CA2122627C - Long wavelength chemically reactive dipyrrometheneboron difluoride dyes and conjugates - Google Patents

Abstract

This invention relates to derivatives of dipyrrometheneboron difluoride fluorescent dyes that have an absorption maximum at wavelengths longer than about 525 nm, and are chemically reactive with nucleic acids, proteins, carbohydrates, and other bio-logically derived or synthetic chemical materials. The dyes generally have structure (I), wherein at least one of the substituents R1-R7, is a reactive functional group, and at least one of the substituents R1-R7 contains a bathochromic moiety. The ba-thochromic moiety is an unsaturated organic group, preferably heteroaryl or alkenyl. The remaining substituents, which may be the same or different, are hydrogen, halogen, alkyl (containing 1-5 carbon atoms), aryl, arylalkyl, or sulfo. The dyes are used to make novel conjugates with members of specific binding pairs that are ligands or receptors.

Description

IZCV. W>.~:: Et'r~-VtIEVCF(E:.~s U(~ : _~U-L()-~J;3 : ~? t :48 : 5U:3 :3~E-1~
65().x~ +~6J F3J ~la;3J4~fcS: N 6 LONG WAYELE.VGTH C,H>EMICALLY REACTIVE plpYRROMETHENEBORON
DgT.UORmE DYES AND CONII1GATE5 FIELD OF THE INVENTION
- This invention relates to chanically reactive flaoresceat dyes that can be attached to ligands and the re,ulting fluot~sceatly labeled ligaads. In particular, the invzadoarelates to chemically reactive dym that are derivatives of dipyrrbmttheaeboron difluoride dyes (derivatives of 4,4-diflnoro~l-born-3a,4a-diazs-s-inda~ctnt dyes) that have as absorption mazimuat at a wavelength looser rhea about 525 ~ These reactive dyes are used for the Ruorescent libeling of nucleic acids, auelootidcs, carbohydrates, drugs, polymers, proteins, and other biologically derived or synthetic chemical matesiats. , 1s BACKGROUND OF THE lh'VEhZ'ION
Fluoreactat dyes have many neon and are lmown to be particularly suitable for biological applications in which the high detectability of fluoresct;aee is required. Fluorescent dyes are used to impart both visible color send fluoresc~ace to other materials. Many applications utili~
chenucally tractive flaoresceat dyes by chemically attuchinE the dye to react'sve sites on a wide variety of materials such as calls, tissues, proteins, ~tibodies, enzymes, drugs, hormones, lipids, nucleotide', nucleic acids, or natural or synthe;io polymers to make ffuoresceat conjugates. With these synthetic probes, ligands are frequently used to confer a specificity for a biochemical reaction that is w be observed and the fluoxesceat dy provides the nxans of detection. or quantitation of the interaction.
Fluorescence useful for such applications is yenerilly initiated by absorption of light from an external, relatively concentrated light source. The sensitivity of these applications is improvod by having dyes ~ that have high absorb~ce of the exciting light and high fluorescence quantum yield. The apylicaboess are funherrno~re improved by having dyes that resist pbotobleachiag by the exciting light and that have spectral wavelengths in a range that avoids the background from contaminants that any be prfsaat in the samples. For many biological applications it is useittl to have dyes whose fluorescence is ao~ quenched by water, since most biological measurements are made; in aqueous solution.
Certain lasers are particularly useful as a concentrated light source for the excitation of fluorescence.
Ths argon laser has been the most cotamoo. light source for excitation of fluorescnnco, with principal . output at 4$$ am and 514 arn. Now other Ltsera are iacreasiagIy used, such as helium-nt~oa lasers that car be selected to have ma~cimum output at either 543 um, 594 ~, or 633 um;
the krypton laser which bas significant output at 568 am and 647 urn; and light emitting diodes w~ch era available at this time with output commonly above 660 um; resulting in increased demutd for longer wavelength fluorescent dyrs.
~U~3TiTi~T~ ~~~~~°~

2 1 2 ~ ~ 2 "~ PCT/US92/08350 A number of dyes that have previously been found to be fluorescent do not have significant sbsorbance at desired longer excitation wavelengths. Many also have other characteristics which interfere with or limit their usefulness. For example, many known fluorescent dyes are significantly quenched in aqueous solution or are unstable doting the illumination.
Dyes derived from dipyrrometheneboron difluoride have many desirable characteristics. Simple alkyl derivatives of the fluorophore 4,4-difluom-4-born-3a,4a~iarsv-s-indac~e have been described by Treibs & Kreuzer, Difluorborvl-komulexe von dl- and tripygylmeth~en, LIEBIGS ANNALEN
CHEM. 718, 203 (1968) and by Worries, Kopek, Lodder, & Lugtenburg, 8, novel water-soluble fluorescent probe:
~",tius;c tnmine~ac~rr and blolOg'1~~ ~f~9'lCrtICB Of the sodium salt of the 4-sulfonato-3 3' S 5'-tetramethvl-2.2'-pyrromethen-1.1'-BF~comnlex, RECL. TRAV. CHIM. PAYS-BAS 104, 288 (1985) as being highly fluorescent with spectral properties that are similar to fluorescein, with maximum absorbance at about 490 to 510 nm sad maximum emission at about 500 to 530 nm.
U.S. Patent No.
4,774,339 to Haugland et al. (1988) ('339 patent) describes 4,4-difluoro-4-born-3a,4a-diary-s-indacene (dipyrrometheneboron difluoride) dyes including hydrogen, halogen, alkyl, cycloalkyl, aryl, arylalkyl, aryl, and sulfo-substituted derivatives that contain reactive groups suitable for conjugation to biomolecules, that have good photostability, sad which have fluorrscein-like spectra. As described in the '339 patent, and by Pavlopoulos, et al., baser action from a tetramethvlovrromethene-BFI, complex, APP. OPTICS 27, 4998 (1988), the emission of the alkyl derivatives of 4,4-difluoro-4-born-3a,4a-diara-s-indac~e fluorescent dyes clearly overlaps that of fluor~escein. The overlap allows the alkyl derivatives of dipyrrometheaeboron difluoride to be used with the same optical equipment as used with fluorescein bssed dyes without modification of the excitation sources or optical filters.
As a result of having the same spectral characteristics, however, the fluor~esceoce of the known class of alkyl-substituted 4,4 difluoro-4-born-3a,4a-diaza-s-indacenes is not readily suitable for detection in combination with fluorescein or for use in applications where excitation by longer wavelength sourrxs such as the helium neon or krypton lasers or light emitting diodes is r~equir~ed.
Although the '339 patent discloses some dyes with an absorption maximum of greater thaw 525 nm (Table 5), the '339 patent is neither enabling nor prior art for the invention of the subject long wavelength reactive derivatives of dipyrmmeth~eboron difluoride dyes. Of the longer wavelength dyes listed, all were measured in chloroform which slightly enhances the spectral absorption sad shifts the emission maxima to longer wavelength by shout 10 nm (compared to methanol). The '339 patent also discloses a non-reactive dye having an absorption maximum greater than 600 nm (cmpd. 27) which has been found to have as undesirably low quantum yield and very broad absorption and emission spectral band widths.
U.S. Patent 4,916,711 to Boyer, et al. (1990) ('711 pat~t) discloses a method of using derivatives of 4,4-difluoro~-hors-3a,4a-diaza-s-indaceaze dyes, in particular symmetrical ailryl and suifonatad alkyl derivatives, as laser dyes. The '711 patent also discloses a multitude of possible alternatives for substituents of the basic tricyclic structure which can be used for the patented method. The '711 patent, however, is neither enabling nor prior art for the invention of long wavelength reactive derivatives of dipyrrometh~eboron difluoride dyes. The dyes described in the '711 patent are not reactive dyes. In addition, the '711 patent neither recognizes nor recites the effect or advantage of substituents that enhance the long wavelength fluorescence properties of such dyes.
Inventors Kang and Haugland have described various substituted dipyrrometheneboron difluoride dyes and other materials incorporating long wavelength dipyrrometheneboron difluoride dyes but none of these long wavelength dipyrrometheneboron difluoride dues are the chemically reactive dyes of this invention.
The novel dyes described in this invention contain one or more substituent groups coupled to the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene ("BDI") fluorophore resulting in significant absorbance and fluorescence at desired longer wavelengths. The dyes of this invention also have the chemical reactivity necessary for conjugation to the reactive sites commonly found in biomolecules, drugs, and natural and synthetic polymers. The reactive dyes of this invention are combined with a variety of materials to form novel dye conjugates. The novel dye conjugates are desirable for use in combination with conjugates formed from other fluorescent dyes such as fluorescein or alkyl-substituted 4, 4-difluro-4-bora-3a,4a-diaza-s-indacene dyes in that the fluorophore of the novel conjugates can be both selectively excited and detected because of their spectral shift to longer wavelengths, particularly an absorption maximum at greater than 525 nm and an emission maximum at greater than 550 nm. The new reactive dyes have the advantage over other dyes that absorb at these wavelengths, of being electrically neutral, photostable and being, in most cases, highly fluorescent in both organic and aqueous solution with relatively narrow absorption and emission spectra.
SUMMARY OF THE INVENTION
In accordance with one aspect of the invention, a compound of the formula:

R5 \ N. .~R2 R F~B~F R 1 is provided wherein at least one of the substituents R~-R~ is -LmG. where G is a reactive functional group that is a carboxylic acid, a succinimidyl ester, an acyl azide, and RUG 28 2001 89:83 FR ~J. GEORGIR URNCOUUER04 682 8274 TO 18199532476 P.03i03 3a anhydride, an acid halide, an acrylamide, an alcohol, and aldehyde, an azide, and imido ester, a sulfonate ester, a haloacetamide, an alkyl halide, a sulfonly halide, a hydrazine, an isocyanate, an isothiocyanate, or a naleimide group and -f.m, where m = 0 or 1, is a substituted or unsubstituted alkyl (containing 1-5 carbons) or arylene gr4up, or a linking bathochromic moiety; and wherein the remaining substituents R,-R7, any of which may be the same or different, are hydrogen; halogen; alkyl (containing 1-5 carbon atoms); sulfa; arylalkyl, the alkyl portion of which contains 1-S carbon atoms; or aryl which may be further substituted, one or more times, by alkyl (containing 1-5 carbon atoms), or alkoxy groups wherein the alkyl portions of such groups have less than 5 carbon atoms; or a Separate bathochromic moiety; or combinations thereof;
such that at least one of the substituents R~-Ra contains a linking bathochromic or a separate bathochromic moiety that is an ethenyl, dienyl, trienyl, or beteroaryl group that is attached to the compound by a single covalent boztd;
said compound having an absorption maximum at a wavelength longer than about nm in methanol and a quantum yield or heater than about 0.1.
Irl accordance with another aspect of the invention, a dye-conjugate comprising a specific binding pair member sbp and one or snore dye molecules of the formula:
R

\' R
RB ~ t~-.~ ~- 2 ~t is provided, wherein at least one o~ the substituents R,-R7 covalently binds each dye molecule to the specific binding pair member;
** TuTAL PAGE.03 **
ia? 28/08/2001 ~12:U4 r~>tiU4 fi82 U2~f4 i0received 3b wherein, further, at least one of the substituents R~-R~ contains a linking bathochromic moiety or separate bathochromic moiety that is an ethenyl, dienyl, trienyl, or heteroaryl, and which is substituted or unsubstituted; and the remaining substituents R~-R~, any of which may be the same or different, are hydrogen; halogen; sulfo or a salt thereof;
cycloalkyl; alkyl or arylalkyl, the alkyl portion of which contains 1-5 carbon atoms; or aryl, which may be further substituted, one or more times, by alkyl or alkoxy, the alkyl portions of which have less than 5 carbon atoms; or combinations thereof.
In accordance with another aspect of the invention, a dye-conjugate comprising a specific binding pair member sbp and one or more dye molecules of the formula:

R8 \ N.. \ R2 R lrBlF p is provided wherein at least one of the substituents R~-R3 covalently binds said dye molecule to the specific binding pair member that is a nucleotide or an oligonucleotide;
wherein, further, at least one of the substituents R~-R3 contains a liking bathochromic moiety or separate bathochromic moiety that is an ethenyl, dienyl, trienyl, or heteroaryl, and which is substituted or unsubstituted; and the remaining substituents R~-R7 , which may be the same or different, are hydrogen; halogen; sulfo or a salt thereof;
alkyl or arylalkyl, the alkyl portions of which contain 1-5 carbon atoms; or aryl, which may be further substituted, one or more times, by alkyl or alkoxy, the alkyl portions of which have less than 5 carbon atoms; or combinations thereof.
In accordance with another aspect of the invention, a dye-conjugate comprising a specific binding pair member sbp and one or more dye molecules of the formula:

R5 ' \ \ 3 R \ ~ ~~R2 R

3c wherein at least one of the substituents R~-R3 covalently binds said dye molecule to the specific binding pair member that is a protein;
wherein, further, at least one of the substituents R~-R3 contains a linking bathochromic moiety or separate bathochromic moiety that is an ethenyl, dienyl, trienyl, or heteroaryl, and which is substituted or unsubstituted; and the remaining substituents R~-R~, which may be the same or different, are hydrogen;
halogen, sulfo or a salt thereof; alkyl or arylalkyl the alkyl portions of which contain 1-5 carbon atoms; or aryl, which may be further substituted, one or more times, by alkyl or alkoxy, the alkyl portions of which have less than 5 carbon atoms; or combinations thereof;
where each of said dye molecules, when not conjugated to the sbp, has an absorption maximum at a wavelength longer than about 525 nm in methanol and a quantum yield of greater than about 0.1.
DESCRIPTION OF THE DRAWINGS
Figure 1 is a general reaction scheme for synthesis of long wavelength reactive BDI dyes. The general method consists of a formation of pyrromethene intermediates followed by cyclizaton with boron trifluoride in the presence of base to give substituted dipyrrometheneboron diifluoridedyes.
Figure 2 is a graph of the relative spectral separations of selected examples of long wavelength reactive BDI dyes in comparison with an alkyl-substituted BDI
dye, in pH 7 phosphate buffer solution.
Figure 3 is a graph showing the relatively narrow emission band width of a selected long wavelength reactive BDI dye in comparison with another known dye emitting at the same wavelength (in methanol, excited at 540 nm).

