CA2194150A1 - N-heteroaromatic ion and iminium ion substituted cyanine dyes for use as fluorescence labels - Google Patents

Abstract

The present invention relates to iminium ion substitute cyanine dyes having a fluorescence absorbance of between about 500 and 900 nm, a reduced tendency to aggregate and enhanced photostability. The cyanine dyes of the present invention are represented by formula (I) wherein n is 0, 1, 2 or 3; m is 0, 1, 2 or 3; R1 and R2 are taken together to form an aromatic ring or a fused polycyclic aromatic ring; R3 and R4 are taken together to form an aromatic ring or a fused polycyclic aromatic ring; R5 and R6 are independently selected from the group consisting of (CH2)pX where p is 1-18 and X is a functional group that reacts with amino, hydroxy and sulfhydryl nucleophiles; R7 and R8 are independently selected from the group consisting of hydrogen; C1-C10 alkyl groups and where R7 and R8 are taken together to form a five- or six-membered heterocyclic ring; R9 are each independently selected from the group consisting of hydrogen, alkyl and where more than one R9 are taken together to form a five- or six-membered ring; Y is selected from the group consisting of C(CH3)2, S, O and Se; and Z is selected from the group consisting of C(CH3)2, S, O and Se. The present invention also relates to a method for using the cyanine dyes of the present invention to fluorescent label molecules, particularly biomolecules such as antibodies, DNA, carbohydrates and cells.

Description

WO96~009022 I q4 ~ 50 P~ S' ~

N-HETEROAROMATIC ION AND IMINIUM ION
Slr~ ;11 CYANINE DYES FOR USE
~ AS FLUORESCENCE LABELS

FFT ~.TIONSrTTP TO COPENT)ING APPT Tc~TIoNs This application is a ~ " ,l ,. , -1;,~"-in-part of copending U.S. Application Serial No. 08/268,852 entitled IV-HETEROAROMATIC ION AND IMINIUM
ION SUBSTITUTED CYANINE DYES FOR USE AS FLUORESCENCE
LABELS, filed June 30, 1994, which is ill~UllJI ' ~ herein by reference.
Fi~ l ofthe Inventinn The present invention relates to cyanine dyes for use as fluorescent probes. More specifically, the present mvention relates to cyanine dyes substituted with either an N het~,.ualulllati~. ion or an iminium ion, the ion reducing the ~ ;dtiUIl of the cyanine dyes and enhancing the phr t~ cf~hility of the dyes.
.

F ~ n~l of thP InY~ nti--n Fluorescent dyes have a wide variety of uses including the labeling of antibodies, DNA, ~ cuI,olly ' and cells. In order for a fluorescent dye to function as a label, the dye must bind to the molecule or cell to be labeled.
Fluorescent labels are therefore designed to include at least one reactive moiety which reacts with amino, hydroxy and/or sulfhydryl . ~ ~ "pl,;l ~ present on themolecules bemg labeled. Examples of suitable reactive moieties include carboxylic acids, acid halides, sulfonic acids, esters, aldehydes, disulfides, iaulLiu~,y~.~ UUy ' , llwllo~lllulullia~ dichlorotriazine, mono- or di-halogen substituted pyridmes, mono- or di-halogen substituted diazines, maleirnide, aziridines, sulfonyl halides, L,ydlw~y~ ~ '..h.l~ . esters, Ly~u~y~ l l...0 esters, imido esters, hydrazines, a~iduLIiuu~ lyl, azides, 3-a-pyridyl dithio)-,u.. ~ and glyoxal. Additional suitable I

W0 96/00902 r~
21 94 1 50 ~

reactive moieties for use in fluorescent labels are described in U.S. Patent No.5,268,486 which is ;II~,UI~)~ ' ' herein by reference.
Fluorescent dyes commonly have an absorbance range of between about 300 and 900 nm and preferably have a Stokes shift of at least about 20 n~n.
Fluorescent dyes that absorb in the 500 to 900 nm range are preferred because they are spectrally removed from other c...., ,l .1 " ....l ~ that may be present in a biological sample and because they may be used with hlc.~ la;v~ light sources.
Fluorescent dyes that have a high extinction coefficient and a high quantum yield are also preferred.
Fluorescent dyes used for labeling 1 ~;"" "~Ir ~ ;, such as C~ubJllydla;~
proteins and DNA, are preferably water soluble since the 1,;. ""f lf ~ ..1. ~ to be labeled generally have limited solubility in n~ solvents. It is known to increase the water solubility of a dye by adding L~ llul~h;lic groups such as sulfonate groups and hydroxy groups. Examples of water soluble dyes that may be used as fluorescent probes include fluorescein (Coons, et al., J. F~, Med.
(1950) 91 1-13), ~)h~,ull;l;~Jlut~,;lla (Oi, et al., J. Cf 11 13iol. (1982) 93 981) and CyS ~Mujumdar, et al., E;i.~ u~ ('hf m (1993) _105-111).
It is important that the fluorescent dye is 1 ,h, 0~ .~1 ~-l ,1.~ However, dyes with a nuu~ ,,.,-,e absorbance greater than 500 nm tend to be less phf~toc Fluorescent dyes also should not be prone to a~ c~;aliull. Dye a~ .iiull~ also known as "stacking" increases the frequency of lluulc ~ C
quenching which reduces the strength of the nuu.~ ,., signal observed. Most fluorescent dyes are large planar molecules, are hlLIh~ lly hy~u~llub;~, and therefore have a tendency to aggregate or "stack," especially in aqueous solutions. Dyes with a ~ absorbance greater than 500 nm generally have a greater tendency to stack due to their increased si~ and associated lowersolubility. Non-a~;~uc~dtillg, ~ u~ 1 l lf fluorescent dyes with a nucu~ e absorbance greater than 500 nm are therefore needed.
Fluorescent probes are ~ .ulafly prone to stack in high salt solutions and when in high local c, .. ~t~ ,.l ;.. ~ on protein surfaces. For example, W0 96/00902 r~
~ 21 q4 1 50 ~ ,~
yl.l ~ a commonly used laser dye, produces protein-dye conjugates which ~ dul~f~ lly consist of the aggregated dye. Aggregated dyes appcar blue-shifted by visible absorbance spectra. Amino-substituted cyanine dyes, such as IR144, are prone to ~-c~;aLiun in aqueous solutions, even in low-salt solutions (i.e. 0.1 M NaCI). Non-aggregated amino-substituted cyanine dyes have only been found to exist in organic solvents. The absorbance spectra of protein-dye conjugates can be simulated by obtair~ing spectra of the dye in high salt solutions (e.g. 4 M NaCl). Dye a~lCt ~I;iVII may be minimized by cull~LI.~ .g highly ionic dyes such as o~yl~ulru~ taught in U.S. Patent No. 5,268,486 or by using naturally ûccurring fluorescent probes such as phycobiliproteins.

