U.S. patent number 5,346,670 [Application Number 07/895,601] was granted by the patent office on 1994-09-13 for phthalocyanine and tetrabenztriazaporphyrin reagents.
This patent grant is currently assigned to British Technology Group U.S.A. Inc.. Invention is credited to Karen L. Fearon, Clifford C. Leznoff, Barry V. Pepich, George E. Renzoni, Deborah C. Schindele, Louis J. Theodore.
United States Patent |
5,346,670 |
Renzoni , et al. |
* September 13, 1994 |
**Please see images for:
( Certificate of Correction ) ** |
Phthalocyanine and tetrabenztriazaporphyrin reagents
Abstract
Red-shifted, water-soluble, fluorescent,
monomerically-tetherable derivatives having the formula: ##STR1##
wherein, M represents either H.sub.2 or is selected from among the
following metals: aluminum, silicon, phosphorus, gallium,
germanium, cadmium, scandium, magnesium, tin, and zinc. Each
R.sub.1 is independently selected from --XYW, --YW, and --W. X
represents either a carbon, or heteroatom selected from among
oxygen, nitrogen, sulfur, phosphorus, silicon, and selenium; Y
represents a linking group; and W represents a water solubilizing
group. The substituent R.sub.2 is selected from among --A, --Y'A,
--XA, and --XY'A, where A denotes a biological entity such as an
antibody, antibody fragment, nucleotide, nucleic acid probe,
antigen, oligonucleotide, deoxynucleotide, dideoxynucleotide,
avidin, streptavidin or membrane probe, or R.sub.2 is a reactive or
activatable group suitable for conjugating to a biological entity.
Y' is a linking group that tethers the biological entity to the
phthalocyanine or tetrabenztriazaporphyrin macrocycle. Z is either
a nitrogen atom or a carbon substituted with hydrogen, alkyl, aryl,
or aralkyl groups. Z may also be attached to R.sub.2. Also
disclosed are derivatives of the compounds of the above Formula in
which 1-4 of the benzo ring(s) contain 1 or 2 N atoms. Methods of
sequencing DNA and detecting analytes, including cells, using these
derivatives are disclosed, as are kits for carrying out assays for
the analytes and flow cytometry. Methods of detecting DNA using
cationic compounds of the above Formula, wherein R.sub.2 =R.sub.1
and W=-N.sup.+ D.sub.1 D.sub.2 D.sub.3 are also disclosed. Further,
compounds containing Tc, Gd, etc. as the metal in the above Formula
may be used for imaging or therapy.
Inventors: |
Renzoni; George E. (Seattle,
WA), Schindele; Deborah C. (Seattle, WA), Theodore; Louis
J. (Seattle, WA), Leznoff; Clifford C. (Ontario,
CA), Fearon; Karen L. (Woodinville, WA), Pepich;
Barry V. (Seattle, WA) |
Assignee: |
British Technology Group U.S.A.
Inc. (Gulph Mills, PA)
|
[*] Notice: |
The portion of the term of this patent
subsequent to August 4, 2009 has been disclaimed. |
Family
ID: |
27535555 |
Appl.
No.: |
07/895,601 |
Filed: |
June 8, 1992 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
Issue Date |
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398433 |
Aug 29, 1989 |
5135717 |
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366971 |
Jun 14, 1989 |
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61937 |
Jun 12, 1987 |
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946475 |
Dec 24, 1986 |
4803170 |
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398433 |
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241608 |
Sep 8, 1988 |
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Current U.S.
Class: |
422/52; 424/1.65;
424/1.69; 424/1.73; 424/9.363; 435/5; 435/6.12; 435/7.1; 435/7.5;
435/808; 435/968; 436/501; 436/56; 436/808; 534/11; 540/121;
540/128; 540/131; 540/132; 540/135; 540/139; 540/140; 540/145 |
Current CPC
Class: |
C07H
21/00 (20130101); G01N 33/533 (20130101); A61K
41/0071 (20130101); Y10S 435/968 (20130101); Y10S
435/808 (20130101); Y10S 436/808 (20130101); Y10T
436/13 (20150115) |
Current International
Class: |
A61K
41/00 (20060101); C07H 21/00 (20060101); C12Q
1/68 (20060101); G01N 33/533 (20060101); G01N
021/76 () |
Field of
Search: |
;435/5,6,7,968,808
;536/23.1 ;436/800,56,172,501 ;422/52,61
;540/121,128,131,132,135,139,140,145 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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63852A3 |
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Nov 1982 |
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EP |
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142369A2 |
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May 1985 |
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EP |
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Primary Examiner: Yarbrough; Amelia Burgess
Attorney, Agent or Firm: Christensen, O'Connor, Johnson
& Kindness
Parent Case Text
This is a continuation of U.S. Ser. No. 398,433, filed Aug. 29,
1989, now U.S. Pat. No. 5,135,717, which is a continuation-in-part
of U.S. Ser. No. 241,608, filed Sep. 8, 1988, now abandoned. This
application is also a continuation-in-part of U.S. Ser. No.
366,971, filed Jun. 14, 1989, which is a continuation-in-part of
international application No. PCT/US87/03226, filed Nov. 12, 1987,
which is a continuation-in-part of U.S. Ser. No. 061,937, filed
Jun. 12, 1987, now abandoned, and U.S. Ser. No. 946,475, filed Dec.
24, 1986, now U.S. Pat. No. 4,803,170.
Claims
The embodiments of the invention in which an exclusive property or
privilege is claimed are defined as follows:
1. A reagent having the formula: ##STR7## wherein M is selected
from the group consisting of hydrogen, aluminum, silicon,
phosphorus, gallium, germanium, cadmium, scandium, magnesium, tin,
and zinc;
each R.sub.1 is independently --XYW, --O-phenyl, --S-phenyl, --YW,
--W, or hydrogen, and wherein at least one R.sub.1 is --XYW,
--O-phenyl, or --S-phenyl;
X is oxygen, nitrogen, sulfur, phosphorus, silicon, or selenium; or
X is CR.sub.3 R.sub.4, wherein R.sub.3 and R.sub.4 are
independently selected from the group consisting of hydrogen,
alkyl, aryl, and aralkyl, or R.sub.3 and R.sub.4 together form a
carbonyl oxygen; or X is phenyl;
Y is a linking group between X and W when R.sub.1 is --XYW, or
between W and an aromatic ring of said reagent when R.sub.1 is
--YW;
W is a water-soluble group;
R.sub.2 is --A or --Y'A, wherein --A is a biological entity and Y'
is a linking group to --A; or R.sub.2 is a reactive or activatable
group; and
Z is nitrogen.
2. A reagent according to claim 1, wherein R.sub.1 is --XYW or --YW
and wherein Y is a C.sub.1 -C.sub.7 saturated or unsaturated,
straight chain, branched, or cyclic hydrocarbon moiety.
3. A reagent according to claim 1, wherein R.sub.1 is --XYW or --YW
and wherein Y is a polyether, polyamine, polyalcohol, sugar,
peptide, or nucleotide.
4. A reagent according to claim 1, wherein W is --OH, --CO.sub.2 H,
--OCH.sub.2 CO.sub.2 H, --PO.sub.4.sup..dbd., --PO.sub.3.sup.-,
--SO.sub.3.sup.-, --SO.sub.2.sup.-, --SO.sub.2 Cl,
--SO.sub.4.sup..dbd., --NH.sub.2, --NHD, --NHD.sub.1 D.sub.2, or
--N.sup.+ D.sub.1 D.sub.2 D.sub.3, wherein D, D.sub.1, D.sub.2, and
D.sub.3 are independently C.sub.1 -C.sub.10 alkyl, C.sub.6
-C.sub.12 aryl, or C.sub.6 -C.sub.12 aralkyl.
5. A reagent according to claim 1, wherein R.sub.1 is --XYW or --YW
and wherein Y is C.sub.1 -C.sub.3 alkylene and W is sulfonate or
carboxylate.
6. A reagent according to claim 5, wherein --XYW is substituted at
1-4 of the 1, 8, 15, and 22 positions on the macrocycle.
7. A reagent according to claim 5, wherein --XYW is substituted at
1-4 of the 2, 9, 16, and 23 positions on the macrocycle.
8. A reagent according to claim 1, wherein M is aluminum, and at
least one R.sub.1 is --XYW, wherein X is O or S, and W is sulfonate
or sulfonyl chloride.
9. A reagent according to claim 8, wherein Y is phenyl.
10. A reagent according to claim 1, comprising
aluminum phthalocyanine 1, 8, 15, 22-tetraglycolic acid, or
aluminum phthalocyanine 2, 9, 16, 23-tetraglycolic acid, linked to
R.sub.2.
11. A reagent according to claim 1, comprising
2, 9, 16, 23 tetraphenoxy aluminum phthalocyanine,
1, 8, 15, 22 tetraphenoxy aluminum phthalocyanine,
2, 9, 16, 22 tetrathiophenyl aluminum phthalocyanine, or
1, 8, 15, 22 tetrathiophenyl aluminum phthalocyanine, linked to
R.sub.2.
12. A reagent according to claim 1, wherein R.sub.2 is --A or
--Y'A, and wherein A is a nucleic acid selected from the group
consisting of ATP, CTP, GTP, TTP, UTP, dATP, dCTP, dGTP, dTTP,
dUTP, ddATP, ddCTP, ddGTP, ddTTP, ddUTP, and derivatives thereof,
oligonucleotides, and polynucleotides.
13. A kit for sequencing DNA, comprising:
a plurality of containers, each containing a different reagent
according to claim 1,
wherein the different reagents are characterized by having a common
excitation wavelength and different wavelengths of maximum
emission.
14. A kit according to claim 13, wherein there are four reagents,
and wherein each R.sub.2 group comprises a different nucleotide,
deoxynucleotide, or dideoxynucleotide.
15. A kit according to claim 14, wherein said reagents comprise
reagents according to claim 10.
16. A kit according to claim 14, wherein said reagents comprise
reagents according to claim 11.
17. A kit for sequencing DNA comprising:
each of the four dideoxynucleotides ddATP, ddCTP, ddGTP, and ddTPT
as A labeled with a reagent according to claim 1, a sequencing
enzyme, a sequencing primer, each of the four deoxynucleotides
dATP, dCTP, dGTP, and dTTP, and each of the four dideoxynucleotides
ddATP, ddCTP, ddGTP, and ddTTP.
18. A kit for sequencing DNA comprising:
a reagent according to claim 1 wherein A is DNA sequencing primer,
a sequencing enzyme, each of the four deoxynucleotides dATP, dCTP,
dGTP, and dTTP, and each of the four dideoxynucleotides ddATP,
ddCTP, ddGTP, and ddTTP.
19. A kit for sequencing DNA comprising:
a plurality of container, each containing a different reagent
according to claim 1 attached to a different one of the four
dideoxynucleotides ddATP, ddCTP, ddGTP, and ddTTP as A, a
sequencing enzyme, a sequencing primer, each of the four
deoxynucleotides dATP, dCTP, dGTP, and dTTP, and each of the four
dideoxynucleotides ddATP, ddCTP, ddGTP, and ddTTP.
20. A kit for sequencing DNA comprising:
a plurality of containers, each containing a different reagent
according to claim 1 attached to a sequencing primer as A, a
sequencing enzyme, each of the four deoxynucleotides dATP, dCTP,
dGTP, and dTTP, and each of the four dideoxynucleotides ddATP,
ddCTP, ddGTP, and ddTTP.
21. An organometallic reagent useful for magnetic resonance
imaging, having the formula: ##STR8## wherein M is a paramagnetic
metal or a radioactive metal;
each R.sub.1 is independently --XYW, --O-phenyl, --S-phenyl, --YW,
--W, or hydrogen, and wherein at least one R.sub.1 is --XYW,
--O-phenyl, or --S-phenyl;
X is oxygen, nitrogen, sulfur, phosphorus, silicon, or selenium; or
X is CR.sub.3 R.sub.4, wherein R.sub.3 and R.sub.4 are
independently selected from the group consisting of hydrogen,
alkyl, aryl, and aralkyl, or R.sub.3 and R.sub.4 together form a
carbonyl oxygen; or X is phenyl;
Y is a linking group between X and W when R.sub.1 is --XYW, or
between W and an aromatic ring of said reagent when R.sub.1 is
--YW;
W is a water-soluble group;
R.sub.2 is --A or --Y'A, wherein --A is a biological entity and Y'
is a linking group to --A; or R.sub.2 is a reactive or activatable
group; and
Z is nitrogen or --CR, wherein --R is H, alkyl, aryl, or
aralkyl.
22. An organometallic reagent according to claim 21, wherein M is
selected from the group consisting of gadolinium, manganese, and
iron.
23. A fluorescent reagent according to claim 1, wherein R.sub.2 is
--A or --Y'A, and wherein the phthalocyanine macrocycle is
monomeric.
24. A fluorescent reagent according to claim 23 having absorbance
in both the red and blue portions of the spectrum, wherein the
ratio of the relative heights of the maximum red and blue
absorbance peaks of the reagent, A(red)/A(blue), is greater than or
equal to 1.4.
25. A fluorescent reagent according to claim 23, wherein M is
aluminum, silicon, gallium, germanium, scandium, or tin.
Description
TECHNICAL FIELD
This invention relates to phthalocyanine and
tetrabenztriazaporphyrin reagents and their derivatives useful as
fluorescent reporting groups, imaging agents, and also as
therapeutic agents. The fluorescent reagents are useful in nucleic
acid sequence analysis, nucleic acid probe and hybridization
assays, fluorescence microscopy, flow cytometry, immunoassay, and
fluorescence imaging. The reagents may also be useful as
therapeutic agents in photodynamic applications.
BACKGROUND OF THE INVENTION
Fluorescent compounds (fluorophores) have been widely used in
immunoassays, flow cytometry, fluorescence microscopy, and DNA
sequencing. To date, the sensitivity of such assays has been
limited by the spectral properties of available fluorophores.
In particular, automated DNA sequencing has become an important
tool in molecular biology. The most successful strategies utilize
the Sanger dideoxy chain termination method with either a
5'-fluorophore-labeled primer or fluorophore-labeled
dideoxynucleotide triphosphates to generate a series of fragments.
The resultant fragments are separated by electrophoresis. Careful
selection of the enzyme, fluorophore, and reaction conditions has
increased the size of DNA fragments that can be sequenced by such
techniques from a hundred to nearly a thousand bases. For example,
Applied Biosystems Incorporated (ABI) reports the ability to
sequence nearly a 700 base pair stretch of DNA within 13 hours
using a fluorophore-labeled primer. Despite advances in automated
sequencing, the current technology does not allow single-run
sequencing of kilobase and greater lengths of DNA. This limit is
imposed, in part, by fluorophore detection and resolution. Signal
detection could be improved by the use of fluorophores with more
ideal spectral properties.
Recently, DNA sequencing systems have been described based on the
use of a novel set of four chain-terminating nucleotides, each
carrying a different chemically tuned succinylfluorescein dye
distinguished by its fluorescent emission. Prober, J. M., et al.,
Science 238:336-341, 1987; European Patent Application No.
87305848.1.
The effect of peripheral substitution of fluoro and cyano groups on
the electronic properties of silicon dihydroxy phthalocyanine has
been modelled. Hale, P. D., et al., J. Am. Chem. Soc.
109(20):5943-5947, 1987. The calculated wavelength of absorbance
for the parent silicon phthalocyanine was predicted to be 673 nm
while the octacyano- and octafluoro- derivatives had calculated
transitions at 685 and 756 nm, respectively. No mention of
fluorescence is made in the report.
Introduction of phenoxy and thiophenoxy substituents into the
phthalocyanine macrocycle reportedly led to an appreciable red
shift in the long wavelength band in the visible absorbance
spectra. Derkacheva, V. M., and E. A. Luk'yanets, J. Gen. Chem.
