BACKGROUND

Renal cell carcinoma (RCC) is the most common form of kidney malignancy and one of the most deadly forms of cancer. 5-fluorouracil (5FU) has been used to treat RCC. Generally, 5FU is administered by a single intravenous injection or through continuous infusion and is used alone or in combination with other agents. The combination of 5FU and leucovorin (folinic acid), a reduced folate cofactor, has been shown to be much more effective against RCC than 5FU alone (Porta et al. (1995) Oncology 52:487-491; Breul, et al. (1996) Eur. J. Med. Res. 17:339-342). It appears that 5FU the binding affinity for 5FdUMP increases 7- to 8-fold upon prior treatment with leucovorin (Santi et al. (1974) Biochemistry 13:471-481; Evans et al. (1981) Cancer Res. 41:3288-3295).

High doses of 5FU (>500mg/m ² ) can lead to side effects, such as mucositis (Salimen et al. (1995) Eur. J. Cancer 3 IA: 849), stomatitis, diarrhea, and flu-like symptoms (Nerenstone et al. (1987) Gastroenterology Clin, of N. America, 16:603- 612). Similar side effects are seen with many chemotherapeutic agents for treatment of RCC as well as other drugs intended to treat kidney disorders. For this reason it is desirable to identify means for targeting drugs more directly to the kidney.

SUMMARY The ligand molecules described herein bind to and are internalized by renal cells, for example epithelial cells of the proximal and distal tubules. The molecules are useful delivering a therapeutic agent, e.g., 5FU, to renal cells. Thus, the ligand molecules can be attached to therapeutic agent via a linker. When a selected therapeutic agent is linked to one of the ligand molecules described herein, the therapeutic agent is expected to exhibit an improved therapeutic index for treatment of renal disease. The ligands described herein generally includes two or more moieties (X) that bind to a receptor present on a renal cell. Two or more binding moieties (X) can be bound to a single scaffold (M) to create molecules having the formula M(X) ₂ . The scaffold, M, is a multi-functional chemical moiety capable of reacting with two or more X moieties, which can be the same or different. Each X is joined to M via, for

example, an ether, phosphate ester, sulfate ester, phosphoramide, sulfonamide, carboxylic ester, amine, thioether, amide, thioamide, thiocarbamate, guanidine, carbonate, thiocarbonate, and/or carbamate group.

Thus, the ligand molecules can have any of the following general structures:

The presence of multiple (e.g., 2, 3 or 4) binding moieties (X) can significantly improve the binding of the ligand to a renal cell compared to a molecule that includes only a single binding moiety. The ligand can be covalently attached to a therapeutic agent or payload (P) via a linker (L) to create molecules having any of the following structures:

The renal cell binding moieties (X) can include saccharides, e.g., monosaccharides or disaccharides such as lactose; galactose; mannose; fucose; N-acetylactosamine; melibiose; 1,3-alpha and beta linked galactose-glucose; 1,3-alpha and beta linked galactose-N-acetylglucosamine; 1,3-alpha and beta linked galactosamine-glucose; 1,2- alpha and beta linked galactose-glucose; and 1 ,2-alpha and beta linked galactose-N- acetylglucosamine. Various combinations of the monosaccharides listed above could be combined to form disaccharides provided that the ligand does not include a terminal terminal N-acetylgalactosamine. In certain embodiments, X is other than N-acetyl galactosamine. The scaffold (M) can be a derivative of glutamic acid or aspartic acid or can have any of the general structures below where m and n are independently 0, 1, 2, 3 ,4, 5, 6, 7 or 8.

Other Suitable ligand scaffolds (M)include:

Compounds in which the renal ligand is attached to a payload that is a therapeutic agent can be used to treat a renal disorder. For example, where the payload is a chemotherapeutic agent, the compound can be used to treat renal cell carcinoma.

The payload can also be a therapeutic agent for the treatment of kidney disease, a cystic disease (e.g., Cystic Renal Dysplasia, Autosomal Dominant (Adult) Polycystic Kidney Disease, Autosomal Recessive (Childhood) Polycystic Kidney Disease, Aquired (Dialysis-Associated) Cystic Disease, and Simple Cysts); a glomerular disease (e.g., Primary Glomerulopathies which include: Acute Diffuse Proliferative Glomerulonephritis, Acute Nephritic Syndrome, Nephrotic Syndrome, Chronic Renal Failure, Rapidly Progressive Glomerulonephritis, Membranous Glomeruopathy, Lipoid Nephrosis, Focal Segmental Glomerulosclerosis, Membranoproliferative Glomerulonephritis, IgA Nephropathy, Focal Proliferative Glomerulonephritis, and

