WO1999049831A2

WO1999049831A2 - Amplified fluorescent materials and uses thereof

Info

Publication number: WO1999049831A2
Application number: PCT/US1999/000203
Authority: WO
Inventors: Vladimir V. Martin; Alexander Weis
Original assignee: Martin Vladimir V; Alexander Weis
Priority date: 1998-03-28
Filing date: 1999-01-05
Publication date: 1999-10-07
Also published as: WO1999049831A3; AU2311599A

Abstract

Compositions of fluorescent amplifier molecules are provided, as well as synthetic methods for their preparation. The procedures provide for the preparation of gram quantities of fluorescent molecules from available precursors. The fluorescent amplifier molecules include a multi-dimensional, rigid core (C), to which at least two fluorescent moieties are attached by way of a linker (L). Amplifier molecules that further include a 'targeting' molecule (a molecule that functions to direct the fluorescent amplifier to a particular binding site) are also presented, the targeting molecule being attached to the rigid, multi-dimensional core. Signal amplification without steric restrictions of fluorophore mobility is provided, hence reducing and/or avoiding interactions between moieties as well as fluorescence quenching. The advantage of the derivatives of the present invention, in addition to other things, over conventional fluorescent labels is due to the ability to amplify signal upon accumulation of the fluorescent moieties. The new amplified molecules are further disclosed to have the capability to quantitate the fluorescence signal. the compositions may also be employed as nucleic acid fluorescent -in-situ-hybridization probes.

Description

AMPLIFIED FLUORESCENT MATERIALS AND USES THEREOF

The government owns rights in the present invention pursuant to grant number CA62467 from NTH, (e.g., the National Institute of Health) and Grant R44CA62467 from the NIH National Cancer Institute. FIELD OF THE INVENTION

The present invention relates generally to the fields of fluorescent molecules. More particularly, it concerns fluorescent molecules that include a structure that amplifies the intensity of a fluorescent moiety and that may be used in both medical and industrial applications where a fluorescence used at least in part as a substrate is desired.

BACKGROUND OF THE INVENTION

Many diagnostic and chemical agents, particularly diagnostic agents, have molecular structures that comprise an "active group" (i.e., a chemical moiety that is directly responsible for the desired diagnostic or chemotherapeutic effect) connected to another molecular structure(s). These types of molecules are useful for any of a variety of purposes such as, but not limited to, solubility of an agent, absorption of the agent, physiological transport of the agent, such as through biological membranes, biotransformation of the agent, or targeting of the agent to a particular situs in the subject. The vast majority of such agents have only one active group per molecule of the agent.

Fluorescence is the emission of an absorbed quantum of electromagnetic energy. This physical phenomenon takes place in molecules such as fluorescent dyes or fluorophores. Fluorescence is the result of a three-stage process, which starts with excitation of the fluorophore molecule by a light (lamp or laser) to an excited singlet state (absorb tion stage). In the second stage (excited-state lifetime), the excited molecule is subject to interactions with its environment, resulting in the dissipation of energy. The new excited state, resulted from these interactions, emits the photon and the molecule returns to the initial electronic state

(emission stage) (Lakowicz, J.R., Ed. Principles of Fluorescent Spectroscopy, Plenum Publishing (1983)). The techniques of fluorescence registration are well-developed and described in the technical literature (Harris, D.A. and Bashford, C.L., Eds., Spectrophotometry and Apectrofluorimetry: A Practical Approach, IRL Press, 1987). The high sensitivity of the fluorescence registration and environmental dependence of the fluorescence make fluorescent dyes important tools for scientific research. Fluorescence registration enjoys a central role in such fields as molecular biology, biochemistry, and medical diagnostics. Most of these applications are based on use of fluorescent dyes introduced into a biological system. The fluorescent dye can be attached to a biological object (biotarget), such as protein, nucleic acid, cell membrane, etc. by a covalent bond, or, alternatively, can be studied without being covalently attached to biotarget. One type of fluorescent dyes are designed for covalent attachment to biotargets were called fluorescent labels or fluorescent tags. They usually consist of a fluorophore fragment and a chemically reactive moiety (targeting group) capable of forming a covalent bond with certain functional groups of the biotarget. A second type of fluorescent dyes are employed in fluorescent study without being attached to biotargets by covalent bond, and are called fluorescent probes.

Fluorescent probes and labels can be recognized in biological systems by three major methodologies: a) fluorescent spectroscopy that measures the average properties of the bulk sample; b) fluorescent microscopy that investigates the distribution of the fluorescent dye as a function of spatial coordinated in two or three dimensions; and c) flow cytometry that measures fluorescence per cell in a flowing stream. The successful applications of fluorescent dyes in biological studies brought about the demand for both new investigative techniques and for the design and synthesis of new fluorescent dyes. Efforts continue to improve the sensitivity and reliability of the fiuorophore's registration. Along with hardware improvement and data processing sophistication, this could be achieved by increasing the signal level from the probe molecule. Such a signal increase would be advantageous because it enhances the resolution of the registration by providing a higher signal-to-noise ratio and lower background fluorescence.

One way of increasing the signal level from a labeled biotarget is to employ more fluorescent labels for biotarget labeling. However, the number of possible binding sites in biological targets is limited. In addition, attachment of a significant-sized label (such as with fluorescent label) to a biological molecule is observed to affect its physical properties (such as solubility, lipophilicity, pKa, etc.) and biological activity. To avoid the saturation of the binding sites of a biotarget with labels, a novel type of labeling agent, called a molecular amplifier, has been used. These molecular devices are designed to introduce multiple copies of the label to a single binding site of the biotarget. An example of molecular amplifiers delivering paramagnetic labels (metal complexes or stable nitroxide radicals) to create contrast enhancement for medical MRI diagnostics is described by J.F.W. Keana.

In J.F.W. Keana's U.S. Pat. Nos. 5,135,787 and 5,252,317, incorporated herein by reference, amplifier molecules are described. These amplifier molecules are described as having multiple diagnostically or therapeutically active groups (such as, but not limited to, nitroxides or paramagnetic metal-ion chelators). Thus, administering a particular number of molecules of such amplifiers would provide a more enhanced effect than administering an equal number of conventional molecules having only one active group per molecule. Also, fewer individual "particles" need be administered to achieve an acceptable effect when amplifiers are used. This is important in the control of the osmolarity of an administered solution of the agent. More particles can result in a greater imbalance in osmolarity and thus greater pain sensation during administration of the compound. Because amplifiers used for contrast enhancement are generally larger than conventional molecules, amplifiers have a slower, more optimal "tumbling rate" which leads to greater enhancement per paramagnetic center. The applicants have demonstrated in the examples provided below, that the original concept of molecular amplifiers does not work well in the case of the fluorescent probes. The results, included in the Example two (2), describe the synthesis of molecular amplifiers structurally similar to what was used in MRI. However, these molecules have fluorescent fragments instead of paramagnetic moieties. The subsequent results listed in Example five (5) show that the fluorescence yield of the amplifiers drops with the accumulation of the fluorescent moieties. The reason for this is mutual quenching of fluorescence because of the interaction of fluorophores in the multifluorophore molecule. This effect has been intensively studied and well-reported in the literature (Haughland, R.P., Handbook of Flourescent Probes and Research Chemical, 6th Ed., Eugene, O. R: Molecular Probes 14, 19, 31, 1996.). The mutual quenching of fluorescence is also common in multiple labeling of biotargets when the increased number of the labels involved decreases the fluorescent yield (Khanna, P.L., Ullman, E.F., Anal. Biochem., 108:156-161, 1980).