USSR 50:1874-1878, 1980. The sulfur substituted phthalocyanines
were said to be more red-shifted in absorbance than the oxygen
substituted derivatives and, in either case, the 3-substituted
phthalocyanines were reportedly more greatly shifted than the
4-isomers. No fluorescence data was reported. Of the compounds
discussed in the Luk'yanets report, only the metal free derivatives
are potential fluorophores. The cobalt and copper analogs are
nonfluorescent. Metal free phthalocyanines are not capable of being
rendered reactive or water soluble by the techniques described
herein since the metal free specie is unstable to some of these
techniques, such as chlorosulfonation.
The application of aluminum phthalocyanines to simultaneous,
multicomponent fluorescence analysis such as in nucleic acid
sequence analysis, flow cytometry, immuno- or nucleic acid probe
assays requires the preparation of a family of tetherable,
water-soluble derivatives with a common excitation wavelength and
yet different emission wavelengths, with maximal spectral
resolution between each family member. For DNA sequence analysis,
four such fluorophores are desired.
Ideal fluorophores have five characteristics: a readily accessible
excitation wavelength with a large molar absorptivity, a high
fluorescence quantum yield, a large Stokes shift (>50 nm),
emission at long wavelengths (greater than 600 nm), and a sharp
emission profile (full width at half maximum, FWHM<40 nm).
Aluminum phthalocyanine (AlPc) has nearly ideal spectral
properties. Excitation of aluminum phthalocyanine at 350 nm results
in emission at 685 nm with fluorescence quantum yield (.phi..sub.f)
of 0.58. Brannon, J. H., and D. Magde, J. Amer. Chem. Soc.,
102(1):62-65, 1980. Aluminum phthalocyanine (AlPc)is composed of a
highly conjugated macrocycle and a trivalent aluminum atom. The
structure of the parent AlPc fluorophore is shown below. L is a
ligand such as OH when the AlPc is in water. The trivalent aluminum
atom provides axial ligation which serves to reduce aggregation and
thereby increases fluorescence in solution. ##STR2##
In a related application, U.S. Ser. No. 366,971, filed Jun. 14,
1989, the present inventors have disclosed water-soluble
phthalocyanine compounds that are monomerically conjugated to
biochemical moieties.
In therapeutic applications, aluminum phthalocyanine sulfonates
have been determined to be effective in directed cell killing.
Ben-Hur, E. and I. Rosenthal, Photochem. Photobiol. 42(2):129-133,
1985. The advantage that phthalocyanines have over other
photodynamic agents is their large molar absorptivity in the red
region of the visible spectrum. The large molar extinction
coefficient coupled with the transparency of tissue at these red
wavelengths provides for more efficient light penetration and
subsequently more effective treatment of subcutaneous malignancies.
Pursuant to the present invention, aluminum phthalocyanine
derivatives red-shifted from the parent compound will provide an
even greater depth of penetration and enable even more effective
treatments. Derivatives attached to biological moieties such as
probes or antibodies can be targeted to specific cell
populations.
Closely related to the phthalocyanines are the
tetrabenztriazaporphyrins, referred to herein as TBTAPs. Barrett,
P. A., et al, J. Chem. See. 1809-1828, 1937. The only structural
difference is the replacement of the nitrogen at position twenty of
the phthalocyanine with a substituted carbon. No substituted
derivatives of these compounds have been reported to date. Nor have
any tetherable or water soluble analogs been reported. The spectral
and luminescent properties of magnesium and palladium
benzoporphyrins have been reported. Solovev, K. N. et al., Opt.
Spectrosc. 27:24-29, 1969. Neither aluminum, substituted,
tetherable or water-soluble derivatives are discussed.
SUMMARY OF THE INVENTION
One aspect of the present invention involves red-shifted,
water-soluble phthalocyanine and tetrabenztriazaporphyrin (TBTAP)
reagents having the formula: ##STR3## wherein M is H.sub.2,
aluminum, silicon, phosphorus, gallium, germanium, cadmium,
scandium, magnesium, tin, or zinc. Each R.sub.1 is independently
selected from --XYW, --YW, --W, or --H. X is CR.sub.3 R.sub.4,
where R.sub.3 and R.sub.4 are independently selected from hydrogen,
alkyl (preferably C.sub.1 -C.sub.12), aryl (preferably C.sub.6
-C.sub.12), or aralkyl (preferably C.sub.6 -C.sub.12), or R.sub.3
and R.sub.4 together may be a carbonyl oxygen, or X is either
phenyl or a heteroatom preferably selected from among oxygen,
nitrogen, and sulfur. Y is a linking group between X and W or
between a benzo ring of the phthalocyanine or TBTAP macrocycle and
W. W is a water-soluble group. R.sub.2 comprises a biological
entity such as an antibody, antigen, nucleotide, nucleic acid,
oligonucleotide, avidin, streptavidin, or a membrane probe, or
R.sub.2 is a reactive or activatable group suitable for conjugation
to a biological entity. Z is N or C--R, where R is H or an organic
group such as alkyl (preferably C.sub.1 -C.sub.12), aryl
(preferably C.sub.6 -C.sub.12), or aralkyl (preferably C.sub.6
-C.sub.12). When Z is CR, the R.sub.1 and R.sub.2 groups may be
located on any of the four benzo rings of the TBTAP.
In a separate embodiment, the R.sub.2 group located on the benzo
ring in formula I is defined as R.sub.1, and Z=--CR.sub.2, where
R.sub.2 is the same as previously described. Thus, in this
embodiment, the biological entity is located on the meso carbon
atom of the macrocycle rather than on a benzo ring.
In all embodiments of formula I, the linking group Y is preferably
less than 4 atoms in length and may contain aliphatic, aromatic,
polyene, alkynyl, polyether, polyamide, peptide, amino acid,
polyhydroxy, or sugar functionalities. Suitable water solubilizing
groups W include --OH, --CO.sub.2 H, --OCH.sub.2 CO.sub.2 H,
--PO.sub.4.sup..dbd., --PO.sub.3.sup.-, --SO.sub.3.sup.-,
--SO.sub.2.sup.-, --SO.sub.2 Cl, --SO.sub.4.sup..dbd., --NH.sub.2,
--NHD, --NHD.sub.1 D.sub.2, or --N.sup.+ D.sub.1 D.sub.2 D.sub.3,
D--D.sub.3 being independently alkyl (preferably C.sub.1
-C.sub.12), aryl (preferably C.sub.6 -C.sub.12), or aralkyl
(preferably C.sub.6 -C.sub.12). Charged species will have
counterions.
In a preferred embodiment, M is aluminum, each R.sub.1 is --XYW, X
is either an oxygen or sulfur atom, Y is a methylene group, W is a
carboxylic acid, Z is nitrogen and R.sub.2 is --X--CH.sub.2
CO.sub.2 H. The substitution of R.sub.1 occurs at the 1,8,15,22
positions (3 isomer) or at the 2,9,16,23 positions (4 isomer) of
the macrocycle. See FIG. 1 for the phthalocyanine and
tetrabenztriazaporphyrin ring numbering system.
In a particularly preferred embodiment, M is aluminum, each R.sub.1
is --XYW, X is either an oxygen or sulfur atom, Y is phenyl, W is
sulfonate or sulfonyl chloride, Z is nitrogen and R.sub.2 is
--O-phenyl-sulfonate, --O-phenyl-sulfonyl chloride,
--S-phenyl-sulfonate, or --S-phenyl-sulfonyl chloride. The
substitution of R.sub.1 occurs at the 1,8,15,22 positions (3
isomer) or at the 2,9,16,23 positions (4 isomer) of the
macrocycle.
For the tetrabenztriazaporphyrin derivatives, the preferred
embodiments are as described above except that Z is a carbon
substituted with either hydrogen or phenyl substituents. The phenyl
may be unsubstituted or substituted by 1-5, preferably 1-2
substituents selected from among C.sub.1 -C.sub.6 alkyl, halogen
(e.g. Cl, Br, F, I), carboxy, nitro, or other substitutents that do
not substantially interfere with the fluorescence or water
solubility of the molecule.
When Z is --CR.sub.2, the rest of the macrocycle contains 4 R.sub.1
groups, as defined above.
For the divalent metals (M), Cd, Mg, and Zn, no axial ligand (L) is
present. The trivalent metal atoms (M), Al, Ga, and So, have at
least one axial ligand (L). The tetravalent metal atoms (M), Si,
Ge, Sn, have at least two axial ligands (L). Phosphorus (M) will
bear either one or three axial ligands (L).
Reagent kits for detection of single analytes using a reagent
described above are provided, as are kits and methods for
sequencing DNA. Reagent kits useful for simultaneous detection of a
plurality of analytes in solution containing combinations of the
subject reagents, each tethered to a different biological entity,
are also disclosed.
A second aspect of the present invention involves pyrazine
porphyrazines, pyrazine tetrabenztriazaporphyrins, pyridine
porphyrazines, and pyridine tetrabenztriazaporphyrins. These
compounds have the same structure as formula I, with the exception
that 1-4 of the benzo rings contain 1 nitrogen atom (pyridine
derivatives) or 2 nitrogen atoms (pyrazine derivatives). When the
benzo ring contains 1 nitrogen atom, both the 3 and 4 positional
isomers are possible. The 2 nitrogen atoms per ring in the pyrazine
derivatives are generally oriented in a 1,4 arrangement in the
benzo ring. Preferably, all 4 benzo rings will contain either 1 or
2 nitrogen atoms. Mixed derivatives are also possible, in which
benzo tines contain one nitrogen atom (either isomer) and 3-1 benzo
rings contain 2 nitrogen atoms. The R.sub.1 and R.sub.2 groups may
be attached to carbon atoms or nitrogen atoms in the benzo tines,
but attachment to carbon atoms of the benzo rings is preferred.
When X is a heteroatom, --XYW will be attached to a carbon; when X
is CR.sub.3 R.sub.4 or phenyl, --XYW may be attached to a carbon
atom (preferred) or a nitrogen atom of the benzo ring. Examples 12
and 13 herein illustrate preferred compounds of the second aspect
compounds. Additional preferred compounds are analogous to those
identified for the compounds of the first aspect of the present
invention. The compounds of the second aspect of this invention may
be used in the same applications as the phthalocyanine and
tetrabenztriazaporphyrin compounds described above.
A third aspect of the present invention involves cationic reagents
having formula I above, except that R.sub.2 is R.sub.1. R.sub.1, X
and Y are as described above. W is --N.sup.+ D.sub.1 D.sub.2
D.sub.3, wherein D.sub.1 -D.sub.3 are independently hydrogen,
C.sub.1 -C.sub.12 alkyl, C.sub.6 -C.sub.12 aralkyl, or C.sub.6
-C.sub.12 aryl groups, or --N.sup.+ D.sub.1 D.sub.2 D.sub.3 forms a
pyridinium ring. The charged groups may be associated with any
conventional counterion as long as it does not substantially
interfere with fluorescence or synthesis of the reagent. These
reagents may be advantageously used to bind to (stain or label)
oligo- and polynucleotides, especially DNA or RNA, for qualitative
or quantitative determination.
In yet another aspect, the present invention provides intermediates
for the synthesis of the compounds of formula I. For example,
reactive or activatable intermediates in which R.sub.2 in formula I
is a group capable of being covalently attached to a biological
entity are contemplated. R.sub.2 may be directly attached to the
benzo ring or may be linked to the benzo ring by an XY or Y
linkage. Such R.sub.2 groups include --SO.sub.2 Cl; --CO.sub.2 H;
--COX', wherein X' is a leaving group such as
N-hydroxy-succinimide; maleimide; or isothiocyanate. R.sub.2 can
also be a nucleophilic moiety, such as an amino group, for reaction
with reactive groups on the biological entity. A water soluble
group W on the benzo rings may alternatively be conjugated to
biological entities, in some embodiments. The other variables in
formula I are the same as defined herein. These compounds may be
coupled to biological entities by standard coupling reactions. Once
coupled, at least a portion of the reactive group becomes a Y'
group, as defined in connection with formula I.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the phthalocyanine and tetrabenztriazaporphyrin ring
numbering system.
FIG. 2 shows the absorbance and emission spectrum of aluminum
phthalocyanine tetrasulfonate, 1, in water.
FIG. 3 compares the absorbance spectra of the glycolic acid
derivatives, 2 and 3, in water.
FIG. 4 compares the emission spectra of the glycolic acid
derivatives, 2 and 3, in water.
FIG. 5 compares the emission of spectra of the two glycolic acid
derivatives, 2 and 3, in aqueous cetyl trimethylammonium bromide
(CTAB).
FIG. 6 compares the absorbance spectra of the oxygen substituted
aluminum phthalocyanine sulfonates, 4 and 5, in water.
FIG. 7 compares the emission spectra of the oxygen substituted
aluminum phthalocyanine sulfonates, 4 and 5, in water.
FIG. 8 compares the absorbance spectra of the sulfur substituted
aluminum phthalocyanine sulfonates, 6 and 7, in water.
FIG. 9 compares the emission spectra of the sulfur substituted
aluminum phthalocyanine sulfonates, 6 and 7, in water.
FIG. 10 compares the emission spectra of the oxygen substituted
aluminum tetrabenztriazaporphyrin sulfonates, 8 and 9, in
water.
FIG. 11 compares the emission spectra of the sulfur substituted
aluminum tetrabenztriazaporphyrin sulfonates, 10 and 11, in
water.
FIG. 12 shows the absorbance and emission spectra of aluminum 20-H
tetrabenztriazaporphyrin sulfonate, 12, in water.
FIG. 13 shows the absorbance and emission spectra of aluminum
20-phenyl tetrabenztriazaporphyrin sulfonate, 13, in water.
FIG. 14 compares the absorbance spectra of the metal free
phthalocyanine cationic fluorophore, 14a, in water with and without
RNA.
FIG. 15 compares the emission spectra of the metal free
phthalocyanine cationic fluorophore, 14a, in water with and without
RNA.
FIG. 16 compares the absorbance spectra of the aluminum
phthalocyanine cationic fluorophore, 14b, in water with and without
RNA.
FIG. 17 compares the emission spectra of the aluminum
phthalocyanine cationic fluorophore, 14b, in water with and without
RNA.
FIG. 18 compares the emission spectra in water of four aluminum
phthalocyanine sulfonates, 1, 4, 5, and 7, suitable for DNA
sequence analysis.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention, in a first aspect, provides improved phthalocyanine
and related reagents in the form of red-shifted, water-soluble,
monomerically-tetherable derivatives according to formula I.
##STR4##
In formula I, M represents either H.sub.2 or is selected from among
the following metals: aluminum, silicon, phosphorous, gallium,
germanium, cadmium, scandium, magnesium, tin, and zinc. Each
R.sub.1 is independently selected from --XYW, --YW, --W, or
hydrogen. X represents CR.sub.3 R.sub.4, where R.sub.3 and R.sub.4
are independently selected from hydrogen, alkyl (preferably C.sub.1
-C.sub.12), aryl (preferably C.sub.6 -C.sub.12), or aralkyl
(preferably C.sub.6 -C.sub.12), or R.sub.3 and R.sub.4 together may
be a carbonyl oxygen, or X is phenyl, or X is a heteroatom selected
from among oxygen, nitrogen, sulfur, phosphorus, silicon, and
selenium. Y represents a linking group; and W represents a water
solubilizing group. The substituent R.sub.2 is selected from among
--A, --Y'A, --XA, and --XY'A, where A denotes a biological entity
such as an antibody, antibody fragment, antigen, oligonucleotide,
nucleotide, nucleic acid probe, avidin, streptavidin, or membrane
probe. R.sub.2 may also be a reactive or activatable group which is
directly attached to the benzo ring or attached by way of a linker,
such as --X--alkylene-- or --X--phenylene--, where X is defined
above. Z is N or C--R, where R is H, or an organic group such as
alkyl (preferably C.sub.1 -C.sub.12), aryl (preferably C.sub.6
-C.sub.12), or aralkyl (preferably C.sub.6 -C.sub.12). Y' is a
linking group that tethers the biological entity (A) to the
phthalocyanine or tetrabenztriazaporphyrin macrocycle. The
biological entities containing nucleotides or derivatives thereof
are generally triphosphorylated, but mono- and di-phosphorylated
compounds may also be employed. Z is either a nitrogen atom or a
carbon substituted with hydrogen, alkyl (preferably C.sub.1
-C.sub.12), aryl (preferably C.sub.6 -C.sub.12), or aralkyl
(preferably, C.sub.6 -C.sub.12) groups.
