Excited-state dynamics in borylated arylisoquinoline complexes in solution and in cellulo

Exci ed-S a e Dynamics in Bo yla ed A ylisoquinoline Complexes in Solu ion and in cellulo Tingxiang Yang,[a, b] Abha Vala alka ,[a, b] An onio Rome o-A enas,[d] Anindi a Dasgup a,[a, c] Pa ick Then,[a, c] A inash Che i,[a, b] Ch is ian Eggeling,[a, c, e, h] Abel Ros,*[d] Uwe Pischel,*[ ] and Benjamin Die zek-I anšić*[a, b, h] Abs ac : Two ou -coo dina e o ganobo on N,C-chela e com- plexes wi h di e en unc ional e minals on he PEG chains a e s udied wi h espec o hei pho ophysical p ope ies wi hin human MCF-7 cells. Thei exci ed-s a e p ope ies a e cha ac e ized by ime- esol ed pump-p obe spec oscopy and luo escence li e ime mic oscopy. The exci ed-s a e elaxa ion dynamics o he wo complexes a e simila when s udied in DMSO. Agg ega ion o he complexes wi h he ca boxyla e e minal g oup is obse ed in wa e . When s udying he ligh -d i en exci ed-s a e dynamics o bo h complexes in cellulo, i.e., a e being aken up in o human MCF-7 cells, bo h complexes show di e en ea u es depend- ing on he na u e o he ancho ing PEG chains. The li e ime o a cha ac e is ic in amolecula cha ge- ans e s a e is signi i- can ly sho e when s udied in cellulo (360�170 ps) as compa ed o in DMSO (~960 ps) a 600 nm o he complexes wi h an amino g oup. Howe e , he kine ics o he complexes wi h he ca boxyla e g oup a e in line wi h hose eco ded in DMSO. On he o he hand, he li e imes o he luo escen s a e a e almos iden ical o bo h complexes in cellulo. These indings unde line he impo ance o e alua e he exci ed- s a e p ope ies o luo opho es in a complex biological en i onmen in o de o ully accoun o in a- and in e molecula e ec s go e ning he ligh -induced p ocesses in unc ional dyes. In oduc ion Fou -coo dina e o ganobo on complexes a e a ac i e lumino- pho es o bioimaging, due o hei highly emissi e na u e.[1] Consequen ly, he pho ophysical p ope ies, o example ab- so p ion and emission spec a in solu ion, as well as when aken up in o cells ha e been s udied.[2] Spec oscopic s udies e ealed ha he exci ed-s a e p ope ies o such dyes a e o en de e mined by he p esence o in amolecula cha ge- ans e (ICT) ansi ions, which a e highly sensi i e o he local en i on- men o he luo opho e.[2b,3] A sho ened ICT li e ime is p esen in pola sol en s o some o he ou -coo dina e N,Co gano- bo on complexes.[4] The pola i y dependence o he ICT s a es causes a ed-shi ed and weakened emission as he pola i y o he su ounding en i onmen inc eases.[5] Sol en -induced ag- g ega ion also impac s he luo escence o such o ganobo on compounds.[6] In eg a ing an elec on-dono -subs i u ed a ylisoquinoline as a chela e ligand, he ICT s a e, in which cha ge densi y is shi ed o he isoquinoline pa , cons i u es an ‘ene gy sink’ and causes he la ge S okes shi s bene icial o bioimaging.[7] Compounds ollowing such design p inciples ha e been syn hesized and s udied o bioimaging by some o us:[7a,8] Howe e , an in-dep h ime- esol ed pho ophysical s udy o he [a] T. Yang, A. Vala alka , A. Dasgup a, D . P. Then, A. Che i, P o . D . C. Eggeling, P o . D . B. Die zek-I anšić Leibniz Ins i u e o Pho onic Technology (Leibniz-IPHT) Albe -Eins ein-S aße 9, 07745 Jena (Ge many) E-mail: [email p o ec ed] [b] T. Yang, A. Vala alka , A. Che i, P o . D . B. Die zek-I anšić Ins i u e o Physical Chemis y F ied ich Schille Uni e si y Jena Helmhol zweg 4, 07743 Jena (Ge many) [c] A. Dasgup a, D . P. Then, P o . D . C. Eggeling Ins i u e o Applied Op ics and Biophysics F ied ich Schille Uni e si y Jena Helmhol zweg 4, 07743 Jena (Ge many) [d] D . A. Rome o-A enas, D . A. Ros Ins i u e o Chemical Resea ch CSIC-US and Inno a ion Cen e in Ad anced Chemis y, ORFEO-CINQA C/Amé ico Vespucio 49, 41092 Se ille (Spain) E-mail: [email p o ec ed] [e] P o . D . C. Eggeling Membe o he Leibniz Cen e o Pho onics in In ec ion Resea ch (LPI), Jena (Ge many) [ ] P o . D . U. Pischel CIQSO-Cen e o Resea ch in Sus ainable Chemis y and Depa men o Chemis y Uni e si y o Huel a Campus de El Ca men, s/n, 21071 Huel a (Spain) E-mail: [email p o ec ed] [h] P o . D . C. Eggeling, P o . D . B. Die zek-I anšić Jena Cen e o So Ma e (JCSM), Philosophenweg 7, D-07743 Jena Suppo ing in o ma ion o his a icle is a ailable on he WWW unde h ps://doi.o g/10.1002/chem.202203468 © 2022 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH. This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion Non-Comme cial NoDe i s License, which pe mi s use and dis ibu ion in any medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non-comme cial and no modi ica ions o adap a ions a e made. Chemis y—A Eu opean Jou nal www.chemeu j.o g Resea ch A icle doi.o g/10.1002/chem.202203468 Chem. Eu . J. 2023,29, e202203468 (1 o 7) © 2022 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH Mon ag, 06.03.2023 2316 / 287915 [S. 103/109] 1 complexes in he en i onmen o li e human cells has no been epo ed so a . In his con ibu ion, we epo on ime- esol ed spec o- scopic s udies o wo bo yla ed a ylisoquinoline dyes. The complexes bea polye hylene glycol (PEG) chains wi h di e en unc ional e minal g oups (NH2,COOH, Scheme 1) o imp o e hei wa e solubili y.[9] This s udy p esen s a new iewpoin on spec oscopic-mechanis ic esea ch on o gano- bo on luo opho es by obse ing he ul a as ligh -induced dynamics when inco po a ed in o li e human cance cells (MCF- 7) and hence, explo ing he e ec o he dyes’ in e ac ion wi h he biological en i onmen . Resul s and Discussion The dyes 1and 2we e p epa ed in good yields (50–70%) by coupling o he co esponding alkyne (6)[7a] o azide de i a i es (7) o he bo yla ed a ylisoquinolines wi h PEG-azide o PEG- alkyne de i a i es, espec i ely, using a coppe -ca alyzed click eac ion (see Scheme 2). The dyes we e cha ac e ized by 1H, 13C, and 11B NMR spec oscopy as well as by high- esolu ion mass spec ome y (see Suppo ing In o ma ion). The discussion o he pho ophysical p ope ies o he in es iga ed o ganobo on dyes s a s wi h he cha ac e iza ion o hei s eady-s a e abso p ion and emission. The abso p ion spec a o 1and 2show simila shapes (Figu e 1) wi h maxima a 423 and 421 nm (Table 1) in DMSO, espec i ely, i.e., he PEG subs i uen s do no al e he abso p ion and emission spec a o he luo opho e signi ican ly.[10] The spec um o 1in wa e includes a small low-ene gy ail a ~480 nm, which is no p esen o 2. Such sol en dependence can also be obse ed in he emission spec um: in wa e , he emission spec um o 1 becomes mo e s uc u ed wi h a maximum a 521 nm and a shoulde a 550–600 nm (Figu e 1). These spec oscopic di e - ences be ween 1and 2indica e he lowe wa e solubili y o 1 and i s endency o o m agg ega es.[11] The luo escence quan um yields (Φ luo) o 1and 2in wa e a e simila , 0.10 and 0.15, espec i ely. Howe e , in DMSO he compounds exhibi signi ican ly highe Φ luo, i.e., >0.9 (see Table 1). The di e ence Scheme 1. S uc u es o he complexes 1and 2used in his wo k. Scheme 2. Syn hesis o he dyes 1and 2. Fo he compounds 3–6see e e ence[7a]. Chemis y—A Eu opean Jou nal Resea ch A icle doi.o g/10.1002/chem.202203468 Chem. Eu . J. 2023,29, e202203468 (2 o 7) © 2022 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH Mon ag, 06.03.2023 2316 / 287915 [S. 104/109] 1 15213765, 2023, 16, Downloaded om h ps://chemis y-eu ope.onlinelib a y.wiley.com/doi/10.1002/chem.202203468 by Uni e sidad De Se illa, Wiley Online Lib a y on [22/05/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License Φ luo in DMSO and wa e is also e lec ed in he luo escence li e ime kine ics (Table 1 and Figu e S2). O e all, he luo escence li e imes in wa e o 1and 2a e much sho e han hose eco ded in DMSO. In addi ion a biexponen ial decay o he luo escence o he compounds in wa e is obse ed. This migh be