CA2450501C - Asymmetric benzoxanthene dyes - Google Patents

Abstract

A class of asymetric monobenzoxanthene compounds useful as fluorescent dyes are disclosed having structure (I) wherein Y1 and Y2 are individually hydroxyl, amino, imminium, or oxygen, R1-R8 are hydrogen, fluorine, chlorine, alkyl, alkene, alkyne, sulfonate, amino, amido, nitrile, alkoxy, linking group, and combinations thereof, and R9 is acetylene, alkane, alkene, cyano, substituted phenyl, and combinations thereof. The invention further includes novel intermediate compounds useful for the synthesis of asymmetric benzoxanthene compounds having general structure (II) where substituents R3-R7 correspond to like-referenced substituents in the structure of described above, Y2 is hydroxyl or amine. In another aspect, the invention includes methods for synthesizing the above dye compounds and intermediates. In yet another aspect, the present invention includes reagents labeled with the asymmetric bezoxanthene dye compounds, including deoxynucleotides, dideoxynucleotides, phosphoramidites and polynucleotides. In an additional aspect, the invention includes methods utilizing such dye compounds and reagents including dideoxy polynucleotide sequencing and fragment analysis methods.

Description

ASY! C BENZ 19.L1 Y\E DYES
'f~3E TI~VEN~"I I~T
~D OF
This invention rdates g y to fluorescent dye compounds usefiil as nwYleailar probes. More specifically, this invention retates to asymrnetric benzoxanthene dyes usefui as f luorescent ]abeling reagents.

i BACKGROLTND
The non-radioactive detection of biological analiytes is an important technology in Ynodern analytical biotechnology. By eliminating the need foi- radioactive labels, safety is enhanced and the environmental impact of reagent disposal is greatly reduc,ed, resulting in decreased costs for analysis. ples of methods utilizing such non-radioactive detection methods include DNA sequencing, oligonucleotide probe methods, detection of polymerase-chain-reaction products, immunoassays, and the llike.
In many applications the independent detection of multipie spatialiy overiapping analytes in a mixtzsre is required, e.g., stngle-tube multiplex DNA probe assays, immuno assays, multicolor DNA sequencing methods, and the like. In the case of multi-loci DNA
probe assays, 2o by providing multicolor detection, the number of reaction tubes ana.y be reduced thereby simpiifyang the experimental protocols and faiii ' the i turing of appl acation-specific kits. In the case of automated DNA sequencing, multicolor iabeling altows for the analysis of all four bases in a, single lane thereby incr throughput over sangle-color methods and .
e . . ting un ' es associatedwith h inter-tane electrophoretic nnobility variations IKnitiplex detection imposes a number of severe constraints on the selection of dye labels, particularly for arWyws requiring an electrophoretic separation and tr ent with enzymes, e.g., DNA sequencing. First, it is difficult to find a collection of dyes whose eniission spectra are spectralty resolved, since the typical eniission band half-vridth for organic fluorescent dye,,s is Wxu 40-80 nanometers (nm) and the vvidth of the av ' Ae spectrum is Jainited by the excitation llight source. As used herein the term "spectral resolutiion " in reference to a set of dyes means that the fluorescent emission bands of the dyes are sufficientiy distinct, i.e., sufficiently non-overlapping, that reagents to which the respective dyes are attached, e.g.
polynucleotides, can be distinguished on the basis of the fluorescent signal generated by the respective dyes using standard photodetection systems, e.g. employing a system of band pass filters and photomultipfier tubes, charged-coupled devices and spearographs, or the hlce, as exemplified by the systems descn'bed in U.S. Pat. Nos. 4,230,558, 4,811,218, or in Wheeless et al, pgs. 21-76, in Flow Cytometry: Instrumentartion cnd Data Anwdysis (Academic Press, New York, 1985). Second, even if dyes with non-overlapping emission spectra are found, the set may stdl not be suitable if the respective fluorescent efficiencies are too low. For example , in.
the case of DNA sequencing, increased sample loading can not compensate for low fluorescence lo e$iciencies, Pringle et aL, DNA Core Facilities Newsletter, 1: 15-21 (1988). Third, when several fluorescent dyes are used conaurently, sarnaltaneous excitation becomes difficult because the absorption bands of the dyes are widely separated. Fourtk the charge, moleailar size, and conformation of the dyes must not adversely affect the electrophoretic mobilities of the fragments. Finally, the fluorescent dyes must be conipati'ble with the.
chemistry used to create or manipulate the fragments, e.g., DNA synthesis solvents and reagents, buffers, polymerase enzymes, ligase enzymes, and the like.
Because of these severe constraints oniy a few sets of fluorescent dyes have been found that can be used in multicolor applications, partimlarly in the area of four-color DNA
sequencing, e.g., Smith et aL, Nucleic Acids Resecv-ch, 113; 2399-2412 (1985);
Prober et aL, 2o Science, 238: 336-341 (1987); and Connell et al., BiotecWqrres, 5: 342-348 (1987). FIG. 1 shows examples of fluorescent xanthene dyes cuffentiy used as long-wavelength labels ernitring above 550 nm including the two rhodarnine-based dyes TAMRA (22) and ROX (26) and the two fluoresce.in-based dyes HEX (23) and NAN (24).

SUMMARY
The present invention is directed towards our discovery of a class of asymmetric benzoxanthene dyes useful as fluorescent dyes.
It is an object of an aspect of our invention to provide a class of asymmetric benzoxanthene dyes useful for the simultaneous detection of multiple spartially-30" overlapping analytes which satisfies the constraints described above and provide fluorescence emission maxima above 550 nm when illuminated by excitation light having a wavelength of between 480 nm and 550 nm.

It is a further object of an aspect of our invention to provide a class of asymmetric benzoxanthene dyes useful for simultaneous detection of multiple spatially-overlapping analytes which satisfies the constraints described above and whose fluorescence properties may be tuned by manipulation of substituents at a variety of positions.
It is another object of an aspect of our invention to provide methods and intermediate compounds useful for the synthesis of the asymmetric benzoxanthene dyes of our invention.
It is a further object of an aspect of our invention to provide nucleotides and polynucleotides labeled with the asymmetric benzoxanthene dyes of our invention.
It is another object of an aspect of our invention to provide phosphoramidite compounds including the asymmetric benzoxanthene dyes of our invention.
It is another object of an aspect of our invention to provide fragment analysis methods, including DNA sequencing methods, employing the asymmetric benzoxanthene dyes of our invention.
In accordance with an aspect of the present invention, there is provided a compound having the formula:

It6 OH

wherein:
R3 is selected from the group consisting of fluorine, chlorine, sulfonate, amino, amido, nitrile, lower alkoxy, and linking group;
R4-R7 taken separately are selected from the group consisting of hydrogen, fluorine, chlorine, lower alkyl, lower alkynyl, sulfonate, amino, amido, nitrile, lower alkoxy, and linking group; and Y2 is selected from the group consisting of hydroxyl and amine.
These and other objects of aspects, features, and advantages of the present invention will become better understood with reference to the following description, drawings, and appended claims.

BRIEF DESCRIPTION OF THE DRALWINGS
FIG. 1 shows the structures of various fluorescent dyes previously employed as long-wavelength labels, i.e., labels emitting above 550 nm.
FIGS. 2A and 2B depict a preferred synthesis of the asymmetric benzoxanthene dyes of the invention.
FIG. 3 shows a preferred synthesis of oligon.ucleotides labeled with the dyes of the invention.
FIG. 4 shows the excitation spectra of TAlVI1Z.A (22) - and CI-FLAN (2)-labeled oligonucleotides.
FIG. 5 shows a comparison of the quantuni yields of TAMRA (22)- and CI-FLAN (2)-labeled oligonucleotides.
FIG.6 shows a co.nparison of the equimolar emission intensity of TAAlRA (22)-and CI-FLAN (2)-labeled oligonucleotides.
FIG. 7 shows fluorescence emission spectra for members of 4-plexs set of dye-labeled DNA sequencing primers.
FIG. 8 shows a s.mthesis of a 2-fluoro-l,3-dihydroxynapthalene intermediate of the invention.
FIG. 9A and 9B sl,ow the results of a DNA sequencing experiment ernploying an oligonucleotide sequencing primer labeled with a dye compound of the invention.
FIG. 10 shows the results of a microsatelite analysis employing an oligonucleotide PCR prirner labeled with a dye compound of the invention.
FIG. 11A, 1lB and 11C show four preferred synthesis routes for the synthesis of the asymmetric ben-loxansthene dyes of the invention.
FIG. 12 shows three preferred synthesis routes for the synthesis of the 1-substituted, 3-hydroxynapthalene intermediate of the invention.

DETAILED DESCRIPTIOlOT OF THE PREFER.RED EIVIBODIMENTS
Reference will now be made in detail to certain preferred embodiments of the invention.
While the invention will be described in conjunction with. the preferred embodiments, it will be understood that they are not intended to limited the invention to those embodiments. On the contzary, the 'on is ' ded to cover el ,ves, m ca.tions, and equivalents, 'ch may be included within the invemion as defined by the appended chims.
Generally, the present irivemion comprises a novel cim of metric benzoxmithm compounds useful as fluorescent dyes, m iaatean for synthesis of such dyes, reagents employing such dyes as molecuiar labels, and methods utiimg such dyes and reagents in the area of analytical biotechnology. The compounds of the presm invention find particular $pphcation in the area of nmlticolor fluorescent DNA sequencmg and fragtnent analysis.

