1、BIOORTHOGONAL CHEMISTRY:A REVIEWOF ITS DIVERSEAPPLICATIONSIN SCIENCEAND MEDICINE IntroductionBioorthogonal chemistry has made a remarkable scientific impact in recent years and has become an important tool to help answer questions on topics including cancer biology,biomarkers,neuroscience,plant phys

2、iology,parasitology,and virology.Bioorthogonal chemistry refers to a set of fast reactions that can take place in biologic environments with minimal interference to biomolecules or native biochemical processes.16 The following criteria must be met for a reaction to be considered bioorthogonal:The re

3、action must occur at the temperatures and pH of physiological environments.The reaction must provide products selectively and in high yields and must not be affected by water or endogenous nucleophiles,electrophiles,reductants,or oxidants found in complex biological environments.The reaction must be

4、 fast,even at low concentrations,and must form stable reaction products.The reaction should involve functional groups not naturally present in biological systems.This white paper will introduce different types of bioorthogonal reactions,their applications,and trends found by the CAS Content Collecti

5、onTM to provide an overview of its role in the publication landscape.Bioorthogonal chemistry allows for a deeper understanding of the structure and function of biologic systems.We will discuss how methods for drug development and delivery may be further optimized and provide an outlook on the future

6、 of bioorthogonal chemistry.The key types of bioorthogonal reactionsAn overview of the key types of bioorthogonal reactions Staudinger ligation,copper-catalyzed azide-alkyne cycloaddition(CuAAC),copper-free azide-alkyne cycloaddition/strain-promoted azide-alkyne cycloaddition(SPAAC),and tetrazine li

7、gation can be found in Table 1.Bioorthogonal reactionsTarget/reactionAdvantagesDisadvantagesStaudinger reactionAzide-phosphine reaction Azides and phosphines are biocompatible,stable amide linkages producedSlow reactions,phosphines prone to oxidation Staudinger ligation6 Traceless Staudinger ligatio

8、n710 Copper-catalyzed azide-alkyne cycloaddition (CuAAC)1,5 Azide-alkyne cycloaddition with copper catalysts Fast reactions,well-established chemistry,good regioselectivity Despite efforts to stabilize copper catalysts,copper toxicity remains a concernN3NH2N-N2H2OPPh3O PPh3PPh3R1R2R1R2R1R2N PS-N2H2O

9、N3OPh2PSNHPhPhPh2PSHOOOR1R2R1R1R2R2notN3NNNNCCCHCHCu(I)HC C NNR1R2R2R2R1O PNHR1N PN3-N2MeOMeO-MeOHH2OPhPhPhPhPh2POOOTable 1:Overview of key bioorthogonal reactionsBIOORTHOGONAL CHEMISTRY|3Bioorthogonal reactionsTarget/reactionAdvantagesDisadvantages Strain-promoted azide-alkyne cycloaddition (SPAAC)

10、2,11Azide-alkyne cycloaddition without copper catalystsNo use of copper catalystsReactions slower than CuAAC,bulky cyclooctynes difficult to incorporate into biomolecules Modifications for improving reaction kinetics of SPAAC with modified cyclooctynes12 Tetrazine ligation3,4 Strained cyclic alkenes

11、 are most commonly used dienophiles with trans-cyclooctenes13,14Dioxolane-fused cyclooctenes are more stable and less prone to isomerization with dioxolane-fused trans-cyclooctenes15 Tetrazine-dienophile inverse electron demand Diels-Alder reaction (IEDDA)Very fast reactions,kinetics/stability tunab

12、le by altering structures of either tetrazines or dienophile Lower stability in aqueous environmentsR1R2R1R2N3CCNNNCCR1R2R3HHNNNN2R1R3-N2R1R3R2HNNH2R12R3HNNN HNH-N2R3HHNNNNR1R2R3NN HNH-N2R3HHNNNNROHHHHHHHHORROOOHHHHHHHHOROOOHHHHHR1R2BNNNNOHOHOHR1R2NNNNOBOHOHOHHNOHR1R2R1R1R2R2BNNNNNNOBNNNOHOHOHOHOHHN