an indica ion o agg ega ion aking place in wa e , signi ican ly sho ening he li e ime o he emission and ende ing i s decay mo e complex. T ansien Abso p ion (TA) Spec oscopy Be o e conside ing in cellulo ansien abso p ion kine ics o 1 and 2in MCF-7 cells (see below), we eco ded ansien abso p ion da a in solu ion. As bo h complexes show e y simila ansien abso p ion signa u es in DMSO solu ion (Fig- u e S3, S4 and S6), we will discuss he da a ob ained o 2 (Figu e 2a and 2d o s- ansien abso p ion da a). The da a o 1can be ound in he Suppo ing In o ma ion (see Figu e S3). The s- ansien abso p ion da a show wo posi i e ΔAbs bands a ca. 450 and 600 nm, while wo minima a e isible a ca. 420 and 510 nm (Figu e 2a). The nega i e band a ca. 420 nm Figu e 1. No malized UV/ is abso p ion (solid line) and emission (do line) spec a (λex =420 nm) o 1( op) and 2(bo om) in DMSO ( ed) and in wa e (con aining 0.5 ol% DMSO, blue). Table 1. Pho ophysical p ope ies o 1and 2. Dye Sol en λabs [nm][a] λem [nm][b] Φ luo [c] τ luo [ns][d] A[ ] k [s1][g] 1 DMSO 423 501 0.93 7.1 100% 1.3×108 wa e 423 521 0.10 0.5/1.5[e] 62%/38% 6×107 2 DMSO 421 497 0.99 7.2 100% 1.4×108 wa e 421 498 0.15 0.3/1.9[e] 76%/ 24% 8×107 [a] Maximum o he s eady-s a e abso p ion band. [b] Maximum o he emission band. [c] Absolu e luo escence quan um yield measu ed wi h an in eg a ing sphe e. [d] Fluo escence li e ime. [e] A bi-exponen ial decay o he espec i e signal is obse ed. [ ] aiis he ampli ude o he co esponding luo escence li e ime componen , and A is he pe cen age weigh o he ampli ude, A¼ai=ða1þa2Þ�100 %. [g] Radia i e decay a e cons an , k =Φ luo/ τ luo. Figu e 2. (a) s-T ansien abso p ion spec a o 2in DMSO wi h λex =400 nm a di e en delay imes as well as he ansien abso p ion spec um o 1a 10 ps in g ey; (b) Kine ic aces o selec ed wa eleng hs in he 2-ns ime window; (c) Decay associa ed spec a o 2. (d) T ansien abso p ion spec a o 2in DMSO in 10-ns ime window; (e) Kine ic aces o 2a 450, 485, 505 and 600 nm eco ded in DMSO in 10/ns ime window; ( ) Kine ics o he decay a 600 and 700 nm o 2in deae a ed and ae a ed condi ions. The spec a a e smoo hed by he Sa i zky-Gola y me hod o educe he elec onic noise ( he o iginal da a a e shown in Figu e S3, S4, and S5). Concen a ion: 20 μM. Chemis y—A Eu opean Jou nal Resea ch A icle doi.o g/10.1002/chem.202203468 Chem. Eu . J. 2023,29, e202203468 (3 o 7) © 2022 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH Mon ag, 06.03.2023 2316 / 287915 [S. 105/109] 1 15213765, 2023, 16, Downloaded om h ps://chemis y-eu ope.onlinelib a y.wiley.com/doi/10.1002/chem.202203468 by Uni e sidad De Se illa, Wiley Online Lib a y on [22/05/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License coincides wi h he in e ed s eady-s a e abso p ion spec um and e lec s he g ound-s a e bleach (GSB). The nega i e band be ween 470–550 nm spec ally conincides wi h s imula ed emission as seen om he in e ed emission spec um. Du ing he i s ca. 50 ps a e pho oexci a ion, his nega i e di e en ial abso p ion band shi s om 490 o 510 nm. A he same ime he p ominen exci ed-s a e abso p ion (ESA) band a he wa eleng h longe han 550 nm dec eases, while di e en ial abso p ion ea u es a 450 nm and 700 nm inc ease (Figu e 2b). The changes a e highligh ed in he decay-associa ed spec a (DAS) (Figu e 2c), which a e based on mul i-exponen ial i ing o he expe imen al da a. The ime cons an s shown on DAS a e de e mined by global i ing o he ansien abso p ion da a, i.e., he kine ic aces a all p obe-wa eleng hs. The p e iously desc ibed spec al changes a e associa ed wi h wo decay componen s in he i , cha ac e ized by ime cons an s o 0.5 and 7 ps. We in e p e he p ocesses as a elaxa ion o he sys em om he F anck-Condon egion ( ep esen ing a locally exci ed s a e, LE) popula ing an ICT exci ed s a e. In he la e s a e elec on densi y is shi ed om he alkoxynaph halene uni o he isoquinoline moie y o he complex.