SUMMARY OF TT~F INVEN~ON
The present invention relates to cyanine dyes substituted with either an N-l,~ u~ ion or an iminium ion which have a n~.ul~ ~"nce absorbance of between about ~00 and 900 nm, a reduced tendency to aggregate and enhanced ;l;Ly. The cyanine dyes of the present invention are ~ c~llt~ l by the forrnula R2~N+--l~ R ~N+~J R /~--R5 R9 Rg wherein n=0, 1,2Or3;

m=O, 1,20r3;
R, and R2 are taken together to form an aromatic ring or a fused polycyclic aromatic ring;
R3 and R4 are taken together to form an aromatic ring or a fused polycyclic aromatic ring;
R5 and R6 are i- l lJ' "'~ y selected from the group consisting of (CH2 )p X where p is 1-18 and X is a functional group that reacts with amino, hydroxy or sulfhydryl r~ PophilPC
R~ and R8 are ;,..1, l) ...l....ly selected from the group consisting of hydrogen, Cl-CIO alkyl groups and where R, and R8 are taken together to forrn a five- or six- membered L~ v~ , ring;
R9 are each I l l" ...1. .,lly selected from the group consisting of hydrogen, alkyl and where more than one R, are taken together to for n a five-or six- membered ring;
Y is selected from the group consisting of C(CH3 )2, S, O and Se; and Z is selected from the group consisting of C(CH3 )2, S, O and Se.
~ The present invention also relates to a method for using the cyanine dyes of the present invention for lluu~ c Iabeling molecules, pol lL~ulally hir)mnlPclllPc such as antibodies, DNA, c~ul.oLy,' and cells.
RRTFF DE~CRIPTION OF THF DRAWINGS
Figure I depicts the absorbance spectrum of a BHDMAP-protein conjugate.
Figure 2A depicts the spectra of BHCI in low (0.1 M NaCI, 50 mM
phosphate, pH 7) and high (3.8 M NaCI, 50 mM phosphate, pH 7) salt solutions.
Figure 2B depicts the spectra of BHDMAP in low (0.1 M NaCI, 50 mM
phosphate, pH 7) and high (3.8 M NaCI, 50 mM phosphate, pH 7) salt solutions.
Figure 3 depicts the spectra of IR144 in a low salt solution (0.1 M NaCI, 50mMphosphate,pH7)andin~" ' yl r(..,.. :~

wo 96/00902 2 1 9 4 1 5 0 Figure 4 depicts the l.1,.-10,1~ ,ua;Liûn rates ûf several cyanine dyes.
Cy5 and Cy7 are ~y' ' ' dyes of U.S. Patent No. 5.268,486.

DETATT.F.n DF~CPTPTION OF T~TF INVENTION
The present invention relates to a class of cyanine dyes substituted with either an N-L,t~.u~ulllnL;c ion or an iminium ion having a lluul~a~ e absorbance between about 500 and 900 mm. This class of cyanine dyes have the advantage of being ~IhuLvaLalJlc and are not prone to ag~ Livll. The present invention also relates to a method for nuulea~ e labeling molecules using the substituted cyanine dyes of the present invention as fluorescent probes.
The N-h~ t~ ,u~uul~ L;c ion and iminium ion substituted cyanine dyes of the present invention are represented by the formula:

~ ~ ,J z/~ R4 R2 N+--I R9 N+ Rg (C=C)n ~ ~ (C=C)m ~N~R6 Ra Rg Ra Rg wherein n=0, 1,2Or3;
m = 0, 1, 2 or 3;
R, and R~ are taken together to form an aromatic ring or a fused polycyclic aromatic ring;
R3 and R~ are taken together to form am aromatic ring or a fused polycyclic aromatic ring;
R~ and E~6 are ;~ ly selected from the group consisting of w o 96100902 P~ . /,i5 (CH2 )p X where p is I-18 and X is a functional group that reacts with amino, hydroxy and sulflhydryl n--rlPophilPc R7 and R8 are ;..~ 1, .,.L .~lly selected from the group consisting of hydrogen, C1-C10 alkyl groups and where R, and R8 are taken together to form a five- or six- membered h~,t~,lv~ ,lh, ring;
R9 are each h~ y selected from t_e group consisting of hydrogen, alkyl and where more than one R7 are taken together to form a five-or six- membered ring;
Y is selected from the group consisting of C(C H3 )2, S, O and Se; and Z is selected from the group consisting of C(C H3 )2, S, O and Se.
Preferably, (n + m) is less than or equal to 3. Most preferably, n=1 andm=l.
R, - R2 and R3- R4 are both preferably taken together to form a benzene or . ' ' ' ring. The aromatic ring or fused polycyclic aromatic rings formed by Rl and R2 taken together and R3 and R4 taken together may be either ", .~. .h~ or substituted. Substitution of the aromatic ring or rings with electron donating groups, such as primary, secondary and tertiary alkyl groups, may be used to lower the absorbance ~.v~L,~ of the dye relative to an h~ dye, Meanwhile~ c--hctih~til~n of the aromatic ring or rings with electron WitlldlO.V~ g groups such as nihro, cyanate, acid, halide, alkoxy, aryloxy, ester, ether, sulfide, thioether, alcohol, alkene, alkyne and aryl groups, may be used to increase the absorbance wavelength of the dye relative to an "..~.ih~ d dye.
The reactive moieties (X) employed with R5 and R~ may be any functional group that reacts with the amino, hydroxy and/or sulflhydryl mlr.lPorhilPc commonly found on the ~,O.IbUIl,r~ , proteins, DNA or cells to be labeled by the fluorescent dye. Examples of suitable reactive moieties include, but are not limited to, carboxylic acids, acid halides, sulfonic acids,esters, aldehydes, disulfides, i:~vlhiv~ u~ w~v~,lllvlv~
di~,LlvlvL~i~ille, mono- or di-halogen substituted pyridines, mono- or di-W096/00902 r~"
21 ~4150 halogen substituted dia7ines, maleimide, a7iridines, sulfonyl halides, hy~Lu~ . -lr. esters, L~llu~a~ esters, imido esters, hydra_ines, aLidu~ u~ yl, a_ides, 3-(2-pyridyl dithio)-propionamide and glyoxal. Additional suitable reactive moieties for use in fluorescent labels aredescribed in U.S. Patent No. 5,268,486. The reactive moieties used in R5 and R6 are preferably au~ c;LIllldyl esters.
R~ and R8 are preferably taken together to form a h. .~ l u" y .,lic five- or six- membered ring including, for example, pyridinium, imi~7~ m, pyrrolium, pyra7olium, pyra_inium, 1~ ; 1.;1...1, uyl ;.1,.,;, .: ,..., .l.." l"l,.........
purinium and i. ~ ' R7 and R8 are more preferably taken together to form a pyridinium or an; . . . ~ 1; . ring . R~ and R8 are most preferably takentogether to form a 4-d;.ll~ ;Lylalu;uu~Jyli~ l, 4-(4-ul()l~llolillyl) pyridinium, or a l-methylimi~7nli-lm ellh~titll~t The L,~ u~ ,lic ring formed by R, and R8 taken together may be substituted or .. ~ I ;I ... t ~~ R, and R8 may be further substituted by either electron donating or electron willldlawh~c groups in electron ~.-.,-. -. ,;. -~;....
with the aromatic system of the dye in order to influence the nuulc~a~
absorbance wa~ l.clll of the dye. Sllh~titlltinn of R7 and R8 with electron donating groups, such as primary, secondary and tertiary alkyl groups, may be used to decrease the n 1 .. ~ . ~ absorbance wavelength of the dye relative to where R~ and R~ are substituted with hydrogen. Meanwhile, a ' .~ ;. ." of R, and R8 with electron w;~ ;..c groups such as nitro, cyanate, acid, halide, alkoxy, aryloxy, ester, ether, sulfide, thioether, alcohol, alkene, alkyne amd aryl groups, may be used to increase the n. . . ~ absorbance wa~ clll of the dye relative to where R, amd R8 are substituted with hydrogen.
The R9 ,.il .~;1 .... .~ are preferably selected such tbat the carbon atoms situated a and ~' to the iminium ion form part of either a five- or six- membered ring. The five- or six- membered ring may be substituted or I ' ' wo 96/00902 r~
2~ 941 50 --Y and Z may be either C(CH3 )2, S, O or Se. Preferably, Y and Z are C(CH3 )2 . Y and Z serve to keep the cyanine dye relatively planar and provide the dye with nuu~ ,e.

A preferred subclass of cyanine dyes of the present invention includes those cyanine dyes of the formula CH=CI I ~ ~CH=CH \R
R5 (CH2)r wherein R~, R2, R3, R4, R5, R5, R" R8, Y and Z are as specified above and 1 0 wherein r is either I, 2 or 3.
Table I provides the names, structures, absorbance and lluul~au~ ll.,e emission ~a~ L of several cyanine dyes of the present invention and of their chloro-substituted precursors. NHCI and ZFHCI do not have an absorbance maxirnurn in phosphate buffered saline (PBS).

WO 96~00902 r 2~1 94 1 ~0 T~ble 1. Dye Structures and Spectral Data Abs Max ~ in PBS (l ) BHCI ~ 776 N+ ~J N
(CH2)sCOOH (CH2)5COOH

N(CH3)2 BHDMAP ~ 786 N+ N
(CH2)sCOOH (CHz)5COOH
/

~ N

BHMI ~ 792 (CH2)sCOOH (CH2)sCOOH

BHPPY ~N~3 786 (CH2)sCOOH (CH2)sCOOH

~N~

BHMPY ~_~3 786 ( H2)5COOH (~:HZ)5cooH

W0 96/00902 r~l~u.. ~
21 94l 50 --Table I (cont.) Dye Structures and Spectral Data Abs Max in PBS ( ~g ' NHCI N I ,N
(CHz)5COOH (CH2)sCOOH