In a separate embodiment, Z is --CR.sub.2, in which case all of the
variables on the benzo rings of the macrocycle will be R.sub.1
groups; that is, the R.sub.2 group may be attached to the meso
carbon rather than to a benzo ring of the TBTAPs.
In a preferred embodiment, M is aluminum, each R.sub.1 is --XYW, X
is either oxygen or sulfur, Y is methylene, W is carboxylate, Z is
nitrogen, and R.sub.2 is an activatable or reactive group attached
to the benzo ring by way of an XY link. R.sub.2 may preferably be
--XY-activatable group, where X is O, Y is methylene and the
activatable group is --CO.sub.2 H.
In a particularly preferred embodiment, M is aluminum, each R.sub.1
is --XYW, X is either oxygen or sulfur, Y is phenyl, W is sulfonate
or sulfonyl chloride, R.sub.2 is an activatable or reactive group
attached to the benzo ring by way of an XY link, and Z is nitrogen.
These derivatives are referred to as tetrasubstituted aluminum
phthalocyanines. R.sub.2 may preferably be --XY-reactive group,
where X is O or S, Y is phenyl and the reactive group is --SO.sub.2
Cl (located in the ortho, meta, or para positions of the phenyl
ring).
A similar preferred embodiment is exactly as above except that Z is
a carbon with either a hydrogen or phenyl substituent. These
derivatives are referred to as tetrasubstituted
tetrabenztriazaporphyrins.
Based on the synthetic procedures used, some of the present
compounds may occur as mixtures, particularly isomeric mixtures or
mixtures of compounds with different numbers of water solubilizing
groups. Such mixtures are within the scope of this invention.
While the phthalocyanines all share a common absorbance wavelength
in the ultraviolet near 350 nm, the visible absorbance is
substituent dependent. A red shift of the visible absorbance maxima
of phthalocyanines is attained by peripheral substitution with
oxygen (ether) and sulfur (thioether) groups, X in formula I.
Sulfur substitution results in a greater red shift of fluorescent
emission than oxygen substitution, and substitution at the three
positions (1,8,15,22 isomer) provides a greater shift than 4
substitution (2,9,16,23 isomer). The trend observed in the
absorbance spectra is found in the fluorescence spectra. The trend
is also observed in the absorbance and emission maxima of
tetrabenztriazaporphyrins. In view of these observations, a
preferred group of reagents are those in which at least one X is a
heteroatom, although 2, 3 or 4 heteroatoms are also
contemplated.
Substituent W is provided to impart water solubility to the
reagent, preferably at 10.sup.-6 M or lower concentrations. The
aqueous solubility should be maintained at temperatures ranging
from about 4.degree. C. (e.g., for flow cytometric applications) to
about 100.degree. C. (e.g, 67.degree. C. for gene probe
applications). Additionally, W is chosen to provide maximum
monomerism or, in other words, to minimize aggregation of the
fluorophores in aqueous solution. Aggregation of the fluorophores
results in the quenching of fluorescence and thus limits the
sensitivity of the probe and therefore its utility in assay
environments. Monomerism is discussed in greater detail
hereinbelow. Since charge repulsion diminishes aggregation, W is
preferably charged rather than neutral. However, W must not promote
nonspecific binding. Thus, for nucleic acid sequencing, the W
groups should be negatively charged (W is sulfonate, for example)
in order to avoid ionic attraction to negatively charged DNA or
RNA. Conversely, a positively charged phthalocyanine derivative (W
is quaternary ammonium, for example) may be utilized to selectively
stain DNA, RNA, and other negatively charged cellular
constituents.
Guided by the foregoing considerations, the water solubilizing
groups W can be selected from among --OH, --poly--OH, --CO.sub.2 H,
--OCH.sub.2 CO.sub.2 H, --OCHD.sub.1 CO.sub.2 H, --OCD.sub.1
D.sub.2 CO.sub.2 H, --PO.sub.4.sup.2-, --PO.sub.3.sup.-,
--SO.sub.3.sup.-, --SO.sub.2.sup.-, --SO.sub.4.sup.2-, --SO.sub.2
Cl, --N.sup.+ H.sub.3 /--NH.sub.2, --N.sup.+ H.sub.2 D/--NHD,
--N.sup.+ HD.sub.1 D.sub.2 /--ND.sub.1 D.sub.2, and --N.sup.+
D.sub.1 D.sub.2 D.sub.3 with D--D.sub.3 being individually alkyl
(preferably C.sub.1 -C.sub.12), aryl (preferably C.sub.6
-C.sub.12), or aralkyl (preferably C.sub.6 -C.sub.12), amino acids
(such as one selected from the common 20 naturally occurring amino
acids) or peptides (e.g. having from 2-10 residues). In particular,
sulfonate groups (preferably 2, 3 or 4) render the molecule water
soluble over a wide range of pH (2-12). Carboxylic acid groups, on
the other hand, are more sensitive to pH, thus limiting their
versatility and performance in aqueous systems. Below pH 5,
carboxylic acid groups are not ionized and therefore have limited
solubility in water. Both sulfonic and phosphoric acids are ionized
below pH 2. Quaternary ammonium groups are positively charged
regardless of pH. Charged groups will be associated with a suitable
counterion. The counterions are not necessarily limited and may be
any known counterions that do not interfere with synthesis of the
compounds or their desirable fluorescence characteristics.
Substituent Y is a group of atoms that links X with the water
solubilizing group W or the reactive or activatable group R.sub.2.
In a preferred embodiment Y is methylene (--CH.sub.2 --); however,
longer alkyl, aryl, or aralkyl chains are possible (preferably
C.sub.2 -C.sub.12). Longer links may adversely impact water
solubility and increase aggregation in solution leading to a
diminution of fluorescence. Therefore, in a preferred embodiment Y
has about 7 carbon atoms or less. Alternatively, the link Y may be
hydrophilic or even charged to increase both water solubility and
monomerism. Suitable hydrophilic spacers include polyethers,
polyamines, polyalcohols, and naturally occurring sugars, peptides,
and nucleotides. In a particularly preferred embodiment, Y is
phenyl with X at position one and W at position 4 (para
substitution).
Within the above constraints, Y can be selected from among
aliphatic, aromatic, mixed aliphatic/aromatic functionalities,
polyene (cis or trans), mixed polyene and/or aliphatic and/or
aromatic functionalities, alkynyl, mixed alkynyl and/or aliphatic
and/or aromatic functionalities, polyether linked by aliphatic
and/or aromatic and/or alkenyl and/or alkynyl functionalities,
polyamides, peptides, amino acids, polyhydroxy functionalities,
sugars, and nucleotides. The precise nature of Y is unimportant,
and practically any Y group will work as long as it does not
interfere with water-solubility or fluorescence to an unacceptable
degree and it is synthetically accessible.
Substituents R.sub.1 are individually selected from among --XYW,
--YW, --W, and hydrogen. In one preferred embodiment, all three
R.sub.1 groups are --XYW, --YW, or --W, especially --XYW. In
another preferred embodiment, one R.sub.1 is --XYW and the other
two are --YW or --W.
Substituent R.sub.2 may be a biological entity such as an antigen
or an antibody attached to the macrocycle. R.sub.2 may also be an
activatable group or a reactive group; as such, R.sub.2 may be
linked to the benzo ring by X or XY linkers, or may be directly
attached to the benzo ring. In some embodiments, discussed herein,
R.sub.2 may be R.sub.1, in which case no biological entity is
covalently bound to the fluorophore. In other embodiments, R.sub.2
is attached to the meso carbon of a TBTAP or other derivatives of a
triazaporphyrin described herein, and the remaining variables on
the benzo rings of the macrocycle are each R.sub.1. Representative
biological entities (A) include natural or synthetic drugs
(therapeutics and abused), drug metabolites, metabolites, hormones,
peptides, nucleotides (e.g., ATP, CTP, GTP, TTP, UTP, dATP, dGTP,
dCTP, dTTP, dUTP, ddATP, ddCTP, ddGTP, ddTTP, ddUTP, and
derivatives thereof), neurotransmitters, enzyme substrates, DNA or
RNA probes, DNA or RNA (oligo and polynucleotides), DNA/RNA
hybrids, DNA/DNA hybrids, RNA/RNA hybrids, growth factors, antibody
fragments (antigen binding fragments), antibodies (polyclonal or
monoclonal), serum proteins, streptavidin, avidin, enzymes,
intracellular organelles, cell surface antigens, receptors, ligand
binding proteins or associated ligands, membrane probes etc. The
fluorescent moiety (i.e., the macrocycle) is preferably attached to
R.sub.2 monomerically to enhance fluorescence. The particular
nature of the biological entity is relatively unimportant. As long
as the conjugation of the fluorophore to the biological entity does
not destroy utility of the conjugate, it is contemplated to be
within the scope of this invention.
By "membrane probe" is meant a lipophilic organic moiety preferably
having 10 to 30 carbon atoms. In a preferred embodiment, the
membrane probe is a long chain hydrocarbon group. Particularly
preferably, the hydrocarbon group is a saturated C.sub.10 -C.sub.30
alkyl group that may be straight chain, branched or may contain
cyclic rings. The membrane probe may be attached to a benzo ring or
to the meso carbon of a TBTAP.
Preferred linkers Y' for connecting the biological entity to the
phthalocyanine include sulfonamide, amide, ether, thioether, ester,
thioester, amine, and carbon-carbon bonds. For this purpose, the
biological entity should bear a terminal amino, carboxy, .alpha.,
.beta.-unsaturated carbonyl, thiol, sulfonyl chloride, or halide
group for attachment to the phthalocyanine. In turn, the
phthalocyanine should bear a correspondingly reactive group, such
as carboxy, amino, thiol, .alpha.,.beta.-unsaturated carbonyl,
sulfonyl chloride, or hydroxy.
The tether Y' to the biological entity, A, in R.sub.2 is long
enough for optimal recognition of A in typical biological assays.
Displacement of A from the phthalocyanine or
tetrabenztriazaporphyrin can be further enhanced by the use of a
rigid linker containing for example, alkene, acetylene, cyclic,
aromatic or amide groups. The water solubility of the
phthalocyanine may also be enhanced by selection of hydrophilic or
charged groups as part of the linker Y'. Hydrophilic spacers
include polyethers, polyamines, polyalcohols, and naturally
occurring species such as sugars, peptides, and nucleotides. To
reduce aggregation in aqueous solution long, hydrophobic tethers
should be avoided.
The following are illustrative embodiments of some compounds of
formula I of the present invention.
Aluminum Phthalocyanine Tetraglycolates
In one embodiment, the invention provides companion water soluble
aluminum phthalocyanine derivatives. In a preferred embodiment, the
invention provides two aluminum tetraglycolylphthalocyanine
isomers, 2 and 3, each having emission bands red-shifted relative
to aluminum phthalocyanine trisulfonate (referred to as compound 1
herein). The tetracarboxylic acid derivatives may be prepared as
set forth in Example 1 herein. The only difference between the two
phthalocyanines is the position of attachment of the glycolyl group
(--OCH.sub.2 CO.sub.2 H) on the macrocycle. Substitution at the
2,9,16,23 positions provides 2, while 1,8,15,22 substitution gives
3. The carboxylic acid groups present in these derivatives provides
both water solubility and a reactive functionality for tethering
compounds to biological entities. Exemplary biological entities for
coupling to 2 and 3 are: antigens, antibodies or antibody
fragments, receptors, intracellular organelles, proteins, such as
avidin and streptavidin, enzyme substrates, membrane probes,
nucleotides and derivatives thereof, nucleic acid probes, and
nucleic acids.
The absorbance spectra of 2 and 3 in water are shown in FIG. 3.
Both exhibit a common excitation wavelength in the ultraviolet (350
nm) with molar absorptivities around 70,000. As shown in FIG. 4,
the emission maxima for the pair are distinguishable, with emission
wavelengths of 704 nm for 2 and 727 nm and 3. The quantum yields of
fluorescence are 0.55 and 0.43, for 2 and 3 environmental effects.
FIG. 5 presents the emission spectra of 2 and 3.3 in
respectively.
The spectral resolution of the two fluorophores may be affected by
environmental effects. FIG. 5 presents the emission spectra of 2
and 3 in aqueous cetyl trimethylammonium bromide (0.010M CTAB).
While the emission maximum of 3 remains essentially unchanged, a
dramatic red shift to 716 nm occurs for 2.
Oxygen and Sulfur Substituted Aluminum Phthalocyanine
Sulfonates
In a most preferred embodiment, the invention provides a family of
four novel, water soluble, tetherable aluminum phthalocyanine based
fluorophores. The family consists of two pairs of isomeric aluminum
phthalocyanine derivatives. The emission of each fluorophore pair
is unique and distinguishable from the other and all are
red-shifted compared to 1.
The first pair of fluorophores are tetraphenoxy substituted
aluminum phthalocyanines. Phthalocyanine formation from 4-
phenoxyphthalonitrile yields a 2,9,16,23 phenoxy substituted
phthalocyanine. Similar reaction with 3-phenoxyphthalonitrile
results in the formation of a 1,8,15,22 substituted phthalocyanine.
After the incorporation of aluminum, treatment of these derivatives
with chlorosulfonic acid produces reactive sulfonyl chloride
derivatives which may be coupled to biological entities, such as
antigens, antibodies or antibody fragments, receptors,
intracellular organelles, proteins, such as avidin and
streptavidin, enzyme substrates, membrane probes, nucleotides and
derivatives thereof, nucleic acid probes, and nucleic acids. The
hydrolysis of the sulfonyl chloride to the sulfonic acid provides
the water soluble analogs. The absorbance spectra of the sulfonated
tetraphenoxy aluminum phthalocyanines, 4 and 5, in water are shown
in FIG. 6. The emission spectra are shown in FIG. 7. The syntheses
of 4 and 5 and a tabulation of their spectral properties are given
in Example 2.
The second pair of fluorophores are tetrathiophenoxy substituted
aluminum phthalocyanines. As above, 4-thiophenoxyphthalonitrile
provides the 2,9,16,23 substituted phthalocyanine, while
3-thiophenoxyphthalonitrile gives the 1,8,15,22 substituted isomer.
After the incorporation of aluminum, treatment of these derivatives
with chlorosulfonic acid yields a reactive form useful in coupling
to biological entities. Hydrolysis produces sulfonates that are
highly water soluble. The absorbance spectra of the sulfonated
tetrathiophenoxy aluminum phthalocyanines, 6 and 7, in water are
shown in FIG. 8. The emission spectra are presented in FIG. 9. The
syntheses of 6 and 7 and a tabulation of their spectral properties
are given in Example 2. These compounds may be attached to the
above-mentioned reactive or activatable R.sub.2 groups to yield
conjugates that may be used for a variety of purposes, including
sequencing of DNA.
Oxygen and Sulfur Substituted Aluminum Tetrabenztriazaporphyrin
Sulfonates
In an alternative preferred embodiment, a second family of four
novel fluorophores derived from the tetrabenztriazaporphyrin
(TBTAP) system is presented. These fluorophores differ from
phthalocyanines 4-7 above only in position 20 of the ring system.
For the phthalocyanines, position 20 is a nitrogen atom, while for
the tetrabenztriazaporphyrins, position 20 is a substituted carbon
(in formula I, Z is N for phthalocyanines, Z is CR for the
tetrabenztriazaporphyrins). In this embodiment, the 20 carbon is
phenyl substituted.
