WO2015103587A2 - Improved environment sensing cyanine and merocyanine dyes - Google Patents

Improved environment sensing cyanine and merocyanine dyes Download PDF

Info

Publication number
WO2015103587A2
WO2015103587A2 PCT/US2015/010269 US2015010269W WO2015103587A2 WO 2015103587 A2 WO2015103587 A2 WO 2015103587A2 US 2015010269 W US2015010269 W US 2015010269W WO 2015103587 A2 WO2015103587 A2 WO 2015103587A2
Authority
WO
WIPO (PCT)
Prior art keywords
biosensor
binding
formula
target
compound
Prior art date
Application number
PCT/US2015/010269
Other languages
French (fr)
Other versions
WO2015103587A3 (en
Inventor
Klaus M. Hahn
Christopher J. MACNEVIN
Original Assignee
The University Of North Carolina At Chapel Hill
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of North Carolina At Chapel Hill filed Critical The University Of North Carolina At Chapel Hill
Publication of WO2015103587A2 publication Critical patent/WO2015103587A2/en
Publication of WO2015103587A3 publication Critical patent/WO2015103587A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/12—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/12—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D215/14—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/52—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings condensed with carbocyclic rings or ring systems
    • C07D263/54—Benzoxazoles; Hydrogenated benzoxazoles
    • C07D263/56—Benzoxazoles; Hydrogenated benzoxazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/10—The polymethine chain containing an even number of >CH- groups
    • C09B23/107—The polymethine chain containing an even number of >CH- groups four >CH- groups

Definitions

  • Cysnine and rnerocyanirie dyes are characterized by electron donor and acceptor components linked by conjugation, usually a system of double bonds.
  • This esults in a ground state thai xnay be represented as a resonance hybrid of charged and nnefcargsd forms (Sh m i t aL bp, Meiemcyd. Chem, 14:7$ ⁇ WS ⁇ 2008); Kulimeh e al, is$, G m Rev, .7 ⁇ :141 ⁇ $00
  • the present im3 ⁇ 4ntfe3 ⁇ 4 provides ayamrte arid meroeyaprne dyes having iniprove propistties for iisem invaglrig, inelnding dsefdl coa aaii of brightness, photo5!3 ⁇ 4bility arid sol ent-deperideiii fluorescence.
  • the lavgstidafttrthet provides eyanine ars meroeyanine dyes having increased eel I memhraae perfneability for enhanced effectiveness in living cells, ' These dyes retain, brightness and solvent sensitivity while readily crossing cell membrases and showing rrtinhrmi staining of inrxaedlnlai me -brarres.
  • t h e invention relates to a compound of Formula I or Formula It
  • n is .an integer -from 0 to 2;
  • each X is independently riydrogen or m electron with#aw3 ⁇ 4g graup selected; from the group consisting of carbonyl., ⁇ yano > lalagen, rdiro, sutf nyl, and trifltioromethyi;:
  • W is-op omUy s bstit ted C ⁇ al-Hyl
  • Y m& Z are- each independently seized froxn tte groip eonsltiag. of hydrogen, €3 ⁇ 4, & . alky!, a conjygaiabie side chain, and
  • o is an integer from O to 5;
  • Eis selected from, th grow cossi sting ' of;
  • ma is s l cted from th ex i consisting ofi
  • Another aspect of the invention relates to a biosensor comprising a ⁇ wmpOxmd ofFomttihi m Fotnrda 11 asd a blsdit g Baari er having a spedfc affittiiy for the target moiecule s . wherein the biosensor exhibits a detecta le bhaage in a fluo escence: property rfmhng or afte " Hadi&g: to the target molecule,
  • a art r «t$*$ of the invention relates to a method for ckferrainiiig the rese ce or amount of onaor more target molecules 3 ⁇ 4 3 ⁇ 4-s3 ⁇ 4mpl ⁇ ⁇ the method, comprising: (a) providing a biosensor of the invention; fb) contacting the biosensor with a sample suspected of containing one or more target molecules to " Uni the one or mote 1 ⁇ 2 t melee4.000s, If present, to the binding mern3 ⁇ 43 ⁇ 4; (e) ; irradiating the sam le suspected of containing one or more target molecules with electromagnetic radiation t induce the compoarid of Formula For Formula II to fluoresce; and ( ) detecting a. flnoreseenee property of the compound O Fomtuia I or Formula !! to determine the presence or amount of one- or more target molecules in the. sample,
  • An additional aspect of the invention relates to a method of detecting an act vity or a location of one or more target molecule within a. cel , the. method comprising: (a) providing a biosensor oih3 ⁇ 4® invention; (b) contacting die: biosensor wit 3 ⁇ 4 cell suspee&d of containing on or more target molecules to bind the one or more ar et rBoleeoies > if present to the indin member; (e) irradiating ins cell suspected of containing one or more target mokeiifes with electromagnetic: .radiation to induce the compound of Formula l or Formula 11 to tlnoresee; and : (&) detecting one .
  • Another aapee of the invention relates to method of detecting an interaction between an endogenous target m lecule and a oellolar entity, the method comprising: (a) providing a eel! comprising an endbgenous target moleetde: (3 ⁇ 4) providing a biosensor of the invention; (c) observing " n background fluorescence signal ffom the biosensor; (d) sG&taotiiig t6 ⁇ biosensor t.
  • feriher aspect of the Inventio relates to a kit eoispri sing 3 ⁇ 4 biosensor ibr dei ctitig 5 monitor g or obse ing a target nm!ees!e, wherein m biosensor comprises a compound of Fbfmuia I or Formula II and. a. binding member having a specific affinity for the target molecule,
  • Dra i gs are not necessarily drawn to scale.
  • Figs. 2A-2B show the change in excitation maximum (A) and emission inajdmum (B) irom smallest value for set of fdftr solvents,
  • Fig. 4 shows the pho sstabiiity of library dyes. Fhotostability values were calculated as (pbotobleachmg rate of fluorescein) / (photobteac ng rata of dye).
  • « «2I1 Figs, 5A-5B show the in vitro hkOm . assay of dye labeled CBD to Cde4Z
  • B a3 ⁇ 4I om .fluorescence intensity at satu ated binding of Cde42.
  • Fig. shows the complete em ssion spectra tor binding of erocyanme- iafeded CBi MBP fragment at low (0,01 uM) and high (3.0 uM ⁇ eoneentrations of C c42, f#0231
  • Fig. 7 shows the dissociation constant ⁇ 3 ⁇ 4). value calculated for mexoCB hmding wiih Cdc42 m 50 m : NaI3 ⁇ 4PO4, fSOmM NaCI buffer at i>3 ⁇ 4 %$,
  • fiOZfS fiOZfS
  • 10 sho ws merecyaaine dyes that consist of el ectron, donor and accepto components that are linked by as extended polyene system, "the R groups indicate the various positions where aeeto ymeihyl ester derivatives integrated to screen for eempounds with sell me bmns permeability and. m nimal membrane sw ung.
  • Fig, 11 shows florescence intensity of each dye in tire initial screening, set, M cetts were incubated for 30 mm at 37 °C In seruni ⁇ ftee media containing 10 fi io a given test dye and 1 pMHoec st 33342.
  • Pkironie F 27 was added to the initial D SO stock solutions of dyes to give a final concentration k the Incabation solution of ⁇ 0,01 %, Fluorescence Intensity (FY) v lues were recorded at the peak of the emissio curve .for each dye when excited at the exei ion maximum wavelength. Values- ere normalized based on relative intensity of Hoschst (exe. 351 m * em, 440 im).
  • Dlgf Fig, 12 sho s the plot of Snoreseenoe intensit fe dye 4 (3 ⁇ 4A )*BA(AM) vs. dye S ( ⁇ (2 ⁇ ) ⁇ ) at various incubation concentrations. Ceil were ineiibaied in medraf containing the test dye at 37 ? C for 30 minutes, The dye solution was aspirated and. the sells were washed with medium, trypsirused, and suspended in fresh medium.
  • the fluorescence intensity (Ff) of the cell suspension was obtained for each dye, Vahies were normalized for cell number based on the relative intensity of the nuclear stain Hoeehst 33342, which w3 ⁇ 4s; rne ded with the dye m each test solut on. ⁇ 0 )
  • Fig. 13 shows i ⁇ dis ribmioa or dyes iiiMEF cel ls. Cells were scaled
  • Fig. 15 shows m xse: embryonic ibreblasis treated with r»eroli3 ⁇ 4 or 1S9a- mero 166 conjugate.
  • FIGs. 16A 6B show the design and synthesis of the small moleede based biosensor CaMero.
  • A Overview of the small Tuofceiife based biosensor approach Aetlvation ofme rotehi of irrerest (POI) llows: hmding: of the sensor, ydiieitmsnlis;
  • FIG. 20 shows rimary murine neurons incubated with CaMero s ow increase in $uorese3 ⁇ 43 ⁇ 4c3 ⁇ 4 3 ⁇ 4t mit fp!3 ⁇ 4 3 ⁇ 4 -stiritui3 ⁇ 4ti ii, Cells were stlmrdsted with & €3 ⁇ 4 (75 rnM), Hot s Of average tluoresoenee i tensit in the cell body region vs ⁇ ilrne for CaMero d Ca ero-NC (n
  • iS f ig. 22 shows a line scan analysis of eel! edge intensity using
  • earboxyf!uorescein diacetate volume indicator. Edge itrtexmty gradient apparent with CaMero with and withou co reateem with clozapine. Mo Intensity gradient is seen with m CaMero C probe,
  • the tern "sbosj when refemng to a value Is meant t c& p variations df » .
  • Cyanins and merooy& ne dyes can he tigsd. as fluorescent labels lor omoleePie&j S eh as proteins or antibodies ⁇ for histoehemica! studies r
  • n is an Integer from 0 to 2;
  • ⁇ aeh X is. inde en entl feydfOge or electron ith ra ing giesup se!sotec Iromihs grovp coim ⁇ tiiig- fOTiKmy ⁇ .ejf.ano ⁇ l ⁇ i ⁇ g ⁇ nitro,,s3 ⁇ 4i£briyl s and
  • D is selected ftoniihe gfo3 ⁇ 4) oos sfi ' s of;
  • Y and Z are each iadepesdeHt!y selected flota r&e gtdiip consisting of hydrogen,
  • Q is integer from to 5;
  • B is seleeisfi! from f e. gfoivp eousistlng of:
  • the invefitki encompasses compounds having any one of the -described D m3 ⁇ 4 es coupled with- any one of the A moieties.
  • the im?emion further encompasses -com ounds having any one of the B: moieties coupled with, a y one of the G moieties ⁇
  • the compound does not contain electron witi drs in .group on the poiymethine chain connecting A mi D or E and G 4 e.g.* each X is a hydrogen, I» other embodiments, t e compound contains a single electron idiirawing group, «, ⁇ . , one X is an electron withdrawing group.
  • the electron withdrawing group s attached to the poiymethine chain carbon mat is connected to A or G.
  • the electron withdrawing group is attached to 3 ⁇ 4 carbon in the middle of thepoiymethine cha3 ⁇ 4 .e &, a carbon fhat is not connected to A, D f ts, or G. la further .embodiments, the compound contains two or more electron -withdrawing groups. In these embodiments;, one or more of the electron withdrawing groups may be attaelied to a poiyttietfeke ena n carbon thai Is oonuected to A, D, E. r G, Placing electron-wiadfawing groupa it selected positions on the pcipae&ine chain, retees phoiobieaelung of cyanine and merocyanme dyes,
  • the conjugatable side chain of Y and-Zor Z comprises a linker and a reactive group and -is one that is capable of conjugating the compo n to. a binding member ha in an affinity fo target niolficnle as described below.
  • the compound comprises one conjugatable side chain,. 3 ⁇ 4 other ib
  • the co p u d does not comprise a conjiigatabie side cfoaiii.
  • a is; ft: integer &3 ⁇ 4a 1 to S:
  • U is O or S:
  • R 3 is a ie viog iro ;
  • R 3 ishydrogen, Lf " , Na * or Kx .
  • R* 3 B. 4 s ;3 ⁇ 4n4 ' R s are each ijrsdepenidectly Cj ,g sikyl;
  • R is a halogen, iodide.
  • M Isa&t one of Y and Z is. eonjngaiable side chain and the co 3 ⁇ 4poiisd. contains o l am emj&gatable side cfeala.
  • :&e Oonjugat3 ⁇ 4bie side eharn is on the or E moiety, .e. . , fee Y in the D or E moiety, !a other OT3 ⁇ 4Gdimesl3 ⁇ 4 the eonj gata le side chain is on.
  • the compoiiftds comprise one or mm sobsiit ents that re penneal3 ⁇ 4Bty enlianeers, le ⁇ sabsiitueats mat increase the ability of tli cempownd to cross edl membranes.
  • the permeability enhancer is
  • o is: integer from 0 to 5;
  • ⁇ is 0 or S
  • R 4 is bydmgen or optionally substituted Q-s alkyh
  • die permeability enhance? is an aeeioxymfctbyl ester, e.g..,
  • the compound comprises on3 ⁇ 4 or more permeability er&aneers, e:g,, 1, 2, 3, 4, or 5 peti3 ⁇ 4eablliiy euhsm3 ⁇ 4rs s 1, 2 ;I :or 3 permeability eimascers, ⁇ certain i cdi ents, the one. or more permeability erikaneef s ate attac ed to h ⁇ or E moiety, la certain embodiments, the ne or more peimeabiliiy eriiaBoers are a3 ⁇ 4ached t the A or Q moiety. In esrtairi embodiments > .
  • the compound comprises at least two permeability efihaneers and the perm ⁇ a ' hil iy- etifeaKcers are attached to both the D or E moio!y and tits A or Q moiety, in .s m embodiments, the compound does mot comprise a ermeability enh nce?.
  • the eompomnd comprises a COM] ugatsble side chsra one or permeability enhancers.
  • the Y in me D or E .moiety is a eosj «gatab!e: side chain: 1 and at least one Z is-
  • the Ym ftie D E moiety is kviirogsn _aad ai least one Z i&
  • one Z is a « ⁇ jiigat3 ⁇ 4h!3 ⁇ 4 side c-hsm and the Y ia the
  • R is .halogen, I
  • the com ound is selected from .$mf ⁇ B ⁇ ing list or ⁇ any s3 ⁇ 4)coi3 ⁇ 4biaatio3 ⁇ 4 thereof. Ea of these corapopn4& amy farther comprise a
  • the compound is se cted from the following list or any sy3 ⁇ 4com 3 ⁇ 4g on feereof
  • tie compound Is seieetd ftom the following: list or my siifecoMfeiMiiof liereoL
  • Each of k se compouiids m furthsE comprise a con
  • a “derivative” refers to a chemical compound that s derived from or obtained from a parent eomp uisd arid contains esssmal elements of fee parent compound (e ⁇ . , ability to flu m ⁇ but t3 ⁇ 43 ⁇ 4e i
  • Sueh fusiesional groups can be added to a parent compound,: fw e a le, to improve- rite molecule's solubility, absorption, o gieal half life, fiuorsSGefit properties, and the like, or to decrease the toxicity of fie molecule, eliminate or attenuate any undesirable side et!eei of the molecule, and the like.
  • Hi substitution of one or more tunotional groups ⁇ 3 ⁇ 4, the: donor and/or aeeeptorring strocture can improve the solubility of tile dye. in particular solvents, -stick as water. For e ample, as described in If Patent
  • the presently disclosed dyes can 3 ⁇ 4e designed to have enhanced water solubility by attaching subsfi uent groups on the ring structure that sterieally block aggregation without unduly increasing
  • tie presently disclosed dyes may have one or more groups arranged in. ibr example, a tetragonal geometry from rings of the dye. I» many eases, the substituent groups- iaclude groups with one or more carbons providing ste ⁇ c hind ance to ring stacking* and/or polar to weak ionic cha aater to enhance water solubility.
  • the subsiituems oft one o more r ng structures of the cornpouBd of Formula % or Formula ⁇ can be any fenotionai group that inhibits stacking of the aromatic frogs and br enhances water solubility.
  • Such s bsiituent groups ean provide sterie Mndcra e to ting stacking, thereby inhibiting aggregation, whhOM reducing water solubility of the dye
  • the subsiituent group can have an aliphatic nature, e.g. , a straight-chain o branched alky! group.
  • the substitueni group can have a polar or weakly charged character.
  • the presently disclosed dyes can be : derivatized with side chains to prevent aggregation, improve solubiiiiy, afibet interactions with, proteins, enable covaknt -attachment to proteins, and improve cell membrane permeability,
  • the presently disclosed compound of formula I o formula ⁇ are enyirotiffienially sensSim
  • the term "mviroma-entally sensitive" i relation, to: a. eompo nxl ox -Formula I of Formula ⁇ refers to dye in which a fiuotescenee signal from the dye changes when the dye is ex osed to a change in:enveO meni 5 for example, a bydrophobicity, hydrogen bonding, polarity, or core3 ⁇ 4rm ii na! change.
  • afcorescenee signal ;f3 ⁇ 4om a compound of Forrnuk I or formula ⁇ detectabiy changes upon exposure to a change in solven s change in hydrogen bonding, change in the hydroph bicity of the environment, Changed polarit or polaiizatiort, or change affecting the conformation of the dye, one embodiment, the fluorescence signal .from: the compound of Fonr ia I or formula II increases when the dye is exposed to a environment that is more hydrophobic.
  • the fluorescence signal provided by the compound of Formula I or Formula II increases when the dye is exposed: to 3 ⁇ 4& ⁇ environmeni - h re there is increased hydrogen bmdi3 ⁇ 4g : between the dye and a component of the en vironment S3 ⁇ 4e-h.
  • an increase in hydtophabieit or an increase In hydrogen bonding can occur when 3 ⁇ 4*: presently disclosed biosensor oompr ng a coxppotiad of Fcami!a ⁇ or.
  • Formula II Mads to a target protein or subcellular component.
  • the fluorescence signal provided by the compound of Formul a I of ormula Jl • • decreases when the dye is exposed to an envhonraeni that is more hydrophilk
  • hi f m3 ⁇ 4er era odimeSts s the fi orescence signalprovided by the compound of Formula ! or Formula ! decreases when the dye is exposed to an environment that has less hydrogen: binding.
  • the presently disclosed dyes can have m extended K iiterioa structure having a polarize groond state, which allows the presently disdosed dyes to respond to changes in solvent polarity, Such dyes can exhibit a strong solvetK-dcpendent excitation wavelength shift, while retaining a bright
  • dyes most have e tended conjugation, which can reduce water solubility and lead to self- aggregation.
  • the dyes designed for use m. living cells were specifically selected to " be insensitive to i fc eavwD&rnent. because
  • the ii ompbor exhibits an increase in a first excitation or enrission mvele3 ⁇ 4gt
  • a ratio between the first wavelength and the- second wavelength cm be eafcrtlated to determine the amount of target molecule: in the: sample under test;
  • Such self referencing can correct for variati ns in excitatlors.sonree Intensity a3 ⁇ 4d other so3 ⁇ 4ces of noise and instability I fee biosensor i&sut requiring: a reference dye.
  • a single flnorophore can be ⁇
  • the presently disclosed su ject matter provides a biosensor co prising a compound of Forrnul& 1 or Formula ⁇ aa4 a binding n ei iber hayin a s! ee fie, affinity for a targe molecule thateaa be used to detect and quantify the presence and/or activities of one or more target molecules.
  • the biosensor can bind to oue or more target molecules and the detection of such binding events can be used irt hoffio eous assays, , assays in which the fluorescent properties of the dye can undergo a ehaage as a resaliof the binding event, and for live-eel! imaging.
  • bioseosws can be used: to; detect and or quantify diverse protein activities, including chang n s bcellular locations, conformational changes, • «pt a$29S. sl teSj-pes feraii.sl tiO. al: masHfte&iianS, and/or small ligand binding of pmi m in vivo *
  • bkding wit a target inolecufc.
  • the term '' roducing a. detectable signal refers to the hiilty to recognise a change in aproperty of a reporte group, e.g. , a fluorophoie s in a man e t3 ⁇ 4at enables the detection ⁇ f Hga ⁇ d-proieif Isnding,
  • Ths present i vention provides methods which can use specific binding: partners for a particular target molecule of ' interest.
  • a specific hmding partner or member, as used herem s Is a metuher of a specific bl mg pair,
  • a target molecule is .3 ⁇ 4 reciprocal member of a specific binding pair.
  • a osens ' o ' r can traris!ate a bhidihg event into a directly measurable fluorescent pro ert .
  • Such changes in one or More fluorescence properties include, but are not limited to, an. increase or decrease m fluorescence intensity, a shift in exeltatios or emission maxima, shape oftiie excitation or emission band profiles, a change in fluorescence lifetime, a change in anisotropy, a change in polarfstioo, and combinations thereof.
  • the pr ducing of a detectable signal can be reversible or son-reversible
  • the signal- roduclBg event includes oo s, programmed, anrf episodic mc3 ⁇ 43s s ,inckding one-tlrne or renss e app!inations.
  • the reversible signal* produein event can -fee instantaneous 3 ⁇ 4r can be time-dependent,, m long as a correlation with fcpresence or epncentraliori of target, molecule Is esiab!isbed.
  • ftuerophore includes a moiety of a larger • molecule or :eom3 ⁇ 4gate that can be induced to emit fluorescence when irradiated,, i.e. , excited, by electromagnetic radiation ⁇ fa appropriate wavelength. More
  • the intensity and the wavelength of the li ght emitted * as well m o r fluorescence properties includedin , but not limited, o, fluorescence lifetime, anisntropy, polarisation, and conihinations thereof, depend on the identity of the fluorophpre and its chemical enviroitt enC:
  • a ilucrophote can inciuds a flnorescent molecule, such as the presently disclosed compounds of Formula f or Formula ill
  • com oun s of Formula ⁇ o Fonnuia H include a reactive group, sufestitiients Y and which can be C0j3 ⁇ 4i3 ⁇ 4a i3 ⁇ 4 w «b another ma!ecole f swel as a blsding meiBber, .e.g., one member of a specific binding pain, which : has an aflMiy for a target- moiseale of interest, e,g ⁇ the oi ie member of a binding pair,
  • Such c lligates can. be nsed as a biosensor com opsd to detect the presence, location, co &rmatiofi j .aeiivatlon siate ;
  • the binding of the two or more sab mts can be fctoo gb.
  • a derivati ve ca include an reaction product of the derivative, for example, the reactioa product of the derivative with, an smiao acid reridae. Accordingly, in seme embodiments the presemly disclosed dyes can !neit cie aromatic ring structure having a eonjiigatabie sideo3 ⁇ 4a3 ⁇ 4 that can be popj gated, e*g.
  • a son-iimMfig example of I -derivative is an ester or amide of a parent compound having a earboxylic acid functional gtoup t fi SJ fo exam le, derivatives of th presently disclosed com oun s of Formula I -or Formula II- can 1 ⁇ 4 adie .with s adnim yi es er conjug ⁇ hl rideehain for aEadioieitt to lysme or hh an iodoaeetyl linker for eetive reactioa it cysteine,.
  • the reactive compounds of Formula 1 or Fo.mii.sla II can be synthesized from starting materials having an amino group, which oars he used for attachment of a s de chain comprising 3 ⁇ 4 reactive linker, group at the en of the synthesis, Accordingly, in some
  • eystein « ⁇ seteeiive iedo acetyl groups can be attached to the dye ring: structitce for covending nding to # binding membe or binding domain, thereof,
  • the presently disclosed dyes ean include a thiol- reactive group that can be conjugated to the thiol moiety of a cysteine amino acid i3 ⁇ 4sMue in a aater-al or nnnatma! protein.
  • thiol-reacttve group refers to a suhstit ent. group that can react with, a thiol moiety to form a earhon-sulihr bond.
  • Fxamples of suitable thiol -' eaeSve gronps that can be introrluced into the presently disclosed dyes include a h lo-as et l group, resort a halo-acetamido group.
  • the haio-acetyl group includes an iodoaeery! group.
  • md didiionyridyi goups are suitable tot use 3 ⁇ 4 the 3 ⁇ 4se$ disclosed subject rr-auer.
  • the residue to whieb -the dye is attached is a cysteine.
  • one ormore snob rsid es can include conjngatab!e. sideobain-i eaetive groups other than cysteine * fJoBittgatable sidec aia-reaciive groups can iscfede my chemic . : &neiic-fial. oaps on the dye 3 ⁇ 4 the hindiag member, or both, thatean ieact wit selected ca3 ⁇ 4 aiafc3e si jec 3 ⁇ 4s to fbfo ose or more chemical bonds.
  • a binding member or binding domain can wvid e0 ⁇ g3 ⁇ 4t3 ⁇ 4bk : j eeh ⁇ ft-r ⁇ a6tive .
  • l&Q l Gonj3 ⁇ 4gataMe ddechains can be: a3 ⁇ 4 type suitable to react with
  • Corysgaia e sldeeh&ins can include a flexible aliphatic or pol mer ehamof suitable length and hydroplhHciiy to bridg between linked molecules, Bivalent linker groups haying the same or different, !ihtesr cbenhstries at each end also can be used, Conjuga3 ⁇ 4o!e si eebains.
  • cap include one of more protective group t protect the Imfcergronp dnring storage , handling or other chemi steles, T3 ⁇ 4e protective gronp can be removed under defined conditions to allow completion of the eon) ligation reactioii.
  • ⁇ ⁇ 08 ⁇ intelligent design of linkage reactions can be accom lished with the select on of a targe! reactive group in a binding member m binding: domain, or engineering of a desired: reactiye groap nto t3 ⁇ 4e blading member or binding domain;.
  • a bi ding member or binding domain can n&iurally have a cysteine residue (reactive, for exainpk, i h a haleacetyl linker at an appropriate location, at ea be routateri to include such a cysteine residue.
  • a:Mndmg membe or a binding, domain csn have a small i mber of cysteine rssi nes.
  • Binding, members or binding domains can be modified or mutated to contain a single or a small .number of cysteine residues by nrdcedures known in the art.
  • Binding members suitable: for use with the presently disclosed subjec matter can include any moleeiiies that hind tp a target rnofeeaie wim : sellable specificity, B hiding m mb rs typiealf ihcl:3 ⁇ 4de binding a ioss of arlmity molecules known in the art ine eohngf but not limited s antibodies, antibody fragments, leucine zi e s, stones erdiarieers, complementary determining regions (CIDRs) v to single chain variable fragments (seFv3 ⁇ 4 receptors, ligands, spi raers, lectins and one of seyeral proteins in a prof em complex or a protein pair, Binding members: can comprise either membe of a binding pair , $,g, , pai pf proteins in a protein-protein Interactiem with the binding member being identified as the member
  • Binding members or bindin domains thereof can be binding. egions of, 3 ⁇ 4 cjtant l , feE/slmdwcrsions oFthe affinity rnoleeide, fragments of the affinity molecule, or the smallest portion of the affinity molecule providing binding useful in the detection of a target molecule.
  • 3 ⁇ 4r can hay ⁇ specific affinity to endogenous ⁇ e.g.* constitutive or mdueili!e f but not recombinant) peptides of a ceil. , 3 ⁇ 4
  • the: binding membe is a small molecule that has affinity for a target .
  • molecule e,g., a protein
  • the small molecule is one that cannot b s produced - y genetic means, is not a peptide or protein
  • the small molecule is a CDS3 ⁇ 4sCmtid ' 1 ⁇ 4vin a molecular wei ht of less than about K)00 3 ⁇ 4 ,g,, less* taakmt 8 0 Da or about 600 Da
  • the small molecule binds a specific proteiri 3 ⁇ 4 . apicrteiniB a specific
  • the small molecule is eoi3 ⁇ 4u 3 ⁇ 4t «£l to a:
  • the sm l! molecule is conjugated to any dye thai is kao ra to be or later identified to be useful for visualizatiiMi in living cells or animals, ,g ri to visualize interactions between proteins and small .molecules,.
  • the dye may be one that is permeable to cell membranes to enhance its nsefiifriess in livin ceils or animals.
  • the dye may be one 3 ⁇ 4at ean l?e visnaJksd by micrdscopy, e.g one thai emits a fluorescent or other dprah
  • tie bindingmember is a binding protein.
  • binding protein refers to a protein, that when conjugated with a • fiuorophore, interacts with a specific target molecule in a manner capable of from when. a. target molecuie is present or abse t s , when a target molecule is-, presen in varying concentrations over time.
  • binding membe can ..comprise a binding domain:, Binding ctomains suitable fbr nse with the presently disclosed biosensors can comprise polypeptide, peptide, or nucleic acid sequences.
  • binding domains can be single stranded DMA (sDNA , double stranded DMA (dsO A), RNA S nucleic acids -with modified bases, and the like, m one embodiment, the binding domain can be an oligonucleotide probe andthe: target eaa be a eoisp!m entary target nucleic acid,.
  • Afi t specificity of peptide binding domains can be provided by a short, sequence of amino acids (c.g 5 a se uence of amine acids: comprising 3 pM residues,
  • Binding domains comprising peptides can ave natural ammo: acid side clams, modified side- chains, or the like, that provide reactive groups specifically reactive with compounds of Fotmiufa I or Formula 1L in some embo ments, the compound of Formula I or Formul can have reactive groups specifically reactive with linker groups present on the binding domain to link the dye to the domain, he position of a dye on a domain can be detertnined by the location of a reaetive group o mker. moiety on the domain. In some ernbodhiients, the binding domain has one or more cysteine residues reactive -with groups on the dyes, for exam le, iodoaeeiyl groups on fee dyes,
  • the presently disclosed biosensors can incorporate binding dom ins of naturally oecvuTing proteins having specific binding activity.
  • the binding domains can include, for example, 3 ⁇ 4iOengt ⁇ i .affinity proteks, m3 ⁇ 4mt>e:rs of proieboproiem Interaction pairs ® portions thereof), Fv aiUibody fragments, aptatners, Mi antibody fragments, and the like, !n on ⁇ embodimenh the biosensors .comprise binding domains that are members of the imtmmoglobaiin.fenily of proteins,, or derivatives thereoE For exa le, the balding domain, can e a complete imnmnoglobnlin, fragment, single chain va iable Segment (seFv3 ⁇ 4 a heavy o light chain variable region, a CDR peptide sequence, a3 ⁇ 4d/or the life.
  • a antibody er antibody feagmsni can h3 ⁇ 4 used in a binding domain to which compounds of Formula 1 or Formula 1! can be attached to form: a biosensor
  • An antibody suitable for use with the presently disclosed subject matter can: bo in any of a .variety of forms, including a whole immimoglobnlbL an antibody fragment such as Fvdonating,Fab 5 : a3 ⁇ 4d s milar fragments, a single chain andbodv whicli ind!ne s the variable domain complementarity determin3 ⁇ 4g regions (CDR).
  • antibody fragment refers to a portion of s ful!- length anti od * generally the antigen binding or variable region.
  • antibody fragments include Fab, J?ab F(a % -jaiid Fv fragments. Papain digestion of antibodies produces two Identical antigen binding fragments, called Fab fra m nts, each with a single an igen binding site, and a residnal Fe fragment, Fab fragments t ns. have an intact light chain and apportion of one heavy chain.
  • Pepsin treatment yields an F(ab3 ⁇ 4 fragment that has two antigen binding fragments that are capable of cros8 ink3 ⁇ 4g anti en, and re si dual f gment that is termed a pFc' fragment
  • Fab' fr gments are obtained after reduction of a pepsin digested, antibody, and consist of an intact light chain and a portion of the: heav chain.
  • Two Fab' fragments are obtained, per antibody molecule, Fab fragments differ from Fab fragrnents by the addition, of a few residues at: the eafboxyl terrranns of Iht heavy chain CHI domai . including one or more cysteines from the antibody hinge region.
  • Fv a small antibody fr ment thai contains a complete antigen recognition and: binding site.
  • This region consists of a di.rn.er of one heavy and one light chain variable domain in a : tight, non-covending association ⁇ %3 ⁇ 4.
  • Antibody fragm e-nis usefully incorporated i nto the presently disclose biosensors can include, but are notessenited to,; single CDRs, VH iegions s; Vh regions, Fv fragments, F(ab) amd F( 3 ⁇ 4 fragments, ⁇
  • Additional fragmen s c3 ⁇ 4n include diahodks * linear antibodies, .sing!e--ehain ssfitibody molecules, and xmMi specific antibodies formed from antibody fragments, Aatibody fragments? suitable for n-se wit the prese ly disclosed binding ddnralm oars include, natural,, synt!ie ie, of- recombinant versions.
  • Single chain antibodies are genetically engineered Molecule eom ⁇ ainmg.3 ⁇ 4 variable region of a light chain mi a yaiiabfc region of the 3 ⁇ 4eavy chain, linked by a Mt e polypeptide linker as a genetically fused single chain molecule.
  • Such single chain antibodies also are referred to as ''single-chain Pv" or "scFv" antifeody fragments.
  • the Fv poiypeptii e ferther comprises a pol ⁇ eptide [inker between the VM and. 3 ⁇ 4 domains that enables the scFv to form :tbe desire struct nm fdf ajfigen binding.
  • the binding domain can be a single eha variable i3 ⁇ 4gment (scFv) of an antibody wife, attachmen site for a dye withi a C R3e ion of the s3 ⁇ 4Fv 3 a modified ftagment of a natnraiiy ooe ttxing protein, or another ontity that binds ⁇ a specific state of the targeted protein or polypeptide.
  • scFv eha variable i3 ⁇ 4gment
  • a compound of Formhlai or Formula 1! can he attached to the se!eoted apFv at any convenient site.
  • snch antibody fragments can be smaller and more stable than Mi-length antibodies.
  • Snch a tibody i igrnents can include about 4 amino 'acids, 5 ammo 3 ⁇ 4ci4 ⁇ 3 ⁇ 4 6 " amino acids/? amm acids, 9 amin -wMs, afeo ' ai 12 ⁇ amino acids, about IS amino :aeids, about I? amino apids.abou !. ⁇ amino acids, about 20 amino acids, about 23 amino acids, -about 30 aniino adds, or more,
  • a antibody iragmeni can liave,any upper m limit so long as it i lias similar or improved properties relative: to an antibody that binds with specificity to a target molecule. For example.
  • smaller antibody fragments can h e less than about 200 ammo aelds, less than about 175 amino aeid3 ⁇ 4 less than about 150 amine acids, i>r less than about 120 amino acids if the antibody frngnient is related to a light chain antibody su nnitv
  • larger antibod frag meats can 3 ⁇ 4tave less than about 425 amino- acids, less i3 ⁇ 4an about 400 amino acids, less than about.375 amino acids, less, tha about 350 amin -acids, tesrihan about 325 mi o ac ds or less than about 300 amino adds- if he: antibody fra ent is rel ated to a ' heavy c n anti body subunit 1 S
  • Antibodies add antibody fragments directed against selected targets can % prepared by techniques- commonly kaown in th art, in some embodiments, antibody ⁇ fragments can be prepared from MMength: antibodies.
  • the tenn "moBoclona! antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, In other words, the individual .mti ⁇ ies witiin'isiiig .
  • Mcmociemal antibodies are highly specific, being directed against a single antigenic site, I rthep, in contrast to polycional antibody preparations thai typically include -different antibodies directed against different determinants; (epitopes), each monoclonal antibody is directed against a single de&imin&nt on the: antigen, In addition to thei specificity, th monoclonal antibodies are advantageous in that they are synthesized by the hybridorna. culture, uneontaniinated by other .
  • the raodiiier "B:ionocl >nal w indicates iris character of the aMibody as being obtained from a s3 ⁇ 4bs3 ⁇ 4ffinaiiy homogeneous; popniatio.n of antibodies, and is mi to be construed as requiring ro ucti n of the antibody by any particular method..
  • the monoclonal antibodies herein specifically include, "chimeric" antibodies In.whi h a: ot-ion of the v anoYor light chain is identical of homologous to correspondin q &s :aMibodles derived ftortia. partlcniar species or belonging to :pariicular aritibod class or subclass, while the remaln lcr of the ehain(s) are identical or homologous to corresponding se ue ces in antibodies derived from, another species or elo gi g to another ⁇ antibody- class or subclass.
  • Fragments of such antibodies also can be used so long as ifaey exhibit t3 ⁇ 4e desired biological activity * See If Pat 4$m, ; M t u ei ., roe, N fl M. Set ⁇ V/ ;685I-S5 (1984).
  • antibody f agments are also kno o in the art (see for example. Harlow and Lane, Antibodies; ⁇ Laboratory Manual Cold Spring Harbor Laboratory, Ne Yerfc. ( .1 88) incorporated herei b reference).
  • Am body fiiagmea is suitable for use with the resently disclosed subjset raatter can be prepared by proteolytic hydrolysis of tte antibody or by expression of nucleie acids encoding the antibody ffagm af ia a suitable host!
  • Antibody fragments can be obtained by pepsin or papain, ⁇ digestion of whole antibodies conventional methods. For example,
  • variable chains can fee linked by an miefffioleeniar disnlSde bond or cross-linked by chemicals such as giutaraldehyds,.
  • the Fv fragments comprise V H asd Vp chains connected by a peptide linker:.
  • These single-chai afitigen binding proteins C3 ⁇ 4Fv) are prepared by Constructing a: strpetwrai gene comprismg Bbi A s quences encoding the ⁇ p and 3 ⁇ 4 domains connected by an oligonucleotide.
  • the straetural gene is hiserted into an expr ssions vector, which is subsequently introduced Into a host cell such as E.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide hridgirig the two ⁇ ' domains .
  • Methods for producing SFVB are described, tor exsmpiefby Whitlow etal, . Metho s; a Companion to Meiho m-Bmym&k& Vol page 97 (1991); Bird et aL Science 1 ⁇ 2:423426 (198 ?); l ⁇ & at, IIS. Pat . 4,946,778; and ?
  • CDR peptides can be obtained by eloning: or eonsirneting genes encoding the CDS. of m. antibody of interest. Such genes are prepared, for example, by using the- polymerase chain reaction to synthesize the variable region ir3 ⁇ 43 ⁇ 4 RNA f anUbody-producing ceils, See, for example*.
  • sequences for manipulation by genetic engineering techniques known in the art Aj&i&o-add. and fciictdciacjd ⁇ eqUe»ces ttas. obtained can be screened for binding ehaMdfeqsti.es: ⁇ desirable I s Sxe presently disclosed biosensors.
  • equences ifeps obtjuned can be logically modified and/or randomly .mutated to generate additional binding domain candidates at can be screened to identify sequences most useful in particular biosensor systems of interest.
  • ⁇ hnraanrzed antibodies are human immunoglobnjing (recipierit antibody) in which residues, from a complementary determining region (CDl ) of the recipient are repiaeed. by residues from a CDIt of a nodmniau specks (donor antibody) such as mouse, rat or rabbit having the desired specificity, afSriity and capacity.
  • immwnogiobn!iii oars be replaced by corresponding non-htmiac residues.
  • Frirtlter no ej humanize anybodies can comprise residues that are fo&ttd neither In the recipient antibody nor in the h iported CDS. or framework se uencer These modifications can be made to.fitrifeer refine and optimize antibody performance.
  • H i In H i
  • ' ki a zed antibodies will comprise substantially all of at least one. and typically two, variable .' ' dom ins, in which all or substantially all.
  • -efths CDR regions c respond to those of .a non-human iimmmo lobulia and al l or substantially ⁇ 3,1 of the PR. regions are those of a bumaft -immunoglobulin co sensus sequence.
  • the huma ized antibody optionally eaa comprise at least a. portion of an iminnnogiobnlirt constant, region (Fc), typically that of a imaxi- immunoglobuli .
  • a mutant binding domain refers to an amino acid sequence variant of a selected binding domain (e.g.. a CDl ).
  • a selected binding domain e.g.. a CDl
  • mutant binding domains ave at le st 7S1 ⁇ 4 amino acid .sequence identity or similarity th t e amino, acid sequence of the reference bidding domain.
  • mutant binding domains have at least 801 ⁇ 4, more preferably at least 81%, even more preferably at feast 90%, and mosi pceferably at least .95% m oq ms identity or similarity t& the amino aci seqvsHee of the reference itdiiig domain,
  • affinity ma i i3 ⁇ 4i ft using phage display ca .be utilized as one method .for generating mutant binding domains.
  • Affi ni ty maturation using phage display refers to a process described ip Low an t u i chemi ry 30(45); 10S32- 1.0838 (199:1), see also Hawkins t L M&IMM.
  • this process can be described briefly as involvi g mutation of several bmdirig drains or antibody hyperyariable regions at a n mber of different sites with the goal of generatirrg all possible mi e acid subs ituti ns at each site.