SUMMARY OF THE INVENTION

The presented invention introduces a novel type of fluorescent materials that are designed to possess three major characteristics: (1) providing one-site multiple labeling;

(2) reducing mutual quenching interactions between fluorophores; and (3) reducing the interaction between fluorophore and biotarget. The latter effect leads to a quenching of the fluorescence in fluorophores, attached to a biotarget.

The present invention, in a general and overall sense, concerns the synthesis and characterization of improved fluorescent molecules having a central rigid core. In some embodiments, the central rigid core comprises an amide prepared from adamantane tetracarboxylic acid and fluoresceinamine. The invention also provides synthetic methods for amplified fluorescent molecules in preparative (gram) scale from commercial precursors. The fluorescent properties of the prepared amplified units and fluorescein derivatives in some embodiments include fluorophores attached to the center core through a spacer molecule. These embodiments of the invention are focused on the effect of the space between each of the fluorescent moieties. These fluorescent molecules are positioned as to minimize and/or alleviate the quenching effect that is characteristic of other conventional fluorescent molecules. The fluorescent properties of the conjugates are improved through use of linker molecules of different lengths in attaching fluorescent moieties to the rigid core. The linker molecule may be rigid or flexible.

In some embodiments, the composition of the present invention comprises the following general structure:

wherein C is a three-dimensional molecular core, and extending from said three-dimensional core is a polyfunctional fluorescent moiety, FI. By way of example, the core may comprise adamantane, Cubane, fullerine, or a combination thereof. The compounds may include any number of polyfunctional fluorescent moieties, from at least two to as many as twenty. In some embodiments, there will be included 3, 4, 5, or 6-20 polyfunctional fluorescent moieties. A linker (L) may be included in other embodiments of the invention. The linker functions at least in some embodiments of these molecules to attach the FI to the C.

The FI, in some embodiments, comprises a fluorophore such as fluorescein, Texas Red, Lucifer Yellow, MCA Blue, rhodamine, or a combination thereof. The two fluorescent FI may be the same or different fluorescent moieties. L, in some embodiments of the various chemical structures defined herein, may be further defined as -CO-NH-; -C₆H₄-; -NH-SO₂-; - NH-SO₂; -CO-O; -NH-; -O-; -S-; NH— C(S)-; NH-CO-NH-; NH-C(S)-NH-; -O-C(O)-; -O- and -C(O)-.

In the above aspect, of the present invention, the molecule may be defined as comprising a structure:

wherein C is a three-dimensional molecular core, L is a linker, and FI is a fluorescent moiety. In yet another aspect, the composition of the present invention comprises the following general structure:

wherein C is a three-dimensional molecular core, L is a linker, and FI is a fluorescent moiety.

In yet another aspect, the composition of the present invention comprises the following general structure:

wherein C is a three-dimensional molecular core, L is a linker, FI is a fluorescent moiety, and X is a targeting group. In the above model, the FI moiety may all comprise the same fluorescent moiety, or they may all be different fluorescent moiety. In particular embodiments, the various constituents of the composition may be defined as follows:

C = Adamantane, Cubane, fullerine, or any three-dimensional molecular core capable of supporting the attachment of a linker molecule, L. X = Targeting group comprising COON, NH₂, SO₂, Cl, O₂, protein, peptide, biotin, antibody, polynucleotide chain, or a combination thereof. Responsible for bringing the molecular amplifier probe to preferable environment or to form a chemical bond with a

desired biotarget. FI = Fluorophore comprising fluorescein, Texas Red, Lucifer Yellow, MCA Blue, rhodamine, or a combination thereof.

L = Linker group that is capable of connecting the fluorophore, FI, to the core and connects the targeting group, X, to the core which may be further defined as: -CO-NH-; -C₆H₄-;

-NH-SO₂-; -NH-SO₂-;

-CO-O; -NH-; -O-; -S-; NH— C(S)-; NH-CO-NH-; NH-C(S)-NH-; -O-C(O)-; -O- or -C(O)-.

wherein:

C = Adamantane, Cubane, fullerine, or any three-dimensional molecular core capable of supporting the attachment of a linker molecule, L; X = Targeting group comprising COON, NH₂, SO₂, Cl, O₂, protein, peptide, biotin, antibody, polynucleotide chain, or combination thereof; L = Linker group connects the fluorophore, FI, to the core and connects the targeting group,

, X, to the core; and FI = Fluorophore comprising fluorescein, Texas Red, Lucifer Yellow, MCA Blue,

Chodamine, or a combination thereof.

Materially, the function of the core is to prevent the fluorescent moieties (FI) of the molecule from interacting with each other and with a target biomolecule. The rigid core will also function to prevent the fluorescent molecules from intercalating into nucleic acid. Examples of the linker (L) that may be used in the practice of the invention include those which are created through a chemical reaction of the core molecule and a starting material. Examples of these types of molecules are presented below:

Linker group formed

I I

1 . -C- NH₂ % FI-CO-Y p> -C- NH-CO-FI Amide

I I carboxylic acid active derivative,

Y= alogen, H-hydroxysuccinimide residue, imidazole, C₆F₅0-, etc.

I I

2. -C- CO-Y % FI-NH₂ ^ -C- CO-NH-FI Amide

I I

3. -C- OH % FI-CO-Y ► -C- O-CO-FI Ester

I I

4. -C- CO-Y % FI-OH ► -C- CO-O-FI Ester

5. -C- NH₂ % Fl-S0₂-Hal ► -C- NH-S0₂-FI Sulfamide

| Hal=halogen |

I I

6. -C- NH₂ % Fl-Z ► -C- NH-FI Amine

| Z=halogen, |

OTs, etc. » 1

7. -C- NH₂ % FI-N=C=0 - -c- NH-CO- Urea

1 NH-FI

1 I 1

1 1

8. -C- NH₂ % FI-N=C=S - ^ -c- NH-CS- Thiourea

1 1 NH-FI 1 1

9. -C- OH % Fl-Z -c- O-FI Ether

1 1

10 -C- SH % Fl-Z w -C- SH Thioether

I 1

11 -C- Z % FI-C₆H₅ - k ^ -c- C₆H₄-FI Arylidene

I 1 derivative

As used throughout the description of the various molecules and structures of the present invention, the following abbreviations are used:

C = Adamantane, Cubane, fullerine, or any three-dimensional molecular core capable of supporting the attachment of a linker molecule, L;

X = Targeting group. Responsible for bringing the molecular amplifier probe to preferable environment or to form chemical bond with a desired biotarget. (X=COON, NH,, SO₂, Cl, O₂, protein, peptide, biotin, antibody, polynucleotide chain, or a combination thereof); Fl = Fluorophore. Responsible for registration with spectrafluorimeter.