13、OHR1R2NOHHNOHHONOHHDIBODIBACBCNCO2MeCH2OHNOHHDIBODIBACBCNCO2MeHHDIBODIBACBCNCO2MeCH2OHVinylboronic acids react rapidly with otherwise stable tetrazinylphenols,allowing rapid in vivo tetrazine ligations with stable precursors with vinylboronic acids16,17Table 1:Overview of key bioorthogonal reactions

14、Biomolecule class Examples of incorporation/labeling methodsProteinsNon-canonical amino acids Residue-specific18 Site-specific1921 Protein/peptide tagging Enzyme-based labeling2224 Self-labeling,e.g.,SNAP-tag,HaloTag25,26GlycansMetabolic oligosaccharide engineering27LipidsClickable azide or alkyne r

15、eactive groups28Modified substrate analogs29,30Nucleic acidsAzide-modified nucleoside triphosphates31,325-vinyl-2-deoxyuridine33Alkenyl deoxynucleoside and alkynylnucleosides29 Antisense oligonucleotides34Pretargeting moietiesAntibody labeling,e.g.,tetrazine based cycloaddition4Positron emission tom

16、ography(PET)imaging35Trans cyclooctene construct36How are bioorthogonal handles incorporated into biologic systems?Precursors bearing a bioorthogonal handle that can be integrated into molecules such as proteins,glycans(carbohydrate-based polymers;Figure 1),lipids,nucleic acids,and antibodies are fe

17、d into cells or organisms.Some of the more commonly used methods are summarized below in Table 2.Table 2:An overview of bioorthogonal incorporation methods by types of biomoleculesBIOORTHOGONAL CHEMISTRY|5Figure 1.Glycans attached to the extracellular domain of a cell surface receptorSignalingAsnAsn

18、Sialylatedbiantennarycomplex-typeN-glycanSialylatedtetraantennarycomplex-typeN-glycanLewisY antigen446332622224444444466633443GlycanGlcNAcGalNAcMannoseFucoseGalactoseSialic acidPublication trends in bioorthogonal chemistry:insights from the CAS Content CollectionCAS is a leader in scientific informa

19、tion solutions,curating,connecting,and analyzing the valuable data disclosed in scientific literature globally to help accelerate breakthroughs.Our team of scientists and AI experts are at the heart of our collection,covering over 150 years of discoveries to build the highest quality and most up-to-

20、date collection of scientific information in the world.The CAS Content Collection was analyzed to determine the number of publications discussing proteins,DNA,RNA,lipids,carbohydrates,glycans,and others(Figure 2).Figure 2.Bioorthogonal chemistry publication volume from 2010 to 2020.*Inset graphic de

21、picts the total volume of bioorthogonal chemistry publications for comparison*2010 was selected as an initial reference point because it was the first year in which the number of documents containing“bioorthogonal chemistry”increased significantly relative to the previous year.Approximately 90%of th

22、e total number of documents containing the term“bioorthogonal”or“bio-orthogonal”have been published since 2010.The total volume of bioorthogonal chemistry publications is shown in the inset graph for comparison.While as expected,the publication volume for each type of biomolecules shows steady growt

23、h in the years 20102020,publications related to protein modification account for the largest portion of documents.The relative volume increased in each class at about the same rates over the decade(data not shown).Taken together,this is indicative of the growth of bioorthogonal chemistry research si

24、nce 2010.3504003002001002010 2012 2014 2016 2018 2020030025020001620182020Number of PublicationsYearProteinsDNARNALipidsCarbohydratesGlycansOthersBIOORTHOGONAL CHEMISTRY|7The numbers of documents discussing specific uses of the method(and the total number of documents published

25、 in the CAS Content Collection discussing bioorthogonal chemistry,inset graph)were obtained,as shown in Figure 3 above.Imaging was the single biggest use of bioorthogonal chemistry between 2010 and 2020,followed by pharmaceutical applications.The use of bioorthogonal chemistry for labeling was repor