  • the bindin dsmaln mutants thus generated are displayed i a monovalent fashion, from filamentous, phage particles m fusion proteins. Fusions are generally made to the gene HI product of Ml 3.
  • the phage e pressing the various mutants CM be cycled through several ou s of selection for tits trait of interest, e.g., binding affinity or selectivity.
  • the presently disclosed: subject matter provides methods of manipulating binding entity or antibody polypeptides or the nucleic 3 ⁇ 4eids encoding tiiern to generate binding entities, .aniibodies and antibody fragments with improved binding properties, that recognize selected targets .
  • portions of an existing binding entity or antibody involve fusing a nu leie acid encoding a polypeptide that encodes a binding omais to nucleic acid encoding a phage eoat protein to generate feeomMoaiit traeleie aei encodin a fusmB: iot 1 ⁇ 2 s mutating the reeom inaat nueiele acid encoding the fusion; pro ein t generate mutant nucleic aeid encoding a mutan fusion, proteiru expressing the mutant fusion protein on the surface of a lage, and selecting phage that bind to a target
  • the .presently disclosed subject matter provides antibodies, antibody ff agme s, and binding entity pol peptides: that tm recognise and bind to selected ta get molecules.
  • Tk s. methods for man!pttlatiiaj those antibodies, antibody t1 ⁇ 4gmeots, and binding entity polypeptides, cm be nsed to optimi e their binding roperties or other desirable properties (e.g. y stability, sk3 ⁇ 4 ease of use ,
  • the biosensor molecule includes an HIY-l
  • a f agment of WASP which hinds oniy to tie aci!yated state n G3 ⁇ 442 can be used detect Cde42 activation, as also des ribed in IIS, Patent Application Pubnealion No, 200 ⁇ / ⁇ 92994 ⁇ : to Halm.
  • Such biosensors ca be used to deieetand or monitor he level of Cd3 ⁇ 442 activity in vitro. In celt lysaies, and in livin cells.
  • methytransferase W& can be used to detect, the enzyme.
  • the compound iifiyoperazmc which binds -selectively to the active conformation of caimodnlin, om be used detect es!mcdul m activity.
  • the presently disclosed biosensors can be ikked to a target mokeule.
  • the dye can be linked to tire target molecule at a position that jesuhs in a. e!mnge in a signal irons the dye upon changes to fee target con ormation ligand binding, to the target, protein-protein inieractioris with tl e target, phosphorylation of ' the- igxget, or: postiranslabonai.
  • the presently disclosed dyes can be 3 ⁇ 4ti3 ⁇ 4ehed to a protein of interest, wherein an aciivi ty y location.; and/or conformational change in the protei can result in a change in the fluorescence of the dye,
  • 3 ⁇ 4t proteins within mnitiprotein complexes can be Moaitored isl situations whore other types of biosensors, ibr exarn iCj those requiring a drj ak to find the target protein, would ! ⁇ blocked,
  • the presently disclosed biosensors can allow the detection from a. single dye, e,g, s without the use of two fl orophores, as is FRET,, to. provide direct excitation and a brighter signal.
  • two o more Of the presently disclosed compo unds of Formula I or Formula II having different emissions wavelengths can. he used to ensHe imaging of mnliiple protein activities in the same eel! simu!ianeoiis!y.
  • it can he desirable to provide more than one dye molecule on the binding domain of the biosensor to facilitate monitorin of a binding event,
  • the presently disclosed subject matt r provides methods to examine protein activit s . sir uctur3 ⁇ 4 o protein-protei interactions t
  • the presently disclosed methods and biosensors ean report protein localizaiiott s protein activation and/or report specific aspects of changing protein structure or ter3 ⁇ 4cilo3 ⁇ 4
  • the biosensors can be used in living: cells ari 3 ⁇ 4 for ho ogoneons assays.
  • the activity of a protein, in a complex mkmre 3 ⁇ 4 such as ceil iysate, can he determined by adding ami defecting the biosensors ltfepnt addrtiunal steps, such as wash steps, and die like.
  • the presently disclosed subject matter provides a biosensor comprising one or more binding domains havi g a specific ⁇ affinity fqr one or more target molecules in a specific state pfp osphory!atidn
  • the binding domain of the biosensor can have a specific .affinity for a binding, site ⁇ produced upon a protein that protein binds to a goanosine triphosphate, thereb activating the signal tra»s ⁇ iion protein
  • the biosensor can ha e a signif!emtf y lower affinity for the: inactive form of the protein (e.g,, the protein bound to ga nosino diphosph at ⁇ instead of guanoslne triphosphate).
  • J3 ⁇ 4. «s,for example, a dye of the biosensor, ,g vie compound of Formula I or Formula 3 ⁇ 4 .cm occupy a position fee binding domain pear the bindin site soeh that, upon binding to its target, the binding environment alters the fluoreseettt signal from the dye. Further, th position can provide a detectable signal change in the dye without significantly hlhi tia m biological activity of the targeted protein. Deieeifcm of the acti vated target in this embodiment does not require conformational changes in the ta get or the presence of third molecntes interacting With the target
  • Interactions- S3 ⁇ 4eh interactions can be high affinity interactions, such as ; for example, interactions' between a gens and antibodies, or the can he lower affinit
  • M o iopSj. snob as, for ⁇ sfk lsi ⁇ cfi ⁇ between enzymes and subsu3 ⁇ 4te3 ⁇ 4 signal cascade members, or members of protein complexes. Is- a protein-protein interaction. hetw ⁇ en two ⁇ binding members * one binding member can be considered the l rger and the otic? binding, member the biosensor probed 5 While the distinction betwee target and- probe ears he arbitrary, because the two intemcj with each other these terra a are used, to facilitate discussion of protein interaction.
  • the target can be the m:olee3 ⁇ 4le to be detected and the probe can be a bipdkg member introduced to interrogate a sample for the presence of the target in a state or form of interest.
  • one member of the binding pair can have a bindin domain complimentary to a binding she o f the second binding pai member.
  • Out or both of the protein-protein interandon pair members can include an attached com ound of Po ⁇ la I or :F ⁇ ri3 ⁇ 4ui II.
  • the protein-protein barreracdon pair members can be falh!eng3 ⁇ 4 naturally occurring proteh3 ⁇ 4 synthetic analogs of naturally occurring proteins, recombinant analogs of naturally occurring proteins * or f agments thereof
  • the presently disclosed biosensors capable of having protein -protein Interactions can have compounds of Formula I or Formula II at one or more positions on one or both binding members s ch that the dyes are positioned between the 3 ⁇ 4 binding members dur ng lad ng, ⁇ such embo iments, binding e n cause a ⁇ 3 ⁇ 4iecteMe change the signal :from tie dye without ' significantly inhibiting the binding interaction crizwee3 ⁇ 4 t e3 ⁇ 4nding members, Suitable dye positions caw be identified by screening alternative positions for im rov signal and/or binding i etis ⁇ B 3 ⁇ 4e resently disclosed biosensor systems,
  • the biosensei? m 3 ⁇ 4ave specific affinity &f a target molecule in a specific conformation hound to a specific ii and, or with a specific pesttfansiatlonai modification other than pbosphdry on.
  • Modular biosensors are senso s: of unified design that allow cer n compoB nis ⁇ he changed,. e,jp s .to change the target .specificity and/or signal character. Modular biosensors typically provide for con venieni alteration of bindin domain speoirleity/.
  • amaffinfy molecule cm be expressed fyom a ⁇ recombinant expression instruct, such as an expression veepm
  • the genetic construct can include unique endonnelease sites bracketing the region encodin ⁇ lie binding domain so that alternate binding aomaius ca b readily inserted into tbe construct for expression as part of a biosensor.
  • the modular binding domains can be selected, e.g., iom a library of binding domains.
  • tbe modules can be encoded scFv dorsains.
  • he: modular systems cm include ailnlty Molecules with alternate biridiiig domain sit3 ⁇ 43 ⁇ 4 .alternate dye linkage sit.es > a!temaie dye linkage reactive molecules, alternate linked targets, alternate linkers, and/or the like.
  • binding domain camb Of broad specificity, and tbe target domain different k nase consensus sequence peptides ⁇ can. be switched in tbe modular design,.
  • Ceil behavior can: he regul ated through trausient activation of protein activities at specific subcellular locations.
  • the ability to study translocation of proteins has been enhanced by advances In the microscopy of fluorescent protein analogues living; cells, in man cases, however localized protein, activities am controlled not Ijy translocating proteins to the site of action, but by localized activation of a small portion of the protein pool. See Ha a- and- Tontebidne, Cum eW o , (2002); - «s Tr ds ⁇ J7:203 : ⁇ 2il
  • the dyes exhibit man properties that make, them .suitable for detecting target niolecnles mi their mtemeti ' Oiis fa living cells,
  • the dyes are, for example, %ight with long, way ⁇ lengths outside of cellular autofluorescence background ixeqiteocies and that are less damaging to cells. Addition or deletion of parts of the aromatic system, or snbstituent groups thereon, can shin excitation and/or emission wavelengtha of the dyes so that more than one event can be monitored in a cell at the same: time.
  • the dyes have increased cell membrane petem&ea blity with minimal staining of intracellular membranes.
  • the method comprising; (a) providing the biosensor of the present invention; (b) contacting the biosensor with, a etdi suspected of containing one or more target meleeu!es to bind the one or more target molecules, If present, to the binding member; (c irradiating the cell suspected of containing one or more target .molecules with electromagnetic radiation to induce the ⁇ compound of Formula I or Formula ⁇ to fluoresce; and (d) detecting one or more oil (i) a fluorescence property of the compound of ormn!al or Formula II* (if) a change in fluorescence propert of the compound of Formula I or Formula II; (iii) a location of a fluorescence of the compound of Formula I or Formula II; and (iv) combinations thereof to determine the an activity or location of one or more target molecules in the cell providing a biosensor comprising a dye of Formula (I) or Formula (il); (h)
  • target molecules can be detected in freshly isolated cells from mammals (e.g., humans), insects, fugal, or bacterial cells.
  • mammals e.g., humans
  • insects e.g., insects
  • fugal or bacterial cells
  • blood cells such as B cells, T cells, monocytes, and neutrophils, and the like
  • stably maintained cell lines such as CHO, HEK-293, L-ceils, 3T3 cells, COS, or THP-i cells can be investigated using the presently disclosed methods.
  • Useful information can be obtained from any ty e of cell using the presently disclosed biosensors and methods.
  • mammalian cells such as human cells or animal cells, that naturally or recombinantly express human proteins can be evaluated to identify potential human therapeutics, observed for interactions between biomolecu.es, and/or studied for the effects of ligands, drugs, and other molecules on mammalian and human systems.
  • bacterial or fungal cells can be used to screen for potential antibiotic or anti-fungal agents,
  • well characterized cell lines known to provide predictive models of human cell functions can be used to obtain results correlated with human systems in pharmaceutical and medical research.
  • Exemplary ceil lines useful in sack research include, for example, COS cells, CHO cells, HE -293 cells, R L-L Jurka U93.T, and YB-1 cells.
  • the cells, t ⁇ 3 ⁇ 4e otritored can be p ovided in either iffimobil izei form 01 as- a sus sion culture, immobilized cells, sucfe-as, .e > . cell law s, tissue slices * or libraries, can be Mo it ed, &gi,.hy irrieroseopy, soakers, or with imaging systems.
  • the immobilized cells ca be mo jiored live or fixed fbr detection of target -molecules Infilled cells.
  • cell deusihes of monitored sus e sions generally ange roa abont 1 eeil ni, to about 30 DCis/nL in, e.g., a react on vessel or detection channel.
  • ceil densities typically range Mm.
  • hese ranges can vary depending upon, e.g.., the cell types used, the ty e of biosensor employ ed > the type of interaction to be studied, fee relativ adherence of tie cells to the vessel surfaces-, as well as each other factors.
  • biosensors that arc capable oFsmdyixrg proteins in living, eel
  • the dyes can be directly attached to proteins, enabling conformational changes to be ollowed in vivo for proteins incorporated m large molecular machines.
  • Sarin vitm applications the dyes exhibi an increased photostablihr and provide a substantially brighter s nal than current solvent-sensitive iluorophorea. Tims, they can enhance sensitivity for studying high* affinity binding Interacti ns, small amounts -of protein in high-teoughpnt assays, or protein changes that produce only small effects on. other d es.
  • the presently disclosed .biosensors can h sed Mxi and/or j» vivo to detect target molecules of interest In some em odiments, the biosensors cm be
  • biosensor ' having; a specific
  • biosensor to facilitate, the translocation or internalization of that biosensor irons
  • Bead ⁇ iage loading procedures are deseAed in MeMei! el at J Cell Biol 8dSM5&4 (1984); and MeNeiiand Warder, J Cel!Sci ⁇ :.$0 ? ⁇ (1987).
  • binding domains disclosed herein can be introduced into cells in expression plasm-ids * e..g, s . by transduction or other forms- f transfermatiOB, Once inside the living cells, the binding domain can be translated from: the nticieie aeid to a mnctional peptide.
  • Com o nds of :F xraula I or Formula can enter the cell e.g. , by inje$;ios v or dif3 ⁇ 4siofi ; to become imked to the expressed feeling domain.
  • the presently disclosed dyes also a 3 ⁇ 4e k rod eed isiio cells by other methods Imovm iri the art, isciuding, tat not limited to vir l-based delivery systems, -including viral-based sroopartides, and lipid delivery vehicles, mef3 ⁇ 4dmg: liposomes, md. 3 ⁇ 4
  • the : presently disclosed subjec matter provides .metk fe 3 ⁇ 4>r ent!fynj-g t&e tiva3 ⁇ 43 ⁇ 4>fi state of endogenous proteins in Ikin .cells.
  • the presently disclosed hiosensorsicaii permit foundedrti lcation, qtisBtiiloatios, and ⁇ resolution, of the spatial, te ⁇ mi.and ' -camp ⁇ entaireguia ion of reoeptor pbosphfirylatiofi and activation dtiring various processes, for example, endofiytosis, la aaother embodlmeiit, the presently disclosed Mosensors an i methods can perrriit ohservatioa of epidermal growth facto receptor (ICIPR) eifeets n the development ⁇ mi prog ressloE of breast eai
  • ICIPR epidermal growth facto receptor
  • the: presently disclosed subject matter provides a method of detecting 3 ⁇ 4 Interaction, between mendoge»ous tar e moleerde mm a cellular entity, the meted eoaiprisirtg: ( ) f rirv!dmg a eel! comprising end3 ⁇ 4 enou& target molectde; ( )p
  • th ee!Mar ent ty is selected from the grosp eorislstlBg oF& eslhdar nucleic acid, a protein, a pep id , m enzyme, a receptor, a cytokine, a eytoskektoa, arid a signal feaasd3 ⁇ 4ction ; proteto.
  • the binding tnea ber of tie ⁇ >1 ⁇ 4 binds fee target molee de at a ph spho ylation site, la some embo me3 ⁇ 4ts ?
  • the binding member tea specific- affinity for a eoaiormatio ⁇ ligattd Interaction, or
  • the detecting a change In fluorescence com rises ⁇ an if lrig a rotebi ametmb locating a protein ⁇ detecting a eon&rmational change In the target moieeiB3 ⁇ 4 cleteetkig aetivadon of fee large! moleeule, er detecimg phosphorylation of the target olecule..
  • binding interactions can oeeur between a biosensor and one or m ore target molecules or components of the ceil.
  • a 'target molecule f interest' 5 is atBoIeeute tbat is by one of skill la the art and is selected fa interaction with a presently disclosed .
  • a target itioleenie often: comprises an endogenous unlabeled sndfc untagged component of a test so! u ioa o cell Endogenous -pompose-Kts be, e,g ⁇ > expressed by the cellnaturaly, or present as a result of introduction of an appropriate genetic construct within t e sell
  • naeieie acid or protein target molecules can be expressed in the cell, either nattrraily (e. . ; wHistitutiveiy) o by ind3 ⁇ 4 tion of an appropriate genetic construct lritrod3 ⁇ 4e
  • the presently d sclos d: s bject matter provides a method for determining the presenc e or am u t of one or more target molecules in a sample, the me hod comprising: (a) providing the biosensor of t e inventi on; (b) contacting the biosensor with a sample suspected of containing one or more target molecules to bind the one or more target molecules, if present, to t e binding member; (e irradiaimg the sample .suspect d of containing one or m re target raokoules wish electromagnetic radiation to indace the compou d of Forinnla I or Foron la 11 to flitoresee; arid, (d) detecting a Snorescence roperty Of the compound of Formula I or Formula II to determine the presence or amount of one or more target molecules in
  • the method further comprises eotitihuo3 ⁇ 4sIyj 3 ⁇ 4) contacting the biosensor- with, the sample suspected of containing one or more target moleeufes; (b) irradiating the sample with eiectromtgnefe radiation; and (c) cleiecimg As fhiorescence property of the nompennd of Formula I or Formnia IL
  • %araple if Includes: any Bauid or " fluid sample, including a sample derived i3 ⁇ 4om a biologicafsource, suc as a physiological Stud, including whole blood or hole blood componen s, such, as red blood eells f white blood cells, platelets, serum and plasma; arche t urine; saliva; sweat; milk; synovial fluid; ert!cjoeal fluid; ainaiotk fluid; peicexehrcspinal Said; lymph Suid; lung embolism; cerebrospinal fin3 ⁇ 4 pericardial fluid; 3 ⁇ 4 ⁇ mc«y «
  • physiological fluids * other liquid samples;, sucluas ater, food products asd the for the periwman&
  • a solid material suspected of containing the analyte also can be used as the test sample.
  • somflr t Stap ⁇ 3 ⁇ 43 ⁇ 4 ⁇ it might be beneficial to modify a solid test sample to fo m a liquid meohura or to release ; the apalyte ⁇
  • the sample can be pre-trea ed prior to use, such as preparing; plasma from blood, diluting viaeous fluids., or the like.
  • Such .methods of treatment can. involve filtration, distillation, conc3 ⁇ 4nkaiio3 ⁇ 4 isactivation of interfering c .u3 ⁇ 45Gim ls f and the addition of reagents..
  • the sample can be arty sample obtained, ffoirs a subject Hie term
  • subject r-eiers to as- o anis , t ssue, or ⁇ ell f om which a sample ca he obtained,.
  • a subject c include a human subject i r medical pnr 3 ⁇ 4Gses ⁇ such: a$ diagnosis aad or treatiftefit of a condition or disease, or an animal subject ihf medical, veterinary
  • the subject is a mammal or a rnammaHaii cell. ' More preferably, the subject is a human or a human cell.
  • Human subjects i:Eciude s bat are not limited to, fetal:, aeonaial, infant, juvenile ⁇ and adult subjects.
  • a "subject" can include a patient afaiieted with or suspected of being afflicted with a condition or disease.
  • subject matter further provides a packaged composition sueh as a kit or other container for delecting, monitoring o.r ot3 ⁇ 4.er ise observing a: target molecule, 3 ⁇ 4e kit or container eap.&old a biosensor comprising a eofflpouad of Formula. I or Formula. IX and instructi ns for: using the biose sor for
  • kits cotiia kg dyes cm cant in,mstFU(;tioss for att ching a dye t a binding domain selected by one of skill k the art.
  • kits also can c m ise containers with, solutions or tools useful for .manipulating or using the presently disclosed dyes or biosensors.
  • tools include buffers:, reaction tubes, reagents for coupling ooTn oimds of Formula I or Formula I to selected binding d&m m mm the like.
  • the kit can contain a seiution of solventa ' and/or buffers to facilitate cotipliag -of 3 ⁇ 4 compound of Fomruia I or Formula ⁇ to a selected binding domain and/or a solution of mereaptoetharjol for queseMng the dye-binding domain eonlugatiort reaction, i3 ⁇ 4e kit a! so can.
  • melode a cont ker of buffer m roughly neottai pH sodinm bospba e buffer, pH 7.5),
  • group wiii generally 1 ⁇ 2ye the structure that is recognized in the art as corresponding to a group 1 ⁇ 4V&g ' ..3 ⁇ 4t name, ualess specified otherwise herein.
  • electron withdrawing group refers to a atom o a group of atoms that dra : electrons a ay from a reaction center, Exam les include, without liispation, carbony
  • l ving group refers to a charged or uncharged atom or gro up of atoms thai: departs Airing a $afcsi3 ⁇ 4ttta w displ ⁇ i ⁇ ⁇ ,.
  • examples include, ithont limitaiiou, haio ns, such as eh!bro ⁇ broP o, and iodo; lko IdCj hiflate, tosyiate, mesylate, torosulfonate and optionally substituted N- hydroxysPoeiniMide.
  • the- terra 3 ⁇ 4o3 ⁇ 4 ttgatalne $ ⁇ 3 ⁇ 4 ⁇ eh®&" efers to side chain comprising, consisting essentiall o3 ⁇ 4. or consisting of a linker and a jeaeiivs grou at the end of the linker that can be used to conjugat a dye compound to a binding member.
  • alfcyl refers to C W o inclusive, linear (?. & ⁇ ⁇ straight-chain' branched, or cyclic, saturated or at least partially and in some cases felly uns tu ated ( .a s a!kenyl and alkynyl) hydrocarbori chains, including for exarnple, methyl, 3 ⁇ 4 3 ⁇ 4 propyh isopropyl ;; butyl, Isobutyh fert-buiyl, psntyh hexyh oeiyi e&en k propenyk btitenyk pesienyk toertyk oetenyl, 3 ⁇ 4uiad3 ⁇ 4nyl, opynyi, butyByl, pem n l 1 ⁇ 2syriyi ⁇ neptynyf, a alenyl groups, "fif
  • aikyl groap having I to. abmd.6 carbon atojns i. e ⁇ , a alkylX 3 ⁇ 43 ⁇ 4 5, or ⁇ earboii atoms, s 3 ⁇ 4tgher alky! refers to an a!kyi group havmg : abpj.fl I to a out 20 carbon, atoms, .e>& > ⁇ » JG, 12,
  • C ⁇ ' siid ⁇ io ⁇ kl refer to a rion-at3 ⁇ 4rn ic mono- or .multicyelk ring system of boM 3 to about 10 carbon atoms, e.g. % 4, S 6, 7, or 1Q carbon atoms, ' 11 ⁇ 4 eydoatkyl group: can be p iwally : partiall unsaturatd
  • the cyekoa!ky! group also can be optionally sahs l3 ⁇ 4ted with an aikyl gronp substifaent as defined feereirn oxo ? mSfm alkyiene.
  • sn3 ⁇ 4stitnsnt is hydrogen, alkyk .stibstitnied aikyk aryk o substituted ajyi* thus providing a heisrp ye!ic.: group, Re resentative monocyelio cyc!oalfcyl rings molade cyo eity!, ov! hexyL tad cyolohepiyl Muliioyclfc
  • the cyeloheteroa-tkyl ring e optionally fused to or o3 ⁇ 4erw!se attached to olner cyelobeieroalkyi rings asd% Bon-a piahe hydrocarbon mm, Represeniative cyelohete &ifcyl ripg systems itfehtde* hnl are mi limited to pyriolidii y!, pyrroHnyh iffilda Bliil nyii iffiidazollnyl, iryrazoi inyi.
  • alfcesyl refers to a straight or bmnebed.
  • aryP is used hsrefe to refer to an aromatic substiinent that can fee a single sfomstie ring, or irrohiple aromatic rings that are fused: together, I ed -eo.vale ly, or Jinked to a &m m grou ,, sue - , n% Hot limM to, a methylene o e 1 ⁇ 2fte:.me.ieiy he common linking group also dan he a e rkmyJ, as in
  • r l 5 specifically encompasses heterocyclic aromatic compounds.
  • the aromatic gs ⁇ £m comprise phenyl, napfeli l hiphsnyi diphe3 ⁇ 4ylehtet,
  • aryf s means a cyclic aroniatie comprising about 5 to about 10 carbon atom% e,g,, 5> 6, 7, 8, or 10 ca.rbon atoms, and -including S- and 6-me bered hydrocarbon and heterocyclic aromatic rings,
  • the aryl group can be optionally sabsti ied (a "f absented aryl") with one or more aryl gronp s3 ⁇ 4hstiiu3 ⁇ 4ats 5 which can he.
  • the .same or diSetettt, w erein s ⁇ aryl group subsfitnent includes alkyl, substituted alkyi, aryl substituted aryl aralky!, hydroxy!., aikoxyl, aryloxy!, aralkyloxyl, carboxyl, aeyl !halo, niiro 5 alfcoxycarboriyl, afyloxyearbonyl, aralkoxyeatbor l s acyioxyi, acy!aisln , 3 ⁇ 4royianino> carbamoyl, alfcy!carfeamoyl, lialkylearbamoyi, aryithio, atkyhhi% aikylene, and -NS.'R", wherein R' and R* can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted atyh and aralky!.
  • ary! groups include, but ate not limited to, phenyl, iuran, th3 ⁇ 4phene 5 pyrrole, pyrau, pyridine, Md ⁇ o ⁇ ffiii ⁇ idazoi ⁇ soiaagoi , i3 ⁇ 4oxa3 ⁇ 4sie 4 p a3 ⁇ 4»l3 ⁇ 4 : ⁇ yra3 ⁇ 43 ⁇ 4e. triaano s pyriiBidine., guinoline s isoqukolme, indole, earbazole, and the like,
  • hoteroaryP refers to an aromatic ring s stem, such as, but not li itedto a S ⁇ ⁇ r 6 ⁇ merob3 ⁇ 4r liftg system, including ane or more heternatorna, which can be the same or different, and are selected from, the group consisting sfK, O, and S.
  • Theheteroaayi ring can be feed or othe ise a tached ⁇ am or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings, or heterocycloalfc i rings,
  • Representative heieroaryl ring systems include, bat are not limited, to. pyridyl, pyrimidyl, pyrrolyl, : pyrazolyl, m>ly% ®tm ⁇ y . isoxaaplyl, 3 ⁇ 4d a2;olyl f thiaxolyl, isothl o!yl, imld&zoM, for n i, thknyh qumoh h jso ms lia l, indolmyL indo!yL cu Oiihesyl bcfizoihiazolyl, eaasjforanyl bensinif dasoiyl benasosffi&ol i, bewzopyraiso!yh triazolvL ie3 ⁇ 43 ⁇ 43 ⁇ 40ly), and the lite
  • a ring: steot re for ex m le, hut act limited to a 3 -ear bora a 4 ⁇ earbe:n, a Smai'heri, a 6-carboi3 ⁇ 4 aiid the iike ⁇ aliphaiio and/or aroni tie cyclic compound, including a saturated, ring structure, a pariiahy satui-ated ring structure, and an unsaturated ring structure as doimed herein, comprising a substlt ent R group, wherein the A grou can he present o absent, and when p ese t, one or more R groups eameach he sub tete «f.
  • n3 ⁇ 43 ⁇ 4 the stactee above where n is i to :2 w uld comprise compound groups including, but not limited to:
  • a dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line
  • representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure,
  • acyl refers to an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent ⁇ i.e, , as represented by RCO ⁇ , wherein R. is an alkyl or an ary! group as defined herein).
  • RCO ⁇ substituent ⁇ i.e, , as represented by RCO ⁇
  • acyl specifically includes arylacyi groups, such as an acetylfuran and a plienacyl group.
  • acyl groups include acetyl and benzoyl
  • alkoxyl refers to an alkyl-O- group wherein alkyl is as previously described
  • the terra “alkoxyl” as used herein can refer to C 3 ⁇ 4> inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyL isopropoxyh butoxyl, t-butoxyl, and pentoxyl
  • alkoxyalkyl refers to an alkyl -O-aikyi ether, for example, a methoxyethyl or an ethcxymethyl group.
  • Aryloxyl refers to an aryl-O-- group wherein the aryl group is as previously described, including a substituted aryl
  • aryloxyl as used herein cm refer to phenyloxy! or hexyloxyl, and alkyl, substituted alkyl, halo, or a!koxyl substituted phenyl oxyi or hexyloxyl,
  • alkyl-thio-alky refers to an alkyl-S-alkyl thioether, for example, a methylthiomethyl or a methylthioethyl group.
  • Araik l refers to . aryl-a!fcy iigrou bgreta axyl m0 a!kyl are as previously described, and included substituted ⁇ w ⁇ mi substituted alkyi
  • f ft3tOS Aralk$3 ⁇ 4s f- refers to . ⁇ ara&y i-Q- group wherein the aralkyl gou is m previousl described, An exemplary aralkylo&y] grou is beirzylo yL
  • ⁇ Q 6 ⁇ "Afcoxycarbouy!” refers to ⁇ skyl-O-CO- group
  • alkoxycatbonyl group is ' beBzyloxycarbonyl
  • C rbamoyr' refers to an 3 ⁇ 4.N ⁇ €G ⁇ grottp, "AlkylcarbarnQyl" tsfers to & ' N-CQ-- grmip wfeereia orse of.R and W is bydrogen and te other of R & if alky! and or substituted aikyl as reviousl described; ⁇ fjiak iw w ' *omb*;$» a ' -CO- group wferein eao ⁇ fii and R ⁇ Is independently a!kyl and/or sihsihit aikyl as previously deseribed,
  • cykayF refers to an acyl- ⁇ - ⁇ group berein aeyl is as previously described.
  • *1 ⁇ 4 ⁇ 8 ⁇ , ' and "alky!anlao” refer to specific K-sribsti ed organic radicals with a3 ⁇ 4yl and aikyl substituent groups respectively.
  • fi 3 ⁇ 4ky3 ⁇ 4mino S5 refers: to a— 3 ⁇ 4 group wherein R Is m aikyl group aad/or a spbstil ted alky! grou as previously described.
  • alfcylamms groups irieiude mgtiiyiamiao, etbylamine, g3 ⁇ 44 the like,
  • Diaikylanisno refers to an - f group wherein each of R and W independently an aikyl group and/or a substituted aikyl group previously described.
  • Exemplary dia.kylami.TO groups include ethylmethylarriiriO, dimsthyiamiao, and diel&ylanrino. ⁇ 0
  • f 1)213) &c$miw& ' refers to an ae i- ⁇ H--- r u wherein aey 1. : is as pre vionsly described.
  • ⁇ Atoylammo refes to art aoyl-NB- group wherein aroyi is as .
  • a ⁇ Quuterasry pitrogen aiors ! M is a ntt og i atom bound to fou atoms* for e3 ⁇ 4 i3 ⁇ 4 ⁇ le : , fou carbon atOHis, atid ': hvitsg « positive e!irge available for hindjrig ionically to m. jioa for the remaining vale&ce, A tiaternajy .nitrogen &L k desi 3 ⁇ 4ateii herein as "
  • %ydr ⁇ xyalkyl ,s refers .3 ⁇ 4 an alky] group substituted with an -OH group.
  • ⁇ ifouyi refers to the -SO?- . group,.
  • Tire i3 ⁇ 4ll vs3 ⁇ 4ag ' fi3i3 ⁇ 4m.ples-:-8-r « differed 1 3 ⁇ 4r 3 ⁇ 4y -of lltettatiOfi-aiiid mi by way of limitation.
  • CBD- fern Wiskoit Aldrleh Syndrome Protein was covendedjy deti atM: with dye tp generate a l ⁇ sor, wi P ⁇ m $&pm $ mi methods prepared CBD iused to Mali)se>feliidmg protein for enhanced solubility (GBD- :MB 5 ⁇ pL of a 132,5 M stock solution in .0LmM.!
  • bacteria pellet was collected by oeaMfegsdoa (Beckniam model J-68) at 4000 trail for m m , ,4 °C, and. stored a -80 a C pnBv to purification.
  • Do3 ⁇ 4or a3 ⁇ 4 cs iw groups ⁇ i ' .behei3 ⁇ 4c»drfe tefeed bf their ietmr eodV3 ⁇ 4ams3 ⁇ 4 derived from th parent heteroeyefe, m order clarify dscttssis (Ernst et&l * Cytometry ⁇ 03 ⁇ (
  • Each acceptor riii was prepar d as its correspoiidfe methyl emA eiier feough ed cah%3 ⁇ 4d reaetioa with malon ideh)3 ⁇ 4 fcis(dimethyl)aeetal(3 ⁇ 4bie2).
  • the f acceptor roup was associated wife ciimlaishec fetl ve brightness, r gardless of the donor group with which it was paired, %eiedaced brightness for Pz-eoata ng dyes may be atiri3 ⁇ 4atahle to an: alternative de-exeitaiion pathway generated through the firestricted rotation of phenyl suhstitnente aroaad the G-M3 ⁇ 4orids (Sharaiy ei a!.,. J, Am. Ck > Si , 9 ⁇ 119- 12 ( ?t) o o!eartrond was: observed beri fe solvents wens ordered based on.
  • TBA Yield - 55%, :i H HM3 ⁇ 4 (400 MHz,. CDC3 ⁇ 4 584.1 (3 ⁇ 4 J ⁇ 1 .8 Hz, IH), 7,60- 7.45 to, 214).4.51 (q, /- 7.0 H3 ⁇ 4 41% 3.96 3 ⁇ 4313 ⁇ 4 1.28 (d3 ⁇ 4 12,8, 6.5 !3 ⁇ 4 6B); 13 C HMR (100 Mlfe, CDC14817:9.3,.172,6, 161.0.160.4, 159,2, ! ! 1,5, 106,7, 77.2, 59,1, 3,8, 43,2, 12,7, 12,6,
  • Si3 ⁇ 4l Yield - 72%. 3 ⁇ 4 M (400 MHz, DMIQ 3 90 °C) ⁇ 7,87 (d, J ⁇
  • CBD binds sefcctively to the activated, CfTP-bound conformation of C c42, Upon binding, a solvent-sensitive dye 05 CBD c3 ⁇ 4n respond to a change in environment as it is htoogiii near Cdc42.
  • VVater 599 i 830 1 143S0G i 0.00- eOH I 801 ⁇ 634 138G0Q i 0,01 140Q uOH I ..60? ! 639 16000.0 I 0.06 9000
  • C e42 biosensors were prepared with each of the four new cory ugaiabte dyes as well as the previously reported ri3 ⁇ 4er «22i»
  • CBB was fased to Cerulean fluorescent protein lot r do image analysis and. mutation 27!
  • C was Included for conj g tion- of the dye (Hedgson et a!,, . Methods Emytnol :140 ⁇ 156 (2006); Hodgson et ⁇ 3 ⁇ 4 > Citrr. Pwtoc. Ceil. Dial pp. 1-26, Chapter 14, Unit 14,1 I (2010)).
  • the biosensors were ⁇ kf0injec e43 ⁇ 4to-3 ⁇ 4oi ⁇ ?i3 ⁇ 4b?yoia!? ' fibroblasts ⁇ MEFs) rid used to examine the Cdc42 activity in eonsfitptive cell prptrusip is : refraetioos (Fig, B The biosensors based on mvtofii,.
  • the reactive ⁇ dyes could t he dissolve m ' Irigfciy nonpolar solvents, u the parei3 ⁇ 4 eornpound I»80 s been reported to reach a brightness value (QY x ⁇ ) of 123,000 m octauol, which is mong the largest values reported for organic dyes (Lavis et l,ACS Chem. mi 142455 (20083).
  • MIB 313 mouse embryonic fibroblasts were maintained in 10% C ( 1 ⁇ 4 at ' 3 C in Dulheceo's modified Eagle's medium ( MEM, CMigro) with im fetal bo vine serum (HyCione . , Thermo Scle rdfic.) nd; 2 mM GkhaMax (Slbco, Life Technologies). The ceils were plated on coverslips coated iii fibroneetiti (Sigma-Aldrich) overnight,.
  • Mass spectra were obtained on an Hewlett-Packard 1! 00 high-performance liquid cta>matograpfe equipped with a I I 0Q ioass-seleetiyc detector (MS-ESI), IJV -visible spectra w re obtaiiieci wi h a Hew!ett-Paefcard 8453 diode array spectrophotometer, Emission and excitation spectra, were obtained 3 ⁇ 4s1 ⁇ 4g a. Spex. PJuorolog spectrofluo ornster at 23*0.
  • the flask was sealed, evae3 ⁇ 4aied, : a»i flushed wife argon and G3 ⁇ 4C13 ⁇ 4 (350 m was added via eamuda, Tdeifeyiamirie ( 8,3 mt :s 275 mrrrol ⁇ was added nd the reaction was: stirred at room temp for 45 min, The flask was cooled 1 ⁇ 4 an ice bath and ethyl !s oyanate (20.8 L, 262,5 m aol) was added quickly dropwise. The reaction was allowed to gradually equilibrate to room temp.
  • hydrochloride (0,624 g f 2,41 mrna3 ⁇ 4 and: sodioan acetate (0, 108 g, 2,41 nrmot] were added together to an oven-dried flask.
  • the H ask was sealed, evacuated, and argon Hushed and aeehe anhydride (2.2 mlT) was added.
  • the flask was added to a preheated oil hath at 135 *C and stirred for 1 h.
  • M -ES1 mh 640. i (j ' + B f requires 640.3 ⁇ ,.
  • erocyanines are characterized by the prsserice of electron donor and acceptor components that are linked by conization, usnaly a. system of double ' bonds. These dyes are especially well salted for use In living cells or «ii.rn.als because they are very bright, they can be toned to emit at wavelengths l gge diae, those of cellular autoi orescence, and they can. exhibit ⁇ ⁇ ) ⁇ > substantia!, fiuorescenee: ehaages response t selvsift ewitomneut (KtjJinich e al , Russian Chem. Rev. 78: ⁇ 4 ⁇ ⁇ 64 (2009); Loving et at* Trends Bioiechnol
  • esters mask c a ts, pennttriag membrane translocation uriti! they are. cleaved to produce earboxyhc acids.
  • ester mnnher aud orientation on membrane permeabilit and staining of intracellula membranes
  • rep res email ve parent meroeyan!ne dye were yn hesized with AM. esters at different positions (Fig, lb).
  • the di-earboxy indoienine salt 9 was reacted with 8 under mildly basic conditions to give a merocyanine product thai after in stallation of the -two. aee oxyntethy! esters and hotodeproieehon of ;D 3 ⁇ 4B provided M «r ⁇ &
  • MSP cells were incubated with either the dye alone or compound 16 for 20 nrinutes 5 washed, and imaged.
  • Cells treated with mere-l 66 showed taiiforrn .fluorescence intensity tnroughoul . the ceil, ydth o apparent organelle or lipid speciik interactions ⁇ Fig > IS).
  • the iigand-dye conjugate: 1 ⁇ showed distinct nuclear localization, which is consistent with the localization expected to esult from binding, of the probe to 9a, a weihcbameteri3 ⁇ 4d slone: methyhransxemse (Shiakai. aL ems Dev. 25:281 -788 (201 !),
  • t e eell imagiagi Live ceil imaging was. carried oat on an Olympus 1X11 m v&s ps with IjPLFL 40X oil ol3 ⁇ 4ecti ⁇ 3 ⁇ 4 A 1.3) and mercury lamp excitation (T03 HBO balh). Filters nsed ⁇ ⁇ CaMero, CaMero-Ne, mi the dye alone were 545/50 excitation: and 620/60 emission:. Excitation was throirgh m MD 2.0 (1 % transmission) neutral density filter, typically aslng 50 ms exposure.
  • W ole nhnal ex er ments Treatment solutions of the probes were prepared as follows; 8.0 pL stock solution of probe (CaMero or CaMero--NC at 20 M in DMSO), 8.0 gL 20% iv/v Flu onic I S? in DMSO, 15 gL stock solu ion of
  • tissue samples were: imaged after beginning from the crypt of Lleherkiihn, and extending 3 ⁇ 4ough to the peripheral smooth: muscle layer using incremental steps in the 2 plane of 4 uM (30 f ames In total),
  • Ga ' ero and (m ero-NOsampiea were acquired within ⁇ proximatel 1.0 minntes of each other using identical anqnlsition parameters (laser power and F T gainj.
  • CaMero aire CFPdlifeAct images are displayed using different contrast settings for each ax al ' position; however, the same contrast settings have 4?een. applied, to ⁇ images: of the CaMero and CaMsro-NC probes.
  • the rrhKt re was stirred ibr 1 h at 0 ⁇ % allowed to equilibrat to T atid stirred for 1 J- h. ater as : added (25 mi.) rd th orgaue layer was separated, dried with gS0 4 , iitee aad the filtrate was concentated, isolated 0.89:3: g 3 ⁇ 4uant) pale amber oil that appeared pure by NMR analysis. The product was carried forward ithout faxtber memelrlcatiou. 3 ⁇ 4 KMR .
  • om&OM 89 (1.52 g, 3.33 mmol) was added to a.
  • the flask was sealed with -a -se tus*, argon Husk d, -mi the reaction mmture wa heated to 60 °C far 16 h.
  • the organic layers were combined, washed with waterpCX tti/j: and brine (200 rriL), dried wild Ma 3 S3 ⁇ 4 filtered, and eoricemfated.
  • the crude product was suspended k a mminuuri amount of C3 ⁇ 4C3 ⁇ 4 and loaded onto 24 g SIO column.
  • The. -column was elated with 0 - 351 eClB in C3 ⁇ 4 €3 ⁇ 4 o er 20 mis, Maltt prodtsct.
  • the balloon was left open to the system and the reaction mixture was stirred :st RT for 1:6 h, The teaetion mixture was filtered through a sand-topped 0ELlTE M column nd the column was rinsed with methanol (75 mL), The filtrate w3 ⁇ 4s concentrated, diluted in (3 ⁇ 4(3 ⁇ 4, dried with N%S0 s and filtered with C3 ⁇ 4C1 ⁇ 4 washes.
  • Bml 7:437444 (201 1): Qm ei l r m#cheMistty* - 4?:98d- 96 ' (20t . 3 ⁇ 4bsnt el d.. Science 305:1615-1619 (2004)).
  • the affinity reagent such as a prot&ir x grnent or arstibody , hinds selecti vely to the active state of its target and this binding event is associated with a measurable change 3 ⁇ 4 fluorescence.
  • Affinity reagent based sensors ai!brd access to endogenous, trnmodiled protein targets.
  • r i3 ⁇ 4e recognition copiponent of the sensor, we sought to take, advantage of the known blading selectivi y of the SiaaM rnoloeufe triftuo eraKiiiss (TFP) ibr die active sBiif msiicJB of calmodulin (GaM) (Massotn et &L Biochemistry- Us 29:671- 681. p- ⁇ 90)).
  • the calcium-binding protein cateodnhB..
  • P 2 Y 3 ⁇ 4 leads to elevation of intracellular calcium levels v
  • CaM3 ⁇ 4o ⁇ NC along with a second, non-environment sensing small moiecizie fluorophore known to distribute evenly throughout the eel! that could be used as a control for fmctuation in cellular volume
  • Stin iarlon of CaMero treated 3 ⁇ 4Y 6 astrocytoma cells with UDP generated a significant increase in ilnoreseenee Intensity, where s 1 no change In intensity was observed in CaMero-NC treated cells 1% 1 1 ⁇ 2 .
  • Biosensors applied in living cells have provided val able insight Into the dynamics of signaling networks, enabling quami!i cation o the kinetics ami localization of protein activity. That cai mdailn may play 3 ⁇ 4c3 ⁇ 4 diverse roles within the cell toc «t4 ⁇ i3 ⁇ 4 6ostt jl3 ⁇ 4ily associated witl understanding the molecular mechanisms by bich calcium signals are integrated Into specific cellular responses.
  • biosensor based o small molecule that is specific for the active state of calmodulin can be attached t&- an envitonmenpsensk dye, introduced into cells using non-iitva.sive methods and be applied therein, to monitor target protein activation dynamics,.
  • Membrane permeable dyes merolt 7 5 msr l68, and m «r l69 were synthesized as sbo n in schemes 8-10.
  • the Celite cafce was el ted on an
  • Isomer At 3 ⁇ 4 KMi (400 3 ⁇ 4 C;0 ⁇ 3 ⁇ 4) g 8,53 (d, J - 13,6 H3 ⁇ 4 IK) 8.20 (ci, J- 12,4 H3 ⁇ 4 III).7,65 ⁇ 7,53 (m, 3HJ, 7.26 - 7.20 (o.2H), 6.86 (dd s .