1. Asjj=etric Benzoxanthene Dve Comp unds t In afrst aspect, the present invention comprises a novel class of asycnmetic benzoxanthene dye compounds having the general ciw: e shown in Formula I
iYa.unediately below. {At1 molecular structures provid herein are intended to ewmpass not oniy the exad electronic structure prese.med, but also include all resonarat stauceures and protonation states thereof.) R~ 3 YX O ;Y2 -RA
Rg Rg R' RS
Rs FORMULA I
In Foimula I, Yi and Y2 are either iaidividually hydro oxygen, . amme, . ``um or oxygem. When Yy is hydroxyl and Y2 is oxryg the compo d is analogous to fluormeein, wlule when Yl is arnine and Y2 is imminiwn, the compound is analogous to rhodamine.
Preferably Y$ is lry xyl and Y2 is oxygen.
Moieties Rl Rg are substituents used to anodulate various properties of the dyes by modifying the electronic struct:ure of the ground and emted states of the e.
In particular, varying moieties Ri-Rg affects the spectmi char stics, chemical stability, and photostability of the compounds. Substituents Rg R3 and Itp are particularly important in defining the properties of the compounds of FoTnmla I. For ommple, it has been observed that placing a fluorine atom at one of possttions Ri R3leads to increased chemical and photostabflity, and that if R9 is substituted plienyl, maldng substituents X2 and Xs chlorine leads to narrowex emission bands. (See below for the d 'on of sub ents X2 and Xs.) Prefembly, substituents Rz-Rs are hydroger4 fluorine, c;hlorine, lower allcyl, lower alkene, lower allcyne, sulfonate, sulfone, amino, imnaininiutra, arnido, nitrde, ary1, lower alkoxy, Iiniang group, or combinations thereofa where as used herein the term "linking group" refers to a ctionality capable of r g with "compimumtary fianct-o ' anached to a reagent, such reacdon forming a"` >e" co g the dye to the reagent. More will said about particular Iinidng groups, complementary functionalities, and ` e.s in a subsequent sectaon of this disclosure. Preferably, R, is lower alkoxy, chlorine, fluorine, or hydrogen; R2 is lower alkyl, fluorine, or chiorine; and R3 is lower aikyl, or fluorene. More preferably, one ofRi, Rj and R3 is fluorine. In a pazti y preferred embodiment, at least R3 is fluorine.
As used herein, the tensa "lower 1" denotes straigh.t-c h and branched hydrocarbon moieties containing from I to 8 carbon atoms, i.e., methyl, ethyl, propyl, isopropyl, tert-butyl;
isobutyl, sec-butyl, neopentyl, tert-pentyl, and the like. `I.ower substitued a1kyP' denotes a lower alkyl including electron-withdrawing substituents, sucli as halo, o, nitro, sulfo, or the lilce. "L.ower haloalkyP" denotes a lower subsituted alkyl -with one or more halogen atom substituents, usually fluoro, chloro, bromo, or iodo, "Lower allcene" denotes a hydocarbon containing from I to 8 carbon atoms wherein one or more of the carbon-carbon bonds are double bonds, wherein the non-double bonded carbons cornprise lower alkyl or lower substituted alkyl. "Lower e" denotes a hydocarbon co g from 1 to 8 cmton atoms wherein one or more of the embons are bonded with a tsiple bond, wherein the non-triple bonded carbons comprise lower aikyl or lower substituted alkyt, "Sulfonate"
refers to moieties including a sulfur atom bonded to 3 oMen atoms, including mono- and di-salts thereo4 e.g., sodhm sulfonate, potassiuan sulfonate, disodium sulfonate, and the like. " =
o" refers to moieties including a nitrol;en atom bonded to 2 hydrogen atoms, lower a1ky1 moieties, or any combination thereof "Amido" refers to moieties including a carbon atom double bonded to an oxygen atom and single bonded to an amino moiety . "N'rtrile" refers to moieties inciuding a 30- carbOn atom triple bonded to a nitrogen atom. "Lower AlL-oxy" refers to a moiety including lower allryl single bonded to an oxygen atom. "AryP" refers to single or mulfiple phenyl or substhuted phenyl; e-g., benzenc; naphthalene, anttmcene, biphenyl, and the lice.
Pref ly Tt9 zs acetylerr, lower WkyL lower alkene, cyano, phenyl or subsutted `
phecayl, heterocyclic aromatic, or combinations thereot the substituted phenyl having the stnlcaure:

Xs wherein Xi-Xg taken separately are hydrogen, chlorine, fluo e, lower alkyl, carboxylic acK
sulfonic acad, -CHZ I3, or g group. As used herein, the tam '`heter clic aro "
refers to aromatic moieties having a heteroatoin as part of the cycdic structure, e.g., pyrole, to indole, and the lzlce. Preferably, Xr is carboxylic acid, mffonic acid, or H,; X2 and Xg taken separately are hydrogen, cHome, fluoririe, or lower aikA and X3 and X4 taken separately are hydrogen, chlorine, #luorue, lower allcyl, carboxylic acld, sulfonic acid, or linidng group. More preferably, X2 and Xs are chlorine. In an additional preferred embodimot, one of X3 or X4 is linldng group. Preferably, X, is carboxylic acid. Iri an additional preferrad embodiment parn y suited to forming phosphoraznidite compounds including the dye compound of the invention, one of X, or Xs is a moiety wltich is capable of fo a cyclic ester or cycHc ether, e.g., carboxylic acid, sulfonic acid, or -M2 FÃ, or any othea group that will form a spirocyclic systern, i.e., bicyclic compounds having one carbon atom coninort to both rings, e.g., spiro[4.5]decane.
Preferably the linla~ng group of the mvention is isoothrocyanate, sulfonyl chiorlde, 4,6-dichloro amine, su nidyl ester, or other ac1ve xylate whenever the complementary functionality is amuie. Preferably the iliniang group is maleunide, halo acetyl, or iodoacetamide whenever the coinplerra ationality' as affiydryl. See R.
Haugland, Molecukr Probes Handbook cf F7uorescent Probes and Research Chemicab, Moleculu probes, Inc. (1992). In a particularly prefecred embodimazt, the linking group is an actir-ated N-hydroxysuccnirnidyl (NHS) ester which arr coanplensen functionality, whece to form the ester, a dye of the inventioo. including a carboxylate Unldng group is reacted with dicyciohexylca "`de and N-hydroxysuccinimide to form the NHS
ester. See FIG. 3.
Several alternative generalized methods may be used to synthesize the asymmetric benzoxanthene dye compo ds of the present invention, four of Nvhich wiU be descri here with reference to FIG. 11. In a first prefen-ed method referred to in FIG. 11 as Route A, compound 27 is reacted with 1,3-dihydroxy or 1,3-aminohydroxy benzene derivative 28 and 1,3-dihydroxy or 1,3- ohydroxy napht ene derivative 29 employing equal equivalents of each under acid catalysis and heat to give asymmetric dye compound 30.
Preferably compound 27 is a cyclic or straight chain anhydri4e, e.g., LVG is C014;
ester, e.g., where LVCi is OR where R is lower alloyl, phezlyl, or sulfonate;
or acid chloride, e.g., where LVG is chlorine or other halogen.
In an alternative prel:erred synthesis method referred to as Route B in FIG.
11, compound 27 is reacted with 2 equivalents of a 1,3-dihydroxybenzene derivative, i.e., Yg is hydroxy, or a 1,3 aminohydroxyberzene derivative, i.e., Y, is amino, 28 to give s etric xanthene dye 31. Cornpound 31 is then decomposed by base hydrolysis to form intermediate benzoyl condensation product 32. Condensation product 32 is then .reacted under acid catalysis and heat with compound 29 to give asymmetric dye 30, where 29 is 1,3-dihydroxynaphthalene when Y2 is hydroxy, or 1,3-aniinohydroxynaphthalene when Yx is amino.
In yet a third generalized synthesis method, referred to as Route C in FIG.
11, compound 27 is reacted with 1 equivalent of 28 with heat to give intermediate benzoyl condensation product 32. Compound 32 is then reacted with 29 under acid catidysis and heat to give asymmetric dye 30.
In a fourth generalized synthesis method, referred to as Route D in FIG. 11, equal , equivalents of compound 33, compound 28, and compound 29 are reacted under acid catalysis and heat to give asynmnetric xanthone intermediate 34. Preferably 33 is a carbonate, e.g., LVC'r is OR where R is preferably lower alkyl or phenyl; or formate, e.g., where LVG is halogen and Glt where R is preferably lower alkyl or phenyl.
Compound 34 is then reacted with an anionic organometallic Rg derivative to give the asymmetric dye 30, e.g., R~Li, I29IvIgX where X is halide, e.g., Br, Cl, I, and the like.

FIGS. and 2B show the synthesis of a set of p' ly preferred asyrnrneffic dye compounds of the anventdon. In this a 1,3 chhydroxynapthalene d' e, such as 1,34hydro ene (9b) or 2-fluoro-l,3 ydro ene (9a), is 1 equivaieiit of a ph 'c ydride dera e, e.g., 3, chloro elletic acid anhydride (10a), and one eq ent of a rcxnol ded ve (11m, 11b, 11it, or 11.d), and heated for 16 hours in neat carganic acid, e.g:, MeS0 -under a'rgon. The crude: dye then precip 'by addition to an ice Iwater e and isolated by fihration. ecrude dye is then fluther purified into 2 isomers 1 and 2 by pmP e layer chroniatography.
Tinsubstituted derivatives of the etric benzounthene dyes dJor R3 is io may be reacted furthei- with halogenating reagents, e.g., co .es y a ' le sources of positive fluorine, NaOC.I, NaOH / Br2, NaOH / 4 to produce quantitatively halogenated derivatives, e.g., lt2 =R3 = Ol, Br, L or F afler adractive workup with 10 %
I3CI / EtOAc, drying vAth Na2SO4, filterang, and concon 'g in vacuo. S : in FIG. 2B.

lI. Substituted Naphthalene lnteUnediates Tn a second aspect, the present invention mpnws novel interrn e compounds useful for the synthests of the a "c b xanthene compounds of the subject invention, such int e having the general structure shown in Formula II ianrnediately below. In p cular, the intennediate compounds of the invention enable the synthesis of a e3ric benzoxanthene compounds with regio-set 've incorporation of' substatuents, e.g., halogen atoms, at the 2-posation of 2-siibstituted asynunetric benzoxanthene compounds, where the 2-position corresponds to the position in the compounds of p'o I and U.