(FI fluorescein, Texas Red, Lucifer Yellow, MCA Blue, Rhodamine, or a combination thereof); L = Linker group. Responsible for connecting the fluorophore, FI, to the core and connecting the targeting group, X, to the core. L= -CO-NH-; -C₆H₄-; -NH-SO₂-; -NH-SO₂-; -CO-O; -NH-; -O-; -S-; NH— C(S)-; NH-

CO-NH-; NH-C(S)-NH-; -O-C(O)- and -C(O)-.

The fluorescent molecules of the present invention in some embodiments may further include a linker (L) extending from the core without an attached targeting group. In other embodiments, the linker may include another fragment that will impart specificity of attachment of the fluorescent molecule to a desired target. Representative targeting groups (not intended to be limiting) include: polypeptides, antibodies, proteins, nucleic acids, carbohydrates, fatty acids, surfactants, glycerides, porphyrins, enzyme-inhibitors, and steroids.

The advantages of the various multiple polyfunctional group containing molecules of the invention include, by way of example:

Prevention of the formation of fluorophore-fluorophore collision complex;

Prevention of the probes from intercalation interaction with DNA strains;

Permitting the use of core for active group attachment;

Labeling single target site with fluorescent unit(s); and

Preparation using available starting materials and employing relatively developed chemistry.

• Prevention of the probes from quenching interactions with biotarget.

The fluorophores FI presented above could be in protected, inactive or masked form, and are able to regain fluorescent properties upon the physical (by light, EM radiation, electron beam, heat) or by a chemical treatment or enzymatic reaction. The ability to produce influx of the fluorescence upon treatment permits the use of the presented compounds in a variety of registration and diagnostic techniques, including ELISA (Vann, W.F., Sutton, A; Schneerson, R., Meth. Enzymol., 1990, 1984, 537), ELF (Haughland's Handbook on Mol. Probes, p.117, 1996; U.S. Pat. No. 5,316,906; U.S. Pat. No. 5,443,986;) and others.

It is envisioned that the invention, among other uses, may provide application in: A. Medical diagnostics. The probe will be connected to a polynucleotide chain to form a complimentary complex with particular polynucleotide sequences, providing diagnostic information (X=polynucleotide chain). Advantages over a conventional FISH probe include: 1) an amplified signal; 2) an increase in selectivity; and 3) a reduction in the amount of test biomaterial (blood, bone marrow) needed to run a diagnostic screen. B. Biomedical imaging including fluorescent microscopy. The probe will be connected to an antibody or receptor target. It will be directed to a desired environment providing imaging of the biotarget (receptor, tumor, etc.). The advantages over conventional molecule probes include: 1) an amplified signal; 2) a higher signal-to-noise ratio; 3) an increase of contrast of image; and 4) an increase in sensitivity.

C. Flow cytometry. The probes will be connected to an antibody, hap ten, receptor target, or polymer microsphere. Also, it can be employed in masked form and set to produce fluorescent response to enzymatic transformation. The probes will be used to sort individual cells based on the enzymatic activity or to spot the presence of specific biotargets. The advantage over conventional probes are: 1) an amplified signal; 2) a higher signal-to-noise ratio; 3) higher sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG 1 HPLC chromatogram of the conjugates' mixture.

FIG 2 Absorbance of compounds 9-12 @ 490 nm at pH 8; ♦ - fluorescein; ■ =

compound 9; ▲ = compound 10; • = compound 11; and x = compound 12. FIG 3 Excitation absorbance of compounds 9-12 @ 490 nm at pH 8; ♦ = fluorescein;

■ = compound 9; ▲ = compound 10; • = compound 11; and x = compound 12.

FIG 4 Emission data of compounds 9-12 @ 510 nm at pH 8; ♦ = fluorescein; ■ =

compound 9; ▲ = compound 10; • = compound 11; and x = compound 12. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Following long-standing patent law convention, the terms "a" and "an" mean "one or more" when used in this application, including the claims.

The various molecular embodiments of the invention may be prepared to include a

"targeting" moiety specifically intended to attach to a biologically important target molecule.

Examples of the biologically important target molecules are listed in Table 1.

TABLE 1 Biological Target Molecules polypeptide diglyceride antibody triglyceride nucleic acid steroid carbohydrate porphyrin fatty acid enzyme inhibitor surfactant

These and other biologically important molecules can be targeted by attaching the "targeting" moiety to a linker (L), the linker then being attached to the core (C) of the molecule. This can be readily accomplished by attaching the targeting material such as a polypeptide via chemistry as disclosed herein or by chemistry known in the bioconjugate art.

Since many antibodies (which are polypeptides) include lysines, an amplifier according to the present invention can be readily attached to a monoclonal antibody and thus be given a "targeting" capability (i.e., rendered capable of being taken up by, retained by, or bound to a particular situs in the body to a substantially greater degree than to other sites in the body).

With monoclonal antibodies, the corresponding target situs will depend on the particular immunospecificity of the monoclonal antibody.

The present invention is particularly capable of being attached to nucleic acids, carbohydrates, and fatty substances. For example, a boronic acid target group enables the amplifier to bind selectively to vicinal diol groups on carbohydrates or on carbohydrate portions of certain proteins or cells. As another example, a fatty acid or other substantially hydrophobic targeting group will be included, rendering the amplifier to which the molecule is attached particularly capable of attaching to peptides, polypeptides, and other biomolecules having substantial hydrophobic domains (such as the serum albumins). According to the present invention, such binding of amplifiers to serum albumins facilitates imaging of intravascular structures and vascular dynamics. According to the present invention, other potential hydrophobic targets for such compounds include any of various membrane structures, both extracellular and intracellular. The molecule can also be fashioned to have a net charge, thereby facilitating electrostatic attachment of the compound to yet another biomolecule of choice. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

PREPARATION OF THE ADAMANTANE 1. 3. 5. 7-TETRACARBOXYLIC

ACID TETRACHLORIDE AND REACTION WITH 5-AMINO FLUORESCEIN

In this example, one to four fluorescein moieties were connected to the rigid adamantane core. Molecular modeling study showed that adamantane substituted in the bridgehead positions could provide the necessary template for building probes with non-interacting fluorophores. This template, if employed as a central core of the molecular amplifier, could accommodate from one to four fluorophores, or up to three fluorophores and a targeting group. Also, because of the adamantane-base probe's overall size, the proposed design should provide additional advantages in DNA probing especially in fluorescense-in-situ-hybridization (FISH) diagnostics, by reducing the unwanted stacking of the aromatic fluorophores into the helical nucleic acid structure.