As in the most preferred embodiment, the family of four aluminum
TBTAP fluorophores consists of two pairs of oxygen and sulfur
positional isomers. Reaction of benzylmagnesium bromide with each
of the four phthalonitriles, 4-phenoxyphthalonitrile,
3-phenoxyphthalonitrile, 4-thiophenoxyphthalonitrile, and
3-thiophenoxyphthalonitrile provides TBTAP ring systems which are
metalated with aluminum and sulfonated to provide compounds 8-11,
respectively. The preparation of the aluminum 20-phenyl
tetrabenztriazaporphyrins and the tabulation of their spectral
properties are given in Example 3.
The emission spectra of the aluminum tetraphenoxy TBTAP derivatives
are shown in FIG. 10. Similarly, emission spectra of the
tetrathiophenyl derivatives are shown in FIG. 11. As with the
aluminum phthalocyanines, the TBTAP sulfur analogs are red-shifted
relative to the oxygen counterparts, and the 1,8,15,22 isomers are
red-shifted compared to the 2,9,6,23 isomers.
Aluminum Tetrabenztriazaporphyrin Sulfonates
Two novel, water soluble aluminum tetrabenztriazaporhphyrins are
also described. These compounds are derived from phthalonitrile and
are therefore unsubstituted. Reaction of methylmagnesium bromide
with phthalonitrile and subsequent aluminum incorporation produced
aluminum 20-H TBTAP. Similar reaction of phthalonitrile with
benzylmagnesium bromide followed by the incorporation of aluminum
gave aluminum 20-phenyl TBTAP. Both of these derivatives were
rendered reactive to reactive groups on biological entities (e.g.,
--OH, --NH.sub.2, --SH) by treatment with chlorosulfonic acid.
Hydrolysis of the reactive sulfonyl chloride provides the
corresponding sulfonates, aluminum 20-H TBTAP sulfonate, 12, and
aluminum 20-phenyl TBTAP sulfonate, 13. The absorbance and emission
spectra of 12 and 13 in water are presented in FIGS. 12 and 13,
respectively. The preparation and spectral summary are provided in
Example 4.
Aluminum Phthalocyanine Tetraquaternary Ammonium Derivative
Two novel, cationic phthalocyanines, 14a and 14b, are also
described. In addition to negatively charged, water soluble
aluminum phthalocyanine derivatives, positively charged derivatives
are presented. Unlike the aforementioned carboxylated and
sulfonated phthalocyanines, the trimethyl ammonium functionalized
phthalocyanines were found to be nonfluorescent in water despite
their great water solubility. Examination of the absorbance spectra
indicated a high degree of aggregation. We found, however, that
disaggregation of the cationic fluorophore was achieved in the
presence of an anionic surfactant (sodium dodecylsulfate, SDS;
typical concentration, about 0.01M). Accompanying the
disaggregation was a concomitant increase in the fluorescence
emission. Contacting a solution of the aggregate fluorophore with
RNA resulted in a similar fluorescent enhancement. The absorbance
and emission spectra of 14a and b in water and in the presence of
RNA are shown in FIGS. 14, 15, 16, and 17. The preparation of 14a
and b and RNA binding experiments are presented in Example 5.
In a second aspect of this invention, there are disclosed
derivatives of the phthalocyanines and tetrabenztriazaporphyrins in
which 1-4 of the benzo rings of formula I contains one or two N
atoms. When two N atoms are contained per benzo group or groups,
they will generally be in a pyrazine relationship (i.e. in the 1,4
positions of the benzo ring). While both phthalocyanine and
tetrabenztriazaporphyrin pyridine/pyrazine derivatives are
contemplated, Z in formula I is preferably N. R.sub.2 may be
attached to a meso carbon, in which case the benzo rings of the
macrocycle will each have an R.sub.1 group attached thereto.
Spectral Properties of Phthalocyanines,
Tetrabenztriazaporphyrins and pyrazine and Pyridine Derivatives
Thereof
The emission wavelength (685 nm) of the trisulfonate derivative of
aluminum phthalocyanine, 1, elicited by excitation at 350 nm, is
red-shifted from the emissions of endogenous fluorophores in
physiological solutions. The red emission wavelength of 1 is one of
the greatest advantages of this fluorophore. Since emission is
shifted away from that of endogenous fluorescence (400-600 nm),
background is reduced. Reduction of background leads to a higher
signal-to-background ratio and greater sensitivity. This advantage
may be realized regardless of where excitation is effected so long
as there is absorbance at the excitation wavelength. Excitation of
1 at 325 nm (helium cadmium laser), around 350 nm (Hg lamp source
or argon ion laser), 633 nm (helium neon laser), 647 nm (krypton
ion laser), or 670 nm (diode laser) leads to emission at 685
nm.
Excitation of 1 at 325 nm or approximately 350 nm leads to emission
with more than a 300 nm Stokes' shift. This Stokes' shift can lead
to further reduction in background and greater sensitivity.
Fluorescence measurements indicate that aluminum phthalocyanine
trisulfonate 1 is detectable at concentrations as low as 10.sup.-15
M. Linear dynamic range studies indicate a working range of over
nine decades and superior detection limits when compared to
fluorescein and rhodamine B. Red emission of 1 coupled with the
advantage of a large Stokes' shift leads to a 100-fold increase in
signal-to-background relative to that of fluorescein.
The application of these reagents to simultaneous, multicomponent
fluorescence analysis such as nucleic acid sequence analysis, flow
cytometry, immunoassays, or nucleic acid probe assays requires the
formation of a family of derivatives. These derivatives must be
water soluble, have common excitation wavelengths yet emit at
different wavelengths. In addition, the emission bandwidths of each
derivative must be narrow (full width at half maximum (FWHM)<40
nm) and resolvable from other members of the family.
Aluminum phthalocyanine (AlPc) based fluorophores in particular
have several advantages over dyes currently used for all of these
applications.
First, emission spectra of AlPc derivatives suffer less background
interference. Interferences attributable to Rayleigh, Tyndall or
Raman scatter can be reduced by more than 100 fold due to the large
Stokes' shift (>about 300 nm) and long wavelength emission
properties of phthalocyanines. Aluminum phthalocyanines emit in the
red (>680 nm) at wavelengths beyond endogenous fluorescence
(400-600 nm). By contrast, the fluorescein and rhodamine
derivatives currently marketed for nucleic acid sequence analysis,
flow cytometry, immunoassay and nucleic acid probe assays have only
20-40 nm Stokes' shifts and emit at wavelengths less than 550
nm.
Second, aluminum phthalocyanine based fluorophores have greater
separation between emission wavelength maxima. The range of
emission maxima for known fluorescein families is only 21 nm with a
typical separation of 6 nm between each dye. In contrast, the
phthalocyanine family spans about 50 nm with an average separation
between family members of greater than 15 nm.
Third, aluminum phthalocyanine based fluorophores have sharp
emission bands. The full width at half maximum for fluorescein
based dyes ranges from about 32-37 nm with significant red tailing.
By comparison, phthalocyanine based fluorophores have bandwidths
from about 21-30 nm (with the exception of 7, FWHM=39 nm) with
little red tailing.
In summary, all of these properties make aluminum phthalocyanine
based fluorophores ideal candidates for multicomponent analysis
with application to nucleic acid sequence analysis, flow cytometry,
immunoassays, and nucleic acid probe assays. Generally, to realize
this potential, the aluminum phthalocyanine based fluorophores must
be monomerically tethered.
The emission spectra of four aluminum phthalocyanine sulfonates (1,
4, 5, 7) selected for nucleic acid sequence analysis are presented
in FIG. 18.
As noted above, fluorescence emission of phthalocyanines and TBTAPs
may be enhanced by rendering the fluorophores monomeric rather than
aggregated. The degree of monomerism of a metallophthalocyanine or
TBTAP in aqueous solution is a function of the metal. Divalent
metals which cannot bear axial ligands tend to stack and exhibit
reduced monomerism. Trivalent and greater metals are less prone
toward aggregation due to axial ligation and are therefore more
fluorescent in solution. The most preferred metals for fluorescent
reagents are therefore aluminum, gallium, scandium, silicon,
germanium, and tin.
Metallophthalocyanines and TBTAPs suitable for magnetic resonance
imaging applications would bear paramagnetic metals such as iron,
manganese, and gadolinium. Here the metals are in the plus three
oxidation state.
Metallophthalocyanines and TBTAPs suitable for radioactive imaging
and therapeutic applications would bear radioisotopes of metals
such as copper, cobalt, gallium, and technetium. The radionuclides
are gamma-emitters and are sensitive imaging probes.
In connection with U.S. Ser. No. 366,971, filed Jun. 14, 1989, we
discovered an empirical relationship between the spectroscopic
properties (in terms of the relative heights of the maximum blue
and red absorbance peaks) of these compounds to their relative
quantum yield.
Early in our investigations of aluminum phthalocyanine sulfonates
we observed that the blue absorbance was independent of the state
of aggregation and hence the emission yield. In contrast, it was
possible to follow the onset of aggregation by changes in the red
absorption band. In general, we found that the A(red)/A(blue) ratio
decreases with decreasing relative quantum yield. In addition, the
behavior of the protein-bound dye is shifted toward a lower
relative quantum yield, but very nicely paralleled the free dye in
solution. This shift or decrease in quantum yield presumably arises
from the hydrophobic nature of the protein environment rather than
aggregation quenching.
The most preferred embodiments of the phythalocyanine conjugates of
the invention, in terms of monomeric binding, have an
A(red)/A(blue).gtoreq.2. Such conjugates are readily prepared by
Method 3 (see below).
Preferably, the A(red)/A(blue) ratio of the subject conjugates
should be .gtoreq.1.75, and such conjugates are readily prepared by
Method 2.
Phthalocyanine conjugates having A(red)/A(blue) ratios between
about 1.5 and 1.75, while suitable for some purposes, have
relatively limited sensitivity and so would not be useful.
Conjugates having A(red)/A(blue) ratios of less than 1 are
considered to be not suitable for use as fluorescent markers.
The phthalocyanine and tetrabenztriazaporphyrin conjugates of this
invention display similar tendencies in terms of their monomeric
binding and its relation to the A(red)/A(blue) ratio. However, some
of the species disclosed in this invention have much stronger blue
absorbances, e.g., compound 4, while others show diminished red
absorbance, e.g., compound 12, as monomers in aqueous environments.
As a result, the most preferred methods of conjugation yield a
range of A(red)/A(blue) from 1.4 to 2.0, depending on the
fluorophore.
Exemplary methods for preparing monomeric conjugates are provided
below. While these methods are illustrated with aluminum
phthalocyanine, it is to be understood that these methods may be
applied to other phthalocyanines and to tetrabenztriazaporphyrins,
which are disclosed herein. Example 6 describes the coupling of the
reactive forms of the red-shifted aluminum phthalocyanines to
streptavidin.
Phthalocyanine Metalation
1: In the first method, aluminum phthalocyanine may be coupled to a
large molecule by a tether linker. The tether linker may be any
small bifunctional organic molecule. The tether linker may be 2 to
12 atoms in length. Preferably, the tether linker is 7 to 12 atoms
in length and sterically hindered. A long sterically hindered
tether ensures that aluminum phthalocyanine is displaced from the
biological entity and that individual aluminum phthalocyanine
moieties on the large molecule are displaced from one another. The
tether linker method may be utilized in conjunction with Methods 2
and 3.
Method 2: Aluminum phthalocyanine may be coupled to large molecules
with the use of an aqueous solvent containing a disaggregated
organic such as DMF. Use of the disaggregant helps to ensure that
aluminum phthalocyanine is bound in a monomeric rather than
aggregated state.
Method 3: In a third method, aluminum phthalocyanine may be coupled
to large molecules by preincubation of the fluorophore in a
disaggregating medium followed by coupling of the fluorophore to a
large molecule in an aqueous solvent containing a disaggregating
organic solvent such as DMF. The preincubation is preferably
performed by mixing a reactive derivative of aluminum
phthalocyanine with dimethylformamide for one hour at 30.degree. C.
prior to conjugation in a disaggregating medium. The preincubation
of fluorophore in a disaggregating organic solvent (e.g., DMF)
prior to conjugation in a disaggregating medium is the first
disclosure of such a method for generating monomeric conjugates
with any fluorescent species including phthalocyanines and
porphyrins.
In a third aspect of this invention, there are disclosed cationic
phthalocyanine and tetrabenztriazaporphyrin derivatives having
formula I, except that R.sub.2 =XYW, R.sub.1, X, and Y being as
described above and W=--N.sup.+ D.sub.1 D.sub.2 D.sub.3, wherein
D.sub.1 -D.sub.3 are independently H, alkyl, aralkyl or aryl, or W
may be a pyridine group. D.sub.1 -D.sub.3 are preferably H, C.sub.1
-C.sub.10 alkyl, C.sub.6-12 aralkyl or C.sub.6-12 aryl. The
counterion of these compounds may be any one that is stable and
synthetically accessible, and that does not interfere with water
solubility or desirable spectral properties. Exemplary negative
counterions are I.sup.-, Br.sup.-, Cl.sup.-, F.sup.-, borate etc.
Exemplary positive counterions are Ca.sup.+2, Mg.sup.+2, Na.sup.+,
K.sup.+, quaternary ammonium, etc.
These compounds may be used to detect DNA and RNA, generally by
nonspecific binding to the DNA or RNA. Fluorescent detection of the
compound bound to the DNA or RNA may then be carried out by
standard fluorescent measurement components.
Uses of the Disclosed Reagents
In general, the reagents of the first and second aspects of the
present invention may be used in combination with binding partners
(or ligands) capable of specifically binding with a target
substance, particularly an analyte. Once the binding partner
specifically binds to an analyte or target of interest, the reagent
(referred to as a reporter group in this context) is detected by
fluorescence measurement and the presence of and/or amount of the
analyte can be determined. The reporter group may be covalently or
noncovalently bound to the binding partner and may be attached
either prior to or after the analyte and binding partner are caused
to interact and bind.
In one embodiment, the reporter group is covalently linked to the
binding partner before the binding partner and the analyte are
caused to interact and bind.
In another embodiment, the binding partner is caused to interact
and bind with the analyte and after binding the reporter group is
covalently or noncovalently attached to the binding partner. For
example, the binding partner may be conjugated with biotin moieties
and the reporter groups may be attached to avidin or streptavidin.
Other specific binding pairs may also be used to join the binding
partner and the reporter group.
As the binding partner/analyte pairs, the following are
representative, preferred embodiments:
nucleic acid probe or primer (e.g., DNA or RNA having 5-10,000
nucleic acid
bases)/complementary target DNA or RNA
enzyme/substrate
antibody/antigen (free or bound to other structures, such as a
cell)
DNA or protein-binding protein/DNA or protein
lectin/carbohydrate
ligand/ligand binding protein
In the above examples, the precise nature of the binding partner
and analyte is relatively unimportant. All that is required is that
the binding partner and analyte be capable of specific binding to
each other and that a reagent as described herein be attachable to
the binding partner, either before or after binding to the analyte
and either covalently or via a second specific binding pair, e.g.,
a tightly binding pair such as avidin:biotin, streptavidin:biotin,
and maltose binding protein:maltose.
In another preferred embodiment, more than one analyte is
determined simultaneously using a corresponding number of binding
partners each attached to a different reagent according to the
present invention for detection. The different reagents are
required to have substantially nonoverlapping emission spectra for
separate detection. The combinations of different reagents used in
a particular assay may all be of the same general type (e.g.
phthalocyanines or TBTAPs) or mixtures of reagent types (e.g.
phthalocyanines and TBTAPs). The fluorescence maxima must occur at
different wavelengths, preferably separated by at least about 7
nm.
For simultaneous use of fluorescent reagents, the fluorophores must
be readily distinguishable for quantitation or quantifiable by
ratioing methods.
For Sanger DNA sequencing, a sequencing primer is modified with an
amino group at the 5' terminus or each of the four
dideoxynucleotides is labeled with one of each of four fluorescent
reagents.
For flow cytometry, cell surface antigens expressed by certain
subsets of cells may be labeled either directly or indirectly with
a fluorescent reagent and antibody or antibody fragment. The number
of cell subsets that may be labeled and quantitated is determined
by the number of unique fluorescent labels employed.