N(CH3k NHDMAp~--~? S25 (CH2)sCOOH (CH2)5COOH

NHMI ~~~1 832 (CH2)sCOOH (CH2)sCOOH

¢~3 BPCI N~ N 798 (CH2)sCOOH (CH2)sCOOH

N(CH3)2 BPDMAP O~ ~ 816 ( H2)sCOOH (CH2)5COOH

W0 96/00902 2 1 9 4 1 5 0 r~ r - I "~

Table 1 (cont.) Dye Structures and Spectr31 Data Abs Max ~h~ in PBS (nn~

F~F
(CH2)5COOH (CH2~5COOH

N(CH3)2 F~F
~CH2)sCOOH (CH2)5COOH

W096/00902 P.l/~.,. i //~

Table I (cont.) Dye Structures and Spectral Data Abs Max h~ in PBS (r~

~S ~3 (CH2)sCOOH (CH2)sCOOH
N(CH3~2 ' ~ S~

(CHz)5COOH (CH2)sCOOH

N

(CH2)sCOOH (CH2)sCOOH

~s~<S~3 --(CH2)sCOOH (CH2)sCOOH
~O~
N

N~S~3 (CH2)5COOH (CH2)sCOOH

W096/00902 r~ 115 ~ 21 941~0 ~

Table I (cont.) Dye S~uctures and Spectral Data Abs Max ~2m~ S~ jn P3S ~nn~

~S ~ <S~

~CH2)5COOH (CH2)sCOOH

N(CH3)2 (CH2)5COOH ~CH2)5COOH

(CH2)5COOH (CH2)5COOH

~-(CH2)5COOH (CH2)5COOH

(CH3)2 ~N~S~3 (CH2)5COOH (~H2)5COOH

Wo 96/00902 Pcr/uSg5/08778 2l q41 50 Table 1 (cont.) Dye Structures and Spectral Data Abs Max ~ in PBS ~nl) ZFHCIJ~ ~ C N~0--F

(CH2)sCOOH (CH2)sCOOH

N(CH3)z ZFHDMAP~ ~ ~ <N~3--F

(CH2)sCOOH (CH2)sCOOH

~ 21 9~1 50 Table I (cont.) Dye Structures and Spectral Data Abs Max jn P~S ~nl) 0~ ,~<Sel3 (CH2)sCOOH (CH2)sCOOH
N(CH3k ~Se~ Se,~;3 N+ ~ J N
(cH2)scooH (CHz)sCOOH

~N
N;
~s<Se,¦3 N+ ~J N
(CH2)sCOOH (CH2)sCOOH

Se~Se, N+ J N
(CH2)sCOC H (CH2)sCOOH

~N~

;~<Se,l~ _ N+ ~J N
(CH2)sCOOH (C H2)sCOOH

W096/00902 21 9~1 50 ~111 'I "~

Table I (cont.) Dye Structures and Spectral Data Abs Max ~:h~ in PBS (I~n~

0~ lie~ Se~

(CHz)sCOOH(CH2)5COOH

¢~CH3)z (CH2)sCOOH(CH2)sCOOH

Sel~g (CH2)sCOOH(CH2)sCOOH

Se~s<Se,l3 (CH2)sCOOH (CH2)sCOOH

N(CH3)2 D~s<Se,~]

(~H2)scooH(CHz)sCOOH

wo 96/00902 2 1 9 4 1 5 0 r~ "~

Tablc I (collt.) Dye Structures and Spectral Data Abs Max ~h~ jn PBS ~n~

Cl se~f~N13 F

(CH2)5COOH (CH2)5COOH

N(CH3)2 N~

F~<Sel3_ (CH2)sCOOH (CH2)~COOH

Wo 96/00902 r~ X~5~

Table I (cont.) Dye S~uctures and Spectral Data Abs Max ~m~ ~h~ in PBS (llrrl) ~LN+~N~J
(CH2)sCOOH (CH2)sCOOH

¢,~CH3)2 [~t~N' N N~
(CH2)sCOOH (CH2)sCOOH

¢N

N~N~ --(CH2)sCOOH (CH2)sCOOH

N

~o<~~3 ~~
(CH2)sCOC11 (CH2)sCOOH

N

~[N = N~
(CH2)sCOOH (CH2)sCOOH

W0 96/00902 2 1 9 4 1 5 0 P~ "~
.

, ~able I (cont.) Dye Structures and Spectral Data Abs Max inpRlC( g~O~<o~3 N+ N
(CH2)5COOH (CH2)5COOH

N(CH3)2 ~~~ S<O~
N+ _ J N
(CH2)sCOO H (CHZ)sCOoH

~0~
N+ N
(CH2)sCOOH ~CH2)sCOOH

~N~N~3 (CH2)sCOOH (CH2)sCOOH

N(CH3)2 ~0~o~
N+ N
(~H2)sCOOH (~H2)sCOOH

W0 96100902 P.~

Table I (cont.) Dye Structures and Spectral Data Abs Max ~ in PBS (nn~) ~

O C 0~
F N+ ,N F
(CH2)5COOI ~ (CH2)6COOH

N(CH3)2 F~ N ~N ~3--F
(CH2)5COOH (CH2)5COOH

W096/00902 21 941 50 r~ ). s~

The cyanine dyes of the present invention have been found to possess enhanced l.l.. .lu- ~"lily and are not prone to 5lg~gsltif~n Without being boundby theory, it is believed that the N-L~,tc.ualulllalic ion and the iminium ion inhibits a~ c~jjdtiull of these dyes. In addition to inhibiting a~,~ lc~aiiull~ the N-L~,t~,Lualulllaiic ion and the iminium ion are also believed to contribute to the phot~rt~llility ofthese dyes.
The following examples set forth the synthesis and physical ~1, - ,.. s ,,~:1.... of some of the cyanine dyes of the present invention. Further objectives and advantdges of the present invention other than those set forth above will become apparent from the examples which are not intended to limit the scope of the present invention.

FXAMPLF~

1.Synthesis of BHDMAP
N(CH3)2 BHDMA~ \ N I ~

0~N+~N~J
(CH2)sCOOH (CH2)sCOOH

The stepwise synthesis of BH-DMAP, shown above, is provided in Examples l(a)- I(d).

w0 96100902 P~~

I(a). SynthesisofI-(5'-.,O.l~u~lyl~ yl)-2,3,3.trim~lLyl;.,ll..l,":..."
bromide.