Among other advantages, the adamantane core presents a relatively well-developed chemistry for derivitization. The proposed synthetic route to the targeted compounds with the adamantane central core and fluorescein fluorophores include a reaction of adamantane- 1, 3, 5, 7-tetracarboxylic acid tetrachloride with 5-aminofluorescein.

For the synthesis of the starting tetrachloride 5, the procedure of Newkome, et al, J. Org. Chem., 1992 was modified for scaling up 5-7 times to obtain about 7.5g of key adamantane- 1,3, 5, 7-tetracarboxylic acid 4 in a single run. The yields were close to those reported in the literature (Newkome, et al, 1992). The synthesis of this intermediate 4 (Scheme 1) starts with conversion of dimethyl malonate 1 into Meerwein's ester 2 by treatment with formaldehyde in methanol in presence of triethylamine, following by heating with sodium methylate. Cyclization of the ester 2 into compound 3 was achieved by heating with methylene bromide in the presence of base in a Parr bomb at 125-130°C for 6 h. The two carbonyl groups in the cyclic intermediate 3 were removed by Wolff-Kishner reduction. This reaction required heating at 200-250°C in a closed bomb and also resulted in hydrolysis of the methyl esters to yield the target adamantane tetracarboxylic acid 4. Acid 4 was determined to be pure by NMR spectroscopy and had a melting point close to that reported in literature

COOMe COOMe

2

j. 1 CH₂0/Et₃N, 2 NaO Me/MeOH, n. CH^R^NaOMe. Me OH, 125-1 0° C

JIΪ. iySTH_j, NaMe/MeOH, 200-250°C, jv. Sθα₂, P Cl (Newkome, et al, 1992).

Scheme 1

The acid 4 was converted into tetrachloride 5 by heating in plain thionyl chloride at reflux temperature. Acid chloride 5 was obtained upon evaporation of the volatiles as a crystalline material that was used in the next step (Scheme 3) without purification.

The key intermediate for the tetrachloride 5, acid 4, was also prepared through photochemical carboxychlorination of adamantane- 1, 3-dicarboxylic acid 6. The procedure gives tetrachloride 5 as a primary product. In order to purify this compound, it was converted without isolation into tetraester 7. This stable intermediate was purified by liquid chromatography and then hydrolyzed under basic conditions to give the acid 4 (Scheme 2). The acid 4 was converted into tetrachloride 5 by heating in plain thionyl chloride at reflux temperature. Acid chloride 5 was obtained upon evaporation of the volatiles as a

crystalline material that was used in the next step (Scheme 3) without purification.

The key intermediate for the tetrachloride 5, acid 4, was also prepared through photochemical carboxychlorination of adamantane- 1, 3-dicarboxylic acid 6. The procedure gives tetrachloride 5 as a primary product. In order to purify this compound, it was converted without isolation into tetraester 7. This stable intermediate was purified by liquid chromatography and then hydrolyzed under basic conditions to give the acid 4 (Scheme 2).

i. C1COCOC1, hv, n. Me OH, in. NaOH/ILO MeOH Scheme 2

This sequence was published by A. Bashir-Hashemi, et al (Tetrahedron Lett., 1995, 36,

1233-1236) as a short communication with only few experimental details provided. It was developed into a synthetic procedure that provides production of tatraacid 4 in preparative quantities. Compared to the conventional method (Scheme 1), the photochemical procedure is faster, uses less chemicals, and does not employ high pressure - high temperature reactions.

The reaction between adamantane- 1,3, 5, 7-tetracarboxylic acid chloride 5 and amino fluorescein 8 was performed in presence of pyridine in chloroform - N- methylpyrrolidone mixture at low temperatures (Scheme 3). Due to a lack of discrimination

These oligomeric by-products from side-acylation of the fluorescein phenolic hydroxyls were cleaved by mild basic hydrolysis of the reaction mixture in aqueous triethylamine. Triethylammonium salts of the products 9 - 12 were precipitated and separated from the excess

5-aminofluorescein 8 by ion-exchange chromatography on Dowex-50 (in H*^" form), to give the mixture of adamantane (N-fluorescein) amides 9 - 12.

HPLC analysis of this mixture on reverse-phase column showed that mixture contained

3.0% of mono amide 9, 19.8% of di amide 10, 39. 5% of triamide 11 and 35.4% of tetraamide 12 (Figure 1) (percentages correspond to the peak area monitored @254 nm, not the actual amount of components). The mixture also contained about 2.5% total (uncorrected) of several unidentified impurities. Most of the tetraamide 12 was precipitated from the mixture by treatment with aqueous

2-propanol, in which the solubility of this compound is very limited, and thereby separated.

Further separation of the fluorescein amides by column chromatography was complicated by their high polarity. However, samples of monoamide 9 and diamide 10 were prepared using reverse-phase preparative TLC or LC for separation. To achieve separation in preparative scale the mixture was treated with acetic anhydride in the presence of pyridine to get compounds 13 - 16 with acetylated fluorescein moieties. This mixture was separated by flash- chromatography to get the individual acetylated di-, tri- and tetraamides 14-16. Acetylated monoamide 13 was not isolated in a pure form, but as a component of the mixture of several polar compounds. Acetylated compounds 14 - 16 are stable to both TLC and column chromatography, if the eluent contains about 1% of acetic acid. The compounds 14-16 were converted to targeted compounds 9 - 12 by mild hydrolysis in aqueous ammonia. Monoamide 9 required additional purification by reverse-phase chromatography and slowly decomposed upon storage. However, other unprotected compounds, especially triamide 11 and tetraamide 12 were obtained after deprotection in analytically pure form (about 100% pure on HPLC) and are very stable to storage. The final compounds 9-12 were characterized by spectral data (NMR, UV; IR), HPLC and ES MS. When five-fold excess of starting amine 8 was employed, the final yields of the products were as follows: monoamide 9, 6%; diamide 10, 20%; triamide 11, 26%; tetraamide 12, 18%.