~ ~

~
Ol:l FO ' MULAII

Substituents R3-R7 in the stnicture of Formula lI correspond to Eke-numbered su`~bstituents in the structure of Formula I described above, and YZ is hydroxyl or amine.
Preferably, R3 is fluorine and 'Y2 is hydroxyi.
FIG. 12 shows three alt ve generafized synthesis schemes for the synthesis of the substituted naphthalene int iediates of the invendon. In a first method indicated as Route A.
in FIG. 12, substituted ester enolate derivative 35 is reacted with activated hoirloph 'c acid ester derivative 36 to give A-leeto-ester derivative 37, e.g., by spontaneous loss of C02when R' is carboxylate. Preferably in compound 35, R' is hydrogen, carboxylate, or halogen and R is lower aflql. Preferably in compound 36, LVG is halogen, Iv1'-hydroxysuccinimide, phenoxade, hydroxybenzotriazole, or carboxylate. Compound 37 is then cyclized under base catalysis and heat to give substituted 1,3-naphthalene diol 38, i.e., Y2 is OI-L
In a second preferred synthesis method indicated Route B in FIIG. 12, compound 35 is reacted with activated phenylacetate derivative 39, where LVG is as;
described above for compound 36 in Route A, to give 0-lceto-ester derivative 40, e.g., by spontaneous Ioss of Ct)a when R' is carboxylate. Compound 40 is then cyclized under acid catalysis and heat to give substituted 1,3- phthalene diols 38, i.e., Y2 is OI-L
In a third preferred synthesis method indicated as Route C, compound 35 is reacted with cyano-phenyl acetate derivatives 41, where LVG is as described above for compound 36 in Route A, to give cyano 0-keto-ester derivatives 42, e.g., by spontaneous loss of CO2 when R' is carboxylate. Compound 42 is then cyclized under base catalysis and heat to give substituted 1-amino-3-hydroxynaphthalenes 38, i.e., Y2 is NH2.

III. Lments tJtilizinng Dge Cornpounds In another aspect, the lpresent invention comprises reagents labeled Y~ith the ` etric benzoxanthene dye compounds of Formula 1. Reagents of the invention can be vktuaUy anything to which the dyes of the invention can be ; hed. Preferably the dyes are covalently hed to the reagent. Reagents include proteins, polypeptides, polysaccharides, nucleotides, nucleosides, polynucleotides, lipids, solid supports, organic and inorganic polymers, -fo-and combinations and as Iages th t such as chrornosomes, nuclei, II g cells, such as bacteria, other microorganisms, n Ilan celts, tissues, and the lice.

A: blucleotide Itea,gents A preferred class of reagents of the present invention comprise nucleotides and nucleosides vahgch incorporate the asymmetric benzoxanthene dyes of the invention. Such nucleotide reagents are par-d ly useful in the context of lab `
gpolynucleotides formed by enzymatic synthesis, e.g., nucleotide traphosp es used in the context of PCR
plif cation, Sanger-type polynucleotide aencing, and naclc- on r `ons.
As used herein, nucleoside" refers to a compound consi g of a p e, d ' e, or pyrirnadine nucleoside base, e.g., ad e, guanine, cytosine, thymine, d `
e, uraciL d anosine, and the like, Iinked to a pentose at the 1' position, including 2'-deoxy and 2'-hydroxyl forrns, e.g. as dwzribed in Komberg and Baker, DNA Repli n, 2nd Ed.
( San Francisco, 1992). The t n"nucIeotide" as used herein refers to a phosphate amter of a nucleoside, e.g., triphosphate esters, wherein the most co' on site of esterification is the hydroxyl group attached to the C-5 position of the pentose. " ogs" in reference to nucleosides include synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g. described generally by Scheit, Nucleofic3e Analogs (John Wiley, New York, 1980). The term "labeled nucleoside" refers to nucleosides which are covalently attached to the dye compounds of Formula I through a e.
Preferred nucleotides of the present invention are sho` vn belaw in Formula wher W3 I-I2 ~ B-D

FO TI.A

B is a nucleotide base, e.g., 'uraciL cytosine, d enine, and d e. Wi and W2 taken separately are I-I or OR W3 is O

PP IP ~ 0 -F -O-F -P - -p -P -P

9 . . , - . , or g 0 0 --O-P-O-P-O-P- . O
I P P
O O 0 inclu ` g associated couraterior.as if present~ e.g., Ii, Na, and the like. D is a dye compound of Formula I. In one p cularly preferred e dianent, the nucleotides of the present invention are dideoxynucleotide triphosphates having the structure shown in Forrnula ffi.1 below, inclu ` g associated counteiions if present.

0-~~-0-~ ~ Iiz 13-D
I P O ~ O O

HI IH
H H
FOltil 1 Labeled dideoxy nucleotides sLich as that shown in Formula UI.1 fis.-id particulu plication as chain te g agents in 3ariger DNA sequencing rr- ods. In a second parti( ly preferred embodiment, the nucleotides of the present invention are deoxynucleotide triphosphates having the strur-ture-shown in Forrnula .2 below, including associated counterions if present.

~Ii2 Es-10 HI I~[
OII Ii I,abeled deoxynucleotides such as that shown in Formula .2 find particular application as means for labeling polymerase on products, e.g., in the polynierase chain r on.

When B is purine or 7-d urine, the sugsr moiety is attached at the N9-position of the purine or deaaapnrine, and when B is p. dane, the sugar moiety is attached at the N'-position of the pytiniidine.
ne linkage ' B and D is attached to D at one of positions RI-Itg .
Preferably, the linkage is not attached at ltx-It3. When the dyes of the invention are rynthemed from trunelleac anhydride, . R9 is preferably substituted phenyl and the hnkage is attached to the dye at one of the X3 or Y" positions of thee substituted phenyl, the other position being a hydrogen aton'L
When B is a purine, the linlcage linldng B and D is aaached to the 8-position of the purine, when B is 7-d taryne, the ' e is attached to the 7-posrtion of the 7-deazapurine, and when B is pyrimidine, the ' e is attached to the 5-position of the pytimidine.
NUcleoside labeling can be accompUshed using any of a large number of known nucleoside Iabeliag t `ques ' usmg known ` es, iinking groups, and associated complementary functionalities. The ' e Binldng the dye and nucleoside should (i) be stable to oligonucleotide synthesis conditions, (Bi) not interfere with ol agonucleotide-target hybridiaation, (iii) be compatible with relevant enzymes, e.g., popymersses, ligases, and the like, and Civ) not quench the fluorescence of the dye.
Preferably, the dyes are covalently linked to the 5-carbon of pyrimid me bases and to the 7-carbon of 7-deazapurine bases. Sevami suitable base labeling procedures have been reported that can be used vvith the invention, e.g. Gibson et al, Nucleic Acids Reseocji, 15:6455-6467 (1987); Gebeyehu et a!, Nucleic Acids Research, 15: 4513-4535 (1987);
Haralambidis et a1, Nucleic Acids Research, 15: 4856-4876 (1987); Nelson et al., Nucleosides cod Nucleotdcles, 5(3): 233-241 (1986); Bergstrogn, et al., JACS, 111: 374-375 (1989); U.S.
Patent Nos. 4,855,225, 5,231,191, and 5,449,767.
Preferably, the es are acetylenic arrido or alkeraic ainido linkages, the linkage between the dye and the nucleotide base being formed by. r g an activated I3 hydroxysuccinunide (~W) ester of the dye with an alkynylaniiino- or allcenyYi ao-derivatized base of a nucleotide. More preferably, the resulting linicage is 3-(carboxy)amino-l-propynyl or 3-amino-I-propyn-1 yl (Formula II 3). 5everal preferred 3uYlkages for ' the dyes of the invention to a nucleoside base are slaown below in Formulas TII.3, IdI.4, and III.5.

--C=C--CH2-NH-.C--FORMUI.A IIZ.3 C=C--CHZ-~iH--~--(CH~5-I~i-C
- --F ' III.4 - .C=CH C ` (CH2)s- ..,_C --FO A. YIi.5 The synthesis of allcynylamino-derivatized nucleosides is tau:ght by Hobbs et al. in U.S. Patent No. 5 151 507, and Hobbs et aL, J. Org. Chem., 54:3420 (198:9). Briefly, the alkyn.ylamino-derivatized nucleotides are formed by placing the approptiate balodideoxynucleoside (u,sually 5-iodopyrirrradine and 7-iodo-7-d pu.rine dideoxynucleosides as taught by Hobbs et al.
(ccted above)) and Cu(.)) in a flask flushing with argon to remove air, add u1g dry Dw, 1s followed by addition of an aikynylaFrtine, trietbyl-arnine and Pd(0). The reaction nabare can be stssred for several hours, or until tlun layer chromatography indicates consumption of the haiodideoacynucleoside. When an unprotected alkynylamine is used, the alkynylamrno-nucleoSide can be isolated by ooncentratang the reacdon rn%acture and chromattographing on silica gel using an eluting solvent whir,h contains arrmoniutn hydroxide to neutraiize the hydrohalide generated in the coupling reaction. When a protected allcymylarnane is used, methanoVmethylene ohloride can be added to the reaction e, followed by the bicarbonate form of a strongly basic anion exchange resin. The slurry can then be stured for about 45 minutes, fikered, and the resin finsed with additional meehanoUmetdrylene ohioride. The coYnb'ned filtrates can be concentrated and purafied by flash-chromalography on silica gel using a methanol-m yiene chloride gsadient. The triphosphates are obtained by srtandard terlsniques.

D. Phosõphoramidite Reagents Another preferred ciass of reagents comprise phosphoramidite compounds which incorporate the asymmetric beazoxanthene dyes of the invention. Such phosphorarrmidite reagents are particu2arly useful for the automated chemical synthesis of polynucleotides labeJed with the asynnnetric ber=xanthene dyes of the invention. Such phosphorarnadite compounds when reacted with a 5'-hydroxyi group of a nucleotide or polynucleotide form a phosphite ester io jirilcer which, in tum, is o)ddized to give a phosphate ester linlcer, e.g., U.S. Patem Nos.
4,458,066 and 4,415,732.

