Review
`
`pubs.acs.org/bc
`
`Click Chemistry and Radiochemistry: The First 10 Years
`†,#
`Jason S. Lewis,*,†,∥,‡
`and Brian M. Zeglis*,†,§,∥,⊥
`§,#
`Jan-Philip Meyer,
`Pierre Adumeau,
`†
`‡
`Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center,
`Department of Radiology and
`1275 York Avenue, New York, New York 10065, United States
`§Department of Chemistry, Hunter College of the City University of New York, 413 East 69th Street, New York, New York 10028,
`United States
`∥
`Department of Radiology, Weill Cornell Medical College, 520 East 70th Street, New York, New York 10065,
`United States
`⊥
`Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, 365 5th Avenue, New York, New York
`10016, United States
`
`ABSTRACT: The advent of click chemistry has had a profound influence on almost all branches of chemical science. This is
`particularly true of radiochemistry and the synthesis of agents for positron emission tomography (PET), single photon
`emission computed tomography (SPECT), and targeted radiotherapy. The selectivity, ease, rapidity, and modularity of
`click ligations make them nearly ideally suited for the construction of radiotracers, a process that often involves working
`with biomolecules in aqueous conditions with inexorably decaying radioisotopes. In the following pages, our goal is to provide
`a broad overview of the first 10 years of research at the intersection of click chemistry and radiochemistry. The discussion
`will focus on four areas that we believe underscore the critical advantages provided by click chemistry: (i) the use of prosthetic
`groups for radiolabeling reactions, (ii) the creation of coordination scaffolds for radiometals, (iii) the site-specific radio-
`labeling of proteins and peptides, and (iv) the development of strategies for in vivo pretargeting. Particular emphasis will
`be placed on the four most prevalent click reactionsthe Cu-catalyzed azide-alkyne cycloaddition (CuAAC), the strain-
`promoted azide-alkyne cycloaddition (SPAAC), the inverse electron demand Diels-Alder reaction (IEDDA), and the
`Staudinger ligationalthough less well-known click ligations will be discussed as well. Ultimately, it is our hope that this
`review will not only serve to educate readers but will also act as a springboard, inspiring synthetic chemists and radiochemists
`alike to harness click chemistry in even more innovative and ambitious ways as we embark upon the second decade of this
`fruitful collaboration.
`
`■ INTRODUCTION
`
`A decade and a half have passed since Kolb, Finn, and
`Sharpless published the landmark review that introduced the
`concept of click chemistry.1 In the intervening years,
`the
`influence of click chemistry has grown by leaps and bounds.
`To wit, the number of publications with “click chemistry” in
`the title has grown from 6 in 2003 to 252 in 2009 to 2014 in
`2015!2
`In the words of the original authors, the criteria for a click
`chemistry ligation are as demanding as they are straight-
`forward:1
`
`“The reaction must be modular, wide in scope, give very high
`yields, generate only inoffensive byproducts
`that can be
`removed by non-chromatographic methods, and be stereo-
`specific (but not necessarily enantioselective). The required
`process characteristics
`include simple reaction conditions
`(ideally, the process should be insensitive to oxygen and
`water), readily availably starting materials and reagents, the
`use of no solvent or a solvent that is benign (such as water)
`or easily removed, and simple product isolation.”
`
`Received:
`September 30, 2016
`Revised: October 26, 2016
`Published: October 27, 2016
`
`© 2016 American Chemical Society
`
`2791
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`DOI: 10.1021/acs.bioconjchem.6b00561
`Bioconjugate Chem. 2016, 27, 2791−2807
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`Downloaded via GEORGE WASHINGTON UNIV on February 28, 2025 at 04:17:21 (UTC).
`
`See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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`Petitioner GE Healthcare – Ex. 1034, p. 2791
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`

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`Bioconjugate Chemistry
`
`Review
`
`Figure 1. Schematics of the (A) Cu-catalyzed azide−alkyne cycloaddition reaction, (B) the strain-promoted azide−alkyne cycloaddition, (C) the
`inverse electron demand Diels−Alder cycloaddition, and (D) the Staudinger ligation.