[7a] Simila assignmen s ha e been done o ela ed N,Co ganobo on chela es.[7a,12] This ICT s a e is emissi e, as indica ed by he s ong nega i e di e en ial abso p ion band in he spec al egion o he complex’s luo escence a 500–510 nm. I s decay appea s o be biexponen ial wi h wo e y simila ly shaped DAS in he kine ic modelling (see Figu e 2c). While he sho e componen , e lec ing he decay o he ICT s a e, is associa ed wi h a cha ac e is ic ime cons an o 960 ps, he esidual decay is oo slow o be app op ia ely modelled om he s- ansien abso p ion da a eco ded on a 2 ns delay line. Howe e , ansien abso p ion da a eco ded on a 10-ns delay line e eal a 7.4 ns decay componen du ing which he s imula ed emission ea u es disappea (see Figu e S5), while a new ESA band a 485 nm o ms (see Figu e 2d, 2e and Figu e S5). Thus, we associa e he 7.4 ns p ocess wi h 1ICT!3ICT in e sys em c ossing (ISC) and hence, quenching o he emission.[12] The o ma ion o a iple s a e is co obo a ed by he oxygen sensi i i y o he ansien , obse ed on he μs imescale (Figu e 2 ): he popula ion o he long-li ed s a e is e ec i ely quenched unde ae a ed condi ions; he li e ime unde deae - a ed condi ion is 15 μs, whe eas i sho ens o ca. 1.9 μs in ae a ed DMSO. To es ima e he o de o magni ude o he ISC quan um yield (ΦISC) om he ansien abso p ion da a we ega d he ansien abso p ion signal eco ded a e 7 μs. Conside ing he single ICT li e ime o 7.4 ns, he exci ed-s a e popula ion emaining a e 7 μs will be he popula ion o he iple s a e (see he Suppo ing In o ma ion o de ails). Taking in o accoun he numbe o pho ons pe pulse and he molecula abso p ion c oss sec ion as well as he expe imen ally obse able GSB a 7 μs, we conclude ha ΦISC is ca. 0.2 o 1 and 0.1 o 2. This de ines a conse a i e es ima e o ΦISC. Taking in o accoun he e o ma gin o Φ luo and ΦISC (ca. 10– 20% e o ), i emains clea ha no o he exci ed-s a e p ocesses in e e e in he decay o he ICT s a e, because bo h quan i ies add up o abou 100%. The main exci ed-s a e pa hways, discussed abo e, a e summa ized in Scheme 3. In acellula Pho ophysics To in es iga e he impac o he local en i onmen on he exci ed s a e elaxa ion p ocesses when 1and 2a e aken up in o human b eas cance cells, MCF-7 cells we e dosed wi h 20 μM o ei he o he dyes. The up ake o 1and 2in o MCF- 7 cells was moni o ed by La ice-SIM mic oscopy a e a 3-h incuba ion pe iod (Figu e 3 and Figu e S8). The compounds show e ec i e pe meabili y in o he cells and a e mainly localized in he cy oplasm. Simila obse a ions we e made o o he ou -coo dina e o ganobo on complexes wi h he po en- ial o be used in bioimaging.[7a,13] Figu e 3 u he indica es ha Scheme 3. Pho ophysical model o 2in DMSO. Figu e 3. Cellula accumula ion o 1(a) and 2(b), scale ba : 10 μm. (c) and (d) a e he zoom-in images a he selec ed posi ions o 1and 2, espec i ely (scale ba : 2.5 μm). Rep esen a i e La ice-SIM images o li e MCF-7 cells. Chemis y—A Eu opean Jou nal Resea ch A icle doi.o g/10.1002/chem.202203468 Chem. Eu . J. 2023,29, e202203468 (4 o 7) © 2022 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH Mon ag, 06.03.2023 2316 / 287915 [S. 106/109] 1 15213765, 2023, 16, Downloaded om h ps://chemis y-eu ope.onlinelib a y.wiley.com/doi/10.1002/chem.202203468 by Uni e sidad De Se illa, Wiley Online Lib a y on [22/05/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 1and 2exhibi a endency o bind o he nuclea memb ane, as highligh ed by he s ong la ge ing-like cen al pa e n in he La ice-SIM images, which is sligh ly mo e p onounced o 1 compa ed o 2. Apa om he nuclea memb ane s aining, he images disclose di e en cy osolic pa e ns o bo h com- pounds, sugges ing ha hey bind o sligh ly di e en componen s wi hin he cellula en i onmen . 