26、ted in nearly as many documents as its use in pharmaceuticals,though labeling is likely to represent a variety of uses not otherwise characterized.Similar numbers of documents used bioorthogonal chemistry in hydrogels or diagnostic agents,for mechanistic or mass spectrometric studies,Figure 3.Docume

27、nts related to bioorthogonal chemistry and specific uses in the CAS Content Collection between 2010 and 2020.Inset graphic depicts the yearly publications in bioorthogonal chemistry over the same periodpull-down experiments,or in attaching molecules to the surfaces of cells.The relative rapid increa

28、se in pharmaceutical work using bioorthogonal chemistry may be a consequence of its use in drug delivery methods,but more significant increases are likely to come only if bioorthogonal chemistry drug delivery methods are found to be effective in clinical trials.Over the period studied,documents in t

29、he CAS Content Collection increased by roughly 50%,while documents in the primary literature citing bioorthogonal chemistry increased approximately fourfold,consistent with a developing interest in bioorthogonal chemistry and with the availability of further opportunities for the use of bioorthogona

30、l chemistry.25060040020000020001620182020Number of PublicationsImagingPharmaceuticalsDiagnosticsLabelingMechanistic studiesCell surface atachmentHydrogelPulldown assaysMass spectrometryOthersBioorthogonal chemistry in the real world:an appraisal of scienti

31、fic and medical applicabilityThe main advantage of using bioorthogonal reactions is the size of the tag,which is smaller than tags such as green fluorescent protein(GFP),and thus less likely to interfere with cellular pathways and mechanisms.Reactants in bioorthogonal reactions are cell-permeable an

32、d can be used in live organisms,which provides the advantage of studying processes in a native environment.Many of these methods are also more convenient than traditional methods.Here,we will discuss the applications of bioorthogonal chemistry:imaging,enzyme tracking and identification,and drug deli

33、very.Protein imagingBioorthogonal non-canonical amino acid tagging(BONCAT)and fluorescent non-canonical amino acid tagging(FUNCAT)techniques use non-canonical amino acids(ncAAs),which are pulse-fed to cultured cells or organisms.During BONCAT,proteins labeled with ncAAs are tagged using affinity tag

34、s to enable new protein purification,while FUNCAT utilizes fluorescent tags to enable visualization of newly synthesized proteins (Figure 4).BONCAT and FUNCAT have been used to study proteomics in a wide variety of microbial,plant,and animal models and have the advantages of being used in living mod

35、els to track protein spatial activities and mechanisms without interfering with other biological processes.27,37 Some examples of applications are:Gut microbiome:BONCAT was used to analyze the microbial ecology of translationally active intestinal bacteria.38 Genetic disorders:The BONCAT method enab

36、led identification of eleven potential biomarkers that may play a role in Fragile X syndrome(FXS)pathophysiology.39 Stem cell therapy:Mutant mesenchymal stem cells(MSCs)were analyzed via FUNCAT.40 Neuroscience:FUNCAT proximity ligation assay in rat hippocampal neurons aided in elucidating mechanisms

37、 around how soluble amyloid precursor protein (sAPP)differentially regulates the synthesis and cell surface expression of AMPA receptors in hippocampal neurons.41 BIOORTHOGONAL CHEMISTRY|9Figure 4:Overview of the BONCAT and FUNCAT workflows(adapted from Hinz et al.2013)37R1R1R1R2During nascent prote

38、insynthesis,bioorthogonal handle-conjugated ncAA was incorporated into the proteins.Purification,identificationor quantification Fluorescent imagingAffinity tagFluorescent tagBONCATFUNCATproteinproteinproteinproteinproteinproteinproteinproteinnewproteinnewproteinnewproteinnewproteinnewproteinnewprot