`expanded dramatically.24−27 This growth means that an
`exhaustive review covering every instance in which click
`chemistry has been applied to nuclear imaging would almost
`certainly be an exhausting read. Instead, in the pages that follow, it
`is our goal to highlight the most interesting, exciting, and
`useful points of intersection between click chemistry and nuclear
`medicine. More specifically, we will focus on the use of click
`chemistry for (i) radiolabeling reactions with prosthetic groups,
`(ii) the creation of novel chelation architectures, (iii) site-specific
`bioconjugation, and (iv) in vivo pretargeting. Taken together,
`we believe that these four areas underscore how the rapidity,
`efficiency, selectivity, modularity, and bioorthogonality of click
`chemistry have empowered radiochemists to create innovative
`agents for imaging and therapy. Ultimately, we sincerely hope
`that this review not only informs the reader about research at the
`intersection of chemistry and radiochemistry but also inspires new
`and seasoned researchers alike to apply this remarkably useful
`chemical technique to the development radiopharmaceuticals.
`
`A handful of reactions that satisfy (or, at the very least, come
`close to satisfying) these criteria have been uncovered, including
`nucleophilic ring opening reactions with epoxides, aziridines, and
`aziridinium ions; the formation of ureas, oximes, and hydrazones
`via nonaldol carbonyl chemistry; and oxidative and Michael
`additions to carbon−carbon double bonds.3 Yet one particularly
`powerful reaction has emerged as the canonical click ligation and
`has proven remarkably useful in myriad applications: the copper-
`catalyzed [3 + 2] cycloaddition between an azide and a terminal
`alkyne (Figure 1A).4,5 More recently, Bertozzi and others have
`pioneered a subset of click reactions that boast an additional
`boundary condition: bioorthogonality.6−9 Bioorthogonal click
`ligations satisfy all of the requirements of standard click reactions
`but are also inert within biological systems. Not surprisingly,
`these reactions are hard to come by, yet a handful (most notably
`the Staudinger ligation, the strain-promoted azide−alkyne cyclo-
`addition reaction, and the inverse electron demand Diels−Alder
`cycloaddition) have been developed and proven powerful in the
`hands of chemical biologists, biochemists, and biomedical
`scientists (Figure 1B−D).7,10−16
`Click chemistry has had a paradigm-shifting influence on
`a wide range of chemical fields, from drug development17,18 and
`polymer chemistry19,20 to chemical biology21 and nanoscience.22
`However, it is hard to imagine a field that has more to gain from
`harnessing click chemistry than radiochemistry. The principal
`reason for this lies in what makes radiochemistry unique: the
`inexorable physical decay of radioisotopes during synthesis.
`As a result, radiolabeling reactionsand especially radiolabeling
`≈ 20 min)
`reactions using short-lived isotopes such as 11C (t1/2
`≈ 68 min)must be rapid and efficient to maximize
`and 68Ga (t1/2
`yield as well as selective and clean to eliminate time-sapping
`purification steps. Furthermore, the widespread use of bio-
`molecules as targeting vectors has also placed a premium on
`bioconjugation reactions that are both selective and unencum-
`bered by water. Finally, the proliferation of an ever-growing list of
`prosthetic groups and radiometal chelators has made modularity
`a critical feature of radiosynthetic protocols as well. Remarkably,
`all of these traits can be found in click chemistry ligations.
`In light of these benefits, it is somewhat surprising that the first
`publications describing radiopharmaceuticals synthesized using
`click chemistry came rather late: a 2006 work from Mindt et al.
`describing the use of click chemistry to create coordination
`scaffolds for 99mTc and a 2007 report from Wuest and co-workers
`detailing the use of the CuAAC reaction to create an 18F-labeled
`variant of neurotensin(8−13).23 Yet in the years since this
`somewhat belated start, work at the nexus of these two fields has
`
`■ RADIOLABELING WITH PROSTHETIC GROUPS
`
`One of the first reported, and still most extensively employed,
`applications of click chemistry to radiochemistry lies in the use
`of “clickable” prosthetic groups for radiolabeling. The ever-
`increasing use of imaging agents based on biomolecular vectors
`has put a premium on radiosynthesis strategies that are both
`mild and selective. Put simply, peptides, proteins, and antibodies
`should be radiolabeled under aqueous conditions at room
`temperature to ensure that their structural integrity is preserved,
`yet critically, many radiolabeling reactions require elevated
`temperatures, nonaqueous solvents, or (at the very least) pH
`conditions outside of the physiological norm. This is especially
`true for 18F-radiofluorination reactions, which often require
`organic solvents and high temperatures.
