Abstract
Herein, we present the synthesis and application of a fluorogenic, large Stokes-shift (>100 nm), bioorthogonally conjugatable, membrane-permeable tetrazine probe, which can be excited at common laser line 488 nm and detected at around 600 nm. The applied design enabled improved fluorogenicity in the orange/red emission range, thus efficient suppression of background and autofluorescence upon imaging biological samples. Moreover, unlike our previous advanced probes, it does not require the presence of special target platforms or microenvironments to achieve similar fluorogenicity and can be generally applied, e.g., on translationally bioorthogonalized proteins. Live-cell labeling schemes revealed that the fluorogenic probe is suitable for specific labeling of intracellular proteins, site-specifically modified with a cyclooctynylated, non-canonical amino acid, even under no-wash conditions. Furthermore, the probe was found to be applicable in stimulated emission depletion (STED) super-resolution microscopy imaging using a 660 nm depletion laser. Probably the most salient feature of this new probe is that the large Stokes-shift allows dual-color labeling schemes of cellular structures using distinct excitation and the same detection wavelengths for the combined probes, which circumvents chromatic aberration related problems.
Highlights
Fluorescent methods are distinguished amongst the techniques dedicated to the sensitive and accurate detection of cellular and subcellular events in vivo
Direct modification of the original HELIOS frame is rather limited and does not allow ready extension of the π-system, for the 3rd position is already taken by the quenching phenyl–methyl tetrazine moiety
We studied the possibility of extending the π-electron system of ultra-fluorogenic HELIOS probes and concluded that the we presented the synthesis and application of a fluorogenic, large Stokes-shift, bioorthogonally applicable, membrane-permeable tetrazine probe
Summary
Fluorescent methods are distinguished amongst the techniques dedicated to the sensitive and accurate detection of cellular and subcellular events in vivo. Due to recent hardware developments in super-resolution microscopy, it is rather the lack of suitable probes, which is considered as the major limitation of further improvements [1,2]. There is a clear need for advanced probes suitable for selective targeting and in vivo super-resolution imaging of intracellular structures. Such membrane permeable, improved probes should address challenges such as selective conjugatability, autofluorescence, and background fluorescence [3]. Suppression of background fluorescence of non- bound probes, on the other hand, is more challenging and often requires extensive washing cycles following staining. Steurcmhsaonfarpepacrotiaocnhkisinuesteicds,robuiotcinoemlypaintibthileityla,bseylninthgestcicheamcceesssoifbivliatryi,ouansdprcootmeinpsa,tiibnilciteylluwloithor GinCEv,ivsotra[6in–8p,1r0o–m1o2]t.ed azide–alkyne cycloaddition (SPAAC) of azides and strained alkynes [13] and the invInertseermelescotfrorenadcteimonankdinDetiieclss,–bAioldcoemr (pIEaDtibDilAity),resyancttihoentiocfatcecterassziibnielistya,nadnsdtrcaoimnepdatailbkielintyesw/ailtkhyGnCesE, [1s4tr]aianreprtohmeotmedosatzirdoeb–uasltkybnioeocrythcloogaodndailtiornea(cStPioAnAs.CB) eosfidaezsid, eassanwdestarnaidneodthaelkrsynheasv[e13]reacnednttlhye dienmveornssetrealetectdr,ofnudncetmioannadl gDrioeulsp–sAoldf ethre(ISEPDADAAC) raenadctIiEoDn DofAterteraazctiinoenssa, ni.de.,sttrhaeinaezdidaelkaenndetsh/aelkteytnraezsi[n1e4] maoreietthiees,maorestarbolebutostmboiodourltahteog(io.en.a, ql ureeanccthio)nflsu. oBreessicdeensc,eabsywvearainodusomtheecrhsahnaivsme rse(cee.ng.t,lyendeermgyotnrsatnrsafteerd, [1fu5–n1c7ti]o, nraoltgartoiounp-sinodfutcheedSPrAelAaxCaatinodn IE[1D8D,1A9],reaocrtiopnhso,tio.ein.,dthueceadzideeleacntrdonthetrteatnrsafzeirne[2m0o])i.etiUesp,oanre traabnlsefotormmaotidounlaotfeth(ie.eq.,uqeunecnhcinhg) flbuiooorretshcoegnocneablyunvaitriionuas smpeeccihfiacnliisgmatsio(ne.gre.,aecntieorng,ythtreafnlusfoerres[1c5en–1c7e], rerointasttaiotens-i(nSdcuhceemdere1l)a. xTahtiiosntw[1o8-,i1n9-]o,noer pcohmotboiinnadtuiocnedofelbeicotorortnhtorgaonnsfaelrh[a2n0]d)l.eUs pwoansterxanpslofoitremdaitniotnheof dtehseigqnueonfcchoiunmg bairoinor[t2h1o]g, oBnOaDl uIPniYt i[n16a],sppehceifincoxliagzaitnioen[1r7ea],ctrihoond, athmeiflneuo[r2e2s]c,eanncde rceyiannstiantee-sb(aSscehdem[2e3]1,). bTiohoisrtthwoog-oina-ollnye acpopmlibcianbalteiofnluoofrboigoeonritchopgroonbaels.haItndwleass woabsseerxvpeldo,itehdowinetvheer,dtehsaigtnthoef cfoluomraesricnen[2c1e], inBcOreDaIsPeYu[p16o]n, pbhieonoorxthaozginoen[a1l7]l,igrhatoidonam, in.ee.,[2f2lu],oarnodgecnyiacintiyn,e-dbraasmedat[i2c3a]l,lybiodoerctrheoagseosnatlolywaaprdpslictahbele bflioulogroicgaelnlyicpprerfoebreres.d Irtedwraasnogbesoefrvtheed,spheocwtreuvmer,(Ftihgautrteh1e).flTuoordeastcee,nthce minocrsetarseemuaprkoanbbleioeoxratmhopgleonofal sulicghatifoluno, ir.oeg.,eflnuicoropgroebneicsityin, dtrearmmasticoafllfyludoercersecaesnescetoiwncaredassteh,eabrieoluogltircaallvyioplreetfe(rUreVd)/rbeldueraenxgceitoafbtlhee usltpreacftluruomrog(Fenigiuc rHe E1L).IOToS d(haytep,etrheemmisosisvterelimgatrikoanb-ilneietixaatmedpolertohfosguocnhalflsueonrsoingegn) ipcrporboebserseipnorteterdmsbyof Wfleuiossrleesdcernceetinacl.reianse2,0a1r4e u[2lt1r]a. vEimoliestsi(oUnV)o/fbltuheeesexccitoaubmleaurilntr-abflauseodrogperonbicesHEwLaIsOpSr(ahcytipcearlleymfiusslilvye qluigeantcihoend-inbiytiattheed boirothoortghoongaolnsaelnhsianngd)lperotebteraszrienpeo. rUtepdobnysWpeecisifsilcedrearcettioanl. winit2h01t4ra[n2s1-]c.yEcmlooiscstieonnesof (TthCeOses)c,o3u–m4 aorridne-brsasoefdmpargonbietsuwdeasinpcrraecatsiecailnlyflfuollryesqcueennccehwederbeyotbhseebrvioeodr,twhohgicohnaalllhoawneddlentoet-rwaazsinhe. flUuoproenscsepneceifiicmreaagcitniognowf iethpitdraenrms-caylcglorocwtethnefsa(cTtoCrOrse)c, e3p–4toorrsd(eErGs oFfRm) augsninitgudTeCiOncrleaabseeleind flaunotir-eEsGceFnRce awnteibreodoibesse.rved, which allowed no-wash fluorescence imaging of epidermal growth factor receptors (EGFR) using TCO labeled anti-EGFR antibodies
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