1shows many oundish pa e ns, cha ac e is ic o s aining o he memb ane o cy osolic esicles, whe eas 2elici s do ed pa e ns indica ing s aining o he ma ix o such esicle. The luo escence li e imes o 1and 2in cellulo we e ob ained by luo escence-li e ime imaging (FLIM). 1and 2 ea u e a simila dis ibu ion o emission li e ime in he ange o 4–8 ns and an a e age emission li e ime o ~6 ns (Figu e 4). This in cellulo emission li e ime co esponds o he luo escence li e ime o he dyes in DMSO (ca. 7�0.2 ns), which indica es ha he adia i e pa hways o 1and 2a e no signi ican ly a ec ed by he local en i onmen in cellulo. No iceably, he emission li e imes do no esemble hose eco ded in wa e , indica ing ha he dyes’ endency o agg ega e in wa e is coun e balanced in he cellula en i onmen ,[7a,14] and hence, emission li e imes esembling hose o isola ed 1and 2a e obse ed. To in e oga e he ul a as dynamics, i.e., wi h he highe lying elec onic s a e o he exci ed-s a e po en ial ene gy su aces o he dyes in MCF-7 cells, 1and 2we e exci ed a 400 nm in cellulo and he pho oinduced dynamics we e s udied a a ious p obe wa eleng hs in he isible spec al ange. This echnique is a he singula and has been used o only ew examples epo ed in he li e a u e.[15] The da a and insigh s ha a e ob ained a e o high alue o dye design and p o ide a e y de ailed pic u e o he dyes’ pho ophysics in he ac ual cellula en i onmen . To check i he cells we e damaged by he ligh -induced p ocesses, ypan blue s aining was used as a li e/ dead cells agen a e each o he measu emen s. No signi ican numbe o cells s ained by ypan blue was obse ed (Fig- u e S15), e lec ing he only ma ginal pho ocy o oxici y o he complexes unde he expe imen al condi ions used. Figu e 5 shows he ansien abso p ion kine ics eco ded om li e MCF-7 cells dosed wi h 1( i s ow) o 2(second ow). The hi d ow in Figu e 5 epo s on con ol measu emen s, essen ially showing he ansien abso p ion backg ound, which is ob ained om li e uns ained MCF-7 ( he small ea u e a pulse o e lap s ems om he cohe en a i ac ).[16] The highes signal- o-noise a io o he in cellulo ansien abso p ion kine ics is ob ained wi h a p obe wa eleng h o 600 nm. This is in line wi h he ansien abso p ion spec um o 1and 2 eco ded in DMSO, which peaks a his wa eleng h. Howe e , also a di e en p obe wa eleng hs he kine ics can be esol ed, i.e., p obing a 580 nm as well as 650 nm yields posi i e di e en ial abso p ion kine ics in cellulo. These expe i- men s allowed o cap u ing exci ed-s a e abso p ion kine ics o he LE and ICT s a es (Figu e 5). The kine ic aces o 1a e in good ag eemen wi h hose eco ded in DMSO a hese h ee p obe wa eleng hs, indica ing ha he o e all na u e o he LE s a e and ICT s a e is no impac ed by he local en i onmen p o ided by li e MCF-7 cells in his pa icula example. On he con a y, di e ences a e obse ed when compa ing he an- sien abso p ion kine ics eco ded o 2a 600 nm in cellulo and in DMSO. The kine ics indica e ha he sub-ns cha ac e is ic ime cons an e lec ing he as componen o he decay o he emissi e ICT s a e appea s sho ened om abou 960 ps in DMSO o 360�170 ps (Figu e S13). The signal- o-noise a io o he kine ics eco ded a 650 nm is insu icien o conclude i he Figu e 4. FLIM mic og aphs o 1(a) and 2(b), espec i ely (Scale ba : 5 μm). (c) Fluo escence li e ime dis ibu ion o 1and 2. Figu e 5. Kine ic aces o 1( i s ow) and 2(second ow) a selec ed wa eleng hs we e measu ed wi h li e MCF-7 cells (cyan line), and eco ded in DMSO (blue line). The dash black line is he i da a. The con ol expe imen s (only MCF-7 cells wi hou complexes) a e shown on he hi d ow (g ey line). Chemis y—A Eu opean Jou nal Resea ch A icle doi.o g/10.1002/chem.202203468 Chem. Eu . J. 2023,29, e202203468 (5 o 7) © 2022 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH Mon ag, 06.03.2023 2316 / 287915 [S. 107/109] 1 15213765, 2023, 16, Downloaded om h ps://chemis y-eu ope.onlinelib a y.wiley.com/doi/10.1002/chem.202203468 by Uni e sidad De Se