Y. Non-nucleotide Phosphorwnid2te lteagents: GenmuUy, in one aspect, the phosphorarnidite reagents of the invention have the structure of Formula lV
imrnediately below, $2,, N-P-O-X-Y-A
I

F AIV
where X is a spacer ann; D is an asymmetric xarr.athene dye of Fomuila I or a protected derivative thereot Y is a lmkage formed with a Iinldng group on the dye; Bi is a phosphite ester protecting group, and B2, and B3 taken separately are lower allcyl, lower allcene, lower aryl having betweea 1 and 8 carbon atonLs, aralkyl, or cycioailcyl c,flntaining up to 10 carbon atoms. Non-nucleotidic phosphoramidites as shown in Formuia lV are particularly well suited for labeling the 5'-end of a chernicalty-synthesized polynucleotide through the sugar-portion of the nucteotide.

Spacer X and ' e Y rnay take a variety of forms, however, the structure X-Y
must be such that (i) it is stable to DNA synthesis conditions, (u) does not interfere with oligonucleotide-target hybridization, and (~iii) does not quench the $uorescence of the dye to which it is attached, e.g., U.S. Patent I+tos. 5,231,191, 5,258,538, and 4,757,141, 5,212,304.
Preferabiy X is bnear or cyclic lower aDcA I'snear or cyrlic substwted " lower aIkyl, polyethlene oxide, lower aryi having between I and 8 carbon atoms, peptide, or polyether.
Preferably the lunkage Y is amido, sulfonamido, urea, urethane, or thiourea.
In one particailariy preferred eanbodiment, the ' eY is anido and the spacer X is Iinear alkyl having the stncture below in Formula Ii1.1 1rT-P-0-m(CFI2)n NH--C-- D
v I

Fl7 A TV.1 where n is from 2 to 30, preferably from 2 to 10, and more preferably from 2 to 6. In a second particularly preferred embodiment, the linkage Y is asxudo and the space:r X is linear polyethylene oxide having the sscructure shown below in Formula IV.2 Bx~
1l7--~- O--(CEi2CFi2O)n--C~-I2C~i2-W~T C-I~
%O

FO A IV.2 where n is from 2 to 30, preferably from 2 to 10, and more preferably from 2 to 6.
Preferably, B2 and B3 taken together form an allcyl chain co ' up to 5 carbon atoms in the principle chain and a total of up to 10 carbon atoms with both tenninal valence bonds of said chains being attached to the nitrogen amrl. Alternmtiveiy. B2 and R3 takm together with the nitrogen atom form a saturated nitaogen hetemcycie wluch corrtains one or more heteroatoms vJwted from the group co '' g of nitrogen, oxygen, ,a.nd sulfur.
Preferably, B2 and B3 taken separately are isopropyl, t-butyl, isobutyl, or secrbutyY, and B2 and B3 taken togethex is morphollino.

Bi is a phosplute ester protecting grovp which prevezats unwanted edarision of the polynucleotide to which the phosphomnidite is attached. B1 is stable to polynucleotide synthesis conditions yei.zs able to be resnoved from the po cleotide product with a reagent that does not advmsely affect the integrity of the polynucleotide or the dye.
Prefersbly, Bi is methyl, A-cyanoetltyl, or 4-nitrophenylethyl. B2 and B3 taken separately are isopropyl, t-butyl, isobutyl, or secbutyl, and B,P and B3 en together is moiphollino.
The ' linking Y and D is attached to D at one of posations Ri I.9 .
Preferably, the linkage is not attached at Ri-R3. When the dyes of the invention are synthesized from thmelletic anhydride, RA is preferably substituted phenyl and the lie is attached to the dye at one of the X3 or X4 positions ofthe substituted phenyL
Such phosphor-amidite compounds may be synthesized by lcnown methods.
Generally, the synthesis proceeds as follows. Phenolic hydroxyls of the dye are protected with dye-protecting groups that r,an be removed with a DNA synthesis deprotection agent, e.&, arnmonia, etianolansine, methylaznine/ oniuzn hydroxide mix:tures, and of t butylaznine/water/methanol (1:2:1), e.g., see U.S. Patent No. 5,231,191. Dyes so protected are referred to herein as "protected derivatives" of the dye. Preferred protecting groups include esters of benzoic acid or pivalic acid. The linking group of the protected dye, e.g., carboxylic acid, is then activated, e.g., with carbod[iimide, and reacted with an alcohol linker derivative, e.g., an amino alcohol, e.g., ethanolanaine,. hexanol amine, or the like, in 2o N,N-dimethylformamide (DNdl), or another like aprotic solvent to yield a protected dye with a free alcohol functionality, e.g., alcohol-amide derivative. The free alcohol is then reacted with a phospliitylating agent using standard procedures, e.g., di-(N,N-diisopropylamino)methoxyphosphine in acetonitrile containing catalytic amounts of tetrazole diisopropylamine, to yield the phosphoramidite, e.g., U.S. Patent No. 5,231,191.

2. Nucleotidic Phosphoramidite Reagents: GenerWly, in a second aspect, the phosphoranudite reagents of the invention have the structure of Formula V
inunediately below, BS--O-CH2 0 B. .D

u Bi FORMULA V
where B' B3 are as described above, Bs is hydrogen or a hydroxyl protecting group, B is a nucleotide base, and D is an asymmetric benzoxanthene dye of Fonaaiila I, or a protected derivative thereof. Nucleotide phosphoramidites such as shown in Formula V are partimflariy well suited for the intern.al labeling of chenzically=synthesized polynucleotides.
When B is purine or 7-deazapurine, the sugar moiety is attached at the N9-position of the purine or deazapurine. Altematively, when B is pyrimidine, the sugar moiety is attached at the W-position of the pycimidine. B and D are linked through a' e formed by the re$ction of a' ` g group and its (cornplecnentaty ctionality, such ' es between dyes and nucleotide bases have been described in detait above. If B is a puYine, the iinlcage is attached to the 8-position of the purine, while if B is 7-dea7apurine, the linlCage is attached to the 7-position of the 7-deazapurine. If B is pyrarnidzne, the ' e is attached to the 5-position of the Pyrimidine.
Bs refers generally to hydrogen or an acid-cleavable hydroxyl protecthzg group.
Preferably, Bs is the triphenylznethyl radical and its eleatron-donating-substituted derivatives, where, as used herein, the term "electron-donating" denotes the tendency of a substituent to release valence electrons to neighboring atoms in the molecule of which it is a part, i.e., it is electropositive with respect to neighboring atoms. Preferably, electron-donating substituents include mino, lower alkyl, lower aryl having betwween I and 8 carbon atoms, lower alkoxy, and the like. More preferably, the electron-donating substituents are methoxy.
Eacemplary trityLs include 4,4'-dimethoxytrittgi4 i.e. bis(p-anisyj)phenyhnethyl, .
anono:nethoxytrityt, a-naphthyldiphenylmethyl, tri(p-m xyphenyl)niethyl, and the like. Attacbrnent and cleavage cimditions for these and other trityls can be found in Greene and Wuts, Protectwe Graups Yn .
Orgargc S}nhesis, 2nd Edition (John ey, New York 1991).
Generally, the nucleotide phosphoramidites of the anvention may be synthesiz,ed as follows. A nucleoside b' a hydroxyl pro group on the 5'- hydroxyl a protected complementary functional'aty on the base is selecdvely deprotected to expose only the complementary functio " Next, a protect.ed dye (as " dmcnbed above) is actdvated by converting ':. a linlang group into its reactme ' forrn. The activated linking group of the dye is then reacted with the complementary functionality of the nucleo ' to form the dye labeled nucleoside that bears protecting groups on the 5'-hydroxyl (and on the 2'-hydroxyl for the case ofRNA,)and on the phenolic groups of the dye. The dye labeleti nucleoside is fim reacted wih a phosphitylating agent as desciibed above to produce the nucleotide phosphorarnidite.
In a preferred method 'where the complementary functionality is amine and the hnking group is carboxyt, the synthesis proceeds as follows. A. protected nucleoside bearing a hydroxyl protecting group on the S'- hydroxl, e.g., a trityl group, and a protected amino-nitrogen complementary functionality on the base is selectively deprotected to expose the amine, such selective deprotection serving to deprotect only the amine functionality without deprotecting the protected 5'-hydroxyl moiety. A protected dye (as descn bed above) is activated by converting a carboxy linnlciing group irato its NHS ester with dicyclohexyl . carbodiunide and N
hydroxysuccininude. The NHS ester is reacted with the amino group of the nucleoside to form the dye labeled nucleoside that bears protecting groups on the 5'-hydroxyl (and on the 2'-hydroxyl for the case of RNA) and on the phenolic groups of the dye. The dye labeled nucleoside is then reacted with a phosphitylating agent as descn-bed above.