Figure 4 is a graph showing the increased photostability of representative long wavelength reactive BDI dyes in comparison with an alkyl-substituted BDI dye, in CH3CN solution.
DETAILED DESCRIPTION OF THE INVENTION
This invention describes novel fluorescent dyes that are reactive derivatives of the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene ("BDI") fluorophore that can be chemically reacted to bond with the reactive sites present in many materials to form novel fluorescent conjugates.
The BDI dyes generally have the structure of formula I:

R
\ ~Bs~R2 p7 Fr F R

At least one of the substituents R1-R7 on the BDI fluorophore, is -LmG where G is a reactive functional group that allows the BDI fluorophore to be coupled to other synthetic or natural molecules. Preferably there are one or two reactive functional groups on the fluorophore; more preferably only one reactive functional group.
The most common reactive functional groups are carboxylic acids and derivatives thereof including succinimidyl esters, acyl asides, anhydrides, and acid halides.
Other reactive groups include acrylamides, alcohols, aldehydes, amines, azides, imido esters, sulfonate esters, haloacetamides, alkyl and aryl halides, sulfonyl halides, hydrazines, isocyanates, isothiocyanates, and maleimides. Table 1 lists common reactive functional groups and the reactive sites with which they most commonly react. The tabulation is not all inclusive since with the appropriate choice of solvent, temperature and catalysts, other functional groups can be made to react.

4a REACTIVE GROUPS REACTNE SITES

acrylamides olefins acyl azides amines, thiols alcohols acid derivatives aldehydes amines, alcohols amines carboxylic acids, halides, aldehydes and ketones anhydrides amines, alcohols azides photoaffinity reagents carbonyl halides amines, alcohols, phenols, thiol halides alcohols, amines, thiols, carboxylic acids haloacetamides thiols, imidazols, phenols, amines hydrazines aldehydes, ketones, acid derivatives imido esters amines isocyanates amines, thiols, alcohols, phenols isothiocyanates amines, thiols, phenols maleimides thiols, amines succinimidyl esters amines, thiols sulfonyl chlorides amines, phenols, alcohols sulfonyl fluorides active sites of esterases The reactive functional group is directly attached to the BDI fluorophore or is attached through a spacer -Lm-, where m = 0 or 1. When the reactive functional group is directly attached to the BDI fluorophore, m = 0 (L is absent). The spacer L
is a substituted or unsubstituted alkyl (containing 1-5) 2122~~'~
carbons) or aryl group. Alternatively, L is a linking hathoehremie meiecy i.e.
a bathochromic moiety that links the fluorophon and a reactive functional gmuh such that the reactive functional group is s aulxctitu~snt on the linking bathqchrotaic moiety. Proferably L, when present, is a straight chain unsubettitutad alkyl group that contains 1-d carbons, an unsubstituted phenyl group. or a linking S bathochroenie moiety. In ox embodiment of the invention. L is a straight chain unsubstitutesd alkyl group containing 1-5 cat~ons and G is a cucciairnidyi taster.
At least one of the subatitttents R~-R, contains a hathoehrorttie moiety. The inclusion of a bathochmtnic moiety in any rrttbstituent shifts the fluorercence of the compound toward the rtd part of the spectrum sad gives the fluctrnphore an absorption tarutimum of greauet than about 525 an:. 'typically the batbochsomic moiety also ,gives the flttorophora as emission matcimum greater tbaa about 550 am.
Typically, the bathachramic moiety is an unsaturated organic group such as a substituted or unsubstituced ethemyl, dianyl, trienyl or heteroaryl group.
A bathochromic moiety ttsay be presort as a subsatituent separate froth the reactive functional group (separate bathochrontic moiety). At least orw, and as many as siz, of the substituants, can be separate bathochmmic moieties any of which they be'ehe same ar different. A
bathochromic moiety they. also or altnmettive;ly be included in the spacer (L) cottaectiag the reactive functional group to tho BDI fluorophore (tinlcing bethuchromic taoiety). One or more bathexhiotnlc moieties may he included only as linking moieties, or only separately, car cnmhinstioas thereof, but a bathochromic moiety must be included in at least one of the substituentr R,-R,. With seven subslitueat locations, there they be as rrsny ax seven bathochroenic r~ieties, some or all of which may each be the same or dsfFersttt. Vb'here the dye compound contsina seven bathochromic moieties, at least ~,ne bethochromic moiety and as many a seven centaiu(s) a reactive functional group. Ptefenbly there are no more than four bnthnchromic moieties included in the substihsents R,-R,, souse or all of which may contain reactive functional groups as aubstituents. Typically, because of the symmetry, at least one of the: groups R,, R;, R,, or R, contains a bathochrotaic moiety, which may contain a rextive functional group as a substituent. For east of synthesis, the bathocframic moiety is preferably present as a ruhstituent separate from the reactive group.
In eithra case, the batltochrnmic moiety is directly attached to the BDI
fluorophore by a covalent hoed.
In one emboditner~t of the invcntien, theta is a singly bathochrotnic moiety that is heteroaryl. '!he trrtst heteroaryl, xa used throughout this document, means :n aromatic heterocyclic substituant. When a heteroaryl group is piraent as a subaittunt, it is art aromatic heterocyclic moiety that contains at last one and as trtnrty as three hNero atom (a non-carbon atom forming the ring structuts) that is N, O, or S. The hetuoaryl group stn contain one, two, or three hatrrc~ stems. The heteroaryl group can be a tangle ring :fracture or a fused two- er three-ring structure. A ring can be a 5- or 6- membered ring.
Examples of heteroatyl substituents an pyrrok, thiophate, or fursirt (single ring, single helero atom), or oaszole, isoxa~tole, oxadiazote, or imidasale (single ring, multiples 6etesro atoms). Alternatively, the beteroaryl group is a multi-ring structure wntainit:g onC ur mare hClcrm atumtr fur esatnple, the heteroaryl subatitueutt is benzoxntole, benzothia~la, or hen zirnidanole (multi-ring, oral tiple hetero atoms).
or best~furan or indole (rttalti-ring. single hetero atom). The term hetemary) incttrdes substituted derivatives of the heter~oaryl substituentx, such as alkyl-, aryl-, arylalkyl-or 6etamaryl-substituted ~~~~~~~ ~r~.y'tu pyrrolyl, furyl or thieayl. The bathochromic beteronryl group airy ulyu contain a reactive functional group xs a substitueat.
In another embodiment of the invention, the single bathochronvc moiety is an alkeayl gmup.
Preferahly the alkenyi group is ethenyl. dienyl or trieayl. The alkcnyl group is unsubstituted or optionally contains substituents, which mny be the carne or diffarant that are hydrogen, halogen, alkyl (containing 1-5 carbon atoms), cyano, carbozylate ester, carbbzanude, aryl or hetzroaryl, or polyethrnyl !o Corm a conjugated dieayl or tcie!ay1 cubstituenr, w~hic6 in tu,ra racy be substituted with substitwnts, which nay lx th. carne or different, that ire hydrogen, halogen, alkyl (containing 1-5 carbon atoms), cyaoo, ester, amide, aryl or hskeroaryl groups. In one embodiaxnt nt least one of the uocaturated organic 1t) groups is an alkeayl group that is etheayl, butadieayl or hezatriatyl, that optionally contains subatitueata, which may be the satfle or different that an hydrogen, halogen, alkyl (containing 1-5 carbon stoma), cysno, carbozylate ester, carbozamide, aryl or heteruaryl. The bathvchromic alkenyl group may also contain a reactive functional group as ~ substituent.
In yet another embodiment of the invention, the fluorophore has 2-d bathochnoauc substihieats, which may be the aatrre or different, that aro substituted or tmsubstitutec!
ethenyl, dianyl, trienyl, or haternuyl groups. One or two of the bethochromic substituants may contain reactive functional groups, which array be the sttnae or different, xs substituents.
'The t~emaiader of the substituents R,-R, that are neither reouivs functional grtrups nor bathochromic tubstituents, which retraining substitunntx rruty txs the same or differznt, are hydrogen, halogen, cycloalkyl or alkyl (containing 1-5 carbons), aryl, arylalkyl {the alkyl portions of which contain 1-5 carbon atoms). or sulfo, alone or in enmbsnYtian, as long as they do not destroy the reactivity of the dyes.
Prefernhly, the remaining substituents ate hydrogen. In one embodiment of the invention, either R, or Rc, or R= sad Rd, and sul fo.
preferred aryl groups jn any of the described subctitueats are phenyl, 1-naphthyl, 2~aphtliyl, 1-pyrenyl, 9~anthryl, std their alknzy suhstitutadderivativts wherein the xlkyl portions of such derivatives have less than 5 carbon atoms. Any aryl nr 6eteroaryl group in any aubxtitusnt may be further substituted one or er~ore timer by alkyl (evnlaining 1-5 carbons): or alkozy groups, the alkyl portions of which have leis than 5 carbon atones: or combinations thereof. Any alkyl group in any substitueai may be fruitier substituted by an ester or anode substitueat.
Tsble Z contains a ample of representative long wavelength reactive dyes and their key precursors.
The examples are preferred, representativE compounds only and are not intended to be exclusive.
~UA9TITUT'~ ~~~T

mz~s~7 Table I
R; R= R, R, R, R7 pound ' 1 (CH,hCO,CH, H H H H THI*

Z (CHzy,CO,H H H H H THI*

3 (CH~,COSucc* H H H H TH1*

4 (CHI=C(=O)NHNH; H H H H THI*

8 (CH~CO,CH, H H H H FUR*

6 (CH~CO,H H H H H FUR*

7 (CH,hCO,CH, H H H H PYR*

8 (CH~CO,H H H H H PYR*

9 (CH~C0,,5ucc* H H H H PYR*

CH3 (CH~l-CO=-CH, H H PYR*
CH, 11 CH, (CHI=- CH, H H PYR*
CO=H

11 THI* ' N . H (CHI:-CH, CH, .
CO;CH, 13 ' THt* 1i H (CH,)1CH, CH, COiH

14 (p)-CtH,-OCHzCOzCH,H H H H THI*

(p)-Chi,-0CH,CO,H H H H H THI*

16 (p)-C,H,-0CH=COlSucc~'H H H H THIw 17 (p)-C,H,-OCHZCO=CFI,H H H H FUR

18 (p)-C,H,-0CH=CO=H H H H H F'UR*

19 (p)-C,H; OCH,CO,CH,' H H H H PYR' 10 (p)-C,H,-0CH:CO.H H H H H PYR*
t I I I I ._ "

_-_ S ~ ~~Q'T1 T 1 1 ~rw- ,pr ~ --21~~~~°~
21 (p)-CeH; OCHiCO,Succ~H H H H PYRE

(DrC.Hr4CHzC0,CH, H H H H STw ?3 MCMST* H H H H ST"

?d CMST* , , H H H H ST*

25 (p)-C,Ii,-OCH,CO~CH,H H ~ H H PHBD

Z6 fp)-C,H,-OCH2COZH H ~ H H H PHBD

27 (CI~i~,COlCH, H H H H PHBD'~

Z8 (CH>~CO~i H H H H PHBD*

(CH~zCOrSucc; H H H H PHBD~

30 (E)-CH ~ CHCO=CHy H H H CH, CHy 31 (E)-CH=CEiCO=H H H H CH, CH, 3I (E)-CH=CHCO Succ~ H H H CH, CH, 33 (E)-CH=CHCO~CH, H H H CaHf CdH, 34 (E)-CH ~ CHCO~H H H H C,H, CtH, .