Chronic Glomerulonephritis); a systemic disease (e.g., . Lupus Erythematosus, Diabetes Mellitus, Amyloidosis, Goodpasture's Syndrome, Polyarteritis Nodosa, Wegener's Granulomatosis, Henoch-Schonlein Purpura, Bacterial Endocarditis, hypertension, hypotension); a hereditary disease (e.g., Alport's Syndrome, Fabry's Disease, Wilm's tumor, and von Hippel Lindau disease); or a tubulointerstitial disease (e.g., Infections/Toxins: Acute Pyelonephritis (infections-bacterial, viral, parasitical), Chronic Pyelonephritis (reflux nephropathy), Acute Hypersensitivity Interstial Nephritis (Drug Induced), Analgesic Nephritis, Heavy Metals (lead, cadmium); Metabolic/Immunologic: Urate Nephropathy, Nephrocalcinosis (hypercalcemia nephropathy), Hypokalemic Nephropathy, Oxalate Nephropathy, Transplant Rejection, Sjorgren's Syndrome; Physical/Miscellaneous: Chronic Urinary Tract Obstruction, Radiation Nephritis, Balkan Nephropathy, Nephronophthisis (medullary cystic disease)).

Among the therapeutic agents that can be linked to a renal ligand are: CpG 7909; BAY 43-9006: Raf kinase inhibitor; BAY 59-8862; Carboplatin, paclitaxel (Taxol); trastuzumab (Herceptin); Interferon-alpha; CCI-779: Rapamycin derivative targeting mTOR; AG-013736: Antiangiogenesis; filgrastim; ABX-EGF; interleukin-12; GTI-2040: Antisense complimentary to the R2 component of ribonucleotide reductase; FK228: Depsipeptide inhibiting class I histone deacetylases; erlotinib (TARCEVA): Protein tyrosine kinase inhibitor (EGFR-TK); BMS-247550: Epothilone B analog, nontaxane antimicrotubule agent; pegfilgrastim, Cisplatin; ABT-510; ABT-751; calicheamicin; doxorubicin; vinblastine; 5-fluorouracil; capecitabine (Xeloda): Thymidylate synthase inhibitor; gemcitabine; methotrexate

The following abbreviations are used in the examples below: ATP, adenosine triphosphate; CPG, controlled pore glass support; DIPEA, diisopropylethylamine; D- MEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; D-PBS, Dulbecco's phosphate buffered saline; DTT, dithiothreitol; EDAC, l-ethyl-3- [3(dimethylamino)propyl] carbodiimide; EDTA, ethylenediaminetetraacetate; FCS, fetal calf serum; GaINAc, N-acetylgalactosamine; MEM, minimal essential medium with Earle's salts; SMCC, N-hydroxysuccinimidyl 4 (N-methylmaleimido)cyclohexyl-l carboxylate; Tris, tris(hydroxymethyl)amine.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and

advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic drawing of the synthesis of a molecule described herein. FIG. 2 is a schematic drawing of certain conjugates described herein in which the therapeutic agent is 5FU.

FIG. 3 is a schematic depiction of the synthesis of the dye-labeled oligonucleotide and conjugate synthesis.

FIG. 4 is an image of ligand-dye conjugate uptake by RPTEC cells. FIG. 5a is an image of ligand-dye conjugate uptake by primary adenocarcinoma cells, ACHN.

FIG. 5b is an image of un-conjugated dye uptake.

DETAILED DESCRIPTION

In order to be therapeutically useful a drug must be present in the appropriate concentration and at its appropriate site of action. Moreover, many drugs must pass across the cell membrane for proper absorption, distribution, biotransformation, and eventual elimination. The full therapeutic potential of many candidate drugs may never be realized because they cannot reach their intended targets within the cell. To compensate, elevated doses of a drug must be administered indiscriminately throughout the body to achieve the necessary intracellular concentration, but may induce life- threatening toxicities may occur. Efficient delivery of a therapeutic agent across the cellular membrane to its intended specific target cell and its molecular target may reduce the dose of the therapeutic required for treatment while reducing the toxic effects. Described herein are organ-specific, cell-specific ligands that employ the bi- antennary substructure of a complex oligosaccharides. These ligands can be used to deliver a therapeutic agent to renal cells.

Example 1: Synthesis of a Kidney Ligand

Described below is the synthesis of a kidney ligand. The synthesis of this molecule, YE(ahLac) ₂ is depicted schematically in FIG. 1. Other ligands containing galactose, mannose, fucose or other saccharides can be generated in a similar manner.

