Note: Descriptions are shown in the official language in which they were submitted.
1 337754
RED-SHIl~TED PHTHALOCYANINE AND
TE:TRABENZTRIAZAPORPHYRIN REAGI~NTS
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
-2- 1 3 3 7 7 5 4
kilobase and greater lengths of DNA. This llmit 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 succinylfluoresceln dye distinguished by its fluorescent
emission. Prober, J.M., et al., Sclence 238:336-341, 1987; European Patent
The effect of peripheral substltution of fluoro and cyano groups on the
electronic propertles of slllcon dihydroxy phthalocyanlne has been modelled.
Marks, T.J., et al., J. Am. Chem. Soc. 109:5943-5947, 1987. The calculated
wavelength of absorbance for the parent silicon phthalocyanine was predicted to
be 673 nm whlle 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. Luk'yanets, E.A. and V.M. Derkacheva, 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 solubleby 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 ofa 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 absorptlvity, a high fluorescence quantum yield, alarge 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).
.
_ 3 _ 1 337754
Alumlnum phthalocyanlne (AlPc) has nearly ldeal
spectral propertles. Excltation of alumlnum phthalocyanlne at
350 nm results ln emlsslon at 685 nm with fluorescence quantum
yleld (~f) of 0.58. Brandon, J.H., and D. Madge, J. Amer.
Chem. Soc., 102 62-65, 1980. Aluminum phthalocyanine (AlPc)
ls composed of a hlghly con~ugated macrocycle and a trivalent
alumlnum atom. The structure of the parent AlPc fluorophore
is shown below. L ls a llgand such as OH when the AlPc ls ln
water. The trivalent aluminum atom provldes axlal llgatlon
whlch serves to reduce aggregatlon and thereby lncreases
fluorescence ln solutlon.
N~N
~ ~ -N ~
In therapeutlc appllcations, aluminum phthalocyanlne
sulfonates have been determlned to be effective in directed
cell killing. Ben-Hur, E. and I. Rosenthal, Photochem. Photo-
biol. 42:129-133, 1985. The advantage that phthalocyanines
have over other photodynamic agents ls their large molar
absorpivity in the red region of the vlsible spectrum. The
large molar extinctlon coefflclent coupled wlth the transpar-
6283g-1163
- 3A - l 337754
ency of tlssue at these red wavelengths provldes for more
efflclent llght penetration and subsequently more effective
treatment of subcutaneous mallgnancies. 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 treat-
ments. Derivatlves attached to blological moletles such as
probes or antibodies can be targeted to specific cell popula-
tions.
Closely related to the phthalocyanines are the tetra-
benztriazoporphyrins, referred to herein as TBTAPs. Linstead,
R.P. et al., J. Chem. Soc. 1809-1828, 1939. The only struc-
tural difference is the replacement of the nitrogen at posi-
tion twenty of the phthalocyanine with a substituted carbon.
No substituted
62839-1163
-4- l 3 3 7 7 5 4
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,
5 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:
~
20 wherein M is H2, aluminum, silicon, phosphorus, gallium, germanium, cadmium,
scandium, magnesium, tin, or zinc. Each R1 is independently selected from
-XYW, -YW, -W, or -H. X is CR3R4, where R3 and R4 are independently selected
from hydrogen, alkyl (preferably C1-C12), aryl (preferably C6-C12), or aralkyl
(preferably C6-C12), or R3 and R4 together may be a carbonyl oxygen, or X is
25 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. R2
comprises a biological entity such as an antibody, antigen, nucleotide, nucleic
acid, oligonucleotide, avidin, streptavidin, or a membrane probe, or R2 is a
30 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 C1-C12), aryl
(preferably C6-C12), or aralkyl (preferably C6-C12). When Z is CR, the R1 and
R2 groups may be located on any of the four benzo rings of the TBTAP.
In a separate embodiment, the R2 group located on the benzo ring in
35 formula I is defined as R1, and Z = -CR2, where R2 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.
_5_ I 3 3 7 7 5 4
In all embodiments of formula 1, 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, -CO2H, -OCH2CO2H, -PO4=,
3 3 2 ~ S02Cl, -S04, -NH2, -NHD,-NHD D or N+D
D3 being independently alkyl (preferably Cl-C12), aryl (preferably C6-C12), or
aralkyl (preferably C6-C12). Charged species will have counterions.
In a preferred embodiment, M is aluminum, each R1 is -XYW, X is either an
oxygen or sulfur atom, Y is a methylene group, W is a carboxylic acid, Z is
nitrogen and R2 is -X-CH2CO2H. The substitution of R1 occurs at the 1,8,15,22
- 10 positions (3 isomer) or at the 2,9,16,23 positions (4 isomer) of the macrocycle. See
FIGURE 1 for the phthalocyanine and tetrabenztriazaporphyrin ring numbering
system.
