selective sensing of tyrosine phosphorylation in peptides ...€¦ · reviewarticle selective...

Review ArticleSelective Sensing of Tyrosine Phosphorylation in PeptidesUsing Terbium(III) Complexes

Jun Sumaoka123 Hiroki Akiba24 and Makoto Komiyama23

1Department of Applied Chemistry School of Engineering Tokyo University of Technology 1404-1 KatakuramachiHachioji Tokyo 192-0982 Japan2Research Center for Advanced Science and Technology The University of Tokyo 4-6-1 Komaba Meguro-ku Tokyo 153-8904 Japan3Life Science Center of Tsukuba Advanced Research Alliance University of Tsukuba 1-1-1 Ten-noudai TsukubaIbaraki 305-8577 Japan4Department of Bioengineering School of Engineering The University of Tokyo 7-3-1 Hongo Bunkyo-ku Tokyo 113-8656 Japan

Correspondence should be addressed to Jun Sumaoka sumaokajnstfteuacjpand Makoto Komiyama komiyamamakotonimsgojp

Received 9 March 2016 Accepted 28 April 2016

Academic Editor Gunther K Bonn

Copyright copy 2016 Jun Sumaoka et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Phosphorylation of tyrosine residues in proteins as well as their dephosphorylation is closely related to various diseases Howeverthis phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteinsand covers only 005 of the total phosphorylation Accordingly highly selective detection of phosphorylated tyrosine in proteins isan urgent subject In this review recent developments in this field are described Monomeric and binuclear TbIII complexes whichemit notable luminescence only in the presence of phosphotyrosine (pTyr) have been developed There the benzene ring of pTyrfunctions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center Even in the coexistence ofphosphoserine (pSer) and phosphothreonine (pThr) pTyr can be efficintly detected with high selectivity Simply by adding theseTbIII complexes to the solutions phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation byprotein tyrosine phosphatases can be successfully visualized in a real-time fashion Furthermore the activities of various inhibitorson these enzymes are quantitatively evaluated indicating a strong potential of the method for efficient screening of eminentinhibitors from a number of candidates

1 Introduction

In nature enzymatic phosphorylation and dephosphoryla-tion of proteins control many biological events Cellularpathways regulated by these enzymatic modifications ofproteins are so versatile In the course of signal transductionin cells for example SerThr and Tyr residues in proteins arereversibly phosphorylated and dephosphorylated resultingin desired modulation of the activity of relevant enzymes [12] In terms of the importance of these enzymatic reactions anumber of elegant chemical sensors to detect them inproteinshave been already reported In most of these sensors phos-phate residue(s) of phosphoserine (pSer) phosphothreonine(pThr) and phosphotyrosine (pTyr) in proteins is selectively

bound as the recognition target so that these three types ofphosphorylations are detected at similar sensitivity withoutsignificant discrimination [3ndash11] Valuable information onthe roles of protein phosphorylations in biological systemshas been obtained The molecular designs of these sensorsand their practical applications have been the subjects ofmany excellent reviews [12ndash21]

In contrast with these overall detections of phosphoryla-tions of Ser Thr and Tyr in proteins this review focuses onselective detection of phosphorylation of Tyr alone (Figure 1)This Tyr phosphorylation by protein tyrosine kinases (PTKs)and protein tyrosine phosphatases (PTPs) accounts for only005 of the total phosphorylation in cells (the majority ofphosphorylation occurs on Ser or Thr) but takes a crucial

Hindawi Publishing CorporationInternational Journal of Analytical ChemistryVolume 2016 Article ID 3216523 14 pageshttpdxdoiorg10115520163216523

2 International Journal of Analytical Chemistry

OH OPOPhosphorylation

Dephosphorylation

PTKs (protein tyrosine kinases)

PTPs (protein tyrosine phosphatases)

OminusOminus

Figure 1 Phosphorylation of tyrosine residue by protein tyrosinekinases (PTKs) and its dephosphorylation by protein tyrosinephosphatases (PTPs) for the regulation of biological functions ofproteins

role in the regulation of highly important biological functions(differentiation adhesion cycle control endocytosis andmany others) [22 23] In epidermal growth factor receptor(EGFR) its autophosphorylation of a Tyr residue triggerssignal-cascade in cells [24 25] In the downstream therework several Src family kinases which are also controlled bytheir Tyr phosphorylations and in turn phosphorylate Tyrresidues in other proteins [26ndash28] If Tyr phosphorylationis excessive or insufficient serious problems are induced tothe living Therefore PTKs and PTPs are regarded as maintargets in drug discovery [29ndash34] For many years a numberof laboratories developed elegant optical sensors to evaluatethe activities of these enzymes In some of them substratepeptide was conjugated (or fused) to a probe molecule (egTb(III) complexes [35ndash40] Mg(II) complexes [41ndash47] Ca(II)complex [48] Zn(II) complex [49] Cd(II) complex [50] pep-tide derivatives [51 52] and others [53 54])Theother sensorsinvolve noncovalent interactions between a substrate and aprobe (eg Tb(III) ion [55ndash62] Eu(III) complex [63 64]platinum(II) complex [65] and Tb(III) complexes [66ndash69])

Among all the probes investigated lanthanide ions andtheir complexes have been widely and successfully employeddue to their unique light-emitting properties [70ndash77] Thephotoluminescence from these ions has unusually longlife-time (in the order of micro- to milliseconds) and thusthe background signal can be minimized with the use oftime-resolved spectroscopy Alternatively the kinase reac-tions were followed by the disappearance of ATP (source ofthe phosphate group for pTyr) [78 79] whereas the phos-phatase functions were monitored by the production ofphosphoric acid [80] However these analytical methods areoften complicated by the perturbation signals from otherphosphate-containing solutes ATP-dependent reactionsandor phosphate-producing processes in the specimens Inaddition to these chemical sensors antibodies specific to pTyrare widely being used at present for practical applicationsbut their usage has been hampered by high costs rather poorstability and other factors Accordingly chemical probes thatdirectly visualize PTKPTP activity and produce unbiasedsignals are required for further developments of the field

This paper reviews recent developments in optical meth-ods to selectively detect pTyr in proteins The primary con-cerns are high sensitivity of pTyr detection and its sufficientspecificity (with respect to pSer and pThr which exist muchmore abundantly in biological systems) As emission probeslanthanide ions (especially TbIII ion) and their complexes areused By combining unique properties of the emission fromthese metal ions with so-called ldquoantenna effectrdquo the back-ground signals are minimized and only the signal from pTyris selectivelymonitored [67]The detection activity on pTyr isfurther promoted by forming binuclear TbIII complexes [68]With the use of these chemical sensors phosphorylation ofpeptides by PTKs and their dephosphorylation by PTPs arefollowed in a real-time fashion [38 68 69] Applications ofthe methods to screening of efficient inhibitors on PTKs andPTPs are also presented

2 Principle of Selective Detection of pTyr byTb(III) Complexes

The emission from lanthanide ions is intrinsically weaksince the corresponding f-f transitions are Laporte-forbiddenHowever this luminescence is enormously strengthenedwhen a chromophore (ldquoantennardquo) is placed near the lantha-nide ions and transfers its photoexcitation energy to theseemission centers [70ndash77 81ndash91] By combining lanthanidecomplexes with antenna molecules elegant systems to detectvarious anionic guests have been already prepared Sophis-ticated examples include the analysis of carboxylic acidderivatives [85ndash100] halide ions [95ndash98 101ndash108] nitrate ions[96 97 101ndash105 109] and hydrogen sulfate ion [110] Further-more phosphate ion [95ndash99 108ndash116] pyrophosphate [113ndash115] ATP [113ndash115 117ndash121] and other molecules containingphosphate [120ndash122] were also detected by using lanthanidecomplexes Upon the binding of the phosphate group(s)to the complexes the chemical environments around thelanthanide(III) ions were altered inducing a change in theluminescent property of the ionsWith this strategy howeverhighly selective detection of pTyr is rather difficult sincecoexisting phosphate groups in solutions (eg pSer pThrATP and DNA) could show similar effects [123ndash125] Thesefactors aremore critical when a TbIII ion (without any ligand)is used note that nucleotides and nucleic acids are alsoeminent antenna (vide infra) [55 56]

One successful solution to these problems (improvementof the selectivity of pTyr detection with respect to (i) non-phosphorylated Tyr (ii) pSer and pThr and (iii) other coex-isting phosphate-containing biomolecules) is presented inFigure 2 This strategy developed in our laboratory [67] isbased on the fact that both the benzene ring and the phos-phate group are definitely required for the efficient photolu-minescence First the benzene ring of pTyr in the target pep-tides is used as an antenna to enhance the emission from theTbIII center The irradiated light (Ex) is first absorbed by thisbenzene ring and the excitation energy is then transferred toTbIII (ET) Finally the metal center emits luminescence fromits photoexcited state (Em) On the other hand the phosphateis essential to bind to TbIII and places the benzene ring near

International Journal of Analytical Chemistry 3

OPO

Tb

pTyr

ETEx

Em

Ominus

Ominus

Figure 2 Mechanism of selective detection of enzymatic phos-phorylation of Tyr by TbIII complex The photoexcited energy(Ex) absorbed by the benzene ring (antenna) is transferred tothe TbIII (ET) resulting in enormous promotion of the intensityof luminescence emitted from this metal ion (Em) Accordinglythe emission is evident only for pTyr which fulfills both of thetwo requirements for the mechanism (notable antenna effect andsufficient binding activity towards the TbIII)

the metal ion as the emission center Among the coexistingsolutes (Tyr Ser Thr and their phosphorylated products)only pTyr possesses both notable antenna effect (benzenering) and sufficient binding activity towards the TbIII com-plex (phosphate group) Thus the selectivities (i) and (ii) arefulfilled Furthermore the selectivity (iii) to other coexistingphosphate-containing molecules is accomplished by using abulky ligand which suppresses the access of these moleculesto TbIII Furthermore nonspecific background signals can beremoved by using time-resolved spectroscopy and analyzingonly long life-time components of the luminescence emittedfrom TbIII