Synthesis of 1.2,2'.3.3'A4',6,6'-Nona-O-acetyl-D-lactose (2) A solution containing 4g (11.7 mmoles) β-lactose (1) in 30 mL of anhydrous pyridine was treated with 30 mL (290 mmoles) of acetic anhydride. The solution was stirred at room temperature for 24 h, then the concentrated under diminished pressure to approximately one half of the original volume. Next, the mixture was poured onto ice cold H ₂ O. The resultant sticky colorless mass was dissolved in CHCl ₃ (100 mL) and washed with H ₂ O (2 x 300 mL) and 10% HCl (100 mL). The organic phase was then dried (Na ₂ SO ₄ ) and concentrated under reduced pressure and dried in vacuo overnight to give the product as a colorless solid, 5.4 g (79%); ¹ H NMR (CDCl ₃ ): δ 1.98 (s, 3H, CH ₃ CO-), 2.02 (s, 3H,

CH ₃ CO-), 2.05 (s, 3H, CH ₃ CO-), 2.15 (s, 3H, CH ₃ CO-), 2.16 (s, 3H, CH ₃ CO-), 4.01 (t, J = 5.7 Hz, 1 H, H-5), 4.14 (m, 2 H, H-6), 4.44 (dd, J = 9.18 Hz, 9.25 Hz, 1 H, H-

4)

Synthesis of l-(6'-Benzyloxycarbonyl)aminohexyl 2,2',3,3'A4',6,6'-octa-O- acetyl-D-lactose (5)

A solution containing 2 g (3.53 mmoles) of l,2,2',3,3',4,4',6,6'-nona-C>- acetyl-D-lactose (2) in 20 mL of glacial acetic acid was treated with 20 mL of 30% HBr in acetic acid solution and stirred at room temperature for 2 h. The mixture was then poured onto cold CHCl ₃ and washed with ice-cold H ₂ O, then drained onto ice cold saturated NaHCO ₃ . The CHCl ₃ layer was drained, dried (Na ₂ SO ₄ ) and concentrated in vacuo. This residue was analyzed by ¹ H NMR spectroscopy and used immediately for the next step without purification. 1-bromo-l-deoxy- 2,2',3,3',4,4',6,6'-octa-0-acetyl-D-lactose (3): ¹ H NMR (CDCl ₃ ): δ 1.98-2.15 (m, 21 H, 7 x CH ₃ CO-), 4.09 (m, 2 H, H-6), 4.45 (t, J = 6.4 Hz, 1 H, H-5), 5.01 (dd, J = 3.9 Hz, 10.6 Hz, 1 H, H-4), 5.36 (dd, J = 3.3 Hz, 10.6 Hz, 1 H, H-3), 5.48 (m, 1 H, H- 2), 6.66 (d, J = 3.9 Hz, 1 H, glucosyl anomeric H-I).

A solution containing l-bromo-l-deoxy-2,2',3,3',4,4 ^l ,6,6'-octa-(9-acetyl-D- lactose (3), 0.88 g (3.5 mmoles) of 6-(benzyloxycarbonyl)aminohexanol (4), 0.88 g

(3.5 mmoles) OfHg(CN) ₂ , and 0.33 g of Drierite in 15 mL Of CH ₃ NO ₂ and 15 mL of toluene was stirred for 16h at room temperature. The reaction mixture was then filtered and the filtrate was concentrated in vacuo. The residue was dissolved in CHCl ₃ (100 mL) and washed twice with aq. 1 M NaCl and once with 0.5 M KBr. The organic layer was dried (Na ₂ SO ₄ ) and concentrated under reduced pressure. Purification of the crude mixture was accomplished using gel filtration (LH 20 Sephadex column, 2.5 x 100 cm, packed with 95% ethanol, loaded ~l-2 mL crude). Elution of the column with 95% ethanol provided fractions that contained product as identified by silica gel TLC (50% ethyl acetate in hexanes) with 15% H ₂ SO ₄ /ethanol charring test and UV detection. These fractions were combined and concentrated under reduced pressure to give the product, l-(6'- Benzyloxycarbonyl)aminohexyl 2,2 ^l ,3,3',4,4',6,6 ¹ -octa-O-acetyl-D-lactose (5), as a colorless foam. 822 mg (27%); silica gel TLC R _f 0.58, 0.70 (ethyl acetate); ¹ H NMR (CDCl ₃ ): δ 1.25-1.69 (m, 16 H, 2 x CH ₂ -CH ₂ -CH ₂ -CH ₂ -), 1.90-2.31 (m, 42 H, lactose 14 x CH ₃ CO), 3.13 (m, 4 H, 2 x N-CH ₂ -), 3.40-5.35 (series of overlapping multiplets, 36 H, 2 x 0-CH ₂ -, lactose ring 10 x CH (H-I through H-5), lactose 2 x CH ₂ H-6, benzylic CH ₂ -), 7.30 (s, 5 H, phenyl H). Synthesis of ZEfahLac)rbis(hepta-acetate) (7) A solution containing 530 mg (0.61 mmole) of l-(6'- benzyloxycarbonyl)aminohexyl 2,2',3,3',4,4',6,6'-octa-0-acetyl-D-lactose (5) in 50 mL of 95% ethanol was exposed to 1 atm OfH ₂ in the presence of 10% Pd/C (130 mg). The conversion was complete in 3h as judged by silica gel TLC (R _f 0.00, (50% ethyl acetate in hexanes)). The catalyst was removed by filtration through Celite 545 and the filtrate concentrated under diminished pressure. The colorless residue (6) (460 mg, 95%) was used immediately for the next reaction.