For immunoassay, each of any number of fluorescent reagents may be
attached to a different antigen, antibody, or antibody fragment.
For example, a simultaneous thyroid immunoassay test panel may be
performed by labeling triiodothyronine (T3) with one fluorescent
reagent, thyroxine (T4) with a second fluorescent reagent, and
anti-thyroid stimulating hormone (anti-TSH) with a third
fluorescent reagent.
For probe assays, any number of fluorescent reagents may be
attached to a different nucleic acid probe to perform simultaneous
probe analysis. The number of probes that may be detected as the
result of a single hybridization step is determined by the number
of fluorescent reagents utilized.
In a preferred embodiment, the reagents of the first or second
aspects are used to sequence nucleic acid molecules or fragments.
The most common approach for DNA sequence analysis is the Sanger
dideoxynucleotide sequencing method. For single lane gel DNA
sequence analysis, a family of four aluminum phthalocyanine
derivatives is required. The derivatives may be used to label
either sequencing primers or each of the four dideoxynucleotides
(ddNTP's). Surprisingly, although related compounds are known to
generate singlet oxygen which can degrade DNA, the present
compounds may be effectively used to sequence DNA without
degradation.
In the labeled primer strategy, a single primer is labeled with
each of four different fluorescent labels. Four separate Sanger
sequencing reactions are performed with one of each of the labeled
primers, template, sequencing enzyme, deoxynucleotides (dNTP's),
and one of each of the four ddNTP's. Once extension and termination
are complete, the four reactions are pooled and loaded onto a
single lane of sequencing gel. Since each extended primer is
terminated with one of the four ddNTP's and labeled with one of the
four dyes, the base sequence may be determined by scanning the
fluorescence emission directly off the gel.
Alternatively, one may use labeled chain terminators such as
dideoxynucleotides rather than labeled primers. Using this
approach, all four of the sequencing reactions may be performed in
a single vessel and then loaded onto a single lane of the
sequencing gel.
The macrocycles involved in the present reagents are larger than
the corresponding fluorescein or rhodamine reagents previously used
for sequencing and are relatively more planar. As a result, it was
unpredictable whether the fluorophore labled primer of this
invention would be compatible with the sequencing enzyme.
Researchers at corporations that develop sequencing fluorophores
predicted trouble with both sequencing enzyme compatibility and
electrophoretic mobility of the sequencing primer and fragments.
Empirically, the fluorophore labeled primer was found to be
compatible with the sequencing enzyme and the electrophoretic
mobility of the dye labeled primer and sequencing fragments is not
significantly different from that of the amino modified primer or
sequencing fragments.
All of the phthalocyanine labeled primers and the 20H and 20 Ph
TBTAP labeled primers have been found to have similar
electrophoretic mobility. This was an unexpected result, especially
considering that various fluorescein and rhodamine labeled primers
have significantly different mobilities. Uniform mobility of
primers suggests uniform mobility of fragments. This greatly
simplifies the sequencing procedure and analysis, as complex
empirical correction factors and equations will not have to be used
as extensively or at all.
The present invention also provides kits containing reagents as
disclosed herein for performing assays for analytes, for DNA/RNA
staining, for DNA sequencing, etc. The kits will generally contain
one or more containers of reagents of the present invention, and
may contain other chemicals, controls, etc., as may be necessary or
desirable. For example, for DNA sequencing kits, there will
preferably be four containers of chain terminating
dideoxynucleotides conjugated to phthalocyanine or
tetrabenztriazaporphyrin moieties, as disclosed herein, additional
containers of deoxynucleotides, especially dATP, dTTP, dGTP, and
dCTP, a container of a DNA polymerase, a container of template DNA,
and a container of a primer DNA. The labeled chain terminating
dideoxynucleotides are selected so that their fluorescence emission
spectra are distinguishable, i.e. substantially non-overlapping. By
"substantially non-overlapping" is meant that the emission spectra
have wavelengths of maximum emission that are separated by at least
about 7 nm, preferably at least about 10-20 nm.
An alternative DNA sequencing kit may have a container of
fluorophore-labeled primer (a reagent of the present invention),
containers of deoxynucleotides, e.g., dATP, dTTP, dGTP, dCTP;
containers of chain terminators, e.g., ddATP, ddTTP, ddGTP, ddCTP,
ddUTP; and a container of a DNA polymerase.
For simultaneous detection of more than one cell type or different
markers on different cell subsets using flow cytometry, two or more
reagents with maximum spectral resolution are required. Use of at
least two different fluorophores having nonoverlapping emission
maxima allows the user to perform two color analyses. Two or more
color analyses are generally effected by labeling subsets of cells
using antibodies specific for each cell type, either indirectly
(e.g., via intervening biotin:avidin binding) or directly (i.e.,
covalently) attached to a fluorescent reagent or dye as disclosed
herein.
AIDS testing may be performed by simultaneous analysis of two T
cell subsets within a sample of peripheral blood containing
lymphocytes. A ratio of T-Helper cells (one color) to T-Suppressor
cells (the second color) of other than 2:1 is an indicator of AIDS
infection. In conjunction with clinical symptomology, this two
color analysis is used for AIDS diagnosis. See Example 16.
Multicomponent immunoassay allows for the simultaneous detection of
more than one analyte. Cost and time considerations make this a
preferred method for many clinical applications. A single patient
sample may be used for detection of a panel of therapeutic drugs,
abused drugs, infectious disease agents, hormones or any
combination thereof if each of the analytes or antibodies specific
for each of the analytes is labeled with a different fluorescent
dye.
Multicomponent probe assays enable detection of infectious disease
agents, cancers and genetic abnormalities. Since there are probe
libraries available for detection of many agents and abnormalities,
cue would like to have as many fluorophores that may be excited
with common wavelengths as possible. In this application, each
probe specific for regions of chromosomes associated with disease
agents, cancers, or genetic abnormalities (leading to birth defects
or genetic diseases) is labeled with a different fluorophore. The
cancers treatable or detectable by the present reagents are not
necessarily limited and any one for which a therapeutic or
diagnostic agent has been developed may potentially be treated or
diagnosed using the appropriate fluorophores described herein.
The reagents disclosed herein, particularly those of the first and
second aspects, may also be used for photodynamic therapy employing
standard methods. See Example 15.
The following Examples are presented to illustrate the advantages
of the present invention and to assist one of ordinary skill in
making and using the same. The following Examples are not intended
in any way to otherwise limit the scope of the disclosure or the
protection granted by Letters Patent hereon.
EXAMPLE 1
The Preparation of Aluminum Phthalocyanine Tetraglycolates
Tetrasubstituted phthalocyanines derived from monosubstituted
phthalonitriles are necessarily an inseparable mixture of four
isomeric products. The product phthalocyanines arise from the
differences in orientation of the phthalonitrile during the
cyclization process. Cyclization of a 4-substituted phthalonitrile
leads to the formation of 2,9,16,23-tetrasubstituted
phthalocyanine, as well as three other tetrasubstituted isomers,
namely, 2,9,16,24; 2,10,16,24; and 2,9,17,24. Similarly, the
cyclization of a 3-substituted phthalonitrile provides the
corresponding 1,8,15,22-tetrasubstituted phthalocyanine along with
three other tetrasubstituted derivatives, 1,8,15,25; 1,11,15,25;
1,8,18,25. Recognizing this, we have for simplicity designated
tetrasubstituted phthalocyanines derived from 3-substituted
phthalonitriles as 1,8,15,22 and phthalocyanines derived from
4-substituted phthalonitriles as 2,9,16,23. See FIG. 1 for
macrocycle position numbering.
Tetrasubstituted aluminum phthalocyanines may be prepared from
monosubstituted phthalonitriles. Nitro displacement from either 3-
or 4-nitrophthalonitrile with oxygen or sulfur nucleophiles provide
the corresponding phthalonitriles in good yield. The oxygen or
sulfur reagent used in the nitro displacement may impart to the
phthalocyanine water solubility and tetherability, or may be
further elaborated to provide these required properties. Reagents
such as hydroxyacetic acid and thioacetic acid may provide
appropriately functionalized phthalonitriles directly (X is O or S,
Y is CH.sub.2, and W is CO.sub.2 H). Alternatively, tetraoxy or
tetrathio substituted phthalocyanines may be treated with an
alkylating agent such as methyl bromoacetate to provide the fully
functionalized phthalocyanine.
The Preparation of Aluminum Phthalocyanine 2,9,16,23-Tetraglycolic
Acid (2)
Treatment of 4-nitrophthalonitrile with neopentyl alcohol and
potassium carbonate in dimethylformamide gave
4-neopentoxyphthalonitrile in 90% yield. The metal free
2,9,16,23-tetraneopentoxyphthalocyanine was formed in 40% yield
from the corresponding diiminoisoindoline upon reaction of the
phthalonitrile with ammonia in methanol followed by reflux in
N,N-dimethylaminoethanol. Leznoff, C. C. at al., Can. J. Chem.
63:623-631, 1985.
The metalation of 2,9,16,23-tetraneopentoxyphthalocyanine was
accomplished by treatment with ten molar equivalents of trimethyl
aluminum in methylene chloride. Smooth conversion occurs at room
temperature in eight hours. The product was isolated after an
acidic aqueous extractive workup to yield aluminum hydroxy
2,9,16,23-tetraneopentoxyphthalocyanine in essentially quantitative
yield.
Cleavage of the neopentyl group is accomplished upon reaction with
boron tribromide in benzene as generally disclosed by Rosenthal,
I., et al., Photochem. Photobiol. 46(6):959-963, 1987. The cleavage
product, aluminum hydroxy 2,9,16,23-tetrahydroxyphthalocyanine, is
a versatile intermediate which may be treated with a variety of
alkylating agents to provide a family of tetraalkoxy substituted
phthalocyanines.
Alkylation of the tetrahydroxy derivative with methyl bromoacetate
and potassium carbonate (forty molar equivalents of each) in
refluxing methanol affords the tetra methyl ester derivative. The
alkylated product may be directly hydrolyzed to the tetracarboxylic
acid by heating in a solution of 0.5M methanolic potassium
hydroxide. Aluminum hydroxy 2,9,16,23-tetraglycolylphthalocyanine
was isolated by precipitation, 2, from an aqueous acid
solution.
The Preparation of Aluminum Phthalocyanine 1,8,15,22-Tetraglycolic
Acid (3)
The synthesis of aluminum hydroxy
1,8,15,22-tetraglycolylphthalocyanine, 3, was analogous to that
described above for 2.
The absorbance and emission spectra in water are shown in FIGS. 3
and 4, respectively. The effect of cetyl trimethlyammonium bromide
(CTAB) on the emission spectra of the two isomers is shown in FIG.
5.
Tabulated below is a comparison of the spectral data for 1, 2, and
3 in water.
______________________________________ Phthalocyanine Absorbance
Emission Quantum Yield ______________________________________ 1 673
nm 683 0.60 2 692 704 0.55 3 720 727 0.43
______________________________________
EXAMPLE 2
The Preparation of Oxygen and Sulfur Substituted Aluminum
Phthalocyanine Sulfonates
Tetrasubstituted oxygen and sulfur substituted aluminum
phthalocyanine sulfonates are described in Example 2. The four
tetrasubstituted reagents of Example 2 are prepared from
monosubstituted phthalonitriles. The following is a detailed
description of the preparation of a family of four aluminum
phthalocyanine based reagents. The presentation is organized into
sections which detail phthalocyanine preparation, phthalocyanine
metalation, reactive phthalocyanine formation, and water soluble
phthalocyanine formation. Within each section a detailed procedure
is given for one member of the family of four reagents followed by
a comment on the procedures for the other three reagents. Any
differences in procedure are highlighted.
Phthalocyanine Preparation
2,9,16,23-Tetraphenoxyphthalocyanine
To 1.0 g (4.55 mm) 4-phenoxyphthalonitrile in 10 mL
3-methyl-1-butanol was added 5 mL lithium 3-methyl-1-butanoxide
(prepared by the dissolution of 10 mg lithium metal in 5 mL of the
alcohol). The resulting solution was heated at reflux under
nitrogen for six hours. The solvent was removed in vacuo and the
crude product was taken up in 50 mL methylene chloride. The
solution was washed with 3-50 mL portions 1N aqueous hydrochloric
acid, dried over sodium sulfate, filtered and concentrated. The
product was then redissolved in 10 mL methylene chloride and
precipitated by the addition of 100 mL methanol. The product was
collected by filtration, washed with 500 mL methanol, and dried in
vacuo. The product, 0.53 g (0.60 mm, 52%), was isolated as a blue
powder. The spectral properties are tabulated below.
1,8,15,22-Tetraphenoxyphthalocyanine
In a procedure analogous to that described above,
3-phenoxyphthalonitrile produced
1,8,15,22-tetraphenoxyphthalocyanine in 45% yield. The spectral
properties are tabulated below.
2,9,16,23-Tetrathiophenylphthalocyanine
In a procedure analogous to that described above,
4-thiophenylphthalonitrile produced
2,9,16,23-tetrathiophenylphthalocyanine in 51% yield. The spectral
properties are tabulated below.
1,8,15,22-Tetrathiophenylphthalocyanine
In a procedure analogous to that described above,
3-thiophenylphthalonitrile produced
1,8,15,22-tetrathiophenylphthalocyanine in 87% yield. In this case,
the product was isolated by precipitation from methylene chloride
without the addition of methanol. The spectral properties are
tabulated below.
The following table summarizes the absorbance and emission
wavelengths for the metal free phthalocyanines prepared as
described above. The spectra were recorded as methylene chloride
solutions.
______________________________________ Phthalocyanine Absorbance
Emission Quantum Yield ______________________________________
2,9,16,23 oxy 700 nm 705 nm 0.25 1,8,15,22 oxy 716 723 0.29
2,9,16,23 thio 711 719 0.40 1,8,15,22 thio 723 738 0.26
______________________________________
Trimethylaluminum Metalation Method
Aluminum Hydroxy 2,9,16,23-Tetraphenoxyphthalocyanine
To a solution of 500 mg (0.60 mm)
2,9,16,23-tetraphenoxyphthalocyanine in 200 mL dry methylene
chloride under nitrogen at room temperature was added dropwise ten
equivalents, 3.0 mL (6.0 mm) of a 2.0M solution of
trimethylaluminum in toluene. The reaction mixture was stirred at
room temperature for 24 hours and then quenched by the careful
addition of 10 mL distilled water followed by 1 mL 1N aqueous
hydrochloric acid. The solution was then separated and the organic
layer was washed with 3-20 mL portions 1N aqueous hydrochloric
acid. The methylene chloride solution was dried over sodium sulfate
and concentrated to dryness. The product, aluminum hydroxy
2,9,16,23-tetraphenoxyphthalocyanine, was isolated as a blue solid,
230 mg (0.25 mm, 41%). The spectral properties are tabulated
below.
Aluminum Hydroxy 1,8,15,22-Tetraphenoxyphthalocyanine
In a procedure analogous to that described above, aluminum hydroxy
1,8,15,22-tetraphenoxyphthalocyanine was prepared and its spectral
data tabulated below.
Aluminum Hydroxy 2,9,16,23-Tetrathiophenylphthalocyanine
In a procedure analogous to that described above, aluminum hydroxy
2,9,16,23-tetrathiophenylphthalocyanine was prepared and its
spectral data tabulated below.
Aluminum Hydroxy 1,8,15,22-Tetrathiophenylphthalocyanine
In a procedure analogous to that described above, aluminum hydroxy
1,8,15,22-tetrathiophenylphthalocyanine was prepared and its
spectral data tabulated below.
The table below summarizes the absorbance and emission wavelengths
and relative quantum yields of the aluminum phthalocyanines
prepared as described above. The spectra were recorded in
dimethylformamide.