39、einR2Nucleic acid imagingRNA expression MicroRNA-21(miR-21)expression is associated with a variety of human cancers,notably breast cancer.Bioorthogonally-labeled oligonucleotide probes were hybridized to miR-21 whereby a fluorogenic response was observed in live cells containing miR-21.The method ha

40、s also been optimized further with the use of fluorophores attached to RNA probes to track mRNA expression in live Chinese Hamster Ovary(CHO)cells.This method has the advantage of lower background signal,deeper tissue penetration,and less tissue damage than previous methods.The visualization of newl

41、y synthesized RNA in cells has posed a challenge in the past,however a bioorthogonal labeling-primed DNA amplification strategy has proven successful in observing how new RNA is organized in cells,which may help to shed further light on pathologic processes.42,43Glycan imaging Bioorthogonal chemistr

42、y has proven to be an important tool for the understanding of the structures,localization and biological functions of glycans.Glycans are oligosaccharides attached to peptides,proteins,and lipids,as well as on cell walls allows their use in visualizing cell types selectively(Figure 1).44 Glycan meta

43、bolic precursors include many bioorthogonal functionalities,including azides,terminal alkynes,and strained alkynes,among others.6,45,46 The glycans can be visualized using the appropriate bioorthogonal partner,e.g.,azides are visualized with phosphine-containing esters or thioesters by Staudinger or

44、 traceless Staudinger ligations,and terminal alkynes,or strained alkynes are visualized using CuAAC or SPAAC,respectively.47 Importantly,some methods of glycan imaging can also be used in living animals.The applications of these labelling methods include:Host-pathogen interactions:Labeling of commen

45、sal bacteria,which were then injected and monitored in vivo to study intestinal pathologic processes.48 Lysosome biology:Lysosomal labeling through incorporation of the azide-labeled sialic acid and treatment of cells with dibenzocyclooctyne (DBCO)-bound dyes to study processes pertaining to cell de

46、ath.49 Tumor biology:Azide-containing galactosamine or mannosamine metabolic precursors were contained within a metal-organic framework(MOF),ZIF-6,which used methylimidazoles as structural components.The MOFs were then encapsulated in membrane fragments from tumor cells,allowing in vivo labeling of

47、tumor cells,imaging of multiple tumors of different cell types,differential imaging of breast cancer cell types,and selective imaging of cells.50 BIOORTHOGONAL CHEMISTRY|11Lipid imagingLipid imaging has been used to study cellular homeostasis,wherein cellular phospholipase D activity was visualized

48、in HeLa cells by tagging them with an azidotetramethylrhodamine conjugate via CuAAC,which is an easier method compared to lipid extraction.28 A trans-cyclooctene-containing ceramide lipid and a highly reactive tetrazine-tagged near-IR dye were used to visualize the Golgi apparatus in HeLa cells via

49、3D confocal and stimulated emission depletion(STED)microscopy.Furthermore,to track complex biosynthesis and trafficking of phosphatidylinositol(PI),two azidoalkyl-myo-inositol probes were employed to label PI lipids in Candida albicans and human T-24 bladder cancer cells.5153Plant physiology can als

50、o be studied through the use of functional C19 alkyne cholesterol and oxysterol analogs were fed to NIH-3T3 cells,and the cells labeled by CuAAC of the alkynylsteroids with a fluorescein azide.This method allowed sterol function to be examined using high-resolution microscopic imaging.54Enzyme ident

51、ification and trackingBioorthogonal chemistry is useful for understanding biologic systems through activity-based protein profiling(ABPP).ABPP is used for characterization of a set of proteins or a single protein with a specific enzyme activity.55 It enables us to look for enzymes in a living model

52、that are not yet characterized without killing the cell.The advantage of using bioorthogonal functionality is that the tags are smaller and will interfere less with the native functionality of enzymes.Drug deliveryBioorthogonal chemistry has been studied as a method to control the release,localizati

53、on,and formation of drugs in vivo,which is a crucial step in optimizing drug efficacy and safety.Several approaches can be used to appropriately deliver drugs to their targets:these include click to release(CTR)and glycan labeling.In situ synthesis of pharmaceutical agents:Bioorthogonal chemistry ma