A solution containing 57 mg (0.20 mmole) of N-CBz-glutamic acid, 55 mg (0.41 mmole, 2 eq.) of 1-hydroxybenzotriazole, and the corresponding amine (0.61 mmole) in 2 mL of dry DMF was treated with 234 mg (1.22 mmoles, 6 eq.) of l-(3- dimethylaminopropyl)-ethylcarbodiimide (EDAC) and 215 μL (2 eq.) N ₁ N- diisopropyl-N-ethylamine and stirred for 20 minutes at 0 ⁰ C. This mixture was allowed to warm to room temperature overnight. Afterwards, the reaction mixture was poured onto CHCl ₃ (100 mL) and washed with H ₂ O (2x 300 mL) and 0.2 Ν HCl (100 mL). The organic phase was dried (Na ₂ SO ₄ ) and concentrated to give the

crude product (330 mg) as a yellow oil. Purification of the product was accomplished by silica gel flash chromatography (28 g column, packed with 50% ethyl acetate in hexanes). The column was eluted with increasing amounts of CH ₃ OH in ethyl acetate (0— »10%), which provided fractions that corresponded to product as judged by ¹ H NMR spectroscopy. These fractions were pooled, then concentrated to give the N,N'-diamide (7) as a colorless oil. 140 mg (41%): R _f 0.22 (ethyl acetate); ¹ H NMR (CDCl ₃ ): δ 1.25-1.69 (m, 16 H, 2 x CH ₂ -CH ₂ -CH ₂ -CH ₂ ), 1.90-2.31 (m, 46 H, Ct-CH ₂ , β-CH ₂ , lactose 14 x CH ₃ CO), 3.13 (m, 4 H, 2 X N-CH ₂ - ), 3.40-5.35 (series of overlapping multiplets, 36 H, 2 x 0-CH ₂ -, α-CH glutamyl, α- CH tyrosinyl, lactose ring 10 x CH (H-I through H-5), lactose 2 x CH ₂ H-6, benzylic CH ₂ -), 7.30 (s, 5 H, phenyl H).

Synthesis of ZYEfahLacVbisfhepta-acetate ^* ) (9)

A solution containing 140 mg (82 μmoles) of ZE(ahLac) ₂ -bis(hepta-acetate) (7) in 15 mL of 95% ethanol was added to a heterogeneous mixture of 70 mg of 10% activated Pd on C in 10 mL of 95% ethanol. This mixture was degassed and purged with H ₂ (3x), then exposed to 1 atm (balloon) of H ₂ for 1 h at room temperature. Progress of the reaction was monitored by silica TLC (ZE(ahLac) ₂ -bis(hepta-acetate)(7) R _f 0.80 (ethyl acetate); product R _f baseline (ethyl acetate)) and judged to be complete after 2 h. The catalyst was filtered (Celite plug) and the filtrate was concentrated to give the amine product (8) as yellowish glassy oil, identified by silica TLC (product: R _f baseline (ethyl acetate); basic KMnO ₄ staining).

This amine (8) was then dissolved in 2 mL of dry DMF and treated with 45 mg (103 μmoles) of Cbz-tyrosine p-nitrophenol ester and 28 μL of diisopropylethylamine (DIPEA) and stirred at room temperature for 16 h. The mixture was subsequently concentrated in vacuo and the resulting tan residue was analyzed by silica gel TLC. Purification of the residue was accomplished by silica gel flash chromatography (27g silica gel 230-400 mesh, packed in 50% ethyl acetate in hexanes). The sample was dissolved in ethyl acetate (500 μL). The column was eluted with linear gradient of CH ₃ OH in ethyl acetate (0— »10%), which , provided fractions that corresponded to product as judged by silica gel TLC and ¹ H NMR spectroscopy. These fractions were pooled, concentrated to give the product

as a colorless oil, 50 mg (32%): R _f 0.18 (ethyl acetate); ¹ H NMR (CD ₃ OD): δ 1.33- 1.61 (m, 16 H, 2 x CH ₂ -CH ₂ -CH ₂ -CH ₂ -), 1.92-2.11 (m, 46 H, Ct-CH ₂ , β-CH ₂ , lactose acetoxy CH ₃ ), 2.85 (m, 1 H, tyrosine CH), 2.99 (m, 1 H, tyrosine CH), 3.04 (m, 4 H, 2 x N-CH ₂ -), 3.40-5.40 (series of overlapping multiplets, 36 H, 2 x 0-CH ₂ - , α-CH glutamyl, α-CH tyrosinyl, lactose ring CH (H-I through H-6), benzylic CH ₂ -), 6.70 (d, J = 8.04 Hz, 2 H, ortho-hydroxyl tyrosine H), 7.06 (d, J = 7.84 Hz, 2 H, ortho-hydroxyl tyrosine H),7.30 (s, 5 H, phenyl H). Synthesis of YEfahLacb (10) A solution containing 50 mg of purified ZYE(ahLac) ₂ -bis(hepta-acetate) (9) in 1 mL of dry CH ₃ OH was treated with 100 μL of 100 mM NaOCH ₃ in dry CH ₃ OH. The solution was stirred at room temperature for 2 h, then neutralized with a few beads of H ₂ O-washed DOWEX 5OW x 8 resin. The resin was then filtered off, and then the filtrate was concentrated under reduced pressure. HPLC analysis and purification of the residue was accomplished on a Rainin reversed phase C 18 column (4.6 mm x 150 mm). The column was washed with a linear gradient system of 50 mM aqueous sodium phosphate (pH 5.8) containing increasing amounts of CH ₃ CN (0-20 min at a flow rate of 1.0 mL/min. The eluate was monitored at 277 ran; the product eluted at 13 min. This product was used directly for the next experiment, yield 31 mg (90%). A heterogenous solution containing 10 mg of 10% Pd on activated carbon in