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

The present invention provides dyes having improved characteristics for imaging applications, biosensors comprising such dyes, and methods of use thereof, including methods for detecting target molecules in a sample and for live-cell imaging. The biosensors can include a binding member, including a biomolecule or fragments thereof, which can interact with target molecules of interest and can be specific to a given conformational state or covalent modification of the target molecule. The presently disclosed dyes can be used for detecting changes in the binding, conformational change, or posttranslational modification of the target molecule.

Description

Figure imgf000002_0001
S ATEMENT Of ΕΪΟΕΙΤΥ
CN! ] This pplication claims the benefit of J.S. Provisional. Application Serial Ho* 61 /9235 37s filed Jan ary 6, 2014, the entire contents of -which are incorporated by reference herein.
SfATi JE T Oi5 lEBE AL SUPPORT
pW2] This iiweniiots. was made with ver ment support under 6rant No,
OMOfOS 17 a arded fcy the National Institutes of BmMi< The government ¾as certain rights in this Invention.
FIELD OF THE I VE TION
M)¾ the res t kvention. rel tes to f!tsPre scen dyes a d Mosensors eOmprising said fJndtescentdyes -^dmethgds-^f 1¾»iT'¾se for detecting a target -.molecule and an. activity of location of a target meieeule within a living cell
Figure imgf000002_0002
unlike biosensors based n Snores cence resonance energy transfer (FRET), their fluorescence is the.profhict of dlmet rather than Indirect: excitation, and 3) n some Mosessar designs the report the conformational changes of endogenous, unmodified proteins (Hahn et ®i M,, Cum Opm. CellSml Μ 67-472 (2002); Toutc kine !,,. J,Am. CMe , Soc. ;/I5;4i 32-41 5 (2003 m t ^Science J05:1615-i§19
Figure imgf000003_0001
sensitive to their environment and have been used e report protein activity in vitro (βοψΜΜ βία!. , Bioarg, M d Chem, Lett. 2!:5(Μ~$ϋ6ί (201 1 ); Loving el al,> Trmds m hml 2S ~ F x applications w vim, a ciye must be capable of providing s t defit signaJMolse to report protein sci dttes at biosenso
e¾nce¾traiions that inimally perturb norma! hysidlogy, This is a product of both the: fluorescence change in response to protein activity, and the brightness of the dye %:e nsed in vitro sometimes undergo impressive changes in fluorescence intensity^ t too dim io be nsed in iving systems. Farther, the membrane perrn.eabi.iiiy of dyes is often msiii!ieient for use in cells,
5f Cysnine and rnerocyanirie dyes are characterized by electron donor and acceptor components linked by conjugation, usually a system of double bonds. This esults in a ground state thai xnay be represented as a resonance hybrid of charged and nnefcargsd forms (Sh m i t aL bp, Meiemcyd. Chem, 14:7$~WS {2008); Kulimeh e al, is$, G m Rev, .7ΐ:141 ϊβΙ $00 The po^mial for resonance
deloealizafio across he polyene syste tenders these dyes especially senshivedo the effects of hydrogen bondin from the surroiaadm solvent Buncel etal,. Ace, Chem. Jo , :226-231 (1990)}, Changes in the local solvation environment can result in targe changes in fluorescence intensity, as well as shifts in. excitation and emission maxima. These changes can be used in quantitative live cell imaging applications to measure the activity of a target protein, usually by attachin the dyes to an 'afiinity reagent* thai binds: only to the activated stats of the targeted protein. When the affinity -reagent binds the activated, endogenous target, tie dye is placed, in ·&. different solvent environment, leading to fluorescence changes (Naihant etal, Science 3&5m 5-1619 (2004); earreti :tt al , Biochemistry. 4ΤΜ5~996 (2008)), Dyes ca also e directly attached to the protein of interest to report eon fooBatlsnai changes. [WIS) Pioneering work feyBrooksr demonstrated thai a sor nce properties of the n¾roe anin¾¾ ¾re strongly affested by varying the terminal donor and acceptor roans: {Broto &L,X Am. Chem, Soc, 67: 1869-1874 1945); Breaker eta;, J ^ . ChentSoc, #7:1875-1889 (1 45); Breaker &i riJ<Am. Chem. oc,
(1951); Brooker ei at,. J Am, Chem. Sac.755326-5332 (1951)), arid later studies confirmed th : prefoimd effect on flaotesoepes prop rties that arises from he use of dit¾i¾t donor arid acceptor components■■(Ma der e h* Science 2?&I23.31236' (1 97); Bobiitz «f «/, s J Am Chem. Sac, 1 / S! 1-2312 (1997); B fa!to ei ai , X Am, Chem, ilP:3365-3376 (1997); Βνά ζ^ at ^An . Rev, hys. Chem, 48:213-24% (1997); M rder ei ai , Am, Chem. Sec iii:30O6-3Og? (1 93)), o thekss, a reladvdy small number of -specific dGaor-sce¾ io eoMbmatfens: have heon synthesized aid fully ebaraoierized it¾ respeet to tbe : fei0 hys.ca! propsrties relevant t live eel! imaging.
[OO07| The presently disclosed subject matter addresses ilyorophwes with the re uisite eomfearlon of brigh¾e¾s md fluorescence responsiveness for use in iivip eel¾ as wel as, increased membrane: permeability.
SUMMARY'
j'ClOflS] The present im¾ntfe¾ provides ayamrte arid meroeyaprne dyes having iniprove propistties for iisem invaglrig, inelnding dsefdl coa aaii of brightness, photo5!¾bility arid sol ent-deperideiii fluorescence. The lavgstidafttrthet provides eyanine ars meroeyanine dyes having increased eel I memhraae perfneability for enhanced effectiveness in living cells, 'These dyes retain, brightness and solvent sensitivity while readily crossing cell membrases and showing rrtinhrmi staining of inrxaedlnlai me -brarres.
P 0 Thus, one aspect of t h e invention relates to a compound of Formula I or Formula It
Figure imgf000004_0001
Figure imgf000005_0001
n is .an integer -from 0 to 2;
each X is independently riydrogen or m electron with#aw¾g graup selected; from the group consisting of carbonyl., ©yano> lalagen, rdiro, sutf nyl, and trifltioromethyi;:
P is sel eeted; fmm the ro p eoBSisifig ©ft
Figure imgf000005_0002
W is-op omUy s bstit ted C^al-Hyl;
m s m teger fhaaS to 4;
Y m& Z are- each independently seized froxn tte groip eonsltiag. of hydrogen,€¾,&. alky!, a conjygaiabie side chain, and
Figure imgf000005_0003
o is an integer from O to 5;
Figure imgf000005_0004
Figure imgf000006_0001
Eis selected from, th grow cossi sting' of;
Figure imgf000006_0002
ma is s l cted from th ex i consisting ofi
Figure imgf000006_0003
Ρ#Ϊ0 Another aspect of the invention relates to a biosensor comprising a ■wmpOxmd ofFomttihi m Fotnrda 11 asd a blsdit g Baari er having a spedfc affittiiy for the target moiecules. wherein the biosensor exhibits a detecta le bhaage in a fluo escence: property rfmhng or afte "Hadi&g: to the target molecule,
{0011} A art r «t$*$ of the invention relates to a method for ckferrainiiig the rese ce or amount of onaor more target molecules ¾ ¾-s¾mpl©} the method, comprising: (a) providing a biosensor of the invention; fb) contacting the biosensor with a sample suspected of containing one or more target molecules to "Uni the one or mote ½ t meleeiiles, If present, to the binding mern¾¾; (e); irradiating the sam le suspected of containing one or more target molecules with electromagnetic radiation t induce the compoarid of Formula For Formula II to fluoresce; and ( ) detecting a. flnoreseenee property of the compound O Fomtuia I or Formula !! to determine the presence or amount of one- or more target molecules in the. sample,
{0012} An additional aspect of the invention relates to a method of detecting an act vity or a location of one or more target molecule within a. cel , the. method comprising: (a) providing a biosensor oih¾® invention; (b) contacting die: biosensor wit ¾ cell suspee&d of containing on or more target molecules to bind the one or more ar et rBoleeoies> if present to the indin member; (e) irradiating ins cell suspected of containing one or more target mokeiifes with electromagnetic: .radiation to induce the compound of Formula l or Formula 11 to tlnoresee; and :(&) detecting one . or m e of: (i) a fluorescence property of die compound of Formula 1 or Formula II: (n). a change i a fluorescence: property of the compound of 'Formula ί or Formula l!; (iii) a loeatloR of a fluorescence of the compoun of formula 1 or Fo mula H; and (tv) combinations xhereo¾ to determine the activity or location of one or more target nio!eoules n the cell
{O 13f. Another aapee of the invention relates to method of detecting an interaction between an endogenous target m lecule and a oellolar entity, the method comprising: (a) providing a eel! comprising an endbgenous target moleetde: (¾) providing a biosensor of the invention; (c) observing "n background fluorescence signal ffom the biosensor; (d) sG&taotiiig t6§ biosensor t. the eel!;, and (e) detecting a change in fluorescence from the biosensor to indicate an interaction between the target Moleeaie and the oeMar entity, f tiftl 4 A: feriher aspect of the Inventio relates to a kit eoispri sing ¾ biosensor ibr dei ctitig5 monitor g or obse ing a target nm!ees!e, wherein m biosensor comprises a compound of Fbfmuia I or Formula II and. a. binding member having a specific affinity for the target molecule,
01 S| Certain embodiments of me presently disclosed subject matter having been stated Bere nabovej which are addressed in whole or in pari by the presently disclosed subject matter, other embodiments will become evident ss the descript n proceeds hen takes i connection, with the■•aeo jnpaayiBg.iixampies and Figures as bes described herein below.
BRIEF DES{3IiFTiON Of THE DHAWINOS 0§i S Having thus described fee presently disclosed subj ect matter in general ierm.¾ reference will now be made to the accompanying Dra i gs, which: are not necessarily drawn to scale.
I?1 1% 1, shows, bri hipess ¥ai¾es at -me ©missies peak m ximwa when., exciting the dye at Its excitation maxiniu n. Values are grouped by donor heteroeyoJe sroi are presented ibr the set ox 4 solvents dfex e, DM S<¾ BuOfh MeCfi) in order of Increasing: hydrogen bondi g capacity
fOW'81 Figs. 2A-2B show the change in excitation maximum (A) and emission inajdmum (B) irom smallest value for set of fdftr solvents,
00i 9| Bigs..3A-3B show the ehange in thel. m of excitation (A) and emission B) from mm- oiaf i^-dioxanej to polar (methaaol) solvent.
fOO ] Fig. 4 shows the pho sstabiiity of library dyes. Fhotostability values were calculated as (pbotobleachmg rate of fluorescein) / (photobteac ng rata of dye). |««2I1 Figs, 5A-5B show the in vitro hkOm . assay of dye labeled CBD to Cde4Z (A) Fold change increase of emission intensity -upon binding of labeled CBD to Cdc42. (B) a¾I om .fluorescence intensity at satu ated binding of Cde42.
erp2¾l was nsed in previously published successfei biosenso applications,
[W2:2 ] Fig.€ shows the complete em ssion spectra tor binding of erocyanme- iafeded CBi MBP fragment at low (0,01 uM) and high (3.0 uM} eoneentrations of C c42, f#0231 Fig. 7 shows the dissociation constant {¾). value calculated for mexoCB hmding wiih Cdc42 m 50 m : NaI¾PO4, fSOmM NaCI buffer at i>¾ %$,
{W \ F gs. 8A-8C sho Cde42 biosensors based on the mm. dyes show the Wi S s of C&42 activation in moving MBF cells. Cells w re injected with. CBD biosensor labeled with mere&i (A¾ ®e o62 {B)¾, or iaero87 {C)> Biosensors show increased Cdc42 activation in areas where the. ceil edge is protruding, as indicated 'by whit arrows. Some activit is also seen at perinuclear compartments arid i the n ci iis of some ceils, consistent with previous observations (Nalbsnt ei al,. Science
|W35J Fig, 9 shows the f m & emission, intensity of the four gonjxigataWe meroeyraii.es over time at different pf i. Each dye was prepared as a 1 $ .μ'Μ soidt n in aqueous sod um phosphateMtrk &&id¼ffer.:
fiOZfS) Fi , 10 sho ws merecyaaine dyes that consist of el ectron, donor and accepto components that are linked by as extended polyene system, "the R groups indicate the various positions where aeeto ymeihyl ester derivatives integrated to screen for eempounds with sell me bmns permeability and. m nimal membrane sw ung.
|0027J Fig, 11 shows florescence intensity of each dye in tire initial screening, set, M cetts were incubated for 30 mm at 37 °C In seruni~ftee media containing 10 fi io a given test dye and 1 pMHoec st 33342. Pkironie F 27 was added to the initial D SO stock solutions of dyes to give a final concentration k the Incabation solution of < 0,01 %, Fluorescence Intensity (FY) v lues were recorded at the peak of the emissio curve .for each dye when excited at the exei ion maximum wavelength. Values- ere normalized based on relative intensity of Hoschst (exe. 351 m* em, 440 im).
Dlgf Fig, 12: sho s the plot of Snoreseenoe intensit fe dye 4 (¾A )*BA(AM) vs. dye S (ί(2Α ) ΒΑ) at various incubation concentrations. Ceil were ineiibaied in medraf containing the test dye at 37 ?C for 30 minutes, The dye solution was aspirated and. the sells were washed with medium, trypsirused, and suspended in fresh medium. The fluorescence intensity (Ff) of the cell suspension was obtained for each dye, Vahies were normalized for cell number based on the relative intensity of the nuclear stain Hoeehst 33342, which w¾s; rne ded with the dye m each test solut on. \0 ) Fig. 13 shows i © dis ribmioa or dyes iiiMEF cel ls. Cells were scaled
Figure imgf000010_0001
market YFP that was stably expressed in the same cell
|W30 Fig, 14 shows the emission spectra for jmr l6 and meroMfc Samples dilated to I J M m respective solvent (BMSO , -eDH, or water if 1 % DMS O), Excitation wavelength: was at sample excitation madnr m minus .3© srm
;|βθ3ΐ Fig. 15 shows m xse: embryonic ibreblasis treated with r»eroli¾ or 1S9a- mero 166 conjugate.
| S2] Figs. 16A 6B show the design and synthesis of the small moleede based biosensor CaMero. (A) Overview of the small Tuofceiife based biosensor approach Aetlvation ofme rotehi of irrerest (POI) llows: hmding: of the sensor, ydiieitmsnlis;
Figure imgf000010_0002
)
.f 35l Figs, I9A-19C show thai Ua ero sepsor shows fluorescence response os stifflwlatioii of xa$e]I Igr¾a c ai release, (A) Astroglial cci incubated with the dye alone or CaMero^biC probe both show uniform intracellular distribution. Ceils itic¾b.ated with Ca em sio eomseieai toealizatioii similar to that of the endoplasmic reticulum marker BR-Traeker, <©} Ratio images of astroglia! bells pre- and pos -stimaiation shfiw Increase in fluorescence intensity (FI) in CaMero treated, cells only, (C) Quantification of FI changes shows modest .change at the whole cell level cine to large changes & cellular morphology. Larger changes in FI a bserved within the perinuclear region (n. ¾
|CH?36J Fig, 20 shows rimary murine neurons incubated with CaMero s ow increase in $uorese¾¾c¾ ¾t mit fp!¾ ¾ -stiritui¾ti ii, Cells were stlmrdsted with &€¾ (75 rnM), Hot s Of average tluoresoenee i tensit in the cell body region vs< ilrne for CaMero d Ca ero-NC (n
037|; f gs,.21 A-21 C show that CaMero sensor shows caimod lia-activation at the eell edge in normally migrating cells, (A.) atio ima es of migrating MEF cells show totalized intensity ¾and at the leading; edge only m CaMero treated cells. Far right panel sho s oom of edge section ; scalo bar for Whole ceil images - 5 ? , m ^ 10 μΜ), <B) Quantification of fluorescence intensivy ratio relative to distance feoxo. the leading edge. (C3 : Plot of fluorescence intensity rati© and calculated cell edge velocity.
iS f ig. 22 shows a line scan analysis of eel! edge intensity using
earboxyf!uorescein. diacetate volume indicator. Edge itrtexmty gradient apparent with CaMero with and withou co reateem with clozapine. Mo Intensity gradient is seen with m CaMero C probe,
[ 0Sf| Figs. 23 A.23C show whole animal treatment with CaMero, (A) Excised hnestlnai tissue shows Intensity of CaMero probe relative to control. (B) Co- locaJization of GFP-LiieAct aetin axk observed with CaMero but not with Ca ero-MC probe in smooth: -muscle cei¼ (C) Plot of fiuorescenee iritejisity over time for CaMero (dark Ike) and Ca ero-NC (light line). The CaMero probe shows periodic changes in intensity consistent wi h the treqneney of tissue contraction. BTAILcii DiiSCRIPTlO
§δ ] I¾e presently disclosed sub] cet mifgr ai iiil e described more fiily ereinafter with reference to the accompanying Figures, in which son c, b t .not all embodiments of t¾e :j>jesejitly discl sed subject matter are shown any
moii fioations and other embodiments of the presently disclosed s ject m tter set forth her k will come to mind to.-one skilled in the art to -which the presently disclosed subject matter pertains having the benefit of Ihe teachings presented in ¾ ¾fe 0j¾.-desc i tip¾s- ¾d' ih$ -a&soeiated Figures, The sibre, it is to fee mdefSfeo tha the presentl disclosed subjeet & Is mi to be limited to specifis embodiments disclosed and that modifications and other embodiments are Intended to he included within the seope of the appended '■ claims; Although specific terms aj-e employed herein, they are used in a genetic and, descriptive sense only and ot for purposes of limitation,
08 1 : Unless the eomext indicates otherwise,, it is specifically intended that the yariouis features of the invention described he ein am be teed k any earnbinaSon. Moreover, the pmsent Invention also contemplates; that in som embodiments of t e indention, any fea ur or combination of "features set ibrth herein can be excluded or omitted, To illustrate, if the specification states that a complex, comprises components A, B and C; it is specifically intended that any of , or C5 or a combination thereof can be onnited and disclaimed singularly or in any cpnabination.
|0#© flnless otherwise defined, ail technical and scientific terms, used herein have the same meaning as co moaiy understood by one of ordinary skill in the ar to whieh this invention belongs. The terminology used in the description ofthe invention herein is for the purpose of describing particular etr odiments only and is not Intended to be limiting of the invention.
§ 31 All p fe!ications, patent applications, pa ents:, patent publications -an other references: cited herein are i co orated hy ieferense in hek entireties for the ieachkp relevant to the sentence and/or paragraph in which the reference is presented,
04.4]· The terms ""an," and "the" refer to "one on more" when need hi this application including me claims. Thus, for example reference to 4¾ sample" includes 1 ¾ a pl alhy of sam l s* unkss the clearly Is to the mm&y (e.g , a .plwalfty of samples),: and so forth.
pt}4S] Also as used herein, "and or " refers to i encompasses any d all possible combinations ©feme or mote of the ass cia ed listed items > as: w l m th iaefc of combinations v/ interpreted is the alternative
0046] Throughout this specification and the- claims* the words "com r se^ "comprises,'* 'and "comprising15 are xm& m&wm- u s sense, except where the context re ires other is e .
Figure imgf000013_0001
MSJ As nsed herein, the tern "sbosj when refemng to a value Is meant t c& p variations df». Irs some embodiments ± 20%, m some embodiments -± VQ¾_S in some esibodimeats ± 5 , in some -embodiments * 1 ¾t in some embodiments ± C 5¾, and in some embodiments ± from the specified a oont, as such vat osis m appropriate to per form the disclose , methods or employ me disclosed compositions.
% Comprmsds
[0049] Cyanins and merooy& ne dyes can he tigsd. as fluorescent labels lor omoleePie&j S eh as proteins or antibodies^ for histoehemica! studies r
imrmm:obasea assays, Such dyes can he atnwd vs lor use as iluorophorcs due, in pari, to their wide range of available wavelengths, large molar extinctiorj coefficients, and high qmmte yields, P«rthe¾ because of the sensitivity of the t oorescence intensity ari wavelength of n my im dyes to solvent polarity and omef: characteristics of the dye environment, the presently disetosed merooysnine dyes can be used as sensors,
Figure imgf000013_0002
a biosensor, of protein activation, protein-protein binding, and protein coniorroaiional ch aies* iSftj One aspect ø£ ihe inventon refetes to. a compound: of F rmula 1 or Formula
Figure imgf000014_0001
Figure imgf000014_0002
herein
n is an Integer from 0 to 2;
©aeh X is. inde en entl feydfOge or electron ith ra ing giesup se!sotec Iromihs grovp coim^tiiig- fOTiKmy^.ejf.ano^l^i^g^ nitro,,s¾i£briyls and
txifly iomoi y!;
D is selected ftoniihe gfo¾) oos sfi's of;
Figure imgf000014_0003
Is optionall ¾i¾l¾x£ed■Cj-g a!kyi
in is as I sieger ftom Q to 4;
Y and Z are each iadepesdeHt!y selected flota r&e gtdiip consisting of hydrogen,
€i a!ky!, a eoirj gatab!e side e'ham5 and
Figure imgf000015_0001
Q is integer from to 5;
U is 0 o S;
.is ¾'i!i-0g€ o opti&Hally :Sttbst¾¾edi:Ci,g alkjl A is selcted from the group consisting of:
Figure imgf000015_0002
B is seleeisfi! from f e. gfoivp eousistlng of:
Figure imgf000015_0003
and
(3 is:se!scf ii from l¾g grou cosisisi&g o£
Figure imgf000016_0001
[0051 j Is specific embodiments, the invefitki. encompasses compounds having any one of the -described D m¾ es coupled with- any one of the A moieties. The im?emion further encompasses -com ounds having any one of the B: moieties coupled with, a y one of the G moieties <
(0052]' In some eo)bodi&ents, the compound, does not contain electron witi drs in .group on the poiymethine chain connecting A mi D or E and G4 e.g.* each X is a hydrogen, I» other embodiments, t e compound contains a single electron idiirawing group, «,§. , one X is an electron withdrawing group. In certain embodiments, the electron wiftfei g. g up- ® .attached to the polyinetkine chain carbon that is- connected to D or E. In certain embodiments,, the electron withdrawing group s attached to the poiymethine chain carbon mat is connected to A or G. I Other- embodiments, the electron withdrawing group is attached to ¾ carbon in the middle of thepoiymethine cha¾ .e &, a carbon fhat is not connected to A, Df ts, or G. la further .embodiments, the compound contains two or more electron -withdrawing groups. In these embodiments;, one or more of the electron withdrawing groups may be attaelied to a poiyttietfeke ena n carbon thai Is oonuected to A, D, E. r G, Placing electron-wiadfawing groupa it selected positions on the pcipae&ine chain, retees phoiobieaelung of cyanine and merocyanme dyes,
£0053 j The conjugatable side chain of Y and-Zor Z comprises a linker and a reactive group and -is one that is capable of conjugating the compo n to. a binding member ha in an affinity fo target niolficnle as described below. In some embodiments, the compound comprises one conjugatable side chain,. ¾ other ib
embodiments, the Dcsm iitmd eoinprfses more t an one cosy ugatable side chain , In softie embodiments, the co p u d does not comprise a conjiigatabie side cfoaiii. |O0S4 la some embo iments, the cosj gatablfc: sde c mfi is selected ixom the group consisting oft:
Figure imgf000017_0001
\ herein;
a is; ft: integer &¾a 1 to S:
p Is an intege tkas ί to 5:
U is O or S:
R3 is a ie viog iro ;
R3 ishydrogen, Lf", Na* or Kx .
R*3 B.4 s;¾n4' Rs are each ijrsdepenidectly Cj ,g sikyl; and
ί¾;3 s Ct,6 alkyl er:C¾.
fO'tfSS] ΪΏ certain mbodments, R is a halogen, iodide. I» some
Figure imgf000017_0002
[ 561 Is oilier e bodi eaiiss the: cosiagaisM© side cham Is
Figure imgf000018_0001
[0057] In some embodiments, M Isa&t one of Y and Z is. eonjngaiable side chain and the co ¾poiisd. contains o l am emj&gatable side cfeala. In some embodiments, :&e Oonjugat¾bie side eharn is on the or E moiety, .e. . , fee Y in the D or E moiety, !a other OT¾Gdimesl¾ the eonj gata le side chain is on. the A of 0 -moiety* fh 'Y in the A or G moiety, k some emb iments, the Y ki¾fe-B or E moiety is hydroge or C;.6 alkyl. and one Z in the com ound,, «.g..» one m the D -or E moiety or one Sift the A or G moiety a corrjugatabfe side chain,
USS] In some embo Smiefits of the inyemtien, the compoiiftds comprise one or mm sobsiit ents that re penneal¾Bty enlianeers, le^ sabsiitueats mat increase the ability of tli cempownd to cross edl membranes. In Formula: I a d Fomm!a 11, Y aad/ot Zmty'. be one or rnore permeability enhancers. In some embodiments, the permeability enhancer is
Figure imgf000018_0002
'wherein:
o is: integer from 0 to 5;
ϋ is 0 or S;
R4 is bydmgen or optionally substituted Q-s alkyh
£.0059) In certain embodiments, die permeability enhance? is an aeeioxymfctbyl ester, e.g..,
Figure imgf000018_0003
la some embodiments, the compound comprises on¾ or more permeability er&aneers, e:g,, 1, 2, 3, 4, or 5 peti¾eablliiy euhsm¾rss 1, 2;I :or 3 permeability eimascers, ΙΛ certain i cdi ents, the one. or more permeability erikaneef s ate attac ed to h © or E moiety, la certain embodiments, the ne or more peimeabiliiy eriiaBoers are a¾ached t the A or Q moiety. In esrtairi embodiments >. the compound comprises at least two permeability efihaneers and the perm^a'hil iy- etifeaKcers are attached to both the D or E moio!y and tits A or Q moiety, in .s m embodiments, the compound does mot comprise a ermeability enh nce?.
§6i| Iri some emtodl ests, the eompomnd comprises a COM] ugatsble side chsra one or permeability enhancers. la certain embodimesis, the Y in me D or E .moiety is a eosj«gatab!e: side chain:1 and at least one Z is-
Figure imgf000019_0001
¾¾J In some embo&mersts, the Ym ftie D E moiety is kviirogsn _aad ai least one Z i&
Figure imgf000019_0002
■ {®M3'I ίύ some embodimems, the Y ta- -the I) or E moiety ¾ bydmgem one 2 is a eosmgatab!e side chain, and at least ou.¾ Z is
Figure imgf000019_0003
16064] In some embodi en s,, he ¥ in the D or E moiety and at least one 2 are 1ft
Figure imgf000020_0001
@951 In some embodimens, one Z is a «∞jiigat¾h!¾ side c-hsm and the Y ia the
D or E rooietv feast o« 2 w
0066
Figure imgf000020_0002
that are
Figure imgf000020_0003
Figure imgf000020_0004
>$| ITS certain eiB od eKts of the compounds: of the ¼v§n†ionf D is
Figure imgf000020_0005
Figure imgf000021_0001
;Pt 9|: In ssrtaki embo!iment y D is
Figure imgf000021_0002
e.g. , hereia R is .halogen, I,
p>07{¾ M some embodiments, the com ound is selected from .$mf©B© ing list or ■any s¾)coi¾biaatio¾ thereof. Ea of these corapopn4& amy farther comprise a
Figure imgf000021_0003
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
cory gstabte daefaam and/or one or moe permeaM!ii en ancers*
Figure imgf000025_0002
Figure imgf000026_0001
|09Τ2] In particular embodiments, the compound is se cted from the following list or any sy¾com ¾g on feereof
Figure imgf000026_0002
Figure imgf000027_0001
f fift¾] fe some 'embements- of these eora o iids, at least om Y is r
Figure imgf000027_0002
θ§75| la pr¾cil¾r embodiments., tie compound Is seieetd ftom the following: list or my siifecoMfeiMiiof liereoL Each of k se compouiids m furthsE comprise a con|«gaiable; sideebaitL
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000029_0002
, (, higfc quan m y ield, as exhibited by some embodiments of the presently disclosed dyes, can provide a strong direct signal from biosensors comprising such es< Such high quantum yields and extinerimi coefficients can allow detec ions ironi very small amounts of biosensor, minimally periafbiftg the biological activity of the endogenous protei being studied,, and enabling high resol te. neiic studies by obtaining siany images before phoio-hkaehing occurs.
[00771 -to some- embodiments, the "doner' ring- structures (D and E) aad or the "accept r 1 ring struc ures (A and 6) of a corr^o and of formula 1 or Formula sail meiude one or mote subs btnera groups, Sneh rin systems eompr sing one more bstiiueat groups nan be te!brred to as ''derivatives'■ of t¾e parent compound* I s. s a derivative of a compound of formula I or Formula JL As used herein, a "derivative" refers to a chemical compound that s derived from or obtained from a parent eomp uisd arid contains esssmal elements of fee parent compound (e^. , ability to flu m^ but t¾¾e i|y'¾QS- OE? or more difierent rmefioaal groups, Sueh fusiesional groups can be added to a parent compound,: fw e a le, to improve- rite molecule's solubility, absorption, o gieal half life, fiuorsSGefit properties, and the like, or to decrease the toxicity of fie molecule, eliminate or attenuate any undesirable side et!eei of the molecule, and the like.
10 )781 In some embodiments. Hi substitution of one or more tunotional groups ø¾, the: donor and/or aeeeptorring strocture can improve the solubility of tile dye. in particular solvents, -stick as water. For e ample, as described in If Patent
Application Publication Fkn ;2W K329946~-†d Halm, the presently disclosed dyes can ¾e designed to have enhanced water solubility by attaching subsfi uent groups on the ring structure that sterieally block aggregation without unduly increasing
Bycirophobielty. Under some circumstances, ik approach•©an. be more desirable than en ianeing yy ier solubility using ghly charged g oups that can affeet protein interaction,
gttft9J Planar meree mne dyes known mine art re thought, to fbrm non»:
Ituoresoent hydrogen conjugates in ater, thereby reducing the e osure of their hydrophobic surfaces to water,. Such aggregation C be decreased by incorporating bulky, non-pianar subslltuents with: tetragonal ge fne!ry in the aromatic rings to make stacking nnf yorabie, litis approach can, provide dyes with, good water solubility -^. wlnle sta ing m smtist3i 4 oi)te i character, . For exa le^, la the presently disclosed I-SO d es, the oni-Of-plane sdbstiiuent groups, can be two alky],
methyl,, groups o nd to a earhon atom -of wa aroamtic.tsiig, or two oxygen atoms houn to a sulfur mm of a heterocyclic ring structure. To this end. tie presently disclosed dyes may have one or more groups arranged in. ibr example, a tetragonal geometry from rings of the dye. I» many eases, the substituent groups- iaclude groups with one or more carbons providing ste^c hind ance to ring stacking* and/or polar to weak ionic cha aater to enhance water solubility.
§SS j Acco mgly, the subsiituems oft one o more r ng structures of the cornpouBd of Formula % or Formula ΪΙ can be any fenotionai group that inhibits stacking of the aromatic frogs and br enhances water solubility. Such s bsiituent groups ean provide sterie Mndcra e to ting stacking, thereby inhibiting aggregation, whhOM reducing water solubility of the dye, ¾ s me embodiments, the subsiituent group can have an aliphatic nature, e.g. , a straight-chain o branched alky! group. In s me embodiments,, the substitueni group can have a polar or weakly charged character. Generally, the presently disclosed dyes can be: derivatized with side chains to prevent aggregation, improve solubiiiiy, afibet interactions with, proteins, enable covaknt -attachment to proteins, and improve cell membrane permeability,
f fiOSl] in some embodiments, the presently disclosed compound of formula I o formula ΙΪ are enyirotiffienially sensSim As used herein, the term "mviroma-entally sensitive" i relation, to: a. eompo nxl ox -Formula I of Formula Π refers to dye in which a fiuotescenee signal from the dye changes when the dye is ex osed to a change in:enveO meni5 for example, a bydrophobicity, hydrogen bonding, polarity, or core¾rm ii na! change. V , afcorescenee signal: ;f¾om a compound of Forrnuk I or formula ΙΪ detectabiy changes upon exposure to a change in solven s change in hydrogen bonding, change in the hydroph bicity of the environment, Changed polarit or polaiizatiort, or change affecting the conformation of the dye, one embodiment, the fluorescence signal .from: the compound of Fonr ia I or formula II increases when the dye is exposed to a environment that is more hydrophobic. In another
©mhodhmmt. the fluorescence signal provided by the compound of Formula I or Formula II increases when the dye is exposed: to ¾&■ environmeni - h re there is increased hydrogen bmdi¾g: between the dye and a component of the en vironment S¾e-h. an increase in hydtophabieit or an increase In hydrogen bonding can occur when ¾*: presently disclosed biosensor oompr ng a coxppotiad of Fcami!a Ϊ or.
Formula II Mads to a target protein or subcellular component. In. < & eix&odlraests, the fluorescence signal provided by the compound of Formul a I of ormula Jl ••decreases when the dye is exposed to an envhonraeni that is more hydrophilk, hi f m¾er era odimeStss the fi orescence signalprovided by the compound of Formula ! or Formula !! decreases when the dye is exposed to an environment that has less hydrogen: binding. Such an increase in hydropbilleity or a decrease in hydrogen binding cat) oocar when a biosensor com osin a compound of Formula I or Formula II is exposed to a aqueous nyi onmeR o when s«eh a biosensor bc«o¾ies Mh un from a target protein or subcellular component.
P082j ¾ some embodiments, the presently disclosed dyes can have m extended K iiterioa structure having a polarize groond state, which allows the presently disdosed dyes to respond to changes in solvent polarity, Such dyes can exhibit a strong solvetK-dcpendent excitation wavelength shift, while retaining a bright
Figure imgf000032_0001
fluoresce at longer wavelengt , dyes most have e tended conjugation, which can reduce water solubility and lead to self- aggregation. The dyes designed for use m. living cells were specifically selected to "be insensitive to i fc eavwD&rnent. because
Figure imgf000032_0002
Figure imgf000033_0001
bid ing enable a biosensor conwisfng a presently disclosed compound of r¾m la 1 or Fomu la II to be self-refereiictftg, la such embodiments, the ii ompbor exhibits an increase in a first excitation or enrission mvele¾gt|t in the presence of a target m tenle arid a decrease- in a second wayeleagit A ratio between the first wavelength and the- second wavelength cm be eafcrtlated to determine the amount of target molecule: in the: sample under test; Such self referencing can correct for variati ns in excitatlors.sonree Intensity a¾d other so¾ces of noise and instability I fee biosensor i&sut requiring: a reference dye. Thus, a single flnorophore can be ^
•used 'to observe a ratioMetric esponse in the fctoseasor. As used iietcirn the term iSratiomeiiie response1 r eaas that the intensities of the first avdeng h and (fee. second wavelength vary such that the ratio of the two emission wavelengths can b used to indica e, interaction with the target mo!ieeule in a sample,
11 Biosensors- Co pri,¾g C« isi|?9¾siis «ί feraaaia I or f "ormiJfa ίϊ
[0086] ¾ some embodiments, the presently disclosed su ject matter provides a biosensor co prising a compound of Forrnul& 1 or Formula ίϊ aa4 a binding n ei iber hayin a s! ee fie, affinity for a targe molecule thateaa be used to detect and quantify the presence and/or activities of one or more target molecules. The biosensor can bind to oue or more target molecules and the detection of such binding events can be used irt hoffio eous assays, ,, assays in which the fluorescent properties of the dye can undergo a ehaage as a resaliof the binding event, and for live-eel! imaging. The presently disclosed bioseosws can be used: to; detect and or quantify diverse protein activities, including chang n s bcellular locations, conformational changes, •«pt a$29S. sl teSj-pes feraii.sl tiO. al: masHfte&iianS, and/or small ligand binding of pmi m in vivo*
Figure imgf000034_0001
bkding. wit a target inolecufc. The term '' roducing a. detectable signal" refers to the hiilty to recognise a change in aproperty of a reporte group, e.g. , a fluorophoies in a man e t¾at enables the detection ©f Hga^d-proieif Isnding,
| 08S| Ths present i vention provides methods which can use specific binding: partners for a particular target molecule of 'interest. A specific hmding partner or member, as used herems Is a metuher of a specific bl mg pair, A "specif c binding pair" refe s to two different molecules where ne of the molecu les throug eheniiea! or physical means specifically hinds the second molecule. In this sense, a target molecule is .¾ reciprocal member of a specific binding pair. Further, specific binding
Figure imgf000035_0001
parameters well: known in the art Art analog of a target molecule also ea» ha e the s me function as the target molecule,
|##8f J . A osens'o'r can traris!ate a bhidihg event into a directly measurable fluorescent pro ert , Such changes in one or More fluorescence properties Include, but are not limited to, an. increase or decrease m fluorescence intensity, a shift in exeltatios or emission maxima, shape oftiie excitation or emission band profiles, a change in fluorescence lifetime, a change in anisotropy, a change in polarfstioo, and combinations thereof. Farther, the pr ducing of a detectable signal can be reversible or son-reversible, The signal- roduclBg event includes oo s, programmed, anrf episodic mc¾3ss,inckding one-tlrne or renss e app!inations. The reversible signal* produein event can -fee instantaneous ¾r can be time-dependent,, m long as a correlation with fcpresence or epncentraliori of target, molecule Is esiab!isbed.
090.1. As used herein, the term "ftuerophore" includes a moiety of a larger •molecule or :eom¾gate that can be induced to emit fluorescence when irradiated,, i.e. , excited, by electromagnetic radiation ©fa appropriate wavelength. More
particularly, a tluorxrphom cm, ¾ a/f«nc i n:¾I poup of a mo iecnle or conjugate that absorbs light of a certain wavelength and ennts light at difereat ¾vslesgth. The intensity and the wavelength of the li ght emitted* as well m o r fluorescence properties includin , but not limited, o, fluorescence lifetime, anisntropy, polarisation, and conihinations thereof, depend on the identity of the fluorophpre and its chemical enviroitt enC: A ilucrophote can inciuds a flnorescent molecule, such as the presently disclosed compounds of Formula f or Formula ill
Of i| In some embodiments, the presently disclosed: com oun s of Formula ί o Fonnuia H include a reactive group, sufestitiients Y and which can be C0j¾i¾a i¾ w«b another ma!ecolef swel as a blsding meiBber, .e.g., one member of a specific binding pain, which :has an aflMiy for a target- moiseale of interest, e,g^ the oi ie member of a binding pair, Such c lligates can. be nsed as a biosensor com opsd to detect the presence, location, co &rmatiofij .aeiivatlon siate;
moditkaiion, binding, and &m Hie, of a target nofeenie.
fiWl] As: used he eim the term. " ¾ ¾agaie; M a varlsikrjs ¾emo£ :refers to raolecwle comprising two more :su¾iniits bo¾d together, optionally t&roiigh a linking group, to fam a single molecular sttuetwe, The binding of the two or more sab mts can be fctoo gb. a direet ebeniicai bond: between tbe Siibusits or through a l k n group, ¾ch.1>mdm in a omy gaie typieally is irfeyerslble. As nsed teem,
Figure imgf000036_0001
,.
io
|0#94| in some <¾i 0ilimeni8s eonjttgatabie s:ideeh¾hm m be provided on compounds of Fottnula f w Formula IL IK sued embodiments, the presently disclosed ex ou ds of Formul I or Formula II can !>e derivatized to make reastive hms which could be site-speeii&ally attached to proteins As vised herein, the term
"dermtized,'' arid variations thereof, is meant to■include my chemical modlfIea£ion5 addition, deletion, or substiteioa to a parent compoS-fld Further, a derivati ve ca include an reaction product of the derivative, for example, the reactioa product of the derivative with, an smiao acid reridae. Accordingly, in seme embodiments the presemly disclosed dyes can !neit cie aromatic ring structure having a eonjiigatabie sideo¾a¾ that can be popj gated, e*g. , cov¾lemIy attaehsdf to an amino acid, for example, an amino aeii residue of a protein. A son-iimMfig example of I -derivative is an ester or amide of a parent compound having a earboxylic acid functional gtoupt fi SJ fo exam le, derivatives of th presently disclosed com oun s of Formula I -or Formula II- can ¼ adie .with s adnim yi es er conjug^ hl rideehain for aEadioieitt to lysme or hh an iodoaeetyl linker for eetive reactioa it cysteine,. Use; of these groups- for SJte-$^eci&- r<>teiai8 in is Imown in the aft S e, e.g., Dent a d As^a , coryug ion, 364-482 (1 98). ar exam l , the reactive compounds of Formula 1 or Fo.mii.sla II can be synthesized from starting materials having an amino group, which oars he used for attachment of a s de chain comprising ¾ reactive linker, group at the en of the synthesis, Accordingly, in some
embodiments, eystein«~seteeiive iedo acetyl groups can be attached to the dye ring: structitce for covaient nding to # binding membe or binding domain, thereof,
t 6f in some embodiments, the presently disclosed dyes ean include a thiol- reactive group that can be conjugated to the thiol moiety of a cysteine amino acid i¾sMue in a aater-al or nnnatma! protein. As used herein, the term "thiol-reacttve group" refers to a suhstit ent. group that can react with, a thiol moiety to form a earhon-sulihr bond. Fxamples of suitable thiol -' eaeSve gronps that can be introrluced into the presently disclosed dyes include a h lo-as et l group, „ a halo-acetamido group. In some embodiments, the haio-acetyl group includes an iodoaeery! group. One of ordinary skill in the. art upon review of the presently disclosed subject matter ou recognize mat other: thiol-re ctive groups known In the art such as maleinride .} /
md didiionyridyi goups, are suitable tot use ¾ the ¾se$ disclosed subject rr-auer.
In some en odlmeits, the residue to whieb -the dye is attached is a cysteine. In some embodiments, one ormore snob rsid es can include conjngatab!e. sideobain-i eaetive groups other than cysteine* fJoBittgatable sidec aia-reaciive groups can iscfede my chemic .:&neiic-fial. oaps on the dye¾ the hindiag member, or both, thatean ieact wit selected ca¾ aiafc3e si jec ¾s to fbfo ose or more chemical bonds. For example, natural amino acids, modified or derivati¾ed maim acids, arsdor other residues In a binding member or binding domain can wvid e0^g¾t¾bk:j eeh^ft-r^a6tive.^o¾>¾ ueb as amines, sflf ydiyls, e ^y lie acids, afeobote, aidebydes, and tbiols, ¾lcas eovaienlly bond to lirrker molecules* l&Q l Gonj¾gataMe ddechains can be: a¾ type suitable to react with