3 Selective Detection ofEnzymatic Phosphorylation of Tyr byTbIII Complex-Based Sensors

Based on the strategy depicted in Figure 2 enzymatic phos-phorylations and dephosphorylations of Tyr were monitoredby using TbIII complexes In the first part of Section 31 amonomeric TbIII complex was prepared primarily to showthe validity of the working hypothesis In Section 32 thesensitivity of pTyr detection has been greatly enhancedby forming binuclear TbIII complexes As a result usefultools to monitor enzymatic phosphorylation of Tyr (andits dephosphorylation) have been obtained and used forpractical applications in the following sections Among lan-thanide ions TbIII has been most widely employed togetherwith Eu(III) for biological applications emitting the mostintensive line at around 545 nm

31 Monomeric Tb119868119868119868 Complex Showing Sufficient Selectivityfor pTyr Detection [67] The sample solutions used for thesensing of pTyr always contain many other biological mol-ecules and some of them show notable antenna effects toinduce the emission from lanthanide ions Among themnucleobases and nucleic acids especially deserve attentionFor example guanosine 51015840-monophosphate (GMP) enor-mously enhances the luminescence through the binding toTbIII ion by multicoordination of both the phosphate andthe guanine (N7 and O6) Thus TbIII ion itself is not directlyapplicable to the sensing In order to suppress the emissiondue to these coexisting molecules and accomplish efficientsensing of Tyr phosphorylation an appropriate ligand is nec-essary to prevent the access of these molecules to TbIII ionFor this purpose DOTAM (2210158402101584010158402101584010158401015840-(14710-tetraaz-acyclododecane-14710-tetrayl)tetraacetamide) was used(Figure 3(a)) This well-known ligand for lanthanide ionshas no aromatic ring to work as antenna [126ndash129] and itsTbIII complexes have +3 net charges which are favourableto bind negatively charged pTyr According to the designthe bulkiness of DOTAM should sterically interfere with theinteractions between bulky nucleobases (or nucleic acids)and TbIII On the other hand the effect of DOTAM on thebinding of the phosphate in pTyr to the TbIII is little (or inmuch smaller magnitude) because of its smaller size

Exactly as designed pTyr notably increased the intensityof luminescence from TbIII-DOTAM complex (blue bars inFigure 4) Apparently the phosphate residue of pTyr satis-factorily interacted with the complex despite the bulkinessof DOTAM and the excitation energy of the benzene ringwas efficiently transferred to the emission metal center Incontrast GMP as well as other nucleotides and nucleic acidshardly promoted the luminescence fromTbIII-DOTAM Fur-thermore nonphosphorylated Tyr pSer and pThr inducedonly marginal increase as expected from the mechanism inFigure 2The effect of either phenylalanine or tryptophanwasnegligible Thus TbIII-DOTAM complex is sufficiently effec-tive in detecting pTyr selectively even in the coexistence ofvarious analytes which otherwise produce undesirable noisesof nonnegligible intensity Using this complex the tyrosinephosphorylation in a nonapeptide (Ac-Glu-Glu-Glu-Ile-Tyr-Glu-Glu-Phe-Asp-CONH2 P1 peptide [130]) was successfullymonitored with a high signal-to-noise ratio The mode ofinteraction between the metal center in TbIII-DOTAM andthe phosphate group of pTyr was investigated by using phenylphosphate (PhOP) as a model compound of pTyr (note thatit also notably increased the luminescence from the DOTAMcomplex in Figure 4) In the presence or the absence of PhOPthe 119902 value (the number of coordinated water moleculeson TbIII) was determined by luminescence life-time mea-surements Interestingly and importantly 119902 value was alwaysaround 1 whether or not PhOP was binding to the TbIII-DOTAM complex In other words one water molecule wasoriginally coordinated to TbIII in the TbIII-DOTAM com-plex and this water molecule was never removed from TbIII

when the TbIII complex interacted with PhOP Thus it has


O

N

N N

NTb

O

O

O

TbIII2-DOTAM

H2N

H2N

NH2

NH2

(a)

HN N

H

O

N

N N

N

Tb

O

O

OO

N

N

N

N

Tb

O

O

O

n

TbIII2-L2

n = 2

TbIII2-L1

n = 1

H2N

H2N

H2N

NH2

NH2

NH2

(b)

Figure 3 Structures of mononuclear DOTAM-TbIII complex (a) and binuclear complexes TbIII2-L1 and TbIII

2-L2 (b) used for selective

detection of pTyr

300 330 900

010

PhO

PSe

rTh

rTy

r

Tbli

g onl

y

Glu

cose

-PpThr

pTyr

Trp

AMPpS

er

GM

PCM

PAD

PAT

P

UM

P

20304050607080

Tb-DOTAM

Lum

ines

cenc

e int

ensit

y (545

nm)

Tb2-L1

Tb(III) as TbCl3

Figure 4 The luminescence intensity at 545 nm of TbIII-DOTAM(blue bars) and TbIII

2-L1 (red bars) in the presence of various

phosphorylated and nonphosphorylated amino acids nucleosidederivatives and PhOP (a model compound of pTyr) Conditions[TbIII complex] = [additive] = 100 120583M pH 70 (10mM HEPESbuffer) 120582ex = 2625 nm For the purpose of comparison the resultsusing TbIII ion without ligand are also presented (yellow bars)Note that nucleotides (UMP GMP CMP and ADP) showed notablesignals and thus selective detection of pTyr was unsuccessful

been concluded that the interaction between TbIII-DOTAMand PhOP (and thus pTyr also) is an ion-pairing rather thandirect coordination of the phosphate to TbIII Neverthelessthe benzene ring of pTyr is placed in a sufficient proximity ofTbIII and satisfactorily works as antenna

32 Binuclear Tb119868119868119868 Complexes for Promoted Detection Sen-sitivity on pTyr [68] As shown in the previous sectionmononuclear TbIII complex has eminent selectivity for pTyrdetection However the sensitivity is still rather limitedprimarily because of its poor binding of phosphate groupIn order to further increase the detection sensitivity ofTbIII-DOTAM on pTyr its binuclear complexes (TbIII

2-L1

and TbIII2-L2) were developed (Figure 3(b)) In the ligands

used two DOTAM groups were connected by appropriatelinkers of different length These complexes as well as TbIII-DOTAM show intrinsically minimal luminescence in theabsence of pTyr (no antennamoiety is available) Importantlythe luminescence from these binuclear TbIII complexes wasgreatly enhanced when pTyr was added to the solution (redbars in Figure 4) Moreover the pTyr-induced enhancementsof luminescence from these binuclear complexes were fargreater than pTyr-induced enhancement of luminescencefrom the mononuclear complex TbIII-DOTAM (comparethe red bar with the blue bar in Figure 4) The origins ofremarkable enhancements for the binuclear TbIII complexeswere investigated in detail using a model compound PhOP inplace of pTyr When [TbIII complex] = [PhOP] = 100 120583M theluminescence from TbIII

2-L1 is stronger than that from TbIII-

DOTAM by more than 10-fold By analyzing the relationshipbetween the luminescence intensity and the concentration ofTbIII complex in terms of Michaelis-Menten type equationthe dissociation constant of TbIII

2-L1PhOP complex was

determined to be 29 120583M This value was 110 times smallerthan the corresponding value for TbIII-DOTAM Thus thebinuclear TbIII complexes have superior photoemission activ-ity mainly because they bind PhOP (and thus pTyr also)more efficiently Apparently the doubled positive chargesof these binuclear complexes (+6) are responsible for thetighter interactions with the negatively charged phosphate


Src injection

10

15

20

25

Relat

ive l

umin

esce

nce i

nten

sity

400 800 1200 16000Time (s)

Src 4120583gmLSrc 04 120583gmL

Figure 5 Time-dependent change of the luminescence intensityfrom TbIII

2-L1 for the phosphorylation of P1 by protein tyrosine

kinase Src At time = 0 Src kinase was added to the solutioncontaining other species and the reaction was started [Src] = 4(pink) and 04120583gmL (navy) [P1] = 5 120583M [TbIII

2-L1] = 100120583M

[ATP] = 5120583M and [MnCl2] = 1mM The excitation at 2625 nm

and the emission at 545 nm Reprinted with permission from [69]Copyright 2014 Springer

of pTyr (note that the electrostatic interactions are primarilyresponsible for the binding vide ante) Furthermore the TbIIIcenter and the benzene ring of pTyr are in sufficient proximityfor energy-transfer to occur smoothly In addition to theseenhancements in fluorescence intensity the selectivity ofpTyr detection of the binuclear TbIII complexes with respectto other cosolutes in solutions is kept sufficiently high andcomparable with that of the mononuclear TbIII-DOTAM Byusing these binuclear TbIII complexes Tyr-phosphorylatednonapeptide (P1-pY) was clearly distinguished at pH 7 fromnonphosphorylated nonapeptide (P1)

4 Real-Time Monitoring ofEnzymatic Tyrosine Phosphorylation andDephosphorylation [68 69]

By using TbIII2-L1 the time-course of Tyr phosphorylation

of peptides by PTKs can be straightforwardly monitoredin real-time For example phosphorylation of the tyrosineresidue in the center of a nonapeptide P1 by Src kinase wasanalyzed in Figure 5 To the solution containing TbIII