1 mL of 95% ethanol was treated with a solution containing 31 mg (24.3 μmoles) of ZYE(ahLac) ₂ in 3 mL of methanol and exposed to 1 ami of H ₂ at room temperature for 3 h. The catalyst was removed by filtration through a Celite 545 plug. The filtrate was concentrated under diminished pressure to give the product as a colorless residue, 26 mg (95%). HPLC analysis and purification of the residue was accomplished on a Rainin reversed phase Cl 8 column (4.6 mm x 150 mm). The column was washed with a linear gradient system of 50 mM aqueous sodium phosphate (pH 5.8) containing increasing amounts Of CH ₃ CN (0-20 min at a flow rate of 1.0 mL/min. The eluate was monitored at 277 ran; the product eluted at 4.7 min.

General Methods for Example 1

¹ H NMR spectra were recorded on a Bruker 300 MHz spectrometer and are referenced to CHCl ₃ at 7.26 ppm, H ₂ O at 4.80 ppm, CH ₃ OH at 3.3 ppm, or DMSO-

H6 at 2.49 ppm. Mass spectra were obtained from Scripps Institute, La Jolla, CA. ΗPLC (reversed phase) was performed on Beckman 126/166 Diode Array System or a Varian Model 9050 pump employing a 7125 Rheodyne injector in conjunction with Varian Model 9010 variable wavelength detector. A Rainin/Varian Microsorb MV reverse phase Cl 8 (4.6 x 100 mm) column was used for all analytical work. All column chromatography was carried out using silica gel (230-400 mesh) obtained from Merck. R _f values were measured on glass-backed silica gel TLC plates (Merck 0.25-mm thickness, 20 x 20 cm). All chemicals were obtained from Aldrich Chemical Company, unless otherwise specified. Anhydrous dimethylsulfoxide (DMSO), l-ethyl-3-[3-(dimethylamino)propylcarbodiimide (EDAC), D-galactose (Gal), D-mannose (Man), D-lactose (Lac), 1- methylimidazole, and dithiothreitol (DTT) were purchased from Aldrich Chemical Co. and were used without further purification. N-Acetyl-D-glucosamine (GIcNAc) and N-acetyl-D-galactosamine (GaINAc) were purchased from Sigma Chemical Co. (St. Louis, MO). Diisopropylethylamine (DIPEA) was purchased from Aldrich Chemical Co. and was distilled from potassium hydroxide prior to use. Pyridine and 2,6-lutidine were distilled fromjo-toluenesulfonyl chloride to remove trace amines, and then from solid KOΗ pellets to effect drying. Dimethylformamide (DMF) was dried by distillation from CaH ₂ and stored over 4A molecular sieves. Tetrahydrofuran (THF) was distilled from sodium prior to use with benzophenone as the indicator. SepPak™ Cl 8 cartridges were purchased from Waters-Millipore Corp.

The C6 disulfide thiol modifier synthon, chemical phosphorylation synthon I and the Universal-Q controlled pore glass (CPG) solid support were purchased from Glen Research, Inc.(Sterling, VA). All other reagents for the automated synthesis were purchased from Glen Research, Inc. Size-exclusion gel chromatography solid packing materials (G-IO and G- 15 Sephadex) were purchased from Amersham Pharmacia Biotech. Reverse phase high performance liquid chromatography (RP-HPLC) was carried out using Microsorb Cl 8 column purchased from Rainin Instrument Co., Inc. Semi-preparative RP-HPLC was accomplished using a Varian Semi-Preparative Cl 8 Dynamax-IOOA (Walnut Creek, CA)(IO mm i.d. x 250 mm L) in conjunction with the appropriate guard column (Varian Dynamax-IOOA (Walnut Creek, CA)). Dithiothreitol (DTT) was

purchased from Aldrich Chemical Company (Milwaukee, WI) and was used without further purification. N-N'-Diisopropyl-N-ethylamime (DIPEA) was purchased from Aldrich Chemical Company (Milwaukee, WI) and was redistilled from calcium hydride prior to use. Waters SepPak Cl 8 cartridges were purchased from Millipore Corp. The purified material was stored at 4°C as an aqueous solution.