54、y be useful in assembling drugs from smaller precursors.By creating drugs as and when they are needed,drugs could be more effective and less toxic;the scope of pharmacologic intervention could also be expanded.These methods have been explored for over 30 years and range from the use of decanal and N

55、-amino decylguanidine to form a hydrazone which caused breakdown of biologic membranes resulting in cell death56 to generation of proteolysis-targeting chimeras(PROTACs)in cells.This is caused by cycloaddition of a thalidomide-substituted tetrazine with trans-cyclooctene-substituted inhibitors of BR

56、D4 and ERK1/2,leading to complete degradation of BRD4 and ERK4 in human cells.57Glycan labeling:Administration of a labeled carbohydrate to an organism generally leads to incorporation of the label throughout the organism rather than in target cells;however,methods exist that can specifically target

57、 tumor cells.For example,solid tumors selectively retain lipid nanoparticles(LNP)owing to the enhanced permeability and retention(EPR)effect:angiogenesis in tumors forms leaky blood vessels which allow nanoparticles to be taken up by tumors more easily than normal tissue,and then retained.58 One stu

58、dy generated LNP containing azide-labeled galactosamines using folate ligands.Owing to the presence of increased folate receptors in tumor tissue,LNP internalization occurred,followed by cargo release into the tumor cells.Tumor membranes incorporated azide-functionalized dibenzocyclooctyne,triggerin

59、g an immune response when tumor cells were exposed to human sera.This method is under development;however,it may have important future applications.Incorporating cancer cell-specific ligands into LNP can be used to improve their selectivity for cancer cells.59,60Click to releaseThe CTR method is a p

60、romising technique using bioorthogonal chemistry to control the timing and location of drug release,resulting in a drug that is selectively toxic to target cells(Figure 5).6168Trans-cyclooctene(TCO)and tetrazine moieties have been most often used in CTR methods because of their rapid reaction.TCO or

61、 tetrazine moieties must be substituted with a targeting or localizing moiety so that one component is localized near the desired target,and the drug must be connected by an oxygen or nitrogen atom to a TCO moiety by an allylic ether,carbamate,or ester.Importantly,the tetrazine-containing component

62、should be minimally toxic,while the drug-TCO conjugate should be substantially less toxic(and active)than the free drug,and the drug must also be cell-permeable.Injection oftetrazine-containingpolymer DrugDrug administrationDrug administrationAdministration oftetrazineNNNNNNNRNNNNNRFigure 5.“Click-t

63、o-release methods as implemented by Shasqi(upper pathway)and researchers in the Robillard group(lower pathway)for the treatment of cancer in mouse xenograft modelsCalifornian company Shasqi used a CTR approach employing a sodium hyaluronate functionalized with tetrazine moieties that was injected ne

64、ar solid tumors in mice.Caged doxorubicin(conjugated to a TCO moiety)was administered to the mice and was shown to be 80-fold less toxic than doxorubicin alone.The total tolerated dose of the caged doxorubicin was approximately 19 times the tolerated overall dose of free doxorubicin.The lives of mic

65、e with tumor xenografts were extended by approximately 16 days.A Phase I clinical trial using this method is currently underway.67 Initial results from the trial in nine patients showed no dose-limiting toxicity and showed better antitumor responses from five of the patients than in any of their pre

66、vious treatments.68 BIOORTHOGONAL CHEMISTRY|13Another recent study investigated the diabody CC49.A diabody is an antibody possessing only the variable chains,and CC49 targets tumor-associated glycoprotein 72(TAG72),which is found in many solid tumors and is not internalized by tumors.A diabody of ca

67、ged monomethyl auristatin E(MMAE;a highly potent antimitotic agent)was constructed following the reaction of a mutant CC49 with a maleimide-substituted derivative of MMAE.Tumor xenografts in mice took up 629%of the diabody upon intravenous administration.The diabody half-lives were 5.5 days.Subseque