illa, Wiley Online Lib a y on [22/05/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License same obse a ion can be made a his wa eleng h (see Figu e S14). By p obing he exci ed-s a e kine ics a 515 nm we expec ed o see also con ibu ions om s imula ed emission based on he ansien abso p ion spec a eco ded in DMSO. Fo 2in li e cells, sligh indica ions o such a nega i e di e en ial abso p ion signal a e obse ed, while he low signal- o-noise a io does no allow o such conclusions when s udying 1in MCF-7 cells. The di e ence in he decay o he ICT s a e when compa ing 1and 2(see de ia ions in he ansien abso p ion kine ics measu ed in cellulo a 515 and 600 nm) p o ides an indica ion ha he di e en local en i onmen s o he dyes in MCF-7 cells (i.e., 1is loca ed in he memb ane o cy osolic esicles, while 2 is ound in he esicle) lead o sligh ly di e en ICT s abili ies and hence li e imes. Appa en ly, only he highe lying elec onic pa s o he po en ial ene gy su ace seem o be a ec ed by he (sligh ly) di e en localiza ion wi hin he cells. The adia i e li e ime, on he o he hand, e lec ing he ime o ISC, which ul ima ely quenches he luo escence o he complexes, emains he same o 1and 2. Conclusion In his con ibu ion ou -coo dina e o ganobo on N,C-chela es we e s udied wi h espec o hei exci ed-s a e elaxa ion pa hways in sol en s and when inco po a ed in o human cance cells (MCF-7). The di e en e minal g oups (NH2o COOH) o he PEG chains, ha we e a ached o he dyes, do no a ec he exci ed-s a e elaxa ion pa hways o he dyes when s udied in DMSO. A sligh ly di e en dis ibu ion o he dyes wi hin he MCF-7 cells was no ed, which led o al e a ions o he decay kine ics o he emissi e ICT s a e. Despi e his obse a ion, luo escence li e ime imaging e eals he same dis ibu ion o emission li e ime o bo h dyes (which ma ch he emission li e imes o he dyes in DMSO). Howe e , he as elaxa ion componen o he ICT s a e appea s o be longe in 1 compa ed o 2when bo h sys ems a e aken up in o MCF- 7 cells. This indica es ha he wo PEG chains a ec he localiza ion o he dyes in cells, causing hem o expe ience a dis inc mic oen i onmen , which in u n leads o sligh di e - ences in he slow s uc u al elaxa ion o he emissi e ICT s a e. Expe imen al Sec ion The s a ing ma e ials 3–6(Scheme 2), 2-azidoe hyl me hanesul o- na e, and he PEG-linke s we e syn hesized acco ding o he p ocedu es desc ibed in he li e a u e.[7a,17] Fluo escence li e imes in solu ions we e measu ed wi h a Hama- ma su HPDTA s eak came a. The exci ed wa eleng h was se a 400 nm wi h a epe i ion a e o 400 kHz. The esponse unc ion is ca. 250 ps. Sample we e measu ed in 1 cm qua z cu e es. T ansien abso p ion spec a we e ob ained wi h a home-buil se up.[18] 400 nm pump pulse o ca. 110 s pulse du a ion wi h 500 Hz epe i ion a e and a whi e-ligh wi h 1 kHz epe i ion a e a e ocused on he sample posi ion. The pola iza ion be ween he pump and p obe beam is se a he magic angle (ca. 54.7°). The sol en samples we e measu ed in 1 cm qua z cu e es. Fo he in cellulo expe imen s, he single wa eleng h p obes (515, 580, 600 and 650 nm) a e employed ins ead o he whi e ligh . The cellula samples we e measu ed in glass bo om μ-dishes. The MCF- 7 cells we e ea ed wi h inal concen a ion o 20 μM o 1and 2in media. The MCF-7 cells we e measu ed in 1 mL Hanks’ balanced sal solu ion (HBSS). Following he measu emen s, he cells we e ea ed wi h T ypan Blue solu ion and imaged by a combina ion o a Raspbe y Pi came a and Ca l Zeiss Axio e 25 mic oscope wi h he objec i e EC Plan-Neo lua 10X/0.30M27 (Figu e S15). Nanosecond ansien abso p ion spec oscopy was used o ob ained he kine ics a long delay ime. The pump pulse was se a 430 nm p oduced by Su eli e Nd:YAG lase sys em wi h a pulse du a ion o 5 ns and a epe i ion a e o 10 Hz. The whi e-ligh p obe is p o ided by a xenon a c lampand samples we e measu ed in 1 cm qua z cu e es. Oxygen- ee