C. Polvnucleotide Reagerats Yet another preferred class of reagents of the present invention coanprise polynucleotides labeled with the asymmetric benzoxanthene dyes of the snvention. Such labeled polynucleotides are useful in a nuniber of irrYportant contexts including as DNA sequencing praniers, PCR primers, oligonucleotide hybridization probes, and the like.
As used herein, the terms "polynucleotrde" or "oligonucleotideg' refer to iinear polymers 30' of natLUal or modified nucleoside monomers, includiing double and single stranded deoxyribonucleosides, n bonucleosades, a-anorneric forms thereot and the like.
Usuatiy the nucleoside monomers are linked by phosphodlester linkages, where as used herein6 the term "phosphodiester ' e" rteferrs to phosphodiester bonds or analogs thereof znclu phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphor d' enoate, phosphoroanilothioate, phosphoranilidate, phogphoranudate, and the Eke, = g assodated counterions, e.g., K NFI4, Na, and the like if such counteraons are present.
The polyinwleotfides range in size form a few monomeric units; e.g. 8-40, to several thousands of monomeric units.
Whenever a polynucleotide is represented by a sequence of leftecs, such as "AT~'iCCTQ" it will be understood that the nucleotides are in 5 ->3 order from Ieft to ri& and that w denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "7"' denotes 1o thymidine, uniess otherwise noted.
The labeled polynucleotides of the invention include a nucleotide having the formula:
Z3 - CI3z 0 B-D

H H

FORMTJI.A V'I
where B is a 7-deazapurine, purine, or pyrrinzidine nucleotide base. .Z, is H
or OIi. Z2 is K OH, BPOa, or Nuc, wlierein Nuc refers to a nucleoside or polynucleotide. The nucleoside of Formula VI and Nuc are linked by a phosphodiester linkage, the linkage being attached to the S"-position of Nuc. Z3 is I3, BFO3, or Nuc, wherein Nuc and the nucleoside are linlced by a phosphodiester linkage attached to the 3'-position of Nuc. D is a dye compound of Formula. I. Base B is attached to the sugar moiety and to the dye compound as described above for the nucleotide phosphoramidite reagent of the invention.
As defined, the labeled nucleotide of Formula VI can be the 5'-terminal nucleotide, the 3'-terminal nucleotide, or any internal nucleotide of the polyraucleotide.
In one prefen-ed embodiment, the labeled polynucleotides of the prmnt invention include multiple dyes located such that fluorescence energy er takes place between a donor dye and an acceptor dye. Such anulti-dye polynucleotides find appilication as sp y tunable sequencing primers, e.g., Ju et ai., Proc. A~'crtt Acad Sci. USA 92: 4347-4351 (1995), and as hybridization probes, e.g., Lee et aI.Nucleic AcidsResecrch, 21: 376I-3766 (1993).

Labeled polynucleotides may be ryritheized either 'cally, e.g., using a DNA
polymesase or 3igase, e.g., Stryer, .8ioohemasty, Cbapter 24, VV:H. Freeman and Company (1981), or by chemical rynthesis, e.g., by the phosphorarnidite method, the phosphite-tsiester method, and the Iike, e.g., Ciait, Olfgorrucleotide S s, IRI., Press (1990).
Labels may be introduc,ed during enzymatic synthesis utili-6ng tabeled nucleotide triphosphate monomers as described above, or ' introduced during chemiral synthesis usng labeled non-t,ucleotide or nucleotide phosphorarnidites as desmibed above, or may be introduced subsequent to rynffiesis.
Generally, if the Wwled polyamcleotide is made using enzymatic synthesis, the foIlowing to procedure may be used. A template DNA is denatured and an oligonucleotude primer is annealed to the template DNA.. nfmture of deoxynucleotide triphosphates is added to the reaction including dG'I"P, dATP, dCTP, and dTI'P where at kast a fraction of one of the deoxynucleotides is labeled with a dye compound of the invention as descril>ed above. Next, a polyxnerase enzyme is added under conditions where the polymerase enzyme is active. A
labeled polynucleotide is formed by the incorporation of the labeled deoxynucleotides during polymerase strand synthesis. In an altema 've enzymatic synthesis method, two primers are used instead of one, one pruaaer s~.omplementary to the + strand. and the other conzplemeatary to the -strand of the target, the potymerase is a thenmostable polyrnerase, and the r 'on tempm-atum is cycled between a d on temperature and ara extension temperature, thereby exponentially synthesizang a labeled complement to the target sequence by PCR, e.g., PCR
Protocols, Innas et al. eds., Acadernic Press (1990).
Labeled polynucleotides may be chemically syethesized using the phosphoramidite method. Detailed descfiptians of the cheinistry used to forin polynucleotides by the phosphoramidite method are provided elsewhere, e.g., Caruthers et al., U.S.
Pat. No.
4,458,066; Caruthers et aL, U.S. Pat. 23o.' 4,415,732; Caruthers et al., Genetic Engineering, 4: 1-17 (1982); Users Mo:nsad I~I d.392 sand 394 Polytaucleotide Synthesizers, pages 6-1 through 6-22, Applied Biosystems, Part No. 901237 (199 Y).
The phosphoramidite method of polynucleotide synthesis is the preferred method because of its efficient and rapid couplisag and the stzbility of the starting materials. The synthesis is performed with the gro ` g polynucleotide chain attached to a solid support, so that excess reagents, which are in the iiquid phase, can be easily removed by filtration, thereby el' ting the need for purification steps between cycles.
The following briefly describes the steps of a typical polynucleotide synthesis cycle using the phosphoramidite method. First, a solid support incltiding a protected nucleotide monomer is treated with acid, e.g., trichloroacetic acid, to remove a 5'-hydroxyl protecting group, freeing the hydroxyl for a subsequent coupla ng reaction.
Aln activated interanediate is then fo ed by simultaneously addiing a protected phosphoramidite nucleoside monomer and a wacid, e.g., tetrazole, to the reaction. The weak acid protonates the nitrogen of the phosphoramidite forming a r "ve interrnediate.
IVucleoside addition is complete within _30 s. Next, a capping step is perforrned which tern,zinates any polynucleotide chains that did not undergo nucleoside addition. Capping is preferably done with acetic anhydride and 1 inethy dazole. The internucleotide ' ge is then converted from the phosphite to the more stable phosphotriester by oxidation using iodine as the preferred oxidizing agent and water as the oxygen donor. After oxidation, the hydroxyl protecting group is removed with a protic acid, e.g., trichloroaceiic acid or dichloroacetic acid, and the cycle is repeated until chain elongation is complete. After synthesis, the polynucleotide chain is cleaved from the support using a base, e.g., arnmonium hydroxide or t-butyl aniine. The cleavage reEcction also removes any phosphate protecting groups, e.g., cyanoethyl. Finatly, the protecting groups on the exocyclic amines of the bases and the hydroxyl protecting groups on the dyes are removed by treating the polynucleotide solution in base at an elevated temperature, e.g., 55 'C.
Any of the phosphoramidite nucleoside rnonorners may be dye-labeled phosphoramidites as described above. If the 5%terminal position of the nucleotide is labeled, a labeled non-nucleotidic phosphorarnidite of the invention may be used during the final condensation step. If ~tn intemal position of the oligonucleotide is to be labeled, a labeled nucleotidic phosphoramidite of the invention may be used during any of the condensation steps.
Subsequent to synth the I cI tide may be labeled at a number of positions 3o including the 5'-t us, e.g., Oligo cleo ' s and Aram- , Ea;Ycstein ed., Chapter 8, IRL
Press (1991) and Orgel et al., Nuclcac Acids R cJr 11(18): 6513 (1983); U.S.
Patent No..

5,118,800; the phosphodiester backbone, e.g., ibid., Chapter 9; or at the 3'-terminus, e.g., Nelson, Nucleic Acids Research 20(23): 6253-6259, and U.S. Patent Nos.
5,401,837 and 5,141,813. For a review of oligonucleotide labeling procedures see R. Haugland in Excited States o,f Biopolymers, Steiner ed., Plenum Press, NY (1983).

In one preferred post-synthesis chemical labeling method mi oligonuleotide is Lib-Jed as follows. A dye including a c,a:. ycy linkir4 group is corrverted to the n-hydroxysuccanxrnide ester by r ' g with appro ' y 1 eqWvalent of 1,3-dis,yciohexyl ..de and appro ' y 3 equivalents of n-hydroxysuccanimide in dry ethyl acetate for 3 houTs at room temp .'T'he reaction nii:ture is washed with 5 / IiCl, dried over esiLtrn mffate~
fiitered, and concentrated to a solid vvhich is r ed in DMSO. The DMSO dye stock is then added in excess (10-20 x) to an aminohexyi derivatized oligonucleotide in 0.25 M
bicarbonate/c,arbonate buffer at: pH 9.4 and allowed to react for 6 hours, e.g., U.S. Patent No.
4,757,141. The dye labeled oligonucleotide is sepamted frorn unreacted dye by passage through a size-exclusion chroma.tography colurm eluting with buffer, e.g., 0.1 molar triethy e acetate (TEAA). The fraction containing the crude labeled olligonucleotide is fin-ther purified by reverse phase HPLC employuig gradient elution.