68、nt tetrazine(substituted with an oligo(ethylene glycol)-linked tetraazacyclododecanetetraacetic acid DOTA chelate)administration,which then reacted with the MMAE diabodies,resulted in a treatment that was 103-fold more toxic to tumor cells than the diabody construct alone.65,66Advantages and disadva

69、ntages of CTR methods:Polymer-based CTR allows for the delivery of a much larger amount of drug to the target cells versus that of an antibody,while the dosage of caged drug is limited only by its toxicity.The functionalized polymer is likely to be easier and cheaper to prepare than an antibody.Anti

70、body-based CTR does not require manual localization to a solid tumor,since the antibody can target any cancer cell possessing the requisite antigen.However,polymer-based CTR can trigger the immune system to act against tumors of the same type distant from the injected polymer.The use of a CTR antibo

71、dy-drug conjugate also allows the use of drugs whose toxicity could not be tolerated as free drugs,even in caged form.The antibody based CTR requires the existence of antibodies to the antigen of interest and requires that antigens are not internalized by the tumor cell,allowing a wide variety of an

72、tibodies to be used in the method.Antibody-based CTR can also be used for imaging tumors.Other bioorthogonal chemistries developed to control drug release in vivo include vinyl ethers as prodrugs and metal-based decaging reactions.69,70Looking towards the future:the potential of bioorthogonal chemis

73、tryBioorthogonal chemistry has grown remarkably in the last two decades and has found more widespread use in recent years.Notable developments and applications in the field are summarized in Figure 6.There are several interesting possibilities for the future applications of bioorthogonal reactions o

74、r for their use to improve or simplify techniques already being practiced.They can be used to explore development of reactive partners with improved biologic stabilities,or to simplify methods by eliminating the need for catalysts,which may reduce toxicity to living organisms.Multiple labeling could

75、 allow for easier exploration of biologic mechanisms and more reliable diagnostic agents.A potential refinement of bioorthogonal reactions is also improvement of light-activated chemistries to minimize damage to organisms and improve penetration into tissues.Such advancements may allow imaging from

76、deeper within living organisms.For instance,tetrazine ligation require UV light for effective labeling,which can damage organisms and has limited penetration into tissue.Recent developments such as in situ generation of tetrazines from dihydrotetrazines using near-IR light may help to address this l

77、imitation.These avenues may uncover cellular mechanisms that we know very little about,or that we have never encountered before.2000Staudinger Ligation2012Genetic Encoding byTetrazine Ligation2016In-situ DrugAssembly2003ABPP2004Strain-PromotedClick Chemistry2002Copper ClickChemistry820182

78、0232004In-vivo CellSurface GlycanImaging2006BONCAT2013First Report of Click to Release2017Cancer DrugTargeting2021Click to Release*2010FUNCAT2008TetrazineLigation2021Microneedles2018Click to Release*2013Figure 6.A timeline of developments in and uses of bioorthogonal chemistry*From antibody-drug con

79、jugates with systemic tetrazine administration*From a systemically-administered masked drug with a polymer-bound tetrazineFurthermore,generating standardized reagents would make bioorthogonal chemistry more easily incorporated into diagnostic or pharmaceutical agents.Addition of new bioorthogonal ta

80、gs as well as improvements in tagging methods may facilitate its broader use,such as in applications which require simpler tagging methods,or which tolerate less disturbance.Using native linkages would reduce changes in behavior caused by the linking groups and might make possible the synthesis of c

81、omplex molecules or antibody-drug conjugates with improved properties under milder conditions.Importantly,bioorthogonal reactions can help us learn more about emerging areas such as glycosylation where it can be used to help uncover the active role glycans have in cellular mechanisms.A promising opp

82、ortunity for bioorthogonal reactions is also in their use to aid improved control over localization of drug delivery.As mentioned above,this can be done through techniques such as CTR.BIOORTHOGONAL CHEMISTRY|15SummaryFor a detailed review of biorthogonal chemistry and its applications,see our public

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