2-L1

and P1 as well as ATP and MnCl2(essential factors in this

enzymatic reaction) Src tyrosine kinase was added and thenthe luminescence at 545 nm (5D

4rarr7F5transition) wasmea-

sured The luminescence intensity increased time-depend-ently reflecting the Tyr phosphorylation The magnitude ofincrease in luminescence intensity is exactly consistent withthe difference in the concentration of P1 Without the sub-strate peptide the luminescence was never enhanced When

Shp-1 injection

08

09

10

Relat

ive l

umin

esce

nce i

nten

sity

200 400 6000Time (s)

Shp-1 10ngmLShp-1 100ngmL


2-L1 for the dephosphorylation of P1-pY by Shp-1 tyrosine

phosphatase [Shp-1] = 100 (pink) and 10 ngmL (cyan) [P1-pY] =10 120583M and [TbIII

2-L1] = 100 120583M The excitation at 2625 nm and

the emission at 545 nm Reprinted with permission from [69]Copyright 2014 Springer

Tyr-phosphorylated P1 was used as the substrate the lumi-nescence was strong from the beginning and not enhancedeven after the addition of Src kinase In order to confirmthe validity of the method furthermore the rate of thisenzymatic phosphorylation was independently determinedusing TAMRA-labeled P1 There the reaction was stoppedat several reaction times and the products were analyzed bypolyacrylamide gel-electrophoresis The results of these twomethods fairly agreed with each other as expected

Similarly the reverse reactions of the phosphorylationsdephosphorylations of a tyrosine-phosphorylated peptideby PTPs were also visualized by TbIII

2-L1 in real-time

(Figure 6) Here the peptide as kinase substrate was simplysubstituted with the corresponding phosphorylated peptideWhen Shp-1 tyrosine phosphatase was added to the solutioncontaining both P1-pY andTbIII

2-L1 the luminescence inten-

sity gradually decreased The magnitude of luminescencechange was exactly dependent on PTP concentration TheTbIII2-L1 binds relatively weakly to the pTyr residue and

does not much disrupt the phosphatase reactions Still morecomplicated sequential reactions of PTK and PTP were alsomonitored in one-pot fashion (Figure 7) When nonphos-phorylated P1 was first phosphorylated by Src kinase theluminescence intensity gradually increased Then (eg 1500seconds later) Shp-1 phosphatase was added to the solutionThe luminescence decreased due to the dephosphorylationof P1-pY After 300 seconds Src kinase was again added (atthe same time sodium orthovanadate Na

3VO4was added

to the reaction mixture to deactivate Shp-1 phosphatase)The luminescence intensity increased again Apparently thesecond Src kinase reaction was successfully monitored evenwhen the mixture was so complicated and contained many


+Src+Src

+Shp-1

10

15

20

Relat

ive l

umin

esce

nce i

nten

sity

500 1000 1500 2000 2500 30000Time (s)

Na3VO4

Figure 7 One-pot visualization by TbIII2-L1 of the sequential

reactions of a PTK (Src kinase) and a PTP (Shp-1 phosphatase) Tothe reaction solution Src kinase Shp-1 and Na

3VO4(inhibitor of

Shp-1) were added at the timings shown by the arrows Reprintedwith permission from [69] Copyright 2014 Springer

components (the products of the foregoing phosphorylationand dephosphorylation reactions the deactivated Shp-1 andother remaining reagents)

Most of previous kinetic studies on these enzymaticreactions involved either radiolabeling of the peptideATP orchemical labeling of peptidewith chromophoresThe labelingprocedures are time-consuming and still more importantlycould cause nonnegligible perturbations on the enzymaticreactions Compared with these methods the present meth-ods using the TbIII complexes are advantageous in that nolabeling is required and kinetic information can be straight-forwardly obtained Simply by adding the TbIII complexes tothe reactionmixture andmonitoring the photoluminescencethe time-courses of reactions can be directly obtained insitu in real-time Accordingly detailed kinetic results areprecisely obtained even when the enzymatic systems arehighly complicated and the reaction rates do not strictly obeysimple Michaelis-Menten equation (eg allosteric control inthe enzymatic systems and inhibition by other products)

5 Quantitative Evaluation of PTK andPTP Inhibitors Using TbIII-Based ChemicalSensor [69]

There are many kinds of protein tyrosine kinases (PTKs) andprotein tyrosine phosphatases (PTPs) in our bodies Each ofthem takes an important role in the corresponding reactionand is strongly related to various diseases Thus inhibitorson a predetermined enzyme among these PTKsPTPs havebeen regarded as promising targets for drug discovery and thesubject of growing interest This section presents the applica-tion of binuclear TbIII complexes to screening of inhibitorsfrom a pool of candidates As described above the binuclearTbIII complexes can visualize the enzymatic phosphorylation

Table 1 IC50of PTK inhibitors

IC50(nM) Dasatinib Gefitinib Imatinib Staurosporine

Src 12 plusmn 096 gt5000a mdashb 310 plusmn 28Fyn 26 plusmn 22 gt2000a gt10000a 260 plusmn 19EGFR 1400 plusmn 110 22 plusmn 24 mdashb 2300 plusmn 820aNot determined due to poor curve fitting of weak inhibitors bInhibitionwas not observed

and dephosphorylation in a real-time fashion Accordinglythe inhibition activity can be evaluated in terms of bothkinetic aspects and static aspects providing new kinds ofinformation to these fields In this section the specificity andactivity of well-known inhibitors on PTKs and PTPs weredetermined by using TbIII complexes and compared with theliterature data primarily to confirm the validity of method

51 Inhibitors on PTK Using TbIII2-L1 as a chemical sensor

the inhibitory effects of PTK inhibitors on three kinds ofPTKs (Src Fyn and EGFR) were analyzed Among thePTK inhibitors investigated staurosporine is a general kinaseinhibitor with minimum selectivity [131 132] On the otherhand gefitinib is effective only in inhibiting EGFR [133] anddasatinib strongly inhibits the other two PTKs [134] Imatinibis inactive to all of them [135] To the solution containing P1MnCl

2 TbIII

2-L1 and a PTK gefitinib was added and the

reaction was started with the addition of ATPThe inhibitoryeffects were visually analyzed in terms of the real-time kinet-ics Gefitinib drastically suppressed the enzymatic reaction ofEGFR (its target PTK) depending on its concentration (Fig-ure 8) When [gefitinib] = 25 nM the activity of EGFR wasreduced to approximately half In contrast this inhibitor wasmuch less effective to the other two PTKs (Src and Fyn) Evenwith [gefitinib] = 25 120583M (1000-fold larger than EGFR case)they showed sufficient activity The inhibition specificity wascompletely identical with the known specificity

Still more quantitative assay of the activity of inhibitorsin terms of the half maximal inhibitory concentration (IC

50)

was also successful (Figure 9) By the use of time-resolvedluminescence spectroscopy high signal-to-noise ratios wereaccomplished for various combinations of PTKs and theirinhibitors and clear sigmoidal dose-response relationshipswere obtained From these curves the IC

50values of these

inhibitors to the corresponding enzyme were determined(see Table 1) The specificity of all the inhibitors investigatedfairly agreed with that reported in the literature [131] Thepresent method should be very powerful and promising forvarious applications especially when a number of substratesenzymes andor inhibitors are analyzed to screen eminentinhibitors from a pool of candidates

52 Inhibitors on PTP The inhibitory effects of Na3VO4

[136] and 120572-bromo-41015840-hydroxyacetophenone [137] on PTPs(Shp-1 and PTP1B) were investigated using TbIII

2-L1 The

luminescence intensity gradually decreased as the enzymaticdephosphorylation proceeded and the concentration of pTyrdecreased Upon the addition of inhibitors the magnitude


N

N

HNO

O

NO

F

Cl

(a)

EGFR0

08

09

10

11

12

13

14

Relat

ive l

umin

esce

nce i

nten

sity

200 400 6000Time (s)

250nM25nM

25nM

(b)

Figure 8 Real-time monitoring by TbIII2-L1 on the gefitinib inhibition of P1 phosphorylation by protein tyrosine kinase EGFR (a) The

structure of the inhibitor gefitinib In (b) gefitinib was added to the solution containing P1 MnCl2 EGFR kinase and TbIII

2-L1 and the

reaction was started with addition of ATP Gray control without EGFR red control without gefitinib Reproduced with permission from[69] Copyright 2014 Springer

00

05

10

15

minus10 minus8 minus6 minus4log([inhibitor]M)

Relat

ive r

ate o

f rea

ctio

n (sminus

1 )

Figure 9 Plots of the rates of EGFR kinase-catalyzed phospho-rylation of P1 versus the concentrations of various inhibitors Therates of phosphorylation of P1 by the kinase were determined in thepresence of dasatinib (red) gefitinib (blue) imatinib (green) andstaurosporine (black) by the method in Figure 8 The IC

50values

of the inhibitors calculated by fitting these sigmoidal curves arepresented in Table 1 together with the values on Src and Fyn kinasesReproduced with permission from [69] Copyright 2014 Springer

of this decrease became smaller Consistently the change inluminescence intensity by PTP-induced dephosphorylation

of P1-pY depended on the inhibitor concentration The IC50

values calculated from the sigmoidal curves were completelyconsistent with known selectivity of these inhibitors

6 Attempts to Improve the DetectionSensitivity Conjugation of Binuclear TbIII

Complexes to Substrate Peptides [38]