`Radiolabeled prosthetic groups provide an efficient way to
`circumvent these issues. Prosthetic groups are radiolabeled
`reactive small molecules that can be appended to biomolecules
`under benign conditions. Until recently, the vast majority of
`prosthetic groups have relied upon reactions with natural amino
`acids (most notably, couplings between N-hydroxysuccinimidyl
`(NHS) esters and lysines and Michael additions between
`maleimides and cysteines).28−30 Yet prosthetic groups of this ilk
`present a number of problems. Most concerning is the complete
`loss of regiochemical control during the labeling of a peptide or
`protein containing more than one lysine or cysteine. This, of
`course, can only be remedied by yield-sapping separations or the
`addition of time-consuming protection and deprotection steps.31
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`2792
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`DOI: 10.1021/acs.bioconjchem.6b00561
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`

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`Bioconjugate Chemistry
`
`Review
`
`Figure 2. An assortment of radiolabeled prosthetic groups used for the synthesis of radiopharmaceuticals via the (A) copper-catalyzed azide−alkyne
`cycloaddition, (B) strain-promoted azide−alkyne cycloaddition, (C) inverse electron-demand Diels−Alder cycloaddition, and (D) traceless Staudinger
`ligation.
`
`On top of this, both NHS esters and their isothiocyanate cousins
`are unstable under aqueous conditions, and maleimide−thiol
`linkages are prone to reversible substitution reactions in vivo.32
`In response to these limitations, radiochemists have increas-
`ingly turned to “clickable” prosthetic groups. Not surprisingly,
`the canonical CuAAC ligation leads the pack. In this regard, the
`relative age of the reaction certainly plays a role. Yet another
`critical advantage of the CuAAC ligation is that its “footprint” 
`a 1,2,3-triazole ring  is unlikely to perturb the structure or
`activity of the vector: the heterocycle is both relatively small and
`a rigid stereoisomer of an amide linkage. At this junction,
`we would be remiss if we did not mention the CuAAC reaction’s
`the ruthenium-catalyzed azide−alkyne
`lesser-known cousin:
`cycloaddition (RuAAC).33 The RuAAC reaction produces 1,5-
`disubstituted 1,2,3-triazoles as opposed to the 1,4-disubstituted
`1,2,3-triazoles created by the Cu-catalyzed cycloaddition. Even
`though it is regarded as a “click reaction”, the RuAAC ligation
`requires organic solvents, elevated temperatures, and inert gas
`atmosphere. Furthermore, the 1,5-disubstituted 1,2,3-triazoles
`produced by the reaction areunlike 1,4-disubstituted 1,2,3-
`triazolesmetabolically active and can be degraded via enzymatic
`N3 oxidation to produce highly reactive and potentially toxic
`metabolites.34 Given both of these issues, it is not surprising that,
`to the best of our knowledge, the RuAAC reaction has not been
`applied to the synthesis of radiopharmaceuticals.