In a particularly preferred embodiment, M is aluminum, each R1 is -XYW, X
is either an oxygen or sulfur atom, Y is phenyl, W is sulfonate or sulfonyl chloride,
l 5 Z is nitrogen and R2 is -O-phenyl-sulfonate, -O-phenyl-sulfonyl chloride, -S-
phenyl-sulfonate, or -S-phenyl-sulfonyl chloride. The substitution of R1 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 orphenyl substituents. The phenyl may be unsubstituted or substituted by 1-5,
preferably 1-2 substituents selected from among C1-C6 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 -CR2, the rest of the macrocycle contains 4 R1 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 Sc, 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.
1 337754
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 1-3
benzo rings contain one nitrogen atom (either isomer) and 3-1 benzo rings contain
2 nitrogen atoms. The R1 and R2 groups may be attached to carbon atoms or
nitrogen atoms in the benzo rings, 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 CR3R4 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
l 5 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 R2 is R1. R1, X and Y are as described above. W is
-N+D1D2D3, wherein D1-D3 are independently hydrogen, C1-C12 alkyl, C6-C12
aralkyl, or C6-C12 aryl groups, or -N+D1D2D3 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- andpolynucleotides, 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 R2 in formula I is a group capable of being covalently
attached to a biological entity are contemplated. R2 may be directly attached tothe benzo ring or may be linked to the benzo ring by an XY or Y linkage. Such R2groups include -SO2Cl; -CO2H; -COX', wherein X' is a leaving group such as N-
hydroxy-succinimide; maleimide; or isothiocyanate. R2 can also be 8 nucleophilicmoiety, such as an amino group, for reaction with reactive groups on the
_7_ l 3 3 7 7 5 4
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
5 formula I.
Brief Description of the Drawings
FIGURE 1 shows the phthalocyanine and tetrabenztriazaporphyrin ring
numbering system.
FIGURE 2 shows the absorbance and emission spectrum of aluminum
l0 phthalocyanine tetrasulfonate, 1, in water.
FIGURE 3 compares the absorbance spectra of the glycolic acid derivatives,
2 and 3, in water.
FIGURE 4 compares the emission spectra of the glycolic acid derivatives, 2
and 3, in water.
FIGURE 5 compares the emission of spectra of the two glycolic acid
derivatives, 2 and 3, in aqueous cetyl trimethylammonium bromide (CTAB).
FIGURE 6 compares the absorbance spectra of the oxygen substituted
aluminum phthalocyanine sulfonates, 4 and 5, in water.
FIGURE 7 compares the emission spectra of the oxygen substituted
aluminum phthalocyanine sulfonates, 4 and 5, in water.
FIGURE 8 compares the absorbance spectra of the sulfur substituted
aluminum phthalocyanine sulfonates, 6 and 7, in water.
FIGURE 9 compares the emission spectra of the sulfur substituted aluminum
phthalocyanine sulfonates, 6 and 7, in water.
FIGURE 10 compares the emission spectra of the oxygen substituted
aluminum tetrabenztriazaporphyrin sulfonates, 8 and 9, in water.
FIGURE 11 compares the emission spectra of the sulfur substituted
aluminum tetrabenztriazaporphyrin sulfonates, 10 and 11, in water.
FIGURE 12 shows the absorbance and emission spectra of aluminum 20-H
tetrabenztriazaporphyrin sulfonate, 12, in water.
FIGURE 13 shows the absorbance and emission spectra of aluminum 20-
phenyl tetrabenztriazaporphyrin sulfonate, 13, in water.
FIGURE 14 compares the absorbance spectra of the metal free
phthalocyanine cationic fluorophore, 14a, in water with and without RNA.
FIGURE 15 compares the emission spectra of the metal free phthalocyanine
cationic fluorophore, 14a, in water with and without RNA.
-8- I 3 3 7 7 5 4
FIGURE 16 compares the absorbance spectra of the aluminum
phthalocyanine cationic fluorophore, 14b, in water with and without RNA.
FIGURE 17 compares the emission spectra of the aluminum phthalocyanine
cationic fluorophore, 14b, in water with and without RNA.
FIGURE 18 compares the emission spectra in water of four aluminum
5 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 1.