As described above both phosphorylation of tyrosine byPTK and dephosphorylation by PTP are visually monitoredsimply by adding the binuclear TbIII complexes (TbIII

2-

L1 and TbIII2-L2) to the reaction mixtures The method is

simple straightforward and useful However the detectionsensitivity is in some case rather small especially when thepeptide substrates are positively charged There the associa-tion of peptides with the positively charged TbIII complexesis suppressed by electrostatic repulsion and thus the energy-transfer from pTyr to TbIII does not satisfactorily proceed(note that bindings are primarily due to electrostatic inter-actions) As a general strategy to overcome these drawbackssubstrate peptide and the TbIII complex were covalentlyconnected and the intermolecular association between themwas converted to more efficient intramolecular one In orderto facilitate the synthesis of these conjugates a new binuclearTbIII complex (TbIII

2-Lc1yne Figure 10(a)) was prepared by

attaching an alkynyl group to TbIII2-L1 Separately an azido


HN N

H

ON

N N

NTb

O

O

OO

N

N

N

NTb

O

O

O

NHO

O

TbIII2-Lc1yne

H2N

H2N

H2N

NH2

NH2

NH2

(a)

HN

NH

O

N

N N

NTbTb

O

O

O

O

N

N

N

N

O

O

O

NHO

O

N NN

NH 2

O

S

Cys (linker peptide) Abltide

H2N H2N

H2N

NH2 NH2

NH2

(b)

0

10

100

1000

[Abl] (ngmL)

20 40 600Time (min)

0

1

2

3

4

5

6

7

Lum

ines

cenc

e int

ensit

y (a

u)

(c)

Figure 10 (a) TbIII2-Lc1yne and (b) its conjugatewithAbltide peptide prepared by click reaction In (c) phosphorylation ofAbltide by tyrosine

kinases Abl was monitored in real-time using the conjugate by measuring time-resolved luminescence at 545 nm The concentrations of Ablwere 1000 (red) 100 (blue) and 10 ngmL (green) Conditions [the conjugate] = 5 120583M [ATP] = 100120583M [MgCl

2] = 1mM and [NaCl] =

75mM Reproduced with permission from [38] Copyright 2015 American Chemical Society

groupwas bound through a linker to a cysteine residue whichwas additionally introduced to the peptide substrate Theconjugate was easily obtained by click reaction between thealkyne in TbIII

2-Lc1yne and the azido group in the peptide

(Figure 10(b))This strategy can be applied to various peptidesubstrates without significant limitation in the structures

TheTyr phosphorylation ofAbltide (Glu-Ala-Ile-Tyr-Ala-Ala-Pro-Phe-Ala-Lys-Lys-Lys a well-known substrate for Ablkinase) [130] by Abl kinase was monitored (Figure 10(c))At the pH for the phosphorylation this peptide bears netcharge of +2 and the interaction with TbIII

2-L1 was too

weak All the attempts to monitor its phosphorylation usingnonconjugatedTbIII

2-L1 complexwere unsuccessful Accord-

ingly a cysteine residue was introduced to the N-terminusof Abltide and an azido was bound thereto The conjugationof this modified peptide with Tb

2-Lc1yne by click chemistry

proceeded smoothly When the resultant conjugate wastreated with Abl kinase in the presence of ATP the lumi-nescence intensity increased time-dependently due to thephosphorylation of Tyr When the peptide concentration was300 nM the signal-to-noise ratio was 151 being sufficient fordetailed quantitative analysis of the reaction The signal for


the Tyr phosphorylation was clearly observed even when thepeptide concentration was decreased down to 50 nM Similarresults were obtained when Src was used as PTK and theTyr phosphorylation of Abltide was successfully monitoredIn addition to the N-terminus conjugation the Tb

2-Lc1yne

can be also conjugated to the C-terminus of Abltide Theadvantage of the present conjugation strategy is conclusive

The effect of the distance between the TbIII complex andthe pTyr on the monitoring activity of the conjugate wasanalyzed by changing the length of linker peptide between theN-terminus of Abltide and the cysteine residue (1 to 5 aminoacids) The rate of phosphorylation was almost the same forall the five conjugatesTheTbIII complex bound to theAbltideimposesminimal steric hindrance on the enzymatic reactionFurthermore the luminescence increased in almost the samemagnitude (10-fold) upon the phosphorylation Apparentlythe structure of the complex formed between the Tb

2-Lc1yne

and the pTyr residue in the peptide is similar irrespective ofthe length of the linker peptideThis ensures the applicabilityof this strategy to various substrates without strict structuralrestriction

7 Conclusions

The importance of phosphorylation of tyrosine residues inproteins and their dephosphorylation has been well recog-nized and chemical sensors to monitor this phosphorylationselectively have been attracting interests Recently significantprogress has been made in this field A DOTAM complexof TbIII showing very high selectivity to pTyr has beendeveloped The photoemission from TbIII-DOTAM complexis notable only when pTyr exists in the solutions There thebenzene ring of pTyr functions as an antenna and transfers itsphotoexcitation energy to the TbIII ion as the emission centerAccordingly the emission is selective to pTyr since non-phosphorylated tyrosine cannot efficiently bind to the TbIIIcomplex and neither phosphoserine nor phosphothreoninecan satisfactorily provide an antenna effect Furthermorethe binding of bulky cosolutes (eg nucleotides and nucleicacids) to TbIII is suppressed by the steric hindrance ofDOTAM By the use of time-resolved luminescence analysisonly the long life-time luminescence from TbIII is analyzedand high signal-to-noise ratios are accomplished

Binuclear TbIII complexes (TbIII2-L1 and TbIII

2-L2) in

which two TbIII-DOTAM complexes are connected throughthe linkers in the ligands are far more effective in the detec-tion of pTyr than the monomeric TbIII complexThe increasein the sensitivity is primarily ascribed to the stronger bindingof pTyr to these complexes due to enhanced electrostaticinteractions between them With the use of these complexesas sensors phosphorylation of tyrosine by protein tyrosinekinases and dephosphorylation by protein tyrosine phos-phatases are visualized in situ in a real-time fashion Further-more the activities of various inhibitors on these enzymes arequantitatively evaluated by the TbIII complexes This methodshould be useful in screening highly eminent inhibitorsfrom a number of candidates These enzymes take crucially

important biological roles so that the information obtainedby these studies should lead to development of new drugs forthe therapy of relevant diseases By immobilizing these TbIIIcomplexes to some solid supports the applications of thepresent methods should be further facilitated and widened

Disclosure

Current address of Makoto Komiyama is World PremierInternational (WPI) Research Center for Materials Nanoar-chitectonics (MANA) National Institute for Materials Sci-ence (NIMS) 1-1 Namiki Tsukuba Ibaraki 305-0044 Japan

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

This work was partially supported by a Grant-in-Aid forSpecially Promoted Scientific Research (22000007) and aGrant-in-Aid for Scientific Research (A) (15H02189) fromMinistry of Education Culture Sports Science and Technol-ogy Japan

References

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[2] P Cohen ldquoThe origins of protein phosphorylationrdquoNature CellBiology vol 4 no 5 pp E127ndashE130 2002

[3] T Sakamoto A Ojida and I Hamachi ldquoMolecular recognitionfluorescence sensing and biological assay of phosphate anionderivatives using artificial Zn(II)ndashDpa complexesrdquo ChemicalCommunications no 2 pp 141ndash152 2009

[4] A Ojida Y Mito-Oka M-A Inoue and I Hamachi ldquoFirstartificial receptors and chemosensors toward phosphorylatedpeptide in aqueous solutionrdquo Journal of the American ChemicalSociety vol 124 no 22 pp 6256ndash6258 2002

[5] A Ojida Y Mito-oka K Sada and I Hamachi ldquoMolecularrecognition and fluorescence sensing of monophosphorylatedpeptides in aqueous solution by Bis(zinc(II)-dipicolylamine)-based artificial receptorsrdquo Journal of the American ChemicalSociety vol 126 no 8 pp 2454ndash2463 2004

[6] E Kinoshita M Takahashi H Takeda M Shiro and T KoikeldquoRecognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complexrdquoDalton Transactions no 8pp 1189ndash1193 2004

[7] E Kinoshita E Kinoshita-Kikuta K Takiyama and T KoikeldquoPhosphate-binding tag a new tool to visualize phosphorylatedproteinsrdquo Molecular and Cellular Proteomics vol 5 no 4 pp749ndash757 2006

[8] A Riechers F Schmidt S Stadlbauer and B Konig ldquoDetectionof protein phosphorylation on SDS-PAGE using probes witha phosphate-sensitive emission responserdquo Bioconjugate Chem-istry vol 20 no 4 pp 804ndash807 2009


[9] T Zhang N Y Edwards M Bonizzoni and E V Anslyn ldquoTheuse of differential receptors to pattern peptide phosphoryla-tionrdquo Journal of the American Chemical Society vol 131 no 33pp 11976ndash11984 2009

[10] B Schulenberg R Aggeler J M Beechem R A Capaldi andW F Patton ldquoAnalysis of steady-state protein phosphorylationin mitochondria using a novel fluorescent phosphosensor dyerdquoJournal of Biological Chemistry vol 278 no 29 pp 27251ndash272552003

[11] B Schulenberg T N Goodman R Aggeler R A CapaldiandW F Patton ldquoCharacterization of dynamic and steady-stateprotein phosphorylation using a fluorescent phosphoprotein gelstain andmass spectrometryrdquo Electrophoresis vol 25 no 15 pp2526ndash2532 2004

[12] E Pazos and M E Vazquez ldquoAdvances in lanthanide-basedluminescent peptide probes for monitoring the activity ofkinase and phosphataserdquo Biotechnology Journal vol 9 no 2 pp241ndash252 2014