`Moving back to the topic at hand, an extensive body of
`work has emerged on the design, synthesis, and optimization of
`radiolabeled CuAAC-ready building blocks. Much, although
`not all, of this work has focused on 18F.35−38 Indeed, a variety of
`radiosynthetic methods have been employed to create azide- and
`alkyne-bearing 18F-labeled prosthetic groups (Figure 2A).37,39,40
`These tools and the CuAAC reaction have been harnessed with
`great success in the radiolabeling of a wide variety of vectors,
`including phosphonium ions,41 peptides,42−50 oligonucleotides,39,47
`and proteins.27,47 This application of the CuAAC reaction is not
`without its flaws, however. These stem primarily from the two
`reagents needed to facilitate the cycloaddition: Cu(I/II) cations
`and a sacrificial reductant. The latter, most often ascorbic acid, can
`inadvertently reduce particularly fragile peptides and proteins.27
`The Cu cations can be even more of a problem. Peptides
`and proteins (specifically serine, histidine, and arginine residues)
`can coordinate Cu2+ ions, resulting in structural and functional
`
`alterations to the peptide.51 For example, Pretze et al. observed
`the accidental formation of Cu−peptide complexes following the
`CuAAC-mediated ligation of an 18F-labeled, alkyne-containing
`prosthetic group to an azide-bearing SNEW peptide.45 The
`coordination of the oxidative Cu(I) species can also lead to dramatic
`alterations to the chelating amino acid residues, as demonstrated
`very recently.52 These issues are compounded even further for
`radiometal-containing constructs. In these cases, not only can the
`chelator capture the copper catalyst and prevent the reaction from
`happening, but residual Cu2+ ions can also outcompete the far less
`abundant radiometal cations for coordination by the chelator.53 On
`top of these coordination-related concerns, the presence of Cu+ can
`also increase the likelihood of undesired side reactions such as
`Glaser couplings or the formation of copper-acetylides.45,54,55 Some
`of these issues can be ameliorated through the use of Cu+-stabilizing
`chelators such as THPTA or N-heterocyclic carbene complexes
`of Cu+; however, these reagents can create their own set of
`complications.56−58
`In light of the limitations of the CuAAC ligation, researchers
`have turned to a handful of “second generation” click reactions
`that are both bioorthogonal and catalyst-free. The most obvious
`place to start is the strain-promoted azide−alkyne cycloaddition
`(SPAAC). The SPAAC reaction is an azide−alkyne cycloaddi-
`tion in which ring strain built into a cyclic alkyneoften a
`dibenzocyclooctyne (DBCO)drives the reaction and eliminates
`the need for a catalyst.59,60 Campbell-Verduyn et al. were among
`the first to use this approach for radiochemistry, creating a series
`of 18F-labeled bombesin derivatives via the reaction of a DBCO-
`modified peptide with an array of 18F-bearing, azide-containing
`prosthetic groups.61 Following a similar strategy, another
`laboratory modified a series of ανβ
`3-targeting RGD peptides
`with DBCO and radiolabeled them using an [18F]fluoro−
`−azide prosthetic group.50,62 In a creative twist, the authors
`PEG4
`scavenged excess unlabeled peptide using an azide-grafted resin,
`allowing them to achieve specific activities of up to 62.5 GBq/μmol.
`Critically, all of the 18F-labeled peptides bore biological affinity
`comparable to their unlabeled cousins and were shown to be
`effective for the visualization ανβ
`3-expressing U87MG xenografts
`(Figure 3). Of course, radiolabeling via the SPAAC reaction goes
`both ways: several laboratories have created 18F-labeled cyclo-
`octynes for the radiofluorination of azide-modified small
`molecules, sugars, and peptides (Figure 2B).63−65
`
`2793
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`Bioconjugate Chemistry
`
`Figure 3. Coronal PET images of a NOD/SCID mouse bearing a
`GLP-1R-positive insulinoma xenograft (white arrow) collected 0.5, 1, 3,
`and 5 h after the injection of an 18F-labeled Exendin-4 radiotracer
`synthesized using a “clickable” prosthetic group. Adapted and reprinted
`with permission from Wu et al., copyright 2013 by the Society of Nuclear
`Medicine and Molecular Imaging, Inc.
`
`The SPAAC reaction has also been used for radioiodinations
`and radiometalations. Choi et al., for example, used a DBCO-
`bearing cRGD peptide and a prosthetic group composed of a
`−azide moiety grafted to an 125I-labeled pyridine to create
`PEG4
`an 125I-labeled cRGD.66 Evans et al. labeled an azide-modified
`DOTA with 68Ga for the radiometalation of several DBCO-
`modified peptides.53 Likewise, the Anderson group has conjugated
`DIBO-bearing copper chelators to an azide-modified cetuximab
`antibody and an azide-bearing somatostatin analogue.67,68
`Despite its utility,
`the SPAAC ligation has one critical
`limitation: its dibenzocyclooctatriazole “footprint”. The work of
`Hausner and co-workers provides a particularly useful cautionary
`example.69 Here, the authors radiolabeled an azide-modified
`A20FMDV2-peptide using an 18F-labeled variant of DBCO.