Example 2 Synthesis of the Renal Cell Conjugate

A conjugate having a dye-labeled oligonucleotide was prepared and used to test renal cell uptake (Figure 3).

An oligonucleotide (12) was synthesized on a solid support using an automated synthesizer and employing commercially available reagents. The oligonucleotide conjugate synthesis is outlined in Figure 3. The solid support was first modified with the "Amino T Modifier" synthon, and then to that the thymidine synthons were added. The total chain-length of the oligonucleotide was 19 residues including the Amino T residue. Each oxidation step was carried out using Beaucage reagent to install the phosphorothioate internucleotide linkage. This linkage was used primarily to impart nuclease resistance to the oligonucleotide. Upon completion of the oligonucleotide chain, the 5 '-end was further modified with the "Thiol Modifier" (Glen Research, Inc.). Finally, the oligonucleotide was removed from the solid support by brief hydroxide treatment and purified while retaining the terminal dimethoxytrityl group. The purified oligonucleotide was allowed first to react with the linker-ligand (11), and then to the tetramethylrhodamine dye. The ligand linker was attached using the chemistry known as the Michael reaction in which the freshly generated thiol modified oligonucleotide (the Michael donor) reacts with the maleimide containing ligand- linker (SMCC-YE(ahLac) ₂ (ll)(the acceptor). Yields are typically greater than 95%.

The dye conjugation was carried out in basic aqueous solution, employing a saturated NaHCO ₃ solution. The dye, TAMRA NHS ester (Glen Research) was purchased as the N-hydroxysuccinimidyl ester. This form of the dye permitted easy modification of the 3 '-terminal oligonucleotide amino group. The reaction was generally complete within 2 h, and was purified by size-exclusion gel filtration

(eluted with 20% ethanol in H ₂ O, G-25 Sephadex). The yield was typically 30%. This method is in that any dye so activated could be substituted. The final conjugate structure was confirmed by mass spectroscopy.

Synthesis of SMCC-YEf ahLactose)? (Ill A solution containing 20 mg (60 μmoles) of N-hydroxysuccinimidyl 4-(N- methylmaleimido)cyclohexane-l-carboxylate (SMCC) in 400 μL of anhydrous DMSO was added to a buffered pH 8.6 solution containing 34 mg (~20 μmoles) of crude YE(ahLac) ₂ (10) in 10 mM borate-buffered saline. The mixture was stirred at room temperature for 3 h, then purified and desalted by size exclusion gel filtration (G- 10 Sephadex, 0.5 x 33 cm) using 20% ethanol in H ₂ O. Fractions were collected and those which corresponded to the product by ¹ H NMR spectroscopy was combined and concentrated under diminished pressure. HPLC analysis and purification of the residue was accomplished on an Rainin reversed phase Ci ₈ column (4.6 mm x 150 mm). The column was washed with a linear gradient system of 50 mM aqueous sodium phosphate (pH 5.8) containing increasing amounts OfCH ₃ CN (0-20 min at a flow rate of 1.0 mL/min. The eluate was monitored at 277 nm; the product eluted at 9.4 min. The yield was determined by UV spectroscopy (A ₂₇₇ = 0.350/1 mL aliquot; 26 mL solution, 45%)

Synthesis of Modified Oligonucleotide (12) The polydeoxythymidine oligonucleotide sequence with the Amino T modifier

(Glen Research, Sterling, VA) was synthesized via an automated synthesizer using amidite intermediate at 0.067 M concentration (5 mL per 50 mg phosphoramidite). The nucleotide molar extinction coefficient for oligonucleotide was calculated to be 154,400 L cm ^"1 M ^"1 . The modified oligomer was synthesized using the corresponding phosphoramidites and methylphosphonamidites from a commercial source (Glen

Research). The 5 '-disulfide linker was then introduced into the oligomer by coupling a C6-disulfide cyanoethyl-phosphoramidite synthon (Glen Research) using phosphoramidite chemistry at the final coupling step of the solid-phase synthesis. When necessary, the sulfuring agent 3H- l,2-benzodithiole-3 -one- 1,1 -dioxide (Beaucage reagent, Glen Research) was substituted for the low moisture oxidizer to effect sulfurization of the phosphite to give the phosphorothioate according to standard established procedures(R.P. Iyler, W. Egan, J. B. Regan, S. L. Beaucage, J. Am. Chem. Soc. 1990, 112, 1253-1254). The oligomer was synthesized without the removal of the