eonjpgatabk sd^ain- eiw oii s to form a jnteige eween selected binding members to compounds oFPormuk ί or f cania IL Represeiriaiive eonjngatable sidechalns suitable ibr use with the presently disclosed su ject matter ipehide, hut are not limited to, side ains reactive: with pib¾a?y ar es (e.g., :hydfOJiys;aeeiaimide3 car osyi, HHS estep o!fb-KHS ester, kofe yaiiate, doester, epoxide), side chains reactive with sm'&ydfyl groups (e.g,, maloimides: baioaoetyls, ditMo yndyts¾ side ebains reactive ith aldehydes, and o&er eabony! gerups e. , hydrazines, :hy¾ia ides| deehains: reactive with csrbpxyi group ($,gr, ethyloiethyianhno pr©py!ca¾odi!mide (E£M¾ md sldeo ns eactive with Moortbogoaai; reactive groups such as-termioai aiky B^ c el alk ne, alkene, tetrazme, l^dl ole f . ., azide, RtroHe, nitrite oxide), Corysgaia e sldeeh&ins can include a flexible aliphatic or pol mer ehamof suitable length and hydroplhHciiy to bridg between linked molecules, Bivalent linker groups haying the same or different, !ihtesr cbenhstries at each end also can be used, Conjuga¾o!e si eebains. cap include one of more protective group t protect the Imfcergronp dnring storage , handling or other chemi steles, T¾e protective gronp can be removed under defined conditions to allow completion of the eon) ligation reactioii.
f #099 $ Firrthef based on sequence data and he: crysal structure or binding, domains of the binding: members, one or more compounds- of Formula I or .Form ula If can be attached to provide the /presentl disclosed biosensors. For e m ie, based on the sequence data .and the crystal stature of aires Fab fragment, several: eyanine: and merocyanuae d es can be attached to a series of one or more different residue ositions to create a scaali but. intelligently designed l b ry of hioserisot candidates. The resultant library ca ¾e sc eened, as can he appreciated those in the art, to identify sapdHates with desired properties, e,g, , of signal srrengfri and binding, affinity,
\ Ϊ 08} intelligent design of linkage reactions can be accom lished with the select on of a targe! reactive group in a binding member m binding: domain, or engineering of a desired: reactiye groap nto t¾e blading member or binding domain;. For example, a bi ding member or binding domain, can n&iurally have a cysteine residue (reactive, for exainpk, i h a haleacetyl linker at an appropriate location, at ea be routateri to include such a cysteine residue. Alternatively, a:Mndmg membe or a binding, domain csn have a small i mber of cysteine rssi nes. Binding, members or binding domains can be modified or mutated to contain a single or a small .number of cysteine residues by nrdcedures known in the art.
A. Rep ttiMim Binding Members
I' IOI.J Binding members suitable: for use with the presently disclosed subjec matter can include any moleeiiies that hind tp a target rnofeeaie wim: sellable specificity, B hiding m mb rs typiealf ihcl:¾de binding a ioss of arlmity molecules known in the art ine eohngf but not limited s antibodies, antibody fragments, leucine zi e s, stones erdiarieers, complementary determining regions (CIDRs)vto single chain variable fragments (seFv¾ receptors, ligands, spi raers, lectins and one of seyeral proteins in a prof em complex or a protein pair, Binding members: can comprise either membe of a binding pair , $,g, , pai pf proteins in a protein-protein Interactiem with the binding member being identified as the member introduced into an assay systera to probe for a target: molecule. Binding: members or bindin domains thereof can be binding. egions of, ¾ cjtant l , feE/slmdwcrsions oFthe affinity rnoleeide, fragments of the affinity molecule, or the smallest portion of the affinity molecule providing binding useful in the detection of a target molecule. In some em¾Q im∞¼¾e i id3i» mei» ¾r can hay© specific affinity to endogenous {e.g.* constitutive or mdueili!ef but not recombinant) peptides of a ceil. ,¾
102] la some embodiments, the: binding membe is a small molecule that has affinity for a target . molecule, e,g., a protein, la some embod ments:, the small molecule is one that cannot b s produced - y genetic means, is not a peptide or protein, la some Sibb dimeni¾ the small molecule is a CDS¾sCmtid'¼vin a molecular wei ht of less than about K)00 ¾ ,g,, less* taakmt 8 0 Da or about 600 Da, la some embodiments, the small molecule binds a specific proteiri¾. apicrteiniB a specific
Figure imgf000040_0001
passive trarssporh In. some esAodimenis, the small molecule is eoi¾u ¾t«£l to a:
compound of Formula I or Formula 11 ¾ other embodime s, the sm l! molecule is conjugated to any dye thai is kao ra to be or later identified to be useful for visualizatiiMi in living cells or animals, ,gri to visualize interactions between proteins and small .molecules,. In some embodiments^ the dye may be one that is permeable to cell membranes to enhance its nsefiifriess in livin ceils or animals. In. -some embodiments, the dye may be one ¾at ean l?e visnaJksd by micrdscopy, e.g one thai emits a fluorescent or other dprah
(0103] in some embodiments, tie bindingmember is a binding protein. As used he ein,, th term "binding protein" refers to a protein, that when conjugated with a •fiuorophore, interacts with a specific target molecule in a manner capable of
Figure imgf000040_0002
from when. a. target molecuie is present or abse ts, when a target molecule is-, presen in varying concentrations over time.
[0 84f The binding membe can ..comprise a binding domain:, Binding ctomains suitable fbr nse with the presently disclosed biosensors can comprise polypeptide, peptide, or nucleic acid sequences. For exam le, binding domains can be single stranded DMA (sDNA , double stranded DMA (dsO A), RNAS nucleic acids -with modified bases, and the like, m one embodiment, the binding domain can be an oligonucleotide probe andthe: target eaa be a eoisp!m entary target nucleic acid,. In another
Figure imgf000040_0003
ά{} enhancer proiei¾ taigai The presently disclosed^ compounds of For uk I or Fo ruk ΪΪ cm be linked to nucleic acids B .any technique known in the art, such as by reaction of Ikfccr roups on ie dye to r act ve groups -available n .modified bases oil the Budefc add
[01.05] Afi t specificity of peptide binding domains can be provided by a short, sequence of amino acids (c.g 5 a se uence of amine acids: comprising 3 pM residues,
¾ 5, 6, 7, ¾ ¾ 1¾ 11, ¾ 13, 14, 15, 16, 17,. IS, 1¾ or 20 residues), or the specificity can rely on. contributions of amino acid side ehaiws brought in proximity by the primary, secondary, tert ary, aad/or quaternary strwteal conformations of one; o ore affinity proteins*
{MM) Binding domains comprising peptides can ave natural ammo: acid side clams, modified side- chains, or the like, that provide reactive groups specifically reactive with compounds of Fotmiufa I or Formula 1L in some embo ments, the compound of Formula I or Formul can have reactive groups specifically reactive with linker groups present on the binding domain to link the dye to the domain, he position of a dye on a domain can be detertnined by the location of a reaetive group o mker. moiety on the domain. In some ernbodhiients, the binding domain has one or more cysteine residues reactive -with groups on the dyes, for exam le, iodoaeeiyl groups on fee dyes,
f¾¾#7! The presently disclosed biosensors can incorporate binding dom ins of naturally oecvuTing proteins having specific binding activity. The binding domains can include, for example, ¾iOengt§i .affinity proteks, m¾mt>e:rs of proieboproiem Interaction pairs ® portions thereof), Fv aiUibody fragments, aptatners, Mi antibody fragments, and the like, !n on© embodimenh the biosensors .comprise binding domains that are members of the imtmmoglobaiin.fenily of proteins,, or derivatives thereoE For exa le, the balding domain, can e a complete imnmnoglobnlin, fragment, single chain va iable Segment (seFv¾ a heavy o light chain variable region, a CDR peptide sequence, a¾d/or the life.
|§M8f In spme embodiments, : a antibody er antibody feagmsni can h¾ used in a binding domain to which compounds of Formula 1 or Formula 1! can be attached to form: a biosensor, An antibody suitable for use with the presently disclosed subject matter can: bo in any of a .variety of forms, including a whole immimoglobnlbL an antibody fragment such as Fv„,Fab5: a¾d s milar fragments, a single chain andbodv whicli ind!ne s the variable domain complementarity determin¾g regions (CDR). and the like forms, all .of which fail under fee broad term ^a tibo y*5* as nsed herein. The ose of any specificity of as. antibody, polyclonal or monocional, s intended to iall nnder the scope of the presently iseased s¾b|ect taatter , la some et bodimentSj in the content of methods described herein, antibody, or fragment thereof is itsed. that is imm¾ ?ospscifie for a selected target
fOil i)] As used herein, the term "antibody fragment" refers to a portion of s ful!- length anti od * generally the antigen binding or variable region. Examples of antibody fragments include Fab, J?ab F(a % -jaiid Fv fragments. Papain digestion of antibodies produces two Identical antigen binding fragments, called Fab fra m nts, each with a single an igen binding site, and a residnal Fe fragment, Fab fragments t ns. have an intact light chain and apportion of one heavy chain. Pepsin treatment yields an F(ab¾ fragment that has two antigen binding fragments that are capable of cros8 ink¾g anti en, and re si dual f gment that is termed a pFc' fragment, Fab' fr gments are obtained after reduction of a pepsin digested, antibody, and consist of an intact light chain and a portion of the: heav chain. Two Fab' fragments are obtained, per antibody molecule, Fab fragments differ from Fab fragrnents by the addition, of a few residues at: the eafboxyl terrranns of Iht heavy chain CHI domai . including one or more cysteines from the antibody hinge region.
{01.10} Fv Is a small antibody fr ment thai contains a complete antigen recognition and: binding site. This region consists of a di.rn.er of one heavy and one light chain variable domain in a: tight, non-covaient association {%¾. dimet}> It Is in ibis eonSgnration tbat th three C0E of each variable domai m to define an antigen binding she on the stirfaee Of fe %r¾ dimsr, Collectively, the six CD s confer antigen binding specificity to the antibody. Even a single variable domain (or half of an F coinprising only three: C Rs specific for an antipn), ho ever, has the ability to recognize and bind antigen, although at a- lower affinity than, the entire binding site . Antibody fragm e-nis usefully incorporated i nto the presently disclose biosensors can include, but are not ihnited to,; single CDRs, VH iegionss; Vh regions, Fv fragments, F(ab) amd F( ¾ fragments, ^
J01 ii] Additional fragmen s c¾n include diahodks* linear antibodies, .sing!e--ehain ssfitibody molecules, and xmMi specific antibodies formed from antibody fragments, Aatibody fragments? suitable for n-se wit the prese ly disclosed binding ddnralm oars include, natural,, synt!ie ie, of- recombinant versions. Single chain antibodies are genetically engineered Molecule eom†ainmg.¾ variable region of a light chain mi a yaiiabfc region of the ¾eavy chain, linked by a Mt e polypeptide linker as a genetically fused single chain molecule. Such single chain antibodies also are referred to as ''single-chain Pv" or "scFv" antifeody fragments. Generally* the Fv poiypeptii e ferther comprises a pol^eptide [inker between the VM and. ¾ domains that enables the scFv to form :tbe desire struct nm fdf ajfigen binding. For a deseripii m of scFv. see Plnakthtm, in The Pk rmmalog of Monoclonal Antiboifes, vol 113, Rosenburg and Moore eds. Springer- Verlag, Ν.Υ,, pp. 269-3 IS (19 4). pil2| In some e h dimeftts, the binding domain can be a single eha variable i¾gment (scFv) of an antibody wife, attachmen site for a dye withi a C R3e ion of the s¾Fv3 a modified ftagment of a natnraiiy ooe ttxing protein, or another ontity that binds ίό a specific state of the targeted protein or polypeptide. Any available scFv can be tised. provided that it binds to a selected, target wit snf ent affinity to permit detection of a complex formed betwee the scF and the selected target, A compound of Formhlai or Formula 1! can he attached to the se!eoted apFv at any convenient site. The■■florescence- the attached: dye, however, can be Influenced, by lis attachment, position, Single: chain variable : fragment (soFv) binding domains can be useful in modular biosensors in which binding domam m& t target modules connected 'with a- linker can be replaced with alternate versions to provide new desired specificities to the sensor, in some embodiments, soFvs can "be combined with somponnds of -'Fnrmtil^ 1 or Formula 11 for methods of probing living cells, fft!iS] Antibody fragments suitable ffe ws wit the presently disclosod sabjeet matter do not have to he hlength antibOilles.. Such smibody fragra ems, however, eaii haye similar or improved immunological or Other jtojserties relative to a full- length. antibody. For example, snch antibody fragments can be smaller and more stable than Mi-length antibodies. Snch a tibody i igrnents can include about 4 amino 'acids, 5 ammo ¾ci4§¾ 6" amino acids/? amm acids, 9 amin -wMs, afeo'ai 12 ^ amino acids, about IS amino :aeids, about I? amino apids.abou !.§ amino acids, about 20 amino acids, about 23 amino acids, -about 30 aniino adds, or more,
f 8II In g neral, a antibody iragmeni can liave,any upper m limit so long as it i lias similar or improved properties relative: to an antibody that binds with specificity to a target molecule. For example. smaller antibody fragments can h e less than about 200 ammo aelds, less than about 175 amino aeid¾ less than about 150 amine acids, i>r less than about 120 amino acids if the antibody frngnient is related to a light chain antibody su nnitv Moreover, larger antibod frag meats can ¾tave less than about 425 amino- acids, less i¾an about 400 amino acids, less than about.375 amino acids, less, tha about 350 amin -acids, tesrihan about 325 mi o ac ds or less than about 300 amino adds- if he: antibody fra ent is rel ated to a 'heavy c n anti body subunit 1 S| Antibodies add antibody fragments directed against selected targets can % prepared by techniques- commonly kaown in th art, in some embodiments, antibody ■fragments can be prepared from MMength: antibodies. Methods for the preparatio of polyclonal antibodies are available to those skilled h the art. See, ibr example. Green t d....Production of Polyclonal. A isera, in: Immunochemical Protocols (Mansop, ed.¾ pages 1-5 {Humana Press); Coligan t &h> Production of Polyclonal Aotisem In Rabbits, Rats Mice and Bamsters, in* Cm-rent Promcais in Immunology, secticn:2 A I (1 92), Mch-aie incorporated herien by reference in tbeir entirety, Sneh polyclonal antibodies can be cle ve ; e.g , by e'hemieai or enzymatic treatmeM to prepare antibody fragme ts useful ½- tbe resenily disclosed, methods,
i Monoclonal antibodies, and fragments thereof,, also can be used in th presently disclosed metliods, The tenn "moBoclona! antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, In other words, the individual .mti^ies witiin'isiiig . he^o d tioa m -identical: except for occasional natmil occtirring mutations- in: some antibodies that can be present in minor amounts, Mcmociemal antibodies are highly specific, being directed against a single antigenic site, I rthep, in contrast to polycional antibody preparations thai typically include -different antibodies directed against different determinants; (epitopes), each monoclonal antibody is directed against a single de&imin&nt on the: antigen, In addition to thei specificity, th monoclonal antibodies are advantageous in that they are synthesized by the hybridorna. culture, uneontaniinated by other .
44
¾mn¾no i0b«ii»s» 'the raodiiier "B:ionocl >nalw indicates iris character of the aMibody as being obtained from a s¾bs¾ffinaiiy homogeneous; popniatio.n of antibodies, and is mi to be construed as requiring ro ucti n of the antibody by any particular method.. The monoclonal antibodies herein specifically include, "chimeric" antibodies In.whi h a: ot-ion of the v anoYor light chain is identical of homologous to correspondin q &s :aMibodles derived ftortia. partlcniar species or belonging to :pariicular aritibod class or subclass, while the remaln lcr of the ehain(s) are identical or homologous to corresponding se ue ces in antibodies derived from, another species or elo gi g to another■ antibody- class or subclass. Fragments of such antibodies also can be used so long as ifaey exhibit t¾e desired biological activity* See If Pat 4$m, ; M t u ei ., roe, N fl M. Set <V/ ;685I-S5 (1984).
ilT] The prepa aiioii of monoc!anai ¾ritibodies Is conventional. See, for example, Kohle m^ ilste^ Natwt,.2$6' 495 (1975); CoKgati ei al., sections 2.5,1- 2.6.7; and Harlow e al , in: Antibodies; A Laboratory M&mai,. page 726 (Gold Spring Harbor Fn :f Ct§$% which are incorporated herein; by reference in. their entirety. Monoclonal antibodies can be isolated and purified fkaa hybridama cu!tnres b a. variety of established teehiiiqnes,. Such isolation techniques kehide affinity chroniatography with Protein- A- Sepharase, size-exclusion chromatography, and ion* exchange chromatography. See, s,g, , Coligan et al , sections 2,74-2-.?, 12 and sections
Barnes et al, Purification of Imrattaogiobulin G 0$Q%w M tm&m M&kcMla i io Vol 16, pages 79-164 (l¾mana Press 1:992})>
fftllSj Methods of .In vitro and i vivo rnampu!ation of antibodies are available to those skilled in the art. For example,, ho mono clonal antibodies to be used In accordance with: the presently disclosed subjec matter can be made by the hybridoma ruetliGd or eau be made b ; i& U® %ih®d g., as described in U.S. Pat. No, -316,567, which is keorporated: ¾rein ¾y efere ce in Its entirety Monoclonal antibodies suitable to use with the presently disclosed, subject matter also- can be Isolated from, phage antibody libraries Using the techniques described, in Clacfcsou et ■at, Nature∞62 -62« (1991), as we!! as i Marks et aL Mtf Mol M S (1991%
Methods of making antibody f agments are also kno o in the art (see for example. Harlow and Lane, Antibodies; Ά Laboratory Manual Cold Spring Harbor Laboratory, Ne Yerfc. ( .1 88) incorporated herei b reference). Am body fiiagmeais suitable for use with the resently disclosed subjset raatter can be prepared by proteolytic hydrolysis of tte antibody or by expression of nucleie acids encoding the antibody ffagm af ia a suitable host! Antibody fragments can be obtained by pepsin or papain,■digestion of whole antibodies conventional methods. For example,
Figure imgf000046_0001
fragments. Alternatively, enzym t c cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for exana ¾ iti ΙΪ& Fab Mas, 4,036,945 and 4,331,647, and feftrepoes contained therekn • hibfe' patents ar mcoi orsied hereib. by reference in their entireties,
ΡΪΙ9] Other methods of cleaving mndbodies; such m separation of heavy chains 'to form monovalent light-heavy chain fragm nts, lurtber e!eavage of iragtrjsntS Qr other enzymatic, cheraieaL or genetic te$miq¾cs also can ¼ nsed, so long as the fragme ts- bind to the antigen thai is recognized by the intact ainihody. For example, Fv fragments comprise an sssocIaiioH of ¾ and % ehains. l¾i§ association can be aoneovaiento? the variable chains can fee linked by an miefffioleeniar disnlSde bond or cross-linked by chemicals such as giutaraldehyds,. Preferably, the Fv fragments, comprise VH asd Vp chains connected by a peptide linker:. These single-chai afitigen binding proteins C¾Fv) are prepared by Constructing a: strpetwrai gene comprismg Bbi A s quences encoding the ¥p and ¾ domains connected by an oligonucleotide. The straetural gene is hiserted into an expr ssions vector, which is subsequently introduced Into a host cell such as E. coli, The recombinant host cells synthesize a single polypeptide chain with a linker peptide hridgirig the two ¥' domains . Methods for producing SFVB are described, tor exsmpiefby Whitlow etal, . Metho s; a Companion to Meiho m-Bmym&k& Vol page 97 (1991); Bird et aL Science 1^2:423426 (198 ?); l^ & at, IIS. Pat . 4,946,778; and ? ei: aL Bio/Technology //; 1271-77 (1993);
Figure imgf000046_0002
onus") are often γ©ίγ¾4 in- anlig¾ rseo'gnit on and femding. CDR peptides can be obtained by eloning: or eonsirneting genes encoding the CDS. of m. antibody of interest. Such genes are prepared, for example, by using the- polymerase chain reaction to synthesize the variable region ir¾¾ RNA f anUbody-producing ceils, See, for example*. Larriei i>, Methods; a Comp nio to Methods in £mymo g) f ΘΙ22] The amino acid sequence of monoclonal antibodies, polyclonal .antibodies, 'fragments & §of, and such, can be determined by amino acid sequencing m t ods ¾¾0 n in the art. ^roiao acid sequences, of astlbodies and fragments of interest can. evaluated, for' h¾d¾g sequences., s b as, -β » CDS. sequences, ase in the presently disclosed biosensors. Desired sequences can be directly synthesi ed or translated ink? nucleic aeld. sequences for manipulation by genetic engineering techniques known in the art, Aj&i&o-add. and fciictdciacjd §eqUe»ces ttas. obtained can be screened for binding ehaMdfeqsti.es:■ desirable I s Sxe presently disclosed biosensors. Optionally , equences ifeps obtjuned can be logically modified and/or randomly .mutated to generate additional binding domain candidates at can be screened to identify sequences most useful in particular biosensor systems of interest.
[0123] Human and fcamanized forms of nondinrnan s rnnrlne) antibodi s also are suitable for rise with the presently disclosed stibjeet matter . S¾eb uniani¾ed- antibodies are chimeric inrr >globttlinsf amrmnoglobuii chains or fragments thereof : su¾b as P seFv? Bab, Fa¾VF(ab')¾- or otaer aatl gen-binding subsequences of antibodies) that contain: ni mal .s^tieafe -deii'^d- fro aO¾«b «¾t¾}: .immuimgiobiiliii, For the most part,, ^hnraanrzed antibodies are human immunoglobnjing (recipierit antibody) in which residues, from a complementary determining region (CDl ) of the recipient are repiaeed. by residues from a CDIt of a nodmniau specks (donor antibody) such as mouse, rat or rabbit having the desired specificity, afSriity and capacity.
f 0134) In some embodiment , Fv framework residues of the human
immwnogiobn!iii oars, be replaced by corresponding non-htmiac residues.
Frirtlter no ej humanize anybodies can comprise residues that are fo&ttd neither In the recipient antibody nor in the h iported CDS. or framework se uencer These modifications can be made to.fitrifeer refine and optimize antibody performance. In H i
general, 'ki a zed antibodies will comprise substantially all of at least one. and typically two, variable .''dom ins, in which all or substantially all. -efths CDR regions c respond to those of .a non-human iimmmo lobulia and al l or substantially §3,1 of the PR. regions are those of a bumaft -immunoglobulin co sensus sequence. The huma ized antibody optionally eaa comprise at least a. portion of an iminnnogiobnlirt constant, region (Fc), typically that of a imaxi- immunoglobuli . For further details, see Jones et aL Ntt m: 52/ 522~S25-098¾ J&elcisraaaa tt-ttl, Nature JJ2:323~52 03m); fi& , Cu Qp. Struct 8wl 2 -5 (1992); ffe!mes ei m.: J. Immunol 155:21 2-2201 (1997) arid Ya$ ¾ni t at, Ληη Aihrg , Asthma & Immunol
1&Ϊ2Β] In the immune system, specific a&iibodies are selected and amplified from a large library (affinity maturation). The camMnatorial techniques employed In it!ti tm e cells can be m micked by irmtageaesis sad .generatio of conibinatorial libraries of binding entities. 'Variant binding entities,, antibody fragments and antibodies therefore also can be generated, through d½play ype technologies, Such display -t e- technol gies include, for example, phage display, retroviral display, rlhosoma! display, yeast display arid other techniques. Techniques available in the art ean .be used ibr generating libraries of binding: entities, for screenin those libraries and the selected binding entities can be subjected to additional niai raiion, such as affinity matu at on, Wright and Harris, supra, Hanef and Bucihau, FNAS USA
#ί *3 84¾><1 ?) .(iteomal displa ^ Parmfey and Smith, Gm$ 73:305-318 (i988) (phage disp] ay)t Scott, TIBS 17:241 -245 (l 992), C Ma ei al, PNAS USA #7: §378~§ 82 (1 90), Russel tfuel Acids Research 2/: 10814085 ( 9S HoganboGis etat,, Immunol Renews . 5iM3-68 (1 92):? Chis e!i and McCafferty, )| | Methods far mutating: antibodies, CDSs or binding domains can be used to optimize then affinity -Selectivity, binding strengih aad of other desirable properties, A mutant binding domain refers to an amino acid sequence variant of a selected binding domain (e.g.. a CDl ). In general, ope or more of the amino aeid residues n. the nuiiant binding domam is different from what is present in the reference binding domain, 'S¾eh-!»S3ta»t: .a0- ibod¾s 'aecessrf . iav less- th¾n. 1G0¼ sequence identity or similarity with the reference amino acid seqnerice. in general, mutant binding „„
48
domains ave at le st 7S¼ amino acid .sequence identity or similarity th t e amino, acid sequence of the reference bidding domain. Preferably, mutant binding domains have at least 80¼, more preferably at least 81%, even more preferably at feast 90%, and mosi pceferably at least .95% m oq ms identity or similarity t& the amino aci seqvsHee of the reference itdiiig domain,
Ι0Ϊ27] For example, affinity ma i i¾i ft using phage display ca .be utilized as one method .for generating mutant binding domains. Affi ni ty maturation using phage display refers to a process described ip Low an t u i chemi ry 30(45); 10S32- 1.0838 (199:1), see also Hawkins t L M&IMM. 254: 88 96 199 % While not strictly limited to the following description this process can be described briefly as involvi g mutation of several bmdirig drains or antibody hyperyariable regions at a n mber of different sites with the goal of generatirrg all possible mi e acid subs ituti ns at each site. The bindin dsmaln mutants thus generated are displayed i a monovalent fashion, from filamentous, phage particles m fusion proteins. Fusions are generally made to the gene HI product of Ml 3. The phage e pressing the various mutants CM be cycled through several ou s of selection for tits trait of interest, e.g., binding affinity or selectivity. he rsptaats of interest are isolated and sequenced, Such, methods are described in more detail in U,S. ϊ¾ί, N«s, 5;!75f j:53?35 6,290,957 arid Carmmgharn tvl, EMMO J. iSfllj m (1 94),
101 8:1 I¾ereiore, in one embo i ent, the presently disclosed: subject matter provides methods of manipulating binding entity or antibody polypeptides or the nucleic ¾eids encoding tiiern to generate binding entities, .aniibodies and antibody fragments with improved binding properties, that recognize selected targets . Such methods of rrmt iiag: portions of an existing binding entity or antibody involve fusing a nu leie acid encoding a polypeptide that encodes a binding omais to nucleic acid encoding a phage eoat protein to generate feeomMoaiit traeleie aei encodin a fusmB: iot ½s mutating the reeom inaat nueiele acid encoding the fusion; pro ein t generate mutant nucleic aeid encoding a mutan fusion, proteiru expressing the mutant fusion protein on the surface of a lage, and selecting phage that bind to a target
f«3¾ Accordingly, the .presently disclosed subject matter provides antibodies, antibody ff agme s, and binding entity pol peptides: that tm recognise and bind to selected ta get molecules. Tk s. methods for man!pttlatiiaj: those antibodies, antibody t¼gmeots, and binding entity polypeptides, cm be nsed to optimi e their binding roperties or other desirable properties (e.g.y stability, sk¾ ease of use ,
130| I» one embo imen, the biosensor molecule includes an HIY-l
neutralizing antibody Fab ftagmem (X5), which binds to HIV envelope protein gr l 20 after foaning a complex wi& the .host ceil receptor CD4> Ihe.preparaiion of such, biosensors is descrihed in U,S. Patent Application. Publicatio .No> 3006/0029946 to Hah% which is incorporated herein by reference in its entirety;
|f 131| In one em gdim iit, a f agment of WASP, which hinds oniy to tie aci!yated state n G¾42, can be used detect Cde42 activation, as also des ribed in IIS, Patent Application Pubnealion No, 200δ/ίΙ92994δ: to Halm. Such biosensors ca be used to deieetand or monitor he level of Cd¾42 activity in vitro. In celt lysaies, and in livin cells. In another embodiffieirt, a selective !igand fo the OMA
methytransferase W& can be used to detect, the enzyme. In a ftuther embodiment, the compound iifiyoperazmc, which binds -selectively to the active conformation of caimodnlin, om be used detect es!mcdul m activity.
01321 As provided here nabove, k. some embodiments, the presently disclosed biosensors can be ikked to a target mokeule. In embodiments wherein the presently disclosed biosensors comprise a target mo!eeule of kterest, the dye can be linked to tire target molecule at a position that jesuhs in a. e!mnge in a signal irons the dye upon changes to fee target con ormation ligand binding, to the target, protein-protein inieractioris with tl e target, phosphorylation of 'the- igxget, or: postiranslabonai.
modification of the target,. The presently disclosed compounds of Formula ΐ or Formula If can be linked to the target molecule in biosensors using the techniques of dye !kkage k own in die art and described: in IJ.S, Patent Application Pubiieati on .No, 20Ο6/ΘΟ299 6 to Hahn. Suitable; positions tor I ge provide, f examp ¾.
improved signal intensit or minimal perturbation of normal biological activity can be identified using screening techniques also described in IJ Jb Patent Application Publication; No. 2006/0029946 to Bafen
£01331. la some embodi e ts^ the presently disclosed dyes can be ¾ti¾ehed to a protein of interest, wherein an aciivi tyy location.; and/or conformational change in the protei can result in a change in the fluorescence of the dye, In. some embo ime ts^ ¾ the protein of interest cm be subject to a chan ed, phosphorylation state find/or protein- !igaad interaction, where the ligand can he a small molecule or a secosd protein, Compounds of Formula I sr Formula can he envalenil att ched to tile protein of. interest to provide a sigaal associated with the phosphorylat on state and/or profem-proteln intetaotiom This type of d ectiun is In contrast to previously described detections, ee, Hahn l, J,, m, Chem. Sac. 135:4132-4145 (3003), herein the biosensors detect & confo matio al change in the target protein induced by the achun of a third element, In the presen ly disclosed methods, detection can he extended to . conformational changes induced by phssphoryia on and does not require mduedon of ¾ confema ioriai change in,¾ protein © inest. One advantage of the presently disclosed methods is ¾t proteins within mnitiprotein complexes can be Moaitored isl situations whore other types of biosensors, ibr exarn iCj those requiring a drj ak to find the target protein, would !κ blocked,
|0i34| Further, the presently disclosed biosensors can allow the detection from a. single dye, e,g, s without the use of two fl orophores, as is FRET,, to. provide direct excitation and a brighter signal. In some embodiments, however, two o more Of the presently disclosed compo unds of Formula I or Formula II having different emissions wavelengths can. he used to ensHe imaging of mnliiple protein activities in the same eel! simu!ianeoiis!y. Thus., hi sums embodiments, it can he desirable to provide more than one dye molecule on the binding domain of the biosensor to facilitate monitorin of a binding event,
The presently disclosed subject matt r provides methods to examine protein activit s. sir uctur¾ o protein-protei interactions t The presently disclosed methods and biosensors ean report protein localizaiiotts protein activation and/or report specific aspects of changing protein structure or ter¾cilo¾ The biosensors can be used in living: cells ari ¾ for ho ogoneons assays. For exam le, the activity of a protein, in a complex mkmre¾ such as ceil iysate, can he determined by adding ami defecting the biosensors ltfepnt addrtiunal steps, such as wash steps, and die like. 10136] i some embod e ts^ the presently disclosed subject matter provides a biosensor comprising one or more binding domains havi g a specific■affinity fqr one or more target molecules in a specific state pfp osphory!atidn, For example, the binding domain of the biosensor can have a specific .affinity for a binding, site ^ produced upon a protein that protein binds to a goanosine triphosphate, thereb activating the signal tra»s ©iion protein, Optionally, the biosensor can ha e a signif!emtf y lower affinity for the: inactive form of the protein (e.g,, the protein bound to ga nosino diphosph at© instead of guanoslne triphosphate). "J¾.«s,for example, a dye of the biosensor, ,g„ compound of Formula I or Formula ¾ .cm occupy a position fee binding domain pear the bindin site soeh that, upon binding to its target, the binding environment alters the fluoreseettt signal from the dye. Further, th position can provide a detectable signal change in the dye without significantly hlhi tia m biological activity of the targeted protein. Deieeifcm of the acti vated target in this embodiment does not require conformational changes in the ta get or the presence of third molecntes interacting With the target
(@137| The presently disefosed biosensors als can detect pt tefc mtela
Interactions- S¾eh interactions can be high affinity interactions, such as; for example, interactions' between a gens and antibodies, or the can he lower affinit
in M o iopSj. snob as, for ^ sfk lsi^cfi^ between enzymes and subsu¾te¾ signal cascade members, or members of protein complexes. Is- a protein-protein interaction. hetw^en two■binding members* one binding member can be considered the l rger and the otic? binding, member the biosensor probed5 While the distinction betwee target and- probe ears he arbitrary, because the two intemcj with each other these terra a are used, to facilitate discussion of protein interaction. Generally, as use herein, the target can be the m:olee¾le to be detected and the probe can be a bipdkg member introduced to interrogate a sample for the presence of the target in a state or form of interest. In a representative protein-protein biosensor, one member of the binding pair can have a bindin domain complimentary to a binding she o f the second binding pai member. Out or both of the protein-protein interandon pair members can include an attached com ound of Po∞la I or :F©ri¾ui II. The protein-protein feteracdon pair members can be falh!eng¾ naturally occurring proteh¾ synthetic analogs of naturally occurring proteins, recombinant analogs of naturally occurring proteins* or f agments thereof
[6138] The presently disclosed biosensors capable of having protein -protein Interactions can have compounds of Formula I or Formula II at one or more positions on one or both binding members s ch that the dyes are positioned between the ¾ binding members dur ng lad ng, ϊη such embo iments, binding e n cause a <¾iecteMe change the signal :from tie dye without' significantly inhibiting the binding interaction beiwee¾ t e¾nding members, Suitable dye positions caw be identified by screening alternative positions for im rov signal and/or binding i etis©B ¾e resently disclosed biosensor systems,
|G139j in some embodiments, the biosensei? m ¾ave specific affinity &f a target molecule in a specific conformation, hound to a specific ii and, or with a specific pesttfansiatlonai modification other than pbosphdry on.
In s-onie embodimen s, the presently disclosed subject matter provides a B ¾Mat hiosess r. Modular biosensors are senso s: of unified design that allow cer n compoB nis ΐο he changed,. e,jps.to change the target .specificity and/or signal character. Modular biosensors typically provide for con venieni alteration of bindin domain speoirleity/. For ^x&mple, amaffinfy molecule: cm be expressed fyom a ■recombinant expression instruct, such as an expression veepm The genetic construct can include unique endonnelease sites bracketing the region encodin {lie binding domain so that alternate binding aomaius ca b readily inserted into tbe construct for expression as part of a biosensor. The modular binding domains can be selected, e.g., iom a library of binding domains. Optionally, tbe modules can be encoded scFv dorsains. he: modular systems cm include ailnlty Molecules with alternate biridiiig domain sit¾¾ .alternate dye linkage sit.es> a!temaie dye linkage reactive molecules, alternate linked targets, alternate linkers, and/or the like.
Alternately,, the binding domain camb Of broad specificity, and tbe target domain different k nase consensus sequence peptides} can. be switched in tbe modular design,.
III. Detecting Presence a«d/«r Activity of Target MoScsmlet hi Living Celts
$14 ί| Ceil behavior can: he regul ated through trausient activation of protein activities at specific subcellular locations. The ability to study translocation of proteins has been enhanced by advances In the microscopy of fluorescent protein analogues living; cells, in man cases, however localized protein, activities am controlled not Ijy translocating proteins to the site of action, but by localized activation of a small portion of the protein pool. See Ha a- and- Tontebidne, Cum eW o , (2002); - «s Tr ds ύέΗΒϊβί J7:203:~2il
(200 ί » S ch: behaviors typically are not pare t when studying protein
teaTistoeations or ita: sing m vitm 'biochemical approaches, Further, the outcome of signaling protein acti ation can depend: on subtle variations in activation kinetics that are not discernible in the population averages generated '.by biochenheal feehalq es.. For precise quant? fioa io of rapid- setivatioH kinetics and of the level of protein activation, it also is necessary to meas r protein cuity in living cells. See Woofers eidl, Trmds Ceil Biol llOM . 1 (2003 )> Wl!Mams etah, Nawre
561 (19g5); .Betridge, J. Biol Chem 2#¾9583~9586 (1990). Accordingly, a need exists for methods ..for monitoring protein movements in living ceils.
$142f ¾e presently disclosed dyes exhibit man properties that make, them .suitable for detecting target niolecnles mi their mtemeti'Oiis fa living cells, The dyes are, for example, %ight with long, way ©lengths outside of cellular autofluorescence background ixeqiteocies and that are less damaging to cells. Addition or deletion of parts of the aromatic system, or snbstituent groups thereon, can shin excitation and/or emission wavelengtha of the dyes so that more than one event can be monitored in a cell at the same: time. The dyes have increased cell membrane petem&ea blity with minimal staining of intracellular membranes.
[0143] The presently disclosed dyes can be detected in ceils by observing changes
Figure imgf000054_0001
method of detecting an activity or a location of one or more target molecule within, a cell, the method comprising; (a) providing the biosensor of the present invention; (b) contacting the biosensor with, a etdi suspected of containing one or more target meleeu!es to bind the one or more target molecules, If present, to the binding member; (c irradiating the cell suspected of containing one or more target .molecules with electromagnetic radiation to induce the■■compound of Formula I or Formula Π to fluoresce; and (d) detecting one or more oil (i) a fluorescence property of the compound of ormn!al or Formula II* (if) a change in fluorescence propert of the compound of Formula I or Formula II; (iii) a location of a fluorescence of the compound of Formula I or Formula II; and (iv) combinations thereof to determine the an activity or location of one or more target molecules in the cell providing a biosensor comprising a dye of Formula (I) or Formula (il); (h) contacting the biosensor with a cell suspected of containing one or more target molecules to bind the one or more target molecules, if present, to the binding member; (c) irradiating the cell suspected of containing one or more target molecules with electromagnetic radiation to induce the dye of Formula (I) or Formula (II) to fluoresce; and (d) detecting one or more of: (i) a fluorescence property of the dye of Formula (Ϊ) or Formula (II); (ii) a change in a fluorescence property of the dye of Formula (I) or Formula (H); (iii) a location of a fluorescence of the dye of Formula (I) or Formula (II); and (iv) combinations thereof, to determine the an activity or location of one or more target molecules in the cell.
[0145) The presently disclosed methods and biosensors cm be useful for detecting target molecules in or on the surface of various types of biological cells, including, iBarnmaIianf bacterial,, fungal, yeast, insect, and plant cells, In some embodiments, target molecules can be detected in freshly isolated cells from mammals (e.g., humans), insects, fugal, or bacterial cells. For example, blood cells, such as B cells, T cells, monocytes, and neutrophils, and the like, can be probed with the presently disclosed biosensors. In other embodiments, stably maintained cell lines such as CHO, HEK-293, L-ceils, 3T3 cells, COS, or THP-i cells can be investigated using the presently disclosed methods.
[0146] Useful information can be obtained from any ty e of cell using the presently disclosed biosensors and methods. For example, mammalian cells, such as human cells or animal cells, that naturally or recombinantly express human proteins can be evaluated to identify potential human therapeutics, observed for interactions between biomolecu.es, and/or studied for the effects of ligands, drugs, and other molecules on mammalian and human systems. In another example, bacterial or fungal cells can be used to screen for potential antibiotic or anti-fungal agents,