`While in vitro experiments confirmed that the 18F-labeled peptide
`retained its affinity and specificity for ανβ
`6-expressing cells, in vivo
`imaging suggested that the bulky and hydrophobic benzocy-
`clooctatriazole footprint introduced by the SPAAC ligation led
`to dramatic changes in the pharmacokinetics of the tracer and
`significantly impaired its uptake in ανβ
`6-expressing xenografts.
`The inverse electron demand Diels−Alder (IEDDA) cyclo-
`addition between tetrazine (Tz) and a dienophile, most
`commonly trans-cyclooctene (TCO) but also norbornene
`(NB), has also provided fertile ground for the development of
`prosthetic groups. Like the SPAAC ligation, the IEDDA reaction
`is bioorthogonal and proceeds without a catalyst. The principal
`advantage of the IEDDA ligation is its extraordinary speed
`(vide infra), which makes it particularly well suited for applica-
`tions with short-lived radioisotopes. In 2010, the laboratories of
`Fox and Conti reported the first 18F-labeled TCO (Figure 2C).70
`This prosthetic group was used for the rapid (t < 5 min)
`radiolabeling of a range of tetrazine-bearing peptides, including
`RGD and the GLP agonist Exendin.71−73 The 18F-labeled
`Exendin proved particularly promising, enabling the PET
`imaging of GLP-1R-positive insulinoma xenografts in mice.
`The same 18F−TCO was also used to great effect by Weissleder
`and co-workers for labeling a Tz-bearing analog of the PARP1
`inhibitor AZD2281. In this work, however, the authors added a
`creative wrinkle: removing unlabeled AZD2281−Tz using a
`TCO-coated magnetic resin.74,75 Finally, a number of 18F-labeled
`tetrazines have also been synthesized, but the in vivo use of
`radiopharmaceuticals created using these moieties has thus far
`remained somewhat sparing.76,77
`The utility of the IEDDA reaction extends beyond radio-
`fluorination.53 To wit, a handful of radioiodinated tetrazine
`constructs have been successfully developed (Figure 2C).
`Albu et al., for example, synthesized an 125I-labeled tetrazine and
`conjugated this building block to a TCO-modified anti-VEGFR2
`
`Review
`
`antibody.78 Interestingly, in vivo studies using this tracer revealed
`an additional benefit of this approach: the 125I-labeled antibody
`proved to be more than 10-fold more stable to deiodination over
`48 h compared to traditionally radioiodinated analogs. More
`recently, Choi et al. used a similar strategy for the radiolabeleling
`of both a cRGD peptide and human serum albumin (HSA).79
`The 125I-labeled HSA displayed impressive in vivo behavior, with
`a deiodination rate reduced by 50-fold compared to constructs
`created via traditional radioiodination. In 2011, Zeglis et al.
`employed the IEDDA reaction to create a modular strategy for the
`bioconjugation of a trastuzumab−TCO immunoconjugate with
`Tz−desferrioxamine (for 89Zr4+) and Tz−DOTA (for 64Cu2+).80
`More recently, Kumar and co-workers harnessed the IEDDA
`reaction to circumvent the incompatibility of antibodies with
`the high temperatures required to radiolabel the CB-TE2A-1C
`chelator with 64Cu.81 To this end, the authors modified the
`chelator with a norbornene moiety and grafted tetrazine onto
`an anti-PSMA antibody (YPSMA). After radiolabeling of the
`chelator-NB building block with 64Cu at 85 °C, the 64Cu−CB-
`TE2A1C-NB synthon was attached to YPSMA−Tz under mild
`conditions, and the 64Cu-labeled radioimmunoconjugate was
`successfully deployed for the PET imaging of PSMA-expressing
`tumors in a murine model of prostate cancer.
`Although the rapidity of the IEDDA reaction provides a
`marked improvement over the sluggish SPAAC ligation, it fails
`to solve one of the latter’s major issues: a bulky, hydrophobic
`footprint. As we have discussed, the SPAAC reaction leaves a
`benzocyclooctatriazole moiety in its wake. The IEDDA ligation
`creates an equally large footprint: a bicyclic [6.4.0] ring system.