35 (E)-CHsCHCOrSucc* H H H CeH, C,H, 36 CBMOXET* H H H CH, CH, 37 CMMOXET* H H H CH, CH, 3a (CH~,COiCHy H H H H (E)-CH=
CHCHy 39 (p)-C,H,-OCH,CO=CH,H H H H (E)-CH=
CHCH, 46 (prC,H,-0CH,COyI-I H H H H (E)-CH-CNCHy 41 MCST . H H H CH, CH, ~U83TITU'~' L tET

2~.2262"~
sri 42 HMPHETx H H H CH, CH, 43 CMPHETw . H H H CH, CH, 44 (CH~j,CO,CH~ H H H H NART*

LS (CH~,CO,CH, H H H H ST*

4i (CH~,CO=H H H H H ST*

47 (CH~ZCO=Succ H H H H ST*

48 (CH>,C(=O)NH(CH~-NHzH H H H STi 49 (CHI=C(=O)NH(CH,~NH-H H H H ST*
C( = O)CH,I

50 (CHI=C(=Q)NH(CH~,-NH-H H H H ST*
C(=O~Hz-Maleim;

S1 ' (CH,),C(=O)NH(CH,y=-NH-H H H H ST*
C(= O~(p)-C6Ei,~HO

s2 {CH~iC(=O~'~IH(CH~,-NH-H H H H ST*
C(=O)'(P)-CeHrNt 53 (CH,~~C(=O)NH(CH~,-NH-H H H H ST*
.C(=O)-CH=CH, 54 (CH~~C(=O)NH(CHJ=- H H H H ST*
N=C=S

55 (CHI C( a O)-O-C( H H H H THI i = O)-O-CH,CH, 56 (CH~,C(=OrNs ~ H H H H THI'~

ST (CH~zC(=O)-F H H H H THI*

SS (CHI=C(=O)NH(CH~,-CH,H H H H THI*

59 ST SO,Na H H SO~Na ST

60 ST SO,CI H H S01CI S1 61 (CH~,C(=O)NH(CH~,- H H H H TH1*
NH=

6Z (CHI=C(=O)NH(CH,~, H H H H THI*
NHC(=O)CH=I

Rs (CHI COiH H H H CHI CH, tSubstituents R,-R, correspond to the farmulx in the text above; R, is hydrogen in sill of the structures lieted in Table t.
'wi'be namrs sad chomica) structures of abbreviations used in thie table are shown imtntdiate~y below.
~Alicyl->:.ub~tit~toJ reactive dipyrrorzietheaeboron difluoride dye, ixluded for cornpsrison.
sUH'3TITUT~ ~~ET

THI:2-thieayl, I I FUR:2-furyl, I I PYR:2-pyrrolyl, o H
ST: (E)-2-pheaylethen-1-yl, ~ - ~ PHBD: (E,E)-4-pheayl-1,3-butadien-1-yl, H/C'-~~-C~
H
1~ICMST: fE~2-[4~methozycarbonylmethozyhenyl)ether-1-yl, H
I

CMST: (E~2-{4-(carbozymethozy)phenyl)echen-1-yl, y H

~ICST: (E}-'-{4-(methozycarbonyl)phenyl]e~en-1-yl, y H

CEMOXET: (E)-2-(5-carboethozy-4-methyl-'-~zazolyl~then-1-yl, H~~~ H

CMMOXET: (E)-2-f;5-carbomethozy-4-methyl-2-ozazolyl~then-1-yl, H~~ H

CH

HMPHET: (E)-2-[4-(hydrozymethyl)phenylkthen-1-yl, CMPHET: fE)-2-[4-(chloromethyl)phenyl]ether-1-yl, H~

\AET: f;E~2-(1-naphthyl~then-1-yl, I

WO 93/09185 10 2 ~ 2 2 6 2 7 P~./US92/08350 Succ: succinimidyl, o Maleim: maleimidyl, Table 3 describes physical properties of selected dyes from Table Z.

Compound M.P. (°C) ~~~'m"~ ~~~ RE T.L.C. Solvent (nm)* (nm)*
2 170-171 559.6 568 0.29 C

3 70-72 559.4 568 0.65 B

4 164-166 557.2 569 0.36 C

6 139-141 560.8 570 0.24 C

8 158-160 578.2 589 0.37 C

11 200 dec 585.4 599 0.29 C

13 191-193 546.0 564 0.19 C

15 210 dec 591.0 618 0.32 D

16 188-189 588.2 617 0.40 C

20 190 dec 609.0 640 0.31 D

24 175 dec 637.4 652 0.48 D

26 188 dec 621.2 643 0.34 D

28 197-199 582.4 590 0.31 C

29 125-126 581.8 591 0.54 B

31 160 dec 535.2 544 0.17 C

32 203-206 538.0 549 0.48 B

34 163 dac 574.0 589 0.35 C

35 195-196 580.6 598 0.71 C

37 252-253 561.4 570 0.15 A

40 210 dac 571.4 592 0.22 D

42 168-170 560.4 571 0.29 B

43 193-195 560.0 570 0.83 A

44 136-138 569.6 579 0.46 A

45 145-14b 563.2 569 0.22 A

46 195-197 564.4 570 0.32 C

47 204-205 563.4 570 0.76 C

48 120 dec 564.4 570 0.06 D

49 188-189 564.4 570 0.50 C

50 104 dec 564.4 570 0.33 C

51 190-192 564.4 571 0.34 C

52 152-153 565.0 571 0.49 C

53 178-180 564.2 570 0.39 C

54 140-141 564.4 570 0.59 C

56 79-80 558.6 568 0.53 C

58 126-127 559.0 569 0.58 C

59 130 dec 623.2 635 0.17 E

62 167-168 559.0 569 0.37 C

* Absorption (Abs) and emission (Em) maxima meaaced in methanol.
~ A = Chloroform; B = 596 methanol in chloroform: C = 1096 methanol in chloroform; D =
2596 methanol in chloroform; E = 409E methanol in chloroform 2 ~ 2 2 s z 7 p~'/US92/08350 1i Table 4 lists the IH NMR spectral data for selocted dyes from Table 2.

COMPOUND CHEMICAL SHIFT IN PPM IN CDC13 (400 MHz NMR) 2 2.86(t,2H,CH~, 3.38(t,2H,CH~, 6.40(d,lH,ArH), 6.80(d,lH,ArH), 6.99(d,lH,ArH), 7.04(d,lH,ArH), 7.12(s,lH,ArCH=), 7.17-7.20(m,lH,ArH), 7.50(d,lH,ArH), 8.17(d,lH,ArH).
3 2.84(s,4H,2xCH~, 3.11(t,2H,CH~, 3.46(t,2H,CH~, 6.44(d, IH,ArH), 6.81(d,lH,ArH), 6.99(d,iH,ArH), 7.05(d,lH,ArH), 7.13(s,lH,ArCH=), 7.20(t, IH,ArH), 7.52(d, lH,ArH), 8.19(d, iH,ArH).
4 2.64(t,2H,CH2), 3.35(t,2H,CH~, 3.87(bs,2H,NH2), 6.38(d,lH,ArH), 6.81(d,iH,ArH), 6.90(bs,iH,NH), 6.98(d,iH,ArH), 7.05(d,lH,ArH), 7.13(s,lH,ArCH=), 7.18-7.21(m,lH,ArH), 7.51(d,iH,ArH), 8.14(d,lH,ArH).
6 2.87(t,2H,CH2), 3.38(t,2H,CH~, 6.38(d, IH,ArH), 6.61-6.63(m, lH,ArH), 6.96(d, lH,ArH), 6.97(d, lH,ArH), 7.04(d, lH,ArH), 7.10(s, lH,ArCH=), 7.59(d, iH,ArH), 7.69(d, lH,ArH).
8 2.86(t,2H,CH~, 3.35(t,2H,CH~, 6.30(d,lH,ArH), 6.37-6.39(m,lH,ArH), 6.84(d,lH,ArH), 6.88(d,lH,ArH), 6.99(s,lH,ArCH=), 6.99-7.01(m, IH,ArH), 7.05(d, IH,ArH), 7.17-7.20(m, IH,ArH).
11 2.08(s,3H,CH~, 2.31(t,2H,CH2), 2.43(s,3H,CH3), 2.61(t,2H,CH~, 6.17-6.20(m,lH,ArH), 6.65(d,lH,ArH), 6.75-6.77(m,lH,ArH), 6.84(d,lH,ArH), 6.87(s,lH,ArCH=), 6.93~.95(m,lH,ArH), 10.2(bs,lH,NH).
13 2.45(s,3H,CH~, 2.60(s,3H,CH3), 2.74(t,2H,CH~, 3.28(t,2H,CH~, 6.18(s, lH,ArH), 6.74(d, lH,ArH), 7.13(d, IH,ArH), 7.13-7.16(m, lH,ArH), 7.41(d,lH,ArH), 8.02(d,lH,ArH).
14 3.84(s,3H,CH~, 4.71(s,2H,CH2), 6.66(d,lH,ArH), 6.81(d,lH,ArH), 7.00(d,2H,2xArH), 7.04(d, IH,ArH), 7.08(d, iH,ArH), 7.13-7.15(m, IH,ArH), 7.16(s,lH,ArCH=), 7.46(d,lH,ArH), 7.96(d,2H,2xArH), 8.11(d,lH,ArH).
28' 2.68(t,2H,CH2), 3.17(t,2H,CH2), 6.47(d,lH,ArH), 6.97(d,lH, CH=), 7.13(d,IH,CH=), 7.16(d,lH,ArH), 7.18(d,lH,ArH), 7.56(s,lH,ArCH=), 7.61(d,2H,2xArH), the remaining six protons are overlapped between 7.28 and 7.54.
37 2.28(s,3H, CHg), 2.51(s,3H,CH3), 2.64(s,3H,CH3), 3.94(s,3H,CH3), 6.20(s, IH,ArH), 6.83(d, IH,ArH), 6.91(d, lH,ArH), 7.00(d,1H,CH=), 7.10(s,lH,ArCH=), 8.02(d,IH,CH=).
42 2.26(s,3H,CH~, 2.61(s,3H,CHg), 4.71(s,2H,CH2), 6.12(s,lH,ArH), 6.86(d,lH,ArH), 6.95(d,lH,ArH), 7.05(s,iH,ArCH=), 7.27(d,IH,CH=), 7.37(d,2H,2xArH), 7.59(d,2H,2xArH), 7.63(d,lH, CH=).
43 2.25(s,3H,CH~, 2.61(s,3H,CH3), 4.60(s,2H,CH2), 6.12(s,iH,ArH), 6.84(d,lH,ArH), 6.93(d,iH,ArH), 7.05(s,iH,ArCH=), 7.24(d,IH,CH=), 7.38(d,2H,2xArH), 7.57(d,2H,2xArH), 7.63(d,2H,2xArH).
44 2.82(t,2H,CH2), 3.37(t,2H,CH2), 3.71(s,3H,CH3) 6.36(d,iH,ArH), 6.97(d,lH,ArH), 7.06(d,lH,ArH), 7.09(d,lH,ArH), 7.11(s,lH,ArCH=), WO 93/09185 21 ~ 2 s 2 ~ PCT/US92/08350 7.77(d,lH,CH=), 7.86-7.90(m,2H,2zArH), 7.98(d,lH,ArH), 8.18-8.23(m,2H,2zArH), the remaining one olefin proton and two aromatic protons are overlapped between 7.51 and 7.59 as multiplets.
45 2.82(t,2H,CH~, 3.37(t,2H,CH~, 3.72(s,3H,CH3), 6.34(d,lH,ArH), 6.91(d,lH,ArH), 6.93(d,lH,ArH), 7.00(s,lH,ArCH=), 7.02(d,lH,ArH), 7.30-7.42(m,3H,3zArH), 7.37(d,lH,CH=), 7.61(d,2H,2zArH), 7.66(d,2H,CH=).
46~ 2.70(t,2H,CH~, 3.17(t,2H,CH~, 6.51(d,lH,ArH), 7.23(d,lH,ArH), 7.24(d,lH,ArH), 7.34(d,lH,ArH), 7.37-7.41(m,lH,ArH), 7.44-7.50(m,2H,2zArH), 7.49(d,IH,CH=), 7.61-7.64(m,2H,2zArH), 7.63(s,lH,ArCH=), 7.71(d,lH,CH=).
47 2.84(s,4H,2zCH~, 3.31(t,2H,CH~, 3.41(t,2H,CH~, 6.41(d,lH,ArH), 6.93(d, IH,ArH), 6.95(d, IH,ArH), 7.05(d, IH,ArH), 7.09(s, IH,ArCH=), 7.52(d,2H,2zArH), 7.65(d,lH,CH=), the remaining three aromatic protons and one olefin proton are overlapped between 7.32 and 7.43 as multiplets.
48 2.70(t,2H,CH2), 2.71-2.76(m,2H,CH~, 3.24-3.29(m,2H,CH~, 3.35(t,2H,CH2), 6.12(bs,1H,NH), 6.37(d, lH,ArH), 6.93(d, lH,ArH), 6.95(d, IH,ArH), 7.05(d,lH,ArH), 7.08(s,lH,ArCH=), 7.61(d,2H,2zArH), 7.65(d,IH,CH=), the remaining three aromatic protons and oae olefin proton are overlapped between 7.31 and 7.42 as multiplets.
49 2.74(t,2H,CH~, 3.28-3.37(m,6H,3zCH~, 3.60(s,2H,CH2), 6.38(d, lH,ArH), 6.69-6.74(m,1H,NH), 6.96(d, lH,ArH), 6.98(d, lH,ArH), 7.08(d, IH,ArH), 7.12(s,lH,ArCH=), 7.42(d,2H,2zArH), 7.62(d,lH,CH=), the remaining three aromatic protons and one olefin proton are overlapped between 7.33 and 7.46 as multiplets.
50 2.25-2.34(m,6H,3zCH2), 2.71(t,2H,CH~, 4.11(s,2H,CH2), 6.19-6.23(m,lH,NH), 6.38(d,lH,ArH), 6.78(s,2H,2zCH=), 6.89-6.91(m,lH,NH), 6.94(d,lH,ArH), 6.99(d,lH,ArH), 7.07(d,lH,ArH), 7.11(s,lH,ArCH=), 7.61(d,2H,2zArH), 7.64(d,lH,CH=), the remaining three aromatic protoas and one olefin proton are overlapped between 7.32 and 7.43 as multiplets.
51 2.76(t,2H,CH2), 3.30-3.58(m,6H,3zCH~, 6.25(d,lH,ArH), 6.34-6.41(m,IH,NH), 6.82(d,lH,ArH), 6.94(d,lH,ArH), 7.01(s,lH,ArCH=), 7.03(d,lH,ArH), 7.63(d,lH,CH=), 7.55-7.64(m,3H,3zArH), 7.83-8.00(m,4H,4zArH), 9.98(S,1H,CH0), the remaining three aromatic protons and one olefin proton are overlapped between 7.30 and 7.45 as multiplets.
52 2.74(t,2H,CH~, 3.30-3.57(m,6H,3zCH~, 6.22(d, lH,ArH), 6.42fi.54(m,1H,NH), 6.78(d, lH,ArH), 6.88(d, lH,ArH), 6.93(d,2H,2zArH), 6.96(s,lH,ArCH=), 6.98(d,lH,ArH), 7.49-7.54(m,2H,2zArH), 7.54(d,lH,CH=), the remaining three aromatic protons and one olefin proton are overlapped between 7.27 and 7.34 as multiplets.
53 2.43(t,2H,CH2), 2.58(t,2H,CH ), 3.24-3.28(m,4H,2zCH~, 4.29-4.33(m,2H,=CHI, 5.73-5.76(m,IH,CH=), 5.97-6.06(m,IH,NH), 6.28~.35(m,1H,NH), 6.87(d, IH,ArH), 6.90(d, lH,ArH), 7.00(d, IH,ArH), 7.04(s,lH,ArCH=), 7.53(d,2H,2zArH), 7.54(d,lH,CH=).
54 2.74(t,2H,CH2), 3.35(t,2H,CH2), 3.46(t,2H,CH2), 3.59(t,2H,CH~, 6.08(bs,lH,NH), 6.36(d,lH,ArH), 6.95(d,lH,ArH), 6.98(d,lH,ArH), 7.07(d,lH,ArH), 7.12(s,lH,ArCH=), 7.62(d,2H,2zArH), 7.65(d,lH,CH=), the remaining three aromatic protons and one olefin protoaare overlapped between 7.32 WO 93/09185 21 ~ 2 ~ 2 7 PCT/US92/08350 and 7.45 as multiplats.
56 3.32(t,2H,CH~, 3.72(t,2H,CH~, 6.43(d,lH,ArH), 6.81(d,lH,ArH), 7.00(d,lH,ArH), 7.06(d,lH,ArH), 7.14(s,lH,ArCH=), 7.20(t,lH,ArH), 7.52(d,lH,ArH), 8.18(d,lH,ArH).
59' 7.32-7.38(m,2H,2xArH), 7.40-7.49(m,4H,4xArH), 7.53(s,ZH,2xArH), 7.54(s,lH,ArCH=), 7.70(d,4H,4xArH), 7.78(d,2H,2xCH=), 8.48(d,2H,2xCH=).
62 2.72(t,2H,CIi~, 3.30-3.38(m,6H,3xCH~, 3.58(s,2H,CH~, 6.07-6.14(m,lH,NH), 6.41(d, IH,ArH), 6.66~.72(m,1H,NH), 6.82(d, lH,ArH), 7.01(d, lH,ArH), 7.08(d,lH,ArH), 7.15(s,lH,ArCH=), 7.19-7.21(m,lH,ArH), 7.53(d,lH,ArH), 8.14(d, lH,ArH).
* Compounds 28 and 46 were measured in DMSO~ and 59 was taken in CDgOD.
The additional electronic conjugation that results from incorporation of bathochromic moieties into the BDI fluorophore results in new long wavelength reactive BDI dyes that have spectral properties that are significantly shifted from those of the related alkyl-substituted fluorophores, thus permitting their use in multi-color fluorescence applications in combination with fluorescein or alkyl-s substituted BDI dyes. Furthermore, this wavelength shift is usually accompanied by an increase in photostability of the long wavelength reactive BDI dyes and in most cases by an increase in the extinction coefficient of the long wavelength reactive BDI dyes relative to the alkyl-substituted BDI
dyes. Table 5 lists the spectral properties of selected dyes from Table 2.