In formula I, M represents either H2 or is selected from among the following
metals: aluminum, silicon, phosphorous, gallium, germanium, cadmium, scandium,
magnesium, tin, and zinc. Each R1 is independently selected from -XYW, -YW,
25 -W, or hydrogen. X represents CR3R4, where R3 and R4 are independently
selected from hydrogen, alkyl (preferably C1-C12), aryl (preferably C6-C12), or
aralkyl (preferably C6-C12), or R3 and R4 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
30 a water solubilizing group. The substituent R2 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. R2 may also be a reactive or activatable group
which is directly attached to the benzo ring or attached by way of a linker, such
35 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 Cl-C12), aryl (preferably
9 1 3 3 7 7 5 4
C6-C12), or aralkyl (preferably C6-C12). 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~hosphorylated compounds may
also be employed. Z is either a nitrogen atom or a carbon substituted with
5 hydrogen, alkyl (preferably C1-C12), aryl (preferably C6-C12), or aralkyl (pre-
ferably, C6-C12) groups.
In a separate embodiment, Z is -CR2, in which case all of the variables on
the benzo rings of the macrocycle will be R1 groups; that is, the R2 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 Rl is -XYW, X is either
oxygen or sulfur, Y is methylene, W is carboxylate, Z is nitrogen, and R2 is an
activatable or reactive group attached to the benzo ring by way of an XY link. R2
may preferably be -XY-activatable group, where X is O, Y is methylene and the
activatable group is -CO2H.
In a particularly preferred embodiment, M is aluminum, each R1 is -XYW, X
is either oxygen or sulfur, Y is phenyl, W is sulfonate or sulfonyl chloride, R2 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. R2 may preferably be -XY-reactive group, where X is O or S, Y
is phenyl and the reactive group is -SO2Cl (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 hy~L o~en 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
-lo- I 3 3 7 7 5 4
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,
5 preferably at 10-6 M or lower concentrations. The aqueous solubility should bemaintained at temperatures ranging from about 4C (e.g., for flow cytometric
applications) to about 100C (e.g., 67C 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 thefluorophores 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 cellularconstituents.
Guided by the foregoing considerations, the water solubilizing groups W can
be selected from among -OH, -poly-OH, -CO2H, -OCH2CO2H, -OCHD1CO2H,
D CO H po 2- _po3-, -SO3, -SO2, -SO4, 2 3
2 , HD1D2/ ND1D2, and -N D1D2D3 with D-D3 being individually
alkyl (preferably C1-C12), aryl (preferably C6-C12), or aralkyl (preferably
C6-C12), 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 awide 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.
-11- 1 3 3 7 7 5 4
Substituent Y is a group of atoms that links X with the water solubilizing
group W or the reactive or activatable group R2. In a preferred embodiment Y is
methylene (-CH2-); however, longer alkyl, aryl, or aralkyl chains are possible
(preferably C2-C12). 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
0 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 alkynyland/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. Theprecise 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 Rl are individually selected from among -XYW, -YW, -W, and
hydrogen. In one preferred embodiment, all three Rl groups are -XYW, -YW, or
-W, especially -XYW. In another preferred embodiment, one R1 is -XYW and the
other two are -YW or -W.
Substituent R2 may be a biological entity such as an antigen or an antibody
attached to the macrocycle. R2 may also be an activatable group or a reactive
group; as such, R2 may be linked to the benzo ring by X or XY linkers, or may bedirectly attached to the benzo ring. In some embodiments, discussed herein, R2
may be R1, in which case no biological entity is covalently bound to the
fluorophore. In other embodiments, R2 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 R1.
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
-la- 1 3 3 7 7 5 4
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
5 macrocycle) is preferably attached to R2 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 C10-C30 alkyl group that may be straight chain, branched or may
- contain cyclic rings. The membrane probe may be attached to a benzo ring or to5 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, ~ -unsaturated carbonyl, thiol, sulfonyl chloride,
20 or halide group for attachment to the phthalocyanine. In turn, the phthalocyanine
should bear a correspondingly reactive group, such as carboxy, amino, thiol,
~,~-unsaturated carbonyl, sulfonyl chloride, or hydroxy.
The tether Y' to the biological entity, A, in R2 is long enough for optimal
recognition of A in typical biological assays. Displacement of A from the
25 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'. Hydrophilicspacers include polyethers, polyamines, polyalcohols, and naturally occurring
30 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.
-13- I 3 3 7 7 5 4
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 (-OCH2CO2H)
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 sbsorbance spectra of 2 and 3 in water are shown in FIGURE 3. Both
exhibit a common excitation wavelength in the ultraviolet (350 nm) with molar
absorptivities around 70,000. As shown in FIGURE 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
respectively.