5'-DMT group. The oligomer was deprotected using 0.5 M NaOH in a 50% (v/v) CH ₃ OH in H ₂ O and were purified with the trityl-on procedure using a SepPak (20 cc). The SepPak cartridge was pre-equilibrated with CH ₃ OH (30 mL), 50% (v/v) CH ₃ CN in H ₂ O, and then H ₂ O. The NaOH solution containing the oligonucleotide was diluted to 100 mL with 0.5 M Na _x PO ₄ (pH 5.8) and applied to the pre-equilibrated SepPak™ cartridge. The column/cartridge was washed with H ₂ O, then a 5% CH ₃ CN in H ₂ O to elute the failed sequences. It should be noted that if yield of the full-length oligonucleotide was low, one should examine this fraction for product resulting from pre-mature loss of the dimethoxytrityl group (DMT). To remove the CH ₃ CN/H ₂ O mixture from the column, a H ₂ O wash was performed (30 mL). At this point, the full- length oligonucleotide was ready for de-tritylation. This step was accomplished by the elution with an aqueous solution of trifluoroacetic acid (1% solution)(~30 mL). The column should become orangish in color due to the presence of the DMT cation. After 10 min of stopped flow, the column was then flushed with H ₂ O and 2 mL of 0.5 M Na _x PO ₄ (pH 5.8). The column was once again washed with H ₂ O to remove any residual buffer and trifluoroacetic acid. The product was then eluted with 50% CH ₃ CN in H ₂ O. Final purification of the product was carried out on a Rainin semi-preparative reversed-phase Cl 8 column using a linear gradient OfCH ₃ CN in 50 mM aqueous solution OfNa _x PO ₄ (pH 5.8) (2%→50% CH ₃ CN in 30 min) (Rainin 10 mm ID x 250 mm L; (5 μm pores) in conjunction with a linear gradient of buffer B in buffer A (0→100% B in 30 min). Buffer B was 50% CH ₃ CN in aqueous 50 mM sodium phosphate (pH 5.8). Buffer A was 2% CH ₃ CN in aqueous 50 mM sodium phosphate (pH 5.8)). Fractions containing pure oligonucleotide, either with or without thiol- modifϊer synthon, were pooled, and then desalted separately on a pre-equilibrated Sep- Pak™. The disulfϊde-containing oligomer was finally purified using a semi-preparative reversed-phase Cl 8 column. The final yield was calculated from the absorbance at 260 nm using an UV spectrophotometer (for oligonucleotide, a conversion factor: 0.154 O.D. per nmole was used).

Synthesis of YErahLac) _r SMCC-fpsT)i _R T ^NH2 (13) The reduction of the disulfide moiety to the thiol was effected by the treatment of the

5'-disulfide-containing oligomer with dithiothreitol (DTT). Thus, a 1.570.D. ₂₆ o (~40 nmoles) disulfide oligomer was dissolved in 400 μL of freshly prepared and degassed 50 mM DTT

solution in 10 mM sodium phosphate, pH 8. The mixture was incubated at 37 ⁰ C for 2 h. Quantitative reduction was confirmed by reversed-phase HPLC analysis, which shows that the thiol oligomers elute faster than the parent disulfide oligomers. The thiol oligomer was then purified on a Sephadex G-25 column (1O x 450mm) to remove DTT and other salts. Column packing and sample elution were effected by the use of degassed 20% CH ₃ CH ₂ OH in H ₂ O. The G-25 fraction containing the pure thiol oligomer was used immediately in the next reaction to minimize unwanted oxidations and dimerizations.

The G-25 fraction containing 1.56 O.D. 260 (~40 nmoles) pure thiol oligomer was mixed with SMCC-YE(ahLac) ₂ (100 nmoles) immediately after it was collected. The mixture was concentrated to dryness in a SpeedVac™. The residue was dissolved in 100 μL of degassed 50% CH ₃ CN containing 0.1 M aqueous sodium phosphate, pH 7.0. The solution was further degassed in a SpeedVac™ by applying vacuum for about 5 minutes. The solution was then capped tight and incubated at room temperature overnight to complete conjugation. The solution was immediately placed in a SpeedVac™ and concentrated to about 1 mL. The solution was then capped tight and incubated at room temperature overnight to allow conjugation to complete. Both procedures have been found to give quantitative conjugation of the thiol-containing oligomers.

To determine the yield of the conjugation reaction, about 0.5-μL portion of the reaction was dried and analyzed by 20% denaturing PAGE. The mobility of the conjugate was compared with that of the unconjugated oligomer in the same gel. Unlabeled conjugate can also be analyzed in similar fashion by UV shadowing. The PAGE results indicated quantitative conjugation of the thiol oligomer with the neoglycopeptide. The conjugate formation was checked by HPLC analysis. A Sephadex™ G25 column (1.0 cm x 45 cm, fine), eluted with 20% ethanol purified the conjugate. The purified conjugate was then used directly in bioefficacy experiments and other experiments, which do not require a radiolabeled conjugate. Final confirmation of the conjugate was accomplished using MALDI mass spectrometry (Scripps Research Institute); mass spectrum (MALDI), m/z 7725 M . (C ₂₆₆ H ₃₅₃ N ₄₅ Oi ₄₅ Pi ₉ S ₂₀ -Na ⁺ requires 7725.1056)(M ⁺ ).