solu ions we e p epa ed inglo e box. Fluo escence li e ime images we e ob ained om a cus om Abbe io Expe Line lase scanning STED mic oscope wi h 100× /1.4 oil imme sion objec i e lens om Olympus. The MCF-7 cells we e seeded on a glass co e slip wi h 20 μM concen a ion o each o he dyes. The cell samples we e exci ed a 440 nm pulses wi h a epe i ion a e o 40 MHz. La ice-SIM images we e acqui ed on a Zeiss Ely a 7 mic oscope equipped wi h a Plan-Apoch oma 63X/1.4 Oil DIC M27 objec i e and a pco.edge sCMOS ( e sion 4.2 CL HS) came a. The 405 nm lase was used o exci e he dyes in cellulo. Acknowledgemen s This wo k was suppo ed by he Eu opean Union ( ia he ITN LogicLab unded unde he Ho izon 2020 esea ch and inno a ion p og am unde he g an ag eemen No 813920). We hank P o . D . Raine Hein zmann and D . Benedic Die- de ich o p o iding BioLab acili ies and suppo ing he image acquisi ion. The ELYRA 7 (used o p oducing Figu e 3 and S8) was unded by he F ee S a e o Thu ingia wi h g an numbe 2019 FGI 0003 and suppo ed by he Mic o e se Imaging Cen e ( unded by he DFG unde Ge many Âs Excellence S a egy � REXC 2051 � RP ojec -ID 390713860). We u he acknowledge unding by he DFG (P ojec numbe 316213987 – SFB 1278, INST 1757/25-1 FUGG), he F ee S a e o Thu ingia (TAB, TMWWDG, Ad ancedSTED/2018 FGI 0022; Ad anced Flu-Spec/ 2020 FGI 0031), BMBF (Pho onics Resea ch Ge many (FKZ; 3N15713/13N15717) in eg a ed in o he Leibniz Cen e o Pho onics in In ec ion Resea ch (LPI)) and he inno a ion p og am by he Ge man BMWi (ZIM; p ojec 16KN070934 / Lab- on-a-chip FCS-Easy). The Spanish Minis e io de Ciencia e Inno ación (g an s PID2020-119992GB-I00, PID2019-106358GB- C21, and PID2019-106358GB-C22), he Consejo Supe io de In es igaciones Cien í icas (g an 202080I005 o A.R.), and he Jun a de Andalucía/Uni e si y o Huel a (g an UHU-202070) a e hanked o inancial suppo . We hank D . Z. Dom Nguez o assis ance in he ea ly s age o his p ojec . Open Access unding enabled and o ganized by P ojek DEAL. Chemis y—A Eu opean Jou nal Resea ch A icle doi.o g/10.1002/chem.202203468 Chem. Eu . J. 2023,29, e202203468 (6 o 7) © 2022 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH Mon ag, 06.03.2023 2316 / 287915 [S. 108/109] 1 15213765, 2023, 16, Downloaded om h ps://chemis y-eu ope.onlinelib a y.wiley.com/doi/10.1002/chem.202203468 by Uni e sidad De Se illa, Wiley Online Lib a y on [22/05/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License Con lic o In e es The au ho s decla e no con lic o in e es . Da a A ailabili y S a emen The da a ha suppo he indings o his s udy a e a ailable om he co esponding au ho upon easonable eques . Keywo ds: exci ed-s a e elaxa ion · luo escence ·li e cells · o ganobo on dyes ·PEG chain [1] a) R. Hech , J. Kade, D. Schmid , A. Nowak-K ol, Chem. Eu . J. 2017,23, 11620–11628; b) M. V. Pin o, F. M. F. San os, C. Ba os, A. R. Ribei o, U. Pischel, P. M. P. Gois, A. Fe nandes, Cells 2021,10. [2] a) K. Liu, R. A. Lalance e, F. Jakle, J. Am. Chem. Soc. 2019,141, 7453– 7462; b) P. Li, H. Chan, S. L. Lai, M. Ng, M. Y. Chan, V. W. Yam, Angew. Chem. In . Ed. Engl. 2019,58, 9088–9094; c) M. Mas-Mon oya, L. Usea, A. Espinosa Fe ao, M. F. Mon eneg o, C. Rami ez de A ellano, A. Ta aga, J. N. Rod iguez-Lopez, D. Cu iel, J. O g. Chem. 2016,81, 3296–3302; d) F. M. F. San os, Z. Dominguez, J. P. L. Fe nandes, C. Pa en e Ca alho, D. Collado, E. Pe ez-Ines osa, M. V. Pin o, A. Fe nandes, J. F. A eaga, U. Pischel, P. M. P. Gois, Chem. Eu . J. 2020,26, 14064–14069. [3] a) H. Gao, D. Xu, Y. Wang, C. Zhang, Y. Yang, X. Liu, A. Han, Y. Wang, Dyes Pigm. 2018,150, 165–173; b) L. Jiang, Y. Wang, D. Tan, X. Chen, T. Ma, B. Zhang, D.