The spectral resolution of the two fluorophores may be affected by
environmental effects. FIGURE 5 presents the emission spectra of 2 and 3 in
aqueous cetyl trimethylammonium bromide (0.010 M 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. Thefamily 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 a2,9,16,23 phenoxy substituted phthalocyanine. Similar reaction with
3-phenoxyphthalonitrile results in the formation of a 1,8,15,22 substituted
-14- I 3 3 7 7 5 4
I
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 8S avidin and streptavidin, enzyme substrates, membrane probes, nucleotides5 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 FIGURE 6. The emission spectra are shown in
FIGURE 7. The syntheses of _ and 5 and a tabulation of their spectral properties are given in ~mple 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
5 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 FIGURE 8. The emission spectra
are presented in FIGURE 9. The syntheses of 6 and 7 and a tabulation of their
20 spectral properties are given in Example 2. These compounds may be attached to
the above-mentioned reactive or activatable R2 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
25 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 1, Z is N for phthalocyanines, Z is CR for the tetrabenztriaza-
30 porphyrins). In this embodiment, the 20 carbon is phenyl substituted.
As in the most preferred embodiment, the family of four aluminum TBTAPfluorophores 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
35 3-thiophenoxyphthalonitrile provides TBTAP ring systems which are metalated
with aluminum and sulfonated to provide compounds 8-11, respectively. The
-15- I 3 3 7 7 5 4
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 FIGURE 10. Similarly, emission spectra of the tetrathiophenyl
derivatives are shown in FIGURE 11. As with the aluminum phthalocyanines, the
5 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
l o 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.,
l 5 -OH, -NH2, -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 TBrAP sulfonate, 13. The
absorbance and emission spectra of 12 and 13 in water are presented in
FIGURES 12 and 13, respectively. The preparation and spectral summary are
20 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
25 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
30 (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 _ in
water and in the presence of RNA are shown in FIGURES 14, 15, 16, and 17. The
35 preparation of 14a and _ and RNA binding experiments are presented in
Example 5.
-16- l 3 3 7 7 5 4
In a second aspect of this invention, there are disclosed derivatives of the
phthalocyanines and tetrabenztriazaporphyrins in which 1-4 of the benzo rings offormula 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
5 tetrabenztriazaporphyrin pyridine/pyrazine derivatives are contemplated, Z in
formula I is preferably N. R2 may be attached to a meso carbon, in which case
the benzo rings of the macrocycle will each have an R1 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 (4Q0-600 nm),
l 5 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 theexcitation 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 15M. 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 ofderivatives. These derivatives must be water soluble, have common excitation
f 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.
-17- 1 337754
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
5 300 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'
lO 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
15 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 basedfluorophores have bandwidths from about 21-30 nm (with the exception of 7,
20 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
25 based fluorophores must be monomerically tethered.
The emission spectra of four aluminum phthaIocyanine sulfonates (1, 4, 5, 7)
selected for nucleic acid sequence analysis are presented in FI~URE 18.
As noted above, fluorescence emission of phthalocyanines and TBTAPs may
be enhanced by rendering the fluorophores monomeric rather than aggregated.
30 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 thereforemore fluorescent in solution. The most preferred metals for fluorescent reagents35 are therefore aluminum, gallium, scandium, silicon, germanium, and tin.
-lB- I 3 3 7 75 4 62839-1163
Metallophthslocyanines and TBTAPs suitable tor magnetlc resonsnce
Imaging appllcatlons would bear paramagnetlc metals such as iron, manganese,
and gadolinium. Here the metals are In the plus three oxldatlon state.
Metallophthalocyanines and TBTAPs suitable tor radloactlve Imaglng and
therapeutlc appllcatlons would bear radlolsotopes of metals such as copper,
cobalt, galllum, and technetium. The radlonuclides are gamma-emltters and are
sensltlve Imaging probes.
We have discovered an empirical relationship between
the spectroscopic propertles (In terms of the relative helghts of the maximum blue
and red absorbance peaks) of these compounds to their relative quantum yield.
Early In our investigations of aluminum phthalocyanlne sulfonates we
observed that the blue absorbance was Independent of the state of aggregatlon and
hence the emlsslon yleld. In contrast, it was posslble to follow the onset of
aggregatlon by changes In the red absorption band. In general, we found that theA(red)/A(blue) ratlo decreases with decreasing relative quantum yield. In
addition, the behavlor ot the protein-bound dye is shlfted toward a lower relative
quantum yleld, but very nlcely paralleled the free dye In solutlon. This shlft or
decrease In quantum yleld presumably arises from the hydrophoblc nature ot the
protein envlronment rather than aggregation quenching.
The most preferred embodlments of the phythalocyanine conjugates of the
Invention, in terms of monomeric binding, have an A(red)/A(blue) > 2. Such
conjugates are readily prepared by Method 3 (see below).
Preferably, the A(red)/A(blue) ratio of the subject conjug~tes should be >
1.75, and such conjugates are readily prepared by Method 2.
Phthalocyanine con3ugates havlng A(red)/A(blue) ratlos between about 1.5
and 1.75, while suitable for some purposes, have relatlvely limlted sensitlvity and
so would not be useful.