______________________________________ Phthalocyanine Absorbance
Emission Quantum Yield ______________________________________
2,9,16,23 oxy 680 nm 686 nm 0.51 1,8,15,22 oxy 701 703 0.25
2,9,16,23 thio 687 696 0.46 1,8,15,22 thio 713 722 0.30
______________________________________
Aluminum Triacetylacetonate Metalation Method
Aluminum Acetylacetonate 2,9,16,23-Tetraphenoxyphthalocyanine
To a solution of 2.5 g (2.8 mm)
2,9,16,23-tetraphenoxyphthalocyanine in 50 mL dimethylformamide was
added ten equivalents, 9.0 g (28.0 mm) aluminum acetylacetonate.
After stirring at room temperature for one hour the solution was
diluted with 500 mL methanol and the crude product was collected by
filtration, washed with 500 mL methanol and dried in vacuo.
Aluminum acetylacetonate 2,9,16,23-tetraphenoxyphthalocyanine, 1.7
g (1.84 mm, 66%), was isolated as a blue powder. The spectral data
is tabulated below.
Aluminum Acetylacetonate 1,8,15,22-Tetraphenoxyphthalocyanine
In a procedure analogous to that described above, aluminum
acetylacetonate 1,8,15,22-tetraphenoxyphthalocyanine was prepared
in 59% yield. The spectral data are tabulated below.
______________________________________ Phthalocyanine Absorbance
Emission Quantum Yield ______________________________________
2,9,16,23 680 nm 686 nm 0.27 1,8,15,22 697 701 0.20
______________________________________
Reactive Phthalocyanine Formation
Aluminum Hydroxy 2,9,16,23-Tetraphenoxyphthalocyanine Sulfonyl
Chloride
To 96 mg (0.104 mm) aluminum hydroxy
2,9,16,23-tetraphenoxyphthalocyanine was added 1.0 mL
chlorosulfonic acid. The mixture was stirred to effect dissolution,
sealed under argon, and immersed in a pre-equilibrated oil bath at
100.degree. C. The solution was stirred at 100.degree. C. for one
hour, cooled to 0.degree. C., and quenched by the gradual addition
of the crude reaction mixture to 10 g of ice. The solid product was
collected by filtration, washed with 2-20 mL portions of distilled
water and 2-20 mL portions diethyl ether. The solid was then
transferred to a flask and pulverized to a fine solid in 20 mL
diethyl ether, collected by filtration, washed with 2-20 mL
portions diethyl ether, and dried under vacuum. Aluminum hydroxy
2,9,16,23-tetraphenoxyphthalocyanine sulfonyl chloride was isolated
in 89% yield. Spectral data are tabulated below.
Aluminum Hydroxy 1,8,15,22-Tetraphenoxyphthalocyanine Sulfonyl
Chloride
In a procedure analogous to that described above except with a
reaction temperature of 70.degree. C., aluminum hydroxy
1,8,15,22-tetraphenoxyphthalocyanine sulfonyl chloride was isolated
in 54%. Spectral data are tabulated below.
Aluminum Hydroxy 2,9,16,23-Tetrathiophenylphthalocyanine Sulfonyl
Chloride
In a procedure analogous to that described above with a reaction
temperature of 100.degree. C., aluminum hydroxy
2,9,16,23-tetrathiophenylphthalocyanine sulfonyl chloride was
isolated in quantitative yield. Spectral data are tabulated
below.
Aluminum Hydroxy 1,8,15,22-Tetrathiophenylphthalocyanine Sulfonyl
Chloride
In a procedure analogous to that described above except with a
reaction temperature of 80.degree. C., aluminum hydroxy
1,8,15,22-tetrathiophenylphthalocyanine sulfonyl chloride was
isolated in 73% yield. Spectral data are tabulated below.
The table below summarizes the maximum absorbance and emission
wavelengths for the reactive sulfonyl chloride derivatives in
dimethylformamide solution prepared as described above.
______________________________________ Phthalocyanine Absorbance
Emission Quantum Yield ______________________________________
2,9,16,23 oxy 684 nm 693 nm 0.41 1,8,15,22 oxy 704 708 0.14
2,9,16,23 thio 697 703 0.38 1,8,15,22 thio 715 724 0.17
______________________________________
Water Soluble Phthalocyanine Formation
Aluminum Hydroxy 2,9,16,23-Tetraphenoxyphthalocyanine Sulfonate
(4)
A solution of 10 mg of aluminum hydroxy
2,9,16,23-tetraphenoxyphthalocyanine sulfonyl chloride in 10 mL
distilled water was stirred vigorously at room temperature for 48
hours. The resulting solution was concentrated to dryness to yield
aluminum hydroxy 2,9,16,23-tetraphenoxyphthalocyanine sulfonate, 4,
in quantitative yield. The absorbance and emission spectra of 4 in
water are presented in FIGS. 6 and 7, respectively. Spectral data
are tabulated below.
Aluminum Hydroxy 1,8,15,22-Tetraphenoxyphthalocyanine Sulfonate
(5)
In a procedure analogous to that described above, aluminum hydroxy
1,8,15,22-tetraphenoxyphthalocyanine sulfonate, 5, was isolated in
quantitative yield. The absorbance and emission spectra of 5 in
water are presented in FIGS. 6 and 7, respectively. Spectral data
are tabulated below.
Aluminum Hydroxy 2,9,16,23-Tetrathiophenylphthalocyanine Sulfonate
(6)
In a procedure analogous to that described above, aluminum hydroxy
2,9,16,23-tetrathiophenylphthalocyanine sulfonate, 6, was isolated
in quantitative yield. The absorbance and emission spectra of 6 in
water are presented in FIGS. 8 and 9, respectively. Spectral data
are tabulated below.
Aluminum Hydroxy 1,8,15,22-Tetrathiophenylphthalocyanine Sulfonate
(7)
In a procedure analogous to that described above, aluminum hydroxy
1,8,15,22-tetrathiophenylphthalocyanine sulfonate, 7, was isolated
in quantitative yield. The absorbance and emission spectra of 7 in
water are presented in FIGS. 8 and 9, respectively. Spectral data
are tabulated below.
Tabulated below are the maximum absorbance and emission wavelengths
of the oxygen and sulfur substituted aluminum phthalocyanine
sulfonate derivatives in water prepared as described above.
______________________________________ Compound Quantum No.
Phthalocyanine Absorbance Emission Yield
______________________________________ 4 2,9,16,23 oxy 685 nm 697
nm 0.49 5 1,8,15,22 oxy 707 717 0.20 6 2,9,16,23 thio 695 708 0.28
7 1,7,15,22 thio 719 733 0.10
______________________________________
Aluminum Acetylacetonate Tetraphenoxyphthalocyanine Sulfonates.
Water soluble aluminum phthalocyanine sulfonates were prepared from
aluminum acetylacetonate 2,9,16,23- and
1,8,15,22-tetraphenoxyphthalocyanines as described above for the
corresponding axial hydroxy compounds. The absorbance and emission
wavelengths as well as quantum yields in water are tabulated
below.
______________________________________ Phthalocyanine Absorbance
Emission Quantum Yield ______________________________________
2,9,16,23 oxy 641 nm 688 nm 0.003 1,8,15,22 oxy 665 706 0.026
______________________________________
The spectral data summarized above for the acetylacetonate ligated
aluminum phthalocyanine sulfonates contrasts significantly with the
data for the corresponding hydroxylated derivatives. The
wavelengths of fluorescence emission of the acetylacetonates are
roughly 10 nm blue shifted relative to their hydroxy analogs. The
blue shift limits their utility in multicomponent analysis when
used in conjunction with the parent, aluminum phthalocyanine
sulfonate, which emits at 684 nm. The ideal family of fluorophores
for multicomponent analysis will have spectrally resolved emission
bands. The emission of the 2,9,16,23 isomer with axial
acetylacetonate at 688 nm is too close to the parent, 684 nm, to be
effectively resolved. More importantly, the fluorescent quantum
yields for the acetylacetonate derivatives are drastically reduced
to the point where their utility as fluorophores is greatly
impaired.
For the above reasons, the preferred embodiment of aluminum
phthalocyanine sulfonates employs axial hydroxy rather than
acetylacetonate ligands.
EXAMPLE 3
The Preparation of Oxygen and Sulfur Substituted Aluminum
Tetrabenztriazaporphyrins
Tetrasubstituted oxygen and sulfur substituted aluminum
tetrabenztriazaporphyrins are described in Example 3. The four
tetrasubstituted reagents of Example 3 are prepared from
monosubstituted phthalonitriles. The following is a detailed
description of the preparation of a family of four aluminum
tetrabenztriazaporphyrin based reagents. The presentation is
organized into sections which detail tetrabenztriazaporphyrin
preparation, metalation, reactive derivative formation, and
water-soluble derivative formation. Within each section a detailed
procedure is given followed by a comment on the procedures for the
other three reagents. Any differences in procedure are
highlighted.
Tetrabenztriazaporphyrin Preparation
20-Phenyl 2,9,16,23-Tetraphenoxytetrabenztriazaporphyrin
To a solution of 1.00 g (4.59 mm) 4-phenoxyphthalonitrile in 4 mL
dry tetrahydrofuran was added 10 mL dry diethyl ether. The mixture
was cooled to 0.degree. and 4.6 mL of 1.0M benzylmagnesium chloride
(4.6 mm, 1.0 equivalent) in diethyl ether was added. The mixture
was stirred under argon at room temperature for 2 hour. The mixture
was then concentrated to dryness and the purple residue was diluted
with 25 mL quinoline and stirred at 200.degree.-210.degree. for 4
hours. The solvent was distilled under vacuum. The resulting
residue was treated with stirring with 30 mL glacial acetic acid at
90.degree. for 2 hour. The reaction mixture was diluted with 200 mL
methylene chloride and washed first with 3-200 mL portions
saturated aqueous sodium bicarbonate and then with 200 mL 5% v/v
aqueous hydrochloric acid. The organic phase was dried over sodium
sulfate, filtered and concentrated. The crude reaction product was
chromatographed on silica gel eluting with chloroform. The
fractions containing the desired product were combined,
concentrated and twice more chromatographed on silica gel, eluting
with 85% chloroform in hexane to afford 204 mg (19%) 20-phenyl
2,9,16,23-tetraphenoxytetrabenztriazaporphyrin as a deep blue-green
solid. Silica thin layer chromatography eluting with 65% methylene
chloride in hexane gave a homogeneous product with an R.sub.f of
0.54. Spectral data are tabulated below.
20-Phenyl 1,8,15,22-Tetraphenoxytetrabenztriazaporphyrin
In a procedure analogous to that described above,
3-phenoxyphthalonitrile was converted to 20-phenyl
1,8,15,22-tetrabenztriazaporphyrin after heating in quinoline for
40 hours. The product was purified by chromatography on silica gel
eluting with methylene chloride followed by crystallization from a
methylene chloride: hexane (1:1) solution. The product was isolated
in 6% yield as a deep green solid with an R.sub.f of 0.60 on silica
eluting with methylene chloride. Spectral data are tabulated
below.
20-Phenyl 2,9,16,23-Tetrathiophenylbenztriazaporphyrin
In a procedure analogous to that described above for 20-phenyl
2,9,16,23-tetraphenoxytetrabenztriazaporphyrin,
4-thiophenylphthalonitrile was converted to 20-phenyl
2,9,16,23-tetrathiophenyltetrabenztriazaporphyrin after heating in
quinoline for 20 hours. After initial chromatography eluting with
chloroform, the fractions containing the desired product were
combined and twice rechromatographed on silica eluting with 55%
chloroform in hexane. The product was isolated in 21% yield as a
deep green solid with an R.sub.f of 0.72 on silica eluting with 65%
methylene chloride in hexane. Spectral data are tabulated
below.
20-Phenyl 1,8,15,22-Tetrathiophenyltetrabenztriazaporphyrin
In a procedure analogous to that described above for 20-phenyl
1,8,15,22-tetraphenoxytetrabenztriazaporphyrin,
3-thiophenylphthalonitrile was converted to 20-phenyl
1,8,15,22-tetrathiophenoxytetrabenztriazaporphyrin. After initial
chromatography on silica eluting with methylene chloride, the
product was twice more chromatographed eluting with 50% methylene
chloride in hexane. Further purification by crystallization from a
methylene chloride: hexane (1:1) solution afforded the product in
11% yield as a deep green solid. Silica thin layer chromatography
eluting with 65% methylene chloride in hexane gave an R.sub.f of
0.70. Spectral data are tabulated below.
Tabulated below are the absorbance data for the oxygen and sulfur
substituted 20-phenyl tetrabenztriazaporphyrin derivatives prepared
as described above. The spectra were recorded in methylene chloride
solution.
______________________________________ Tetrabenztriazaporphyrin
Absorbance ______________________________________ 2,9,16,23 oxy
656, 694 nm 1,8,15,22 oxy 676, 712 2,9,16,23 thio 666, 704
1,8,15,22 thio 695, 728 ______________________________________
Tetrabenztriazaporphyrin Metalation
Aluminum Hydroxy 20-Phenyl
2,9,16,23-Tetraphenoxytetrabenztriazaporphyrin
To a solution of 200 mg (0.209 mm) 20-phenyl
2,9,16,23-tetrabenztriazaporphyrin in 15 mL methylene chloride was
added 2.0 mL 2.0M trimethylaluminum (4.00 mm, 19 equivalents) in
toluene at 0.degree.. The mixture was stirred at room temperature
for two hours. The mixture was then cooled to 0.degree. and
carefully treated dropwise with 1 mL of distilled water. The
mixture was stirred for 10 minutes and treated dropwise with 2 mL
10% V/V aqueous hydrochloric acid. The reaction mixture was stirred
for 5 minutes, treated with 20 mL 10% V/V aqueous hydrochloric acid
and stirred for one hour. The mixture was diluted with 50 mL
methylene chloride and washed with 50 mL 5% V/V aqueous
hydrochloric acid. The organic phase was drived over sodium
sulfate, filtered and concentrated to afford 183 mg (88%) aluminum
hydroxy 20-phenyl 2,9,16,23-tetraphenoxytetrabenztriazaporphyrin as
a deep blue-green solid.
Aluminum Hydroxy 20-Phenyl
1,8,15,22-Tetraphenoxytetrabenztriazaporphyrin
In a procedure analogous to that described above, aluminum hydroxy
20-phenyl 1,8,15,22-tetraphenoxytetrabenztriazaporphyrin was
isolated in 90% yield.
Aluminum Hydroxy 20-Phenyl
2,9,16,23-Tetrathiophenyltetrabenztriazaporphyrin
In a procedure analogous to that described above, aluminum hydroxy
20-phenyl 2,9,16,23-tetrathiophenyltetrabenztriazaporphyrin was
isolated in 96% yield.
Aluminum Hydroxy 20-Phenyl
1,8,15,22-Tetrathiophenyltetrabenztriazaporphyrin
In a procedure analogous to that described above, aluminum hydroxy
20-phenyl 1,8,15,22-tetrathiophenyltetrabenztriazaporphyrin was
isolated in 97% yield.
Tabulated below are the absorbance wavelengths of the aluminum
axial methyl derivatives in methylene chloride solution and the
emission wavelengths of the axial hydroxy derivatives in
tetrahydrofuran. The quantum yields were determined in
tetrahydrofuran.
______________________________________ Quantum TBTAP Absorbance
Emission Yield ______________________________________ 2,9,16,23 oxy
656, 694 nm 690 nm 0.40 1,8,15,22 oxy 676, 712 704 0.25 2,9,16,23
thio 666, 704 701 0.26 1,8,15,22 thio 694, 728 722 0.19
______________________________________
Reactive Tetrabenztriazaporphyrin Formation
The sulfonyl chloride derivatives of the four tetrasubstituted
aluminum hydroxy 20-phenyl tetrabenztriazaporphyrins were prepared
by treatment with chlorosulfonic acid as described previously for
the corresponding aluminum phthalocyanines in Example 2.