-T. Yang, Chem. Sci. 2022; c) Z. Zhang, P. Xue, P. Gong, G. Zhang, J. Peng, R. Lu, J. Ma e . Chem. C 2014,2, 9543–9551. [4] S. K. Melle up, G. Youse alizadeh, S. Wang, K. G. S amplecoskie, J. Phys. Chem. A 2018,122, 9267–9274. [5] a) M. Kollmannsbe ge , K. Ru ack, U. Resch-Genge , J. Daub, J. Phys. Chem. A 1998,102, 10211–10220; b) R. R. Maa , R. Zhang, D. G. S ephens, Z. Ding, J. B. Gil oy, Angew. Chem. In . Ed. Engl. 2019,58, 1052–1056. [6] a) Z. Zhao, Z. Chang, B. He, B. Chen, C. Deng, P. Lu, H. Qiu, B. Z. Tang, Chem. Eu . J. 2013,19, 11512–11517; b) S. I o, M. Gon, K. Tanaka, Y. Chujo, Na l. Sci. Re . 2021,8, nwab049; c) M. Yamaguchi, S. I o, A. Hi ose, K. Tanaka, Y. Chujo, Ma e . Chem. F on . 2017,1, 1573–1579. [7] a) V. F. Pais, M. M. Alcaide, R. Lopez-Rod iguez, D. Collado, F. Naje a, E. Pe ez-Ines osa, E. Al a ez, J. M. Lassale a, R. Fe nandez, A. Ros, U. Pischel, Chem. Eu . J. 2015,21, 15369–15376; b) Z. Dominguez, R. Lopez- Rod iguez, E. Al a ez, S. Abba e, G. Longhi, U. Pischel, A. Ros, Chem. Eu . J. 2018,24, 12660–12668. [8] Z. Dominguez, V. F. Pais, D. Collado, P. Vazquez-Dominguez, F. N. Albendin, E. Pe ez-Ines osa, A. Ros, U. Pischel, J. O g. Chem. 2019,84, 13384–13393. [9] a) H. Yang, J. J. Mo is, S. T. Lopina, J. Colloid In e ace Sci. 2004,273, 148–154; b) Q. Song, S. Goia, J. Yang, S. C. L. Hall, M. S ani o h, V. G. S a os, S. Pe ie , J. Am. Chem. Soc. 2021,143, 382–389; c) A. W. Bosman, H. M. Janssen, E. W. Meije , Chem. Re . 1999, 1665–1688. [10] F. M. San os, J. N. Rosa, N. R. Candeias, C. P. Ca alho, A. I. Ma os, A. E. Ven u a, H. F. Flo indo, L. C. Sil a, U. Pischel, P. M. Gois, Chem. Eu . J. 2016,22, 1631–1637. [11] a) K. Kuma , J. Hille , M. Bende , S. Nos a i, Q. Liu, M. Edelmann, S. Maie , T. Rammle , F. Wackenhu , A. J. Meixne , K. B aun, U. H. F. Bunz, M. Scheele, ACS Nano 2021,15, 480–488; b) S. Na h, H. Pal, D. K. Pali , A inash, V. Sap e, J. P. Mi al, J. Phys. Chem. B 1998, 10158–10164; c) A. Nagai, J. Miyake, K. Kokado, Y. Naga a, Y. Chujo, J. Am. Chem. Soc. 2008, 130, 15276–15278. [12] F. Bosca, M. C. Cuque ella, V. F. Pais, A. Ros, U. Pischel, ChemPho oChem 2018,2, 34–41. [13] V. F. Pais, P. Rami ez-Lopez, A. Rome o-A enas, D. Collado, F. Naje a, E. Pe ez-Ines osa, R. Fe nandez, J. M. Lassale a, A. Ros, U. Pischel, J. O g. Chem. 2016,81, 9605–9611. [14] a) J. Rumin, H. Bonne ond, B. Sain -Jean, C. Rouxel, A. Sciand a, O. Be na d, J. P. Cado e , G. Bouga an, Bio echnol. Bio uels 2015,8, 42; b) A. Ray, S. Das, N. Cha opadhyay, ACS Omega 2019,4, 15–24. [15] a) K. R. A. Schneide , A. Che i, H. D. Cole, K. Reglinski, J. B uckmann, J. A. Roque, 3 d, A. S umpe , D. Nau oozi, S. Schmid, C. B. Lage holm, S. Rau, P. Baue le, C. Eggeling, C. G. Came on, S. A. McFa land, B. Die zek, Chem. Eu . J. 2020,26, 14844–14851; b) T. Yang, A. Che i, B. Radwan, E. Ma uszyk, M. Ba anska, B. Die zek, Chem. Commun. 2021,57, 6392– 6395; c) B. M. A eline, R. M. Sa le , R. W. Redmond, Pho ochem. Pho o- biol. 1998,68, 51–62. [16] B. Die zek, T. Pasche , V. Sunds öm, A. Ya se , Lase Phys. Le . 2006,4, 38–43. [17] a) S. T. G. S ee , D. N. Chin, G. J. Hollingwo h, M. Be y, J. C. Mo ales, M. C. Galan, Chem. Eu . J. 2017,23, 6953–6958; b) L. S. Wong, S. J. Janusz, S. Sun, G. J. Legge , J. Mickle ield, Chem. Eu . J. 2010,16, 12234–12243; c) T. Anabuki, M. Tsukaha a, H. Ma suu a, K. Takahashi, Biosci. Bio echnol. Biochem. 2016,80, 432–439. [18] R. Siebe , D. Akimo , M. Schmi , A. Win e , U. S. Schube , B. Die zek, J. Popp, ChemPhysChem 2009,10, 910–919. Manusc ip ecei ed: No embe 8, 2022 Accep ed manusc ip online: Decembe 7, 2022 Ve sion o eco d online: Feb ua y 14, 2023 Chemis y—A Eu opean Jou nal Resea ch A icle doi.o g/10.1002/chem.202203468 Chem. Eu . J. 2023,29, e202203468 (7 o 7) © 2022 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH Mon ag, 06.03.2023 2316 / 287915 [S. 109/109] 1 15213765, 2023, 16, Downloaded om h ps://chemis y-eu ope.onlinelib a y.wiley.com/doi/10.1002/chem.202203468 by Uni e sidad De Se illa, Wiley Online Lib a y on [22/05/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License