IV. Methods IJtiliang the Compounds and Reagents of the Invention The dyes and reagents of the present invention are vvelt suited to any methcid u...
fluorescent detection, pardoAarty methods requiring the simultaneous detection of muttiple s'patmUy-overiappmg analytes. Dyes and reagents of the invention are particailarly vwell suited for identifying classes of po3ynucleotides that have been subjected to a biochemuat separation procedure, such as electrophoresis, where a smies of bands or spots of target substances havuig similar physiochernical properties, e.g. szze, confonnatron, charge, hydrophobicity, or the like, are present in a linear or planar arrangement. As used herein, the terrn "bands" includes any spatial grouping or aggregation of analytes on the basis of xi1milar or identical physiochemical properties. IJsually bands arise in the separation of dye-polynucleotide conjugates by electrophoresis.
Oasses of polynucleotides can arise in a variety of contexts. In a prefemed . category of methods referred to herein as -fi-agment anaiysisp or "genetic analysie' methods, labeled polynucleotide fragments are generated through template-directed enzyrna~c syrnhesis using labeled primers or nucleotides, e.g., li 'on or pol e ected primer extension;
the ftagments are subjected to ize-dependent d separatioa process, e.g., electrophoresis or chromatography, and, the separated fragments are detacted subsequent to the sepanation, e.g., by laser-induced fluorescence. In a p 'cularly p erreci ' ent, multiple clasm of polynucleotides are separated simultaneously and the di8'erent dasm are distinguished by spectrally resolvable labels.
One such fragment ysi.s method known as arnphfied fimgment length polyinorpbisim detection (AmpFLP) is based on arnplified fi-agment length polyinorphimLs, i.e., restriction io fragment length polymorphisms that are amplified by I'CIt. These amplified fi ents of varying size serve as linked markers for follo ' genes through fanulies. The cioser the wnplified fi-,igrnent is to the mutant gene on the chromosome, the higher the linkage correlation.
Because genes for many genetic disorders have not been identified, these Iinkage markers serve to help evaluate disease risk or paternity. In the AmpFLPs technique, the polynucleotides may is be labeled by using a labeled polynucleotide PCR primer, or by utilizing labeled riucleotide triphosphates in the PCR
In another such fi-agment analysis method lmown as nick traeaslation, a reacdon is used to replace unlabeled nucleoside triphosp es in a double-smnded DNA molecule with LaWed ones. Free 3'-hydroxyl groups are created within the uiilabeled DNA by nic.lcs" caused by 20 deoxyrn'bonuclease I(DNA,ase I) treatrnent- DNA pol ' I then catalyzes the addition of a labeled nucleotide to the 3`-hydroxyl tominus of the nick. At the same tisney the 5' to 3'-exonuclease activity of this enzyme eliminates the nucleotide unit from the 5'-phosphoryl tenninus of the nick. A new nucleotide with a free 3'-OH group is incorporated at the position of the original excised nucleotide, and the nick is shifted alort,g by one nucleotide `t in the 3' 25 direction. This 3' shift wM result in the sequential addition of new labeled nucleotides to the DNA with the removal of existing unlabeled nucieotides. The nick transiated polynucieotide is then analyzed using a separation process, e.g., electrophoresis.
Another exemplary fi agrnent anaiysis method is base,d on varaable number of tandem repeats, or VNTRs. VNTRs are regions of double-stranded DNA that contain adjacent multiple 30 copies of a particular sequence, with the number of r g'ts being variable.
Examples of VNTR loci are pYNZ22, pMC'TI 18, and Apo B. A subse of VNTR methods are those methods based on the detecdon of rnicaosatellite repeats, or short tandem repeats (STRs), i.e., tandem repeats of DNA characterazed by a short (2-4 bases) repeated sequence.
One of the most abundant interspersed repetitive DNA families iri humans is the (d )n--(dG-d'I )n dinucleotide repeat fauuly (also called the (CA)n dinucleofide repeat family).
There are thought to be as many as 50,000 to 100,000 (CA)n repeat regionLs in the hunm genome, typically with 15-30 repeats per block. Many of these repeat regions are polymorphic in leaigth therefore serve as useful genedc marlcers. Prreferably, in VNTR or STR
methods, label is iaxtroduced into the polynucleotide fi-agments by using a dye-labeled PCR
pruner.
In a particularly preferred fragment analysis method, classes identified in accordance lo with the invention are defined in terms of terminal nucleotides so that a correspondence is established between the four possible terminal bases and the rn ers of a set of y resolvable dyes. Such sets are readily assembled from the dyes of the invention by measuting emission and absorption bandwidths with cornmercially s.vaUable photorneters.
More preferably, the classes arise in the context of the chemical or chain tennination methods of DNA sequencing, and most preferably the classes mise in the conteact of the.
diain tetmination method, i.e., dideoxy DNA sequencing, or Sanger sequencing. This method involves the synthesis of a DNA strand by a DNA polymerase in vitro using a single-stranded or double-stranded DNA template whose sequence is to be: determined. Synthesis iis inifiated at only the one site where an oligonucleotide pzimer anneals to the template. T
he synthesis reaction is terminated by incorporation of a nucleotide analog that will not support continued DNA elongation. The chain-terminating nucleotide analogs are the 2',3'-dideoxynucleoside 5'-tciphosphates (ddNTPs) which lack the 3'-OH group necessary for 3' to 5' DNA
chain elongation. VVhen proper proportions of dNTPs cleoside 5=triphosphates) and one of the four ddNl'Ps are used, enzyme-catalyzed polyrnerization be t ed in a flacdon of the population of chains at each site where the ddN'I'P can be incorporated. If labeled primers or labeled ddNTPs are used for each reaction, the sequence irafoaznation r.azi be detected by fluorescence after sepamtion by high-resolution electrophoresis. In the chain tenaination method, dyes of the invention can be attached to eit.her sec;uen primers or dideoxyr-ucleotides. Dyes caa.i be ed to a cornplernentary functiorml aty on the 5' end of the primer, e.g. foIlowing the teaching in Fung et al, U.S. Pat. No. 4,757,14:1;
on the base of a primer, or on the base of a dideoxynucleotide, e.g. via the alkynylamino linking groups disclosed in Hobbs et al, European Patent No. 0251786.
In each of the above fragment analysis methods labelled polynucleotides are preferably separated by electrophoretic procedures, e.g. Gould and Matthews, cited above; Rickwood and Hames, Eds., Gel Electrophoresis of Nucleic Acids: A
Practical Approach, (IRL Press Limited, London, 1981); or Ostezman, Methods of Protein and Nucleic Acid Research, Vol. 1 Springer-Verlag, Berlin, 1984). Preferably the type of electrophoretic matrix is crosslinked or uncrosslinked polyacrylamide having a concentration (weight to volume) of between about 2-20 weight percent. More preferably, the polyacrylamide concentration is between about 4-8 percent.
Preferably in the context of DNA sequencing in particular, the electrophoresis matrix includes a strand separating, or denaturing, agent, e.g., urea, formamide, and the like.
Detailed procedures for constructing such matrices are given by Maniatis et al., "Fractionation of Low Molecular Weight DNA and RNA in Polyacrylamide Gels Containing 98%
Formamide or 7 M Urea," in Methods in Enzymology, 65: 299-305 (1980); Maniatis et al, "Chain Length Determination of Small Double- and Single-Stranded DNA
Molecules by Polyacrylamide Gel Electrophoresis," Biochemistry, 14: 3787-3794 (1975); Maniatis et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1982), pgs. 179-185; and ABI PRISMS)T'N 377 DNA
Sequencer User's Manual Rev. A, January 1995, Chapter 2 (p/n 903433, The Perkin-Elmer Corporation, Foster City, CA). The optimal polymer concentration, pH
temperature, concentration of denaturing agent, etc. employed in a particular separation depends on many factors, including the size range of the nucleic acids to be separated, their base compositions, whether they are single stranded or double stranded, and the nature of the classes for which information is sought by electrophoresis. Accordingly application of the invention may require standard preliminary testing to optimize conditions for particular separations. By way of example, oligonucleotides having sizes in the range of between about 20-300 bases have been separated and detected in accordance with the invention in the following matrix: 6 percent polyacrylamide made from 19 parts to 1 part acrylamide to bis-acrylamide, fonned in a Tris-borate EDTA buffer at pH 8.3.

Subsequent to electrophoretic separation, the dye-polynucleotide oonjugates ane detected by measuring the $uorescence emission from the dye labeled polyrwdeotidea. To perfonn such detection, the labeled polynucleotides are Muminated by standard means, e.g high intensity mercLuy- vapor lamps, lasers, or. the L'lce. Preferably the illumination meeAS is a laser having an illumination beam at a wavelength between 488 and 550 nm More preferably, the dye-polyinudeotides are dluminaxed by laser light geneniod by an argon ioa lew, pardcutaiiy the 488 and 514 nm einission Gnes of an argon ion laser, or an the 532 emiaaon line of a neodymium solid-stata YAG laaer. Several argo ion la4as are avaa'Wa1e commercially which lase sinuiltaneously at these line.s, e.g. Gyonics, Ltd.
(Snnnyvale, CariE) io Mode12001, or the like. The fluorescence is then detected by a light-sensitive detector, e.g., a photomultiplier tube, a charged coupled device, or the 18ce.

IV. Exam~
The invention will be further clarified by a consideration of the following examples, which are intended to be purely exernplary of the invention and not to in any way limet its scope.
Unless otherwise indicated, ail chemicala weie obtamed from Aldrich Cherual Company (Milwaukea, WI) and used as purchased. 3 Fluororesorcanol (lla) was syntlesiaed from 2,4-dimet}wxyaniline according to the hterature procodure (Perl* J. Cfiem Soc. 110:
1658-1666 (1980)). 2-Chloro-4-n>ethoxyresorcinol (llc) was sytrthe,sized from 3-hydroocy-4 methoxy-benzaldehyde according to U.S. Paterg No. 4,318,846. 3,6-Dic}ilorotrimevitic add was Mahesized according to U.S. Patent No. 4,318,846, and converted to the anhyriride 10a by reHtnong in neat acetic anhydride for 4 hours and precotation of the cooled rnomu+e with diet6y) ether. Ethyl lrydrogen fluoromalonate was syethesiaed from diethyl fluoromalonate according to the literatiu+e (Org. S)n CoA. 4: 417-419 (1963)). Ethyl trbutylp (19) was synthesized according to the litenawre (Tet Lett. 30: 6113 (1980)). 2 Fiuoro-1,3-dihydroxynapthalene (9a) was synthesuxd as described in the present disdosiut.
Dry dichlorornethane (CHzCh) was disged S-om calcnun hydride and tetr8hydrofuraa (TFIF) from fithium a}umirum hydride (L.AH) prior to use. Aboolute ethanol was used as pumbased or dried 3o by distillation from sodium and stored over activated mole;ailar sieves:
Dry ethyl acetate (EtOAc) was dish'lled from PA after pr+o-drying with MgSO4. Dry dimethylformatnide (DMF) was distilled after pre-drying with magnesium sulfate and stored over activated molecuiar sieves.
All reactions were run under anhydrous conditions under dry argoa Reactions were monitored by thin layer chromatography (TLC) (Si'tica gel 60, A254). P7ash chromatography was performed on siica gel 60 (200-400 mesh, Baxter). Final purification of the asymmetric beiuoxa~rtha~e dyes to give the pure isomers, designated "1õ and "2", employed preparatrve TLC on uUCa gel 60 PTLC plates (EM Science) eluting with CH2CIz : MeGH : AtOH (7 : 3: 0.1).
Pure dye isomers were identified by giving a single spot on TLC employing CHzCiz : MeOH
: AcOH (7 :
3: 0.1) and visualizing with short and long-wavelength UV irra.diation. Isomer 2 nms slower on both normal and reverse-phase media lntermediate products were identified by 'FIlVIvIIt spectra on a Varian 300 MHz NMR. Absorption spectra of the purified dyes were recorded on a Hewlett Packard 8451A diode array spectrophotometer, and 13uorescence emission spectra were recorded on a Perkin Elmer. LS 50-B luminescence spectrophotometer. HPLC
purification of dye labeled oligonucleotides was performed on a Perldn Elmer 200 sesies pump, connected to a PE LC 240 fluorescence detector, and a PE LC 295 UV/VIS detector, connected to a 2 channel PE 1022 integrator. Buffers employed for dye labeled oligonucleotide purification and identification include tris(hydroxymethyl) aminomethane / borate / EDTA (TBE), tris(hydroxymethyl) aminomethane / EDTA (TE), triethylammonium acetate (TEAA).
Buffers are stored as 10 x solutions at 0 C and diluted fresh before use. HPLC
purification employed a reverse-phase RP-18 column.