Water Soluble Tetrabenztriazaporphyrin Formation
Hydrolysis of the above sulfonyl chloride derivatives in a
procedure analogous to that described previously for the
corresponding aluminum phthalocyanines in Example 2, provided four,
water soluble aluminum hydroxy 20-phenyl tetrabenztriazaporphyrin
sulfonates. The absorbance and emission wavelengths of the four
tetrabenztriazaporphyrins in water along with the quantum yields.
The emission spectra for 8, 9, 10, and 11 are presented in FIGS. 10
and 11, respectively.
______________________________________ Compound Quantum No. TBTAP
Absorbance Emission Yield ______________________________________ 8
2,9,16,23 oxy 664, 692 nm 695 nm 0.43 9 1,8,15,22 oxy 676, 704 711
0.22 10 2,9,16,23 thio 690, 713 717 0.14 11 1,8,15,22 thio 691, 715
728 0.06 ______________________________________
EXAMPLE 4
The Preparation of Aluminum Tetrabenztriazaporphyrin Sulfonates
Aluminum tetrabenztriazaporphyrins sulfonates substituted at
position twenty with either hydrogen, 12, or phenyl, 13, are
described in Example 4. These water solution and reactive
derivatives have performance characteristics similar to the
aluminum phthalocyanines sulfonates and possess the optical
properties of the aluminum tetrabenztriazaporphyrins. The following
is a detailed description of the preparation of these compounds.
The presentation is organized into sections which detail
tetrabenztriazaporphyrin preparation, metalation, reactive TBTAP
preparation, and water soluble TBTAP preparation. Within each
section a detailed procedure is given for the 20-hydrogen
derivative followed by a comment on the procedure for the 20-phenyl
derivative.
Tetrabenztriazaporphyrin Preparation
Magnesium 20-H Tetrabenztriazaporphyrin
To a suspension of 5.0 g (39.1 mm) phthalonitrile in 25 mL diethyl
ether was added dropwise 1.1 equivalents, 14.3 mL, (43.0 mm) 3.0M
methylmagnesium bromide in diethyl ether. The resulting solution
was stirred at room temperature under nitrogen for two hours. The
ether was removed under vacuum and 25 mL quinoline was added. The
reaction solution was heated at 200.degree. under nitrogen for 16
hours. The solution was cooled and diluted with 1 L methylene
chloride to precipitate the crude product. The crude product was
collected by filtration and extracted with methanol in a Soxhlet
extractor until the extract was colorless. The product, the Soxhlet
residue, was isolated as a blue solid, 2.95 g (5.48 mm, 56%).
Spectral data are tabulated below.
Magnesium 20-Phenyl Tetrabenztriazaporphyrin
In a procedure analogous to that described above, magnesium
20-phenyl tetrabenztriazaporphyrin was prepared. The product was
isolated by dilution of the quinoline reaction mixture with 500 mL
distilled water. The crude product was collected by filtration and
dried in vacuo. The product was purified by chromatography on
silica eluting with hexane: tetrahydrofuran (1:1). Spectral data
are tabulated below.
Tabulated below are the absorbance wavelengths of the magnesium
tetrabenztriazaporphyrin derivatives in tetrahydrofuran prepared as
described above.
______________________________________ TBTAP Absorbance
______________________________________ 20-H 645, 665 nm 20-Ph 648,
670 ______________________________________
20-H Tetrabenztriazaporphyrin
A solution of 1.0 g (1.86 mm) magnesium 20-H
tetrabenztriazaporphyrin in 10 mL trifluoroacetic acid was stirred
for 16 hours. The solution was diluted with 100 mL distilled water
and the solid was collected by filtration. The product was washed
with 500 mL distilled water, 500 mL methanol and dried in vacuo.
20-H Tetrabenztriazaporphyrin, 280 mg (0.54 mm, 29%), was isolated
as a blue solid. Spectral data are tabulated below.
20-Phenyl Tetrabenztriazaporphyrin
A solution of 1.0 g (1.63 mm) magnesium 20-phenyl
tetrabenztriazaporphyrin in 10 mL acetic acid was heated at reflux
for 1 hour. The solution was cooled and diluted with 100 mL
distilled water. The product was collected by filtration and washed
with 500 mL distilled water and dried in vacuo. 20-Phenyl
tetrabenztriazaporphyrin, 115 mg (0.22 mm, 14%), was isolated as a
blue solid. Spectral data are tabulated below.
Tabulated below are the absorbance wavelengths of the
tetrabenztriazaporphyrin derivative in tetrahydrofuran prepared as
described above.
______________________________________ TBTAP Absorbance
______________________________________ 20-H 640, 682 nm 20-Ph 643,
684 ______________________________________
Tetrabenztriazaporphyrin Metalation
Aluminum 20-H Tetrabenztriazaporphyrin
A solution of 100 mg (0.195 mm) 20-H tetrabenztriazaporphyrin in 5
mL quinoline was treated with ten equivalents, 260 mg (1.95 mm)
aluminum trichloride under nitrogen. The solution was heated at
200.degree. for two hours, cooled, and diluted with 100 mL
methylene chloride. The precipitated product was collected by
filtration and washed with 500 mL methylene chloride. Aluminum 20-H
tetrabenztriazaporphyrin, 85 mg (0.15 mm, 76%), was isolated as a
purple solid. Spectral data are tabulated below.
Aluminum 20-Phenyl Tetrabenztriazaporphyrin
To 115 mg (0.224 mm) 20-phenyl tetrabenztriazaporphyrin in 20 mL
methylene chloride was added ten equivalents, 1.12 mL (2.24 mm)
2.0M trimethylaluminum in toluene. The solution was stirred at room
temperature under nitrogen for two hours and then carefully
quenched with 1 mL distilled water followed by 1 mL 1N aqueous
hydrochloric acid. The organic solution was extracted with 3-20 mL
portions 1N aqueous hydrochloric acid, dried over sodium sulfate,
and concentrated. Aluminum 20-phenyl tetrabenztriazaporphyrin, 85
mg (0.15 mm, 68%), was isolated as a blue solid. Spectral data are
tabulated below.
Tabulated below are the absorbance and emission wavelengths, and
quantum yields of the aluminum tetrabenztriazaporphyrins in
dimethylformamide prepared as described above.
______________________________________ TBTAP Absorbance Emission
Quantum Yield ______________________________________ 20-H 649, 670
nm 672 nm 0.69 20-Ph 656, 681 680 0.56
______________________________________
Reactive Tetrabenztriazaporphyrin Formation
Aluminum 20-H Tetrabenztriazaporphyrin Sulfonyl Chloride
A solution of 150 mg (0.26 mm) aluminum 20-H
tetrabenztriazaporphyrin in 5 mL chlorosulfonic acid was heated at
150.degree. for two hours under nitrogen. The mixture as cooled and
carefully quenched on 5 g ice. The product was collected by
filtration, washed with 20 mL distilled water, 100 mL diethyl
ether, and dried in vacuo. Aluminum 20-H tetrabenztriazaporphyrin
sulfonyl chloride, 180 mg (0.189 mm, 73%), was isolated as a blue
powder. Spectral data are tabulated below.
Aluminum 20-Phenyl Tetrabenztriazaporphyrin Sulfonyl Chloride
In a procedure analogous to that described above, aluminum
20-phenyl tetrabenztriazaporphyrin sulfonyl chloride was isolated
in 72% yield. Spectral data are tabulated below.
Tabulated below are the absorbance wavelengths for the aluminum
tetrabenztriazaporphyrin sulfonyl chloride derivatives in
dimethylformamaide prepared as described above.
______________________________________ TBTAP Absorbance
______________________________________ 20-H 655, 677 nm 20-Ph 657,
683 ______________________________________
Water Soluble Tetrabenztriazaporphyrin Formation
Aluminum 20-H Tetrabenztriazaporphyrin Sulfonate (12)
A solution of 9.6 mg aluminum 20-H tetrabenztriazaporphyrin
sulfonyl chloride in 5.0 mL distilled water was stirred at room
temperature for 48 hour. Concentration in vacuo gave aluminum 20-H
tetrabenztriazaporphyrin sulfonate in quantitative yield. The
absorbance and emission spectra in water are presented in FIG. 12.
Spectral data are tabulated below.
Aluminum 20-Phenyl Tetrabenztriazaporphyrin Sulfonate (13)
In a procedure analogous to that described above, aluminum
20-phenyl tetrabenztriazaporphyrin sulfonate was isolated in
quantitative yield. The absorbance and emission spectra in water
are presented in FIG. 13. Spectral data are tabulated below.
Tabulated below are the absorbance and emission wavelengths of the
aluminum tetrabenztriazaporphyrin sulfonates in water prepared as
described above. The quantum yields are also included.
______________________________________ TBTAP Absorbance Emission
Quantum Yield ______________________________________ 20-H 649, 667
nm 672 nm 0.67 20-Ph 653, 672 681 0.59
______________________________________
EXAMPLE 5
The Preparation of Phthalocyanine Tetraquaternary Ammonium
Derivatives
Exemplary cationic phthalocyanines are presented in Example 5, The
derivatives in Example 5 satisfy formula I where M is either
H.sub.2 or aluminum, each R.sub.1 is --XYW, X is oxygen, Y is
ethylene (--CH.sub.2 CH.sub.2 --), W is trimethylammonium iodide, Z
is nitrogen, and R.sub.2 is --XYW, --YW, or --W. The positively
charged tetrasubstituted phthalocyanines are prepared from
monosubstituted phthalonitriles.
The phthalocyanine precursor,
4-dimethylaminoethanoxyphthalonitrile, was prepared by displacement
of nitro from 4-nitrophthalonitrile with 2-dimethylaminoethanol.
Formation of the diiminoisoindoline and subsequent cyclization
resulted in the metal free tetrasubstituted phthalocyanine. The
amino groups were quaternized with methyl iodide. Aluminum was
incorporated by treatment with aluminum triacetylacetonate. The
aluminum phthalocyanine was rendered water soluble by alkylation
with methyl iodide to provide the tetraquaternary ammonium compound
14b.
The absorbance spectrum of 14a in water presented in FIG. 14 shows
nearly complete aggregation. The fluorescence quantum yield is less
than 0.01. However, in the presence of RNA (Torula yeast) a strong
specific binding interaction occurs which results in the
disaggregation of the fluorophore. The absorbance spectrum of 14a
in the presence of RNA, FIG. 14, is indicative of a monomeric
phthalocyanine. The emission spectra for the two solutions are
compared in FIG. 15. The fluorescence enhancement of 14a upon RNA
binding is 450-fold.
The corresponding absorbance and emission spectra for aluminum
derivative 14b are shown in FIGS. 16 and 17, respectively. The
fluorescence enhancement upon RNA binding is 340. No fluorescence
enhancement was observed for either 14a or 14b in the presence of
bovine serum albumin.
Tabulated below are the spectral data for the metal free and
aluminum phthalocyanine derivatives prepared as described above.
The emission wavelength and fluorescence enhancement of the
fluorophores in the presence of RNA are presented. The absorbance
data was recorded with a fluorophore concentration of
5.times.10.sup.-6 M and an RNA (Torula Yeast) concentration of 1.0
mg/mL. The fluorescence data was obtained for these solutions at
100-fold dilution.
______________________________________ Emission Wavelength
Fluorescence Phthalocyanine In Presence of RNA Enhancement
______________________________________ Metal free 720 nm 450
Aluminum 705 340 ______________________________________
Another specific embodiment of the cationic phthalocyanines is the
case where in formula I M, R.sub.1 and R.sub.2 are as described
above, and X=--CH.sub.2 --, Y=--CH.sub.2 CH.sub.2 -- and
W=diethylmethylammonium. The counterion is iodide.
EXAMPLE 6
The Preparation of Fluorophore Streptavidin Conjugates
The preparation of covalent streptavidin fluorophore conjugates is
described in Example 6. The reactive forms of the red shifted
aluminum phthalocyanine derivatives, the sulfonyl chlorides, are
coupled to streptavidin according to procedures analogous to those
previously disclosed. Schindele, D. C. et al., Monomeric
Phthalocyanine Reagents, U.S. patent application Ser. No. 366,971:
1989. The following is a detailed description of the preparations.
While the Example explicitly describes coupling to streptavidin,
other proteins may be coupled by the same methodology.
Direct Coupling of Aluminum. Hydroxy
2,9,16,23-Tetraphenoxyphthalocyanine Sulfonyl Chloride to
Streptavidin
To 15.0 mg aluminum hydroxy 2,9,16,23-tetraphenoxyphthalocyanine
sulfonyl chloride solid was added 300 .mu.L dry dimethylformamide.
The solution was placed in a pre-equilibrated 30.degree. C. dry
bath. After one hour, 20 .mu.L of the dimethylformamide solution
containing the reactive fluorophore was added dropwise to 1.15 mg
streptavidin in 185 .mu.L 0.2M sodium bicarbonate in phosphate
buffered saline pH adjusted to 9.0 and containing 30 .mu.L
dimethylformamide at 4.degree. C. After one hour, the reaction was
quenched by the addition of 250 .mu.L of a 10 mg/mL solution of
lysine in 0.2M sodium bicarbonate in phosphate buffered saline
containing 0.02% sodium azide as a preservative. After stirring for
30 minutes at 4.degree. C., the conjugate was purified by size
exclusion chromatography on Sephadex G-50 in phosphate buffered
saline containing 0.02% sodium azide. Spectral data for the
conjugate is tabulated below.
Direct Coupling of Aluminum Hydroxy
1,8,15,22-Tetraphenoxyphthalocyanine Sulfonyl Chloride to
Streptavidin
In a procedure analogous to that described above, aluminum hydroxy
1,8,15,22-tetraphenoxyphthalocyanine sulfonyl chloride was coupled
to streptavidin. Spectral data for the conjugate is tabulated
below.
Direct Coupling of Aluminum Hydroxy
1,8,15,22-Tetrathiophenylphthalocyanine Sulfonyl Chloride to
Streptavidin
In a procedure analogous to that described above except that a 30
minute incubation at 30.degree. C. was used rather than a one hour
incubation, aluminum hydroxy 1,8,15,22-tetraphenoxyphthalocyanine
sulfonyl chloride was coupled to streptavidin. Spectral data for
the conjugate is tabulated below.
Tabulated below are the absorbance and emission wavelengths for the
fluorophore streptavidin conjugates in phosphate buffered saline
containing 0.02% sodium azide and prepared as described above. The
fluorophore per streptavidin ratio (F/P) was determined by
comparing the absorbance of the protein at 280 nm relative to the
fluorophore absorbance at 350 nm. The quantum yields reported are
per fluorophore.
______________________________________ Phthalocyanine Absorbance
Emission F/P Quantum Yield ______________________________________
2,9,16,23 oxy 678 nm 698 nm 3.7 0.35 1,8,15,22 oxy 704 718 2.4 0.13
1,8,15,22 thio 719 729 3.6 0.03
______________________________________
EXAMPLE 7
The Preparation of Fluorophore Labeled Nucleic Acid Primers
The preparation of covalent fluorophore labeled nucleic acid
primers is described in Example 7. The reactive forms of the red
shifted aluminum phthalocyanine derivatives, sulfonyl chlorides,
are coupled to nucleic acid primers according to procedures
analogous to those previously disclosed. Schindele, D. C. et al.,
Monomeric Phthalocyanine Reagents, U.S. patent application Ser. No.
366,971: 1989. The following is a detailed description of the
preparations.
Aluminum Hydroxy 2,9,16,23-Tetraphenoxyphthalocyanine Labeled
M13mp18 (-21) Universal Sequencing Primer
To a stirred solution of 0.022 .mu.mol aminohexane modified M13mp18
(-21), 5' TGTAAAACGACGGCCAGT 3', Universal sequencing primer in 20
.mu.L 0.5M sodium bicarbonate/0.5M sodium carbonate (pH adjusted to
9.0) was added 1.3 mg aluminum hydroxy
2,9,16,23-tetraphenoxyphthalocyanine sulfonyl chloride in 12 .mu.L
dimethylformamide. After stirring overnight at room temperature in
the dark, the labeled primer was purified by size exclusion
chromatography (Sephadex G-50) followed by polyacrylamide gel
electrophoresis. Spectral data for the labeled primer is tabulated
below.