[0147] In some embodiments, well characterized cell lines known to provide predictive models of human cell functions can be used to obtain results correlated with human systems in pharmaceutical and medical research. Exemplary ceil lines useful in sack research include,, for example, COS cells, CHO cells, HE -293 cells, R L-L Jurka U93.T, and YB-1 cells.
fOt 48 j The cells, t© ¾e otritored can be p ovided in either iffimobil izei form 01 as- a sus sion culture, immobilized cells, sucfe-as, .e >. cell law s, tissue slices* or libraries, can be Mo it ed, &gi,.hy irrieroseopy, soakers, or with imaging systems. The immobilized cells ca be mo jiored live or fixed fbr detection of target -molecules Infilled cells.
ψί 9] In many embodhnerr¾ cells sii fect to target molecule detection wife the presently disclosed ¾io§easors are in suspension,. Suspended, cells Can be cells fxom suspension ceil cultures or ceils, liberated fern, tissues or iayms. Suspended cells are particularly well suited to hsmdling and monitoring in. flnldie systems, such as cell sorters, cell counters* and microt tudic systems, Cell suspensions cm be provided at cell densities appropriate to the handling system and detection inethod that is being employed. Determination, of optimal cell densities is routine far one of ordinal skill in- the art. In th case of o-w-¾ough einhodimsnts, cell deusihes of monitored sus e sions generally ange roa abont 1 eeil ni, to about 30 ceiis/nL in, e.g., a react on vessel or detection channel. In the case of tespnr¾e or muM eil plate based reactions, ceil densities typically range Mm. aboiil 1,000 eelis¾irri¾ to about .100,000 ce s/ in \ Of course, hese ranges can vary depending upon, e.g.., the cell types used, the ty e of biosensor employ ed> the type of interaction to be studied, fee relativ adherence of tie cells to the vessel surfaces-, as well as each other factors.
1-0150] in sum,, fee presently disclosed subject matter provides. 'biosensors that arc capable oFsmdyixrg proteins in living, eel , The dyes can be directly attached to proteins, enabling conformational changes to be ollowed in vivo for proteins incorporated m large molecular machines. Sarin vitm applications, the dyes exhibi an increased photostablihr and provide a substantially brighter s nal than current solvent-sensitive iluorophorea. Tims, they can enhance sensitivity for studying high* affinity binding Interacti ns, small amounts -of protein in high-teoughpnt assays, or protein changes that produce only small effects on. other d es. A AdwinMmUon of Siosmmn Comprising Compounds o Formi r Form la
lSll. The presently disclosed .biosensors, can h sed Mxi and/or j» vivo to detect target molecules of interest In some em odiments, the biosensors cm be
Figure imgf000057_0001
of possible en yme substrates. A presently disclosed, biosensor 'having; a specific
Figure imgf000057_0002
in embodm snts wherein the. resently diseiosed biosensors are administered, to living ceils, binding can take place with targets on the ceil strrfitee, or
Figure imgf000057_0003
the biosensor to facilitate, the translocation or internalization of that biosensor irons
Figure imgf000058_0001
.ΐ0ϋιΒ72?1 (2003-),
|i! 56J Additional iec pfc ies such as eleciroporatioix also can be osed, Examples
Figure imgf000058_0002
Physiol i 55:443-50 (19881
$t } One of skill in the: art also can use bead ringe loading to icteotes the reseriiiy disclosed biosensors: im coils. Bead^ iage loading procedures are deseAed in MeMei! el at J Cell Biol 8dSM5&4 (1984); and MeNeiiand Warder, J Cel!Sci Μ:.$0 ?§ (1987).
fOiSSf Nucleic acids encoding binding domains disclosed herein can be introduced into cells in expression plasm-ids* e..g,s. by transduction or other forms- f transfermatiOB, Once inside the living cells, the binding domain can be translated from: the nticieie aeid to a mnctional peptide. Com o nds of :F xraula I or Formula can enter the cell e.g. , by inje$;iosvor dif¾siofi; to become imked to the expressed feeling domain. o ener a¾ioset¾sor in si u. The presently disclosed dyes also a ¾e k rod eed isiio cells by other methods Imovm iri the art, isciuding, tat not limited to vir l-based delivery systems, -including viral-based sroopartides, and lipid delivery vehicles, mef¾dmg: liposomes, md. ¾
Figure imgf000059_0001
pfesphoryla nm
f0¾$Lf n. one embodiment, the: presently disclosed subjec matter provides .metk fe ¾>r ent!fynj-g t&e tiva¾¾>fi state of endogenous proteins in Ikin .cells., The presently disclosed hiosensorsicaii permit ideirti lcation, qtisBtiiloatios, and ■resolution, of the spatial, te^ mi.and'-camp ^entaireguia ion of reoeptor pbosphfirylatiofi and activation dtiring various processes, for example, endofiytosis, la aaother embodlmeiit, the presently disclosed Mosensors an i methods can perrriit ohservatioa of epidermal growth facto receptor (ICIPR) eifeets n the development ^ mi prog ressloE of breast eaiieer. M f thm embodiment, eo¾p!ex formatio between HlV gplSO and GD4 celt receptors can be mottteas
Accordingly, la some embodiments, the: presently disclosed subject matter provides a method of detecting ¾ Interaction, between mendoge»ous tar e moleerde mm a cellular entity, the meted eoaiprisirtg: ( ) f rirv!dmg a eel! comprising end¾ enou& target molectde; ( )p
observing a ¾ack ro^.d iteaiesceBce si n l fem the blosense ; ζά contacting #te biosensor with the cell; arid (e) detecting a a¾ge lit esoenee from the biosensor to Indicate m Interaedon be ween the target olecnie and the eellolar entity,
$1 1 la some em odiments, th ee!Mar ent ty is selected from the grosp eorislstlBg oF& eslhdar nucleic acid, a protein, a pep id , m enzyme, a receptor, a cytokine, a eytoskektoa, arid a signal feaasd¾ction;proteto. in some embodiments, the binding tnea ber of tie ρκ>¼ binds fee target molee de at a ph spho ylation site, la some embo me¾ts? the binding: member tea specific- affinity for a eoaiormatio^ ligattd Interaction, or |)osttr:¾»Sl¾ onaI modlrlcation of the target Bjoleca!e. In some e bbdimm¾¾ the detecting a change In fluorescence com rises ^an if lrig a rotebi ametmb locating a protein^ detecting a eon&rmational change In the target moieeiB¾ cleteetkig aetivadon of fee large! moleeule, er detecimg phosphorylation of the target olecule..
Figure imgf000060_0001
C. Methods β? Bet ing the- Presence &r moimi &f Target Motemte MSJ in accordance with the presently disclosed subject matter, binding interactions can oeeur between a biosensor and one or m ore target molecules or components of the ceil. A 'target molecule f interest'5 is atBoIeeute tbat is by one of skill la the art and is selected fa interaction with a presently disclosed .
DO
biosensor, A target itioleenie often: comprises an endogenous unlabeled sndfc untagged component of a test so! u ioa o cell Endogenous -pompose-Kts be, e,g<> expressed by the cellnaturaly, or present as a result of introduction of an appropriate genetic construct within t e sell For example, naeieie acid or protein target molecules can be expressed in the cell, either nattrraily (e. .; wHistitutiveiy) o by ind¾ tion of an appropriate genetic construct lritrod¾e|d into the ceil lias,
f §M§| In tome embodiments, the presently d sclos d: s bject matter provides a method for determining the presenc e or am u t of one or more target molecules in a sample, the me hod comprising: (a) providing the biosensor of t e inventi on; (b) contacting the biosensor with a sample suspected of containing one or more target molecules to bind the one or more target molecules, if present, to t e binding member; (e irradiaimg the sample .suspect d of containing one or m re target raokoules wish electromagnetic radiation to indace the compou d of Forinnla I or Foron la 11 to flitoresee; arid, (d) detecting a Snorescence roperty Of the compound of Formula I or Formula II to determine the presence or amount of one or more target molecules in
Figure imgf000061_0001
with a sample suspected of containtng one or more target molecules; mi (c) determining a ratio of the second emission wavelength to the rirst emission
wavelength: to determin the presence or amount of One or more target molecules: in the sample,
|8 8f hi some embodiments, the method further comprises eotitihuo¾sIyj ¾) contacting the biosensor- with, the sample suspected of containing one or more target moleeufes; (b) irradiating the sample with eiectromtgnefe radiation; and (c) cleiecimg As fhiorescence property of the nompennd of Formula I or Formnia IL
01d9;| A used herein, the term %arapleif Includes: any Bauid or "fluid sample, including a sample derived i¾om a biologicafsource, suc as a physiological Stud, including whole blood or hole blood componen s, such, as red blood eellsf white blood cells, platelets, serum and plasma; arche t urine; saliva; sweat; milk; synovial fluid; ert!cjoeal fluid; ainaiotk fluid; peicexehrcspinal Said; lymph Suid; lung embolism; cerebrospinal fin¾ pericardial fluid; ¾§mc«y«|iaai samples; tissue extracts; ceil extracts; a d other constitoenis of the body that are suspected of coj taisiftg the anslyte of interest. In addition, to physiological fluids* other liquid samples;, sucluas ater, food products asd the for the periwman&s of
environmental or food production sssays are suitable for use with the presently disclosed subject matter . A solid material suspected of containing the analyte also can be used as the test sample. In somflrtStap<¾¾§ it might be beneficial to modify a solid test sample to fo m a liquid meohura or to release ; the apalyte^
|Θ17§ I» some embodiments, the sample can be pre-trea ed prior to use, such as preparing; plasma from blood, diluting viaeous fluids., or the like. Such .methods of treatment can. involve filtration, distillation, conc¾nkaiio¾ isactivation of interfering c .u¾5Gim lsf and the addition of reagents..
fOl ?1| The sample can be arty sample obtained, ffoirs a subject Hie term
"subject" r-eiers to as- o anis , t ssue, or ©ell f om which a sample ca he obtained,. A subject c include a human subject i r medical pnr ¾Gses} such: a$ diagnosis aad or treatiftefit of a condition or disease, or an animal subject ihf medical, veterinary
Figure imgf000062_0001
e.g. , igs, hogs, arid the like; e uines, e.g., horses, donkeys, zebras,, and the like; felines,. Including wild and domestic eats; canines, including dogs; lagon phs including rabbits, hares, and the like; and rodents, including mice, rats,, arid the like. Prefeahh , the subject is a mammal or a rnammaHaii cell. 'More preferably, the subject is a human or a human cell. Human subjects i:Eciudes bat are not limited to, fetal:, aeonaial, infant, juvenile^ and adult subjects. Further, a "subject" can include a patient afaiieted with or suspected of being afflicted with a condition or disease.
Thus, the terms "subject'* and "patient" are used interchangeably herein, A subject 0.2
also cas refer to cells ax collections of cells In laboratory or bioproeessing culture is tests for viahd ¾f diff|rentjailo¾ Marker pradneiio¾ expression, and the Me.
IV, B|os«issor Kits offipristeg Campffinis of Fo mula ί or Fo mul IF
|0i72| : T¾& presently disclosed, subject matter further provides a packaged composition sueh as a kit or other container for delecting, monitoring o.r ot¾.er ise observing a: target molecule, ¾e kit or container eap.&old a biosensor comprising a eofflpouad of Formula. I or Formula. IX and instructi ns for: using the biose sor for
Figure imgf000063_0001
of Formula I or formula Π. attd mstme!ians for using me dye. In some embodiments, kits cotiia kg dyes cm cant in,mstFU(;tioss for att ching a dye t a binding domain selected by one of skill k the art.
[0173] lie presently disclosed kits also can c m ise containers with, solutions or tools useful for .manipulating or using the presently disclosed dyes or biosensors. Such tools include buffers:, reaction tubes, reagents for coupling ooTn oimds of Formula I or Formula I to selected binding d&m m mm the like. In o e
embodhaenn the kit can contain a seiution of solventa 'and/or buffers to facilitate cotipliag -of ¾ compound of Fomruia I or Formula ίϊ to a selected binding domain and/or a solution of mereaptoetharjol for queseMng the dye-binding domain eonlugatiort reaction, i¾e kit a! so can. melode a cont ker of buffer m roughly neottai pH sodinm bospba e buffer, pH 7.5),
V. Chemical I raltsoBS i7 | While the following: terms are believed to be well understood by ong of ordinary skill in the art, the Ml owing definitions are set forth to facilitate explanation of the presently disclosed subject matter. 'Unless: otherwise. defined^ all technical and „ ,
63
scientific emis i5sed herein ¾¾v® the same meaning as commonly understood by one of ordinary skill in file art to which this presently described subject matter belongs, |#1?5| ¾to¾gte*ut the specification and, claims, a gives omical formula or name shall encompass ¾I1 optical aad stereoisomers, as well as race ic mixtures where suehisomers and mixtu es exi t
\βϊ ] ¾en the. term "independently selected" is used, the substltuents being referred t (e,g,, R gruts * such as groups. ¾ R¾ and the ¾e. w groups X and Y)> can ¾e Identical or diSereni For example, both ¾ jad ¾.oan be substituted alkyls or 11· can be hydrogen and R¾ can be a substituted aikyi, and the tike,
0i? | A »med Pi tsX!? group wiii generally ½ye the structure that is recognized in the art as corresponding to a group ¼V&g'..¾t name, ualess specified otherwise herein. For the purposes of il!nstraiion, e iaiii- fe resei& e -'R'* and W
Figure imgf000064_0001
[0178] As .used herein ffee term "electron withdrawing group" refers to a atom o a group of atoms that dra : electrons a ay from a reaction center, Exam les include, without liispation, carbony|5 oy m, halogen, n¾ot snliopj and trifipororncthyi groups.
[0179] As used herein the term "l ving group" refers to a charged or uncharged atom or gro up of atoms thai: departs Airing a $afcsi¾ttta w displ^ i^ ^ ,. Examples include, ithont limitaiiou, haio ns, such as eh!bro^ broP o, and iodo; lko IdCj hiflate, tosyiate, mesylate, torosulfonate and optionally substituted N- hydroxysPoeiniMide.
I&t 8fi As used herein, the- terra ¾o¾:ttgatalne $ΐ¾δ eh®&" efers to side chain comprising, consisting essentiall o¾. or consisting of a linker and a jeaeiivs grou at the end of the linker that can be used to conjugat a dye compound to a binding member.
181] As used herein d term "alfcyl" refers to C Wo inclusive, linear (?. & } ^straight-chain' branched, or cyclic, saturated or at least partially and in some cases felly uns tu ated ( .as a!kenyl and alkynyl) hydrocarbori chains, including for exarnple, methyl, ¾ ¾ propyh isopropyl;; butyl, Isobutyh fert-buiyl, psntyh hexyh oeiyi e&en k propenyk btitenyk pesienyk toertyk oetenyl, ¾uiad¾nyl, opynyi, butyByl, pem n l ½syriyi} neptynyf, a alenyl groups, "fifase ed5' refers to an aikyl group m vMebt a lower aikyl group, smh rnethyL ethyl or propyl, is attached to a linear aikyl chain, ifl&mt alkyi" refers do m. aikyl groap having I to. abmd.6 carbon atojns (i. e< , a alkylX ¾¾ 5, or β earboii atoms, s¾tgher alky!" refers to an a!kyi group havmg: abpj.fl I to a out 20 carbon, atoms, .e>&> §» JG, 12,
Figure imgf000065_0001
PnsnM&ried nitrogen atoms,, wherein the nitrogen subsiitueni is hydrogen, lower aikyl ^alsp relerrsd to herein as ''a !aaiinosIkyk'), or aryk
|0!83| Thiis, as used herein, the temrE¾ bstikted aikyl" inc!iidos aikyl. oups s as is md & , I» which one: or more atoms or fu ctional groups of the aikyl grotrp are rep!acei with anoffior atom -or functional .group* including lor exni ie, alfcyk snbsttoteil ikyl, halogen* ax !s sii stituted aryl, alkoxyk hydroxy!, nitro, amnio;, alkylanfino, dialkylamifto, s¾i&te,, and t r pio,
|0£B4$. "C^^' siid ^^io^kl:" refer to a rion-at¾rn ic mono- or .multicyelk ring system of boM 3 to about 10 carbon atoms, e.g.% 4, S 6, 7, or 1Q carbon atoms, '1¼ eydoatkyl group: can be p iwally : partiall unsaturatd The cyekoa!ky! group also can be optionally sahs l¾ted with an aikyl gronp substifaent as defined feereirn oxo? mSfm alkyiene. There tm ¾e opdonaily inserted along the cyclic alky! chain one or more oxygen, mu& or substituted ot ims si eiS -i-itogea atoms, •wfier»i»-Ae-i-irogei3. sn¾stitnsnt is hydrogen, alkyk .stibstitnied aikyk aryk o substituted ajyi* thus providing a heisrp ye!ic.: group, Re resentative monocyelio cyc!oalfcyl rings molade cyo eity!, ov! hexyL tad cyolohepiyl Muliioyclfc
Figure imgf000066_0001
can include one. or more double bends, The cyeloheteroa-tkyl ring e optionally fused to or o¾erw!se attached to olner cyelobeieroalkyi rings asd% Bon-a piahe hydrocarbon mm, Represeniative cyelohete &ifcyl ripg systems itfehtde* hnl are mi limited to pyriolidii y!, pyrroHnyh iffilda Bliil nyii iffiidazollnyl, iryrazoi inyi.
pyra2oIinyk piperidyl, plperazl¾yb fod jisyl* quinuelidipyi, J or hQil ryl, tbiorporpfeolipyi t adia manyl tetts droferasyj, arid the like.
lftl¾7J: The term "alfcesyl" as: used herein refers to a straight or bmnebed.
hydrocarbon of a.designed number of o¾rbc¾ M containing at least one carbon- carbori double bond, Exafap!es 0: s¾lkei3yl?s Include vinyb a¾l 2*me!:!ryi *hepte»¾
Figure imgf000066_0002
„ ,
Inserted akmg the .¾ikyteae:gt a|3«B€-.orSio|¾ o ^ eft, svifdr ot substituted ot ansubshtuiad nitrogen atorns (also referred to herein as ^alkylammoa!kyP'). hsreln
Figure imgf000067_0001
Q, 1, ¾ 3, , ¾ 6, 7, 8, 10, II, 1.2, 13, 14, !5S 16, 17, 18, 19, or 20, and R.is hydrogen or lower aikyl; meth !me&ox ! (-0~€I¾--O-); and ethylenedioxyl (-0- (C¾):~0- An alkykne grpnp ean .have ab ¾r 2 to about 3 earboaaioms and e¾ farther ave 6-20 carbons,
[0191 } '¾ term "aryP is used hsrefe to refer to an aromatic substiinent that can fee a single sfomstie ring, or irrohiple aromatic rings that are fused: together, I ed -eo.vale ly, or Jinked to a &m m grou ,, sue - , n% Hot limM to, a methylene o e ½fte:.me.ieiy he common linking group also dan he a e rkmyJ, as in
benxophesone, Or oxygen, as in diphenyi ether. Or nitrogen, aa in. d!pheny! smme. The term " r l5- specifically encompasses heterocyclic aromatic compounds. The aromatic gs}£m comprise phenyl, napfeli l hiphsnyi diphe¾ylehtet,
iiphs»ylanihe and be Ko es ne, among others. In particular en¾ odimnts> the term "aryfs means a cyclic aroniatie comprising about 5 to about 10 carbon atom% e,g,, 5> 6, 7, 8, or 10 ca.rbon atoms, and -including S- and 6-me bered hydrocarbon and heterocyclic aromatic rings,
:flil921 The aryl group can be optionally sabsti ied (a "f absented aryl") with one or more aryl gronp s¾hstiiu¾ats5 which can he. the .same or diSetettt, w erein s<aryl group subsfitnent" includes alkyl, substituted alkyi, aryl substituted aryl aralky!, hydroxy!., aikoxyl, aryloxy!, aralkyloxyl, carboxyl, aeyl !halo, niiro5 alfcoxycarboriyl, afyloxyearbonyl, aralkoxyeatbor ls acyioxyi, acy!aisln , ¾royianino> carbamoyl, alfcy!carfeamoyl, lialkylearbamoyi, aryithio, atkyhhi% aikylene, and -NS.'R", wherein R' and R* can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted atyh and aralky!.
PX93] T us, m used herein, the term 'isthstiiuted aryl" ineludPs aryi grou s, as defined herein-, in which one or more toms or functional groups: of the aryl gro¾>- are replaced with another atom or fPnctionai gro:ops ncluding for ¾¾sm k, alkyh substituted alayh halogen, atyt, substituted ary , alJeoscy!, y<fe xyl? mir¾ amino, alk iami *, diaikyla iuo, sulfate* md mercap o,
|0194} Specific examples ofary! groups include, but ate not limited to, phenyl, iuran, th¾phene5 pyrrole, pyrau, pyridine, Md ^o^^ffiii^idazoi^ soiaagoi , i¾oxa¾sie4 p a¾»l¾ :^yra¾¾e. triaanos pyriiBidine., guinolines isoqukolme, indole, earbazole, and the like,
[01 US] The term "hoteroaryP refers to an aromatic ring s stem, such as, but not li itedto a S÷ ©r 6~merob¾r liftg system, including ane or more heternatorna, which can be the same or different, and are selected from, the group consisting sfK, O, and S. Theheteroaayi ring can be feed or othe ise a tached ίύ am or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings, or heterocycloalfc i rings,
Representative heieroaryl ring systems include, bat are not limited, to. pyridyl, pyrimidyl, pyrrolyl,: pyrazolyl, m>ly% ®tm≠y . isoxaaplyl, ¾d a2;olylf thiaxolyl, isothl o!yl, imld&zoM, for n i, thknyh qumoh h jso ms lia l, indolmyL indo!yL cu Oiihesyl bcfizoihiazolyl, eaasjforanyl bensinif dasoiyl benasosffi&ol i, bewzopyraiso!yh triazolvL ie¾¾¾0ly), and the lite
|0t 6] A. siruciure represented generally by the fonn.uia; wherein the sing
Sirueture can he aromatic: or automat
Figure imgf000068_0001
as; used herein refers to a ring: steot re, for ex m le, hut act limited to a 3 -ear bora a 4~earbe:n, a Smai'heri, a 6-carboi¾ aiid the iike^ aliphaiio and/or aroni tie cyclic compound, including a saturated, ring structure, a pariiahy satui-ated ring structure, and an unsaturated ring structure as doimed herein, comprising a substlt ent R group, wherein the A grou can he present o absent, and when p ese t, one or more R groups eameach he sub tete«f. on am oruiore ataila c carbon atoms of the ring structure, The presence or absences of the R. group and number of E groups is determined by the value of the integer a. Each R group, if more- thau one, is;
substituted os an available carbon of the ring structure rather dra on another R group. For e n¾¾ the stactee above where n is i to :2 w uld comprise compound groups including, but not limited to:
Figure imgf000069_0001
and the like.
0197J A dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line
representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure,
[Θ198] When a named atom of an aromatic ring or a heterocyclic aromatic ring is defined as being "absent," the named atom is replaced by a direct bond.
[0199] As used herein, the term "acyl" refers to an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent {i.e, , as represented by RCO~, wherein R. is an alkyl or an ary! group as defined herein). As such, the term "acyl" specifically includes arylacyi groups, such as an acetylfuran and a plienacyl group. Specific examples of acyl groups include acetyl and benzoyl, [0200] "Alkoxyl" refers to an alkyl-O- group wherein alkyl is as previously described, The terra "alkoxyl" as used herein can refer to C ¾> inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyL isopropoxyh butoxyl, t-butoxyl, and pentoxyl, [0201] The term "alkoxyalkyl" as used herein refers to an alkyl -O-aikyi ether, for example, a methoxyethyl or an ethcxymethyl group.
[0202] "Aryloxyl" refers to an aryl-O-- group wherein the aryl group is as previously described, including a substituted aryl The term "aryloxyl" as used herein cm refer to phenyloxy! or hexyloxyl, and alkyl, substituted alkyl, halo, or a!koxyl substituted phenyl oxyi or hexyloxyl,
[0203| The term "alkyl-thio-alky!" as used herein refers to an alkyl-S-alkyl thioether, for example, a methylthiomethyl or a methylthioethyl group. [H204| "Araik l" refers to . aryl-a!fcy iigrou bgreta axyl m0 a!kyl are as previously described, and included substituted ~w≠ mi substituted alkyi Exemplary aralkyl groups ciurje en yl, phssy!etiay € ¾t ¾ lme¾L
f ft3tOS Aralk$¾s f- refers to .∞ ara&y i-Q- group wherein the aralkyl gou is m previousl described, An exemplary aralkylo&y] grou is beirzylo yL
\Q 6\ "Afcoxycarbouy!" refers to ^^skyl-O-CO- group, Exemplary
Figure imgf000070_0001
arylo^C on ! groups ipefcde berjrt ' ami ¾ap ¾Ky-erbony
Figure imgf000070_0002
An exemplary
at alkoxycatbonyl group is 'beBzyloxycarbonyl,
i i "C rbamoyr' .refers to an ¾.N~€G~ grottp, "AlkylcarbarnQyl" tsfers to & ' N-CQ-- grmip wfeereia orse of.R and W is bydrogen and te other of R & if alky! and or substituted aikyl as reviousl described; ^fjiak iw w ' *effet*;$» a ' -CO- group wferein eao ©fii and R< Is independently a!kyl and/or sihsihit aikyl as previously deseribed,
20t] cykayF refers to an acyl-Ο-· group berein aeyl is as previously described.
0)21:0$ The tern* "amipo'5 refers to the --NHa grouja and. also lts to- a. nitrogen eontakmg grou as is; kw in t¾e art derved iiom :anmoBia. by the replaeerneni of one or more hydrogen radicals by organic radicals, For exampk, the terras
*¼ογΐ8πηηο,' and "alky!anlao" refer to specific K-sribsti ed organic radicals with a¾yl and aikyl substituent groups respectively.
|011· The term fi¾ky¾minoS5 refers: to a— ¾ group wherein R Is m aikyl group aad/or a spbstil ted alky! grou as previously described. Exemplar
alfcylamms groups irieiude mgtiiyiamiao, etbylamine, g¾4 the like,
02i¾ "Diaikylanisno refers to an - f group wherein each of R and W independently an aikyl group and/or a substituted aikyl group previously described.
Exemplary dia.kylami.TO groups include ethylmethylarriiriO, dimsthyiamiao, and diel&ylanrino. ί0
f 1)213) &c$miw& ' refers to an ae i-^H--- r u wherein aey 1. : is as pre vionsly described.
[0214] ^Atoylammo" refes to art aoyl-NB- group wherein aroyi is as .
• eviousl dss cr ibed.
|02T | A ^Quuterasry pitrogen aiors! M is a ntt og i atom bound to fou atoms* for e¾ i¾}le:, fou carbon atOHis, atid':hvitsg« positive e!irge available for hindjrig ionically to m. jioa for the remaining vale&ce, A tiaternajy .nitrogen &L k desi ¾ateii herein as "
I&21£ The term "crteay V ' refers to ϊ¾¾ gK>«p.
p2i:7f The term 4¾arbo¾yJ" refers to the -COC¾H group,
2 IS| ¾ term "cyano" refes to t e HO¾M group.
f¾21S] The terip'S: S¾ S ¾a e" or '¾alr>gen'*' as used herein refer to- fi ro * ehlofiDj brorao, and lodo■ roups.
[ 220] The ferns '% droxyF ofes to she -OH group,
The term %ydr©xyalkyl,s refers .¾ an alky] group substituted with an -OH group.
:02;¾¾i The terra ¾sroa to" tefers to the—SHgroup.
f©223 The terra %¾; 5ί refers to the =0 group,
224J The term refers to t - Oa group,
10225) l¾e fernr sifrio; 5 refers to a ^ompwfid described: previously herein wherein a carbon or oxygen atom is .replaced by a s i¾ atom..
|¾22o~| The terra * -sulfate*■ refers t the group,
f§227f The term, ^ifouyi" refers to the -SO?- .group,.
|0|2f J The t ing Example® have been included: to provid guidarsoe to one of o-rdiiiar-- skill -in the art ibr prastieiug rppr&Beirtaivo ¾¾hodimeats of the pres¾Ktry disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill cats ¾¾j¾siate:that the. Io ag Examples are intended to be exeTftpiary only and that numerous changes, modifioaloris. and alterations can be employed without departiag -from the scope of the presently disclosed subject •roatfet.. Tire i¾ll vs¾ag 'fi3i¾m.ples-:-8-r« differed1 ¾r ¾y -of lltettatiOfi-aiiid mi by way of limitation.
Figure imgf000072_0001
roiBOi) nd iodoiBetnane (0.44 rniL 7, nan©!) wer a.dded to a small ffliemwave vial and heated to 120 *C f¾r 5 miji. The fflixture wis cooled, filiered, arid ash ¾ke-«¾iIled:et¾er¾o -gm-.Q ! g (80%) pale piak powiety solid. Spectra! data matched those previously reported? ¾ B R (400 Ml¾ D S0) -S 7.98 - 7.87 (m. 1 Bp 7.85 7.7$ inp IO):! 7.67- 7.57 (fp, 2¾ 397 (s; 3H), 2.77 <·ρ 3¾ 1.53 £s, 6H).
HMfiaO- H^ MSO^S- m. 142414 !29.¾ 128.7, 123,2, 1ί$.ί,,.5ΐδ; 34,7,21,7, 14.2. 2311 Ge.it era! rocedure for dbe prepar tion of acceptor heterocyciss as the ■activated -eaoi ethe , Representative example ict eompoaad ht l 4&$ dwm (0, 1 6: g; 1.00 mnio!) was added to small microwave vial with 1, 1,3,3- efeamthoxypropaoe (0823 ml,, S mmo fewed by additon of trilkoiBaeetic acid (7.7 at, Q.10 mof), vial was capped and healed -to 150 °C for 15m¾. The reaction was cooled and 'the precipitate was filtered arid rinsed with ice shilied 3:1 laexanes/E¾0, Isolated GJSS g (74%) brown soM. ¾ N E. (400 MH¾ CDC¾) δ
7J5 (m, 2H¾ 7.76 -■ 7.70 (m} 2H}; 751 (d, J« 12,2 Hz, IH), 7,42 (d, J- 12,3 H¾ 1H>, 7.27 m 12. , 11,6 1H¾ 3.93 (¾3H). %¾ΜΕ(ίΟ M¾ CDC¾) S 191.4, 190.5, 168.9, 145.0, 142.0, 140.7, I S, 134,1, 124.1 , 123.0, 122.7, 103,9, 58.5.
£'0232 ] Geaeral.proc^dwre for the synthesis oft»er.ocya«i»e dyes not bearin reaelrve side ikni , ipresentative example for ί»ϊ¾Ι: Donor I (38 r¾ 0 J 3
¾¾o|)i. sodium acetate (11 r¾ M3 mmo!), a aGcejstar ftt (21 r¾g, iO nimoi) were addedto a small iaicowave vial xi diluted in 1:1 eOHCfiCh (1 HIL), 1¾ vial was capped and heated to ?5 °C for 3 rain. lie reaelioa mhtms was cooled, and eoiieestoted with 230 mg i¾ and eluted on a 12 silica column with - 3% MeOtf in C¾C¾t over 20 rain. Isolated 29 mg (S2% dark blue solid. ¾ MMR (400 H¾ J3 SO) & 8.12 (?., J- 13.1 H¾ IH), 7.82 - 7.63 , 5i¾ 7.52 (dd, J" 17.4, 10,2 Hz, 2K), 7.33 (i, J- - ,7 ¾ !H), 7.23 (d, j- 8.0 ¾ IB), 7.12 ( J- -7,4 i¾ ill), 6,14 d ,/ - 13,5 ¾ 1 Id), 3,49 (s, 3H), 1.62 (s, OH), l3C NME (100 ¾ CDC¾ d 191 16S.L 152.3, 146,6, 143,7, 122, 1 0.SS I40.I, i 3.3.9.133.73128.4S 123¾ 122,2, 122,1, 122,0, 120.8, 120.3, 108.5, 99 X, 47.8, 30,1, 28,5. MS-ESi mfz 356,2 (£M r H requires 356,2),
Figure imgf000073_0001
dissolution, Et3N (7,67 ml, 55.0 mmol) was added" quickly drop wise and. the reaction mixture was stirred at morn isaap under argon for 24 -k The resulting while alpitate wax filtered and reeiysta!lized from metlmsol to give 4.83 g (37%) while solid, ¾ W (400 H¾ DMSO) £ ¾, 9 (s52H), 3 ,50 { j~ 6.0 H¾ 2% 3, 14 -
Figure imgf000074_0001
Tn 5^yjttd l^-i '(4i 4 mL. ¾7·7 maiol) was added a d the flask as added predted oil bab at 150 °G arid the reaction stirred tbr 5 niki. The flask w s cooled to room: temperature, and then further cooled in as Ice bath, Excess liquid was d and fWfeer reroqved b syringe. The residue was niased with:E¾0 (3 X it) mL), item dried uuder vacuum. ¾e predsret was iss ed as 1 ,86 g pittk grarralar solid
Figure imgf000074_0002
dssovecim MF mQ WEder argou. After it mi¾ eceptor FW (0,214 g, i .00
Figure imgf000074_0003
flask was tted w a co¾deaet ap ed - ith dryng tabe andtbe reaction was heated at reflax for b. Th solvent was then emo ed mi tbe crude reaeUon mi¾!are was coacentraied, diluted ia meibaim!, aud s«bmlttel ¾ HPLC eobruiu rj ritkftion to give 0,030 g: (2 i¾ from tht) dark blue erysiliiae solid. ¾ K E (400 MHz. DMSO) & «.1:2 ft J- 13 ,1 I k. !!¾ 7,7 - 7.67 (rm 5H), 7,57 (dd, J■■ 13.1, ¾5 ¾ IK), 7.50 4 J- ,5 Hz, IH¾ 7.38- 7.28 ¾m;.l¾:7,23 d.J^g.O H¾ 1% 7.13 ¾ 7.2 ¾ iH)s 616 {¾ J- 13.4 ¾ ¾ 4,05 - 3,82 (ms 4H}53.52 - 3,36 (rn, 4t% 2,4S - 2.37 (i¾ 2!i).2.00 - 1 ,72 (m, 411), 1.64 (s( 6H). MS-fiSi ms 687.3 ([ - j¾- tequires 687.1), ίθ23?! Genera! pma rniot &e syafeesfe &f ~tne e&pt©ei¾¾ro! derivatives of :<50»i¾gatsbie Me oey ais * Representative xample I i ilye ¾er©6ft; Mei¾ ( , 9≠ m wss ikd to & S raL cortical; vial with DMSO (10 iL) and; 50 m aqoeoHs NasHPO^ H 7-5 (49.9 ; ϊ,); he mixure was sfered and ^mereaptoethanoi (3.4 tL: 4|; iffiol) was added 1¾e vial was^ capped, co ered in fm an stirred at ioer , emp lor 24 !p The leaotio miJCture was s¾¾eete neat to HFLC column purification. The nsia product fractions were ©om&la , eoaeentrgted, an i pM&ed to give 2.1 ; U): dark M¾e po fey solid.
3S1 Qasatttm yeld d«ie ffil ail0as. Qaa tMi yield (QY) v were leiermified using the following e do ; ¾«¾ * Q¾¾i (gradsaJW gradsiti) {tftsmp-- where "grad" is equal to , the slope f a plot reiatkg the irrtegrated emission to ilie a¾S0 anee ^
com m s per sample, and is the refractive iadex: of tlie solveat used for the fiuorescenee readings, Merocyaairie S403■ asd siilfbrfeodanlirie 10132 were used as standards,
1023$! ¾0iosabil y Measa eteeas, Eac sainpk dye aad &ioresce1« stand rd sokiipn wps: prepared at ϊ Ϊ Μ in degassed w-BuOB and placed in a capped 1 ernX 1 cn cuvette, Sarn leS: were irradiated with &¾) W halogen ftmgsteri lamp wiih&n cooling fo maintain OT exsiure of 25 1 duririg tlie study, Absorbaaee mi mm s were taken prior to hradiadPn and at designated intervals- dnring Irradiation.
i CBB-MBF ye ¾MI¾g i C hl mg My. The Cdc42 binding domain (CBD) fern Wiskoit Aldrleh Syndrome Protein (WASP) was covaientjy deti atM: with dye tp generate a l^sor, wi P≠m $&pm $ mi methods prepared CBD iused to Mali)se>feliidmg protein for enhanced solubility (GBD- :MB 5 Θ pL of a 132,5 M stock solution in .0LmM.! ¾HR¾} pH 7.S buffer) was raked' with 5: molaf : equivaleate: eonmgatabfe dy© in DMSO (3 PL of 2Q - 25 mM solution} and stirred .at roam temperamre for 2 h, lie -feactiosi as ,¾u£snched by adding I pL of β-meteap ioethanoL Excess dye was xemoved by passing the actio mixture thro¾gTi l κ 2 era OAS column pre-cqpilahraied with SO n¾M Na¾HP04> pH 75 bnf!er aad wrapped in alurnlnuni il Labeling efficiency and eance-ntradons of
Figure imgf000076_0001
every i m¾> Iraage proeessiag was. carried oat as prev!onsly described (Hodgson ei l, Meilwds E ymol d> 14045a" 0 ) Hodgson et u G r & Cell MM: PP 1-2 Ck¾3t¾f 14, Unit 14 il (2010)},
[G242J roteiii eS»£s$i0E, The Cdc42~K¾diag domain (CBD), derived ft& istoit-Aidtiem sy d'mme pf oteia (WASPS esldiKis 201 -320)S: was muated o iaci de a sir¾¾ie cysteine $27 IC) for dye -attes!hmmt sad ised at its C-tertainas with rn ltose-.¾iadiag protein (MBP) throu h a Q C Q Inte The CBD-MBP fta ment was sabeloned kto a :pBT23b p!asmld to gener te a C-fefmis l 6 £¾ fusion. protein, file B -:Ce«¾ aft fesfon protein was prepared with€49$ mut ida a Pemie . to
Figure imgf000076_0002
sahcloned ist¾ihe QE-8€L p!asmid to generic aa N-iermina! 6 His fesion protein. The resaithig eo str cts were translariBed into the BL2!( E3) strati from. E. li (Siratagene) and. the bacteria was ealtare t 37 245 mm (New Bmftswlei :SdeatiBc} liiaova 4900) in Lenria-Berfajii aiediam ia t¾« preseaee af 100 mz/ L earbeaieillk taadl QBoOS.eached 0,S, PrPteia expression was conducted at 32 *C for 2 hoars artec additioaof 40 JBM isoprPpyl p^D'-i- iio aiaetopyraoside ^TQ). fhe .
bacteria pellet was collected by oeaMfegsdoa (Beckniam model J-68) at 4000 trail for m m , ,4 °C, and. stored a -80 aC pnBv to purification.
[8243]: Pro » proSe iefi, Per lys s, tfe bsetena! pellets were resiispended in m ML of the l sis uffer (50 xijM:N¾¾PP*, pH , 300 mM Ha€¾ arid disrupted by someaiion (Braiisori.:0igi¼i Somites); The supernatant was collected by
ce«tri%$t©» ;{S.O?va!l> m<?f¾l Ί21) at 6000 rpm for 1 , 4 ¾C, iblfowed fey
Figure imgf000077_0001
.„„
77
exc!tafeH ( 03 HBO bulb). Klteis usedlm Cemhm were 430/24 e^eiiabn add 470/24 emission, :Bxc|i de¾ was through aBDl.5 (3% tramm&s¾8i.) neutral density filter, with.200 ins- exposure. For $rtiCl> titters ere 54500 excitation mi 630/45 smiasioa. Excitation was through a ¾.0 (i%T) fitter witb.200 rss exposure, iiasglog ef iH«yo€3 mi Me Sl was carried oat ag 590/30 exctation 630/40 emlssloM, M 2.0 (!·%'¾ asd 200 ms exposure. Images: re acquired with a Coolsnap ES2 camera (Photoaetriss) with a S ny S x 6,45 μΜ/pixel chip using ¾ % 2 rmkg, All image acquistion;, pr mg, aad sftal is was earned out with Metamorph: sciftware,
Bmmp 2
Figure imgf000078_0001
:(Pr¾a rti st ai.^l Chew. Sm. FerMr i'a -- h &iWm)), Do¾or: a¾ cs iw groups \ i'.behei¾c»drfe tefeed bf their ietmr eodV¾ams¾ derived from th parent heteroeyefe, m order clarify dscttssis (Ernst et&l* Cytometry Ϊ03 ΰ (|J½9¾ (t able I;}.: Dotior groups are ordered aecordisrg to their Btooker basidty, . wth isdoleaiae (Ϊ) he&¾ the weakest md ^daeline (Q) the strongest (Broofcer # t¾£ J. jm, Cta. m 23· 1.00-1093 (195% Breaker, vMH Fhys. / :275^ 3 (1.942 ). Donor eomponsats ofthe dyes were activated for c |ugaiion throug qi etmzM ia with iodomethaM 1). Each acceptor riii was prepar d as its correspoiidfe methyl emA eiier feough ed cah%¾d reaetioa with malon ideh)¾ fcis(dimethyl)aeetal(¾bie2).
Tabfe Ϊ, Sy¾feesis of cpiatemized donor !ietefosycles,
Figure imgf000079_0001
tW . Symfeesis; of activated aecs iar ethers.
Figure imgf000079_0002
Figure imgf000080_0001
|C>247} The dyes for Initial screening were synthesized through' .reaction of an acceptor enol ether with a dqriot fti the resenc of J ,2S equivakflts base (Table 3.}. All dyes were made with three lateryemxi efeubls botv between the termiiral bsieroeyeles m this provided ibe optimum compromise between solvent-sensitive fluorescence 'aiid photostahiH y {Mostovnikov et:. l} Zh. Prikl Spekirosk 20:42-41 (1974); Isbchenko, Visp. Kkim, ΐ7ί)8-1743 (1991¾ Following reflux, the dyes were purified by eolinrifi Gbroraa ograpby i>ri¾ s m cases by reeipitaiiop through concenfraiicm of the reaction. mixtate, followed by addition of diethyl ether and washing with MeOlL Donor Q did. not react successfully with acceptors when usfeg NaO A C: Md nstead required the ¾se of the steopger base A^naethy Ipiperi dine .
Table 3. Preparation of i e merocyanine !¾¾ry
Figure imgf000080_0002
(78} S.4 A.{83) S*P¾ .?ø) (77) T -TBA (70) fOft
'"'toiegeu
Figure imgf000081_0001
162:48] Brf tH«ss. and solvaiseiroMisr!i of the dy s. The synthesized library dyes were screened for relative hrigkmess based o» fee fi ereseesee intensity value at the missioa peak: mMixr m (EPM) when the d e is excited at its excitation saxkuffi. Given tfist the emission pest, shapes for ike dyes were generally siralkr? the PM value .provided a str ight£or §i3& meas rement that is useful fer the s
Figure imgf000081_0002
Bttoag hydrogen beading (Hanse , Hansen Solubility Parameters: A User's
Ha dbook Secmi E ion (Hansen, C, M»s,Ed?) pp 1 -26, Chapter 1, CRC Press, Soea it toa Qff7)i Mmscu ei Dan. Kmi M (If 67f'). e 4% Polarity chara teri sties of soivests selected for brightness screening;
Figure imgf000081_0003
|,4«diox¾ne 17.5 1.8 9.0
Figure imgf000081_0004
Dyes with weak® donor groups mck as iii!o!efiine {¾ bensoxa¾:ole { Λ arid be&zotMazole: (S) tended to have the largest EP -values .Fig, I). As a hole, dyes containing the quinoiine (Q) donor group were significantly dimmer than the other dyes. The presence of EA. TBA, or SO acceptor ou s led to the highest brightness levels, regardless of ¾fe-d¾aorcom^oaii»i"# i dye. Conversely,, the f acceptor roup was associated wife ciimlaishec fetl ve brightness, r gardless of the donor group with which it was paired, %eiedaced brightness for Pz-eoata ng dyes may be atiri¾atahle to an: alternative de-exeitaiion pathway generated through the firestricted rotation of phenyl suhstitnente aroaad the G-M¾orids (Sharaiy ei a!.,. J, Am. Ck > Si , 9^ 119- 12 ( ?t) o o!eartrond was: observed beri fe solvents wens ordered based on. dipolar e racieris†ics: but dye brightness in all cases was raaxiroized a-soh¾rrts with. intermediate hydrogea bonding capacity, The a resosB 0 Intensity of several of the brightest dyes FM > f l 06} as -strOagly sofveia-depeadeat As sPlvest-depeadent changes in brigbtoess can e harnessed to report protein. activity (Gislyaai et at,. NM. Chem. BiaL ¾437-44 (2G1 !)), this was iroportaat consideration when seleeting dyes for synthesis of reactive derivatives (see
Figure imgf000082_0001
of re ction v h excited oxygen, species and .sabse¾ae»t disproporioi ioa reactions, w M also be decreased.
>2-52 SefeetioB aad syaftes!s of water soluble, e»»J«g»tat>Ie !«er«ieya»k s< Several dyes dKw dexftaordinar sad seful eonihinafions of brightness,.
•ph.otaste¾il¾ fed s lvesi-dependent fiporeseence. -Weteefois developed these ilii1¾erfcy: fcc rperatipg water :saIu li¾Bg and mmu side cbaias to generate derivatives, dmt cox h& .used fat proteip iabel g> es eoHtaising ihel doaer groap were selected teed es« ibeir pbetostability mA fcecspse the ¾emina!' dimethyl gropp of I is kno n, to assist in limiting dye aggregation thai cm result la redaeed: solubility 4' «s0eaee .(f oat&bkke et≠.. .Am, Chem. So, 123:4132- 145 (2003)).
Despite tfcee sifeaMe phoiosiabilfty of M¾, ibis dye as not selected for iurhet
Figure imgf000083_0001
ft 2S3:f of the water soluble* eo¾u aiarfe: isions of the selected, dyes begaP wil¾ -reaction of l«broisspti3 iam&e wii!x propane m m to give
lm»a propyl)aminopi:Opan.e>-! -^lib ic acid i% The doaorheterocyeie wis tbeii quatenaze with eompoimd 3 apd subse ently coupled with th acceptor & l ether in the presence of e dmaeetic arihydride and m um acetate using a &m~ pot procedure (Taafe dna et t, BiQsmjtigafe Chm* l 1344-1348: (2Q07))..
ReSpxmg ia MeOH'fa? 3 ¾. in the presence: of an exeess of Nal gave mexoeyaaiaes cotita ug iodoaeeCamide :(I4A) io proteip labeling. Consistent with the
·6 ϊή©ή¾ξ^ ¾ mgfocyaQines-.us§i ¾ oi .la (OutjaS si el, Nat Chern. Biol 7:437-444 (¾G11))S these ep3¾ugatb!e dyes wee pamed »ie <>6¾ e oSI, ¾ero€2> : and ifi*r«S7t¾fete tj. Physical eoastants w©¾ obtaiaed fer both the reaPtivs derivatives aedi r bea¾ereaptsetl¾Mi0l addpetat at e!in¾¾aie effects of iodine on qiiappim yield, aftd better .mmic dye attached te protein:. Scftemt .1, S afcsis of water so!iifele, c«m¾gatable raeroe aaiftes.
SA
Figure imgf000084_0001
Figure imgf000084_0002
dysmm solvent
end r*
eiTsisSisi msnm SI DMSG M7 I26G00 46S S 620
M¾OB Ο3Ο0β 2060 SS
BuQH 0.16 132908: 21120 593 6; 6
¾o lOS GO 11 505 60g
D SO 0.3? i 00000 37035 570 595
MeOH 0.07 ί 67000 11690 565 58 ¾
BiiOH 0.17 544080 mm 570 592
«20 0.03 m 565 S6
21 PMSO 0.2G m 220OQ sn 615
MeOH 0.09 140000 590 60¾
BuOK 0.18 i 000 2020 594 61
0.10 M ® 3600 585 608
PMSO 0.34 OO0O 47609 597 620
MeOH 0.06 107860: 6420 62Q
BaOe 0.2! 1 ? 000 23310 •622
HjG Θ...02 s oso 1620 592 616-
- .DMSO 066 24000 315 6IS
SO liSOOO .570 594
- DMSQ 0. 1 ® 58220 5§6 614
- M Q 6 7 105000 2B3S $90 620
BuOH 854 I3400S: 72000 587 61S
OcQH 0.98 125000 123000 53? 61?
- MeOH O.O 143000 1140· 5 515 a(¾ias¾a5i ieMof flaersseejic, error ± 10%, 'Molar CQfffSoktii. error ά: 10%, 'Values BQ as reported w t$Zf ' M&& &>r csmpsrisos |see B!scessica ad Cssclusfcms), Q®M&mi%c$ im feeterocycles;
PS4]. O: Yield - 51%. Ή NMB. (400 MHz, 0MSO) 88.17 - 8.0S (i¾ .2H), 7.83 7,74 (¾ ¾)s 4,08 (s, 3H), 3.0/ (s, 3H); ¾ΝΜ¾{Ι!)0 MH¾ DMSO) § 169,0,. !47.25130.3, 12S. 127,6, 1UA.112.8.32,6, 13.4.
102551 S: Y¾= 93%. l NMR (400 MB¾ DMSO) & 8,48 - 8.42 (r¾ IB).8.29 {4 J- 8.4 H¾ li¾ 7,89 (ddd, J- 8,5, 7.3, U 1¾ Π¾ 7.80 (ddd.J- 8J, 7,3, .0 ¾ 1H),4.21 (s,.3H), 3,18■ (s, 31): ¾KMR (100 MH¾ DlViSO) δ 177.2, 41.5, 129,2, 128,6, 128,0, 124.5, 116.7, 36,3, 17.2,