Intermediate acylated compounds 14-16 were characterized by spectral data and

HPLC-MS combination. EXAMPLE 2 SYNTHETIC PROCEDURES

A. Photochemical carbochlorination. Into 1-L round-bottomed flask, equipped with magnetic stirring bar and effective reflux condenser, terminated with Drierite drying tube, adamantane 1, 3-dicarboxylic acid 6 (5g) was suspended in 600 ml of oxalyl chloride. The mixture was placed into a preheated 70 °C oil bath and refluxed upon stirring until the acid dissolved (from 4 to 6 h). The mixture was cooled down to room temperature (r.t.) and was transferred into a quartz photochemical reaction vessel, equipped with small magnetic stirring bar, cooling finger, connected to a chilling circulator (set for -5 °C) and a gas outlet, terminated with a Drierite tube. The vessel was placed into a Rayonet photoreactor on an air stirrer. The mixture was irradiated for 50 min at -5 °C. It was quickly transferred into 2-L round-bottom flask and was evaporated to dryness. To the evaporation residue, cold (-5 °C) dry methanol was added (HC1 evaluation), and the flask was closed with a Drierite tube and allowed to react at r.t. for 2 h. The mixture was evaporated, re-dissolved in 200 rnL EtOAc, washed in a separation funnel with saturated NaHCO₃ (2 x 25 mL, sat. NaCl (50 mL), dried over MgSO₄ and evaporated to dryness. Chloroform (50 mL) was added and the mixture was evaporated again to remove traces of EtOAc. Chromatography (TLC) of the mixture (developed in plain CHC1₃) revealed traces of starting material (R_f = 0.9), a spot about 0.5 and a major spot of a product at 0.4, followed by faded spot(s) of polychlorinated compound(s). For visualization, a treatment with phosphomolibdic acid solution, followed by heating on an open flame was used. The mixture was loaded on a silica gel column (6 cm x 50 cm bed; 1100 mL silica gel), and eluated (flash speed) with plain chloroform. The results were: 2000 ml - Void; 2000 mL - starting, etc.; 850 mL - void; 1300 mL - upper spot; 150 mL - mixture (product + upper spot); 880 mL - product; 120 mL (traces of product + lower spots). The evaporation of the product fractions followed by treatment with cold hexane (5 mL) (product was cooled, filtered and washed with 2 x 5 mL cold hexanes) to give from 1.27 g (lowest) to 2.04 g (highest); usually 1.7 g yield of the pure (single spot on TLC) tetraester 7.

The mixed fractions and hexane washings were evaporated together. After five runs of these procedure they were re-chromatographed to give about 2 g of the pure product 7. Hydrolysis. In a 250 mL flask, equipped with a magnetic stirring bar and reflux condenser, tetraester 7 (1.0 g, 3 mmol) was suspended in MeOH (30 mL). Sodium hydroxide (30 ml of IN soln., 30 mmol) was added and the reaction mixture was placed into a pre-heated (70 °C) bath and refluxed for 2 h. The mixture was cooled to r.t. and evaporated to 1/2 to remove methanol. The residual clear solution was cooled to r.t. and acidified with 2N HC1 to pH 1 and left overnight at 5 °C to precipitate. Crystalline precipitate of the product 4 was filtered and washed with water (3 x 20 mL). Yield of the tetraacid 4 was about 0.8 g. B. Reaction of tatraacid chloride 5 with fluoreaceinamine 8.

Fluoresceinamine 8 (5.40 g, 15.5 mmol) was dissolved in a mixture of NMP (45 mL and pyridine (3.5 mL, 43 mmol). The solution was diluted with ethanol-free CHC13 (75 mL), cooled to -60 °C and tetrachloride 5 (prepared from 1.01 g, 3.23 mmol of the acid 4 ) in MePh (8 mL) was added dropwise in 15 min. at this temperature under stirring. The mixture was stirred at -60 °C for 1 h, then allowed to heat to r.t. and was poured into H₂0 (300 mL) and Et₃N (20 mL) mixture. The resulting solution was evaporated to 50 ml, more H₂0 (100 mL) and Et₃N (15 mL) was added. The mixture was refluxed for 17 h. The mixture was evaporated to dryness and kept at 75 °C at 0.1 mm Hg to ensure the removal of volatiles. The residue was diluted with CH₂C1₂ (50 mL), then was quickly further diluted under stirring to 1 L volume. The mixture was kept for 16 h and precipitated solid was filtered off, washed with CH₂C1₂ and dried on air to give approximately 5.50 g of crude salts, which were dissolved in 2-PrOH (340 mL) and water (170 mL). The solution was passed through a Dowex-50 column (H⁺ form, 1,5 x 20 cm). The column was eluted with 2-PrOH - H₂0 mixture (2:1) until the eluate became colorless (about 500 mL). The eluate was evaporated and the residue was treated with 0.02 N HC1 (100 mL). Precipitate was filtered off, washed with 0.02 N HC1, H₂0 and dried on air to give a mixture of amides (3.66 g).

Isolation of tetraamide 12. The above mixture of amides (3.61 g) was dissolved in a mixture of 2-PrOH (265 mL) and H₂0 (35 mL). The solution was diluted with H₂0 (235 mL) and cone. HC1 (0.1 mL), and seeded with crystals of the compound 12. The mixture was kept for 2 days and precipitated solid was filtered, washed with 50% 2-Pr (200 mL) and dried on air to give the mixture (1.19 g) of amides, enriched with tetraamide 12. According to HPLC data, this mixture contained 78% of the compound 12, 21% of the compound 11 and 0.7% of the compound 10. This crude mixture was dissolved in a mixture of 2-PrOH (100 mL) and H₂0 (11 mL), heated to 70 °C and diluted with a hot mixture of H₂0 (35 mL) and cone. HC1 (0.1 mL). Precipitated product was collected, washed with 50% 2-PrOH, dried on air and the procedure was repeated to give tetraamide, 0,89 g, which was 96% pure on HPLC. Two additional recrystallizations as above gave the analytical sample of the compound 12 as orange solid: mp. dec. above 350 °C; IR (KBr): 850, 1112, 1175, 1206, 1259, 1452, 1502, 1607, 1725 and 3413 cm^"1: UV (H₂0, pH 8): 239 (5.22), 490 (5.35); Η NMR (DMSO-d₆) 52.30 (br.s., 12H), 6.52 - 6.62 (m, 16H), 6.66 (s, 8H), 7.24 (d, J = 8 Hz, 4H), 8.04 (d, J = 8Hz, 4H), 8.44 (s, 4H), 9.94 (s, 4H, N-H), 10.13 (br.s., 8H, OH). HPLC (30% to 100% B in 20 min): 15.6 min (98.3%). MS, m/e: Calcd (M+l)⁺ for C₉₄H₆₀N₄O₄ 1629.4; Found: 1630.6. Separation of the protected amides (13-16). The above filtrate after separation of the compound 12 was evaporated to give 2.88 g of a mixture of amides. According to HPLC analysis it contained 8.4% of the compound 9, 30.3% of the compound 10, 48.7% of the compound 11 and 8.5% of the compound 12. This mixture (2.57 g) was stirred in Ac₂O (50 mL) and pyridine (10 mL) for 1 h, and evaporated at 30-35 °C to dryness. Acetic anhydride (10 mL) was added to the residue and the mixture was evaporated again. This was repeated 3 times then once with 90% AcOH to remove pyridine acetate. The residue was flash- chromatographed on a silica gel column (4 x 35 cm; prepared in CHC1₃, containing 1% AcOH) using mixtures from 2.5% MeOH and 1% AcOH in CHC1₃ to 5% MeOH and 1% AcOH in

CHC1₃, and finally 10% MeOH and 1% AcOH as eluent to give protected diamide 14 (0-72g), triamide 15 (1.32 g) and tetraconjugate 16 (0.18 g). Evaporation of the polar fraction gave a mixture of compounds, 0.39 g, which contained mostly acetylated monoamide 13.