Compound ~~b~""X exl0-3 ~E'"~"x Quantum Photostability (am)* (ca; tM-t)*(nm)* Yield(~)$

2 559.2 86.9 568 1.00 0.94 8 578.0 99.2 589 0.74 0.96 28 582.2 158.8 590 0.81 0.96 II

46 564.2 143.2 570 0.90 0.92 Rt 505.0 83.8 512 1.00 0.71 tCompound R is presented as an example of alkyl-substituted dipyrrometheneboron difluoride dyes for comparison.
*Absorption (Abs) and emission (Em) maxima measured in methanol. Extinction coefficients (e) at their absorption maxima in methanol.
$Fluorescence quantum yields (4') in methanol are measured relative to fltwrescein in 0.1 M NaOH
(~=0.92). Integrated fluorescence intensities are corrected for variation of solv~t refractive index.
The integrated fluorescent intensity is also corrected for variation of incident excitation light intensity with wavelength by recording spxtra in ratio mode relative to a rhodamine B/ethylene glycol quantum counter solution. [Dames, et al. J. PHYS. CHEM. 75, 991 (1971)].
~Retention of emission int~sity after continuous illumination at absorption maxima for 30 minutes in acetonitrile. Intensity at 0 minutes = 1.00 (Example 29) The novel dyes have as absorption maximum at greater than about 525 am and, preferably, as WO 93/09185 212 2 6 2 ~ PCT/US92/08350 emission maximum at greater thaw about 550 nm (see Tsble 3). These spoctral properties distinguish the long wavelength reactive BDI dyes from the related alkyl-substituted BDI dyes.
As indicated in Table below, the absorption and emission spectra of the new reactive BDI dyes are shifted to significantly longer wavelengths as compared to the alkyl-substituted dyes. The long wavelength reactive BDI dyes 5 also demonstrate improved photostability (Table 5, Figure 4) relative to the alkyl-substituted dyes; with high extinction coefficients, generally greater than 80,000 cm tM-1 (Table S).
The dyes of this invention also have a substantial quantum yield, i.e. greater than 0.1 as measured in methanol (Table S), which distinguishes these dyes from previously disclosed BDI derivatives having an absorption maximum greater than about 525 nm.
Of further significance is the characteristic that the emission spectra of appropriate combinations of the dyes can be readily resolved (Figure 2). This relatively high degree of spectral resolution is partly the result of the unusually narrow emission band width of most BDI dyes (including the subject dyes) relative to that of other important fluorophores in common use such as a tetramethylrhodamine derivative 5 (Figure 3). Furthermore, the fluorescence of the long wavelength reactive BDI dyes is not usually quenched in aqueous solution. This general insensitivity of the dye to the environment of use increases the utility of these dyes in their applications.
The general scheme for the synthesis of new long wavelength BDI dyes that can be modified to have the desired chemical reactivity falling within the scope of this invention is illustrated in Figure 1. Suitable substituents R~ to R~ (Fig. 1) on the pyrrole intermediates include, but are not limited to, hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, heteroaryl and alkenyl.
The general method consists of an acid catalyzed condensation of a 2-acylpyrrole or appropriately substituted 2-acylpyrrole with pyrrole or substituted pyrrole having a hydrogen on the 2-position used in approximately stoichiometric proportions to give a pyrromethene intermediate.
In g~eral, there are two alternative routes whose choice depends primarily on the availability or ease of synthesis of the 2-acylpyrrole reactants. Suitable acids (QJ~ include, but are not limited to, hydrogen halides, metal salts typically used in Friedel-Craft reactions such as zinc halides, and non-metallic, electron deficient Lewis acids such as boron halides, halides of sulfur acids and phosphorous oxychloride in that such acids contain elements or groups of elements capable of forming an anionic counterion.
Preferred is phosphorous oxychloride, since its use results in moderate to good yields of pyrromethene intermediates. In some cases it may be practical to use boron trifluoride both as the acid component and to complete formation of the BDI dye in a "one pot reaction".
Cyclization of the heterocyclic ring with formation of the BDI dye is completed by addition of boron trifluoride in combination with a suitable base. Boron trifluoride is preferably used as one of its ether complexes due to the ease of handling of these complexes rather than the gaseous reagent. Suitable bases include, but are not limited to, trimethylamine, triethylamine, N,N-diisopropylethylamine, N,N,N',N'-tetramethylethyleaediamine, 1,8-bis(dimethylamino)naphthalene, diazabicyclooctane, diazabicyclounderxae, 4-dimethylaminopyridine, 4-pyrrolidinopyridine and other similar bases.
Once prepared, the long wavelength reactive BDI dyes may be further modified by sulfonation, nitration, alkylation, acylation, halogenation and other reactions by methods known in the art, such as is described in Exampk 24 and by U.S. Patent 4,916,711 b Hoyer, at al. (1990);
Worries, et al., RECL. TRAV. CHD~I. PAYS-BAS 104, 288 (1985).
T6e resulting products are generally aluble in organic solvents. Aqueous solubility can be obtained by adding appropriate water solubiliution group: that include sulfonstes, carboxylatec, ammonium wad hydroxyl rewdua to the dyes. In most cases, the solid dyes are eaaly purified by techniques of chrom"tography and/or crystalliution from a suitable solvent. The chemical structure of r~r~atotstive examples of long wavelength roactive BDI dyes have bees confirmed by nuclar magnetic reooanoe spectroaoopy (Table 4).
Once synthesised. the new long wavelength chemically ractive BDI dyes an be used to fluoreceatly label a wide variety of other material:, including cells, tissues, c:rbohydrates, drugs, lipids, nucleic acids, nucleotide, polymers. oligonucleotide, proteins. toxins, viruses, vitamins, and other biologically derived or syntbaic chemical materials. The novel BDI dyes are particularly useful for the fluorescsat labeling of members of specific binding pairs (sbp) to form dye coojugstas which can be owed to detect biological intaactiooc. The sbp member can be a ligand or a receptor. As used in this d~ocumeat, the term ligaad means any organic compound for which a recxptor naturally exists or can be prepared. A recxptor is any compound or composition capable of reoogniriag a spatial or polar organisation of a molecule. e.g.
epitopic or deta~minaat site. Ligaods fx which naturally occurring receptors exist include nadiraf and :yathetic protein:, including avidia and stroptavidia. antibodies. eazyma, and hormouea; nucleotides and nadiral or synt6dic oligonueleotide. including primers for RNA and single- and double-strm~ded DNA;
Gpida; polysaccharide and carbohydrates: wad a variety of drugs, including tbaapeuac drugs wad drugs of abuse; and pesticides. Ligaads wad reoeptorc are complementary mambas of a specific binding pair.
each sbp member having m arra on the surface or in a cavity which specifically binds to and is oomplementuy with a particular spatial and polar orgaaintion of the other.
One embodiment of the invention a a method of pr~aring a fluoreaaat conjugate comprising combining a BDI dye of formula I with the sbp member being labeled. Mother embodiment of the invention is a sbp m~ba labeled with a new ractive BDI dye. Preferably the sbp member is a ligaad, more preferably the ligaod is a nucleotide, oligooucleotide, peptide, protein, lipid polysaccharide or carbohydrate.
To label a sbp member, the new BDI dyes of this reaction are combined with a sbp member of interest which sbp member is capable of reacting with the reactive group (G) on the BDI fluomphore.
Generally, any sbp member containing one or moro of the reactive sits listed is Table 1 an be labeled using the new dyes. Preferred rcacoive sits are amines. thiols or carboxylic acids. The :bp member may be mooovaleat (containing a single reactive site) or polyvalent (containing multiple reactive sites).
When the tbp member lacks a ractive site or rewction at the reactive site inhibits formation of a sbp pair interaction. an appropriate coupling moiety can often be introduced by syntbeis or modifications familiar in the art. For example, the starting material for nuckic acid conjugate is an oligonucleotide B