Con3ugates havlng A(red)/A(blue) ratlos of less than 1 nre consldcred to be
not sultable tor use as ~luorescent markers.
The phthaloeysnlne and tetrabenztriazaporphyrln con3ugate~ o~ this
inventlon dlsplay slmilar tendencles In terms o~ thelr monomerlc blnding and itsrelatlon to the A(red)/A(blue) ratio. However, some of the specles disclosed in
this Invention have much stronger blue absorbances, e.g., compound 4, whlle
others show dlmlnlshed red absorbance, e.g., compound 12, as monomers In
aqueous environments. As a result, the most preterred methods ot con~ugatlon
yield a range ot A(red)/A(blue) trom 1.4 to 2.0, dependlng on the tluorophore.
-19-
1 337754
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
5 streptavidin.
Method 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 ina 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 30C 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 1, exceptthat R2 = XYW, R1, X, and Y being as described above and W =-N+D1D2D3,
wherein D1-D3 are independently H, alkyl, aralkyl or aryl, or W may be a pyridine
group. D1-D3 are preferably H, C1-C10 alkyl, C6_12 aralkyl or C6_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-, Br~, Cl, F, borate etc.
Exemplary positive counterions are Ca+2, Mg+2, Na+, K+, quaternary ammonium,
etc.
-20- 1 3 3 7 7 5 4
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.
-21- I 3 3 7 7 5 4
In another preferred embodiment, more than one analyte is determined
simultaneously using a corresponding number of binding partners each attached toa 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 aparticular 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
l O 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
l 5 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
e mployed.
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 secondfluorescent 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 nucIeic 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.
-22- I 337754
In the labeled primer strategy, a si~gle 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 oneof the four ddNTP's and labeled with one of the four dyes, the base sequence maybe 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 ofthe 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 sequencingfragments 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
-23- I 3 3 7 7 5 4
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 orany 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 fordetection of many agents and abnormalities, one 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
1 337754
-24- 62839-1 16 S
genetlc diseases) Is labeled wlth a different fluorophore. The cancers treatable or
detectable by the present reagents are not necessarily llmited and any one for
whlch a therapeutic or diagnostic agent hss been developed may potentially be
treated or diagnosed using the spproprlate fluorophores described herein.
The reagents dlsclosed hereln, partlcularly those of the flrst and second
aspects, may also be used for photodynamic therapy employlng 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.
Example 1
The Preparation of Aluminum Phthalocyanine Tetraglycolates
Tetrasubstituted phthalocyanines derived from monosubstituted
phthalonltriles are necessarily an inseparable mixture of four isomeric products.
l 5 The product phthalocyanines arise from the differences in orientation of the
phthalonitrile during the cyclization process. Cyclization of a 4-substituted
phthalonltrlle leads to the formation of 2,9,16,23-tetrasubstituted phthalocyanlne,
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 ~-substituted phthalonitrile provides the
corresponding 1,8,15,22-tetrasubstltuted 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 derlved from 4-
substituted phthalonitriles as 2,9,16,23. See FIGURE 1 for macrocycle position
nu mbering.
Tetrasubstituted aluminum phthalocyanines may be prepared from
monosubstituted phthalonitriles. Nitro displacement from either 3- or
4-nitrophthalonltrile with oxygen or sulfur nucleophiles provide thc corresponding
phthalonltrlles In good yield. The oxygen or sulfur reagent used In the nltro
displacement may impart to the phthalocyanine water solubility and tetherability,
or may be further elaborated to provide these required properties. Reagents suchas hydroxyacetlc acid and thioacetic acid may provlde appropriately
functionalized phthalonitriles directly (X is 0 or S, Y is CH2, and W is C02H).
Alternatively, tetraoxy or tetrathio substltuted phthalocyanlnes may be treated
with an alkylatlng agent such as methyl bromoacetate to provlde the fully
~unctionallzed phthalocyanine.
,~
, ,3 .: _ `
-25- I 3 3 7 7 5 4
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
5 ammonia in methanol followed by reflux in N,N-dimethylaminoethanol. Leznoff,
C.C. et 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
lO 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 Leznoff, C.C. et al., Photochem.l 5 Photobiol. 46:959-963, 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
20 potassium carbonate (forty molar equivalents of each) in refluxing methanol
affords the tetra methyl ester derivative. The alkylated product may be directlyhydrolyzed to the tetracarboxylic acid by heating in a solution of 0.5 M
methanolic potassium hydroxide. Aluminum hydroxy 2,9,16,23-
tetraglycolylphthalocyanine was isolated by precipitation, 2, from an aqueous acid
25 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 FIGURES 3 and
30 4, respectively. The effect of cetyl trimethlyammonium bromide (CTAB) on the
emission spectra of the two isomers is shown in FIGURE 5.