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[14] S J Butler and D Parker ldquoAnion binding in water at lanthanidecentres from structure and selectivity to signalling and sens-ingrdquo Chemical Society Reviews vol 42 no 4 pp 1652ndash16662013

[15] K L Haas and K J Franz ldquoApplication of metal coordinationchemistry to explore and manipulate cell biologyrdquo ChemicalReviews vol 109 no 10 pp 4921ndash4960 2009

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[18] V V Zherdeva and A P Savitsky ldquoUsing lanthanide-basedresonance energy transfer for in vitro and in vivo studies ofbiological processesrdquo Biochemistry vol 77 no 13 pp 1553ndash15742012

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[20] D S Lawrence and Q Wang ldquoSeeing is believing peptide-based fluorescent sensors of protein tyrosine kinase activityrdquoChemBioChem vol 8 no 4 pp 373ndash378 2007

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[23] T Hunter ldquoTyrosine phosphorylation thirty years and count-ingrdquo Current Opinion in Cell Biology vol 21 no 2 pp 140ndash1462009

[24] S Morandell T Stasyk S Skvortsov S Ascher and L A HuberldquoQuantitative proteomics and phosphoproteomics reveal novelinsights into complexity and dynamics of the EGFR signalingnetworkrdquo Proteomics vol 8 no 21 pp 4383ndash4401 2008

[25] A Guo J Villen J Kornhauser et al ldquoSignaling networksassembled by oncogenic EGFR and c-Metrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 2 pp 692ndash697 2008

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[32] Z-X Jiang and Z-Y Zhang ldquoTargeting PTPs with smallmolecule inhibitors in cancer treatmentrdquoCancer andMetastasisReviews vol 27 no 2 pp 263ndash272 2008

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[38] H Akiba J Sumaoka K Tsumoto and M Komiyama ldquoClickconjugation of a binuclear terbium(III) complex for real-timedetection of tyrosine phosphorylationrdquo Analytical Chemistryvol 87 no 7 pp 3834ndash3840 2015

[39] AM LipchikM PerezW Cui and L L Parker ldquoMulticoloredTb3+-based antibody-free detection of multiple tyrosine kinaseactivitiesrdquo Analytical Chemistry vol 87 no 15 pp 7555ndash75582015

[40] M S Tremblay M Lee and D Sames ldquoA luminescent sensorfor tyrosine phosphorylationrdquo Organic Letters vol 10 no 1 pp5ndash8 2008

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[42] M D Shults K A Janes D A Lauffenburger and B ImperialildquoAmultiplexed homogeneous fluorescence-based assay for pro-tein kinase activity in cell lysatesrdquoNature Methods vol 2 no 4pp 277ndash284 2005

[43] E Lukovic J A Gonzalez-Vera and B Imperiali ldquoRecognition-domain focused chemosensors versatile and efficient reporters


of protein kinase activityrdquo Journal of the American ChemicalSociety vol 130 no 38 pp 12821ndash12827 2008

[44] E Lukovic E V Taylor and B Imperiali ldquoMonitoring pro-tein kinases in cellular media with highly selective chimericreportersrdquo Angewandte ChemiemdashInternational Edition vol 48no 37 pp 6828ndash6831 2009

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[46] J R Beck A Lawrence A S Tung E N Harris and C IStains ldquoInterrogating endogenous protein phosphatase activitywith rationally designed chemosensorsrdquoACS Chemical Biologyvol 11 no 1 pp 284ndash290 2016

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sensitizationrdquo Analytical Chemistry vol 85 no 5 pp 2582ndash2588 2013

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[77] AThibon andVC Pierre ldquoPrinciples of responsive lanthanide-based luminescent probes for cellular imagingrdquo Analytical andBioanalytical Chemistry vol 394 no 1 pp 107ndash120 2009

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[79] M Schaferling and O S Wolfbeis ldquoEuropium tetracyclineas a luminescent probe for nucleoside phosphates and itsapplication to the determination of kinase activityrdquo Chemistryvol 13 no 15 pp 4342ndash4349 2007

[80] T P Geladopoulos T G Sotiroudis and A E EvangelopoulosldquoA malachite green colorimetric assay for protein phosphataseactivityrdquoAnalytical Biochemistry vol 192 no 1 pp 112ndash116 1991

[81] E G Moore A P S Samuel and K N Raymond ldquoFromantenna to assay lessons learned in lanthanide luminescencerdquoAccounts of Chemical Research vol 42 no 4 pp 542ndash552 2009

[82] L Armelao SQuici F Barigelletti et al ldquoDesign of luminescentlanthanide complexes frommolecules to highly efficient photo-emitting materialsrdquo Coordination Chemistry Reviews vol 254no 5-6 pp 487ndash505 2010

[83] S Lis M Elbanowski B Makowska and Z Hnatejko ldquoEnergytransfer in solution of lanthanide complexesrdquo Journal of Photo-chemistry and Photobiology A Chemistry vol 150 no 1ndash3 pp233ndash247 2002

[84] N Sabbatini M Guardigli and J-M Lehn ldquoLuminescent lan-thanide complexes as photochemical supramolecular devicesrdquoCoordination Chemistry Reviews vol 123 no 1-2 pp 201ndash2281993

[85] T Gunnlaugsson A J Harte J P Leonard and M Nieuwen-huyzen ldquoThe formation of luminescent supramolecular ternarycomplexes in water delayed luminescence sensing of aromaticcarboxylates using coordinated unsaturated cationic heptaden-tate lanthanide ion complexesrdquo Supramolecular Chemistry vol15 no 7-8 pp 505ndash519 2003

[86] T Gunnlaugsson A J Harte J P Leonard and M Nieuwen-huyzen ldquoDelayed lanthanide luminescence sensing of aromaticcarboxylates using heptadentate triamide Tb(III) cyclen com-plexes the recognition of salicylic acid in waterrdquo ChemicalCommunications no 18 pp 2134ndash2135 2002

[87] A J Harte P Jensen S E Plush P E Kruger and T Gunnlaugs-son ldquoA dinuclear lanthanide complex for the recognition ofbis(carboxylates) formation of terbium(III) luminescent self-assembly ternary complexes in aqueous solutionrdquo InorganicChemistry vol 45 no 23 pp 9465ndash9474 2006

[88] J P Leonard C M G dos Santos S E Plush T McCabeand T Gunnlaugsson ldquopH driven self-assembly of a ternarylanthanide luminescence complex the sensing of anions usinga 120573-diketonate-Eu(III) displacement assayrdquo Chemical Commu-nications no 2 pp 129ndash131 2007

[89] S E Plush and T Gunnlaugsson ldquoLuminescent sensing ofdicarboxylates in water by a bismacrocyclic dinuclear Eu(III)conjugaterdquo Organic Letters vol 9 no 10 pp 1919ndash1922 2007

[90] S E Plush and T Gunnlaugsson ldquoSolution studies of trimetalliclanthanide luminescent anion sensors towards ratiometric

sensing using an internal reference channelrdquo Dalton Transac-tions no 29 pp 3801ndash3804 2008

[91] J Vanek P Lubal P Hermann and P Anzenbacher JrldquoLuminescent sensor for carbonate ion based on lanthanide(III)complexes of 14710-tetraazacyclododecane-147-triacetic acid(DO3A)rdquo Journal of Fluorescence vol 23 no 1 pp 57ndash69 2013

[92] D Parker and J Yu ldquoA pH-insensitive ratiometric chemosensorfor citrate using europium luminescencerdquo Chemical Communi-cations no 25 pp 3141ndash3143 2005

[93] L Wang B Li L Zhang P Li and H Jiang ldquoAn optical anionchemosensor based on a europium complex and its molecularlogic behaviorrdquoDyes and Pigments vol 97 no 1 pp 26ndash31 2013

[94] S J Butler B K McMahon R Pal D Parker and J WWalton ldquoBright mono-aqua europium complexes based ontriazacyclononane that bind anions reversibly and permeatecells efficientlyrdquo ChemistrymdashA European Journal vol 19 no 29pp 9511ndash9517 2013

[95] C Yang J Xu J Li M Lu Y Li and X Wang ldquoAn effi-ciently colorimetric and fluorescent probe of fluoride acetateand phosphate ions based on a novel trinuclear Eu-complexrdquoSensors and Actuators B Chemical vol 196 pp 133ndash139 2014

[96] M Montalti L Prodi N Zaccheroni L Charbonniere LDouce and R Ziessel ldquoA luminescent anion sensor based ona europium hybrid complexrdquo Journal of the American ChemicalSociety vol 123 no 50 pp 12694ndash12695 2001

[97] L J Charbonniere R Ziessel M Montalti et al ldquoLumines-cent lanthanide complexes of a bis-bipyridine-phosphine-oxideligand as tools for anion detectionrdquo Journal of the AmericanChemical Society vol 124 no 26 pp 7779ndash7788 2002

[98] D Zhang M Shi Z Liu F Li T Yi and C Huang ldquoLumi-nescence modulation of a terbium complex with anions andits application as a reagentrdquo European Journal of InorganicChemistry vol 2006 no 11 pp 2277ndash2284 2006

[99] J Hammell L Buttarazzi C-H Huang and J R MorrowldquoEu(III) complexes as anion-responsive luminescent sensorsand paramagnetic chemical exchange saturation transferagentsrdquo Inorganic Chemistry vol 50 no 11 pp 4857ndash4867 2011

