A more sophisticated example was the development of a fluorescentlabeling 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 proteinsin 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 engineeredLplA acceptor peptide (LAP) catalyzed by mutated versionsof Escherichia coli enzyme lipoic acid ligase (LplA). Forexample, the W37I mutant of LplA catalyzes site-specificThis journal is © The Royal Society of Chemistry 2012 Org. Biomol. Chem., 2012, 10, 9309–9318 | 9311

iocatalysis. The activity and selectivity, including enantioselectivity,of natural metalloenzymes are due to the second coordinationsphere 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 transitionmetal complex is incorporated into a host biomacromolecule,typically a protein 95 or DNA. 96,97The 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 typicallycharacterize 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 moietiescovalently or noncovalently onto proteins. 98However, 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 technologyin 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. 84,90,95,99A 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 synthetictransition 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 cycloadditioncatalysis. 91,92,97b One strategy used a DNA-based catalysisto produce both enantiomers of the adduct product 26 fromdienophile 24 and diene 25 (Fig. 15). 97b Different organometalliccatalysts 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 resultingstructure of the substrate bound copper complex has beenshown to be competitive with conventional homogeneous catalystsin several reactions, further optimization of the Cu-terpyFig. 15 DNA-based catalytic asymmetric reactions and Cu(II) complexesof bi- and terpyridine ligands.Fig. 16 Engineered Cu-metalloenzymes catalyzing Diels–Alderreaction.type catalysts, i.e. to enhance their catalytic activity, is requiredfor synthetic applications.In a different way, directed evolved hybrid catalysts were successfullysynthesized to catalyze the Diels–Alder reaction byintroducing a Cu 2+ 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.ConclusionsThis 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.).9316 | Org. Biomol. Chem., 2012, 10, 9309–9318 This journal is © The Royal Society of Chemistry 2012

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 representsa new way and one of the main priorities for the chemistryand 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 syntheticactivity from a huge amount of protein) or hybrid systems(combination of enzymes and organometallic complexes).AbbreviationsAlexa Fluor® 488Alexa Fluor® 594Alexa Fluor 568Cy3BodipyDansylAcknowledgements4-carboxy-2-(3,6-diamino-4,5-disulfonatoxanthenium-9-yl)benzoate[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]methanesulfonate6-(2-carboxy-5-{[(2,5-dioxopyrrolidin-1-yl)-oxy]carbonyl}phenyl)-2,2,10,10-tetramethyl-8-(sulfomethyl)-10,11-dihydro-2Hpyrano[3,2-g:5,6-g′]diquinolin-1-ium-4-yl]-methanesulfonate2-[(E,3Z)-3-(1,2-[(E,3Z)-3-(1,3-dihydroindol-2ylidene)prop-1-enyl]-3H-indol-1-ium,dihydroindol-2-ylidene)prop-1-enyl]-3Hindol-1-ium4,4-difluoro-4-bora-3a,4a-diaza-s-indacene5-(dimethylamino)naphthalene-1-sulfonylThis work has been sponsored by the Spanish National ResearchCouncil (CSIC). 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