Acetylated amides. Analytical samples of diamide 14 and triamide 15 were prepared by purification on prep. TLC (eluent - 7% MeOH and 1% AcOH in CHC1₃) following by recrystallization from EtOAc. Sample of tetraamide 16 was prepared by recrystallization (twice) from 50% DMF in EtOH.

Diamide (14): mp. dec. above 300 °C; IR (KBr): pending. Η NMR (DMSO-d₆) δ 2.03 (br.s., 12H), 2.28 (s, 12H), 6.93 (br.s., 8H), 7.27 (br.s., 6H), 8.03 (br., 2H), 8.52 (br.s., 2H), 10.03 (br.s., 2H, NH). HPLC (40% to 100% B in 20 min): 17.0 min (95%). MS, m/e: Calcd (M+l)⁺ for C₆₂H₄₆N₂O₂ 1139.3. Found: 1139.5. Triamide (15): mp. dec. above 300 °C; IR (KBr): pending. Η NMR (DMSO-d₆) δ 2.12 (br.s., 12H), 2.28 (s, 18H), 6.93 (br.s., 12H), 7.25 (br., 9H), 8.05 (br.s., 3H), 8.56 (br.s., 3H), 10.30 (br.s., 3H, N-H). HPLC (30% to 100% B in 20 min): 20.3 min (97.7%). MS, m/e: Calcd (M+l)⁺ for C₈₆H₆₁N₃O₂₆ 1552.4. Found: 1553.7. Tetraamide (16): mp dec. above 350 °C; IR (KBr): pending. 'H NMR (DMSO-d₆) δ 2.29 (br.s., 36H), 6.95 (br., 16H), 7.29 (br.s., 8H), 7.39 (d, J=8Hz, 4H), 8.06 (br.d, J = 8Hz, 4H), 10.05 (br.s., 4H, N-H). HPLC (30% to 100% B in 20 min): 22 min (98%). MS, m/e: Calcd (M+2)^{+ +} for C₁₁₀H₇₆N₄0₃₂ 983.2. Found: 984.1.

Deprotection of acetylated amides (typical procedure; triamide 12 used as representative example). A solution of acetylated triamide 15 (0.5 g) in cone. aq. NH₃ (20 mL) and z-PrOH (10 mL) was stirred for 1 h, then evaporated to dryness and re-dissolved in 50% 2-PrOH (60 ml). The solution was filtered and acidified with cone. HC1 (0.5 mL). The mixture was evaporated at 40 °C and the residue was treated with H₂O (20 mL). Precipitated solid was filtered, washed with water and dried on air to give triconjugate 11, 0.42 g (100%) as a red- orange solid: mp. dec. above 350 °C; IR (KBr): pending; UV (H₂0, pH 8): 239 (5.13), 490 (5.29); 'H NMR (DMSO-d₆) δ 2.14 (br.s, 6H), 2.50 (br.s., 6H), 6.52 - 6.62 (m, 12H), 6.67 (s., 6H), 7.23 (d, J = 8 Hz, 3H), 8.03 (d, J = 8Hz, 3H), 8.42 (br.s., 3H), 9.94 (b.s, 3H, N-H), 10.13 (br.s., 6H, OH), 12.70 (br.s., 1H, OH); HPLC (20% to 100% B in 20 min): 14.9 min (100%). MS, m/e, Calcd (M+I)⁺ for C₇₄H₄₉N₃0₂₀ 1300.3; Found: 1300.5. Anal, clcd for C₇₄H₄₉N₃O₂₀ C, 68.36; H, 3.80; N, 3.23. Found: pending. Deprotection of diamide 14 was performed similarly. Crude product 10 was purified on reverse-phase column (RP-C₁₈, using Gradifrac LC system) to give the compound 10 as a red- orange solid: mp. dec. above 350 °C; IR (KBr): pending; UV (H₂0, pH 8): 239 (4.97), 491 (5.16); Η NMR (DMSO-d₆) δ 2.14 (br.s., 12H), 6.52 - 6.62 (m, 8H), 6.66 (s, 4H), 7.23 (d, J - 8 Hz, 2H), 8.01 (d, J = 8Hz, 2H), 8.39 (br.s., 2H), 9.90 (b.s, 2H, N-H), 10.13 (br,s., 4H, OH), 12.61 (br.s., 2H, OH). HPLC (20% to 50% B in 20 min): 22 min (97%). MS, m/e: Calcd (M+l)~ for C₅₄H₃₈N₂O₁₆ 971.2; Found: 971.6. Anal, clcd for C₅₄H₃₈N₂O₁₆ C, 66.80; H, 3.94; N, 2.89. Found: pending.

Deprotection of monoamide 9 was performed similarly from the above mixture of polar compounds. Crude product was purified on reverse-phase C,₈ TLC plate (eluent: 60% 2-PrOH with 0.2%) TFA) to give the compound 7 as a red-orange solid: mp. dec. above 350 °C; IR (KBr): pending; UV (H₂0, pH 8): 239 (4.57), 491 (4.77); 'H NMR (DMSO-d₆) δ 1.86 (br,d., 4H), 1.97 (br.s., 8H), 6.52 6.58 (m, 4H), 6.66 (s, 2H), 7.18 (d, J = 8 Hz, 1H), 7.94 (d, J = 8Hz, 1H), 8.34 (s., 1H), 9.88 (s, 1H, N-H), 10.10 (br.s., 1H, OH), 12.50 (br.s., 3H, OH). HPLC (20% to 100% B in 20 min): 15.4 min (96.2%). MS, m/e: Calcd (M+l)⁺ for C₃₄H₂₇NO₁₂ 642.2. Found: 642.3. Anal, clcd for C₃₄H₂₇NO,₂ C, 63.65; H, 4.24; N, 2.18. Found: pending. MISSING AT THE TIME OF PUBLICATION

EXAMPLE 2

SYNTHESIS OF N-HYDROXYSUCCINIMIDE ESTER OF ADAM ANT ANE-1.3.5.7- TETRACARBOXYLIC ACID (51- FLUORESCEIN) TRIAMIDE

13 102

A mixture of triamide 13 (0.130 g, 0.1 mmol), N,N-diethylamino carbodiimide hydrochloride (water-soluble carbodiimide WSC, 0.039 g, 0.2 mmol), and N-hydroxysuccinimide (0.023 g, 0.2 mmol) in DMF (2 mL) was protected from light and stirred overnight at r.t. under argon. The mixture was evaporated on evaporator at 1 mm Hg and the oily residue was treated with 0.05 N HCl (2 mL). The precipitated product was filtered, washed with 0.05 N HCl (3 x 1 mL), then H₂O (3 x 1 mL) and dried on air to give about 150 mg of the orange solid.