[100] M Schaferling T Aaritalo and T Soukka ldquoMultidentate euro-pium chelates as luminoionophores for anion recognitionimpact of ligand design on sensitivity and selectivity and appli-cability to enzymatic assaysrdquo ChemistrymdashA European Journalvol 20 no 18 pp 5298ndash5308 2014

[101] T Yamada S Shinoda and H Tsukube ldquoAnion sensingwith luminescent lanthanide complexes of tris(2-pyridyl-methyl)amines pronounced effects of lanthanide center andligand chirality on anion selectivity and sensitivityrdquo ChemicalCommunications no 11 pp 1218ndash1219 2002

[102] R K Mahajan I Kaur R Kaur et al ldquoAnion receptor functionsof lanthanide tris(120573-diketonate) complexes naked eye detectionand ion-selective electrode determination of Clminus anionrdquo Chem-ical Communications vol 9 no 17 pp 2238ndash2239 2003

[103] Y Kataoka D Paul H Miyake S Shinoda and H Tsukube ldquoAClminus anion-responsive luminescent Eu3+ complex with a chiraltripod ligand substituent effects on ternary complex stoichiom-etry and anion sensing selectivityrdquo Dalton Transactions no 26pp 2784ndash2791 2007

[104] S Shinoda ldquoDynamic cyclen-metal complexes for molecularsensing and chirality signalingrdquo Chemical Society Reviews vol42 no 4 pp 1825ndash1835 2013

[105] S Shinoda and H Tsukube ldquoLuminescent lanthanide com-plexes as analytical tools in anion sensing pH indication andprotein recognitionrdquo Analyst vol 136 no 3 pp 431ndash435 2011


[106] QWang andH Tamiaki ldquoHighly efficient and selective turn-offquenching of ligand-sensitized luminescence from europiumimidazo[45-f]-110-phenanthroline complex by fluoride ionrdquoJournal of Photochemistry and Photobiology A Chemistry vol206 no 2-3 pp 124ndash128 2009

[107] S J Butler ldquoQuantitative determination of fluoride in purewater using luminescent europium complexesrdquo Chemical Com-munications vol 51 no 54 pp 10879ndash10882 2015

[108] C Tan and QWang ldquoTwo novel europium (III) centered anionreceptors and their naked eye detectionsrdquo Synthetic Metals vol162 no 15-16 pp 1416ndash1420 2012

[109] D F Caffrey and T Gunnlaugsson ldquoDisplacement assay detec-tion by a dimeric lanthanide luminescent ternary Tb(iii)-cyclencomplex high selectivity for phosphate and nitrate anionsrdquoDalton Transactions vol 43 no 48 pp 17964ndash17970 2014

[110] W Xu Y Zhou D Huang et al ldquoLuminescent sensing profilesbased on anion-responsive lanthanide(III) quinolinecarboxy-late materials solid-state structures photophysical propertiesand anionic species recognitionrdquo Journal ofMaterials ChemistryC vol 3 no 9 pp 2003ndash2015 2015

[111] C M G dos Santos P B Fernandez S E Plush J P Leonardand T Gunnlaugsson ldquoLanthanide luminescent anion sensingevidence of multiple anion recognition through hydrogenbonding and metal ion coordinationrdquo Chemical Communica-tions no 32 pp 3389ndash3391 2007

[112] C M G Dos Santos and T Gunnlaugsson ldquoThe recognitionof anions using delayed lanthanide luminescence the use ofTb(III) based urea functionalised cyclen complexesrdquo DaltonTransactions no 24 pp 4712ndash4721 2009

[113] S-H Li C-W Yu W-T Yuan and J-G Xu ldquoA lanthanidehybrid cluster as a selective optical chemosensor for phosphate-containing anions in aqueous solutionrdquo Analytical Sciences vol20 no 10 pp 1375ndash1377 2004

[114] Y-W Wang S-B Liu Y-L Yang P-Z Wang A-J Zhangand Y Peng ldquoA terbium(III)-complex-based onndashoff fluorescentchemosensor for phosphate anions in aqueous solution and itsapplication inmolecular logic gatesrdquoACSAppliedMaterials andInterfaces vol 7 no 7 pp 4415ndash4422 2015

[115] S Nadella J Sahoo P S Subramanian A Sahu S Mishra andM Albrecht ldquoSensing of phosphates by using luminescent EuIIIand TbIII complexes application to the microalgal cell Chlorellavulgarisrdquo ChemistrymdashA European Journal vol 20 no 20 pp6047ndash6053 2014

[116] C M Andolina and J R Morrow ldquoLuminescence resonanceenergy transfer in heterodinuclear Ln119868119868119868 complexes for sensingbiologically relevant anionsrdquo European Journal of InorganicChemistry vol 2011 no 1 pp 154ndash164 2011

[117] S Banerjee M Bhuyan and B Konig ldquoTb(III) functionalizedvesicles for phosphate sensing membrane fluidity controls thesensitivityrdquoChemical Communications vol 49 no 50 pp 5681ndash5683 2013

[118] N Shao J Jin G Wang Y Zhang R Yang and J YuanldquoEuropium(III) complex-based luminescent sensing probesfor multi-phosphate anions modulating selectivity by ligandchoicerdquo Chemical Communications no 9 pp 1127ndash1129 2008

[119] S Mameri L J Charbonniere and R F Ziessel ldquoLanthanideATP interaction in water mediated by Luminescent hemispher-ical-shaped complexesrdquo Inorganic Chemistry vol 43 no 6 pp1819ndash1821 2004

[120] E A Weitz J Y Chang A H Rosenfield and V C Pierre ldquoAselective luminescent probe for the direct time-gated detection

of adenosine triphosphaterdquo Journal of the American ChemicalSociety vol 134 no 39 pp 16099ndash16102 2012

[121] E A Weitz J Y Chang A H Rosenfield E A Morrow andV C Pierre ldquoThe basis for the molecular recognition and theselective time-gated luminescence detection of ATP and GTPby a lanthanide complexrdquo Chemical Science vol 4 no 10 pp4052ndash4060 2013

[122] M D Best and E V Anslyn ldquoA fluorescent sensor for 23-bis-phosphoglycerate using a europium tetra-N-oxide bis-bipy-ridine complex for both binding and signaling purposesrdquoChemistrymdashA European Journal vol 9 no 1 pp 51ndash57 2003

[123] P K-L Fu and C Turro ldquoEnergy transfer from nucleic acidsto Tb(III) selective emission enhancement by single DNAmis-matchesrdquo Journal of the American Chemical Society vol 121 no1 pp 1ndash7 1999

[124] D P Ringer S Burchett and D E Kizer ldquoUse of terbium(III)fluorescence enhancement to selectively monitor DNA andRNA guanine residues and their alteration by chemical mod-ificationrdquo Biochemistry vol 17 no 22 pp 4818ndash4825 1978

[125] M D Topal and J R Fresco ldquoFluorescence of terbium ion-nucleic acid complexes a sensitive specific probe for unpairedresidues in nucleic acidsrdquo Biochemistry vol 19 no 24 pp 5531ndash5537 1980

[126] S Amin J R Morrow C H Lake and M R Churchill ldquoLan-thanide(III) tetraamide macrocyclic complexes as syntheticribonucleases structure and catalytic properties of[La(tcmc)(CF

3SO3)(EtOH)](CF

3SO3)2rdquo Angewandte Chem-

iemdashInternational Edition vol 33 no 7 pp 773ndash775 1994[127] S Amin D A Voss Jr W D Horrocks Jr C H Lake

M R Churchill and J R Morrow ldquoLaser-induced lumines-cence studies and crystal structure of the europium(III) com-plex of 14710-tetrakis(carbamoylmethyl)-14710-tetraazacy-clododecane The link between phosphate diester binding andcatalysis by lanthanide(III) macrocyclic complexesrdquo InorganicChemistry vol 34 no 12 pp 3294ndash3300 1995

[128] A Rodrıguez-Rodrıguez D Esteban-Gomez A de Blas etal ldquoSolution structure of Ln(III) complexes with macrocyclicligands through theoretical evaluation of 1H NMR contactshiftsrdquo Inorganic Chemistry vol 51 no 24 pp 13419ndash134292012

[129] Z Baranyai I Banyai E Brucher R Kiraly and E TerrenoldquoKinetics of the formation of [Ln(DOTAM)]3+ complexesrdquoEuropean Journal of Inorganic Chemistry vol 2007 no 23 pp3639ndash3645 2007

[130] Z Songyang K L Carraway III M J Eck et al ldquoCatalyticspecificity of protein-tyrosine kinases is critical for selectivesignallingrdquo Nature vol 373 no 6514 pp 536ndash539 1995

[131] MW Karaman S Herrgard D K Treiber et al ldquoA quantitativeanalysis of kinase inhibitor selectivityrdquo Nature Biotechnologyvol 26 no 1 pp 127ndash132 2008

[132] T Anastassiadis S W Deacon K Devarajan H Ma and JR Peterson ldquoComprehensive assay of kinase catalytic activityreveals features of kinase inhibitor selectivityrdquo Nature Biotech-nology vol 29 no 11 pp 1039ndash1045 2011

[133] F Ciardiello and G Tortora ldquoEGFR antagonists in cancer treat-mentrdquoTheNew England Journal of Medicine vol 358 no 11 pp1160ndash1174 2008

[134] J CMontero S Seoane AOcana andA Pandiella ldquoInhibitionof Src family kinases and receptor tyrosine kinases by dasa-tinib possible combinations in solid tumorsrdquo Clinical CancerResearch vol 17 no 17 pp 5546ndash5552 2011


[135] R Capdeville E Buchdunger J Zimmermann and A MatterldquoGlivec (ST1571 imatinib) a rationally developed targetedanticancer drugrdquo Nature Reviews Drug Discovery vol 1 no 7pp 493ndash502 2002