EXAMPLE I
Synthesis of Asynzmetric Benzoxanthenes Compounds 1-7 in FIGS. 2A and 2B were synthesized by reacting a 1,3 dihydroxynapthalene derivative, such as 1,3-dilrydroxynapthalene 9b or 2 9uoro-1,3-dlydroxynapthalene 9a (0.2 mole), with 1.1 equivalent of the phthallic anhydride derivative 3,6-dichlorotrimelletic acid anhydride 10a, and one equivalent of a resorcinol derivative 11 (0.2 mole), ila, llb, lle, or lld depending on the final product desired, and heated for 16 hours in neat MeS03H (3 ail) at 110 C under Argon The crude dye (a mixture of regioisomers in reactions employing 10a) was precipitated by add.ition to an ice / water mDcture and isolated by filtration. The crude dye was purified into 2 isomers 1 and 2 by preparative thin layer chromatography eluting with a mixture of CH2ClZ : MeOH : Acetic Acid (70 : 30 :1).
The insset in FIG. 2B shows that R2 and/or R3 unsubstituted (Rz- R3= H) derivatives of the asymmetric benzoxanthene dyes, shown for isomer 2 of dye 5, react frttxr with s halogenating reagents (NaOCI, NaOH / Br2, NaOH / Iz) at 0 C for 3 hours to produce quantitatively the halogenated derivatives such as 8 (R2= R3= Cl, Br, I, F) after extractive workup with 10 % HCI / EtOAc, drying with Na2SO4, filtering, and co~ in vacuo.
EXAMPLE 2 ' Synthesis of Dye-labeled Oligonucleotides The synthesis of dye labeled oligonucleotides of the invention wiU be descnbed with reference to FIG. 3. CI-FLAN, dye 2, was converted to the n-hydroxysuccaiuRride ester 12 by re=acting with 1.2 equivalents of 1,3-dicyclohexylcarbod'umide and 3 equivalents of n-1S hydroxysuccinimide in dry ethyl acetate for 3 hours at room tempetature.
The reaction mixtum was washed with 5 % HCI, dried over magnesium sulfate, filtered, and concentrated to a solid which was resuspended in DMSO ( 10 mg dye / 50 L DMSO). The DMSO dye stock (5-L) was added in excess (10-20 x) to an aminohexyl derivatized -21M13 oligonucleotide primer (1x103 M) in 0.25 M bicarbonatelcarbonate buffer at pH 9.4 and allowed to react for 6 hours. The aminohexyl derivatized primer was prepared by automated soIid-phase DNA
synthesis using Aminolink-2 in the last cycle (PE p/n 400808). The dye labeled oligonucleotide was separated from unreacted dye by passage through a Sephadex G-25 colwnn eluting with 0.1 molar triethylamine acetate (TEAA). The fraction containing the crude labeled oligonucleotide was purified by reverse phase HPLC employing gradient elution from 8% AcCN in 0.1 M
TEAA to 25% over 25 minutes using an RP-18 chromatography column. The pure dye ]abeled oligonucleotide 13 was lyophTmed to a solid and resuspended in I x TE buffer pH 8.4. The concentration of the dye labeled oligonucleotide was deteimined by UV
absorption at 260 nm assuming additive extinction coefficient values of 6,650 for T, 7,350 for C, 11,750 for G, and 14,900 for A, and the relative contribution of the dye absorption at 260 nm detennhied from spectra of the fim dye mwmnvd in the same twffer.

Comparison of the Excitation Spectra of TAMRA (22) and Cl-FLAN (2) Labeled Oligonucleotides from Example 2 Excitation spectra were recorded for each dye in 1 x TBE buffer at pH 8.4.
Dyes where present at an equimolar concentration (ca. 1 x 1e M). The emission intensity was recorded at 7,,,Em for each dye. FIG. 4 shows that for excitation at 488 nm the relative excitation efficiency of Cl-FT.AN is approximately 2.5 times that of the TAMRA dye, whfle for excitation at 514 nm, the relative excitation efficiency of Cl-FLAN is approximately 1.5 times that of the io TAMRA dye.

Comparison of the Quantum Yield of TAMRA (22) and Cl-FLAN (2) Labeled Oligonucleotides from Example 2 FIG. 5 shows emission spectra the fluorescen.se emission intensity of a TAMRA
(22) labeled -21M13 oligonucleotide'and-a Cl-FLAN(2) labeled -21M13 oligonucleotide excited at the absorption maxima of each dye. The oligonucleotides were prepared as in Example 2. The data demonstrate a 600/o greater quantum yield for the Cl-FI.AN (2) labeled oligonucleotide as compared to the TAMRA (22) labeled oligonucleotide. Spectra were recorded in 1 x TE buffer at pH 8.4 at a concentration resulting in an equal ~,.Abs of 0.05 for each labeled oligonucleotide. Emission spectra were recorded for each dye with irradiation at ~,.Abs for each dye.

Comparison of the Molar Emission Intensity of CI-FLAN (2) and TAMRA (22) Labeled Oligonucleotides Emission spectra of equimolar concentrations (ca. 1x10-6 M) of a TAMRA (22) labeled oligonucleotide and a Cl-FLAN (2) labeled oligonucleotide dissolved in I x TE
buffer at pH
8.4 were measured by irradiating each oligonucleotide at 488 nm and 514 nm, and adding the spectra to approximate the radiation of a multiline argon laser. FIG. 6 shows that the fluorescence intensity of the Cl-FLAN (2) labeled oligonucleotide is over 2 times greater than that of the TAMRA (22) labeled oligonucleotide.

Multiplex Dye-labeled Oligonucleotide Set Long-wavelength fluorescence emission of a Cl-FLAN (2) labeled oligonucleotide -21M13 sequencing primer was compared with the emission from -21M13 sequeneing primers labeled with 6-FAIvI, TET, and, HEX 23 dyes, where 6-FAM refers to 6-carboxyfluorescein, "TET " refers to 6-carboxy-4,7,2',7'-tetrachlorofluorescein, and "HEX" refers to 6-carboxy-4,7,2',4',5',7'-hexachlorofluorescein. Primers were labeled as described above in 1o Example 2. The excitation wavelength was 490 nm Emission spectra were run in 1 x TE
buffer at pH 8.4 and nonnaliz.ed to equal intensity (ca. 1* 106 M). FIG. 7 shows that the 573 nm emission maxima and the narrow width of the emission spectrum of the Cl-FLAN (2) labeled oligonucleotide makes the Cl-FLAN (2) labeled oligonucleotide specbrally resolved from the emission spectra of the other 3 dyes in the set. Such spectral resolution indicates the suitability of a dye set including, FAIvI, TET, and HEX labeled oligonucleotides with the Cl-FLAN (2) asymmetric benzoxanthene dye.

Synthesis of a 2-Fluoro-1,3-Dihydroxynapthalene Intermediate See FIG. 8. Commercially available homopthallic anhydride (14) (100 gm) was reacted with ethanol (300 mL) under acid catalysis (0.5 mL TFA) to produce a 95 %
yield of the intermediate ethyl ester 15 after refluxing for 3 hours, concentration to a sotid, and recrystalization from toluene. Intermediate 15 (10 gm) was then reacted with 1.1 equivalents of oxalyl chloride in CHZCIz (200 mL) for 4 hours at room temperature to produce an 80'/o yield of acid chloride 16 as a crude solid after concentration at room temperature under high vacuum.
Crude 16 was suspended in THF and reacted by either of the following two methods with fluoro acetate equivalents to produce compound 20.

Metliod A: The potassium salt (17) of ethyl 8uoroacetate (3 equivalents), formed by reaction of ethyl fluoroacetate and potassium t-butoxide at 0 C in THF, or the magnesium salt of ethyl hydrogen 8uoromalonate '(18) (1.5 equivalents), formed by reaction of isopropyl magnesium bromide (2 equivalents) and ethyl hydrogen fluoromalonate at -60 C, were added slowly to the THF suspension of 16 and allowed to react for 6 hours at 0 C.
The reaction was quenched by adding 5 % HCI, extr-acted (3 times) with EtOAc, the organic layer was dried, concentrated, and the resulting crude mixture purified by flash chromatography employing gradient elution from 6:4 hexanes/CH2C12 to 100 % CHZCl2 giving 35 to 50 %
yield of compound 20.

Methad B: The phosphorous ylid 19 was slowiy added to the TBF suspension of 16 at -70 C, then allowed to warm to room temperature and react for 16 hours. The reaction was quenched by addition of 5 % NaHCO3 and stirred for 6 hours. The reaction was extracted with 1o THF / water (3 times) and the product was isolaied as for Method A to produce intermediate 20 in >50 % yield. Purified 20 intra-molecularly cyclized under base catalysis (2 equivalents NaOEt) to a cyclic intermediate 21 which decarboxylated in situ to give the 2-fluoro-1,3-dihydroxynapthalene (9a) in 50% yield. Alternatively, the cyclic intermediate 21 can be isolated in >80 % yield when employing potassium t-butoxide in TI3F and decarhoxylkated to 2-fluoro-1,3-dihydroxynapthalene (9a).