`Both structures have the potential to interfere with the bio-
`logical activity and pharmacokinetics of vectors, particularly
`small molecules and short peptides. The traceless version of the
`Staudinger ligation offers an exciting alternative (Figure 4A).
`This ligation relies on an initial reaction between a phosphine-
`based moiety and an azide followed by a rearrangement that
`produces a simple amide linkage. Along these lines, the radio-
`labeling of peptides with 18F has been achieved via the reaction
`between (diphenylphosphanyl)methanethiol thioester-bearing
`peptides and an 18F-labeled azide as well as that between a
`radiolabeled 2-(diphenylphosphanyl)phenol ester with an azide-
`bearing peptide (Figure 2D).82−84 Unfortunately, however,
`the traceless Staudinger ligation requires high temperatures
`(90−130 °C) to achieve speeds that are compatible with short-
`lived isotopes. This undoubtedly limits its utility with fragile small
`molecules, peptides, and proteins; however, we are optimistic
`about the potential applications of this elegant transformation with
`longer-lived isotopes.
`Finally, a handful of other, less-well-known click ligations have
`made sparing yet interesting appearances in the literature of
`prosthetic groups. In 2012, Zlatopolskiy et al. reported the
`formation of a reactive nitrone from 18F-fluorobenzaldehyde
`and phenylhydroxylamine.85 The authors showed that
`this
`18F-labeled nitrone could undergo a [3 + 2] cycloaddition with
`a maleimide, resulting in quantitative conversion in less than
`15 min at 80 °C (Figure 4B). It must be said, however, these
`reaction conditions leave much to be desired when it comes to
`labeling biomolecules. Later the same year, the same group probed
`the potential of cycloaddition reactions between nitriloxides
`and dipolarophiles (Figure 4C).86 An 18F-labeled nitriloxide was
`synthesized from 18F-p-fluorobenzaldehyde and reacted with a
`series of dipolarophiles, producing quantitative conversions in
`<10 min at 40 °C. However, these reactions were performed in
`alcohol, and no data was presented regarding the feasibility of this
`
`2794
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`DOI: 10.1021/acs.bioconjchem.6b00561
`Bioconjugate Chem. 2016, 27, 2791−2807
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`Petitioner GE Healthcare – Ex. 1034, p. 2794
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`

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`Bioconjugate Chemistry
`
`Review
`
`Figure 4. Schematics of an assortment of click chemistry ligations (beyond those depicted in Figure 1) used for prosthetic group radiolabelings: (A)
`traceless Staudinger ligation, (B) nitrone−alkene cycloaddition, (C) nitrile−oxide cycloaddition, (D) 1,2-aminothiol−cyanobenzothiazole
`condensation, and (E) phenyloxadiazole methylsulfone−thiol conjugation.
`
`transformation under aqueous conditions. Recently, other groups
`have harnessed the reactivity of 2-cyanobenzothiazoles toward
`1,2-aminothiols to radiolabel peptides and proteins containing
`N-terminal cysteines (Figure 4D).87,88 To this end, 18F-labeled
`2-cyanobenzothiazoles were synthesized and appended to RGD
`and diRGD peptides bearing N-terminal cysteines as well as a
`genetically engineered variant of
`luciferase with a cysteine
`at the N-terminus. Lastly, just this year, Chiotellis et al. have
`explored phenyloxadiazole methylsulfones (PODS) as more
`stable alternatives to maleimides for conjugations with thiols
`(Figure 4E).89 In this work, an 18F-labeled PODS was used to
`radiolabel both a cysteine-bearing peptide and a cysteine-modified
`affibody, and the resulting constructs were used to HER2-positive
`tumors in a mouse model of breast cancer.
`
`■ CREATING COORDINATION SCAFFOLDS
`
`The use of click chemistry to create radiometal chelation
`architectures provides one of the best examples of the unique
`modularity conferred by this synthetic approach.90,91 Easily
`the best known of these methods, dubbed “click-to-chelate”
`by its inventors, was introduced in 2006 by Mindt et al.