~ ~ sr(cH2)5coo Br~ (CH2)5cOOH

A mixture of 1,3,3~ ylhldOlill~ (3.8 g, 23 mmol) and 6-acid (4.6 g, 23 mmol) were stirred under nitrogen at 110~C for 12 h. The red solid was triturated with 2 x ~0 mL refluxing ethyl acetate followed by 20 mL refluxing acetone. The pink powder was filtered and air dried. Yield: 6.3 g, 18 mmol, 77%. 'H NMR: (CD30D) ~ 1.54 (m, 2H), 1.61 (s, 6H), 1.71 (m, 2H), 2.00 (m, 2H), 2.34 (t,J=7.3Hz, 2H), 4.53 (t, J=7.7Hz, 2H), 7.62-7.9 (m, 4H). The 2-methyl protons are acidic and exchange with the deuterated solvent.

W0 96/00902 r "~ x ~ 21 941 50 1 (b). Syntnesis of N-[S-anilino-3-cb~oro-2,4-(propane- 1',3 '-diyl)-2,4-pentadien-l-ylidene]-anilinium chloride.

O O Cl ~ Cl H~N(CH3)~ Poc~ ~ b CH2a2 H2o ¦ oHc~b~oH -- PhHN+~NHPh A " "~ , of the procedure taught by Reynolds in Reynolds, et al., J.Or~ l~h~m (1977)42885wasused. Asolutionofdllll~,;hylr",.,.-.."~1f (7.6 S g, 100 rnmol) and ~ ,LIulu~ ,L~ (2 rnL) was cooled in ice with stirring under nitrogen. Phua~llullla u~y~,llul;;i~, (12 g, 75 mmol) in dichlulvlll~ ~le (2 rnL) was added dropwise over 10 rnin. Cy~ (2 g, 20 mmol) in dichlvlvlll~ (3 mL) was added dropwise over 10 min. The solution mrned yellow. The solution was refluxed for 3 h and became orange.
The solution was poured over 50 g of ice. The organic layer was separated and discarded. Aniline (5 g, 54 rnmol) was added. A dark purple precipitate formed " '~o The solid was filtered and washed with I N
HCI and air dried. Yield: 1.9 g, 5.3 mmol, 27~/~.

w0 96100902 r~

l(c). SynthesisofBHCI.

1~ + PhHN+f~NHPh , ~
~3r~ (CH2)5COOH E~OH (CH2)sCO (CH2)3COOH

A solution of 1-(5'-c~bu~ u~yl)-2~3~3-llull~lllyli~ linilmn bromide (100 mg, 0.28 mmol), N-[5-anilino-3-chloro-2,4-(propane-1',3'-diyl)-2,4-pentadien-l-ylidene]anilinium chloride ( 50 mg, 0.14 mmol), sodium acetate (50 mg, 0.36 mmol) and ethanol (20 mL) was refluxed for 30 min. The solution was ' to dryness and the residue was trituated twice with 5 ml of 2N
HCI. The residue was dried in vacuo to give a dark oil. Yield: 90mg, 0.12mmol,43~/O. 'H- NMR (CD30D) li 1.51 (m,4H), 1.7 (m, 4H), 1.74 (s, 12H), 1.87 (m,4H),1.97 (m, 2H), 2.35 (m, 4H), 2.75 (m, 4H), 4.18 (m, 4H), 7.32 (m, 2H), 7.4-7.55 (m, 8H), 8.45 (m, 2H).

W0 96/00902 r~ s ~ / /8 ~ 2 t q4 ~ 50 l(d). Synthesis of BHDMAP.

N(CH3)2 ~CH3)2 ~
(cH2)sc (cH2)scooH DNIF (cH2)5c (cH2)5cooH

To a solution of BHCI (10 mg, 0.014 mmol) in DMF was added 4-dull~,llyl~u;llu~y ' (8.6 mg, 0.071 mmol). The reaction was monitored by S HPLC analysis on a POROS R2 column, 4 X 100 mm (20% to 80% acetonitrile vs. 0.1 M l~ - 1 l acetate over 4 min, 5 mL/min). After 15 h at ambient t~ lul~, the reaction was complete. The solution was then ' to dryness and dissolved in dichlu.w~ e (0.15 mL). Ethyl acetate (I mL) was added. Ihe dark precipatate was separated by c~ntlifi~g~linn Yield: 11 mg, 0.13 mmol, 93%. IH-NMR(CD3OD): ~ L41 (s, 12H), 1.48 (m, 4H),1.69 (m, 4H), 1.85 (m, 4H), 2.11 (m, 2H), 2.36 (m, 4H), 2.84 (m, 4H), 3.49 (s, 6H), 4.21 (m, 4H), 6.42 (d, J=11.8Hz, 2H), 6.94 (d, J=11.8Hz, 2H), 7.3-7.5 (m, I OH), 8.30 (d, J=6.7Hz, 2H). Fluulea~ ,e emission maximum: 807 mm.

W096/00902 P~,l/IJ.~.. I 1/~1 ~

2. Synthesis of NHMI.

5 ~&[

(CH2)sCOOH (CH2)sCOOH

The stepwise synthesis of NHMI, shown above, is provided in Examples 2(a) - 2(c) 2(a). Synthesis of 4,5-benzo-1-(5~ w~u. .I~.yl)-2,3,3-n' ~ bromide.

-W_N f Br(CH2)5COOH ~N+
Br~ (CH2)sCOOH

A mixture of 4,5-benzo-2,3,3-uh.l.,ll.yl;l.dolil.~ (0.5 g, 2.4 mmol) and 6-. acid (0.5 g, 2.6 mmol) were stirred under nitrogen at 110~ C for 2 h. The black tar was triturated with 20 mL acetone. The gray powder was filtered and air dried. Yield: 0.8 g, 2 mmol, 83%. 'H NMR: (CD30D) ~ 1.59 ( m, 2H), 1.73 (m, 2H), 1.85 (s, 6H), 2.06 (m, 2E~), 2.35 (t, J=7.0 Hz, 2H), 4.64 (t, J=7.7 Hz, 2H), 7.7-8.3 (m, 6H). The 2-methyl protons are acidic and exchamge with the deuterated solvent.