[136] J A Gordon ldquoUse of vanadate as protein-phosphotyrosinephosphatase inhibitorrdquo Methods in Enzymology vol 201 pp477ndash482 1991

[137] G Arabaci X-C Guo K D Beebe K M Coggeshall andD Pei ldquo120572-Haloacetophenone derivatives as photoreversiblecovalent inhibitors of protein tyrosine phosphatasesrdquo Journal ofthe American Chemical Society vol 121 no 21 pp 5085ndash50861999


OH OPOPhosphorylation

Dephosphorylation

PTKs (protein tyrosine kinases)

PTPs (protein tyrosine phosphatases)

OminusOminus

Figure 1 Phosphorylation of tyrosine residue by protein tyrosinekinases (PTKs) and its dephosphorylation by protein tyrosinephosphatases (PTPs) for the regulation of biological functions ofproteins

role in the regulation of highly important biological functions(differentiation adhesion cycle control endocytosis andmany others) [22 23] In epidermal growth factor receptor(EGFR) its autophosphorylation of a Tyr residue triggerssignal-cascade in cells [24 25] In the downstream therework several Src family kinases which are also controlled bytheir Tyr phosphorylations and in turn phosphorylate Tyrresidues in other proteins [26ndash28] If Tyr phosphorylationis excessive or insufficient serious problems are induced tothe living Therefore PTKs and PTPs are regarded as maintargets in drug discovery [29ndash34] For many years a numberof laboratories developed elegant optical sensors to evaluatethe activities of these enzymes In some of them substratepeptide was conjugated (or fused) to a probe molecule (egTb(III) complexes [35ndash40] Mg(II) complexes [41ndash47] Ca(II)complex [48] Zn(II) complex [49] Cd(II) complex [50] pep-tide derivatives [51 52] and others [53 54])Theother sensorsinvolve noncovalent interactions between a substrate and aprobe (eg Tb(III) ion [55ndash62] Eu(III) complex [63 64]platinum(II) complex [65] and Tb(III) complexes [66ndash69])

Among all the probes investigated lanthanide ions andtheir complexes have been widely and successfully employeddue to their unique light-emitting properties [70ndash77] Thephotoluminescence from these ions has unusually longlife-time (in the order of micro- to milliseconds) and thusthe background signal can be minimized with the use oftime-resolved spectroscopy Alternatively the kinase reac-tions were followed by the disappearance of ATP (source ofthe phosphate group for pTyr) [78 79] whereas the phos-phatase functions were monitored by the production ofphosphoric acid [80] However these analytical methods areoften complicated by the perturbation signals from otherphosphate-containing solutes ATP-dependent reactionsandor phosphate-producing processes in the specimens Inaddition to these chemical sensors antibodies specific to pTyrare widely being used at present for practical applicationsbut their usage has been hampered by high costs rather poorstability and other factors Accordingly chemical probes thatdirectly visualize PTKPTP activity and produce unbiasedsignals are required for further developments of the field

This paper reviews recent developments in optical meth-ods to selectively detect pTyr in proteins The primary con-cerns are high sensitivity of pTyr detection and its sufficientspecificity (with respect to pSer and pThr which exist muchmore abundantly in biological systems) As emission probeslanthanide ions (especially TbIII ion) and their complexes areused By combining unique properties of the emission fromthese metal ions with so-called ldquoantenna effectrdquo the back-ground signals are minimized and only the signal from pTyris selectivelymonitored [67]The detection activity on pTyr isfurther promoted by forming binuclear TbIII complexes [68]With the use of these chemical sensors phosphorylation ofpeptides by PTKs and their dephosphorylation by PTPs arefollowed in a real-time fashion [38 68 69] Applications ofthe methods to screening of efficient inhibitors on PTKs andPTPs are also presented

2 Principle of Selective Detection of pTyr byTb(III) Complexes

The emission from lanthanide ions is intrinsically weaksince the corresponding f-f transitions are Laporte-forbiddenHowever this luminescence is enormously strengthenedwhen a chromophore (ldquoantennardquo) is placed near the lantha-nide ions and transfers its photoexcitation energy to theseemission centers [70ndash77 81ndash91] By combining lanthanidecomplexes with antenna molecules elegant systems to detectvarious anionic guests have been already prepared Sophis-ticated examples include the analysis of carboxylic acidderivatives [85ndash100] halide ions [95ndash98 101ndash108] nitrate ions[96 97 101ndash105 109] and hydrogen sulfate ion [110] Further-more phosphate ion [95ndash99 108ndash116] pyrophosphate [113ndash115] ATP [113ndash115 117ndash121] and other molecules containingphosphate [120ndash122] were also detected by using lanthanidecomplexes Upon the binding of the phosphate group(s)to the complexes the chemical environments around thelanthanide(III) ions were altered inducing a change in theluminescent property of the ionsWith this strategy howeverhighly selective detection of pTyr is rather difficult sincecoexisting phosphate groups in solutions (eg pSer pThrATP and DNA) could show similar effects [123ndash125] Thesefactors aremore critical when a TbIII ion (without any ligand)is used note that nucleotides and nucleic acids are alsoeminent antenna (vide infra) [55 56]

One successful solution to these problems (improvementof the selectivity of pTyr detection with respect to (i) non-phosphorylated Tyr (ii) pSer and pThr and (iii) other coex-isting phosphate-containing biomolecules) is presented inFigure 2 This strategy developed in our laboratory [67] isbased on the fact that both the benzene ring and the phos-phate group are definitely required for the efficient photolu-minescence First the benzene ring of pTyr in the target pep-tides is used as an antenna to enhance the emission from theTbIII center The irradiated light (Ex) is first absorbed by thisbenzene ring and the excitation energy is then transferred toTbIII (ET) Finally the metal center emits luminescence fromits photoexcited state (Em) On the other hand the phosphateis essential to bind to TbIII and places the benzene ring near


OPO

Tb

pTyr

ETEx

Em

Ominus

Ominus









O

N

N N

NTb

O

O

O

TbIII2-DOTAM

H2N

H2N

NH2

NH2

(a)

HN N

H

O

N

N N

N

Tb

O

O

OO

N

N

N

N

Tb

O

O

O

n

TbIII2-L2

n = 2

TbIII2-L1

n = 1

H2N

H2N

H2N

NH2

NH2

NH2

(b)



detection of pTyr

300 330 900

010

PhO

PSe

rTh

rTy

r

Tbli

g onl

y

Glu

cose

-PpThr

pTyr

Trp

AMPpS

er

GM

PCM

PAD

PAT

P

UM

P

20304050607080

Tb-DOTAM

Lum

ines

cenc

e int

ensit

y (545

nm)

Tb2-L1

Tb(III) as TbCl3






2-L1








Src injection

10

15

20

25

Relat

ive l

umin

esce

nce i

nten

sity

400 800 1200 16000Time (s)





2-L1] = 100120583M







2-L1





Shp-1 injection

08

09

10

Relat

ive l

umin

esce

nce i

nten

sity

200 400 6000Time (s)









2-L1 in real-time





3VO4was added



+Src+Src

+Shp-1

10

15

20

Relat

ive l

umin

esce

nce i

nten

sity

500 1000 1500 2000 2500 30000Time (s)

Na3VO4



3VO4(inhibitor of












2 TbIII




50)


50values of these




2-L1 The



N

N

HNO

O

NO

F

Cl

(a)

EGFR0

08

09

10

11

12

13

14

Relat

ive l

umin

esce

nce i

nten

sity

200 400 6000Time (s)

250nM25nM

25nM

(b)



2-L1 and the


00

05

10

15


Relat

ive r

ate o

f rea

ctio

n (sminus

1 )


50values








2-






HN N

H

ON

N N

NTb

O

O

OO

N

N

N

NTb

O

O

O

NHO

O

TbIII2-Lc1yne

H2N

H2N

H2N

NH2

NH2

NH2

(a)

HN

NH

O

N

N N

NTbTb

O

O

O

O

N

N

N

N

O

O

O

NHO

O

N NN

NH 2

O

S


H2N H2N

H2N

NH2 NH2

NH2

(b)

0

10

100

1000

[Abl] (ngmL)

20 40 600Time (min)

0

1

2

3

4

5

6

7

Lum

ines

cenc

e int

ensit

y (a

u)

(c)









2-L1 was too








2-Lc1yne



2-Lc1yne


7 Conclusions



2-L2) in



Disclosure


Competing Interests


Acknowledgments


References







































































































































3SO3)(EtOH)](CF
















OPO

Tb

pTyr

ETEx

Em

Ominus

Ominus









O

N

N N

NTb

O

O

O

TbIII2-DOTAM

H2N

H2N

NH2

NH2

(a)

HN N

H

O

N

N N

N

Tb

O

O

OO

N

N

N

N

Tb

O

O

O

n

TbIII2-L2

n = 2

TbIII2-L1

n = 1

H2N

H2N

H2N

NH2

NH2

NH2

(b)



detection of pTyr

300 330 900

010

PhO

PSe

rTh

rTy

r

Tbli

g onl

y

Glu

cose

-PpThr

pTyr

Trp

AMPpS

er

GM

PCM

PAD

PAT

P

UM

P

20304050607080

Tb-DOTAM

Lum

ines

cenc

e int

ensit

y (545

nm)

Tb2-L1

Tb(III) as TbCl3






2-L1








Src injection

10

15

20

25

Relat

ive l

umin

esce

nce i

nten

sity

400 800 1200 16000Time (s)