Tabulated below is a comparison of the spectral data for 1, 2, and 3 in water.
2 1 33775~
-- 6--
Phthslocyanine Absorbance Emission Quantum Yield
673 nm 683 0.60
692 704 0.55
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
0 detailed description of the preparation of a family of four aluminum
phthalocyanine based reagents. The presentation is organized into sections whichdetail 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 lN 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.
-27- I 3 3 7 7 5 4
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, theproduct 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
Phthalocyanine Metalation
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 2.0 M 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
25 distilled water followed by 1 mL lN aqueous hydrochloric acid. The solution was
then separated and the organic layer was washed with 3-20 mL portions lN
aqueous hydrochloric acid. The methylene chloride solution was dried over sodiumsulfate and concentrated to dryness. The product, aluminum hydroxy 2,9,16,23-
tetraphenoxyphthalocyanine, was isolated as a blue solid, 230 mg (0.25 mm,
30 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.
-28- I 3 3 7 7 5 4
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
l O 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
20 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.
25 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
-29- l 3 3 7 7 5 4
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 100C. The solution was stirred at 100C for one hour,
cooled to 0C, 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 70C, 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 100C, 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 80C, 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
_30_ l 3 3 7 7 5 4
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 FIGURES 6 and 7,
respectively. Spectral data are tabulated below.
Aluminum Hydroxy 1,8,15,22-Tetraphenoxyphthalocyanine Sulfonate (5)
l O 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
FIGURES 6 and 7, respectively. Spectral data are tabulated below.
Aluminum Hydroxy 2,9,16,23-Tetrathiophenylphthalocyanine Sulfonate (6)
l 5 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
FIGURES 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
FIGURES 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
~ 2,9,16,23 oxy 685 nm 697 nm0.49
1,8,15,22 oxy 707 717 0.20
2,9,16,23 thio 695 708 0.28
1,8,15,22 thio 719 733 0.10
Aluminum Acetylacetonate Tetraphenoxyphthalocyanine Sulfonates. Water
soluble aluminum phthalocyanine sulfonates were prepared from aluminum
35 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.
-31- l 3 3 7 7 5 4
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
20 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, reactivederivative 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
and 4.6 mL of 1.0 M benzylmagnesium chloride (4.6 mm, 1.0 equivalent) in diethyl35 ether was added. The mixture was stirred under argon at room temperature for 2
-32- l 3377 54
hour. The mixture was then concentrated to dryness and the purple residue was
diluted with 25 mL quinoline and stirred at 200-210 for 4 hours. The solvent was
distilled under vacuum. The resulting residue was treated with stirring with
30 mL glacial acetic acid at 90 for 2 hour. The reaction mixture was diluted
with 200 mL methylene chloride and washed first with 3-200 mL portions
5 saturated aqueous sodium bicarbonate and then with 200 mL 5% v/v aqueous
hydrochloric acid. The organic phase was dried over sodium sulfate, filtered andconcentrated. 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 Rf 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 geleluting 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 Rf 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 Rf of 0.~2 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
~33~ l 3 3 7 7 5 4
purification by crystallization from a methylene chloride: hexane (1:1) solutionafforded the product in 11% yield as a deep green solid. Silica thin layer
chromatography eluting with 65% methylene chloride in hexane gave an Rf of
0.70. Spectral data are tabulated below.
Tabulated below are the absorbance data for the oxygen and sulfur
5 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
1,8,15,220xy 676, 712
2,9,16,23 thio 666, 704
1,8,15,22 thio 694, 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. The mixture was
stirred at room temperature for two hours. The mixture was then cooled to 0
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.
_34_ t 3 3 7 7 5 4
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
~O
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 FIGURES 10 and 11,
respectively.
Compound Quantum
No. TBTAP Absorbance Emission Yield
" 2,9,16,23 oxy 664, 692 nm 695 nm 0.43
1,8,15,22 oxy 676, 704 711 0.22
0 2,9,16,23 thio 690, 713 717 0.14
_1 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 similar35 to the aluminum phthalocyanines sulfonates and possess the optical properties of
the aluminum tetrabenztriazaporphyrins. The following is a detailed description
-35- I 3 3 7 7 5 4
of the preparation of these compounds. The presentation is organized into
sections which detail tetrabenztriazaporphyrin preparation, metalation, reactiveTBTAP preparation, and water soluble TBTAP preparation. Within each section a
detailed procedure is given for the 20-hydrogen derivative followed by a commenton 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) 3.0 M methylmagnesium
bromide in diethyl ether. The resulting solution was stirred at room temperatureunder nitrogen for two hours. The ether was removed under vacuum and 25 mL
quinoline was added. The reaction solution was heated at 200 under nitrogen for16 hours. The solution was cooled and diluted with 1 L methylene chloride to
precipitate the crude product. The crude product was collected by filtration andA extracted with methanol~j,n 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
30 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.
fr~
-36- l 3 3 7 7 5 4
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 andwashed 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.
l O 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 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) 2.0 M
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 lN aqueous hydrochloric acid. The organic solution was
extracted with 3-20 mL portions lN 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.