W0 96/00902 r~

2(b). Synthesis of NHCI.

Q~ Cl NaOAV Q ~ C~ ~
W~N ~ PhHN+~b_NHDh ~N'W
Df (CH,),COOH cr (CH,)5COOH (CH2)~COOH

Amixture of 4,5-benzo-1-(5'-~bu~yl ~Jl)-2~3~3-L~ oli bromide (110 mg, 0.28 mmol), N-[5-anili~o-3-chloro-2,4-propane-1',3'-diyl)-2,~pentadien-1-ylidene]anilininum chloride (50 mg, 0.14 mmol) and sodiurn acetate (100 mg, 0.72 mmol) was dissolved in 20 ml of ethanol. After stirring atroom i~ for 15 hours umder N~, the solvent was removed under reduced pressure. The residue was triturated with a 1: I solution of ethyl acetate and hexane (100 ml) to give a dark green solid. The solid was then washed with 5 ml of IN HCI amd dried in vacuo. Yield: 6S mg, 0.79 mmol, 56%. IH-NMR(CD~OD): o 1.56 (m, 4H), 1.73 (m, 4H), 1.94 (m, 4H), 1.98 (m, 2H), 2.04 (s, 12H), 2.34 (m, 4H), 2.75 (m, 4H), 4.3 (m, 4H), 7.5 (m, 2H), 7.65-8.26 (m, 12H), 8.56 (m, 2H).

WO 9610090~ F~ .' I In 21 q4l ~0 2(c). Synthesis of NHMI.

D~.IF ~
(CH2)5COOH (CH,),COOH (CH~),COOH (CH2~,COOH

A solution of NHCI (lOmg, 0.012 mmol) and N-methylimidazole (20 mg, 0.25 mmol) in 50 ~LI of DMF was allowed to stand for 7 days. The solvent was then removed Imder reduced pressure and the residue was dissolved in 200 ~LI of methylene chloride and I ml of ethyl acetate was added to precipitate theproduct. Yield: 3.2 mg, 0.0034 mmol, 28C/o.

wo 96/00902 P( llVJ3rl 1 1?5 ~ 21 941 50 3. Synthesis of BHDMAP ~ ~1 ester.

N(CH~)~
N~CH,), ¢~

;~o DMF ~CH~)~COOH
~CH:~COOH (CH215COOH ~
0;~
o The reaction sequence for tbe synthesis of BHDMAP-~uu~ hlilllidyl este}
is shown above. To a solution of BHDMAP (2 mg, 2 ~Lmol) in DMF was added S N-hydlu/o.~ (3 llg, 19 mmol) and di~ ~ loh. Aylcarbodiimide ( 2 mg, 10 ~Lmol). The reaction progress was monitored by HPLC on a POROS R2 column (20% to 40C/o acetonitrile vs. 0.1 M lli~,~lyl . l ll l l. .. ,: 1 , acetate over three min, 5 mL/min). After ~ 6 h tbe reaction mixture contained starting dye (17%), monoester (60%) and diester (23%). Acetic acid (20 ~lL) and methanol (0.5 mL) were added and the solution filtered to remove dh,.~.,lvll~".yl~ca. The solution was .~ 1 to dryness and redissolved in DMF (0.5 mL). The c..~ ..., . of the '.yl ester solution was determined by dilution of an aliquot into phosphate buffered saline and " ,. ~ .. ,1 of the optical density at 786 nm.
The extinction coefficient was assumed to be 200,000 cm -~M-l. The O.. ll.. ,.(.. ofBHDMAP~.Ilfill.idylesterwasfoundtobe 12mg/mL.

wo s6/00902 r~l,o,.,~
.

4. Antibody labeling with BHD~AP.

To a solution of mouse IgG (100 IlL, 2.5 mglrnL) was added BHDMAP
~u~ hulll;(lyl ester (1.3 ,uL, 12 mg/mL) and l,iu~u~ buffer (5 IlL, I M, pH
9.2). After 15 min at ambient ~u~ the excess dye was removed by size exclusion gel filtration. The dyelprotein ratio was determined by W-visible absorbance spectra. The extinction coefficient of the protein was assumed to be 170,000 cm -~M ~ at 280 nm. Figure I shows the spectrum of the BHDMAP-protein conjugate.

5. ~' ' spectra of dyes in salt solution.

The tendency of dyes to aggregate on proteins can be simulated by measuring the absorbance of the dye in low and high salt solutions. High salt solutions simulate the C;IIV;IUIUII.~III of the dye in high local ~.. , . . 1 . ,.1 ;. ", on the surface of a protein. Figure 2A shows the spectra of BHCI in low (0.1 M NaCI, 50 mM phosphate, pH 7) and high (3.8 M NaCI, 50 mM phosphate, pH 7) salt solutions. Figure 2B shows the spectra of BHDMAP in low (0.1 M NaCI, 50 mM phosphate, pH 7) and high (3.8 M NaCI, 50 mM phosphate, pH 7) salt solutions. BHCI appears to aggregate even in low salt, and in high salt the absorbance maximum has shifted to shorter wavelength. In contrast, the more ionic dye, BHDMAP, shows little aggregation in low and high salt solutions.
Figure 3 shows the spectra of a related dye, IRI 44, in low salt and rn ~h-l.,Lylr ,",.,,.,.;rl~ solutions. Based on the spectra shown m Figure 3, IR144appears to aggregate in low salt solutions.