1H2S6] TI>: Yield - 98%. ½ NM (400 MH¾, DMSO} § 4,17 (s, 3.% 3.02 (s, 3H}52,81 (¾ 3!¾ ¾ K RCMO MH , D SC^ 81.74.4, 166.7, 41,4339,5, 15.9, 14.9, [02S7| Q: Yield 95%. ¾ 14MR (400 MB¾ DMSO) δ 9.37 (d, J- 6 J 1¾ !H), E,5« - 8.43 (m,.¾.B. ? (d¾d, -8.7, 7.0, 1,4 ifc 1H), 8,12-8,00 (m, 2H) 4,58 (s, 3H}33,01 (s} M);: ¾ N R (100 ¾ PMSO)§ 15S.I, 148,9, 137.6, 134,9, 129,6,. 1284, 126,7, !22,4S 119.5, 45,0, 39,5, 19.6,
Accepto e ol et!sersj
fft258| ΒΛ: Ykld ** 61%. ¾ N. R. (400 MHz, CDC¾) 58.17 - 7,97 (n\ 1¾ 7,58- 7.38 (m, 2H¾ 3.98 (in, % 3.92 (s, 3H), 1.22 (m, 6¾ °C MR (I0O Ml¾ CO€l3)S 171,4,. !:62.45162.2, 157,5, 151.0, Ώ®, 105.8..58,7* 37,2, 36.6, 13.6, 13,5. f 0259 j SO■ Yield « 73% , Spedtea matched fee. reviousl reported
{To »&kine^¥-s Am. ChemS&c, 125:4:132 2003)), ■ K HMR (400 MH¾, CDClj) S 8.04 (d,/= 7,6 ί¾ 1H), 7.9B (d, J- 8.0 H¾ 1H), 7,86 (ΐ,. - 7.6 Hz., 1¾ 7,78 ίΐ, J -7,6H¾ 111.}.7,67 Id, J = 12.S!k, Π-ί), 7.45 (d, J - 12,0 ¾, ffi), 6,45 (t5:J- 12.4 0z, !!% 3.96 (¾ !H),
[0260] TBA: Yield - 55%, :iH HM¾ (400 MHz,. CDC¾ 584.1 (¾ J~ 1 .8 Hz, IH), 7,60- 7.45 to, 214).4.51 (q, /- 7.0 H¾ 41% 3.96 ¾31¾ 1.28 (d¾ 12,8, 6.5 !¾ 6B); 13C HMR (100 Mlfe, CDC14817:9.3,.172,6, 161.0.160.4, 159,2, ! ! 1,5, 106,7, 77.2, 59,1, 3,8, 43,2, 12,7, 12,6,
im&l] J¾: Yield - 70%. H NMR (400 MI¾ CDCb} 87,8S - 7,67 (m, 1 H), 7.50 (i 12,3 ¾ 1H), 7,44 7.27 m, H)S 7,20 -7,08 (x¾ 2¾ 3,91 (s, 311); "CH S (100 MH¾ CDC¾)S 170,¾ 164.4 (2(¾ 152.1, 137.2 (2€), 129.1, 129,0 (2C), J 26,3, 126.2, 122.4122.3, i ! LO, 104.1, 58.?. lbray dyes:
[0262 trfcA; Yield - 89%. ¾NMR (300 MHz, :BMSO}88.12 (di, J- 13. L 6,5 Hzs 2H), 7.71 ( J~X ®Z, IK), 7.5t(¾ J- 6.8 I¾ I 7,4.0 -7.21 (ia,2H), 7.21 ~1M (m, IB), 6.13 (d,.J- 13,5 H¾ IH), 3,84 (q, J- 6,8 Hz, 3H), 3.51.s, 3H), 1.62 (s, 6H), 1.10 tc, §.8, 1,6 Hz, 6H). MS-ESI 394,1 f|M + H]'5' requies 394.2). I026S] KSO: Yield ^ 97%. ¾ NMR (400 M¾ CIX¾) 5 S.00 id. J - 7,4 H¾ IB), 7,94 |d, 7- 7.4 ¾, IH), 7,83 -7737 (m, 4H)S 7.34-7.26 (ra, 2H), 7.11 (t, 7- 7.5 H¾ IH)S 6,92 (df J- 7.9 Hz, IHJ» { J- 13.2 IH), 5,89 <ds J- 12.8 H . HI), 3,46···· 331 (%,3¾ L62(s, 6ΪΠ. MS-ESI .^¾.392-0 ¾ ÷ H rs iires 392,1). f§264 I-tB A: Yield * 93%. Ί1 NMR (400 MH¾ CDC!j) 58.17 ~ S.04 (m, 111), 7.95 - 7.81 (m, 2H)S 736 - 7.2S (s¾ 2H), 7.16 (di? J- 7.5, 0.7 B¾ H), 6.97 (47- 7.9 Hz, IK), 6.05 - 5.90 (nu 1¾4.58 C¾J- 6,9 ]¾ 41% 3.38 is, 3¾ 1.62 (s, 6¾ 1.38- L26 (¾ S-ESI wfe-41(U {[Αΐ^-Ή]* quires 10 ).
[ 2551 I-Pz: Yield. - 87%. Ή NMR 6400 MHz, CDi¾) δ 7,76 (dd,,7 - 25,0, 13,0 HE, 2K), 7,57 (1 J 3,1 Hzs IH), 7.48 - 7.40 <m, 4H), 7,33 - 7,26 (m? 6H), 7.14 - 7,D7 (M, 3H¾ 6.91 {47- 7,8 Hz, IB), $S7 (47∞: 13,0 !¾ IH), 3.36 (¾ 3.H), 1.65 ¾ 6ii), MS-ES1 m/z 462.1 tf. + Hf i¾ wKS 462,2).
[0266] -Phf; Yield - 59%, fH NMR (400 MH¾ DMSO) § 7. 1 ft J - 12.5 E¼ IH), 7,70 - 7,56 (m, 6H57.55 - 7.3.2 (in, 4H), 6.10 {¾, J^ 13,5 Hz, 1H); 3.72 <s? 3H), S« .¾& 130.1 (fM + Hf requires 330.1).
(§267] MA; Yield - 29%. ¾ MMR (30 MIR CDCh) δ 8.08 (d,7- 12.6 H , IH), 7.85 (id, 7 25.3, 3,1 Hz, 2H), 7,40 <d, 7- 8.0 Hz.1¾ 7,35 - 7.24 (m, 2H), 713 - 7.06 (·«, 1H¾.5.4S (d,J- 1Ϊ, Μ¾ IH), 4.Ο2{%7-7.0, 1.3 ¾ 41-1), 3,53 (s, 3Ή)> ί 25 (dt, 7 -7.0, 3.0 Hz, JH). MS-HSi m/t 3611.C{ :- H]f'i¾q«kes 368.2), 026S) O-SO: Yield - 29%.41 NMR (400 MHz, DMSO) δ 8,01 ¾ J- 13 ,0 Hz, 19}, 7,92. J- 6,5 H¾ IH), 7.85 -7,76 (m, 3B), 7,72 (d,J- g;2 H¾ IH), 7.69- 7,57 (m, '2H)v?,4g ft J- 7.2 B¾.1H)S 7,4! & 7- 7,2 ¾ HI), 6.55 (t, /- 13.1 Hz5 I H)56.26 id, J - 13,5 H¾ li¾ 3.75 ( , 3H), MS-BSI z 366.1 ([M + Hf fequires; 366.1). S6
[$M ] O- BAr Yield %5 , ¾ N M (400 MHz, I5MSO) 8 $.04 7.9$ (m,
1 H), 7.83·· 7.72 (m, 4H)S 733 (¾ J - 7. , 1,3 Hz. H¾ 7.48 (di, J - 7.6, L2 Hz, 1H)S 6, 2 (d, J- 13.8 ¾ 1¾ 4,4 (q, 6.9 H¾ 0), 3.83 (s, 3H}? 1.17 ft, J - 6.9 H¾ 6H). MS-ESI A 384,1 ([M + .Hf s^wres.384,1).
[0270] 0«P ¾: Yield -78%.1H NMR (400 MH¾ C£K¾} S 7.81 (1 -7 - 12.8 H¾ ffi)t 7.66 (¾ 13.2 ¾ IH 731 ¾ J- 13.2 Hz, 1H)57.46- 7,36 (m, SB), 7.31 - 7.21 {x&, 6H), 732-73)5 irn, 3H)554265,../- 1:231¾ Hi), 3.51 (s;3B). MS-ES1
437.0 ({ Iff requires 436.2}>
[0271] Si¾l: Yield - 72%. ¾ M (400 MHz, DMIQ390 °C) δ 7,87 (d, J~
Figure imgf000087_0001
.1H}57,94 - 713 :(¾ 4H), 7.63 ( - ,6 HzJH), 7.51 ( J % Bz, lH)i ,82(dJ J - 13.5 Hz, iHjS: 4.43 (q,J~ 6.8 Hz.. ¾ 3,946;, 3H), 1 /* O 1¾ 6H), MS- ESI ®¾ 400,1 f[M Hf requires 400.1),
[0275] S-F¾: Yield. 70%. ¾ NM (4δ.0 Ηζ, CD(¾}67.66 (d; J« 12.9 Hz, 1H), 7.55 (t J - 1,1,3 Hz, 2H), 7.46 - ,37 (ro, 6H), 7,31 - 7.22 (m, 5H)57.18 (A 3= 8.0 H¾ Ii¾ 743 ~ 7.04 (m52¾ 6.02 $, ~ 12.3 Hz, 1!¾ 3.61 (s, 3H), MS-ESI ^ 452,0 (( -f Β req ires 452.1).
27SJ TB~V t. Yield - 82%, 1H K R { 00: M¾ DMSO) S 7.68 - 7.40 (m, 5¾ 738 (i J= S..4 ¾ 2K), 632 (d; J «· 123 Hz, 1¾ 334 (s, 3B)/237 (s, 3B). MS-ESI 31 L0 ijM ÷ H]f rs um.31 L), P277| TD-BA: Yield - 61%. Ή NMR (40§Μί¼ DMSO) & 7,85 - 7.70 (H¾
1H); 7.67 -7.47 (m, 2H}} 644 6.19 (m, 1I¾ 3 M (s, 3H), 3,82 (¾ J - 6.9 i¾ 4H),
2.58 (s, 3¾ 1.07 {ts J- 6.9 H¾ 6H)» MS-ESI Μ&Μ9Λ <[M + Hf requires 349.1).
[02781 TB-S0: Yk - 77%. ¾ MMR (400 ΜΒ¾ DMSO) 87.88 id, J - 6.2 B¾ :1B¾ 7.84 -7773 G¾ 3H), 7.68 ft J- 12.6 ¾ Hfc 7.56 (d,.J~ 13.§ ¾ IB), 6,45 ¾ j= 14.0 H¾ 2B)53,88 (s? 3H),.2,60 (s, 3H), MS-BSI m/z 347.0 £[ Hf requites .347.0),
j027y TIV!BA: Yield - 7G9 lil H S. (400 Mf¾ KMSO) 67.76 - 7,55 (m> m, 468 - 48 IB), 4.42 (% 6.9 ¾S,4H¾ 3,96 (s, 3M)? 2.6 (s, 3H), 1.16 (i, J- 6.9 H¾, )H). MS -ESI JM Z 365.0 ([M + Hf requires 365.1).
TOO] » : Yield - 53%, ¾ NMR (400 MHz, CDC½) S 7.58 (d> J - 13.0 H¾ III), 7,50 - 7.40 (mx H)S 731 - 7.24 (m, 4H), 7.1.5 (d, J= 12.7 ¾ 2R)r 7.11 - 7.05 (m, 2H), 5.75 (4, J« 12.7 :¾ ]¾ 3.69 (s, 3E¾ 2.49 (s, 3H), MS-ESI 417.1 (j + Hf requires .417.1),
10281] Q-F ; Yield ~ 56%, 1H NMR.: (40Q: MHz, DMSO) § 8.5701 J - 8,5 Hz, l.H)s 8,27 (d; J- 7.3 Hz, !¾' 8,14 <, J- 12.01k, !¾, 7,91 (m, 2H), 7,70 ~- 7,47 ( . m, 7.35 (si, J™ 13.8 ,Hz, ¾ 7,96 (d, 13.6 i¾. IH), 4,05 s, 3H). MS-lSl 340,1 ([M ··· .11]" requires 340,1).
282I Q~BA: Yield - 45%. !il R (400 ¾ CDCh) δ 8,0 (¾ ~ 8.6 Hz, 13-E 8.04 id, J - 13.2 H¾. lK% 7,93 (r, </- 12,8 Hz, ill), 7.79 (t, ~ 12.7 Hz, IB), 7.69 (ddd, J~ 8,5, 7,1, 1.4 Hz, 1H)57.46 (ddi J 8,3, 7,1, 1,1 Hz, IH), 7.42 - 7.38 (¾ !H),.7,28 (d,J- 7,4 ¾, HI), 6.95 £ iH)s 6.75 (d,j- 12.8 B¾ 1%
4,02 (¾ J= 7.0 ¾ 4B), 3.82 (s: 3H), 1.24 (d J - 7.3, 6.8 H¾ 6H). MS-ESI m.l.(| +H quires 378,2).,
{mm} Q 0; Yield - 20%, ¾ NMR (400 MHz, DMSO) 88.65 (d, 8,7 Hz, IH), 8.46 (d J 7,0 Hz, 1H)58,25 --·· 8.15 (i H), g.04 - 7,94 (:¾, 2|¾ 7.86 - 7.82 (m, 1¾ 7.79 - 7,67 (m, 5 H), 7.54·· 7.34 , - 1), 7.19 (d, J- 14.0 H¾ IH), 6.77 ~, 6.51 (m, H), 4,1 (8, 3H). S- SI ??Y¾ 3740 ([M Y¾ requires 376,1).
] Q-TBA; Yield = 69%. ¾ NMR (400 MHz, DMSO) § 8.72 ~ 8.65 (n¾ 2M), 8, 1 - 8.10 :(m, 2H), 8,08 ~ 8.0.0 (m} 2H), 7,86 ~ 7,76 (m, 2U% 7.63 (d5 J- 14.4 His, 7.26 /d,Y- 14 H^JH), 4,45 (%J- 7 Hz, 4H), 4.24 (s? 3H), 1,17 ( J- 6.9 H , 6H). MS-ES! m2394.) (i * H] xeqwlres 394.2), 1$¾85| Q »«: Yield ^ 39%, ¾ H (400 MHz, DM80) δ M id, J - 8.7 Hz, IB), t, « (d - ?>0 Hz, IH), g.32 - 8.11 (¾J* 14,0, 1 i.2 H¾ 10), 7.99 (q, J- S j Hzs 2H), 7,79 (d, J= 7,2 Hz, 1% 7.71 (t,J- 7.3 Hz, I B), 7.49 - 7.21 (ms 10H), 7,fS - 6, 6 ¾s 3M}, 14 {§, 3H MS-ESI 446,2 ( M + H reqnhes 446,2).
Intermediat s for eo¾]¾gaf»l}fe dye syafems?:
10286} INS: ¾ MR (400 MHz, D20) 57,86 - 7,7S (m, 2% 7,73 - 7.65 (m, 2H), 4,80 (¾ 3¾), 4.67 - 4,57 (iH, 2H)S 3,40 - 332 <m5 2H), 3.32,- 25 (m, 2Ρ¾ 3.05 (t, J- 7,3 H¾ H), 2.91 - 8 ¾, HI), 2,50 - 2.35 (&¾ 2H > 2,25 - 24 1 (n¾ 211), 1.61 (s, 6H¾ ¾WR (10O MH¾f !¾0) § 197,7, 141. , 140.5, 130,0, 129,2, 123 5, 117.7, 114,8, 114.6, 54,6, 47.7S 46A 44,6, 44.¾ 23.§f 21.6, 21.1 ,
{0287} Cc!iijugates of the reaotive dyes with the€d©42 binding domain (CBD) of Wiskoit Aidriek Syndrome Protein (WASP) were p epared' arid evaluated, for their ability to ts the aetiva d, ©TP- ound conformation of Cdo42, As s o-wn. m ■p iom s di asing dye^abekd CfiD as a biosensor if e Cd¾42 activity in wire aud m vivo (n aM M al, Scimee i£ 5:1615-1619 (2004)), CBD binds sefcctively to the activated, CfTP-bound conformation of C c42, Upon binding, a solvent-sensitive dye 05 CBD c¾n respond to a change in environment as it is htoogiii near Cdc42.
288J All of the .newly $ re ared dyes exhibited an .increase; in finorescenoe Intensity ©a blndnig to activated Cde42 in vi ,, with me o60 showing a remarkable 14.7 ibid increase (Fig. 5a), The mero61 a»d mero62 dyes exhibited the greatest absolute brightness at sainratkg levels of C c42 (Fig, .5b) but their brightness changes upon bmding Cdc42 mm smaller relative to mer 69 and. »xer«87 dyes due to their much higher initial brightness in aqueous solution (Table.6). Biosensors based on these new dyes showed significant improvements in. both brightness and extent of change iipon. Cds42 binding relative to ibe s»ero221-based biosensor
Figure imgf000089_0001
constant (¾ values calculated for the binding of the meroCBD constructs to Cdc42 ranged mm i ± 62 nM (r&era&CBX to lM & 40 nM (mseroS^CBD) (Fig.7), .These v lues are sirpilar to that of the previously; reported ¾ for r022:l-CBD (150 ± SO nM) fNaihsai et ah, Samee 5ft5:1615 61 #004)) and are slightly higher thau that reported for the unlabeled CBD fragment (7? sfc 9 nM). (Rudolph et a *, J. BioL Chern. 2?3rl806? SO?6 (1998)), This .relatively modest diSermee indicates that the presence of the dye o s not so¾stafii¾ally ert i¾ the overall bln4kg. affinity. a le d: Structure and phoioph sieai properties of Bieto221<
Figure imgf000090_0001
irnvmi ■QY
"VVater" 599 i 830 1 143S0G i 0.00- eOH I 801 ί 634 138G0Q i 0,01 140Q uOH I ..60? ! 639 16000.0 I 0.06 9000
f¾189] To tes the dyes in live cell imagin applicad O , C e42 biosensors were prepared with each of the four new cory ugaiabte dyes as well as the previously reported ri¾er«22i» For these biosensors, CBB was fased to Cerulean fluorescent protein lot r do image analysis and. mutation 27! C was Included for conj g tion- of the dye (Hedgson et a!,, . Methods Emytnol :140~156 (2006); Hodgson et <¾> Citrr. Pwtoc. Ceil. Dial pp. 1-26, Chapter 14, Unit 14,1 I (2010)). The biosensors were ^kf0injec e4¾to-¾oi^^?i¾b?yoia!?' fibroblasts \ MEFs) rid used to examine the Cdc42 activity in eonsfitptive cell prptrusip is : refraetioos (Fig, B The biosensors based on mvtofii,. a*ero62, and m o^ each reported endogeno s: Cit42 activation during proimsron at the cell's leading edge, consistent with previously reports of Cdc42 hehaylor 1%, 8 , Although the M£r06§ bl seo&or had shown the greatest flo escence change upon activation mm, lis signal bleached too rapidly to provide Gdc42 data m viw without rising concentrations thai clearly caused cell coniyactioij and otherwise affected cell behavior. %9 1 For iaagffig, if is eM to deling dye fcri ghhiess as ¾je product of quaoi B yield and extinctiea eoefiicie«t (QY x ε), as this gauges tks total light output per unit lilua instion intensity. By this measure, all: dyes .except m&m Z showed maxim m brightness values comparable to those of ftaorophoKOs irequsntiy used as intracellular mattes kit which have little soivent-depeno½td fluorescence (e.g. Cy5, QY x e - 67500.( ujnn dar■ Λ, Biocott ug CMWL 4Λ&5 1 1 (1993)}, madamine - 61000 (Leyt s aL Bi h . J 2 % (i }% m eGFP **33O'O0 (Patterson ei: ttl, Biop ys, J. 75:2782-2790 fl997)¾>
(02MJ It is difficult to use our solvent- dependent fiuoreseerice data to: predict how ea^h d e wil behave in a gives: application. The hrlghtpess of these dyes o proteins will depend on the ..specific protein envir nmen , and. opttrftigation of petitioning is usually requited to place the Soorophore in m environment mhiwckiug a hydrophobic soly ml where roost dyes: are at their brightest The fluorescence res onse, o msrocyanmes is oonrplex, involving interplay of multiple ihciors including solvent viscosity, local. solverEpo!arity, arid hydrogen. bonding of specific dye he eroatoiss (Han et l CmmP ^CMe 4 Μ «Ιύ94 iWmj L et L, J PhyS. Cke . A, 108:3545-355$ (2®0 );. TouicKkjme at , Pftys* Chen A, ili; 10849-10860 (2007)), Physical constants weps determined for d-mereaptoethar!ei add cte of the dyes, to -.mimic cysteine; eenf agates and eii»«safe the quenching effects of iodine CTo≠ a et a J, Am. Chem- So i2J: 4132- 145 (2003¾> However even these brightness values ma not be as large as hen the dye is in a very 'hydrophobic protein environment. The reactive^ dyes could t he dissolve m ' Irigfciy nonpolar solvents, u the parei¾ eornpound I»80 s been reported to reach a brightness value (QY x έ ) of 123,000 m octauol, which is mong the largest values reported for organic dyes (Lavis et l,ACS Chem. mi 142455 (20083).
f029¾ Al hou h: the solvent, depands ee S udies of the screening library can be useful In guiding the selectio of fluorophores likely to show a strong response to changes k protein ciiv roiimeBt, only direct testing can ulthiraiely determine which, dye will be best &r any given biosensor application. The dyes showed ddfrerest trends ha their response to solvent, implying that cash may sfeow mnque behavior on proteins. Mero62 achieved its i imnrrt brightness m btttanoi, unlike ail the other reactive: dyesf which achieved rnaximprn. brightness InD SO. Fluorescence: in water ^ was. not .measured in. our Initial screening assay becan.se side chains for water solubilization had not bee added to the dyes,. For the reactive dye derivatives,. QY x a values in water were very lo , consistent with previous studies indicating that biosensor response Is dtre to increased shielding from water when biosensors Interact with their targets (Han et al , ChemFt&tGtem 4; 10844094 (2003); Liu: et al , J, Phys. Chem, A. 108:3545^555 (2004); Toutehkme &i aL Λ Chem, A.- Π1ύΟΜ9~ iOSoO (2007)), For the nonreactive versions of compounds that were later converted to reactive forms, solvent-dependent differences in brightness ranged .from three to twenty fold. Fa the reactive versions of these same dyes, mik values in ater Included, brightness varied from seven to twenty-nine fold. The brightness of m ro 0 was low in water that the extent of increase could not be precisel quantified. However, r¾ero6 was among the brightest dyes la organic solvents. Indicating: a potential ibr very l rge changes when it Is used In favorable protein, environments. Hone of the dyes showed a significant pfl-dependent chan ge in iinoresesnee intensity when tested from pFI 5,0 to 8.0 for 6 hours (Fig, 9).
0293) There were several parent fluprophorss that showed not only changes in ilnareBoence intensity, but also changes in excitation and emission maxima. Such fiuorophores are very valuable: for ratio imaging: applications, a the can be used without adding a sectrad fluorophore to the biosensor- however these dyes were not pursued because of their poor photostabiiity.. Additional structural modifications aimed at. reducing the suseeptibiiity of these dyes to photodegradative attack b singlet oxygen may make then? useful tor protein applications in the iianre (Griebeh liar. BummgBg. P ys Chem. ( mO)},
|!¾94] The potential of the new dyes I biosensor applications was demonstrated: by making new versions of dye-based€dc42 biosensors. When substituted for the dye mere22I, 'which, was utilized in a previously published biosensor that had proveu successf l » s , each of the new dyes produced a several fold improvement hrlglihres or change in Tt mreseeoee intensity upomproiem binding- Dyes merolst ieroS2s and nieraS? proved to be superior to the original meroSSI , bat mero60 was difficult to use because of its more rapid photob leaching, 'Oris dye underwent the largest fluorescence change upon protein soil vation, so still ma prove valuable in ;¾
•Sc eening assays and other .ap lication ¾gt requiring; repeated measurements over time.
f §295] In conclusion, the systematic variation of donor and. acceptor groups on. meroeyanme liuorophonm ennpled with the synthesis of reactive and water soluble derivatives., has led to a set o£« w dyes with proven utility for biosensor applications requiring solvent sensitive fluorescence. The combined propertie of these dyes should enable experimentalists to develop biosensors that can effectively report endogenous protein activity al low intracellular biosensor concentrations, thereby reducing perturbation of normal cell physiology. Biosensors based n these dyes may be used to enhance the sensitivity of biophysical measurements across a range ox potential applications In which ¾ dye is directly conjugated to a. protein or peptide to report conxormahonal eharsges Of hgand interactions,
8296| Cel. Culture, MIB 313 mouse embryonic fibroblasts (MEF) were maintained in 10% C(¼ at '3 C in Dulheceo's modified Eagle's medium ( MEM, CMigro) with im fetal bo vine serum (HyCione., Thermo Scle rdfic.) nd; 2 mM GkhaMax (Slbco, Life Technologies). The ceils were plated on coverslips coated iii fibroneetiti (Sigma-Aldrich) overnight,.
fi¾i7] age Aeqm¾tlosu Li ve ceil imaging was. carried out an an Olympus IX l microscope with a: UPLFLN GX oil objective £ A 1.3} and mercury lam excitation (103 W HBO bulb), Filters were 545/50 for excitation and 630/45 for em ssion. 'Excitation was through a HI) 2,1) (1%T). f&er, Images were acquired with a Cooisnap ES2 camera (Photometries) with a Sony d',45 x 6,45 μ, /pixei chip using 2 x 2 binning. All image acquisition, processing,, and analysis was- earned out ith Metauinrph.software, Mage : were shade corrected, background subtracted, and iinsarly contrast stretched to reveal subcellular features as previously escri e .*^ { 9 } Ckeriristry> Alanine methyl ester hydrochloride {S gma- Aldric'h} and maionaide!iyde dlauiilde hydrochloride (TCI America) were purchased and used as received, 1¾e fallowi g compounds were prepared according to literature
procedures: iJ-diethylharhitiuic acid, 1 ^^^S hetr me hyJi do!ium iodide, 1 ,3-
Figure imgf000094_0001
chr matography (TLC) on ;EM Science pre-coated glass-backed plates (silica, gel 60 A Fas*, 0<25 ana thickness), flash ebjotnatograp ry was carried out wit silica gel 60 A (230 * 400 nresh and automated. eb mato raphy was performed on an Iseo
Combiilash Companion, Unless otherwise slated, organic extracts were dried ove commerciall av ilable magnesium, sulfate and he solvents were removed by rotary eva o;rad n. Hi and l3C NMR spectra were recorded on either a Vari&¾ !nova 400 MHz In deuieraied chloroform ·{€;ø€¾) unless: otherwise n«ted and referenced, to the residual solvent peak (for CD<¾ ^ 1H § 7.26 pprm 13 C δ 77,23 ppm for D SO-d* ~ ¾ S 2.50 ppr¾ ¾ δ 39,51 &p ). Mass spectra were obtained on an Hewlett-Packard 1! 00 high-performance liquid cta>matograpfe equipped with a I I 0Q ioass-seleetiyc detector (MS-ESI), IJV -visible spectra w re obtaiiieci wi h a Hew!ett-Paefcard 8453 diode array spectrophotometer, Emission and excitation spectra, were obtained ¾s¼g a. Spex. PJuorolog spectrofluo ornster at 23*0. AO operations with dyes were performed under dim light, Fhotorsaetion were carried oni using a E yonet RM ^ 600 equipped with 350 nm; bulbs, llgh perfemsnec liquid chromatogra hy was performed on a Shi adzu Prominence system ith a Vydae 21 S. F152022 column (15-20 &-| υ1κ&|»&, 300-vL 250 X 22 nam) in. the waiermie hanol or aoei^niirile/T A mktnre: Sol vent A ;(wa e 95 parts, organic solven 5 parts, TFA «.05%), Solvent B (water 5 parts, organic solvent 95 parts, TFA (1,05 %). Standard gradient tor separation of final products is 1.0% Solvent B, 30 mis 90 solvent B, total rati 45 rear
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
|iS99 Metfeyt 3-i3«et¾yI¾r<e¾¾)pr<pa»o:ate (SI), Alanine methyl ester hydrochlori e (34.9 g.250 nnnoJ) was added to a f!arae dried flask. The flask was sealed, evae¾aied,: a»i flushed wife argon and G¾C1¾ (350 m was added via eamuda, Tdeifeyiamirie ( 8,3 mt:s 275 mrrrol} was added nd the reaction was: stirred at room temp for 45 min, The flask was cooled ¼ an ice bath and ethyl !s oyanate (20.8 L, 262,5 m aol) was added quickly dropwise. The reaction was allowed to gradually equilibrate to room temp. Ate stirring tor 16 % the reaction was quenched with half-saturate aq, N¾GI (400 -mL)> The organic layer was separated rid the aq. layer was further extracted wiua C%C¾ (200 mL X 2), The organic layers were combined, ashsfd with -water and ¾ dried with MgS¾5 fih¾reds and
concentrated. The resulting solid was further dried under high vacuum to give 25.2 g (58%) white solid suitable for use without further purification, ¾ NMR. (400 MHz, CdX¾) 6" 5,24 (b % 4,82 (bs, ¾ 3,66 3f¾3.44 (¾/- 6.0 R¾ 21¾ 3,2:0 - 3-12 (ni, 2H)S 2,52 (h J- 6,0 Hfc, 2I¾ 1.10 ¾ J- 7.2 Hz, 3H), °C NMR (! 00 MHz, CDCh) δ 173.6, 15S.4, 51 36.0? 35.4, 4,9, 15,6. MS-ES!»¾ 197.0 i[M 1- Naf requires 197.1).
:[0301¾ Mettiyl:3-{3~e^^
yi) rnpaniiate $¾). Compound Si (24,9 g5143 itrmol) and malonic acid 15,6 g, 1 SO nirnoi) were suspended in glacial AeOH (200 raL), 'he mixture was heated to 60 ¾ and stirred at that mp fo IQ rxtin. e^D (53 ,9 ml,, 572 rniftol) was then added in bulk: and the reaction Was heated further to 100 dC and sdited or 1 ,5 h, The reaction mbqrure was concentrated ami set at room temp open to air . The resulting crystals ere ie-c saUKed from 3:1 hexanes/BtOAc (50 ml,), The re-crystallized solid: was iirer and washed wnd ice cold E¾0 (TOO urL A 2} to gAe 23.9 g (69%) hite solid suitable for use la the next step. ¾ NMR (400 MHz, CDf.¾)h 4,18 (tf J~ IX 2H), 3,92 (¾ /" 7.2 !fe, 2H13.67 ¾ 3H), 3,65 2ft), 2,62 it ~ 7,2 f¾ 2H), 1,20 it J -7, i¾3H), ¾NMI :I00Ml¾eDC¾)g: i7).5; 164.7,, 164.4, 151.2, 52,1, 39.9, 37,9, 57.6, 32.5, 1 .4 S-BSI mz 40.9 ((M - ΗΓ requires 241.1).
|03¾1]. A3'¾hyf-¾ f6 no^
Compound S2 ( D g, 34. mraol) was diluted in THF (70 ml) and DI (35 ml) was added, UDe¾Q (5,75 g, 4.00 eq) was added and the re action was stirred at room ¾mp for 16 h, The Sas wss set in Mi ice bath and the reaction wss quenehed. with.6 N a , HQ (27 L) to a pH of - . The rcactioa mxtre ws concentrated and the restating precipitate was alered, washed with DI $2x) d ice chilled E¾0 (2x), The product w¾s dried imder vacuum tes give 7,30 g (93 ½) white solid that required no further purification. ¾ H R.(400 MHz» DMSO~E¾) 12.36 (bs, IH), 3. , (h J 7.81¾ 2H), ,75 (q, J - 72 Hz, 2j¾ 3.7 ! (s, 2¾ 2.45 (t, ,/ = 7..8 I¾ :2C¾ 1.08 (s J**12 ¾, 3H). ¾NMR (100 MHz, DMSO- } § 172.2, 165,6, 165,4, 151.4, 40 ; :.3:6,7, 36,1732.0, .119. MS-ESt 226.8 (031 - H; equire 227. j). 13621 3n(3~e%l-: ^^
eaylaeeisimdo}®!^ aekl (7).
Compound (0,500 g.2.1 nimoi), .7(3^ et ^^^
hydrochloride (0,624 gf 2,41 mrna¾ and: sodioan acetate (0, 108 g, 2,41 nrmot] were added together to an oven-dried flask. The H ask was sealed, evacuated, and argon Hushed and aeehe anhydride (2.2 mlT) was added. The flask was added to a preheated oil hath at 135 *C and stirred for 1 h. The reaction naxarre was cooled to room iernp and diluted with diohioromei ane, "J'¾e organic layer was washed with sataraed aodinni bicarbonate, saturated ammonium chloride, and brine, and dried with sodium sulfate. The organic layer was filtered, concentrated, mid loaded seat onto a 120 g silic column, The eohnrffi was elated with 0- 75% tOAe in exa es over 25 rnin. Main product iraedons were combined and concentrated to give 0,387 g (44%) yellow •solid as m a proximate 1 :.l mixture of S and Isom rs. Isomer A: 1HNMR.(4(K) MH¾:DMSO-i¾) h 12,28 (hs, I¾ B (d,J - 13.6 fe, i¾ 8.2 (d, 7 - 12,4¾ 1B77.65 - 7.53 ( , 311), 7.44 ~ 7.36 (rn, 211), 6.70 (1, J- 13,0 H¾ 37), 5.86 it. J~ 7,8 H% 2H), 3.80: (q, J- 7.2 I¾ 2E) 2.37 7,8 H¾ 2I¾ 2.09 (s, 3H), 1.0? (i - 7.0Hz,3H). °C MR(i00MH¾D SO-4i)5172,3, 169.9, 161.5, 161.0, 5?,¾ 150,3, 137.8, 130.3, 129,5, 128.¾ 110.5, 309,0, 37.0, 36.2, 35.5,32.2, 233, 13,0. Isomer B; 71 NMR (400 V!I- , E i O^ S 12,28 (hs, IB), 8.67 (& J~ 13.6 Hz, IH), g,22 (d, 7- 12.4 ¾ IH).775 -7,53 fa 3 it), 7.44 ·~7.3δ(κι: 213, 671 (t, 13.0 Hz, \ HT 3.99 it.../- 7.8 ¾ 2H), 3.68 (¾ 6.8 I¾ 2H); 2.46 (t, 7- 7,8 Hz;, 2H), 2.09 (s, 3H 0,9:9 (ts .7- 7.0 H¾ 3H). ¾ NMR [100 Ml¾ DMSO- ) §· 172.2, 169,9, 161.43161.1, 157,2, 150,3, 137,8, 130.3, 129,6, 128,2, 110.5, 109,0, 37,0, 36,3, 35.5.32.323.3133. MS7ESI m 400.0 ([M* - H7 requires 400.2). Φ).2i3 ^f!^etb i-5-e:ate>¾ 0k «e: (11)0 g, 4.92 m oY) and htomopapknlc sc ( .828 g, 5.41 ffimol) were added to a:iiine-dried 2~neeked flask fitted with ¾ condenser and rubber septum. The flask was sealed, e acuated, and argon flushed, and ίί-dichlorobenzene (10 mL) was added, The suspension was heated to 150 PC ibr !# h under argon. l¾e reaction mis inre was cooled gradually to 0 °C and filtered. The precipitate was washed with o!horcrilbrm (3 x 2G mL) and dri d, under high vacuum to give 1 J 67 g pak ted solid that was earned on to th next ste without farther purifieaiion, MS-RSI m/z 27S J (|M ~ Br'; tepnirss 276.1).
C0304J )~2^HwI>3"d ¾^^
yIid¾«) srf~ ~CT - M acid (S3)< 13* l¾ h i<-5^ g, 0,500 mm&l) was added to a flame dried flask fitted with a. eosdensof ., A.1 ; 1. mixture of eOH and CHC¾ (5 mL) was added and the Sash was ipash d with argon., 5 - C hoxy p23 etr m^ (0.2G7 ¾ 0,600 &!) was added, followed quickly by NsOAo (0.049 g; 0.690 mrool). l re rsaotion was allowed to stir for 16 h at reflux. Solvent was removed and the residue was fedissolve Ί» CHC¾ and loaded onto silica. The silica eake was elated on a 112 g sllka column using O— 5%: MeOB in Ci-k The. product was isolated as 0.075 g (34%) purple powdery solid, iNM!^OOM 12.75 (bs, 1H); 8.21 (¾/- 12.8 ¾ M),
Figure imgf000101_0001
i^et%Mpio¾^ (2), Compound S3 (50,0 mgs 0,114 rnmol) was diluted in.2,5 piL si dio apetQnit?iIe and the flask was flushed with argon, Dlkopropy!etbylainlpe (30.0 pL, 0,171 minol) was added, followed addition of hrerpo¾eihylaeetato: (i3,:0 !> B.B7rimaol) and the reaction was stirred at room ienxpetaPtre for 16 h, The motio was concentrated and the .reside was: tedissotved in nhtamtm amount of diohlorpmeihane. The crude mixture was eluted on a 12 g silica eoluam with.0 - 50% hexanes in EiOAe, The produei was isolated as 52 rag (Wn) purple sticky solid, ¾ M R (409 i¾ DM.SQ-c¾) S 123 ¾ J ~: 12J Bz; ! B), 8.15 (t, J - 13,2 Hz, IB), §,00 (d, J- 1 ,6 ¾ IB), 7,95 (dd, J~ 8. , ! .6 H¾ I H.}s 7,7S (1, J- 12,8 H¾ 1HV 7.2S (a, J- 8,4 Hz, IH).6.14 (d, « 13,2 Hz, 1H), 5,92 (s, 2H), 3.90 ~ 3.S0 («I, 4!¾ 3,46 3H), 2.11 <s, 3% 1.65 (s 611), 1,1 ~ 1.07 (m, 6H), MS-ESl?a¾ 510,2 ([M + Hf' requires 510,2).
Figure imgf000102_0001
1>2 ,3--I'eir m lind0lium iodide (0,151 g 0.500 mmol) and compound 7 (0,300 g5 0,75 io!) were d uied in 1 : 1 CHCip eOB (5 rni.) arid MaOA (0,051 g, 0,621 mmol) was ad ed lit bulk, he. reaction was stirred at om Wm for 16 h and concentrated, Th concentrate was prepared a a silica cake and uted on a 24 g silica column with 0 - 8% MeOH in 0¾(¾ over 25 mm. Pure product containin fractions were eornbmed and concentrated to give 0.123 g (56%) dark blue solid, 3H N fl (400 MHz, DMSQ-i¾ 612,27 Qss, t% 8,1.7 - 8,03 (m 2H), 7.72 {di, J 1 .2, 4.0 Hz. itip 751 (d, J - 7,2 Hz, IB), 7,35 (dp./- 7,2, 1.2 R¾ IH), 7,30 - 725 ( , IB), 7,16 (dt, ./ - .2, ! .2 Hz, 111), 6,16 (d, J 13.6 Hz, ί B), 4.03 (1, J - 7.6 i¾ 2H), 3.S4 (q, J -6.8 Hz, 2B\ 3.51 (s, 3B), 2.47 (pJ- 7.6 Hz, 2H% 1,62 (s, 6B), 1.10 (r ./
- &.S Hz, 3H). MS-Eil 438.1 f[M iff requires 43S,2),
(03071 Aeeift^ methyl 3-((1).3»¾ίΙϋ I~2? 4§ 3dexo~SK(2Es E« "(l
trimeftrylmdofe
y})pro$m i% (3)v Compound S (33.8 mg, 0-07? mmol) y*$ diluted to 2 ml, anhydrous AC$ ίύ the flask was flushed Willi argera, Diisopropyled^iarnine (20,2 μΐ,, 0.116 impol) was added, followed by ad'diti¾R.o ¾rornof&e iaeeiae. (11,4 pL, Ο,Πβ .ι¾«ιό!) and the reactio.fi was stirred at roam. temperature for 16 h. The reaction was concentrated and the residue was redlssofved in a minimum ampum of C¾C¾, The crude mixture was eluted o a 12 g silic column, with.0 ~ 50% hexanes i EtO Ac, The product was isolated as 33.8 rug (93%) dark violet solid , ¾ NMR (400 MHz, DMSO~r¾, 50 °C) 5817 - 8.03 ( p 2B), 7.80 - 7.65 (op IH), 7.50 (d, J- 7,2 ¾ 1 hi), 7.35 (dp /- 7,2, 1,2 ¾ III), 7,26 (d,J- 8.0 H¾ Hi), 7.16 (dp J ·- 7.2, 1.2 Hz, IH), 6,15 (d, J- 14,0 ¾ IH), SM ¾ 2H), 4,0 (t. J~ 7.4 Hz, 2H)53,85 (q,.J- 7.2 Hz, 2H), 5.52 Op 3H), 2,63 (p J - 7,2 Hz, 211), 2.07 p 3H).1.63 (s, 6H), i .11 (p /
- 7.0 ¾ 3H). MS-ESI f«k 510,2 f[ ÷ Fl requires 510,2). $308] K -5-(<21 BH^^ yO repsask acid (SS), n(2 3arboxyetbyi)-23 lm>niid (47 mg, 0,150 rnrnoi) compound 7 (90 rag, 0.223 mmoi) were dd ted in 1:1
CHCyMeOH (L5 mL) and aOAe (1 g tag, 0,225: mmoi) was added in bulk.. The resnikm was stirred at roo temp ibr 16 h and concentrated* The residue was diluted in C1¾C¾} prepared as a silica cafees and eiuts o¾ a 24 g silica eoiumn with 0 -~ 8% MeOH ln.C¾t¾ over 25 mm. Pure product eoaiaitdng Itactsans were eombirsed and concentrated to five 27 ¾ (36%) dark Mae solid. ¾· NME (400 MH¾ DMSCHd¾) § 12,35 (bs5 1¾ 8:. f -~ 8.0S (ms 2Hj, 7.74 (o¾J- 13,1 s 43 Hz, !!¾ 7,50 ( ds J - 7.5, 1 ,2 H/, H i): 7,32 (dt5 J - 7,4¥ 1 .2 Hz, I K). 7.26 d,J- S. F!¾ 1H)5 7,14 (dt, 1.2 liz, IH), 6.23 (d, J~ 13.3 life, il¾ 4,23 (i, J- 7,2 Bz, 2B), 4.03 & - 7,8 Hz, 2H); 3.84 (¾: j - 7.2 Hz, 21!), 2.66 (t, J - 7.2 fife, 2H), 2,47 (t, 7 - 7.6 Hz, 2H), 1,62 (¾ 6B), 1,10 (dt J - 6.8, 2,0 H¾ 3H). MS-ESi mh 406,2 ( M + H requires 496.3), [0309j A^toM t 3 :(E)-S^
Figure imgf000103_0001
t loxt t i5 &ydreps Tiii¾!dla-l(2H)-yI)| o a¾oa e (4), Compound SS (14 ra , 0,028 am lj was diluted m I mL anhydrous ACM arid the flask was i s¾ed with arg a, Diisopyopyietbyiaadae (20 pL, 02 13 mraoQ: was added, followed: afler 5 min by addition of bromomeihyla.eetale (i I ^L, fl.l 13 mmoi) and the reaction was stirred ¾i room temperature for 1 a. The reaction was eofteentrated and the residue was redisso!ved in a fpinlmpm: amount of C¾C½> The crude ιρΙχίϋΓ© was eluied on a 12 g silica eolppm with 0 - 3¾ MeOH in€¾<¾, The product was isolated a¾ 16 mg (89%) dark violet solid, Ή MMR (400 MHz, DMSO--;¾) 8 8 6- «.10 (m} 2H)S 7,75 (dt, J « 1:3,0, 9,0 l iz, IH), 7.50 (d. - 7,6 Hz, IH). 7,33 (t, J~ 7.6 Hz, 1 H), 7.25 (3,, J =- 8.0 H¾ I H), 7.14 (1, J~- 7.4 ¾ IH), 6,23 (dd, J~ 1 .2, 5.2 Hz, I H), 5.66 ¾ 2H), 5.63 (s, 2H)SA27 ft, .-/- 6,8 Hz, 21:1), 4.07 (1, ./ - 6.8 Hz, 2H), 3, (q, j~ 7,2 Hz, 214),.2,82 (i, J « 7.2 Hz, 2H); 232 (i, J- 7.2 H¾ 2H), 2.08 ( Ml 2.02 (s, 311), 1 ,62
Figure imgf000103_0002
dh«etby!isd0il¾«~5~carboxylle dd (S6). Compound 9 (0278 gs 0.500 mmoi) and aOAc (0,051 g5. 0,625 mmo!) ere diluted m id M«0H ;fiC¾p.O ml) and
Figure imgf000104_0001
oVj was added, The flask was fitted w th a condenser md flushed with a gon mid the reaction was heated to reflux lor 16 h. T&e reaction was concentrated and the rcsidne was diluted in C¾€l2:> prep red as a siBea cak and slated -on a 2 g silica eo!nmrs with 0 8% MeOH in C¾C¾: over 25 mis, Pure product containing fractions were■combined and concentrated to give 53 rug (2:1%); dark bine solid. Hi NMR (400 M¾ DMSG^) S 12.61 (hs.2B), §,22 (d, J - 13.2 I¾5 \ H). g, 13 (t, J « 13.4 Hz, 1% 7.9? (». 1% 7 9) fd, J~ 8, H¾ IH), 7.79 (t, J ** 13,0 F¾ 1.H), 7,26 (d, J- 8,4 ¾, 1 B), 4,20 (t J - 6 J H¾ 2H), 3,9 ·- 3,80 (rn, 211), 2,65 (t, J ·- 6,8 ¾ aft), 1.63 (s? 611), Li l (†, 7- 6 3 Hz, SHI 5¾ NMR (100 ¾ OMS0- ) 6 177.1 . 16K3X 167.0, 1 2.0, 161.3 156.4, 155.7, 150,6, 146,1, 140.2, 130.4, 125,0, 1.23.0, 122.6, 109-4 i 1.5, 73735 353, 31.2, 27.4, 133. MS- ESI /z 493,9 ([M - 11]" requires 494.2).
fOSU] ¾-AcetexyMfefIwU di¾¾t¾j¾ Cumnonnd : (3d mg, 0,073 mrnci) was diluted til 2, rat aiiliydrous Α and the flask was flushed with argon.
lisDjrropylet ^ amine (51 μί,? 0.291 nimol) was added, followed after 5 mm by addition: of broru.am¾tj:iylacetate (28 μί.¾ 0,291 rool) and the reaeiinn was stirred at rOQfn tefaperattire for 3 k The reaction: was ccneentra ed and the residue was redissolved In a minimum arnotmt of C¾¾Cii. The crude mixture was e!nted on a 12 g silica colnmn with 0 ~ 3% MeOH In CJ¾C¾, Tire product was isolated as 31 mg (67%) dark violet solid. ¾ NMR, (400 MHz, DMSQ-ife) 8.25 (d, 7 - 12.8 I¾ 1HX 8, 14 (t J- 13, K¾ IB), 7.99 (¾ </~ .6 b, 1H)} 7.93 (d4 J- 8.4, 1. H¾ 110, 7,81 (u J * 13.2 1½, I B), 7,28 01 8.4 H¾ 113), 6 22 (d, J - 13.2 H¾ 11 ¾ 5.92 (s,: 2B), .5.62 (s, 2B), 4.23 (;, J - 6.8 Hz, 2Η ·, 3.90 ... 3,80 (m, 4¾ 2.81 (1. ./ - 6.8 H¾ 2H), 2.10 (s, 31-h, 2.01 (s, 3I¾ 1.64 (s, 6H), 1, 1 5 ·- 1 ,08 (m., OH). °C MMR ( 100 MHz? MSQ~£¾} S 169.6, 1:69,4, 169,1, 1.67,8, 164.2. 162,0, 161, 3, 156,6, 155.2, 150.5, l.4?4 14:0,4, 130.9, 123.1. 22.2, 109.4 105.9, 01,7, 79,6, 79,2, 7,0, 38,4, 35.9,. 35.3, 30.8, 30.7. 27.3, 20.5, 20.4, 13.3. M -ES1 mh 640. i (j' + B f requires 640.3},.
Figure imgf000105_0001
The fesfc- as sealed.: evacuated, and argon. flushed,. Die lGr me ha (12,5 xtsL) was added, J Iowed by HS -dieye!qliexylcatbedlimide. The reaction was stirred at roam temperature md&r argon for 16 h, The ^eiion mix ure wm hen filleted through a short pad of CEI ETM. The filtrate was conesatraled and Stteted Through s 0,45 Μ nylon filter, This filtrate was loaded omo- a 40 g silica eo!iimr! and ie column was elated with 0 ~>50% BiOAc w kexanes. The prodaat was Isolated m 0,538 g (82%) dark orange fbaiii soli , Mixture of Z I m (1 : 1 mi Isomer A: ¾ MR (400 MI¾, DMS0-i¾) 8 8.50 (d5: J~ 13.6 ¾ IB¾ S.I6 (d,/- 12.8 J¾ IK), 7,71 (s,:
1H), 7,64 7,49 ¾ 3J¾ 7.25 ■ 7,19 (nn 2tt%lM (a, H¾ 6,80 (t, J- 12,5 F¾ III),
5.45 (5, 2H), 4.17 0>J- 7. .H¾ 3H), 3.98 - 3.9! (m, 2I¾ B. 7 (s;3H)? 193 (¾ 3M), 2,67 ¾ J- .4 H¾ 2 ¾ 2.01 is. 3.R), 1,11 ( -J- .0.Hz, 31 ), isomer B: II N R (400 MHz, DM80H¾} S B.50 d, J- 13.6 ί¾ 1H}5 8.13 (d, J- 12.8 H¾ III), 7,70 (¾.
Figure imgf000105_0002
2.76 ( J- 7.4 1¾ 20), .2.01 (s, 3D); 1.10 { ·: . J- 7.0 H¾ 3H). MS-BSI m/¾ 595.1 ¾
Figure imgf000105_0003
Compmmd § (1.784 ¾ 3.00 mraoi), compoxtnd 9 (1,603 g, 4.5Q mmoi), and NaOAe (0.369 g, 4,50 rr mol) were added to a flame dried flask and dilu ed I 1 ; I
MeOB/CH£¾ (30 l, The reaction was heated to reilax and siirrsd r¾der argon i¾r 1 o k Th reaction mixture wis ao«c.eRiraisd witlx 1 g silica and residdng cake was elided on an 80 g silica eo!unm with 0 - 5% MeOH in C¾€¾. The mam product wa Isolated as 0,385 g (17%) dark amber foamy solid. j H 'MM (400 MHz, 0MSO a¾ δ 12.60 (bs, 211), 8.2.2 8,07 (m, % 7.77 (d, ,7- 1.6 !¾ IB), 7.91 (dd, J - 8.4
¾ 1 .6 ¾ IB) 7 M i■■- 7,69 (rn 1 II), 1M (s, l.H),- 7.2-7 (d} J- 8.4 ¾ 1H), 7,1 d, J - 3,6 H¾ .iH), 6.23 (CJ- 10.0, 2.4 I¾, I H), 5.37 (s, 2B), 4.21 (i, 7 - 6.6 ¾ 2H)> 4.11 (dfc,J'~ ?.¾ 2,6 i¾ 2!¾ 3,90 (s, 3H13.E7 (s, 3I¾ 3.85 - 3,77 (flu 211), 2.72 - 2,6! (mr % I .63 I ,08 (t 7- 7.0 I¾ 3H). MS-BS! m4735 J. C[M + Hf req ires 735.3),
Figure imgf000106_0001
irtoxotef a&ydro^^
Figure imgf000106_0002
feilowBci fey addition of romomethyi aeetaie (144 pL:i 1.47 mmoi). The reaction was allowed to stir under argon: Tor 16 k Sdve i was removed and the residue was redissolved in l¾OAc, The organic layer was washed with water and brine, dried, with MgSC ii filtered, and concentrated. The residue: was prepared as a silica Cake and. ei ted OK a 2 g silica, column with 0■- !SWEtO Ac m hexanes ove 25 totu. Main prodxtet Iso edas 0.163 g (51%) dark bine solid, Ή M (400 MHz, CDCb) 6 8.07 (dd, .7- 12 4.2 ¾; IB), 8,05 (dd, 7 - 8 , L.5 Hz, 1)1), 7.97 (<1 - 1.5 H¾ 1Η),7,88-7^Η3η ϊ1Η)ί?^0-7.74(«ϊ> IK).7.71 (d,7-4.3 Ik, Π!).7.08 (d.7- 8.6 B¾ IH), 6.96 aid, 7 - 8,5, 3.7 H¾ I S>9 (s, 211), 5.87 (da, 7 - 12.2, 3,2 ¾ Hi). $.72 (4,7-2.8 Ik, 7Π).5.54 id.7- 3.6 ik, 211), 4.55 (dt, 7-7,4, 3,2 Hz, 21 i), 473 ( .7- 7.0 ¾ 211).4.0001,7-4.4 Hz, 31% 4,00 ^ 3.95 (on 2H) 3,96 (d, J~ 1.9
Ik, 3H12.S4 2,?67m54¾ 2.14 (s, H), 2.09 id, -4. 1¾ 311), 1,65 (d, 7 - 1,3
Ez, 6H).1 ,22 (t, 7 - 7.0 Ik, 3H). MS-HSi -v/z 879.0 ([ - Β Γ requires 179,3). |631Si M rorg, Com omid 11 (132 tag, ΰ 150 mm¾I) was diluted in degassed ACM (75 aiL). The flask was sealed, evacuated, and atgcw Hushed, he reaction was Irradiated at 365 nju for 24 & The reaction mixture ws concentated and the residue was loaded In niinimun amoun of Ci¾CI¾ onto a 12 g silica eolumti arid elated with 0 · 5% MeOB in ϋ!¾α2. Isolated W tug (86%) desired product as a dark blue solid, 5H7 MR (400 Ml¾, D SO U 512,31 (bs, IH), 8.24 (ed, 7- 13.0.3.3 Hz, I H), 875 (i..7- 13,1 Ok, ! H), 7.99 (d,7- ).S ¾ !!¾ 7,9 (dd, -1,4, 1-7 Hz, IH). 7.81 dtJ- 13,153,6 ¾ IB), 7.29 id, 7 - 8,51k. IB), 6.23 id.7- 12.91k, UD, 5.72 (s, 21:1), 5.62 (s.211), 4.24 (1, =·· 6.6 Hz, 211K 4.03 (T, 7— 7,4 ¾ 2HT3.88- 3, 0 (on 211), 2.81 it, 7- 6.9 I k.211).2.49 - 2.43 f 2H72,10 ¾ 3H)52,0! (¾ 31¾ ΊΜ (s, 6I¾ 1 (t» J'~ ?J6 Hz, 3H), HRMS m/s 614.2408 f [M Hi" equires 684.2405).
|§3M] i-(2-AgIdoetlyI)-3.~e¾y!5sre¾ (S8)< 2-Azic!oe I m! e (2, 15 g, 25.0 mal) was diMed in THE (25 mL) in m oven dried flask under argon and the solution was sooted in a cold water bat , Ethyhsocyarraie (1.98 mL, 25.0 mniqQ was added dronwise and the eac ion was stirred for 16 h. The reaction was concentrated and ail residual solvents were removed under high'va nmi. to give 3.0:2 g (77%) oft- white solid ssitabie tor use without farther purification, Hi NMR (400 . ¾ CDCk) B 5.53 (hs; i¾ 5.:26 (¾, W% 3M -3,31 3.22 - 3,1 (m, 211), 1.1 1
7,2 Hz, 3R). u0 NMR (100 MHz,€DC¾) 6 158.8, 52.0, 39.9, .313, IS.6., MS-ES! «fc 158,0 ([M - Hf requires 158.1 ).
i ii} l~(2~A¾ide:eth^ (S9), Com ound (l .5? g, 10.0 m ol) and iadoaic acid (1.09 g, 1.0,5 nxmol) were aided in a flarne dried rla.sk and. the flask was sealed, evacuated, and argon, flushed, AcOH (14 mL) was added and the flask was heated to 60 aC for 1.0 rnin, Acetic anhydride (3,8 mL> 40,0 mmo).} was added, and he reaction was■ further heated to 95 °C attd stirred tor 1 h. Be reaction was idea concentrate and the residue was loaded in a. minirnnTS amaiml. of CILGb onto a silica column and. elided with 0 ~ 2.¾ MeOB in •€¾Cb. Main product containing fractions were combined and concentrated to give a pale amber oil. that became a waxy solid on cooling. Isolated IM g (83%), ¾ NMR (400 M¾ CDCia) § 4.13 ft, J - 6.0 1¾ 211), 3.95 (q, J - ?..?. Hz, 2H), 3.70 (s, 2tH 3.52 (t, J- 6,0 Hz, 2U), 1.22 (t5 J- 7,2 H¾ 3H}.. ¾N ll (100 MHz, CDC¾) S IMS, 64 151.3, 48,8, W..5, 39.¾ 37.7, 13.4. M8^BS1 m/z 223.9 ( l - Hj" requires 224.1),
Figure imgf000107_0001
MTOoietrA ^^
{SIO}. Compound $ f 1 2$ g5 5,00 rnol)! malonal ehyde dianitide hydrochloride (1.423 , 5.50 mmoi); and sodium acetate (0.45 i ¾ 5.50 nrrnoi) were diluted in acetic anhydride (6,0 mL), The reaction mixture was added to a pre-hea.ted oil bath at 1 DO ¾C and stirred, for I h. The reaction was cooled and added in. portion to saturated aq. KaHCOj (50 int..). The aq. layer was extracted with. EtDAc (3 X 50 nil.,). The organic layers were corubined5 washed with brine, dried with MgSO*, filtered, and eaneeutraied, The residue w s diluted m Uf¾Cl;? aud concentrated with CHLrfETM. Ί¾ CEIiTE« cake was elided o m -&Q g silica column, with 0 -· 50% BtOAe .in hexanes over tela, llhe product was isolated ax 0,479 g C¾ %) yellow solid as an approximate !;!. Hiixtnre of ii and Zls&mers. Isomer A: ¾ NMR (400 l¾ CD<¾) d 8,52 (d, /- 13.6 Hz, IH), 8J9 (d: J- 12,8 Hz, 1H}} 7,6 ■■■■ 7.54 (m, 311), 7,26 ~ 7.22 (tx m 6M t j ^ 13,2 Hz, Hi),.4.17 (i; J - 6.2 Hz, 2H), 3 ,87 ( , J- 753 ¾ 2H), 3.52 ft ,/ - 6,01¾ 211), 2,0! (s, 3H), 1,1 ft ./ - 7,0 B¾ 3H). ¾ NMR (100 MHz, CDC )S 170.0, 162.1, 161 A 158.8, 151,1, 150,6, 138.0, 131.0 (2Q, 130,4, 128,1 (2CX 111.6S 110.5, 49,0, 40,5, 36.6, 23,6, 13.5. Isomer B. lH MR (400 Hz,
CDC½) δ 8.52 (d, 13,6 H¾ IH), 9 (4J:::! 12,4 H¾ 1HX 7.64 7,54 (m, 3B),
7.26■··· 7.22 (ru, 2H),6tS3 ft 13. U ¾ IH)S 4.04 ft J ~ 6,4 Hz, ,2H)/5.9S ft, J- 7. 1¾ 211), 3.43 ft J- 6.2 Hz, 2H¾ 2,01 ft, 30), 1 ,21 7.0 Hz, 3H). °C MME (100 MHz, CDClj) δ 170.0, 162.5, 161,7, 158.8, 151.1, 150,5, 138.0, 131.0 (2C), 130.4, 128.1 (2Cft 111.6, 110.6, 48.9, 3 , , 37 A 23.7, 13,1 MS-ESI m/z 41B.4 (iM + NaT Acquires 419,1); »xo£ i& ydro yyM
Figure imgf000108_0001
add ($>i!}. C m ound SI
(0,458 g, 1.155 mmol) and eompasttv f Μ7 g, 1.733 mrnol) were diluted in hi MeDH/eilClj (7.5 ml.), The reaction Bask was lopped with ¾. condenser with argon Met and healed for 16 . The reaction was concentrated and re h!uted.in EiOAe and 1 M aq. NaOH (25 nL) was added. The aq. layer was washed with EtOAe ( X 25 ML), cooled in an. ice bath, and acidified with 2 RCL he aq. layer was the SKtmeted with i2¾C . The orgaihe layers were combined, dried with M:gS€>4, Stem, and concentrated. Tle residue: was prepared as a ΤΚΠΤΕ'^ cake sr>d elated oft. 40 g siiiea: eelrsran with 0 - 5% MeOH m C¾Ct¾ Main product isolated as. 0,134 g (22%) dark Mae solid, ! II NMR (400 MHz, DMSO~ds) S 12.58 (bs, 2H1 S.25 (ft J- 13.2 Hz, IH), 8,18 ft J- 13,2 B¾ IH), 7,98 ft, IB), 7,92 (d, J - 8,8 Hz, H), 7,85 ···· .74 (m:, IH),.7,28 (d, 8,4 Hz, IH), 6.31 - 6.21 (ru, IH), 4.30 -4,15 (m, 21 D, 4. id -400 (re, 2H),.3.92 -3.78 ( , 21ft 3.49 ft J- 5.81fef 2H)S 2,66 it J - 6,8 H¾2H), 1.64( 6Hft 1.11 ft 6,8 T¾ 3H), M.$~E$l /z 536,9 ([M + H requires 537.2). Mi o C Compound Sli (56 ¾ 0.094 m tii) Was diluted ACN (3.0 m ) md b tmmihyl acetate (37 uLf 0.3 S mmo!) was added, followed by addition of NiN~dikQpm yk&≠&mim(€6 ΐ* 0. 8 mmol). The flask w¾§ capped and Utted with an argon inlet Ime and the reaction was stirred at room te , for 16 h, Tns e ction mixture was t en concentrated and re-di hated is. C¾C¾ md prepared as a CELITF5*3 eafcc, l¾e sample was ©bated n a 12 g silica qoluxsm with 0 ~ 50% EtOAc in. bexanes ■ fer 25 mm. Isolated 37*»g.{52%) dark blue solid, ¾ NM!l (408 Mt¾ ΟΧ¾ δ 8.11 (d J- 12,2,2,2 Hz, ΓΗ18.03 (dd. J«8;4, 1.6 Ezf 1H), 7.8 (d5 J- 1,61¾ !H), 7.88■··· 7.72 (ms 211), S id,. - 8,41¼ 1H), 5.95 >κ, 2H}: 5.88 (d: /- 12.4 !¾ !H), 5.69 ¾ 21¾ 4, IS (q? J- 6,4 ¾ 2H¾ 4.11 (a J~ 7.0 Ha, 211), 4.03 - 3.94 (m5. 2i¾ 3.52 it,,/ -62 Hz, 2B¾ 2.77 {1, 7,2 H¾.2B).2.12 (s, 30), 2.07 (s: 3f¾2.0Q (s.3KK 1.3 is, 6H), 1.22 ( J - 7.0, 1.6 Hz, 3H). HEMS ?n/¾ 68 i .2539 ([M Hf requires 684.2520),
13.11] G¾ I%»d - s»t o!f¾ eo¾«gafe (1.2), Mer¾Ii¾ (5.1 mg, 7.5 μηιοΐ) nd G¾ !igMtl (6.2 mg, 11 μιηο!) were diluted m 500 μί, Cl¾Cb. A 15 niM stock solution of tetrakls(8.cetonitrik)eopper(l) hexa¾orop osphate was prepared in CH2CI2. and 00 pL was added to the reaction mixture dtopwlse. After 24 h; the reaction was concentrated ami submitted to preparative C The product Was isolated after lyophiifeation as 6.8 mg (74%) dark blue solid. MS-lSl in 1228,2 ([M ÷ Hf requires 1228,6},
