Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2000-07-19
2002-04-09
Park, Hankyel T. (Department: 1648)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C436S525000, C424S179100
Reexamination Certificate
active
06369206
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to small organometallic probes, processes for making the small organometallic probes, and applications of the small organometallic probes. In particular, the small organometallic probes of the present invention are preferably less than 0.02 micron (0.02 &mgr;m) or 20 nanometers (20 nm) in diameter, and comprise a metal cluster compound having a solid metal core, with organic groups attached to the metal core so as to impart desirable physical and chemical properties to the organometallic probes. Alternatively, the organometallic probes may comprise a metal colloid having organic groups attached to the outer surface of the metal colloid. The metal clusters or colloids may also be functionalized with other molecules attached that can be used for targeting and detecting another substance, generally, a biologically significant substance, such as an antibody, a protein, or lipid bilayer. The metal in the metal clusters or colloids is gold, platinum, silver, palladium or combinations thereof.
In one specific embodiment, the metal in the cluster is palladium or platinum and the attached organic groups are covalently attached through 1,10-phenanthroline moieties. Also disclosed is a new method for making the small organometallic probes. The method consists of a chemical procedure wherein the metal cluster compounds are prepared directly by reaction of a mixture of a salt of the cluster metal and the coordinating organic groups with a reducing agent in solution.
BACKGROUND OF THE INVENTION
Previous work by others has also described the preparation of gold and silver colloids. Such colloids do not have a fixed number of metal atoms and vary considerably in size. For example, the metal colloids can vary in size from 1 nm to 2 &mgr;m in size and may contain from about 10 metal atoms to thousands of metal atoms, depending on size. It was found that a number of proteins, such as IgG antibodies, could be adsorbed to these sol particles.
Gold colloids have been most commonly described. These conjugates have been used in electron and light microscopy as well as on immunodot blots for detection of target molecules. These conjugates have many shortcomings. Since the molecules are only adsorbed onto the colloids, they also desorb to varying extents. This leads to free antibody which competes for antigen sites and lowers targeting of gold.
Furthermore, the shelf life of the conjugates is compromised by this problem. The ‘sticky’ colloids also tend to aggregate. If fluorescence is used to detect the target molecules, the gold particles quench most of it. Also, the gold colloids must be stabilized against dramatic aggregation or ‘flocculation’ when salts are added by adsorbing bully proteins, such as bovine serum albumin. Due to the effects of aggregation and bulky additives, the penetration of immunoprobes into tissues is generally <0.5 &mgr;m. Access of the probes to internal cell structures, e.g., nuclear proteins, or to cells deeper in a tissue sample, is impeded by these properties.
Colloidal gold immunoprobes are also used in diagnosis on immunoblots. The sensitivity of these detection schemes is also reduced by problems relating to detachment of antibodies from the gold which results in a short shelf life and non-specific gold binding causing problems with background signal. The gold prepared in standard ways also has low activity due to few adsorbed antibodies and denaturation of some antibodies during adsorption.
Various metal cluster containing organic shells have also been previously described, such as Au
11
(PPh
3
)
7
Cl
3
(PPh
3
=triphenylphosphine), and Pd
561
L
36
O
200
(where L=1,10-phenanthroline). These metal clusters have a fixed number of metal atoms in their metal cores which range in size from ca. 0.8-2.4 nm. Most of these metal clusters are based upon reduction of metal-triphenyl phosphine or the use of 1,10-phenanthroline.
Examples of larger cluster complexes (greater than 1 nm in size) have also been reported such as clusters having the formula M
55
(PPh
3
)
12
X
6
(Ph=phenyl or m-phenylsulfonyl) where M=gold, platinum and rhodium and X=halide.
For example, Barlett, P. A. et al, in “Synthesis of Water-Soluble Undecagold Cluster Compounds . . . ,”
J. Am. Chem. Soc.,
100, 5085 (1978), describe a metal cluster compound (Au
11
) having a core of 11 gold atoms with a diameter of 0.8 nm. The metal core of 11 gold atoms in the undecagold metal cluster compound is surrounded by an organic shell of PAr
3
groups. This metal cluster compound has been used to form gold immunoprobes, for example, by conjugating A
11
to Fab′ antibody fragments as well as other biological compounds.
Another metal cluster compound which has been used as a probe is Nanogold™ available from the assignee of the present application. Nanogold™ has a metal core with 50-70 gold atoms (the exact number not yet being known but believed to be 67 gold atoms) surrounded by a similar shell of organic groups (PAr
3
) such that Ar is an aryl group into which a reactive group such as a primary amine, a maleimide, or a N-hydroxysuccinimide ester may be incorporated for conjugation to biologically significant entities including antibody IgG molecules and Fab′ fragments, proteins, lipids, hormones and oligonucleotides. Nanogold™ and the smaller undecagold cluster, which contains 11 gold atoms, have been used as probes for detecting and identifying biomolecules. The metal core of Nanogold™ is 1.4 nm in diameter. The production of Nanogold is described in pending application Ser. No. 988,338, filed Dec. 9, 1992, of James F. Hainfeld and Frederic R. Furuya.
Another class of cluster complex compounds having Pt or Pd as the metal core and further having a core ranging in diameter from 1.8 to 3.6 nm is prepared by reduction of metal acetate in acetic acid by molecular hydrogen in the presence of 1,10-phenanthroline ligands. The ligated cluster is then carefully oxidized with air to neutralize the exposed metal atoms and render the compounds air-stable.
Complexes prepared by the above method and characterized by electron microscopy in order to determine the size of the metal core include a 1.81 nm core diameter platinum compound of proposed formula [Pt
309
phen
36
O
30±10
] (see Scmidt, G., Morun, B., and Malm, J.-O.;
Angew. Chem. Int. Ed. Eng.,
1989, 28, 778), a 2.43 nm core diameter palladium compound of proposed formula [Pd
561
phen
36
O
200
] (de Aguiar, J. A. O.; Brom, H. B.; de Jongh, L. J., and Schmid, G.;
Z. Phys. D.:Atoms, Molecules and Clusters,
1989, 12, 457), and a mixture of 3.16 and 3.6 nm core diameter palladium compounds with proposed formulae [Pd
1415
phen
60
O
~1100
] and [Pd
2057
phen
84
O
~1600
] respectively (Schmid, G.; Harms, M.; Malm, J.-O.; Bovin, J.-O; van Ruitenbeck, J.; Zandbergen, H. W., and Fu, W. T.;
J. Amer. Chem. Soc.,
1993, 115, 2046), where phen is either 1,10-phenanthroline or bathophenanthroline, (1). The proposed formulae are based upon the extension of the crystal packing of metal atoms within known smaller clusters outward in discrete layers.
Although the preparation and properties vary for these metal cluster compounds having organic shells, many of these can only be synthesized in low yields, derivatization for use in coupling to biomolecules is expensive in time and effort, and again in low yields, and many of the cluster compounds are degraded rapidly by heat or various chemical reagents.
SUMMARY OF THE INVENTION
In accordance with the present invention, a new class of metal cluster compounds, and a process for making such compounds, are described. The compounds may be generally described as organothiol metal clusters, wherein the metal core is comprised of gold, platinum, silver, palladium or combinations of these metals. Prominent among the disclosed organometallic compounds is a large palladium and platinum cluster compound. The metal core of the compounds, wherein Gold is the prominent metal, is about 1.4 nm in diameter
Hainfeld James F.
Leone Robert D.
Park Hankyel T.
Proskauer Rose LLP
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