`(Figure 5).92−94 This strategy employs the CuI-catalyzed azide−
`
`Figure 5. “Click-to-chelate” approach: a variety of prochelators
`exhibiting electron-donating groups undergo the CuI-catalyzed azide−
`alkyne cycloaddition with an azide to from a tridentate ligand that can
`coordinate an organometallic [M(CO)3]+ synthon.
`
`alkyne cycloaddition (CuAAC) reaction to attach small molecule
`“pro-chelators” to peptides and small molecules. However,
`the 1,2,3-triazole produced by the click ligation becomes far
`more than just a simple link between the subunits of the
`construct. Indeed, the heterocycle forms an integral part of a
`tripodal coordination scaffold capable of the rapid chelation
`
`in which M can be the γ-emitting
`of [M(CO)3]+ synthons,
`radiometal 99mTc (t1/2 = 6.01 h) or the β-emitting radiometal
`188Re (t1/2 = 16.98 h). In this way, “click-to-chelate” facilitates
`the creation of a chelator and its subsequent radiometalation
`in a rapid, robust, and reproducible one-pot reaction. This is
`particularly important given the mercurial coordination chem-
`istry of 99mTc.
`In their initial proof-of-concept report, the authors created
`seven different tripodal scaffoldsincluding N3, N2S, and N2O
`ligand architecturesusing a series of azide-modified small
`molecules. Subsequent labeling with M(CO3) [M = Re, 99mTc]
`synthons resulted in a series of highly stable,
`low-spin
`d6-complexes despite differences in the size, molecular charge,
`and hydrophilicity of the prochelator.92−95 The creation of a
`99mTc-labeled variant of folate using “click-to-chelate” provides
`an excellent example of the approach (Figure 6). The 1,2,3-
`triazole ring formed in the first phase of the reaction between
`the azide-bearing folate construct (1) and the alkyne-modified
`amino acid (2) not only connects the pro-chelator to the folate
`vector but also serves as an essential part of the N2O coordina-
`tion scaffold for the [99mTc(CO)3]+ moiety. The incubation of
`the chelator-bearing construct with [99mTc(CO)3(H2O)3]+
`reproducibly yields 99mTc-labeled folate (3) in high yield and
`specific activity.92
`In subsequent work, this technique was applied to peptides
`as well as an array of other biologically active small molecules
`such as sugars, nucleosides, and steroids.96−100 Fernandez et al.,
`for example, developed a 99mTc-labeled glucose derivative as an
`imaging probe for glucose metabolism.97 Similarly, Struthers et al.
`developed an elegant one-pot “click-to-chelate” synthesis of a
`99mTc-labeled thymidine analogue as a SPECT surrogate for the
`clinically successful proliferation marker 18F−FLT.98 Taken
`this work clearly demonstrates that 99mTc-labeled
`together,
`tracers created using the “click-to-chelate” methodology demon-
`strate in vivo behavior that is comparable, and in some cases
`superior, to the current “gold standard” chelators for [99mTc-
`(CO)3]+: Nτ-derivatized histidine and Nα-acetylated histidine.
`Indeed, studies using 99mTc-labeled folate revealed that the
`click-to-chelate approach furnished compounds in purities and
`radiochemical yields equal
`to those achieved using tradi-
`tional radiolabeling techniques. Furthermore, in this work, the
`click-to-chelate approach did not alter biodistribution patterns
`or pharmacodynamic parameters such as receptor affinities and
`selectivities. Finally,
`the superiority of
`the click-to-chelate
`methodology becomes most obvious in the context of syntheti-
`cally challenging molecules. In the case of the azide-modified
`
`2795
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`DOI: 10.1021/acs.bioconjchem.6b00561
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`Petitioner GE Healthcare – Ex. 1034, p. 2795
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`

`

`Bioconjugate Chemistry
`
`Review
`
`Figure 6. Advantages of the one-pot “click-to-chelate” approach are particularly apparent in the context of synthetically challenging probes such as this
`99mTc-labeled folate radiopharmaceutical (3).