[03251 and others have optimized roeroeyanine dyes for live cell imaging applications {Toiiichkine ei-<zl £ Am. Ohem. Soc.125:41324145 (ac>03); .M¾oN$'.te a ai , Bio n^g. Ch m, 24$ 15-223 (2013); Totdchfee aL Org left. 775OT7 (2007); To te¾me f ί, Mio j g. Chem, io':! 344- 1348 (2)007); ¾ linkh et Russian Chem, Rev..78: 1 1-16 (2009)). erocyanines are characterized by the prsserice of electron donor and acceptor components that are linked by conization, usnaly a. system of double' bonds. These dyes are especially well salted for use In living cells or «ii.rn.als because they are very bright, they can be toned to emit at wavelengths l gge diae, those of cellular autoi orescence, and they can. exhibit < ξ)ί> substantia!, fiuorescenee: ehaages response t selvsift ewitomneut (KtjJinich e al , Russian Chem. Rev. 78:Ί4ί ~ί 64 (2009); Loving et at* Trends Bioiechnol
(2010); Toaiohkins ei a IMys. Ck i A ; ././: 10S49- 1 Q860 (2007); Bimc l et &L, Accounts Chem, m 3:226--23 I (1990); Bondarev e el, J. luminescence Π4 7$~ 18» (2007) Kulinich et i.5 'Russian J 6m. Chem> M; 1441 457 (2006)),
ereeyanlne dyes have been used in several different biosensor designs thai were able to report the aetivit of endogenous i stems (Nalbant !,s S m 30S: 1615- 1619 (20W): Garrett M.aL> mochs istry 4? 9Μ-92$ (2008); j¾htt : Biol, Chew. 20:20335-20345 (1990); Habn ei i? ., Bi phys. J, J/:A248-A248 (1 90); Golvanigi d., A¾r/, C'taft J¾o/, 7:437444 (2011))..
[!)323| Like almost all bright, long; wa lKRgib l¾orophores5 water somhfe em$ &¾¾e dyes are poorly membrane permeani, so: require cheancai modification t allow tfaern to enter cells through passive iraasioeatkm If water solubility is reduced to enhance metrmmue passage, the less polar dyes tenet to stain intracellular membranes such as di Oolgl apparatus, rn¾ocbondrlaS: and endop!asrnic reticulum, One method lor achieving cell permeability has been the use of aceioxyii ethyl (AM) ester gtopps that lead to charged carboxyiate side chains upon hydrolysis by mtraceilular esterases ζϊ$ &%Μΐι*Μ. 2Pd;52?-528 (1981)), The esters mask c a ts, pennttriag membrane translocation uriti! they are. cleaved to produce earboxyhc acids. To investigate the effect of ester mnnher aud orientation on membrane permeabilit and staining of intracellula membranes, several versions of a rep res email ve parent meroeyan!ne dye were yn hesized with AM. esters at different positions (Fig, lb). Based a our earlier work optimi¾ingmerocyamnes for live eel) biosensors, the indolenine-haibituric acid (I ~B A) scai!bld was chases (an iodolenine ring (I) linke to a barbituric acid (BA by four earbons). This dye she ed, a favo able eombmatrofi of brightness. photostabHi , fluoresceuee wave lengths, and solvent■sensitivity (Toutehidne & ,?/.. ,/ A , Chem, oc, 125 4132-4145 (2003); MaeHeym a/ a!. , Bioconjiig, Chem, 24:215-22 (2013)). We w¾main*4 t soBemteie peiMea ili an snbceliuiar distribution of the underivatlzed paren meroeyanine I (Kg. if), two versions of the dye with single AM esters (on the donor Ϊ component (2) or on die acceptor BA component (3)), and two versions of the dye containing two AM esters (on the donor and the accepto (4), and with both esters on. the donor {§}), , ; o
10324) Mouse embryonic fibroblast cells were incabalod in. medium containing the test cdm ouB l at 37 **C' for 30 mirmtes, rhl!owed by aspfet oa of the dye solu ion aid washing with medium. The cells were then ttyps lzed, suspended in medium., and t¾e Siiareseeaoe intensity of the eeti suspension was obtained far dye, Values were normalized tor edl B ei based ø¾ the relative mie&sity of the nuclea stan Hoechst 33342* which was included w tfe the dy in each test solution. As expected, the unmodified merocyaTtlne dye 1 showed negligible import in o cells (F g. II). The etocyaninss contammg, a. single AM ester both showed similar
improvements in fluorescence uptake relative to I. However, the addition of a iwo AM esters provided an evert greater enhancement f import. Compound S> which contains two AM esters on the donor component, was slgmfioantly hri ghter than compound 4, which has an AM ester on both the indolenine and barbituric acid rings. Subsequent studies showed that eomponnd 5 also had more eff ent Import relative to 4 at lower concentrations (Fig, 12),
$325] import ot! s dye 5 showed uniform distribution within the cells, unlike the other dyes which stained m† Csllnlar membranes and produced fluorescent vesicles (Fig. 13). Subeelinlax distribution of the dye was compared to the distribution of molecules thai are foiown to show uniform distribution (stably expressed yellow fluorescent protein). The dyes arid these control nraleenles were visualized in the same cell The combination of intrmsic fluorescent properties and efficient uptake fax dye $ ' led to exceptionally bright images irelativs to other dyes> Cells tolerated the presence of this dye and a pea ed healthy fo the dilation of the S minute
experin epts. Having both AM esters on the donor component of o was advantageous as it provided ready synthetic accessibility to dyes with different acceptor
components: varying the acceptor has proven useiul when tallothig iluor phores to different applications MaeN in et aL gioco ug. Chent, 24:215-223 (2013)). Based on these results. :n erocyanine 5 was chosen as tire lead structure to carry forward for further development into a eonjugatable version.
0326] Synthesis of mmtfft, a conjngatable version of dye S bearin a earhoxyllo acid side chain, began from the barbituric acid derivative 6h obtained In 3 steps from j aianlnc methyl ester hydrochloride (Scheme Compound 6 was first converted to the activated diene 7 and the free carhuxytle acid wa then coupled with the Π ] dimethoxy vitso &z l ( B) photoiabile protectin group to give compound 8 (Bochet J€hem, Soc.-Perkin TranmcHom 1 125* 142. (2002); M ® .., J Org, C¾em. 07:5567-557? (2002)). The di-earboxy indoienine salt 9 was reacted with 8 under mildly basic conditions to give a merocyanine product thai after in stallation of the -two. aee oxyntethy! esters and hotodeproieehon of ;D ¾B provided M«r§ & The azide comaiftmg merocyanme .m ro Sj for use in click, chemistry, was prepared thorough an, analo ous: route involving reaction of eompoimd 9 h an a ide- eontaining barbituri acid, but without the ijeesdio u ilke protecting group chemistry. The emission spectra of mero76-a«.d tar i illustrate the signifieani so!veni- ensitivlty of these dyes (Fig, 14), Large increases in fluorescence intensity are observed: hen moving rorn water to less polar sol vests. Both dyes maintained good brightness ana solvent seasidviry characteristics relative to the nomderivatked parent structure (Table 7} (MaeNevin ei ΐ, Bloeanfng. Chem, M 5-223 (2013)),
3271 We sought to demonstrate the yem ! of these dyes for me in live ceil imaging: applications, A, selective ligand for the DNA-nietbyltransfoage G%. (Yedadl e oi.f N ,. Ckem. Biol .1011, 7:648-648 (2011)) was t¼ctionaiized with an aikynyi side chain and conjugated to meroMS via click chemistry to give com ound 10
(Scheme »), MSP cells were incubated with either the dye alone or compound 16 for 20 nrinutes5 washed, and imaged. Cells treated with mere-l 66 showed taiiforrn .fluorescence intensity tnroughoul . the ceil, ydth o apparent organelle or lipid speciik interactions {Fig> IS). In. contrast, the iigand-dye conjugate: 1 ø showed distinct nuclear localization, which is consistent with the localization expected to esult from binding, of the probe to 9a, a weihcbameteri¾d slone: methyhransxemse (Shiakai. aL ems Dev. 25:281 -788 (201 !)),
j$328] In conclusion, we have developed new merpcyanme dyes thai are able to freely cross eei akr membranes and distribute uniformly without appreciable staining of cellular membranes, We have further prepared these dyes with reactive side chains m& demonstrate that they can he conjugated to a small tnolecules passively diffi se into ceils, and there be used to observe the distribution of the small molecule as it binds to its ligand. We believe the dyes described bete have the potential for use in a variety of ap lications that aim to. monitor protein localization and dynamics in live cells and animals.
Figure imgf000113_0001
xam ale 7 f 03 91 la vitro ¾ssays:. Stock calci um scfciioas were prepared aging 1.00 M NaCi, SO mM Ttis-HCI, pR 7,5 and buffered with the appro iate nient ofEOTA to maintain fhe targe See calcium eoneenhatkm at an ionis- strength of 0.05 using the mimt calculator located at: m xe elai0r< St fo ^
Figure imgf000114_0001
experiments we e ten in triplicate ½ 3:i6-well plates. For calcium titration and re rsihiHiy ex erimente, t¾ and probe (GaMera or CaMero-TiC) concentrations were, hoife 1.0 Μ and ike stack B TA solution w s prepared at 200 μΜ,.
flBSOj Cell cdtefc &»<J. treatment wl& imaging probes t Human 13¾N1 •astro^ oas¾ &eii . aiid ΉΜ 3 3 moase embryoriie fibroblasts (MEF) stably expressing YPet Inorop ore were mal mgd In ! 0% C02. at 3?'eC in Dnlbeeeo's modified Eagle t mediu DMEM, Cellgto) with 10% fetal bovine serum (HyCIone, Thermo SeieriiSe) and 2 m i GlntaMax (Gibeo, life Technologies). Cells were plated 3-4 hoars efore imaging on eoversiips coated with fibroneetin 2:0 ue/mi. (Sigrna- A!drich) o vernight Cells wees ncubated with the test probe (5,0 pM) fer 20 mm in 10% CO, at 37 C m haaging medium TRAM'S F~I2 ( ) Medium (SKU
$$mm$ hh lbeo, Life Ted¾alogies) with $% fetal bovine m . (HyCioae, Themio Scientific) and 2 mM GiutaMax (Giboo, Life Tee¾nQiogiee) , The cells were then gently washed with inaaging medium (2 X 2 mLl ami imaged In the same .medium,
[0331] t e: eell imagiagi Live ceil imaging was. carried oat on an Olympus 1X11 m v&s ps with IjPLFL 40X oil ol¾ecti\¾ A 1.3) and mercury lamp excitation (T03 HBO balh). Filters nsed ί τ CaMero, CaMero-Ne, mi the dye alone were 545/50 excitation: and 620/60 emission:. Excitation was throirgh m MD 2.0 (1 % transmission) neutral density filter, typically aslng 50 ms exposure. For YPets filters were 500/20 for excitation and S35/30 for emission, Excitation, was through a HD 2.0 (1% transnilssion) filter with §00 nis exposure. Excitation, was through a MD 13 (5%T) neutral density filter with .500 ra exposure. Images were acquired with a Conlsnap ES camera (Photometries) with Sony 6.45 x 6.45 μ.Μ ixel chip using 2 x 2 binning. All image acquisition,, processing., and anal Bia was carried oat with Metamorph soSware, 'Ratio images were generated from unprocessed images fellowing shade correction* background subtraction, masking, registration for image alignment, and: pholobieae ing correction. The display threshold of ratio images was restricted to the pixels■ .falling: between S ·■ 95% of the overall frequency msto:grarnt.
[0332] W ole nhnal ex er ments : Treatment solutions of the probes were prepared as follows; 8.0 pL stock solution of probe (CaMero or CaMero--NC at 20 M in DMSO), 8.0 gL 20% iv/v Flu onic I S? in DMSO, 15 gL stock solu ion of
Figure imgf000115_0001
administered to Q- P-LifeAcf mice via tail -vein i»j<sc¾¾o» GFF-LifeAct mice. Mies were allo ed to recover for a period of 1 · 2 hours, sacrificed by cervical dislocation, and short sections fVI enffof the intestine were removed at a distance of
•approximately: 10 - I S cm from the siomadh, washed once in PBS, and placed Into Fish Ringefs solution In glass bottom, dishes (Matek) for Imaging. Tissue was imaged using a LsV eii TB M scope attached to a Kiko Eclipse la inverted stand with an Olympus 0x, 0.95 NA water immersion objective. Two photon excitation of CaMero/Ca ero- C was achieved with, an optical parametric oseiHator laser timed to 1110 urn and emission was detected us.bg a 630 ¾0 emission 111 tor for
C¾ e o/ ero" ■ ami 549/1 S filter for the SHG signal of eoi!sgem Hoechst was excited, with a la-sapphire laser tuned to §00 rtm and emission was collected using a 435/40 gher. For localization studies, tissue samples were: imaged after beginning from the crypt of Lleherkiihn, and extending ¾ough to the peripheral smooth: muscle layer using incremental steps in the 2 plane of 4 uM (30 f ames In total), For Imaging of muscle contraction, Ga 'ero and (m ero-NOsampiea wer acquired within ^proximatel 1.0 minntes of each other using identical anqnlsition parameters (laser power and F T gainj. CaMero aire CFPdlifeAct images are displayed using different contrast settings for each ax al' position; however, the same contrast settings have 4?een. applied, to■images: of the CaMero and CaMsro-NC probes. iOH- beset adse S9), Fkplieuazine dlbydrjcMoiide (I5 ΊΜ g> 1.96 mmoi) was diluted hi sturted aqueous sodium bicarbonate (SO tuL) aud stirred jfer 1 mm. The aqueo s layer was extracted with C iC (3 x. SO mid. Organic layers were combined, washed with: satite aq, Na£I? dried wltli g O¾ and filtered. The filtrate was .eoi-ee«trated to ive 0.S5 g pale amber oil (quaut) that was carried i¾rward directly to the fieMt step,.
[)334] 1¾e etude redict (0.84S g, 1.93 mtaol) was diluted €!¾¾ (25 rat) n oven dried lOO nL round -bottom flask The . flask was sealed with, a rubber sept up flushed with argon, aud chilled in m i bath,. ITIeihylarmue (0,41 ? mL, 2.99 mmo!) was added., fell owed by dropwlse additiou of methases lfo yl chloride (0.233 niL 2.99 wmol). The rrhKt re was stirred ibr 1 h at 0■% allowed to equilibrat to T atid stirred for 1 J- h. ater as: added (25 mi.) rd th orgaue layer was separated, dried with gS04, iitee aad the filtrate was concentated, isolated 0.89:3: g ¾uant) pale amber oil that appeared pure by NMR analysis. The product was carried forward ithout faxtber puilrlcatiou. ¾ KMR . (40 H¾ CD€¾:} h 7.24 ~ 7.08 (m, 4H)S 7.03 (¾ !l¾ 5.97 - 6.SS <m, 2¾ 3.9S (t, J~6$ !¾ 2S)S 3.S6 i - ?,! ¾ 211), 2,70■ { 7.1 Hz, 213), 2.62 - 2,25 iro.10H}5 L9t - 1,87 (m, 2H). nQ MR (100 M!¾ CDGbj I45A 144.5, 130.1, 129.7 (q, J- 32 ihp, 127 , 127.7, 127.6, 1243 (q. - 270 Hz), Π .2, 123.2, 119.1 (q, J~ 3,9 Hz), 116. |, 112,1 (q, J- 3,8
55.5, 533 (four C), 45,1, 4], 0; 24.3. MS-ESI m/z 456,2 {IM Hif requires
456.1).
Figure imgf000117_0001
P335] tO- ^^-AsMo^^^
i#M~ he530iMg¾!¾e: plCS), om&OM 89 (1.52 g, 3.33 mmol) was added to a. S h m dried rotmi-boltorn fiasfc and diluted in DMF (20,0 aiL), Sodium iodide (0,599 gs 3,99 mm&ty as added, followed by sodium az e (0.433 g, 6 mmoij. The flask was sealed with -a -se tus*, argon Husk d, -mi the reaction mmture wa heated to 60 °C far 16 h. Aqueous L!Gl (1 |¾ w¾ 5 Q isL) as added and the aqueous kyef was extracted wtt i BtO Ac (3 x SO tnt). The organic layers were combined, washed with waterpCX tti/j: and brine (200 rriL), dried wild Ma3S¾ filtered, and eoricemfated. The crude product was suspended k a mminuuri amount of C¾C¾ and loaded onto 24 g SIO column. The. -column was elated with 0 - 351 eClB in C¾€¾ o er 20 mis, Maltt prodtsct. cofitamitig: fractions were combined, concentrated, and dried uoder vacuum to give 0.890 g (58%) pale amber oi lE (40Q MH¾. CDCif) δ 2.22 ~ 7,09 n, 4H), 7,04 (s, Hi), 6.92 - 6 (on 2H)S 3.96 (t, J - 6.8 i¾ 1 H). 3.32 (t J - 6T ¾ Hi), .2,56 J - 6.1 Hva 2H), 2.54 2.20 (m, 8H}. 1.97 - K <rm 2H). '•'C !S R (100 Mife, COC¾) 0 145.9. 144.5, 130.0, 129.7 (q, J - 32 Hz), 127,8, 127.7, 127.6, 124.3 (0. 7 - 271 >-¾ 124.2, 123.2, 1 19.1 (¾ 7- 3, H% 116,1, 1 12.1 (q, 7 - 3.8 Hz), 52,2, 55.5, 3.3 (four C¾ 48.4, 5.5, 24.3, MS7BSI mM 463.2 ( + B requires 463,2},.
ί¾332¾ 2~ K3~(2"{ rIih¾o o^
l-y }etliaa»!»ainlEe £2), Ι¾Μιη¾ϊ on carbon (10 wt, %, 0,060 g) was added to a SO mi, flame dried two-a eck round-bottom flask,. One neck of the flask was sealed with a rubber septum fitted with an argon line and. the ether with glass sto e . The flask s: evacuated and argon flushed two times. Compound 3 (0,305 g, 0,659 inmol) was diluted ½ anhydm-us methanol (10 mL) and ded1 to the reacti n flask, The glass stopper was loplaced with a hydrogsn containing halloom The argon line was removed and the flask, was flushed with hydrogen 0 x). The balloon was left open to the system and the reaction mixture was stirred :st RT for 1:6 h, The teaetion mixture was filtered through a sand-topped 0ELlTE M column nd the column was rinsed with methanol (75 mL), The filtrate w¾s concentrated, diluted in (¾(¾, dried with N%S0 s and filtered with C¾C¼ washes. The filtrates were concentrated and loaded i a rnu num amou of C¾C% onto a 1,2 g Si02 oo trmi and elated with 0 - W% MeOH (containing 1% '¾) m C¾Ci2 over 25 min, ain peak comainmg fractions were combined, concentrated and dried, under high vacuum to gi e 0.242 g (84%) colorless oil Hi w (400 H¾€DC¾ 37.2 - 7.0? (my 4¾ 7.03 (s IB), 6S7 - 6.88 (ms 2H), 3.95 (t, 7- .8 H¾ 2H), 2.7h (ts </- 12 ¾ 2H)52.63 - 2.20 (a, 12r¾ 2.0! - 1,85 (m, m% 1.41 (b¾ 2hL). ¾ M 1O0ME¾ CDCI3} δ 145.9, 44.5, 130.0, 1.29.7 <qs J - 32,1 H¾), 127=8, 127,7, 127.6, 124.3 (q, J « 271 Hz), 124.2, 123.2.,
Figure imgf000119_0001
MeOH (1.0 ml,} in a 1.0 ran round-bottom flask open to air. Sodium perbdate (0.028 g, 0,132 ravnol.) was dissolved in a 3:1 eOH/1,0 M aq. HC1 solution (2.0 alt) with onieaiion and added id the reaction flask quickly dropwise, ¼ reaction mixture Was: stirred for 4 km. ET, Saturated aq. Na¾0¾ (2,0 mL) was added and the mixture was con entrated. Some additi nal water as added mi the aqueous layer was extracted with€¾C¾ (10 mL x 4), Organic layers were combined, washed with saturated aq, aCL dried with Mg 0, tuteed, and eqnc raied to give 0,040 g (77%) clear oil t at oamed on drying. The product required no further purification. ¾ MMR (400 MH¾ CDCh) 38.0 (d, 7- 8.01?/, IH).7.95 (dd, ../ - 7,7, i.5 !¾ 1 Ii), 7.72 is.1¾ 7,68 :~ 7,62 (rn, IHh 7.59 (d, 7 " 8.3 Bz, H¾ 7.47 (d57 - 8.0 Hz, 111), 7.30 (t J - 7.4 ¾ ί H , 4.50:- 4.31 ( , 21¾ 2.79 (97- 6.2 Hz, 2H): 2.69: --2.27 (n¾ 12i-l), 2,12 - 1.99 (m, 2H), 1 ,63 (b¾ 2H). ¾ NMR. (100 MHz, CDC¾) δ 138.7, 1311, 134.4 (q57 - 32.3 \{y)f 13:3.1, 132.2, 131,5, 127,2, 124,6, 123,5 (q, J~ 272 ife), 122.6, 17,9 (q, ,/ - 3,5 Hz), 116.5.113.2 (q,7- 4.0 i3¾ 61,1, 54.6.53.4 (two C), 53,3 (two C), 45,6, 38,8, 23,9. MS-E3I..««& 453,2 ([M + ΒΫ requires 453.2),
[@3:3S] CsMera, Gor po and $ro7$ ( 0 mg,.0.01.. mrnol), compound 2 (9 , 6 tag, 0,022 nrmo¾ an T 'U (7,0 mg, 0.022 mmoi) were added to & 5 nl conical vial with spin vane and dissDived in DMF (0.70 niL), foll wed by addition ofEt3N (3.1 μΕ, 0,022 nimol). The vial was capped and covered in foil and the reaction was stirred at RT for 16 h. The reaction mixture was. -submitted to preparative EPiX. ai , product containing fr ctions were combined, concentrated, and lyophiimed. Isolated 13,0 mg (Si ) dark bine powdery solid. ¾ NkOl (400 MH¾ DMSO) § 8,24 . ft J- 12.2 Hz, 1H}S. 8.20 - M (m, 2H), 7.99 (s, 1 B), 7,94 (ds J= 8,4 K¾, 1% ?J1 £dd, J~ 24,9, 12.9 Ι¾ 1H), 7,39 d, J :;: 7,9 Hz, IH}, 7,32 - 7.26 (ms 4¾ 7,22 (d, 7.S fix, 1% 7. 10 (dd, ,/ - 7.9, 2.7 Hz . 1I¾ 7,03 & ·/ - 7,4 Hz, Ϊ M), 6.22 (¾J- 15.6. 13.1 l lz. !I¾ 5,936s, 2H), 5.62 (d, J- 2.6 Hz, 2¾ .4.3.0 - 4.15 (m} 2H), 4.09 ·· 3.97 (m, 4H)S 3,90 - 3.80 (rn, 2H), 3,35■- 3.22 (m, 2H), 3.1 2.84 (m, ll), 2,81 £t J
- 6,3 Hz, 20), 2.3:7 (0 6,4 jf¾ 2E):; 2.10 (s, 3H), 2,05 » L95 (m, 2H), 2.01 (d, J■■■■■■■■ 2,2 Hz, 3H}> 1.63 (s, SH)> 1.10 (tS: J~ 7,0 Hz, 3H). ER,FTMS m/z 1 102,4218 (:[M + H requites 1 102,4208).
f033 | €¾Mer -MC, Com O tt saef d?¾ ( 10.2 n¾ 0,01 mrnol), compound 3 (1 .2 nig, 0,023 IB O!), and ΪΒϊϋ (7,2 sag, 0.022 mmol) Were added to a S mL comcai vial, with s i vane and dissolve in DMF (0,70 rnL), followed by addition of E%N (3.2 ΐ, 0.023 mmoi), The vial was capped arid covered tail and the reaction ■was stirred at RT for 16 &< The react! G& rnixtare was submitted to preparati e HPLC, M&m pfoduet-cdatainfeg.fracti S- were .eoi¾¾l«ed, concentrated, arid lyophilized. Isolated 12,0 mg (.71%) dark blue powdery solid. *H HMR (400 MHz, DMSO) 8 8.28 7.90 £m, 6H), 7,98 (s, I ri), 7.9/ - 7J9 (ms 1H), 7.86 - 7.73 (m. 31¾ 7.61 (4 J™ 8.1 I¾ IE), 731 (t,J- 7,4 Hz, l i¾ 7.29 ¾ J- 9.0 Hzs I.H), 6,29 ~ 6, 15 (m, M% 5.93 (s,: 211), 5.i2 id. J■■■ 3,4 Hz, 2¾ 4.6 ~ 4,60 (rns.2H), 430 - 4 17 i m; 2¾ 4.02 ft, ,/ - 6.5 H¾ 2ΓΙ), 3.90 - 3.78 (in, 2¾ 3.37 - 3.22 (m, 2H), 3.05 - 2.75 (ro, 6¾ 2.37 a. J
- 6-4 Hz, 7M}> 2,10 (s, 3H), 2.09 - 2,02 (m, 2H), 2,00 (d, J - 2.8 Hz, 3H)5 1.6 (s, 6H), 1.30 U, 7 - 7,0 Hz, 3i l), HR-P fMS ¾ 11 18.4162 ([M + HI " requires
1118.4157).
Iiek.
Figure imgf000120_0001
|( 40] The tremendous importance of spatial and temporal meters to the regulation of cellular signalin eveats is evidenced by di meh ditc ted rales that a given protein ma play in di verse and s met mes opposing cellular processes. Even hen the primary nioleeular constituents of a signaling network are known5 it often remains difficult to midenstatrd the dynamics of their collective behavior without taking into account spado te poral aspects of the endogenous system. Biosensors are o tditat e: took that can be used to provide. msighi into how signaling networks are | ? transientl constructed to produce a specific behave in real time and with, spatial resolution. Despi te the power of these tools, the idespread applicatio of biosensors has been somewhat limited. It has recently been estimated that sensors have become available f r little more thaa 100 of the vastly greater number of potential molecular targets of interest, due in targe part to the complexity associated with, their development ana optimization (Lemke m at , Nature Ghsm. Bk>L 7:48 - 3 (2011 )}, (03 1 One approach to the design o biosensors is through, the use of dye-based aiSnlty reagents (K irnmer οΐ αί, Ckem. ml 2QM7 S6 (2013); Cmtyani t L N ium Che . Bml 7:437444 (201 1): Qm ei lrm#cheMistty* - 4?:98d- 96' (20t . ¾bsnt el d.. Science 305:1615-1619 (2004)). The affinity reagent, such as a prot&ir x grnent or arstibody , hinds selecti vely to the active state of its target and this binding event is associated with a measurable change ¾ fluorescence. Affinity reagent based sensors ai!brd access to endogenous, trnmodiled protein targets. The use of a dye for signal transduction provides a bright signal due to direct excitation, rather ibm indirect excitation as In designs utilizing Fdrstsr resonance energy transfer between, fluorescent proteins. The enhanced signaHo-noise of dye-based sensor relative to other designs also allows for lower excitation light and lower biosensor loading to he used, wM#b minimizes perturbation to normal cellular activity.
However one s gnificant drawback associated with affinity reagent "baaed biosensors: is that they have historically required mieroinleetiom This Is a challenging ieeimique thai requires specialized equipment and many labs are not sufficiently motivated t make this pan of their routine,
19S42J We report here a new type of biosensor, consisting of a small, molecule recognition: component coupled to an environmentall ■■sensitive dye, which extends upon the affinity reagent based biosensor concept to provide several additional advantages (Fig, ϊ , As opposed to a protein fragment or antibody, a small molecule based biosensor can be introduced Into cells through simple passive diti on, Further, one of the bottlenecks at the development of biosensors for new molecular targets is the back of suitable affinity reagents. Small molecule based, biosensors can be constructed from c m ou ds having known activity profiles and can thus potentially provide access to a ide variety of molecular targets for which no natural binding partner is known. This type of biosensor design ha the potential to afford, access to unknown aspects of signaling pathway dynamics k both genetically intractable ceils as well as whole organisms.
11)343] Radons! desi n ai s nt esis of CaMero : For the reporter component ©f the sensor, we chose to work with iirerooyarhn dyes. Morooyanines are oharaeterhserl b the presence of electron donor and acceptor components linked by a system of coigugstien (Kotieteh ei ®L> M s. Ckem. Rev 71: 141-164 (2009))- These dyes are especially well suited for use in li ve cell Imaging as they are very bright, having some of the largest extinction eoerlkiervis reported for organic dyes, they can be tuned, to emit at ¾¾g wavelengths that a oid overlap with cellular
autoflitorescenoe, and they are sufficiently photosiahle to withstand the repeated excitation required in experiments aimed at monitoring protein dynamics over lime (Loving i l, Ίτα Bk chnal 28 ;?3-S3 (2010)). In. addition to these features, nteroeyaxunes are perhaps most notable for their "environnient-sensing" capabilities. Changes in solvation snviroinnest, s¾eh as when goin front a more polar, solvates state to one in which the dye is brought into proximity with: a protein surface, can he associated, with significant changes in fl orescence (Toutchkln et a , J. Am. Qh h Sac. ί25ί4ί32- 1:45. (2003); MscN'evif*. ef Mtpc jug Chem. 24:215-223 (2013)), However;, as is the case with, most organic dyes, the merocyarhn.es are not inherently iiQbm.¾e- 3e i$ jjt «s4 ¾«^ef ?e re ¾i j¾?|--str«¾st¾f.¾i: -optimizs ioa to enable the envisioned passive delivery of the smalt molecule based biosensor. Several analogues of a lead merocyanke compound were prepared with acetoxynrethyi ester functional groups in various: positions and these dyes were screened for cellular membrane permeability and uniformity of intracellular distribution. The best dye among this screening set was farther prepared as a eotrjugatable version with a earbosyhc acid side chain (Scheme $}, This new .dye, m% ti7§, maintained, the good brightness and solvent sensitivity charaeterisiies of its non-mnehonalized parent dye (Tahl 7).
.
Figure imgf000123_0001
" Wats* sxt sa <PI % DMSO,
|0344] r i¾e recognition copiponent: of the sensor, we sought to take, advantage of lire known blading selectivi y of the SiaaM rnoloeufe triftuo eraKiiiss (TFP) ibr die active sBiif msiicJB of calmodulin (GaM) (Massotn et &L Biochemistry- Us 29:671- 681. p-§90)). The calcium-binding protein cateodnhB.. which is ublq itousiy expressed i&euk&tyQik eelis, is involved in m mmm processes throughout the ceil, iaciiidltig growth, proliferation, md motility (Chin ei ai. Trends Ceil iaL 10 322- 3.2:8 (2000)). Calmodulin usdergoea a disiirtet coafr¾fmatlo»a). shift iko a more giofe lat form In its caidum-ftee state, to an open, "dtimfebs!f5 shape once in complex it¾ cakinni ions (Fig.17) (Zhang et l. Mature Str mml 3M 2:758-767 ( 1995)). Tha sd seqoent binding: of iriiuopetaa ae to ca!rnodidia w coptipgeni upon this eon&rpiatioasl change. We t gf s¾e ^ ot¾¾si et t!ia & Massisw t calmoduli acti vation could: be developed tfexongh the construction Of a TFP cepjngate containing
Figure imgf000123_0002
11345] Severn! crystal Btme u.res of the TFP-CaM: cemplex are available
(Y¾»$ms©har ei d. > timute- 8 tiu? £fol 1:795-801 (1.994);
M chemisiry^Us 33:15259-15265 (1994):; Vertess si άί, Biochemistry 37:15300* 15310 (1 98)). la each ease, the structures reveal that the general orientation efths TFP molecule once bound with calmodulin places the N- ihyl sphsth ent of the TFP piperazine moiet towards solvent (tig 16¾), We saw this position of TfP to he | ?, m ideal point for elaboration with a reactive side chain for the attac m nt of die dye, Comxnerclaliy available fh^henasi e hydrochloride (is Fig Ihe) was converted to the ■iV-ethyIgirn¾e-s¾¾s-tit ted pipera?i».e -analo *!® ¾>#T:EP, conipound X, in three steps, Compo n d 2 was then conjugated with the optimized dye nsero/ft to give the small molecule s d biosensor CaMero, Previous screening studies had identified, the s ifox e derivative of TEP as a cotrrpound having slgniflcaihly reduced affinity for CaM relative to TP? itself (Levin el ai., J. Phama i. Εψ. Th 20¾:45 45 : (1 79)), Due to the structural similarity of the two eompouuds5 the sulfoxide derivative was thought t be ideal target for the development ox a eotutoi probe. Treatment of compound 2 with sodium: petiodate in: dilute acid provided the desired sulfoxide derivative compound 3 (Morrow Hefa Chim. Ae 88:962-96? (2005)), Compound 3 was then conjugated with m ro?$ to give the negative control probe,
Figure imgf000124_0001
|!!346| BtotiMttr respps.se e&¾r&etet¾sfes m v¾wu We fust examined the behavior of the two probe molecules in vitro,. The GaMero probe showed a significant increase in fluorescence intensify (S-fold) with increasing calcium concentration (Fig 1.8a). No change in CaMero fluorescence Intensity was observed with increasing calcium concentratio in the absence of calmodulin, This .result demonstrated: that the probe as not acting as ealchrm. indicator but repaired the presence of activated calmodulin to generate a fluorescence response. Minimal changes in fluorescence were observed tor the negative control probe CaMero- C with increasing calcium under the same conditions. Gusduai reduction in calcium concentration below the level required to generate maximal fluorescence response resulted in a corresponding reduction In fluorescence intensity of CaMero (Fig 18b), Purihur, only in the presence of saturating levels of calcium ( Id y.M) did G&Mero show an Increase in rlnoresoence Intensity with increasing concentration o calmodulin: (Fig lie). Thus the fluorescence response of CaMero was not dre result of nomspeeifie binding with calmodulin, The specificity ox CaMero fluorescence response fbr die active,, calcium-bound state of calmodulin, In conjunctio with the demonstrated reversibility of response, indicated that CaMero had the potential to act as a sensor of calmodulin activation, dynamics in more complex systems. ; , 4 i$MJ\ l*eati∞itat ¾a# d naotfc* o CaMero m i^e cells; We ext e .¾mi»sd th intracellular localization of the different probes using live cell imaging.
Localization studies were carried out in B21N 1 astrocytoma cells stably expressing the nucleotide rece or ¾Y$, C lls were briefl incubated in media containing the test eonmormi, then, the eatment media was removed and the cells were washed: and imaged. Treatment with the membrane permeant dye component of the sensor resulted in uniformity of fluorescence a d no apparent organelle specific interactions in the 1321N1 cells- . (¾ if a). The negative control ohe CaMero -NCS which contained both dye and small mo! culs com o en s, proved to maintain membrane permeability and also showed even distribution throttghout the ceils, llreattnent with the active CaMero probe however showed distinct localization In the cells, with maximal fluorescence appearing to be localized generally in the perinuclear region, Co-treatment of ceils: with: a marker for the endoplasmic mileuium (BE) revealed clear co-locaiizaiion with CaMero,
3 §! We next sought to assess the ability of CaMero to report calmodulin activation dyna ie in liv sells throu h modulation of intracdiolar calcium levels. Th f t , receptor can be selectively stimulated with the nucleoside uridine diphosphate (UDP) (Nicholas el al, Mai Pharmacol. 50:224-229 (.1996)). It is well establ shed that activation of P2Y¾ leads to elevation of intracellular calcium levels v|a initiation of phospholipase C mediated cleavage of phosphatidyi-inosif.oh4}:5- bisphesphate (PIP2) to diacyi glycerol and inositol-triphosphate (ipf) and inn simsequent release of calcium from stores located within the ER following IPs binding with ER localized ¾ receptors. Colls wore eo reated with either CaMero or
CaM¾o~NC along with a second, non-environment sensing small moiecizie fluorophore known to distribute evenly throughout the eel! that could be used as a control for fmctuation in cellular volume, Stin iarlon of CaMero treated ¾Y6 astrocytoma cells with UDP generated a significant increase in ilnoreseenee Intensity, where s1 no change In intensity was observed in CaMero-NC treated cells 1% 1 ½ . Average fluorescence intensity when quantified across the entire ceil skewed little change following UDP treatment ( !g 19e). Stimulation resulted In characteristic ceihilar fof n ioB and this increase In overall area served to offset localized increases In fluorescence intensity. When analysis wa constrained to BE rich peirinaclear areas, fluorescence changes were most ekarly e ident This particular localization of irrtsasity eiaage is consistent with the si-gnaling cascade leading to intrace! ar calcium release from the EE, Additional cellular stimulation
experiments were earned/out usin imary mnrlna neurons. Depolar zation following rreatrnsat ith K& respited in Increased tluotescence intensity when an l sed withi the entir cell body region (fig. 20%
£0349] We also observed the behavior of the Ca ero probes in normally migrating mouse embryonic fibroblasts: (MEFs), Fluorescence intensity was most uotkeahiy res t in. perinuclear regions, as previously observed In the 1321ΝΊ cells. However we were surprised to also see a. band of intensity at the ceil edge In CaMero, bat ot Cs srO"NC5 treated cells (Fig. 21a). This apparent area of calmodulin activation appeared predominantly within.3 pM from the cell edge (Fig 211?). The small moleeuk ilnorophore carboxyilnoreseeln. dkeetate (;€FDA) and yellow fluorescen protein (YFP) were both used as volume indicators in separate
experiments In order to control for any potential artifacts due to differentia] localization associated with moieeal r sk .. Experiments using either Indicator gave similar resuMs with respect to both Intensity of signal and depth of range (fig, 22). Fre^reatment of the cells with clozapine to minimize potential off target bindmg of the. probes with eel! surface GFCR did not affect the !oe !!zat!an or mtenslty of signal of CaMero (Fig, : 2 Cell edge vebeliy was calculated1 sech that protruding areas of die ceil were: given a. positive velocity and retracting areas a negative velocity, Correlation analysis of cell edge velocity with respect to fluorescence intensity of CaMero revealed an Interaction between the two varialsks that was.
especially prominent In the positiv direction (Fig 2T«, pes, velocity: p < tXCK) 1 , n™ 58; n.eg. velocity: p < 0.05. n - 56), Although limited correlation between. CaMnro intensity at lower negative velocity vakcs was fo nd, at the more e ljetse ranges of negative velocity,: such as k areas of tail, rpiractiom a strong correlation was present:. Increased Intensity of the probe was clearly visible I these areas.
lD3Sd| " hole animal ir t zM with CaMero; CaMero or Ca ero- C was inkcted Into CIFP-Lik Act mice and sections of Intestine were imaged ex vivo using multi-photos microscopy, CaMero sho ed distinctly more: intense fluorescenc than CaMero-NC in the excised tissue samples taken from, probe treated mice (fig 23a), n
Image stacks wore ac uired starting at the base of the crypt of Lleherknhn (2™ 0 uta, ¾23ii) downward, through the circular (2 -56 pPi) wd longitudinal smootfc muscle layers (-84 pm), which were visualized using GFP-LifeAei (left column). The CaMero localization pattern (right column) corresponds to the fiber direction in each muscle layer. C&Mero-NC did not clearly localise to .smooth muscle cells as indicated by .minimal fluorescence present in those areas, l¾e excised tissue mai ta ns contractility e* vivo for sustained periods. An analysis of JI uonsscenee intensity over time showed an oscillation In intensity for the CaMero probe at a. frequency consistent with that of tissue contraction. No sueh periodic oscillation was observed in the CaMero-NC treated tissue,
[11351] Biosensors applied in living cells have provided val able insight Into the dynamics of signaling networks, enabling quami!i cation o the kinetics ami localization of protein activity. That cai mdailn may play ¾c¾ diverse roles within the cell toc«t4§ i¾ 6ostt jl¾ily associated witl understanding the molecular mechanisms by bich calcium signals are integrated Into specific cellular responses. We have shown that a biosensor based o small molecule that is specific for the active state of calmodulin can be attached t&- an envitonmenpsensk dye, introduced into cells using non-iitva.sive methods and be applied therein, to monitor target protein activation dynamics,.
352) Membrane permeable dyes merolt 75 msr l68, and m«r l69 were synthesized as sbo n in schemes 8-10.
Figure imgf000128_0001
Figure imgf000129_0001
^
10353] l-et&yI-3-Cp:re 4- s!i l- I)srea ξβίϊ), Pm argyiarnine hydrochloride (0, 76 g, 10.92 mmol}- was diluted in THF (10 mL) m an oven dried flask under argon, 'Memy!anrlns (1 J mL, 11,47 mmo!) was added dropwise ana the solution was cooled. In a. cold water bath, Edayi!socyaaats (0,86 mL, 10,92 mrno!) was then added dropwase and the reaction was stirred fk 16 h, The solution was combined with half saturated aq, NHCI (15.mL> and dilnied with€¾>€¾ (15 ml,}. The aqneous layer was extacted with G¾C¾ (3 x 15 mL). Organic layers were combined, washed with brine, dried with MgSO¾ filtered, eQ¾ce»tr¾ted., and dried under vacun . Isolated 0.7.61 g (57%) white solid that required no farther
p ritaion. ¾ϊ NMR (40 MM¾:C C¾) § 5.38 it, J =· .9 ί iz, 111), 5,19 ( d-53) H . !!·¾ 3.97 td< ./ - 5.7 Hz, 20), 3.21 (do.,/- 7,3 H¾ 2ii).2.19 (p J™ 2.71¾ !Hp 1,12 (u.I -7.2 ¾ 3H). ¾ N l (100· MRz, CD ) 8158 A. gi,¾ 71.0.35.4.30.2. 15.6, MS-BS; m/k 127,1,0 (M 7- Bf requires 12709),
Figure imgf000130_0001
C«xnpound SI2 (0.708 g.5.61 wafj. & ittaloflib mid (0.61 ¾ 5,89 mmol) were added to a flame dried flask and the fl ask was sealed, evacuated, arid argon flushed. Acetic acid (8,0 mL) was added and the flask was heated to 60 ¾ for 10 Ma, Acetic anhydride (2.12: raL, 22.4 mmol) was. arlded and the reaction was further heated in 95 °i7 and. stirred for 1 h The reaction, mkture was then concentrate and the residue was loaded in a rninimnxii amount ø£ø!¾£¾ onfo a 24 g silica column and eiuted with Q ···· 2% MpOB in 01¾(¾,. Main prodnet containing fractions were conihined .and concentrated to give 0,852 (78%) amber oil ¾ MR (400 MB¾ GDCkj 64M- (d, J- 2.7211 h 3.96 (q, J S17-0, 2H1, 3.70 (a, 2;!;.222 (q J - 2.6 B¾ lii), 1.22 (t, J ■■■■ 7.0, 311). dC .(lD0 MH¾ C (¾) 8164,1, 163.9.130.6, 77,6, 71.8, 39.8, 37.7: 31,2, 13.4 S-ES! m¾ 19,5,17 ([M + Mf requires 195.08).
Figure imgf000130_0002
6,46 mmol);, and sodium acetate (0,40 g, 6.46 mmol) were diluted In acetic anhydride (7.0 ml,). The reaction mixtue was add d:o a pre-heaied oil hath at 100 °C mid stirred ibr 1 h The reaction was cooled and added in portions to saturated aq, NaHCQa (50 mL), The aq, layer was extracted with EiOAe (3 X SO n¾ The organic layes were combined, washed wife brine, dried with NaS¾ filtered, and concentrated The residue was diluted: in C¾C¾ and concentrated with Ceike. The Celite cafce was el ted on an | silica column with 0 - 5Q% EtOAe in hexgoes over 30 rate, The product, was isolate as 0.928 g (59%) yeOo solid as aft ap roximate 1:1 mmnte of Emd Z isomers. Isomer At ¾ KMi (400 ¾ C;0{¾) g 8,53 (d, J - 13,6 H¾ IK) 8.20 (ci, J- 12,4 H¾ III).7,65 ·· 7,53 (m, 3HJ, 7.26 - 7.20 (o.2H), 6.86 (dds. J™ 12.61¾ !H)54.5S (d, ,/ - ,8 H¾5. ZH), 3.99 (q? J- 7.2 !¾ 2¾ 2.1.8 (t, /- 2.4 Ik. i.H}52.00 (s, 3Ή), 1.21 (t, J · 7.2 Hz, 311), isome B: *H NMR (400 MHz, CDa3):d :g:,S2 (d, J<* 14.0 ¾, 1¾B.20 (d5..«/« 12,4 Hz, H¾ 7.65 - 7.53 (ms 3H), 7.26···· 7.20 (:«, 2¾ 6.85 (da, ,/ - 12.4 H¾ !H).4.69 (4 J - 28 H , 2H).3.t? (¾ J 7.2 Hz, 2H)S 2,14 (tj- 2,6 ¾ lHj,2.Ui (s, 3i¾ 1.13 it,-./ -7.0 H¾ 3H). MS-ESi
388.26 (fM H¾ requires 388.13).
[(B5i¾ (¾ -(^earbosyet^
!" i) mh iro rB^
d««.et itt.do B«-S«c8rb xyic; acid (SIS), Compound $14 (0.868 gs 2.38 ransoi), sodton. aceate: (0,293 g, 3,57 mrno¾ and compound 9 (1.272 ¾ .1.5? .fflffiol) wore diluted lo: 1:1 McOlTCHCi¾ (I S L, The reaction flask was topped with a condense with- gg n inlet and heated to reflux for 16. h. The reaction was concentrated and re ih.ited :BiQAc and m hnal amount of McO!I. l¾e residue was prepared: as ¾ Celite cake and elnted ors a 40 g silica column with 0 ~5¾ MeOM in C¾C¼ o er 36 min. Main product isolated as 0,352 g (29%) dark blue solid, o SH NMR (400 H¾ DMSO-d*) 512.59 (hs, 2H); 8.29 ··■■ 8.14 (m, 2H)57.99 d, J--- 1.6 ¾ 1 % 7.92 (dd. /- 8.4, .1 ,6 Hz, 1 B)? 7.83 ~ 7.73 (m5 W), 7,29 id, /- 8.4 Hz, 1 H)., 6.27 (d,J- 152 H¾ III), 4.55 s, 2¾ 4.23 (u J 6.8 Hz 2B), 3,90-3.80 (m, 2Ii). $,t i ·■■■ 3,08 inn i.% 2.66 (1, 7- 7.1 ¾ 21 n, 1,64 (ss 6H), 1.12 ··■ 1.10 (m, 3H). MS- m mA Sm jn ({M * Najf requires 528.17).
\ S7] Aeeiexyaselby! {£)»·! -S^eclnxym^^^
Figure imgf000131_0001
reaction was stirred at mom tem er t re for 16 h. The reaetfoB mixture was t¾ea. ;eoa-cen rateds re-di!aied in. md prepared as a Ce'Kte cake, The sample was eluied on a 24 g nli eotem with€ -- 50% BtOAe in hexanes over 2S mm, Isolated 0,292 g {71·%} dark bine solid. ¾ MMR (500 MHz, DMSO~d6) δ 8,28 (d, J- 13.0 H¾ 0.SH), 8.2? (d, J - 13.0 Hz, 0.5H), 8,21 (t; ,/ :- 13.0 Hz, 1 H), 8,01 (df J■■■ 1.0 H¾ W% 7.94 (dd J™ 8,5, l.S Hz, 1 H), 7J2 ¾ J- 13.3 Hz, 0,51 i 7.80 (i, 13.0 H¾, 0.SB), 7,32 (d; J- 8.5 i¾ 1 E), 6,2§ (d5 13.0 I¾ 1% 5,92 (s¾ 2H), 5,62 is, 2R;, 4.55 ( . i-2.5 H¾ 2H)5 4,25 a. /- 6.8 ¾ 2H). 3.90 - 3.80 (m, 2Β. 3,14 -- 3,10 H k 2,82 (0 ./ - 7.0 B . 2H), 2.10 (¾ 3Rh 2.0i (s, 3B), 1.64 (s, 6H). 1.12 (O J- 7.0 Hz, 1 .5H), 1 ,1 ! ¾ 7,0 I¾ I.5H), MS-ESl m/z 672,40 ( M - M i ' requires 672,22).
03581 A!taigh the foregoing sublet matter has been described Is some detail by way of Illustration and example for pm oses sf clarity of lindersiandmg, it will be
Figure imgf000132_0001
in the art; Chat certain changes and modifications can be practiced ithiiriho scope of the appended claims,
|t)359j All publications, patent appheadons, tents, a id other references re; herein incorporated by reference to the same extent as if each individual publication, paters! sppUeabon, patenx, and odier reference was specifically and i#dMd:¾&)iy indicated to be incorporated by reference. It v«ill be understood that, aUhougb a n mber of patent applications, patents, and other references are referred to herein, such reference doss not constitute an admission that any of these ocuments m& part of trie common general knowledge m he-at .