According to HPLC this crude product contained about 80% of the compound 102 along with 10% of the starting material 13 and several minor impurities. It was further purified by prep. HPLC to give 97% pure compound 102 as an orange solid: mp dec. above 300 °C; IR (KBr): cm-1; MS, m/e: Calcd (M+l)⁺ for C₉₀H_IOON₃O₂₆ 1567. Found: 1567.7.

Compound 102 was found to be unstable at r.t. and slowly decomposed to release free acid 13, however it can be stored for several weeks at -20°C without detectable changes in IR or HPLC. EXAMPLE 3

SYNTHESIS OF ISOTHIOCYNATE DERIVATIVES.

CONHΠ

102 103

A mixture of N-hydroxysuccinimide ester 102 (0.150 g, 0.1 mmol) and 2-amino (4'-aminophenyl) ethane (0.20 mL, 0.2 mmol) in DMF (2 mL) was protected from light and stirred at r.t. under argon for 24 h. The mixture was evaporated on evaporator at 1 mm Hg and the residue was treated with 0.05 N HCl (2 mL). The precipitated product was filtered, washed with 0.05 N HCl (3 x 1 mL), then H₂0 (3 x 1 mL) and dried on air to give about 210 mg of the orange solid. This crude material (30 mg, apr. 0.02 mmol) was dissolved in a mixture of 2- PrOH (17 mL) and water (3 mL) and 1M solution of SCC1₂ in CHC1₃ (0.15 mL, 0.15 mmol) was introduced. The mixture was stirred for 20 h, evaporated to dryness and the residue was dissolved in MeOH and loaded on two reverse-phase prep. TLC plates. The plates were eluted with 60%) 2-PrOH (with 0.2% TFA) and zone of the product was removed from the plates and washed with MeOH to give 25 mg of the isothiocyanate 102 as a yellow solid. EXAMPLE 4 LABELING OF A HUMAN IMMUNOGLOBULIN MOLECULE WITH MULTIPLE AMPLIFIED LABELS

A 10 mg/mL stock solution of Human IG (Rockland) in 0.02 M phosphate buffer - 0.15 M NaCl, ρH=7.2 (500 μL) was diluted with 300 μL of 0.1 M phosphate buffer (pH=8.0). To the resulting solution an aliquote (75 μL) of 13 mg/mL N-hydroxysuccinimide ester 101 in DMF was added dropwise. The mixture was protected from light and stirred for 6 h at r.t. It was loaded on Sephadex G-25 (fine grade) 1 x 40 cm bed column pre-equilibrated with 0.1 M phosphate buffer, pH =8. The column was eluted 0.3 mL/min with the same buffer and labeled protein was collected in void volume. According to UV spectrum (A₄₉₀ to A₂₈₀ ratio) it contained about 2.8 labels (8.5 fluorescein moieties) per immunoglubulin molecule. The recovery of protein calculated on UV (A₂₈₀) basis was about 90%.

FXAMPLF 5

LABELING OF A HUMAN IMMUNOGLOBULIN MOLECULE WITH SINGLE AMPLIFIED LABEL

A 10 mg/mL stock solution of Human IG (Rockland) in 0.02 M phosphate buffer - 0.15 M NaCl, pH=7.2 (500 μL) was diluted with 300 μL of 0. 1 M phosphate buffer (pH=8.0). To the resulting solution an aliquote (75 μL) of 6 mg/mL N-hydroxysuccinimide ester 101 in DMF was added dropwise. The mixture was protected from light and stirred for 2 h at r.t. It was loaded on Sephadex G-25 (fine grade) 1 x 40 cm bed column pre-equilibrated with 0.1 M phosphate buffer, pH =8. The column was eluted 0.3 mL/min with the same buffer and labeled protein was collected in void volume. According to UV spectrum (A₄₉₀ to A₂₈₀ ratio) it contained about 1.4 labels (4.3 fluorescein moieties) per immunoglubulin molecule. The recovery of protein calculated on UV (A₂₈₀) basis was about 90%. The fluorescence intensity of the sample was very close to the value for IG labeled with the same numbers of mono-fluorescein labels.

These results demonstrate that N-hydroxysuccinimide derivative can be used as labeling reagent for covalent attachment of the amplified trifluorescein moiety to biological targets. This moiety is capable of delivering fluorescent signal of the same intensity as three separate single fluorescein labels, using only single binding site of biotarget.

EXAMPLE LABELING OF AVIDIN WITH SINGLE AMPLIFIED LABEL

To a solution of avidin (5 mg; Sigma) in 800 μL of 0.01 M phosphate buffer (pH=8.0) an aliquote (35 μL) of 60 mg/mL N-hydroxysuccinimide ester 101 in DMF was added dropwise. The mixture was protected from light and stirred on ice for 5 h at r.t. It was loaded on Sephadex G-50 (fine grade) 1 x 40 cm bed column pre-equilibrated with 0.01 M phosphate buffer, pH =8. The column was eluted 0.3 mL/min with the same buffer and labeled protein was collected in void volume. According to UV spectrum (A₄₉₀ to A₂₈₀ ratio), it contained about 1.0 labels (3.0 fluorescein moieties) per avidin molecule. The recovery of protein

calculated on UV (A₂₈₀) basis was about 80%.

EXAMPLE FLUORESCENT SPECTRA OF THE ADAMANTANE DERIVATIVES

To avoid the influence of adamantane carboxy groups, the study of fluorescence was also performed in a buffer solution at pH8. Fluorescent spectra of the adamantane derivatives

9 - 12 taken at pH8 show the excitation maxima at 490 nm (FIG 3) and emission at 510 nm

(FIG 4) with symmetry corresponding to Stocks' law. The intensities of both excitation and emission spectra for adamantane derivatives 9 - 12 were almost linear to the number of fluorescent moieties with slight deviation of tetraconjugate point. REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

1. Dreyer, G.B. & P.B. Dervan, Proc. Natl. Acad. Sci. U.S.A., 78:6633, 1981.

2. Falck, et al., J Am. Chem. Soc, 103:7396, 1981.

3. Frazer, J.D., Homer, S.M., Wolski, S.A. Tetrahedron Lett., 39, 1279-1282, 1998.

4. Gillam, LC. & G.M. Tener, Anal. Biochem., 157:199, 1986.

5. Green, et al, J Am. Chem. Soc., 112:7337, 1990.

6. Haugland, R.P., Handbook of Fluorescent Probes and Research Chemicals. Sixth Ed.,

Eugene, OR: Molecular Probes: 4, 19, 31, 117, 1996.

7. Khanna, P.L., Ullman, E.F., Anal. Biochem., 108: 156-161, 1980.

8. Lange's Handbook of Chemistry. 14th Edition; Dean, J.A., Ed.; McGraw-Hill: N.Y., 1992, p. 8.103.

9. Likhtenshtein, G.I., Biophysical Labeling Methods in Molecular Biology. New York: Cambridge University Press, 1993.

10. Martin, et al, Bioconjugate Chem., 6:616, 1995.

11. Martin, V.V., Keana, J.F.W., OPPL, 27: 117-120, 1995. 12. Newcome, G.R., Nayak, A., Behera, R.K., Moorefield, C.N., Baker, G.R. J Org. Chem.; 57: 358-362, 1992.