`
`Figure 7. The asymmetry of the CuAAC reaction creates two different coordination scaffolds depending on whether the prochelator contains the alkyne or
`azide functionality. The “regular click ligand” (A) is a more effective chelator for [99mTc(CO)3]+ and [188Re(CO)3]+ than the “inverse click ligand” (B).
`
`folate construct, for example, the differences in synthetic effort
`and yield are striking: “click-to-chelate” furnished an 99mTc-
`labeled tracer in 80% overall yield in 8 steps, whereas 10 steps
`were required to muster approximately 1% yield with a histidine-
`based chelator.92
`From a chemical standpoint, it is important to note that the
`inherent asymmetry of the CuAAC reaction means that two
`different 1,2,3-triazoles can be formed when linking the vector
`and the chelator (Figure 7).93,95 In the first, the “regular click
`ligand”, the pro-chelator bears the alkyne moiety while the
`vector contains the azide group, and the N3 atom of the triazole
`participates in the coordination of 99mTc. In the second, the
`“inverse click ligand”, the pro-chelator boasts the azide moiety
`while the vector wields the alkyne group, and the N2 atom of
`the triazole participates in the coordination of 99mTc. Somewhat
`surprisingly, the two different chelation environments display
`quite different behavior when radiolabeled with [99mTc(CO)3]+
`and [188Re(CO)3]+, with the “inverse click ligand” offering
`significantly lower labeling efficiency and decreased in vivo
`stability.93 Although a concrete explanation for this phenomenon
`remains elusive, the most likely hypothesis points to the decreased
`electron density in the N2 position compared to the N3 site.
`Before moving on, it is worth noting that a handful of other
`groups have also used click chemistry in the synthesis of
`radiometal chelators. Bailey et al., for example, used the CuAAC
`reaction in the synthesis H4azapa: a carboxypyridine-based
`chelator for 111In3+ and 177Lu3+ (Figure 8A).91 In addition,
`Bottorff et al. have developed a synthetic strategy to generate
`isoxazole ligands via click chemistry (Figure 8B).101 Yet in the
`end, it is undeniable that the “click-to-chelate” methodology
`
`Figure 8. Structures of the acyclic H2azapa (A) and isoxazole (B)
`chelators for diagnostic and therapeutic radiometals such as 67Ga, 64Cu,
`111In (A), and 99mTc (B), respectively. The isoxazole ligand (B) was
`synthesized via click chemistry using the Cu-free 1,3-dipolar cyclo-
`addition between an alkyne and an oxime.
`
`represents the gold standard in this area. Indeed, this approach
`not only provides a cardinal example of the modularity and
`flexibility provided by click chemistry but also stands as one of
`the most useful and innovative developments in 99mTc chemistry
`of the past decade.2−4
`■ SITE-SPECIFIC BIOCONJUGATION
`
`The selectivity and bioorthogonality of click chemistry have also
`been leveraged for the site-specific modification of proteins and
`antibodies. This process has become ubiquitous in the synthesis
`of biomolecular therapeutics such as antibody-drug conjugates,
`and it is increasingly important in the creation of radiolabeled
`probes as well. Until recently, the overwhelming majority of
`bioconjugation methods were predicated on ligations between
`reactive bifunctional probese.g., N-hydroxysuccinimide-bearing
`chelators or maleimide-modified toxinsand amino acids within
`the biomolecule, most often lysines and cysteines. While these
`
`2796
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`DOI: 10.1021/acs.bioconjchem.6b00561
`Bioconjugate Chem. 2016, 27, 2791−2807
`
`Petitioner GE Healthcare – Ex. 1034, p. 2796
`
`

`

`Bioconjugate Chemistry
`
`methods are undeniably simple, they are far from precise.
`Control over the location and frequency of these ligations is
`impossible because proteins have multiple copies of
`these
`amino acids distributed throughout their structures. As a result,
`these bioconjugation strategies produce constructs that are
`both heterogeneous and poorly defined. Furthermore, random
`conjugation strategies can decrease the reactivity of constructs if
`the cargo is inadvertently appended to the target-binding domains
`of the biomolecule.
`In response to these issues, significant effort has been
`dedicated to the creation of strategies for the site-specific
`bioconjugation of proteins and antibodies. A wide variety of
`methods have been developed,
`including variants predica