W0 96/00902 P.~
~ 21 941 5~

6. Ph: ~ , ' of dyes.

- The p~ t~et~lhility of several cyanine dyes were compared. Cy5 and Cy7 are the penta- and hepta-methine derivatives, Ic~ .,ly, of a class of S aly' ' ~ dyes described in U.S. Patent No. 5,268,486. The structures of BHCI, NHMI and BHDMAP are found in Table 1.
Solutions of dyes in phosphate buffered saline were irradiated in I mL
.."yl.,'c disposable cuvettes with a halogen lamp. The intitial absorbamce of each dye varied from 0.5 to 1.7 O.D. units. The absorbance of the dye solutions at the absorbance maxium of each dye was monitored periodically.
The ~...,. ~ .,1,, l;,." ofthe dye was assumed to be proportional to the opticaldensity of the dye solution in acco}dance with Beer's Law. A plot of the logarithm of the norrnalized absorbance (IntDye]/tDye]0) vs. time is shown in Figure 4. Values of k were determined from least squares analysis and the half-life of each dye tl~-t~nin~d The most stable dye was foumd to be the dye with the shortest .,111, Cy5, whose structure contains five methine groups. The remaining dyes contain seven metbine groups. BHDMAP, NHMI and Cy7 all have similar stabilities. The least stable dye was foumd to be BHCI.
S.. hctitnti-m of the chloride with .L,Il~,Lllyl~l~ ;dlll., to provide BHDMAP
was found to improve the ~ y of the cyanine dye seven-fold.
While the present invention is disclosed by reference to the preferred ...,.1"~.1;.,....1~ and examples detailed above, it is to be understood that these examples are mtended in am illustrative rather than limiting sense, as it is e . ' ' that ,.,., 1; r~ will readily occur to those skilled in the art, which ...~..liri. -I;....~willbewithinthespiritoftheinventionandthescopeof the appended claims.
i

Claims (42)

What is claimed is:

1. A cyanine dye having the formula wherein - n and m are each independently either 0, 1, 2 or 3;
R1 and R2 are taken together to form an aromatic ring or a fused polycyclic aromatic ring;
R3 and R4 are taken together to form an aromatic ring or a fused polycyclic aromatic ring;
R5 and R6 are independently selected from the group consisting of (CH2)pX where p is 1-18 and X is a functional group that reacts with amino, hydroxy or sulfhydryl nucleophiles;
R7 and R8 are independently selected from the group consisting of hydrogen, C1-C10 alkyl groups and where R7 and R8 are taken together to form a five- or six- membered heterocyclic ring;
R9 are each independently selected from the group consisting of hydrogen, alkyl and where more than one R9 are taken together to form a five- or six- membered ring;
Y is selected from the group consisting of C(CH3)2, S, O and Se; and Z is selected from the group consisting of C(CH3)2, S, O and Se.

2. A cyanine dye according to claim 1 wherein R7 and R8 are taken together to form a ring selected from the group consisting of pyridinium, imidazolium, pyrrolium, pyrazolium, pyrazinium, pyrimidinium, pyridazinium, purinium, quinolinium and isoquinolinium.

3. A cyanine dye according to claim 2 wherein R7 and R8 are taken together to form a pyridinium or imidazolium ring.

4. A cyanine dye according to claim 3 wherein R7 and R8 are taken together to form a substituent selected from the group consisting of: a 4-dimethylaminopyridinium, 4-(4-morpholinyl)-pyridinium, and a 1-methylimidazolium substituent.

5. A cyanine dye according to claim 1 where X is independently selected from the group consisting of carboxylic acids, acid halides, sulfonic acids, esters, aldehydes, disulfides, isothiocyanantes, isocyanates, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridines, mono- or di-halogen substituted diazines, maleimide, aziridines, sulfonyl halides, hydroxysuccinimide esters, hydroxysulfosuccinimide esters, imido esters, hydrazines, azidonitrophenyl, azides, 3-(2-pyridyl ditbio)-propionamide and glyoxal.

6. A cyanine dye according to claim 5 where X is a carboxylic acid.

7. A cyanine dye according to claim 5 wherein R7 and R8 are taken together to form a ring selected from the group consisting of pyridinium, imidazolium, pyrrolium, pyrazolium, pyrazinium, pyrimidinium, pyridazinium, purinium, quinolinium and isoquinolinium.

8. A cyanine dye according to claim 1 wherein (n + m) is less than or equal to 3.

9. A cyanine dye according to claim 8 wherein n = 1 and m = 1.

10. A cyanine dye according to claim 1 wherein Y and Z each are C(CH3)2.

11. A cyanine dye having the formula wherein r is 1, 2 or 3;
R1 and R2 are taken together to form an aromatic ring or a fused polycyclic aromatic ring;
R3 and R4 are taken together to form an aromatic ring or a fused polycyclic aromatic ring;
R5 and R6 are independently selected from the group consisting of (CH2)p X where p is 1-18 and X is a functional group that reacts with amino, hydroxy or sulfhydryl nucleophiles;
R7 and R8 are independently selected from the group consisting of hydrogen, C1-C10 alkyl and where R7 and R8 are taken together to form a five- or six- membered heterocyclic ring;
Y is selected from the group consisting of C(CH3)2, S, O and Se; and Z is selected from the group consisting of C(CH3)2, S, O and Se.

12. A cyanine dye according to claim 11 wherein R7 and R8 are taken together to form a ring selected from the group consisting of pyridinium, imidozolium, pyrrolium, pyrazolium, pyrazinium, pyrimidinium, pyridazinium, purinium, quinolinium and isoquinolinium.

13. A cyanine dye according to claim 12 wherein R7 and R8 are taken together to form a pyridinium or imidazolium ring.

14. A cyanine dye according to claim 13 wherein R7 and R8 are taken together to form a substituent selected from the group consisting of: a 4-dimethylaminopyridinium, 4-(4-morpholinyl)pyridinium, and a 1-methylimidazolium substituent.

15. A cyanine dye according to claim 11 where X is independently selected from the group consisting of carboxylic acids, acid halides, sulfonic acids, esters, aldehydes, disulfides, isothiocyanates, isocyanates, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridines, mono- or di-halogen substituted diazines, maleimide, aziridines, sulfonyl halides, hydroxysuccinimide esters, hydroxysulfosuccinimide esters, imido esters, hydrazines, azidonitrophenyl, azides, 3-(2-pyridyl dithio)-propionamide and glyoxal.