21~262'~ 16 ....
(oligo) which has incorporated a reactive residue, typically an amine or thiol, to form a derivatized oligo.
The reactive residue is attached through a carbon chain spacer of a variety of lengths at either a substituted nucleotide base or the 5' ~d of the oligo. The thiol- or amine-derivatizad oligos are typically commercially available with a carbon chain spacer of about 2-12 carbons.
Typically, the reactive BDI dye is coupled directly to the sbp member. For example, if the sbp member is a ligand that has an amino substituent and the Inactive dye has a carboxyl substitu~t, then the ligaad and fluorophore may be directly conjugated to each other by procedures known in the art; for example, an active ester technique. Where the sbp member is a protein, it is less likely to require derivatiution to add additional reactive sites. However, if desired, other groups can be introduced such as by thiolation with succinimidyl 2-pyridyldithiopropionste (SPDP).
A sbp member can be labeled with the BDI dyes by using procedures such as those illustrated in fixamples 25-30. G~erally, the derivatized or reactive sbp member is solubilized in an appropriate solv~t. The conditions required for solubilizing sbp members are generally known in the art. The dye is solubilizad in as appropriate solvent that is mutually miscible with the solvent that is used to solubilize the sbp member. For applications which require use of a water miscible solv~t, appropriate solvents include, for example, methanol, ethanol, dianethylformamide (DMF), dimethylsulfoxide (DMSO), and acetone. For preparation of a conjugate where the sbp is water insoluble, appropriate solvents include, for example, DMF, DMSO, chloroform, toluene, and many other organic solvents.
The temperature at which the reaction for preparing the conjugates of this invention proceeds is not critical. The temperature should be one which is sufficient to initiate and maintain the reaction, without compromising the stability or activity of the sbp member or its conjugate.
Generally, for convenience and economy, room temperature (- 25°C) is suffici~t.
In preparing the conjugates of the present invention, the ratio of r~eactaats is not narrowly critical.
For each mole of reactive dye of formula I, one usually employs about one mole of the sbp member being labeled to obtain a reasonable yield. In some cases it is preferred to employ as excess of reactive dye for ease of reaction and sufficient labeling of the sbp member. For example, in preparing oligo conjugates, 50-100 fold excess of dye has bin found to be appropriate. The combined reactants, dye and sbp member, are incubated for suffici~t time to allow the conjugation to occur, which may range from about 10 minutes to 24 hours, more typically from about 30 minutes to about 12 hours.
Following attachment of the reactive dye to the sbp member, the conjugate is then purified using standard purification techniques, for example, using chromatography, crystallization, etc.
The dye~onjugated ligands are useful in a wide variety of areas as biochemical tools for the detection, identification, and measurement of biological compounds, particularly immunochemically reactive components such as those found in body fluids, cells, and cell components. For example, the labeled proteins can indicate the presence and amount of certain antigens or identify the site of certain immunochemical reactions, using known assay methods and imaging txhniques for detection of the fluorescence signal. The labeled nucleotides and oligonucleotides can be used in research efforts directed at determining the pres~ce or sequ~ce of nucleic acids in gees, using known gene mapping techniques.
The following examples of the synthesis and characterization of the long wavelength reactive BDI

v WO 93/09185 17 ~ ~ ~ ~ ~ ~ ~ P~/US92/08350 dyes and the combination of such dyes with other natural and synthetic materials are intended to illustrate the g~erality of the invention and not to define or limit the scope of the invention.
EXAMPLE 1: 4,4-Difluoro-3-(2-mothoxycarbonylethyl)-5-(2-thienyl)-4-bona-3a,4a~iiaza-s-indacene (Compound 1): To a solution of 500 mg (2.82 mmol) of 2-formyl-5-(2-thi~yl)pyrroleand 435 mg (2.84 mmol) of 2-(2-methoxycarbonylethyl) pyrrole in 50 mL of dichloromethane is added 265 ~I. (2.84 mmol) of phosphorus ozychloride. The reaction mixture is stirred at room temperature for 16 hours and then 2.0 mL (11.5 mmol) of N,N-diisopropylethylamine is added, followed by the addition of 1.5 mL (11.5 mmol) of boron trifluoride etherate. After the whole reaction mixture is stirred at room temperature for 2 hours, it is washed with two 50 mL portions of brine. The organic layer is separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a dark brown solid. The crude product is purified by chromatography on silica gel with chloroform as eluaat to give 380 mg (52 q6) of Compound 1 as a dark purple solid.
The 2-formyl-5-(2-thienyl)pyrrole is prepared from 2-(2-thi~yl)pyrrole by the Vilsmeier-Haack formylation, according to R.M. Silverstein et al., ORG. SYNTH. COLL. Vol. IV, p. 831. The 2-(2 thi~yl)pyrrole needed for this synthesis is prepar~ad in s similar manner as described in Kruse, et al., HETEROCYCLES 26, 3141 (1987).
The 2-(2-methoxycarbonylethyl)pyrrole is prepared as follows: A solution of pyrrole-2 carboxaldehyde (3.9 g, 41 mmol) and (carbomethoxymethylene)-triphenylphosphorane (14 g, 42 mmol) in 160 mL of dry benzene is heated at refluz for 18 hours under a nitrogen atmosphere. After cooling, the reaction mixture is concentrated under vacuum and the resulting oil is purified by silica gel column chromatography, eluting with a mixture of ethyl acetate and hexane (1:9) to give 5.11 g of a mixture of cis- and traps-methyl 2-(2-pyrrolyl)acrylate.
It is then dissolved in 100 mL of methanol in a Purr shaker bottle and to this is added 500 mg 10 96 palladium on charcoal. The mixture is pressurized to 50 psi with hydrogen and shaken at room temperature for 2 hours. The reaction mixture is filtered through a diatomaceous earth pad and the filtrate is concentrated under vacuum to give 5.10 g (82 9b) of 2-(2-methozycarbonylethyl)pyrrole as a pale yellow oil, tH-NMR (CDCIg) 8 2.64(t,2H,CH~, 2.92(t,2H,CIi~, 3.71(t,3H,CHg), 5.93(d, 1H, ArH), 6.12(m,lH,ArH), 6.68(d,lH,ArH), 8.62 (bs,lH,NH).
EXAMPLE 2: 4,4-Difluoro-3-(2-methoxycarbonylethyl)-5-(2-furyl)-4-bona-3a,4a-diaza-s-iadacene (Compound 5): This is prepared in the same meaner as described in Example 1 from 2-(2-methoxycarbonylethyl)pyrrole (240 mg, 1.56 mmol) and 2-formyl-5-(2-furyl~yrrole (250 mg, 1.55 mmol). Compound 5 (295 mg, 55 ~fo yield) is obtained as a dark purple solid.
The 2-formyl-5-(2-furyl)pyrrole is prepared from 2-(2-furyl)pyrrole by the Vilsmeier-Haack formylation as described in ORG. SYNTH. COLL. Vol. IV, p. 831. The 2-(2-furyl)pyrrole needed for this synthesis is prepared in a similar manner as described in HETEROCYCLES
26, 3141 (1987).
EXAMPLE 3: 4,4-Difluoro-3-(2-methoxycarbonylethyl)-5-(2-pyrrolyl)-4-bore-3a,4a-diara-s-indscene (Compound 7): To a solution of 150 mg (1.13 mmol) of 2,2'-bipyrnole sad 205 mg (1.13 mmol) of 2-(2-methoxycarbonylexhyl)-5-formylpyrrole in 10 mL of dichloromethane is added 110 ~L (1.18 mmol) of phosphorus oxychloride. The reaction mixture is stirred at room temperature for 12 hours and then is added 800 pL (4.59 mmol) of N,N~diisopropylethylamine, followed by the addition of 560 pL (4.55 mmol) of boron trifluoride etherate. After the whole mixture is stirred at room temperature for 2 hours, it is washed with two 10 mL portions of brine. The organic layer is separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a dark red solid. The crude product is purified by chromatography on silica gel with a mixture of chloroform and hexane (1:1) as eluant to give 125 mg (329b) of Compound 7 as a darn purple solid.
The 2,2'-bipyrrole is prepared as described in Rappaport et al., J. AM. CHEM.
SOC., 84, 2178 (1962). The 2-(2-methoxycarbonylethyl)-5-formylpyrrole is prepared from 2-(2-methoxycarbonylethyl)pyrrole by the Vilsmeier-Haack formylation according to ORG. SYNTH. COLL.
Vol. IV, p 831.
EXAMPLE44,4-Difluoro-1,3-dimethyl-2-(2-methoxycarbonylethyl)-5-(2-pyrrolyl)~-hors-3a,4a-0iara-s-indac~e (Compound 10): This is prepared in the say method as described in Example 3 from 2,2'-bipyrrole (500 mg, 3.78 mmol) and 2.4-dimethyl-5-formyl-3-(2-methoxycarbonylethyl)pyrrole(795 mg, 3.80 mmol). Compound 10 (760 mg, 54 96 ) is obtained as a dark purple solid.
The 2,4-dimethyl-5-formyl-3-(2-methoxycarbonylethyl)pyrrole is prepared from 2,4-dimethyl-3-(2-methoxycarbonylethyl)pyrroleby the Vilsmeier-Haack formylation as described in ORG. SYNTH. COLL.
Vol. IV, p 831. The 2,4-dimethyl-3-(2-methoxycarbonylethyl)pyrroleis prepared as described in Bullock, et al., J. CHEM. SOC. 1420 (1958).
EXAMPLES: 4,4-Difluoro-1,3-dimethyl-8-(2-methoxycarbonylethyl)-5-(2-thienyl)-4-bore-3a,4a-diaTa-s-indac~e (Compound 12): To a solution of 500 mg (2.39 mmol) of methyl 4-[2-(3,5-dimethyl)pyrrolyl]-4-oxobutanoate and 355 mg (2.38 mmol) of 2-(2-thienyl)pyrrole in 30 mL of dichloromethane is added 230 p.L (2.47 mmol) of phosphorous oxychloride. The reaction mixture is heated under reflux in a nitrogen atmosphere for 2 days. It is cooled to room temperature and then is added 1.7 mL (9.76 mmol) of N,N-diisopropylethylamine, followed by addition of 1.2 mL (9.76 mmol) of boron trifluoride etherate.
After the whole reaction mixture is stirred at room temperature for 4 hours, it is washed with two 30 mL
portions of brine. The orgaaic layer is separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product is purified by chromatography on silica gel with a mixture of chloroform and hexane (1:1) to give 340 mg (3496) of Compound 12 ss a dark red solid.
The methyl 4-[2-(3,5-dimethyl)pyrrolyl]~~xobutanoateis pr~epsred as described in Treibs, et al., LIEBIGS. ANN. CHEM., 702, 112 (1967).
EXAMPLE 6: 4,4-Difluoro-3-[4-(methoxycarbonylmethoxy)phenyl]-5-(2-thienyl~4-bona-3a,4a~iaTa-s-indacene (Compound 14): This is prepared in the same method as described is Example 1 from 2-[4-(methoxycarbonylmethoxy)phenyl]pyrrole ( 100 mg, 0.43 mmol) and 2-formyl-S-(2-thienyl)pyrrole (75 WO 93/09185 212 2 6 2 l PCT/US92/08350 1g -mg, 0.42 mmol). Compouad 14 (68 mg, 3796) is obtained as a dark purple solid.
The 2-[4-(methoxycarbonylmethoxy)phenyl]pyrrole needed for this synthesis is prepared as follows:
To a suspension of 1.0 g (11.9 mmol) of ethanethiol, sodium salt in 10 mL of dry DMF is added 2.0 g ( 11.5 mmol) of 2-(4-methoxyph~yl)pyrrole (prepared in a similar method as described in HETEROCYCLES 26, 3141 (1987)) while the reaction mixture is stirred in an ice bath under nitrogen atmosphere. It is then stirred st 110°C for 4 hours. After the reaction mixture is cooled to room temperature, 1.82 g (11.9 mmol) of methyl bromoacetate is added. After stirring at room temperature for 2 hours, it is poured into ice-water (100 mL). It is extracted with chloroform (3x70 mL). It is dried over aahydrous sodium sulfate sad conceattrated under reduced pt~easure to give a brown solid. The crude product is purified by chromatography on silica gel with chloroform as closet to give 1.92 g (68 96 ) of the 2-[4-(methoxycarbonylmethoxy)ph~yl]pyrroleas as off white solid; m.p. 136-137°, Rf = 0.30 in chloroform,1H-NMR(CDCIg) 8 3.82(s,3H,CHg), 4.65(s,2H,CH2), 6.26-6.29(m, IH,ArH), 6.40-6.44(m, 1H, ArH), 6.81-6.84(m,lH,ArH), 6.91(d,2H,ArH), 7.40(d,2H,ArH).
The Compounds 17, 22, 25, sad 39 are prepared by the reaction of 2-formyl-5-(2-furyl)pyrrole; 2 formyl-5-[(E)-2-phenylethen-1-yl]pyrrol~-formyl-5-[(E,Er4-ph~yl-1,3-butadien-1-yl]pyrrol~-formyl 5-[(E)-proper-1-yl]pyrrole, respectively, with 2-[4-(methoxycarbonylmuhoxy)phenyl]pyrroleas described in Example 6. Compound 19 is prepared by the reaction of 2,2'-bipyrrole with 2-formyl-5-[4 (methoxycarbonylmethoxy)phenyl]pyrrole, which is obtained by the V ilsmeier-Hsack formylation of 2-[4 (methoxycarbonylmethoxy)phenyl]pyrrole, as described in Example 6.
EXAMPLE 7: 4,4-Difluoro-3-[(E)-2-[4-(methoxycsrbonylmethoxy)phenyl]ether-1-yl]-5-[(E)-2-phenylethen-1-yl]-4-bore-3a,4a-diaza-s-indacene (Compound 13): This is prepared in the same manner as described in Example 1 from 2-formyl-5-((E)-phenyleth~-1-yl]pyrrole(75 mg, 0.38 mmol) and 2-[(E)-2-[(4-methoxycarbonylmethoxy)ph~yl]ether-1-yl]pyrrole(100 mg, 0.39 mmol). The Compound 23 (59 mg, 3296) is obtained as a dark blue solid.
The 2-[(E)-2-[4-(methoxycarbonylmethoxy)phenyl]ethen-1-yl]pyrrole needed for this synthesis is prepared in the similar way as described in Example 6 by the reaction of 2-[(E)-2-(4-methoxyph~yl)ethen-1-yl]pyrrole with ethanethiol, sodium salt, and methyl bromoacetate in DMF.
EXAMPLEB: 4,4-Difluoro-3-(2-methoaycarbonylethyl)-5-[(E)-2-pheaylethyl-1-yl]-4-hors-3a,4s-diara-s-indac~e (Compound 45): To a solution of 3.0 g (15.2 mmol) of 2-formyl-5-[(E)-2-phe~nylethyl-1-yl]pyrrole and 2.33 g (15.2 mmol) of 2-(2-methoxycarbonylethyl)pyrrole in 70 mL of dichloromethane is added 1.45 mL (15.6 mmol) of phosphorus oxychloride. After the reaction mixdrre is stirred at room temperature for 18 hours, 10 mL (57.4 mmol) of N,N-diisopropylethylamine, is added followed by the addition of 7 mL (56.9 mmol) of boron trifluroide etherate. It is stirred at room temperature for 3 hours and worked up in the same manner as described in Example 1 to give 3.57 g (6296) of Compound 45 as a dark red-purple solid.
Compounds 27, 38, and 44 are prepared by the reaction of 2-formyl-5-[(E,E)-4-phenyl-1,3-butadien-1-yl]pyrrole, 2-formyl-5-[(E)-proper-1-yl]pyrroleand 2-formyl-5-[(E)-2-(1-naphthylkthen-1-yl)pyrrole, respectively, with 2-(2-methoxycarbonylethyl)-pyrrole as described in Example 8.