Aluminum Hydroxy 1,8,15,22-Tetraphenoxyphthalocyanine Labeled M
13mp18 (-21) Universal Sequencing Primer
In a procedure analogous to that described above, the primer was
labeled with aluminum hydroxy 1,8,15,22-tetraphenoxyphthalocyanine
sulfonyl chloride. The primer was purified by ethanol precipitation
followed by polyacrylamide gel electrophoresis. Spectral data for
the labeled primer are tabulated below.
Tabulated below are the absorbance and emission wavelengths and
quantum yields of the aluminum phthalocyanine labeled primers
prepared as described above in 0.1M aqueous triethylamine
acetate.
______________________________________ Phthalocyanine Absorbance
Emission Quantum Yield ______________________________________
2,9,16,23 oxy 684 nm 696 nm 0.39 1,8,15,22 oxy 704 715 0.18
______________________________________
EXAMPLE 8
Monofunctional Reactive Tetrabenztriazaporphyrin Derivatives
The 20-substituted tetrabenztriazaporphyrins (TBTAP) described
above, like the phthalocyanines, are useful as reagents for
fluorescence analysis. One unique property of the TBTAP system is
the position 20 substituent. By appropriate selection of the
Grignard reagent used in the preparation of the TBTAP (see Examples
3 and 4), a reactive 20-substituent may be synthesized. The
Grignard reagent may either contain the functional group of choice
or be capable of further elaboration to the group of choice. The
resulting 20-substituted TBTAP is then monofunctionally
reactive.
Particularly useful reactive groups as R.sub.2 enable efficient
coupling to biological entities. Preferred reactive groups would
include sulfonyl chloride, carboxylic acid and derivatives, amino,
isothiocyanate, maleimide, and imidate among others.
An example of a useful monofunctionally reactive TBTAP reagent
would be one with an isothiocyanate or N-hydroxysuccinimide ester
moiety at position 20. These reagents may be useful in various
applications such as immunoassays, nucleic acid sequencing, nucleic
acid probe assays, flow cytometry or for selective
functionalization. As an example of selective functionalization,
the isothiocyanate derivative could serve as a fluorescent reagent
in protein sequence analysis utilizing the Edman degradation
process. The isothiocyanate portion of the fluorophore couples to
the N terminus of the peptide to be sequenced which is immobilized
(C terminus) on a solid phase. Degradation of the peptide follows
with the fluorophore labeled terminal amino acid being cleaved from
the peptide. The fluorophore labeled amino acid is then removed
from the immobilized peptide and the amino acid is identified. The
new N terminus of the remaining peptide, now one amino acid residue
shorter, is ready for the next cycle. Repetition of the process
results in the sequential identification of the amino acid residues
of the peptide of interest. Highly fluorescent reagents, such as
phthalocyanines and TBTAPs, would improve the detection limits of
protein sequence analysis and enable the sequencing of smaller
quantities of protein. The advantage of highly sensitive
fluorophores is particularly relevant when only trace quantities of
rare proteins are available.
EXAMPLE 9
Monofunctional Wavelength Modified Tetrabenztriazaporphyrin
Derivatives
The 20-substituent of the TBTAP ring system may be designed to
create the desired optical properties of the TBTAP. As with
peripheral ring substitution detailed above in Examples 2 and 3,
the wavelengths of absorbance and fluorescent emission may be
manipulated by the choice of substituent at position 20. Electron
donating groups are expected to red shift both absorbance and
fluorescence wavelengths while a blue shift is anticipated for
electron withdrawing groups.
Fluorinated 20-substituted TBTAP derivatives such as
trifluoromethyl (CF.sub.3) and perfluorophenyl (C.sub.6 F.sub.5)
may be prepared from commercially available
1,1,1-trifluoro-2-bromoethane and 2,3,4,5,6-pentafluorobenzyl
bromide, respectively. These TBTAP bearing electron withdrawing
substituents are predicted to absorb and emit light at wavelengths
blue of the parent.
EXAMPLE 10
Phthalocyanine and Tetrabenztriazapophyrin Derivatives Bearing
Substituted Phenyl Groups
The tetrasubstituted phthalocyanines and tetrabenztriazaporphyrins
described in Examples 2 and 3 are derived from unsubstituted
phenoxy or thiophenylphthalonitriles. Substituted phenoxy or
thiophenylphthalonitriles may also be prepared and cyclized to the
corresponding phthalocyanines or tetrabenztriazaporphyrin systems.
These modified derivatives may serve to fine tune the optical
properties of the parent tetrasubstituted material.
For example, 3-(4-fluorophenoxy)phthalonitrile may be prepared by
treatment of 4-fluorophenol with 3-nitrophthalonitrile in a
procedure analogous to that which results in the production of
3-phenoxyphthalonitrile. Cyclization of the fluoro substituted
phthalonitrile to the phthalocyanine or TBTAP, will result in the
formation of a species slightly different from its nonfluorinated
parent. The optical properties will also vary slightly from the
parent.
Many substituted phenols and thiophenols are known. By the
methodology described above, many substituted derivatives of
tetraphenoxy- and tetrathiophenylphthalocyanines and TBTAPs may be
prepared.
EXAMPLE 11
Octasubstituted Phthalocyanine and Tetrabenztriazaporphyrin
Derivatives
Octasubstituted phthalocyanines and tetrabenztriazaporphyrins may
be prepared from disubstituted phthalonitriles in procedures
analogous to those described in Examples 2 and 3 for the
preparation of tetrasubstituted phthalocyanines and TBTAPs from
monosubstituted phthalonitriles. The octasubstituted derivatives
may be broadly categorized based on the position of the
substitution. Symmetrical phthalocyanines and TBTAPs are derived
from 3,6- and 4,5-disubstituted phthalonitriles. Less symmetrical
and more difficult to prepare are 3,4- and 3,5-disubstituted
phthalonitriles.
Octaoxy and octathiophthalocyanines derived from 3,6- and
4,5-disubstituted phthalonitriles have been reported. 3,6-octaoxy:
Witkiewicz, Z. et al., Materials Science II, 1:39-45 (1976).
4,5-octaoxy: Metz, J., et al., Inorg. Chem., 23:1065-1071 (1984).
3,6- and 4,5-octathio: Oksengendlee, I. G., et al., J. Org. Chem.
USSR, 14(5):1046-1051 (1978). The sulfur substituted derivatives
absorb at greater wavelengths than the oxygen analogs.
We tabulate below the spectral properties of aluminum
3,6-octamethoxy and 4,5-octamethoxyphthalocyanine. The absorbance
and emission wavelengths and quantum yields were recorded in
dimethylformamide solution.
______________________________________ Phthalocyanine Absorbance
Emission Quantum Yield ______________________________________
3,6-octamethoxy 739 nm 748 nm 0.02 4,5-octamethoxy 672 678 0.21
______________________________________
The 3,6-methoxy derivative exhibits a significant red shift.
However, the fluorescence quantum yield is low. The 4,5-methoxy
derivative is actually blue shifted and retains more of a
fluorescence emission. Both of these derivatives may be further
elaborated to water soluble and reactive reagents by a reaction
sequence completely analogous to that described for the isomeric
aluminum tetraneopentoxyphthalocyanines described in Example 1.
Octasubstituted derivatives composed of four sulfur substituents
and four oxygen substituents may also be prepared as described in
the Examples above. These derivatives may be prepared from
phthalonitriles substituted with both an oxygen and a sulfur
substituent, for example, 3-thiophenyl-5-phenoxyphthalonitrile. The
phenyl groups in the example may be other than phenyl and the
position of the substituents may also vary. The optical properties
of these mixed derivatives is expected to be intermediate between
the octaoxy and the octathio analogs.
4,5-Octasubstituted carbon derivatives may also be prepared. In the
case where the 4,5-substituent is a benzo ring, the system is known
as a naphthalocyanine. These highly conjugated derivatives are
approximately 100 nm red shifted relative to their phthalocyanine
counterparts. Vogler, A. and H. Kunkely, Inorganica Chimica Acta,
44:L209-L210 (1980). Tabulated below are the spectral
characteristics of aluminum phthalocyanine and naphthalocyanine
chlorides in dimethylformamide.
______________________________________ Absorbance Emission Quantum
Yield ______________________________________ Phthalocyanine 671 nm
672 nm 0.60 Naphthalocyanine 768 770 0.11
______________________________________
EXAMPLE 12
Pyrazine Porphyrazines
Closely related in structure to phthalocyanines are pyrazine
porphyrazines. Linstead, R. P. et al., J. Chem. Soc. 911-921, 1937.
Phthalocyanines bear four benzo rings appended to the macrocycle
while pyrazine porphyrazines have four pyrazine (1,4-diazabenzene)
rings. ##STR5## Elaboration of tetra- and octaphenylpyrazine
porphyrazine to reactive, and water soluble aluminum derivatives is
the subject of Example 12. Cyclization of either 5-phenyl or
5,6-diphenylpyrazine 2,3-dinitrile results in the porphyrazine
macrocycle. Metalation with aluminum chloride in quinoline provides
the corresponding aluminum derivatives. Treatment with
chlorosulfonic acid gave the reactive intermediates and hydrolysis
of these produced the water soluble aluminum pyrazine porphyrazine
sulfonates. Tabulated below are the spectral data for aluminum
tetra and octaphenylpyrazine porphyrazine sulfonates in water.
______________________________________ Pyrazine Porphyrazine
Absorbance Emission Quantum Yield
______________________________________ tetraphenyl (pH 10) 641 nm
647 nm 0.71 octaphenyl 651 654 0.95
______________________________________
EXAMPLE 13
Pyridine Porphyrazines
Closely related in structure to phthalocyanines are pyridine
porphyrazines. Linstead, R. P., et al., J. Chem. Soc. 911-921,
1937. Structurally, replacement of the benzo ring in phthalocyanine
with pyridine gives pyridine porphyrazine. ##STR6##
These derivatives may be prepared from either 2,3-dicyanopyridine
or 3,4-dicyanopyridine. Cyclization of 2,3-dicyanopyridine gives
3-pyridine porphyrazine while 3,4-dicyanopyridine produces
4-pyridine porphyrazine. Like pyrazine porphyrazines, the pyridine
porphyrazines absorb at wavelengths blue-shifted relative to
phthalocyanines, with the 3-pyridine isomer blue-shifted relative
to the 4-pyridine porphyrazine. Metalation with aluminum chloride
in quinoline provided the aluminum derivatives.
Application of the oxygen and sulfur substitution methodology
developed for the phthalocyanines and tetrabenztriazaporphyrins as
described in Examples 2 and 3, respectively, will result in a
family of reagents for each of the aluminum pyridine
porphyrazines.
EXAMPLE 14
Imaging and Radionuclide Reagents
The reagents of this invention are organometallic compounds and as
such many different metals may be bound. The macrocyclic ring
systems disclosed are capable of efficient chelation of a variety
of metals useful in image analysis and therapeutic applications,
such as magnetic resonance imaging, radionuclide imaging, and as
radiopharmaceuticals. Active metals for these applications may be
incorporated into the macrocycle and directed to the site of
interest. The targeting of the metal bearing reagent may be a
naturally selective uptake of the reagent by the site of interest,
an antibody directed against an antigen present at the site of
interest to which the reagent is conjugated, a complementary
fragment of DNA to which the reagent is coupled, a membrane probe
to which the reagent is coupled or some other delivery
mechanism.
Paramagnetic metals useful for magnetic resonance imaging contrast
agents include gadolinium, manganese, and iron.
The field of nuclear medicine utilizes radioisotopes, usually
gamma-emitting isotopes, for diagnostic purposes. Radioactive metal
complexes of copper 67, technetium 99, cobalt 57, and gallium 67
have been used as radiopharmaceuticals in both diagnostic and
therapeutic applications.
The reagents of this invention may be useful in the applications
described above by virtue of their metal binding capabilities.
Also, the biological conjugates of this invention will serve to act
as targeting agents for the applications described above.
Representative malignancies that can be treated by the
radionuclides are: leukemia, ovarian cancer, lymphoma, breast
cancer, myeloma, kidney, liver, and colorectal cancer, and the
like.
EXAMPLE 15
Improved Photodynamic Therapeutic (PDT) Reagents
PDT agents (photosensitizers) are selectively taken up by cancerous
tissue and upon irradiation with visible light become activated.
The activated photosensitizers effectively kill cells in their
immediate vicinity presumably by the generation of singlet oxygen.
Spikes, J. D., Photochem. Photobiol. 43(6):691-699 (1986). The
reagents of this invention offer two improvements over the existing
technology. The first advantage lies in the deep red absorbance of
the disclosed reagents and the second in the targeting of these
reagents made possible by their biological binding conjugates.
Phthalocyanines and TBTAPs which absorb in the deep red with large
molar absorptivities will enable treatment of more tissue.
Currently PDT reagents are limited by their relatively blue
abosrbance profiles with respect to depth of penetration of
activating light. Since human tissue is nearly transparent in the
near infrared, PDT agents which absorb in this region will be most
effective. The utilization of red-shifted phthalocyanines and
TBTAPs will enable access to tissues which would be unaffected by
currently employed blue absorbing sensitizers.
The targeting of the photosensitizer is a critical aspect in PDT.
Today, the natural selectivity of photosensitizers for tumorous
tissue is the most commonly relied upon delivery mechanism. The
reagents of this invention, by virtue of their conjugation to
biological entities such as antibodies or oligonucleotides, can
seek out and bind to sites requiring photodynamic treatment. The
conjugation of these deep red absorbing phthalocyanines and TBTAPs
to antibodies (or antigen binding antibody fragments) directed
against cancerous tissue or cancer-associated antigens enables
efficient delivery of the photoactivatable agents to the cancer.
Alternatively, the coupling of red absorbing phthalocyanines and
TBTAPs to a complementary fragment of DNA enables the use of
anti-sense oligonucleotides or DNA probes as targeting agents.
Another targeting method involves covalently attaching the reagent
to a membrane probe, as defined above.
Representative malignancies that could be treated by PDT using the
present reagents are: bladder cancer, skin cancer (melanoma),
esophogeal cancer, brain tumors, other solid tumors, and the
like.
EXAMPLE 16
A representative example of a two color system for AIDS testing
that employs the phthalocyanine based fluorophores is as follows.
Anti-CD4 (helper T cell specific monoclonal antibody) labeled with
phthalocyanine (I) where R.sub.2 is antibody-SO.sub.2 --, Z=N, two
R.sub.1 groups are --SO.sub.3.sup.- the third R.sub.1 group is
hydrogen (Dye I) and anti-CD8 (suppressor T cell specific
monoclonal antibody) labeled with phthalocyanine (I) where R.sub.2
is antibody-SO.sub.2 -phenyl-O, each R.sup.1 is XYW, wherein X=O,
Y=phenyl, W=--SO.sub.3.sup.-. The R.sub.1 and R.sub.2 group, are
located at the 1, 8, 15, 22 positions. (3 isomer, Dye III) are
incubated with peripheral blood lymphocytes. During this
incubation, anti-CD4-Dye I binds to the helper cells and
anti-CD8-Dye III binds to the suppressor cells. Since the T helper
cells are labeled with a fluorophore that emits at one wavelength
(Dye I) and the T suppressor cells are labeled with a fluorophore
that emits at a different wavelength (Dye III) that is both
resolved and red-shifted from that on the helper cells, each subset
of cells may be quantitated simultaneously using a flow cytometer
equipped with optical filters that allow for discrimination of the
two different fluorophores.
While the present invention has been described in conjunction with
preferred embodiments and illustrative examples, one of ordinary
skill after reading the foregoing specification will be able to
effect various changes, substitutions of equivalents, and other
alterations to the reagents, methods, and kits set forth herein. It
is therefore intended that the protection granted by Letters Patent
hereon be limited only by the definitions contained in the appended
claims and equivalents thereof.
* * * * *