-37- I 3 3 7 7 5 4
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 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
FIGURE 12. Spectral data are tabulated below.
30 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 FIGURE 13. Spectral
data are tabulated below.
-38- I 3 3 7 7 5 ~
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 Derivstives
Exemplary cationic phthalocyanines are presented in Example 5. The
derivatives in Example 5 satisfy formula I where M is either H2 or aluminum, each
Rl is -XYW, X is oxygen, Y is ethylene (-CH2CH2-), W is trimethylammonium
iodide, Z is nitrogen, and R2 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 FIGURE 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, FIGURE 14, is indicative of amonomeric phthalocyanine. The emission spectra for the two solutions are
compared in FIGURE 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 FIGURES 16 and 17, respectively. The fluorescence
enhancement upon RNA binding is 340. No fluorescence enhancement was
observed for either 1 or 14b in the presence of bovine serum albumin.
_39_ 1 337754 62839-1163
Tabulated below are the spectral data for the metal free and alumlnum
phthalocyanlne derlvatlves prepared as de~crlbed above. The emisslon wavelength
and fluorescence enhancement of the fluorophores In the presence of RNA are
presented. The absorbsnce data was recorded with a fluorophore concentratlon of
5 X 10 6 M and an RNA (Torula Yeast) concentration of l.0 mg/mL. The
5 fluorescence data was obtained for these ~olutlons at 100-fold dllutlon.
Emlsslon Wavelength Fluorescence
Phthalocyanine In Presence of RNA Enhsncement
Metal free 720 nm 450
Aluminum 705 340
Another specific embodiment of the cationic phthalocyanines ls the case
where in formula I M, R1 and R2 are as described above, and X = -CH2-,
Y = -CH2CH2- and W = diethylmethylammonlum. The counterion Is lodide.
Example 6
The Preparation of Fluorophore Streptavidin Conjugates
The preparation of covalent streptavldin fluorophore conjugates is described In
Example 6. The reactive forms of the red shifted alumlnum phthalocyanlne
derlvatives, the sulfonyl chlorldes, are coupled to streptavidin accordlng to
procedures analogous to those previously disclosed. T he following ls a
20 detailed descrlption of the preparatlons. While the Ex~mple
explicitly describes coupling to streptavidin, other proteins
nlay be coupled by the same methodology.
Dlrect Coupling of Aluminum ilydroxy 2,9,16,23-Tetraphenoxyphthalocyanlne
Sulfonyl Chloride to StreptavidJn. To 15.0 mg aluminum hydroxy 2,9,16,23-
tetraphenoxyphthalocyanine sulfonyl chloride solld was added 300 ~IL dry
dimethylformamide. The solution was placed in a pre-equilibrated 30C dry
bath. After one hour, 20 ~L of the dimethylformamide solution contalnlng the
rcactlve fluorophorc wns addcd dropwlse to 1.15 mg streptavldln In 185 ~IL 0.2 Msodlum bicarbonate in phosphnte buffered sallne plI ad3usted to 9.0 and contalnlng
30 IJL dimethylformamide at 4C. After one hour, the reaction was quenched by
the additlon of 250 ~IL of a 10 mg/mL solution of Iysine In 0.2 M sodlum
bicarbonate In phosphate buffered sallne containing 0.02% sodlum azlde as a
preservatlve. After stlrrlng for 30 minutes at 4C, the con3ugate was purlfied by
slze excluslon chromatography on Sephadex*G-50 In phosphate buffered sallne
contalnlng 0.02% sodlum azlde. Spectral data for the con3ugate Is tabulated
below.
*Trade-mark
1 337754
62839-1163
Direct Coupllng of Aluminum Hydroxy 1,8,15,22-Tetraphenoxyphthalocyanlne
Sulfonyl Chlorlde to Streptavldin. In a procedure analogous to that described
above, alumlnum hydroxy 1,8,15,22-tetrsphenoxyphthalocyanine sulfonyl chlorlde
was coupled to streptavidin. Spectral datn ~or the conJugate Is tabulated bclow.Direct Coupllng of Aluminum Hydroxy 1,8,15,22-Tetrathiophenylphthalocyanlne
5 Sulfonyl Chlorlde to Strepta~idln. In a procedure analogous to that described
above except that a 30 mlnute incubatlon at 30C was used rather than a one hourIncubatlon, alumlnum hydroxy 1,8,15,22-tetraphenoxyphthalocyanlne sulfonyl
chloride was coupled to streptavidln. Spectral data for the con~ugate is tabulated
below.
l 0 Tabulated below are the absorbance and emission wavelengths for the
fluorophore streptavidln con3ugates in phosphate buffered saline containlng 0.02%
sodium azide and prepared as described above. The fluorophore per streptavidin
ratio (F/P) was determlned by comparlng the absorbance of the proteln at 280 nm
relative to the Eluorophore absorbance at 350 nm. The quantum yields reported
l5 are per fluorophore.