WO 93/09185 2 1 ~ 2 ~ PCT/US92/08350 Compounds 30, 36, 37 and 41 are prepared by the reaction of 2-[(E)-2-carbomethoxyethen-1-yl)pyrrole, 2-[(E)-2-(5-carboethoxy-4-methyl-2-oxawlyl~th~-1-yl]pyrrole,2-[(E)-2-(S~arbomethoxy-4-methyl-2-oxazolyl)ethen-1-yl]-pyrrole and 2-[(E)-2-(4-carbomethoxyphenyl)ethen-1-yl]pyrrole, respectively, with 3,5-dimethylpyrrole-2~arboxaldehyde as described in Example 8.
5 Compound 33 is prepared from 2-[(E)-2-carbomethoxyeth~-1-yl]pyrrole and 3,5-diphenylpyrrole-2-carboxaldehyde in the similar way as described in Example 8.
Most of the key intermediates needed for the synthesis of Compounds 22, 23, 25, 27, 30 33, 36, 37, 38, 39, 41, 44 and 45 are prepared from the appropriately selected starting materials. As a typical example, 2-[(E)-2-(4-carbomethoxyph~yl~th~-1-yl]pyrrole needed for the synthesis of Compound 41 10 is 3prepared as follows: A mixture of 1.0 g (10.5 mmol) of pyrrole-2-csrboxaldehyde, 2.5 g (10.9 mmol) of methyl 4-bromoethylbeazoate, 2.2 g (10.9 mmol) of tributylphosphine sad 700 mg of zinc is heated at 100°C in a stream of nitrogen for 15 hours. After cooling to room temperature, the whole reaction mixture is treated with 50 mL of chloroform. It is washed several times (3x50 mL) with water.
The organic layer is dried with anhydrous sodium sulfate and concentrated under reduced pressure. The 15 crude product is purified by chromatography on silica gel with 309b chloroform in hexane as eluant to give 427 mg (189b) of the product as a white solid; m.p. 120-121°C, tH-NMR (CDCIg) b 3.90(s,3H,CH3), 6.26-6.29(m,lH,ArH), 6.42-6.45(m,lH,ArH), 6.69(d,lH,CH=), 6.84-6.88(m, lH,ArH), 7.08(d,1H,CH=), 7.45(d,2H,2xArH), 7.99(d,2H,2xArH), 8.55(bs,1H,NH).
20 EXAMPLE 9: 4,4-Difluoro-3-(2-carboxyethyl)-5-(2-thienyl)-4-hors-3a,4a~iaTa-s-indacene(Compound 2): To a solution of 3.0 g (8.33 mmol) of Compound 1 in 200 mL of tetrahydrofuran are added 100 mL of water and 12 mL of 85 96 phosphoric acid and the whole mixture is heated under reflux for 4 days.
After cooling to room temperature, the reaction mixture is concentrated under reduced pressure to remove most of the tetrahydrofuran. The resulting aqueous residue is extracted with chloroform. The chloroform layer is dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a purple-red oil. The crude product is subjected to silica gel column chromatography. It is first eluted with chloroform to give 0.65 g of an unreacted starting material 1. It is then eluted with 196 methanol in chloroform to afford 1.75 g (6196) of Compound 2 as a dark purple solid.
Compounds 6, 8, 15, 18, 20, 24, 26, 28 31, 34, 40, and 46 which contain a carboxylic acid as a reactive group, are prepared by hydrolysis of the corresponding esters 5, 7, 14, 17, 19, 23, 25, 27, 30, 33, 39 and 45 as described in the preparation of Compound 2 in Example 9.
EXAMPLE 10: Succinimidyl ester (Compound 3): To a solution of 650 mg (1.88 mmol) of Compound 2 in 70 mL of tetrahydrofuraa is added 230 mg ( 1.99 mmol) of N-hydroxysuccinimide followed by addition of 410 mg (1.99 mmol) of N,N'-dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 24 hours. The resulting precipitate is removed by filtration and the filtrate is evaporated to dryness under reduced pressure to give a crude product. It is purified by chromatography on silica gel with chloroform as eluant to give 715 mg (86 96 ) of Compound 3 as a dark purple solid.
Compounds 9, 16, 21, 29, 32, 35 and 47, which contain a succinimidyl ester as a reactive group, WO 93/09185 21 2 ~ ~ ~ S 2'~ PGT/US92/08350 are prepared from the contesponding carboxylic acids 8, 15, 20, 28, 31, 34 and 46 by the similar method as described in the preparation of Compound 3 in Example 10.
EXAMPLE 11: Hydrazide (Compound 4): To a solution of 10 p,L of anhydrous hydrazine in 1 mL of chloroform is added 25 mg of Compound 3 and the mixture is stirred at room temperature for 30 minutes. The reaction mixture is diluted with 15 mL of chloroform, washed with water, dried over anhydrous sodium sulfate and c~centtated under reduced pressure to give a crude product. It is purified by silica gel column chromatography with 296 methaaol in chloroform as eluaat to give 21 mg (9896) of Compound 4.
EXAMPLE 12: Amine (Compound 48): To a solution of 150 ~,L (2.24 mmol) of ethyl~ediamine in 2 mL of chloroform is added a solution of 100 mg (0.22 mmol) of Compound 47 in 3 mL of chloroform dropwise over a period of 10 minutes and the mixture is stirred at room temperature for 1 hour. It is then diluted with 15 mL of chloroform, washed with water (3x20 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product. It is purified by chromatography on silica gel with 1096 merbaaol in chloroform as eluaat to give 75 mg (8596) of the amine 48. Compound 61 is prepared from Compound 3 by the same method as described in Example 12.
EXAMPLE 13: Iodoacetamide (Compound 49): To a suspension of 20 mg (0.05 mmol) of Compound 48 in 3 mL of chloroform is added 10 pL of triethylamine, followed by addition of 12 mg (0.05 mmol) of iodoacetic anhydride and the mixture is stirred at room temperature for 2 hours. After evaporation under reduced pressure, it is purified by silica gel column chromatography with 296 methanol in chloroform as eluant to give 19 mg (6796) of the iodoacetamide 49. Compound 62 is prepared from Compound 61 by the same method as described in Example 13.
EXAMPLE 14: Maleimide (Compound 50): To a suspension of 20 mg (0.05 mmol) of Compound 48 in 3 mL of chloroform is added 10 ~L of chloroform is added 10 ~cL of triethylamine, followed by addition of 13 mg (0.05) of succinimidyl maleimidylacetate and the mixture is stirred at room temperature for 2 hours. It is concentrated and purified by column chromatography on silica gel with 296 methaaol in chloroform as eluant to give 18 mg (67 96 ) of the maleimide 50.
EXAMPLE 15: Aldehyde (Compound 51): To a suspension of 20 mg (0.05 mmol) of Compound 48 in 3 mL of chloroform is added 10 ~cL of triethylamine, followed by addition of 12 mg (0.05 mmol) of succinimidyl p-formylbenzoate and the mixture is stirred at room temperature for 2 hours. It is concentrated and purified by column chromatography on silica gel with 2 96 methanol is chloroform as eluant to give 22 mg (8396) of the aldehyde Sl.
EXAMPLE 16: Azide (Compound 52): To a suspension of 20 mg (0.05 mmol) of Compound 48 in WO 93/09185 ~ 1 ~ z 6 ~ 22 PCT/LJS92/08350 3 mL of chloroform is added 10 ~,L of triethylamine, followed by addition of 13 mg (0.05 mmol) of succinimidyl p-azidobenzoate. After stirring at room temperature for 2 hours, it is concentrated under reduced pressure and purified by column chromatography on silica gel eluting with 19b methanol in chloroform to give 17 mg (63 96) of the aside 52.
S
EXAMPLE 17: Acrylamide (Compound 53): To s suspension of 20 mg (0.05 mil) of Compound 48 in 3 mL of chloroform is added 10 pL of triethylamine, followed by addition of 9 mg (0.05 mmol) of succinimidyl acrylate and the mixture is stirred at room temperature for 2 hours. After concentration under reduced pressure, it is purified by silica gel column chromatography with 196 methanol in chloroform as eluant to give 18 mg (7996) of the acrylamide 53.
EXAMPLE 18: Alcohol (Compound 42): To a solution of 125 mg ( 1.01 mmol) of 3,5-dimethylpyrrole-2-carboxaldehyde and 200 mg (1.00 mmol) of 2-[(E)-2-(4-hydroxymethylphenyl)ethen-1-yl]pyrrolein 15 mL of dichloromethane is added 100 WL (1.07 mmol) of phosphorus oxychloride.
The reaction mixture is stirred at room temperature for 24 hours and then is added 700 ~.L (4.02 mmol) of N,N-diisopropylethylamine, followed by addition of 490 ~L (3.98 mmol) of boron trifluoride etherate. After the whole mixture is stirred at room temperature for 2 hours, it is washed with two 15 mL portions of water. The organic layer is separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a dark red oil. The crude product is subjected to silica gel column chromatography. It is first eluted with 1:1 hexane/chloroform to give 32 mg of the chloride, Compound 43, as a dark red solid. It is then eluted with chloroform to afford 75 mg (219b) of the alcohol, compound 42, as a dark red solid.
The compound 2-[(E)-2-(4-hydroxymethylphenyl)ethen-1-yl]pyrrole needed for this synthesis is prepared as follows: To a solution of 1.0 g (4.4 mmol) of 2-[(E)-2-(4-carbomethoxyphenyl)ethen-1-yl]pyrrole in 20 mL of dry tetrahydrofur~n is added 167 mg (4.4 mmol) of lithium aluminum hydride portionwise while the reaction mixture is stirred at 0°C under nitrogen atmosphere. After addition is complete, it is stirred at room temperature for 15 hours. It is then cooled with an ice-water bath and the excess lithium aluminum hydride is destroyed by the slow addition of 2 mL
water. The reaction mixture is conc~trated under reduced pressure to remove most of the tetrahydrofuraa.
The resulting residue is extracted with chloroform (3x50 mL), dried over anhydrous sodium sulfate and concentrated to give 0.68 g (7896) of 2-[(E)-2-(4-hydroxymethylphenyl)ether-1-yl]pyrroleas an off white solid, m.p. 186-189 °C;
1H-NMR(CDCIg) b 4.69 (s,2H,CH~, 6.24~.27(m, lH,ArH), 6.35~.37(m,lH,ArH), 6.66(d,lH,=CH), 6.81-6.84(m,lH,ArH), 6.98(d,lH,=CH), 7.33(d,2H,ArH), 7.44(d,2H,ArH), 8.36(bs,lH,NH).
EXAMPLE 19: Halide (Compound 43): To a solution of 20 mg (0.06 mmol) of Compound 42 in 5 mL of dichloromethane is added one drop of triethylamine, followed by the addition of 5 mg (0.03 mmol) of cyanuric chloride. The mixture is stirred at room temperature for 1 hour.
It is then subjected to silica gel column chromatography with 1:1 hexane/chloroform as eluant to give 15 mg (7196) of the halide 43.