2-L1] = 100120583M







2-L1





Shp-1 injection

08

09

10

Relat

ive l

umin

esce

nce i

nten

sity

200 400 6000Time (s)









2-L1 in real-time





3VO4was added



+Src+Src

+Shp-1

10

15

20

Relat

ive l

umin

esce

nce i

nten

sity

500 1000 1500 2000 2500 30000Time (s)

Na3VO4



3VO4(inhibitor of












2 TbIII




50)


50values of these




2-L1 The



N

N

HNO

O

NO

F

Cl

(a)

EGFR0

08

09

10

11

12

13

14

Relat

ive l

umin

esce

nce i

nten

sity

200 400 6000Time (s)

250nM25nM

25nM

(b)



2-L1 and the


00

05

10

15


Relat

ive r

ate o

f rea

ctio

n (sminus

1 )


50values








2-






HN N

H

ON

N N

NTb

O

O

OO

N

N

N

NTb

O

O

O

NHO

O

TbIII2-Lc1yne

H2N

H2N

H2N

NH2

NH2

NH2

(a)

HN

NH

O

N

N N

NTbTb

O

O

O

O

N

N

N

N

O

O

O

NHO

O

N NN

NH 2

O

S


H2N H2N

H2N

NH2 NH2

NH2

(b)

0

10

100

1000

[Abl] (ngmL)

20 40 600Time (min)

0

1

2

3

4

5

6

7

Lum

ines

cenc

e int

ensit

y (a

u)

(c)









2-L1 was too








2-Lc1yne



2-Lc1yne


7 Conclusions



2-L2) in



Disclosure


Competing Interests


Acknowledgments


References







































































































































3SO3)(EtOH)](CF
















O

N

N N

NTb

O

O

O

TbIII2-DOTAM

H2N

H2N

NH2

NH2

(a)

HN N

H

O

N

N N

N

Tb

O

O

OO

N

N

N

N

Tb

O

O

O

n

TbIII2-L2

n = 2

TbIII2-L1

n = 1

H2N

H2N

H2N

NH2

NH2

NH2

(b)



detection of pTyr

300 330 900

010

PhO

PSe

rTh

rTy

r

Tbli

g onl

y

Glu

cose

-PpThr

pTyr

Trp

AMPpS

er

GM

PCM

PAD

PAT

P

UM

P

20304050607080

Tb-DOTAM

Lum

ines

cenc

e int

ensit

y (545

nm)

Tb2-L1

Tb(III) as TbCl3






2-L1








Src injection

10

15

20

25

Relat

ive l

umin

esce

nce i

nten

sity

400 800 1200 16000Time (s)





2-L1] = 100120583M







2-L1





Shp-1 injection

08

09

10

Relat

ive l

umin

esce

nce i

nten

sity

200 400 6000Time (s)









2-L1 in real-time





3VO4was added



+Src+Src

+Shp-1

10

15

20

Relat

ive l

umin

esce

nce i

nten

sity

500 1000 1500 2000 2500 30000Time (s)

Na3VO4



3VO4(inhibitor of












2 TbIII




50)


50values of these




2-L1 The



N

N

HNO

O

NO

F

Cl

(a)

EGFR0

08

09

10

11

12

13

14

Relat

ive l

umin

esce

nce i

nten

sity

200 400 6000Time (s)

250nM25nM

25nM

(b)



2-L1 and the


00

05

10

15


Relat

ive r

ate o

f rea

ctio

n (sminus

1 )


50values








2-






HN N

H

ON

N N

NTb

O

O

OO

N

N

N

NTb

O

O

O

NHO

O

TbIII2-Lc1yne

H2N

H2N

H2N

NH2

NH2

NH2

(a)

HN

NH

O

N

N N

NTbTb

O

O

O

O

N

N

N

N

O

O

O

NHO

O

N NN

NH 2

O

S


H2N H2N

H2N

NH2 NH2

NH2

(b)

0

10

100

1000

[Abl] (ngmL)

20 40 600Time (min)

0

1

2

3

4

5

6

7

Lum

ines

cenc

e int

ensit

y (a

u)

(c)









2-L1 was too








2-Lc1yne



2-Lc1yne


7 Conclusions



2-L2) in



Disclosure


Competing Interests


Acknowledgments


References







































































































































3SO3)(EtOH)](CF
















Src injection

10

15

20

25

Relat

ive l

umin

esce

nce i

nten

sity

400 800 1200 16000Time (s)





2-L1] = 100120583M







2-L1





Shp-1 injection

08

09

10

Relat

ive l

umin

esce

nce i

nten

sity

200 400 6000Time (s)









2-L1 in real-time





3VO4was added



+Src+Src

+Shp-1

10

15

20

Relat

ive l

umin

esce

nce i

nten

sity

500 1000 1500 2000 2500 30000Time (s)

Na3VO4



3VO4(inhibitor of












2 TbIII




50)


50values of these




2-L1 The



N

N

HNO

O

NO

F

Cl

(a)

EGFR0

08

09

10

11

12

13

14

Relat

ive l

umin

esce

nce i

nten

sity

200 400 6000Time (s)

250nM25nM

25nM

(b)



2-L1 and the


00

05

10

15


Relat

ive r

ate o

f rea

ctio

n (sminus

1 )


50values








2-






HN N

H

ON

N N

NTb

O

O

OO

N

N

N

NTb

O

O

O

NHO

O

TbIII2-Lc1yne

H2N

H2N

H2N

NH2

NH2

NH2

(a)

HN

NH

O

N

N N

NTbTb

O

O

O

O

N

N

N

N

O

O

O

NHO

O

N NN

NH 2

O

S


H2N H2N

H2N

NH2 NH2

NH2

(b)

0

10

100

1000

[Abl] (ngmL)

20 40 600Time (min)

0

1

2

3

4

5

6

7

Lum

ines

cenc

e int

ensit

y (a

u)

(c)









2-L1 was too








2-Lc1yne



2-Lc1yne


7 Conclusions



2-L2) in



Disclosure


Competing Interests


Acknowledgments


References







































































































































3SO3)(EtOH)](CF
















+Src+Src

+Shp-1

10

15

20

Relat

ive l

umin

esce

nce i

nten

sity

500 1000 1500 2000 2500 30000Time (s)

Na3VO4



3VO4(inhibitor of












2 TbIII




50)


50values of these




2-L1 The



N

N

HNO

O

NO

F

Cl

(a)

EGFR0

08

09

10

11

12

13

14

Relat

ive l

umin

esce

nce i

nten

sity

200 400 6000Time (s)

250nM25nM

25nM

(b)



2-L1 and the


00

05

10

15


Relat

ive r

ate o

f rea

ctio

n (sminus

1 )


50values








2-






HN N

H

ON

N N

NTb

O

O

OO

N

N

N

NTb

O

O

O

NHO

O

TbIII2-Lc1yne

H2N

H2N

H2N

NH2

NH2

NH2

(a)

HN

NH

O

N

N N

NTbTb

O

O

O

O

N

N

N

N

O

O

O

NHO

O

N NN

NH 2

O

S


H2N H2N

H2N

NH2 NH2

NH2

(b)

0

10

100

1000

[Abl] (ngmL)

20 40 600Time (min)

0

1

2

3

4

5

6

7

Lum

ines

cenc

e int

ensit

y (a

u)

(c)









2-L1 was too








2-Lc1yne



2-Lc1yne


7 Conclusions



2-L2) in



Disclosure


Competing Interests


Acknowledgments


References







































































































































3SO3)(EtOH)](CF
















N

N

HNO

O

NO

F

Cl

(a)

EGFR0

08

09

10

11

12

13

14

Relat

ive l

umin

esce

nce i

nten

sity

200 400 6000Time (s)

250nM25nM

25nM

(b)



2-L1 and the


00

05

10

15


Relat

ive r

ate o

f rea

ctio

n (sminus

1 )


50values








2-






HN N

H

ON

N N

NTb

O

O

OO

N

N

N

NTb

O

O

O

NHO

O

TbIII2-Lc1yne

H2N

H2N

H2N

NH2

NH2

NH2

(a)

HN

NH

O

N

N N

NTbTb

O

O

O

O

N

N

N

N

O

O

O

NHO

O

N NN

NH 2

O

S


H2N H2N

H2N

NH2 NH2

NH2

(b)

0

10

100

1000

[Abl] (ngmL)

20 40 600Time (min)

0

1

2

3

4

5

6

7

Lum

ines

cenc

e int

ensit

y (a

u)

(c)









2-L1 was too








2-Lc1yne



2-Lc1yne


7 Conclusions



2-L2) in



Disclosure


Competing Interests


Acknowledgments


References







































































































































3SO3)(EtOH)](CF
















HN N

H

ON

N N

NTb

O

O

OO

N

N

N

NTb

O

O

O

NHO

O

TbIII2-Lc1yne

H2N

H2N

H2N

NH2

NH2

NH2

(a)

HN

NH

O

N

N N

NTbTb

O

O

O

O

N

N

N

N

O

O

O

NHO

O

N NN

NH 2

O

S


H2N H2N

H2N

NH2 NH2

NH2

(b)

0

10

100

1000

[Abl] (ngmL)

20 40 600Time (min)

0

1

2

3

4

5

6

7

Lum

ines

cenc

e int

ensit

y (a

u)

(c)









2-L1 was too








2-Lc1yne



2-Lc1yne


7 Conclusions



2-L2) in



Disclosure


Competing Interests


Acknowledgments


References







































































































































3SO3)(EtOH)](CF

















2-Lc1yne



2-Lc1yne


7 Conclusions



2-L2) in



Disclosure


Competing Interests


Acknowledgments


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