13. Newkome, et al, Aldrichimica Ada., 25:31, 1992.

14. Newkome, et al, J Org. Chem., 57:358, 1992.

15. Reid, T., Baldini, A., Rand, T.C., Ward, D.C, Proc. Natl. Acad. Sci. U.S.A., 89:1388- 1392, 1992.

16. Robins, M.J. & P.j. Barr, Org. Chem., 48:1854, 1983.

17. Sollott, G. & E.E. Gilbert, J Org. Chem., 45:5405, 1980.

18. Stetter, H. & M. Krause, Tetrahedron Lett., 1841. 1967.

19. Stryer, L., Ann. Rev. Biochem., 47: 819-846, 1978.

20. Telser, et al, J Ann. Chem. Soc, 11:6966, 1989.

U.S. Patent (Ref. 1505-37127), Keana, et al, October 26, 1996.

21. Vann, W.F., Sutton A.; Schneerson, R, Meth. Enzymol. (1990) 1984, 537.

22. U.S.Patent No. 5,316,906 23. U.S. Patent No. 5,443,986

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A fluorescent molecule having enhanced fluorescent capabilities comprising at least one fluorescent residue and a rigid organic three-dimensional core, wherein said fluorescent residue is connected to said rigid organic three-dimensional core.

2. A chemical compound comprising: a three-dimensional core; and at least two polyfunctional moieties extending from said three-dimensional core, wherein at least one of the polyfunctional moieties includes a fluorophore FI, and wherein said polyfunctional moieties are attached to said core by a linker L.

3. The chemical compound of claim 2, wherein at least two of the polyfunctional moieties comprise the same fluorophore.

4. The chemical compound of claim 2, wherein the polyfunctional moiety is:

-O-, -OH-, -NH-, -EOOH-, -SH~, --PO-, -SO-, --NCO-, -NCS-, - N, or NHNH--, or a mixture thereof.

5. The chemical compound of claim 2, wherein said fluorophore FI is Lucifer Yellow, MCA Blue, Texas Red, or a combination thereof.

6. The fluorescent molecule of claim 1, wherein the three-dimensional core is a rigid organic caged fragment.

7. The fluorescent molecule of claim 6, wherein said organic caged fragment comprises a carbon atom framework.

8. The fluorescent molecule of claim 7, wherein the carbon atom framework is an adamantane residue or a cubane residue.

9. The fluorescent molecule of claim 6, wherein the organic caged fragment comprises a heteroatom.

10. The fluorescent molecule of claim 9, wherein the heteroatom is N, O, S,

P or a combination thereof.

11. The chemical compound of claim 6, further defined as a tetracarboxylic acid conjugated with fluorescein.

12. The chemical compound of claim 2, further defined as an adamantane conjugate.

13. The chemical compound of claim 2, further defined as 1,3,5,7- tetracarboxylic acid conjugated with fluorescein.

14. The chemical compound of claim 2, further defined as comprising three polyfunctional moieties.

15. The chemical compound of claim 2, further defined as comprising four polyfunctional moieties.

16. The chemical compound of claim 2, further comprising a targeting group comprising a polypeptide, protein, antibody, nucleic acid, carbohydrate, fatty acid, surfactant, glyceride, steroid, porphyrin, inhibitor, biotin, hapten, polymer microsphere, or combination thereof.

17. An amino derivative of adamantane having amino groups on an adamantane core, wherein FI is a fluorescent moiety, L is a linker, and X is a targeting group.

18. The amino derivative of claim 17, wherein FI is further defined as Lucifer Yellow, Texas Red, fluorescein, MCA blue, or a combination thereof.

19. The amino derivative of claim 17 wherein L is further defined as -CO- NH-; -C6H4-; -NH-SO2-; -NH-SO2-; -Co-O; -NH-; -O-; -S-; NHΓÇö C(S)-; NH-CO- NH-, NH-C(S)-NH-; -O-C(O)-; -O-; or -C(O)-.

20. Fluorescent materials, comprising at least one fluorescent dye residues connected to a rigid organic three-dimensional core.

21. A chemical compound comprising:

a three-dimensional core C; and at least two polyfunctional moieties (X and -L-Fl) extending from said three-dimensional core, wherein at least one of the polyfunctional moieties include a fluorophore FI, and wherein said polyfunctional moieties are attached by a linker L to said core.

22. The fluorescent molecule of claim 21 where the three-dimensional core is a rigid organic caged fragment. The skeleton of rigid fragment could comprise of carbon atoms, such as in adamantine, cubane, etc., or could also include heteroatoms, such as N. O. S. P.

23. The fluorescent molecule of claim 22 wherein the rigid organic caged fragment comprises carbon.

24. The fluorescent materials of claim 20 where the fluorophore FI is a residue derived from fluorescent dye.

25. The fluorescent materials of claim 24 wherein the fluorophores comprise fluorescein, rhodamine, Oregon Green, coumarines, Texas Red, BODIPY,

MCA Blue, Lucifer Yellow, Cascade Blue, or a combination thereof.

26. The fluorescent materials of claim 22 where the fluorophore FI is in protected, inactive or masked form, and able to regain fluorescent properties upon physical or chemical treatment or enzymatic reaction.

27. The fluorescent materials of claim 20 where the linker fragment is an organic divalent grouping.

28. The fluorescent materials of claim 20 wherein the linker comprises: -

CHR- -CO-NR-; -NRSO₂._; -COO-; -NR-; -NR-O-; -O-; -S-; -CS-NR-; -NR-CO-NR; -

NR-CS-NR-; -CO-; -CR=CR-; -CR=N-; -N=N-; -C-C-; or C₆H₄, and where the residue

R comprises hydrogen atoms (H), alkyl or aryl (hetaryl) radicals.

29. The fluorescent materials of claim 20 further comprising a targeting group, said targeting group comprising a reactive organic functionality, capable of forming covalent or non-covalent bond with another organic functionality or biomolecule.

30. The fluorescent materials of claim 29 wherein the targeting groups comprise: -COOH, -COX-;-COOR, -CONHR, -NHR, -NOO; -N=C=S, -NR (halo-s- triazine), -N-maleimide; aryl substituted with the above groups; alkyl Cl - C6, substituted with the above groups, or with aryl substituted with the above groups.

31. The fluorescent materials of claim 29 where the targeting group is conjugated with a bioactive fragment comprising a receptor; enzyme, a substrate or inhibitor thereof, a nucleic acid comprising a synthetic oligonucleotide, protein, comprising avidin or streptavidin, antibody, a hapten, comprising avidin or streptavidin derivatives.

32. The fluorescent materials of claim 30 wherein at least two of the polyfunctional moieties -L-Fl comprise fluorophores.