EXAMPLE 1A: laoth;ayanate (Compormd S4): To a surpeoaoo of 30 m= (0.07 tmaol) of Compamd 46 in 5 mL of chloroform is added 20 ~I. of triethylamine, foUowad by the addition of 10 ~I, (0.13 tomol) of thiophos8ate and the mixture is aimed at room temperature for 1 hour. It is concentrated under rndtroed pun and the resultia= resadva a purified by column chromatosrap6y an silica 8el with chloroform as eluant to dive 22 m8 (669G) of the isothiocyaarte 54.
EXAMPLE Il: Anhydride (Coc~ound S~: To a suspension of 30 m8 (0.09 mmol) of Compound 2 in S mL of chloroform is added 15 ~I. of triethylamine, followed by addition of 10 ~I. of ethyl c6loroformate and the couture is stirred at room tampasture for 1 hour.
Without isolation of the anhydride, Compound 55. it is characterized as its n-butyl amide. Compormd Sa.
by dditian of 15 ~L
of n-butylamine to the reaction mixture. The cede residue isolated by evapoation of the reaction mixture is purified by column chromato8raphy ca silica 8e1 with chloroform as eluaat to yield 29 m8 (85 96 ) of the Compound 58.
EXAMPLE 22: Acyl aade (Compound 5~: To a wspeaaioa of 30 m~ (0.09 mmol) of Compound Z
in 5 nsL of chloroform is added 15 ~L of triethylamine, followed by the additiaa of 20 ~L (0.09 mmol) of dipheaylpbosphoryl ar~de and the reaction mixture is stirred at room temperaturo for 3 hours. It is concentrated undo roduced pr~un sad purified by column chromato8rap6y on silics 8e1, elutin8 with chloroform to 8ive 1? m8 (5396) of the acyl a>'de 56.

EXAMPLE ?3: Carbonyl fluoride (Compound Sn: To a solution of 10 m~ (0.03 mmol) of Compound I and 3 ~L of pyridine is 5 mL of dichloromethane is added 2 y~L of cyaauric fluoride and the mixture is stirred at room temperature for 30 minutes. Without isolation of the carbonyl fluoride, Compound 57, it is characterized as its n-butyl amide, Compound 58 (10 mg, 9096 yield) as described in Exar~rple 21.
EXAMPLE IA: Sulfonyl Chloride (Com~povmd 60): To an ice-cooled solution of 30 m= (0.08 mmol) of 4,4ditluoro-3,5-his[(E~2-phenYlethw-1'Yll-4-bon-3a,4a-diaza-s-indaxne(which is prepared from 2~
I(~-2-pb~Yl~'1-YIIPY~leand 2-formyl-5-[(E~2-pheaYlethea~1-yIJPYrroieas described in Example 1) is 3 mL of dichloromethane is added 12 ~L (0.18 tamol) of chlorosulfonic acid and the mixture is stirred at 0'C for r/i hours undo aitroBCa armoap6a~e. T6e resultin8 precipitate is collected by filtruion, dissolved in 1 mL of water and the solution is treated with 20 m8 of sodium bicarbonate. The cede product is purified by chromato8rsphy ova lipophilic Sephaderc*usin8 water for elution sad the desired fractions are combined sad lyophilized to 8ive 12 m8 of Compound 59. To 300 ~L
of thionyl chloride is added 30 ~L of dry dimethylformamide sad the mixture is stirred st room temperature for 10 minutes.
To the above solution is added 10 m8 of Compound 59 and the mixture is stirred a room temperature for 2 hours. The rewltin8 ruction mixture is treated with 10 mL of chloroform It is poured into 10 mL of ice water and shakes vi8orously for a few minutes. The chloroform layer is separated, dried over anhydrous sodium sulfate sad concentrated under reduced pressure to Give ? m8 of the wlfonyl chloride f0 as a dark blue wlid.
*Trademark s WO 93/09185 21 ~ 2 6 2 7 PCT/US92/08350 .r- 24 .r...,.
EXAMPLE 25: Sphingosine Conjugate (Ceramide): To a solution of 5 mg of D-sphingosine (Sigma Chemical Co., St. Louis, MO) in 1 mL of chloroform are added 5 ~L of triethylamine and 9 mg of Compound 47 and the mixture is stirred at room temperature for 12 hours. The reaction mixture is subjected to chromatography on silica gel with 196 methanol in chloroform.
From the desired combined fractions 7 mg of a fluorescent ceramide is obtained: m.p. 77-79 ° C, R~ 0.51 ( 10 % methanol in chloroform), absorption maximum: 564.5 am in methanol, emission maximum: 571 nm in methanol.
EXAMPLE 26: Nucleotide Conjugate: To a solution of 2 mg of 5-(3-aminoallyl)-2'~leoxyuridine-5'-triphosphate, ammonium salt (Sigma Chemical Co., St. Louis, MO) in 200 ~L of water is added a solution of 2 mg of Compound 3 in 200 ~L of acetonitrile, followed by addition of 5 ~L of triethylamine.
After the mixture is stirred at room temperahue for 3 hours, it is purified by chromatography over lipophilic Sephadex using water for elution. The desired fractions are combined and lyophilized to give 2 mg of the fluorescent nucleotide: Rf=0.47 (10:5:9:9/dioxaae:2-propaaol:water: ammonium hydroxide), absorption maximum: 558.6 nm in pH 7 phosphate buffer; emission maximum: 570 nm in pH 7 phosphate buffer.
EXAMPLE 27: Oligonuclootide Conjugate: The 5'-amine-derivatized oligonuclootide of sequence XTGTAAAACGACGGCCAGT is prepared by automated solid phase synthesis on a ABI
380A using the standard 1 ~,M synthesis cycle, where X, the last phosphoramidite (5' ~d), is the commercially available C6 aminolink-TFA obtained from Glen Research Inc., Sterling, VA. After the standard cleavage from the solid phase column and deblocking in concentrated aqueous ammonia (5 hours at 55°C), the oligonucleotide is concentrated on a vortex evaporator to dryness in a 15 mL
plastic c~trifuge tube. The oligonuclootide is desalted with a Bio-Rad IODG desalting column, Bio-Rad Labs, Richmond, CA, using the standard procedure. The oligonucleotide is aliquoted into conveai~t 10 ODU
portions is 2 mL
microcentrifuge tubes and evaporated on a speed-vac. The 5'-amine-derivatized oligonucleotide, 10 ODU
(350 pg, 0.055 pM) dissolved in 250 ~L of 0.5 M NaHC03/Na2C03 pH 9 aqueous buffer in the microcentrifuge tube. The compound 16, 2 mg (3.8 ~M), is dissolved in 200 pL
of DMF and added to the oligonucleotide solution. This is shaken by hand and allowed to sit for 16 hours. 1 mL of 0.1 M
TEAR is added and the solution is passed through a Bio-Rad IODG desalting column substituting 0.1 M
TEAR for deionized water in the standard procedure. The second 2 mL fraction is collected and evaporated in a 15 mL centrifuge tube on a vortex evaporator. The labeled oligonucleotide is then HPLC
purified on a 220 mm x 10 mm 300th C8 reverse phase column, Rainin Instrument Co., Woburn, MA, using the following gradient: Solv~t A - 0.1 M TEAR , Solvent B -acetonitrile. Ramp solv~t B from 15 96 to 95 96 over 40 minutes then hold at 95 9b for 5 minutes. Detection is with a Waters 490 dual wavelength UV-Vis detector ~nitoring 254 am and 590 nm. The appropriate peak is collected and evaporated to give 2.3 ODU (80 p,g, 0.013 WM) 2496 yield.
Example 28: Protein Conjugates: The succinimidyl esters, Compounds 3 and 16, are dissolved to give a cony --~-,~:~~ ~f m ,t,Q dve/mL DMF. Bovine serum albumin (BSA) or other proteins are dissolved WO 93/09185 ~ ~ ~ ~ ~ ~ ~ ~ PCT/US92/08350 to a concentration of approximately 10 mg protein /mL in 0.1 M sodium bicarbonate. Amicon GH-25 desalting a~edis in phosphate buffered saline (PBS) sad 1.5 M hydroxylamine pH
8 are freshly prepared.
The appropriate amount of each dye in DMF corresponding to a molar ratio of 10 (dye/pmtein) is slowly added to the separate protein solutions with stirring. Each reaction is incubated at room temperature for one hour and is the terminated by the addition of hydroxylamine to a final concentration of 0.15 M.
Following incubation for an additional thirty minutes the protein c~jugates are separated from free unrrxcted dye in the two solutions by gel filtration on a GH-25 column equilibrated in PBS. The initial, protein-containing fractions from each eoludon are pooled and the degree of substitution is determined fmm the extinction coefficient for each dye at its absorption ma~timum (559 am for Compound 3 and 590 nm for Compound 1~. An approximate degree of substitution of 3 (dyeBSA) is determined for both cases. The fluoreec~ce of each final protein conjugate is approximately 1096 of the free dye.
R-phycoerythrin, avidin, epidermal growth factor, immunoglobulins, concaaavalin A, wheat germ agglutinin, protein A are conjugated with succinimidyl esters using essentially the same procedure.
Example 29: Peptide Conjugate: To a suspez<sion of 5 mg of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys (Bachem. Inc., Torrance, CA) in 500 ~L of dimethylformamide is added 3 ~L of triethylamine followed by the addition of 3 mg of Compound 3 and the whole reaction mixture is stirrod at room temperature for 30 minutes. The reaction mixture is purified by chromatography over lipophilic Sephadex using water for elution. The desired fractions are combined and lyophilized to give 4 mg of the fluorescent hexapeptide.
Example 30: Carbohydrate Conjugate: To a solution of 5 mg of 6-amino-deoxy-D-glucose hydrochloride (U.S. Biochemical Corp., Cleveland, OH) in 0.5 mL of water and 1 mL of tetrahydrofuran is added 5 ~,L of triethylamine followed by the addition of 10 mg of Compound 3. After the mixture is stirred at room temperature for 1 hour, it is purified by chromatography over silica gel using 1096 methanol in chloroform. From the desired combined fractions 12 mg of a fluorescent carbohydrate is ob4ined: m.p. 99-102°C, Rf=0.53 (2596 methanol in chloroform), absorption maximum: 559.0 am in methanol, emission maximum: 570 nm in methanol.
EXAMPLE 31: Determination Of The Chemical Reactivity Of The Dyes: The chemically reactive dyes that are the subject of this invention are subjxted to incubation in aqueous, mdhaaolic, or chloroform solution with model compounds and their reactivity is demonstrated by thin layer chromatography is a solvent that separates the rractive dye from its products with visual detection of the fluorescence emission. It is demonstrated that n-butylamine reacts to form a new product with 3, 9, 16, 21, 32, 35, 47, 54, 55, 56, and 57, that 2-mercaptoethanol reacts with 43, 49, 50, and 62 that acetic anhydride reacts with 4, 42, 48, and 61, that acetone reacts with 4, that hydrazine reacts with 51, and that brefeldin A
reacts with 2 in the presence of N,N'~icyclohexylcarbodiinude to give as ester. Furthermore, it is demonstrated that the esters such as 1 can tract with hydrazine to give hydrazides such as 4.

WO 93/09185 2 ~ PCT/US92/08350 EXAMPLE 32: Spectral Characteri~tion Of The Dyes: 1H NMR spectra are measured using a General Electric QE-400 MHz spectrometer for solutions in CDCIg (unless otherwise stated), with tetramethylsilane ('TMS) as an internal standard. Chemical shifts are given in ppm from TMS and splitting patterns are designated as: s, singlet; d, doublet; t, triplet; m, multiplet. Results of spectral data for repre~tative new dyes are given in Tsble 4.
Absorption spectra era obtained using an IBM 9429 UV/visible spectrophotometer by dissolving the dye at a conc~tration of approximately SxlO~ M in an appropriate solvent including but not limited to methanol, chloroform, acetonitrile, acetone or hexane. Extinction coefficients (E) of the dyes at their absorption maxima are determined by standard Boer's law calculations. Results of the spectral doterminstion for representative new dyes are tai~ulated in Tsble 3 and Table S.
Fluorescence of new long wavelength reactive dipyrromefh~eboron difluoride dyes ie determined using a Perkin-Elmer Model 650-40 fluorescence spectrophotometer equipped with a Perkin-Elmer/Hitachi 057 X-Y recorder by dissolving the dye st a concentration of shove 1x10'~~ M
(optimum concentration ranges, 10~ -- 10-7 M) in an appropriate solvent including but not limited to methanol, water, ethanol, acetonitrile, acetone, chloroform, tolu~e or hexane. Results of the spectral determination for rrpresentative new dyes are summarized in Table 3 and Table 5. Fluorescence can also be observed for the dyes in solution or on thin layer chromatography (TLC) plates, by visual inspection with illumination by a suitable source that gives off light below 650 nm.
EXAMPLE 33: Photobleaching Studies: Photoetability measurem~ts of new long wavelength inactive BDI dyes are conducted in scetonitrile solution with continuous illumination by a 250 watt xenon arc lamp with 20 nm slit width at the corresponding excitation maxima. After thirty minutes of continuous illumination while stirring at room temp~shu~e in a cuvette of a fluorometer, emission intensity data is collected with 2.5 am slit width st the emission maxima of individual samples.
The results are listed in Table S, together with that of the alkyl-substituted BDI dye, Compound R, for comparison (also shown in Figure 4). Conc~trations of individual samples are pr~eparad to have the same optical density for all of the dyes st the respective excitation wavehgth.
It will be obvious to one skilled in the art that synthesis of many other long wavelength reactive 4,4 difluoro-4-bore-3a,4a-diem-s-indac~e dyes that contain other bsthochromic moieties can be acxomplished using other appropriate pyrrole precursors that contain substituents compatible with the chemistry in Figure 1 and such long wavelength reactive 4,4-difluoro-4-bore-3a,4a-diaza-s-indaceae dyes from other appropriately substituted pyrrole precursors will fall within the description of the invention.