16. A cyanine dye according to claim 15 where X is a carboxylic acid.

17. A cyanine dye according to claim 15 wherein R7 and R8 are taken together to form a ring selected from the group consisting of pyridinium, imidazolium, pyrrolium, pyrazolium, pyrazinium, pyrimidinium, pyridazinium, purinium, quinolinium and isoquinolinium.

18. A cyanine dye according to claim 11 wherein Y and Z each are C(CH3)2.

19. A cyanine dye selected from the group consisting of:

and

20. A cyanine dye selected from the group consisting of:

and

21. A cyanine dye selected from the group consisting of:

and

22. A cyanine dye selected from the group consisting of:

and

23. Method for fluorescent labeling a molecule comprising:
labeling a molecule having am amino, hydroxy or sulfhydryl functional group with a cyanine dye having the formula wherein n and m are each independently either 0, 1, 2 or 3;
R1 and R2 are taken together to form am aromatic ring or a fused polycyclic aromatic ring;
R3 and R4 are taken together to form an aromatic ring or a fused polycyclic aromatic ring;
R5 and R6 are independently selected from the group consisting of (CH2)p X where p is 1-18 and X is a functional group that reacts with amino, hydroxy or sulfhydryl nucleophiles;
R7 and R8 are independently selected from the group consisting of hydrogen, C1-C10 alkyl and where R7 and R8 are taken together to form a five- or six- membered heterocyclic ring;
R9 are each independently selected from the group consisting of hydrogen, alkyl and where more than one R9 are taken together to form a five- or six- membered ring;
Y is selected from the group consisting of C(CH3)2, S, O and Se; and Z is selected from the group consisting of C(CH3)2, S, O and Se.

24. A method according to claim 23 wherein the molecule is selected from the group consisting of antibodies, DNA, carbohydrates and cells.

25. A method according to claim 23 wherein R7 and R8 of the cyanine dye are taken together to form a ring selected from the group consisting of pyridinium, imidazolium, pyrrolium, pyrazolium, pyrazinium, pyrimidinium, pyridazinium, purinium, quinolinium and isoquinolinium.

26. A method according to claim 25 wherein R7 and R8 of the cyanine dye are taken together to form a pyridinium or imidazolium ring.

27. A method according to claim 26 wherein R7 and R8 of the cyanine dye are taken together to form a substituent selected from the group consisting of: a 4-dimethylaminopyridinium, 4-(4-morpholinyl) pyridinium, and a 1-methylimidazolium substitutent.

28. A method according to claim 23 wherein (n + m) is less than or equal to 3.

29. A method according to claim 28 wherein n = 1 and m = 1.

30. A method according to claim 23 wherein Y and Z of the cyanine dye are C(CH3)2.

31. Method for fluorescent labeling a molecule comprising:
labeling a molecule having an amino, hydroxy or sulfhydryl functional group with a cyanine dye having the formula wherein r is 1, 2 or 3;
R1 and R2 are taken together to form an aromatic ring or a fused polycyclic aromatic ring;
R3 and R4 are taken together to form an aromatic ring or a fused polycyclic aromatic ring;
R5 and R6 are independently selected from the group consisting of (CH2)p X where p is 1-18 and X is a functional group that reacts with amino, hydroxy or sulfhydryl nucleophiles;
R7 and R8 are independently selected from the group consisting of hydrogen, C1-C10 alkyl and where R7 and R8 are taken together to form a five- or six- membered heterocyclic ring;
Y is selected from the group consisting of C(CH3)2, S, O and Se; and Z is selected from the group consisting of C(CH3)2, S, O and Se.

32. A method according to claim 31 wherein R7 and R8 are taken together to form a ring selected from the group consisting of pyridinium, imidazolium, pyrrolium, pyrazolium, pyrazinium, pyrimidinium, pyridazinium, purinium, quinolinium, and isoquinolinium.

33. A method according to claim 32 wherein R7 and R8 are taken together to form a pyridinium or imidazolium ring.

34. A method according to claim 33 wherein R7 and R8 are taken together to form a substituent selected from the group consisting of: a 4-dimethylaminopyridinium, 4-(4-morpholinyl)pyridinium, and a 1-methylimidazolium substitutent.

35. A method according to claim 31 where X is independently selected from the group consisting of carboxylic acids, acid halides, sulfonic acids, esters, aldehydes, disulfides, isothiocyanates, isocyanates, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridines, mono- or di-halogen substituted diazines, maleimide, aziridines, sulfonyl halides, hydroxysuccinimide esters, hydroxysulfoccinimide esters, imido esters, hydrazines, azidonitrophenyl, azides, 3-(2-pyridyl dithio)-propionamide and glyoxal.

36. A method according to claim 35 where X is a carboxylic acid.

37. A method according to claim 36 wherein R7 and R8 are taken together to form a ring selected from the group consisting of pyridinium, imidazolium, pyrrolium, pyrazolium, pyrazinium, pyrimidinium, pyridazinium, purinium, quinolinium and isoquinolinium.

38. A method according to claim 31 wherein Y and Z each are C(CH3)2.

39. Method for fluorescent labeling a molecule comprising:
labeling a molecule having an amino, hydroxy or sulfhydryl functional group with a cyanine dye selected from the group consisting of:

and

40. Method for fluorescent labeling a molecule comprising:
labeling a molecule having an amino, hydroxy or sulfhydryl functional group with a cyanine dye selected from the group consisting of:

and

41. Method for fluorescent labeling a molecule comprising:
labeling a molecule having an amino, hydroxy or sulfhydryl functional group with a cyanine dye selected from the group consisting of:

and

42. Method for fluorescent labeling a molecule comprising:
labeling a molecule having an amino, hydroxy or sulfhydryl functional group with a cyanine dye selected from the group consisting of:

and