Claims

THAT WHICH IS CLAIMED:
A com o nd of Formula I or Formula II:.
Figure imgf000133_0001
Figure imgf000133_0002
herein:
•fi is an integer from to ¾
ead X is independently hydrogen or an. electron withdrawing group stkctev irom the grou consisting of earhon.yis cysrio, halogen, Pitro, siisifoiiyl, and trifluorometfryk
D is selected f orn the group. soassiRg of:
Figure imgf000133_0003
W is optorially substituted alkyi
m h> m integer ror!i 0 to 4; Y and Z are each in e endently selected. H rn: the group consisting of
Figure imgf000134_0001
Q is. as i teg r ίτοΐη 0 te 5;
U is O or S;
K4 is hydrogen or optionally substituted Q-g alkyl;
A is selected ίίόπα the gro¾p consisting oi¾
Figure imgf000134_0002
E is se;ected from he gr u consisting of;
Figure imgf000134_0003
G is Bcie ied from the gou eomismg oi;
Figure imgf000135_0001
ehain is selected
Figure imgf000135_0002
iierem:
o is an integer fitsm I to 5;
p is m irsteger from 1 to 5;
U is ϋ or S;
R5 is a leaving roup;
Figure imgf000135_0003
■]¾*„ R4, ajjd R* are each Independently C^. alkyl; arid
R* is alkvi or€F'S. 3> the compound of claim 1 or 2S. wherein at leasione of Y and s a eonjugafcahi© s de chain and the compound eoniams only one eonjogataMe side chain,
4, The compotmd of any one of cl ims 1 -3:, wherein the Y in the D or E moiety is a eonftigatahJe side chain and at least one Z Is
Figure imgf000136_0001
5. ¾ compound ox any one of claims 1-3, wlierem the Y in the D or E moiety is hydrogen i one Z is a confn atable side chain.
6. The compound, of any cms of claims 1-3, wherein the Y in the D or E moiety is hy drogen and at leas one Z is
Figure imgf000136_0002
7. The compound of any one of claims. 1 ~ wherei the Y in the D or E moiet is h drogen one Z is a com ug abl side chain, and. at least one Z is
Figure imgf000136_0003
. The compound of any one of claims 1-3, wherein the ¥ in the D or E moiety ari at least one Z are
Figure imgf000137_0001
9. The βόϊ¾. β'ί.5Πί| of any o¾e of daims l~% wherehi one E is a cory¾gatab!e side ohaia ani the Y k lre D or IS moiety and at feast oiss Z e
Figure imgf000137_0002
10. The com ound of any one of da ns i-¾ wftereiB the eompou comprises at least two Z: moieties that are
Figure imgf000137_0003
!L The coMpouri.il of any oris of claims !-lO, wfvef^fttlie οδήιροοΓκ! co ises at least three moieties that are
Figure imgf000137_0004
12... The coffi Q imd of any ne of elairns 1-11>. w eein onl one K s s electron thdawin group.
13, The: Hiehsd of daf 1 or 2 , feerekt :
Figure imgf000138_0001
A is selected from the grouts eenssstitsg i
Figure imgf000138_0002
The method of cl im 13, w ereis D is
Figure imgf000138_0003
The met od of claim !4S wherei¾ E' is I,
I B, T¾$ meth&d of 1. ¾ hereia the compo nd is sclestc<t fr©ai;
Figure imgf000138_0004
Figure imgf000139_0001
Figure imgf000139_0002
Figure imgf000139_0003
I ?<: The met od i dai.ni 1 , w&erdii;
Figure imgf000139_0004
A Is:
Figure imgf000140_0001
i , Tlia met¾od of claim IT* wiherelfi, at. least one Ύ is:
Figure imgf000140_0002
9:. The metkoi i claim 18, wherein &e: compoimd seleced from:
Figure imgf000140_0003
Figure imgf000141_0001
Figure imgf000141_0002
Figure imgf000141_0003
and
Figure imgf000142_0001
20, A biosensor comprising the eompoiu o of any one of claims 1 9 conjugated to a binding member having an affinity lor a target molecule,
21, l¾e Biosensor of claim 20, wherei the compound is conjugated to the binding member feough. a cor 'of alab!e side enain on the compound.
22, The biosensor of claim 20 or 2 J s wherein the binding mem er is selected from the group consisting of a rucleic aeid, a polypeptide, and a binding protein,
23» The oaeasor of any one of dalm 20-22, erein the binding member a binding domain comprising at least one of an aptarper, a eomplemmtary determining region (€DR>, a VR region, a VL region, a Fv fragment an F(ab) if agme i , an F(ab¾ fragment., an antibody, an. antlBody: fragment, a leucine zipper, a histone, an enhancer, a single chain variable fragafien (soFv). a li nd, a receptor, one p t& in a protein complex:, a lectin, and combinations 'thereof.
24. The Biosenso of a y one of claims 20» 5 wBerern the target molecule, is selected from the gron consisting of a: m kic acid and protein. 2015/103587 . .... PCT/US2015/010269
25. 'Ti . biosensor of any one of claims 20-24, wherein the binding member binds to the target molecule when the target molecule is is a specific conformation, is bound to a specific iigand or has been postirans!ationalty modified,
26. The biosenso of any one of claims 20-25, ¾dicrelE the target molecule has been postttandationall modified by phosphorylation,
27. The: biosensor of any one of claims 20-26, wherein tt e eon onnd of Formula I or Formula I F is conju ate with the binding member at a position on the bindin member selected tmm the gm consisting of a cysteine, a l s ne, an arglriine, a naatra! aits ino acid side chain, a erivatke .amin - acid -s& ^. and an unnatural amine aci side chain,
2B< he biosensor of an one of claims 20-27,: wherein two or mote compounds of Formul I or Formula H are conj gated with the binding member,
29, A biosensor device comprising the biosensor of any one of claims 2(h28(
30, A teagen i for deierm ining the presen ee or amount, of one o more ta get molecules in a sample,, the reagent comprising die biosensor of any one of clanns 20- 28.
31 » A Mi for determining the presence or amount of one or mote ta et oiecuies In a sample, die ki comprising tire biosensor device: of claim 29 or the reagent of claim 30..
32, A method for determining the presence or amount of one o more target molecules in a -sample, the method comprising:
(a) providing the biosensor any one of claims 20-28;
(b) contacting the biosensor with a sample suspected of containing one or more target molecules to bind the one or more target molecules, if present, to the binding member: (c) irradiating the sample suspected of coataln'mg one or mote ta get molecules with electromagnetic radiation to induce the cc po iad of Formaia I. or
Formula II to flu resce; and
•(d) detecting a fh-rorescenee property of the compound of formula. I or Formula II to deiennine the presence or amount or one or more target molecules in the sample.
.13. The melted of claim 32, further comprising:
(a) measuring a. ilnoreseerme intensity M. a first: emission wavelength before
contacting the biosensor ll a sample suspected of containing one or more target molecules;
( ) measuring a fluorescence intensity at a second emission wavelength after contacting the osense with a sample suspected of containing one or more target molecules; and
(e) determinin a ratio of the second emission wavelength to the first emission wavelength to determine the presenee or amoirat o:f one or more target molecules in the sample,
34. The method of cl&m* 33, iuiil er comprising continuously;
(a) contacting the biosensor w th the sample sus ecte of containing one or m ore target molecules;
(b) irradiating the sample "wi†¾ elecu'ornagnetio adiati ns and
(el detecting the fluo escence property of the eompound of Formula 1 or Fonrmla If
35 , A. method of detecting an activity or & looatioo of one or more target molecule within a ceil the method eonmrlsing::
(a) providing the hioseasor of any one f claims 2Q-2 ;.
w eomacting: the iosensor with cell, suspected of containing one or more target molecules to bind the one or more target molecules, if present, to the binding member. (e) Irradiating the cell suspected of containing one or more target molecules with
■electromagnetic radiation to induce the compound of formula I or Formula II to fluoresce; and
(d) defecting one or more of;
(0 a fluorescence p erty of the compound of Formula I or Formula II;
(is) a change a a xlnorescence roperty of the compound of Formula 1 or
Formal a II
(iii) a location of a fjoorescenee of the c mp und of formula I or Formula II; mid
(iv) combinations thereof
to determine th m activity or location of one or more target molecules in the cell,
36. The met o of cla m 35, wherein the targe molecule is selected fr m t e group consisting of a protein, a receptor, a Sgand, and an e zyme,
31, The method of claim 35, wherein the ceil is selected from the group consisting of a li ving cell, ¾ cell lysate, a cell library, o a cell culture.
38. The method of claim 3SS wherein the activity or location of the one or more target molecules is selected ftorn the group: consisting of a phosphorylation state, a su cellular location, an interaction with one or morn subcellular structures, and an Interaction with one or more: Cellular proteins,
39. A method of detecting: an interaction between an myogenous target molecule and ;a cellular entity, the method comprising;
(at provldm a coll coniptising an ©ndogetio s tar$eimokeu!e;
(h) providing the biosensor of any one of cl ims 20-28;
(c) observing a haekgroimd fluorescence signal from the biosensor;
( ) contacting, the biosensor with the cell;, and
(c) detecting a change In fluorescence ίϊήηχ the biosensor to Indicate an
interaction between the target molecule and the cellular entity.
40.. The method of cl im 3-9» wherein the cellular en ty' ¼ selected from the group consisting of a cellular nucleic acid, a protein, a peptide, an en ym , a receptor, a cytokine, a cyta skeleton, and a signal: tisfisdyctino protein.
41 , The method of claim 39. wherein the binding membe of the biosensor binds fes target molneule at a phosphorylation site,
42. Tire method of claim 39. wherein the binding member m a specific affinity for a c Bforrnatlon, a ligand intaraotion, or posttranslationai modification of the target molecule,
43., The method of claim 3% wherein the com oun of Formula.1 w Fornmlg II is linked to the binding member at a site h¼t does not: substantially interfere with binding between the probe and the target biomoleenle.
44, The metitod of cla m 43, wherein the site is selected, by an examinatio of a cr st l structure for the binding membe or the targe mo ecule.-
45> The method of claim. 39. further comprising introducing the biosensor into the ceil by using- a teehmqae selec ed from; the group consisting of dec roporaTiorc transduc ion,: mloroporatiom, and mleroinieetinn.
46, Tkt- method, of claim 39f wherein the detecting a chang in II uoresceftce comprises quantifying s protein amom t locating a -protgin, detecting a
eosfbrmational change In rise tar get molecule, detecting activation of the target molecule, or detecting phosphorylation of he target molecule.
47 , A kit com risin thn biosensor of any one of claims 20-28 for detecting, monitoring or observing a target molecule, wherein the hioaensor comprises a compound of Forumla ! or Formula II and a binding member hav ng a specific affini y for the target .molecule. i 45
48. The kit of dai 4? i ri er comprising instructions for usin the biosensor to detect, monitor, o observe a target molecule.
49- Λ biosensor comprising a compound: having ait finity for a target molecule corrugated to a dye.
50, The biosensor of claim 4¾ wherein the compound has a moiecular eight less to -about 1000 Da,
$ 1 < Tlie biosensor of claim 4 or SO, wherein the dye ¼ a cell membrane permeable dye.
52.; The biosensor of any one. of claim 49-51 , wherein the dys cm be visualized by microscopy.
PCT/US2015/010269 2014-01-06 2015-01-06 Improved environment sensing cyanine and merocyanine dyes WO2015103587A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201461923937P 2014-01-06 2014-01-06
US61/923,937 2014-01-06

Publications (2)

Publication Number Publication Date
WO2015103587A2 true WO2015103587A2 (en) 2015-07-09
WO2015103587A3 WO2015103587A3 (en) 2015-09-11

Family

ID=53494239

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/010269 WO2015103587A2 (en) 2014-01-06 2015-01-06 Improved environment sensing cyanine and merocyanine dyes

Country Status (1)

Country Link
WO (1) WO2015103587A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106565697A (en) * 2016-10-20 2017-04-19 郑州大学 Lysosome positioning fluorescence probe, preparation method, and application in arginine detection in near infrared ratio

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10313173A1 (en) * 2003-03-25 2004-10-07 Bayer Chemicals Ag Optical data carrier with polymer network in the information layer
EP1735453A2 (en) * 2004-03-12 2006-12-27 The Scripps Research Institute Fluorescent signal emitting live cell biosensor molecules and dyes for detection and quantification of protein activities
WO2008027075A2 (en) * 2006-02-16 2008-03-06 The Scripps Research Institute Sulfhydryl-reactive, water soluble dyes
WO2008157762A2 (en) * 2007-06-21 2008-12-24 The University Of North Carolina At Chapel Hill Methods to increase the photostability of dyes
CN104395407B (en) * 2012-03-30 2016-07-13 生命科技公司 For detecting the cyanine dye of the modified bind nucleic acid of active oxygen

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106565697A (en) * 2016-10-20 2017-04-19 郑州大学 Lysosome positioning fluorescence probe, preparation method, and application in arginine detection in near infrared ratio
CN106565697B (en) * 2016-10-20 2019-02-26 郑州大学 A kind of lysosome positioning fluorescence probe, preparation method and the application in near-infrared ratio test arginine

Also Published As

Publication number Publication date
WO2015103587A3 (en) 2015-09-11

Similar Documents

Publication Publication Date Title
Tiede et al. Affimer proteins are versatile and renewable affinity reagents
Shi et al. Label-retention expansion microscopy
AU2017248165B2 (en) Ultra bright dimeric or polymeric dyes with spacing linker groups
Esposito et al. α-Synuclein and its disease-related mutants interact differentially with the microtubule protein tau and associate with the actin cytoskeleton
CN101946171B (en) Fluorescent compounds
US9688743B2 (en) Optical biosensors
JP2022078077A (en) Recognition of cellular target binding by bioactive agent using intracellular bioluminescence resonance energy transfer
Klima et al. Incorporation of sensing modalities into de novo designed fluorescence-activating proteins
CN109553687B (en) Fluorescent probe constructed based on G protein coupled receptor
Hussain et al. One-step site-specific antibody fragment auto-conjugation using SNAP-tag technology
JP2016014033A (en) Quenched dendrimeric dyes for florescence detection
Petazzi et al. Fluorescence microscopy methods for the study of protein oligomerization
CN114539183A (en) Lipid drop targeting and biological thiol sensitive fluorescent probe for cancer cell tissue diagnosis, preparation and application
Sakamoto et al. Whole-Body and Whole-Organ 3D Imaging of Hypoxia Using an Activatable Covalent Fluorescent Probe Compatible with Tissue Clearing
WO2015103587A2 (en) Improved environment sensing cyanine and merocyanine dyes
WO2015102541A1 (en) Optical biosensors for diagnosis and high-throughput drug screening using unique conformational changes of recombinant tagged g protein-coupled receptors for activation
Hovan et al. Förster resonance energy transfer as a tool to study photoreceptor biology
Rode et al. Generation and characterization of virus-enhancing peptide nanofibrils functionalized with fluorescent labels
Lü et al. Live-cell fluorescence imaging of ciliary dynamics
ES2739525T3 (en) Methods to detect interactions in eukaryotic cells using microtubule structures and dynamics
Alam et al. Detecting RNA–protein interactions with EGFP‐cy3 FRET by acceptor photobleaching
Adoff et al. Ligand-directed labeling of opioid receptors for covalent attachment of fluorophores or small-molecule probes
Veshaguri et al. Developing an Assay to Probe Activtion and Conformational Dynamics of β2-Adrenergic Receptor on Single Molecule Level
CN108047241B (en) Spiropyran derivative and application thereof in detection of G-quadruplex DNA and lysosome
Cisneros Use of Enzymes for Amplified Signal Generation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15733108

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15733108

Country of ref document: EP

Kind code of ref document: A2