Synthesis of tetramethylrhodamine-labeled YEfahLacVSMCC-ah(psDi R-T ^{NH" TAMRA} conjugate (14)

A solution containing 25.4 nmoles (0.154 O.D./mL) of YE(ahLac) ₂ -SMCC- ah(psT)i ₈ -T ^NH2 (13) in 400 μL OfH ₂ O was mixed with 100 μL of saturated NaHCO ₃

solution at room temperature. To this solution, N-hydroxysuccinimide ester of 5,6- TAMRA (Glen Research)(5 μL, 0.22 M) in DMSO was added. The reaction mixture was allowed to incubate at room temperature in the dark for 2 h. Afterwards, the reaction mixture was applied to a G-25 Sephadex column (1.0 x 45 cm) for purification. Elution of the column in the dark with 20% ethanol in H ₂ O provided fractions, which corresponded to the largest molecular weight component. This fraction was then reapplied to a G-25 Sephadex column (1.0 x 45 cm), packed and eluted with 20% ethanol in H ₂ O. The fraction corresponding to the largest molecular weight was analyzed by UV spectroscopy (ε _max 260 and 556 nm) and reverse phase HPLC using the diode array analysis (compound eluted at 10.92 min.), yield 8.53 nmoles). The ratio of absorbance values A ₂ 6o/As56 was an indication of the conjugate:dye stoichiometry (ratio 1.10). Final confirmation of the conjugate 14 was accomplished using MALDI mass spectrometry (Scripps Research Institute); mass spectrum (MALDI), m/z 8145 M+Na ⁺ . (C _29I H ₃₇₄ N ₄₇ O ₁₄₈ Pi ₉ S ₂₀ -Na ⁺ requires 8145.2508)(M+Na ⁺ ).

EXAMPLE 3 Cellular Uptake Studies

Cellular uptake experiments using this labeled conjugate described above demonstrate the renal cell selectivity the lactose bi-antennary conjugate and its ability to be internalized effectively by both normal renal cells (Figure 4) and renal carcinoma cells (Figures 5a and 5b). The bi-antennary, lactose-derivatized, phosphorothioate-lmked oligonucleotide conjugate was specifically endocytosed by the RPTEC cells to the extent of 15.8 pmoles per million cells as shown in Figure 4. The measurements were obtained upon the direct quantitation of the intracellular fluorescence as averaged over cells clearly identified on the microscope slide (>15 cells). The cells were incubated with the dye-labeled conjugate for 24 h at 37 ⁰ C. This length of incubation time was to ensure sufficient uptake to observe fluorescence. For easy identification of the cell boundaries and nucleus, Cell Tracker Blue™ CMCA (Molecular Probes) was used.

A bi-antennary lactose ligand-derived, methylphosphonate-linked payload conjugate was applied to Caki-1 cells (metastatic cells from the skin, obtained from ATCC) in culture. The result was minimal or no cellular uptake with non-specific binding to the cell surface by the conjugate. In contrast, when the experiment was repeated using conjugate (14) and cells of a primary renal adenocarcinoma cell

line(ACHN) significant uptake (7.8 pmoles/10 ⁶ cells) was observed (Figure 5a). In contrast, only minimal uptake (< 1 pmole/10 ⁶ cells) was observed when unconjugated oligonucleotide was applied to ACHN cells (Figure 5b). Other renal cell lines that can be used for testing renal cell uptake of conjugates include: Various commercially available cell lines that fit this type are RPTEC, ACHN, 786- O, Caki-2 G-402, 769-P, Hs 835.T, and Hs 926.T.

General Methods for Example 3

Cells (RPTEC and ACHN) were seeded into two-chamber Lab-Tek™ slides and grown overnight in EMEM/ 10% fetal bovine serum (FBS) growth factors. The medium was then removed and replaced with MEM/2% ABS containing the tetramethylrhodamine (TAMRA)-labeled neoglycoconjugate at a concentration of 1 μM. Cells were incubated with the tetramethylrhodamine (TAMRA)-labeled neoglycoconjugate for 24 h. At 22 h, Cell Tracker Blue™ CMCA (Molecular Probes) was added at 25 μM for the remaining 2 h. Chambers were then removed. The cells were rinsed in PBS and the slides were air-dried. Prolong Anti-Fade™ (Molecular Probes) was added under a glass cover slip. The mounting medium is allowed to dry and the slides were examined for blue fluorescence to locate the cells and the red fluorescence to locate the conjugate within the cells. The images were taken on a Leica™ DMRXA microscope with IMAGE Pro Plus 3.0.1.1 Software™ for processing and quantitation of uptake based upon pixel density at fixed exposure time (2 sec). A standard curve (IOD versus concentration) was made to allow extrapolation of the intracellular concentration.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.