Claims (46)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A compound of the formula:

wherein at least one of the substituents R1-R7 is -LMG where G is a reactive functional group that is a carboxylic acid, a succinimidyl ester, an acyl azide, an anhydride, an acid halide, an acrylamide, an alcohol, an aldehyde, an azide, an imido ester, a sulfonate ester, a haloacetamide, an alkyl halide, a sulfonyl halide, a hydrazine, an isocyanate, an isothiocyanate, or a maleimide group and -Lm-, where m = 0 or 1, is a substituted or unsubstituted alkyl (containing 1-5 carbons) or arylene group, or a linking bathochromic moiety;
and wherein the remaining substituents R1-R7, any of which may be the same or different, are hydrogen; halogen; alkyl (containing 1-5 carbon atoms); sulfo; arylalkyl, the alkyl portion of which contains 1-5 carbon atoms; or aryl which may be further substituted, one or more times, by alkyl (containing 1-5 carbon atoms), or alkoxy groups wherein the alkyl portions of such groups have less than 5 carbon atoms;
or a separate bathochromic moiety; or combinations thereof;

such that at least one of the substituents R1-R7 contains a linking bathochromic or a separate bathochromic moiety that is an ethenyl, dienyl, trienyl, or heteroaryl group that is attached to the compound by a single covalent bond;

said compound having an absorption maximum at a wavelength longer than about 525 nm in methanol and a quantum yield of greater than about 0.1.

2. A compound, as claimed in Claim 1, wherein G is a carboxylic acid, a succinimidyl ester, an acyl azide, an anhydride, an acid halide, a sulfonyl halide, an isocyanate, an isothiocyanate, or an imido ester.

3. A compound, as claimed in Claim 1, wherein G is a haloacetamide, a maleimide, an alkyl halide, or a sulfonate ester.

4. A compound, as claimed in Claim 1, wherein L is an unsaturated organic group that is substituted or unsubstituted phenylene, alkenyl or heteroaryl.

5. A compound, as claimed in Claim 1, substituted by 1-4 separate bathochromic moieties where each separate bathochromic moiety is an unsaturated organic group.

6. A compound, as claimed in Claim 5, wherein at least one of the unsaturated organic groups is a heteroaryl group that is a pyrrole, thiophene, furan, oxazole, isoxazole, oxadiazole, imidazole, benzoxazole, benzothiazole, benzimidazole, benzofuran, or indole.

7. A compound, as claimed in claim 6, in which the heteroaryl group is substituted one or more times, by aryl, arylalkyl, heteroaryl, alkyl, or alkoxy groups, or combinations thereof, the alkyl portions of which have fewer than 5 carbons.

8. A compound, as claimed in claim 6 or 7, in which any substituent is further substituted by an ester or amide.

9. A compound, as claimed in Claim 6, having an emission maximum at a wavelength longer than about 550 nm in methanol.

10. A compound, as claimed in Claim 5, wherein at least one of the unsaturated organic groups is an alkenyl group that is ethenyl, butadienyl or hexatrienyl.

11. A compound, as claimed in claim 10, in which the alkenyl group contains substituents, which may be the same or different, selected from hydrogen, halogen, alkyl (containing 1-5 carbon atoms), cyano, carboxylate ester, carboxamide, aryl or heteroaryl.

12. A compound, as claimed in Claim 1, wherein L is a straight chain unsubstituted alkyl group containing 1-5 carbons and G is a succinimidyl ester (-COO(NC4O2H4)).

13. A compound, as claimed in Claim 1, wherein R2 or R6, or R2 and R6 are sulfo or a salt thereof.

14. A compound, as claimed in Claim 1, wherein R1 is L m G; R7 is a separate bathochromic moiety that is an unsaturated organic group; R5 is a separate bathochromic moiety or is a hydrogen, alkyl, sulfo or salt thereof; and the remaining substituents, which may be the same or different, are hydrogen, alkyl, sulfo or a salt thereof.

15. A compound, as claimed in Claim 14, wherein R1 is L m G , wherein L is a straight chain unsubstituted alkyl group containing 1-5 carbons and G is a succinimidyl ester (COO(NC4O2H4));

R7 is a heteroaryl group that is a pyrrole, thiophene, furan, oxazole, isoxazole, oxadiazole, imidazole, benzoxazole, benzothiazole, benzimidazole, benzofuran, or indole;or R7 is an alkenyl group that is ethenyl, butadienyl or hexatrienyl;

R2 and R6, which may be the same or different, are hydrogen, alkyl, sulfo or a salt thereof;

and the remaining substituents, which may be the same or different, are hydrogen or alkyl.

16. A compound, as claimed in claim 15, in which, when R7 is a heteroaryl group, it is substituted one or more times by aryl, arylalkyl, heteroaryl, alkyl, or alkoxy groups, or combinations thereof, the alkyl portions of which have fewer than 5 carbons.

17. A compound, as claimed in claim 15 or 16, in which any substituent is further substituted by an ester or amide.

18. A compound, as claimed in claim 15, in which, when R7 is an alkenyl group, it contains substituents, which may be the same or different selected from hydrogen, halogen, alkyl (containing 1-5 carbon atoms), cyano, carboxylate ester, carboxamide, aryl or heteroaryl.

19. A dye-conjugate comprising a specific binding pair member sbp and one or more dye molecules of the formula:

wherein at least one of the substituents R1-R7 covalently binds each dye molecule to the specific binding pair member;

wherein, further, at least one of the substituents R1-R7 contains a linking bathochromic moiety or separate bathochromic moiety that is an ethenyl, dienyl, trienyl, or heteroaryl, and which is substituted or unsubstituted; and the remaining substituents R1-R7, any of which may be the same or different, are hydrogen; halogen; sulfo or a salt thereof;
cycloalkyl; alkyl or arylalkyl, the alkyl portion of which contains 1-5 carbon atoms; or aryl, which may be further substituted, one or more times, by alkyl or alkoxy, the alkyl portions of which have less than 5 carbon atoms; or combinations thereof.

20. A dye-conjugate according to Claim 19, wherein sbp is a nucleotide.

21. A dye-conjugate according to Claim 19, wherein sbp is an oligonucleotide.

22. A dye-conjugate according to Claim 19, wherein sbp is a peptide or a protein.

23. A dye-conjugate according to Claim 22, wherein the protein is an antibody.

24. A dye-conjugate according to Claim 22, wherein the protein is avidin or streptavidin.

25. A dye-conjugate according to Claim 19, wherein sbp is a drug.

26. A dye-conjugate according to Claim 19, wherein one of R1-R7 contains a heteroaryl group that is a pyrrolyl, thiophenyl, furanyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, or indolyl.

27. A dye-conjugate according to claim 26, in which the heteroaryl group contains substituents that are independently aryl, arylalkyl, alkyl, or alkoxy groups, the alkyl portions of which have fewer than 5 carbons, or by a second heteroaryl.

28. A dye-conjugate as claimed in claim 27, in which the second heteroaryl group contains substituents independently selected from aryl, arylalkyl, alkyl, or alkoxy groups, the alkyl portions of which have fewer than 5 carbons, or by combinations thereof.

29. A dye-conjugate as claimed in Claim 26, wherein the heteroaryl group is 2-pyrrolyl or 2-thienyl.

30. A dye-conjugate according to Claim 19, wherein one of R1-R7 contains an alkenyl group that is ethenyl, butadienyl or hexatrienyl.

31. A dye-conjugate as claimed in Claim 30, wherein the alkenyl group contains substituents independently selected from hydrogen, halogen alkyl (containing 1-5 carbons), cyano, carboxylate ester, carboxamide, aryl or heteroaryl.

32. A dye-conjugate, as claimed in Claim 31, in which the heteroaryl substituent on the alkenyl group contains substituents independently selected from aryl, arylalkyl, alkyl, or alkoxy groups, the alkyl portions of which have fewer than 5 carbons, or by combinations thereof.

33. A dye-conjugate as claimed in Claim 19, wherein one or more dye molecules are linked to the sbp through a bond that is an amide, an ester, an ether, a thioether, a urea, a thiourea, an amidine, or an alkyl amine.

34. A dye-conjugate as claimed in Claim 19, wherein the dye molecule is separated from the sbp by a linker containing 1-5 carbons.

35. A dye-conjugate as claimed in Claim 19, wherein R2 or R6, or R2 and R6 are sulfo or a salt thereof.

36. A dye-conjugate comprising a specific binding pair member sbp and one or more dye molecules of the formula:

wherein at least one of the substituents R1-R3 covalently binds said dye molecule to the specific binding pair member that is a nucleotide or an oligonucleotide;
wherein, further, at least one of the substituents R1-R3 contains a linking bathochromic moiety or separate bathochromic moiety that is an ethenyl, dienyl, trienyl, or heteroaryl, and which is substituted or unsubstituted; and the remaining substituents R1-R7, which may be the same or different, are hydrogen; halogen; sulfo or a salt thereof;
alkyl or arylalkyl, the alkyl portions of which contain 1-5 carbon atoms; or aryl, which may be further substituted, one or more times, by alkyl or alkoxy, the alkyl portions of which have less than 5 carbon atoms; or combinations thereof.

37. A dye-conjugate according to Claim 36, wherein one of R1-R3 contains a heteroaryl group that is a pyrrolyl, thiophenyl, furanyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, or indolyl.

38. A dye-conjugate according to claim 37, in which the heteroaryl group contains substituents independently selected from aryl, arylalkyl, alkyl, or alkoxy groups, the alkyl portions of which have fewer than 5 carbons, or by a second heteroaryl group.

39. A dye-conjugate as claimed in Claim 38, in which the second heteroaryl group contains substituents independently selected from aryl, arylalkyl, alkyl, or alkoxy groups, the alkyl portions of which have fewer than 5 carbons, or by combinations thereof.

40. A dye-conjugate according to Claim 36, wherein one of R1-R3 contains an alkenyl group that is ethenyl, butadienyl or hexatrienyl.

41. A dye-conjugate according to Claim 40, in which the alkenyl group contains substituents independently selected from hydrogen, halogen, alkyl (containing 1-5 carbons), cyano, carboxylate ester, carboxamide, aryl or heteroaryl.

42. A dye-conjugate according to Claim 41, in which the heteroaryl contains substituents independently selected from aryl, arylalkyl, alkyl, or alkoxy groups, the alkyl portions of which have fewer than 5 carbons, or by combinations thereof.

43. A dye-conjugate comprising a specific binding pair member sbp and one or more dye molecules of the formula:

wherein at least one of the substituents R1-R3 covalently binds said dye molecule to the specific binding pair member that is a protein;
wherein, further, at least one of the substituents R1-R3 contains a linking bathochromic moiety or separate bathochromic moiety that is an ethenyl, dienyl, trienyl, or heteroaryl, and which is substituted or unsubstituted; and the remaining substituents R1-R7, which may be the same or different, are hydrogen; halogen; sulfo or a salt thereof;
alkyl or arylalkyl, the alkyl portions of which contain 1-5 carbon atoms; or aryl, which may be further substituted, one or more times, by alkyl or alkoxy, the alkyl portions of which have less than 5 carbon atoms; or combinations thereof;
where each of said dye molecules, when not conjugated to the sbp, has an absorption maximum at a wavelength longer than about 525 nm in methanol and a quantum yield of greater than about 0.1.

44. A dye-conjugate, as claimed in Claim 43, wherein the protein is an antibody, avidin, or streptavidin.

45. A dye-conjugate, as claimed in Claim 43, wherein R3 is a heteroaryl group;
R2 and R6, which may be the same or different, are hydrogen, alkyl, sulfo or a salt thereof;
and the remaining substituents, which may be the same or different, are hydrogen or alkyl.

46. A dye-conjugate, as claimed in Claim 43, wherein R3 is an alkenyl;
R2 and R6, which may be the same or different, are hydrogen, alkyl, sulfo or a salt thereof;
and the remaining substituents, which may be the same or different, are hydrogen or alkyl.