DNA Sequencing Employing Asymmetric Benzoxanthene Compound 2 Automated cycle sequencing was performed using a Perkin-Elmer Catalyst 800 Molecular Biology Labstation (The Perldn-Elmer Corporation, Foster City, CA
(PE)). Four separate Sanger sequenang reactions were run employing the same -21 M13 primer labeled with 6-FAM (C terminator), TET (A terminator), HEX (G tenninator), or Cl-FLAN
2 (T
tetzninator) as described below. A mixture of the four reactions was lo4ded and data was generated on a Perlcin Elmer ABI Prismrm 377 DNA sequencer and associated data analysis software.
Cycle sequencing reactions were performed on the Catalyst 800 Molecular Biology Labstation using the 3.02 platform software. The Catalyst was programmed to deliver 0.6 L of pGEM 3Z+ template DNA at a concentration of 100 ng/ L, and 1.9 L of premix defined below. Sequencing data was generated on an ABI Prism7'`t 377 DNA
Sequencer using a 5% Long Ranger gel (FMC corporation, Rockland, Maine). Each of the four sequencing premixes is defined below in Table I:
TABLE I
A Premix 60cnM Tris pH 9.0; 2.5 mM MgC.12; 4 mM Kc1; 0.04 mM DTT;
4 MEDTA; 0.1 pM TET labeled primer, 0.66U/ L Amplitaq FS;
1.66 U/pL rTth Pyrophosphatase; 0.5 M ddATP; 125 pM dATP; 125 M dCTP; 150 M c7dGTP; 125 pM dTTP.
C Premix 60mM Tris pH 9.0; 2.5 nuM MgC12; 4 mM KCI; 0.04 mM DTT; 4 M
EDTA; 0.1 pM 6-FAM labeled primer, 0.66 U/pL Ampiitaq FS; 1.66 U
/ L rTth Pyrophosphatase; 0.5 pM ddCTP; 125 ivi dATP; 125 M
dCTP; 150 M c7dGTP; 125 pM dTTP.

G Premix 60mM Tris pH 9.0; 2.5 mM MgClz, 4 n=iM Kcl; 0.04 mM DTT; 4 pM
EDTA; 0.1 M HEX labeled primer, 0.66U/11L Amplitaq FS; 1.66 U/ L rTth Pyrophosphatase; 0.375 pM ddGTP; 125 pM dATP; 125 M dCTP; 150 M c7dGTP; 125 M dTTP.

T Premix 60mM Tris pH 9.0; 2.5 mM MgC12; 4 mM Kcl; 0.04 mM DTT; 4 M
IDTA; 0.1 pM FLAN labeled prnner; 0.66U/pL Amplitaq FS; 1.66 U/ L rTth Pyrophosphatase; 0.875 M ddTTP; 125 M dATP; 125 M dCTP; 150 M c7dGTP; 125 pM dTTP.

Cycle sequencing was perfonned on the above rnixuues of template and prem'nces. The cycling conditions on the Catalyst were as follows: one cyde of 96 C for 20 seconds; 15 cycles of 94 C for 20 seconds, 55 C for 40 seconds, and 68 C for 60 seconds; and 15 cycles of 94 C
for 20 seconds and 68 C for 60 seconds.
Following thermal cycling, the four separate reactions were combined into the 1o concentration buffer ( 83% DMSO/25mM IDTA/ 8mg/ml Blue Dexttan ) and concentrated using standard Express Load methods (v 2.02 Catalyst Manual, PE). 2 mL of concentraed sample was loaded onto a weU of the 377 sequencer, run, and analyzed usuig version 1.1 Software. The sequence between base 233 and 263 is shown in FIG. 9.

EXAMPLE 9 .
Ivticrosatellite Fragments Labeled using Cl-FLAN (2), HEX and TET Labeled Primers Separated Simultaneously with ROX Labeled Intennal Size Standards.

s PCR reactions of four loci of a humaa CEPH family DNA using dye labeled primers was performed as described below. ' The PCR products were pooled and electrophoretically separated on a Perldn-Etmer ABI Prism 3777m DNA sequencer (PE).
The unique fluorescent signal of each dye labeled fragment peak was analyzed using GeneScanm Analysis Software v 2Ø2 (PE). Referring to FIG. 10, the red peaks (labeled lo R) correspond to ROX (26) labeled intennal standard fragments, the blue peaks (labeled B) correspond to TET labeled fragments, the green peaks (labeled G) correspond to HEX
labeled fragments, and the black peaks (labeled K) correspond to Cl-FLAN (2) labeled fragments.
The PCR reactions were run on a Perkin-Elmer 9600 thermocycler (PE). A
13 separate reaction was performed for each dye labeled primer employing the following cocktail:

Reaction Components Volume [uL) Dye labeled Primer M'ix (5 M) 1.0 20 DNA (50 ngl L) 1.2 10X PE PCR Buffer II 1.5' dNTP mix (2.5 mM) 1.5 AmpliTaq' (5 unitsl L) 0.12 2.0 mM MgC12 1.2 25 Sterile D.I. Water 8.48 Total M'ix 15.0 The mixtures were ampIified using the following cycling conditions: 1 cycle at for 5 nzinutes; 10 cycles at 94 C for 15 seconds, 55 C for 15 seconds, and 72 C for 30 30 seconds; 20 cycles at 89 C for 15 seconds, 55 C for 15 seconds, and 72 C
for 30 seconds; and 1 cycle at 72 C for 10 minutes.

The amplified PCR Products were pooled by mixing the Cl-FLAN (2) and TET -labeled PCR products (0.5 pL) with 1.0 L of each HEX labeled PCR product to give an overall ratio of mixed dye labeled fragments consisting of 1:2:1 (Cl-FLAN :
HEX : TET).
The pooled PCR fragments were mixed with a loading cocktail, consisting of 2.5 L

formamide, 0.5 L Blue Dextran (50 mM EDTA, 50 mg/mL Blue Dextran), and 0.5 pL
Size Standard (GS-350 ROX, PE p/n 401735). The pooled mixture was denatured at C for five minutes and then loaded onto one gel lane of a PE ABI Prism7j 377 DNA
sequencer. The fragments were electrophoretically separated and detected using an acrylamide gel having the fotlowing characteristics: 0.20 nun thiclcness, 4.25% (wt) acrylamide, 19:1 acrylamidelbisacrylamide (wt/wt), 34-well square tooth comb, lOX TBE
Buffer (89 mM Tris, 89 mM Boric Acid, 2 mM EDTA) pH of 8.3. The instrument was run using Filter Wheel A and the GS 36D-2400 Module which has the following run parameters: EP Voltage of 3000 V, EP Current of 60.0 mA, EP Power of 200 W, Gel Temperature of 51 C and a laser power of 40 mW.

Comparison of the Spectral Properties, Photostability, and Chemical Stability of Rhodamine Dyes, Xanthene Dyes and the Asymmetric Xanthene Dyes of the Invention Table I below summarizes and compares various spectral and chemical properties of the asymmetric benzoxanthene dyes of the invention and other spectrally similar xanthene and rhodamine-based dyes.

TABLE II .

Dye 7..Em Width at Relative Relative Stability in (nm) Half Height Photo- Brightness NILOH-tm nm) stabilitv r HEX 550 32 17 2.4 11 (5) 552 45 2.8 3.9 430 (6) 554 47 - -(4) 564 41 - - -(1) 565 45 5.3 1.6 478 2 CLrFLAN 568 42 5.1 2.1 146 (7) 570 45 1.1 1.6 TAIvIRA 577 39 0.9 1.3 NAN 579 44 0.3 1 52 (3) 583 43 1.7 1.1 14 ROX 594 53 0.5 272 DEB 598 48 0.3 0.3 See FIGS. I and 2 for the structures of the dyes referred to in the table. All data are reported for pure dye isomer 2. All emission spectra were recorded in 1 x TBE buffer at pH 8.4 in dye solutions having an absorbance of 0.05 at X.Abs (ca. 1 x 106 M) at room temperature. Photodecomposition rate was determined for equal volumes of the dyes at initially I absozption unit at ),...Abs and run in pairs at equal volumes under equal high intensity white light irradiation at 35 C in 1 x TBE buffer pH 8.4.
Absorption spectra of aliquots were taken at I hour intervals and the intensities at X,..Em were fitted with first order exponential curves to determine the tLa rate for loss of dye. Relative brightness at l,..Em was determined using 514 nm excitation of dyes at approximately equal concentrations. (7l.Abs= 0.05). For the NH,OH stability measurements the dyes were diluted in concentrated ammonia hydroxide at approximately equal concentrations (..Abs = I) and incubated at 60 C for 20 hours in sealed vials. Absorption spectra of aliquots were taken at 1 hour intervals and the intensities at X..Em were fitted with first order exponential curves to determine the tu2 for dye decomposition.

Those having ordinary skM in the chemical and biochemical arts wM clearly understand that many modifications are possible in the preferred embodiment without departing from the teachings thereof. All such modifications are intended to be encompassed within the following cl3im

Claims (5)

CLAIMS:

1. A compound having the formula:

wherein:
R3 is selected from the group consisting of fluorine, chlorine, sulfonate, amino, amido, nitrile, lower alkoxy, and linking group;
R4 -R7 taken separately are selected from the group consisting of hydrogen, fluorine, chlorine, lower alkyl, lower alkynyl, sulfonate, amino, amido, nitrile, lower alkoxy, and linking group; and Y2 is selected from the group consisting of hydroxyl and amine.

2. The compound of claim 1 wherein R3 is fluorine.

3. The compound of claim 1 wherein Y2 is hydroxyl.

4. The compound of claim 1 wherein R3 is chlorine.

5. The compound of claim 1 wherein Y2 is amine.