Phthalocyanine Absorbance l~mission 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 Nuclelc Acid Primers
The preparatlon of covalent fluorophore labeled nucleic acid primers i9 described
in Example 7. The reactive forms of the red shifted aluminum phthalocyanine
25 derivatives, sulfonyl chlorides, are coupled to nucleic acid primers according to
procedures analogous to those previously disclosed. T he following is a
detailed description of` the preparations.
Alumlnum ~Iydroxy 2,9,16,23-Tetraphenoxyphthalocyanine Labeled M13mpl8 (-21)
30 Universal Sequencing Primer. To a stirred solution of 0.022 ~mol aminohexane
modified M13mpl8 (-21), 5' TGTAAAACGACGGCCAGT 3', Universal sequenclng
primer in 20 ~L 0.5 M sodium bicarbonate/0.5 M sodium carbonate (pH adjusted to
9.0) was added 1.3 mg aluminum hydroxy 2,9,16,23-tetraphenoxyphthalocyanine
sulfonyl chloride in 12 ~L dimethylformamide. After stirring overnight at room
35 temperature in the dark, the labeled primer was purified by slze excluslon
chromatography (Sephadex G-50) followed by polyacrylamide gel electrophoresis.
-41- I 3 3 7 7 5 4
Spectral data for the labeled primer is tabulated below.
Aluminum Hydroxy 1,8,15,22-Tetraphenoxyphthalocyanine Labeled M13mpl8 (-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 ethanol5 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 0.1 M 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 ~15 0.18
Example 8
5 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
20 Examples 3 and 4), a reactive 20-substituent may be synthesized. The Grignardreagent 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 R2 enable efficient coupling to
25 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.
30 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
35 the fluorophore couples to the N terminus of the peptide to be sequenced which is
-42- 1 337754
immobilized (C terminus) on a solid pha~e. 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 g~roups.
Fluorinated 20-substituted TBTAP derivatives such as trifluoromethyl (CF3)
and perfluorophenyl (C6F5) 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
1 337754
-43-
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 w;ll 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
l S 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: Hanack, M. et al., Inorg. Chem.,
23:1065-1071 (1984). 3,6- and 4,5-octathio: Luk'yanets, E.A. et al., J. Org. Chem.
USSR, 14: 1046-1051 (1978). The sulfur substituted derivatives absorb at greaterwavelengths 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.
_44 l 337754
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
5 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. 911-922, 1937. Phthalocyanines bear four
benzo rings appended to the macrocycle while pyrazine porphyrazines have four
pyrazine (1,4-diazabenzene) rings.
~;~
Elaboration of tetra- and octaphenylpyrazine porphyrazine to reactive, and watersoluble aluminum derivatives is the subject of Example 12. Cyclization of either5-phenyl or 5,6-diphenylpyrazine 2,3-dinitrile results in the porphyrazine
45_ l 3 3 7 7 5 4
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. 911-922, 1937. Structurally, replacement of
the benzo ring in phthalocyanine with pyridine gives pyridine porphyrazine.
20~ ~
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 providedthe aluminum derivatives.
Application of the oxygen and sulfur substitution methodology developed for
the phthalocyanines and tetrabenztriazaporphyrins as described in Examples 2 and3, respectively, will result in a family of reagents for each of the aluminum
pyridine porphyrazines.
-46- I 3 3 7 7 5 4
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:
25 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
30 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. 43:691-699
(1986). The reagents of this invention offer two improvements over the existing
technology. The first advantage lies in the deep red absorbance of the disclosed35 reagents and the second in the targeting of these reagents made possible by their
biological binding conjugates.
-47 I 3 3 7 7 5 4
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 thenear 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 commonlyrelied 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 R2 is
antibody-SO2-, Z = N, two Rl groups are -SO3- the third R1 group is hydrogen
(Dye I) and anti-CD8 (suppressor T cell specific monoclonal antibody) labeled with
phthalocyanine (1) where R2 is antibody-S02-phenyl-0, each Rl is XYW, wherein
X = O, Y = phenyl, W = -SO3 . The R1 and R2 group, are located at the 1, 8, lS,
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
-48- 1 3 3 7 7 5 4
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 ofequivalents, 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
10 equivalents thereof.
