thiolan-2-imine

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Pure cholesterol-ethylenediamine (10 mmol) was dissolved in 300 mL of DCM. 100 mmol of 2,2′-dipyridyl disulfide was dissolved in 100 mL of methanol and added to the cholesterol-ethylenediamine solution plus 20 mmol of diisopropylethylamine. 15 mmol of 2-iminothiolane was dissolved in 50 mL of methanol and then drop-wise added to the cholesterol-ethylenediamine/2,2′-dipyridyldisulfide solution over 30 minutes at R.T. Following an overnight R.T. stir, organic solvent was roto-evaporated away using medium vacuum until a minimal sample volume remained. Approximately 800 mL of acetonitrile was added to precipitate the crude product. The solid was then collected by filtration onto a Whatman #5 filter apparatus, rinsed several times with acetonitrile and then air-dried. The crude solid was dissolved in a minimal volume of chloroform and chromatographically purified using a silica column. The purified product presented a single TLC spot (4:1 chloroform/methanol), and a single peak (parent ion M+H=685) was identified by positive mode electrospray. organic solvent was roto-evaporated away Approximately 800 mL of acetonitrile was added to precipitate the crude product The solid was then collected by filtration onto a Whatman #5 filter apparatus rinsed several times with acetonitrile air-dried The crude solid was dissolved in a minimal volume of chloroform chromatographically purified

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 1-L round-bottom flask equipped with a magnetic stir bar and rubber septum was purged with nitrogen and charged with 600 mL of USP-grade dry ethanol, 60 mL of anhydrous THF, 4.95 grams (10.5 mmol, 1.05 equivalents) of dexamethasone-mesylate and 10 grams (19.5 mmol, 4.95 equivalents) of spermine. 2-Iminothiolane, 1.38 grams (10 mmol, 1.0 equivalents) in 3 mL of water, was added dropwise to the solution over 5 minutes with vigorous mixing. The solution changed color from clear to clear light yellow. The reaction was monitored by TLC and by analytical HPLC. After three hours at room temperature, the TLC spot at R1=0 (the charged DS) (Rt=0.7 minutes DS by analytical HPLC) was maximized, and dexamethasone-mesylate (Rt 0.47, 2.8 minutes HPLC) spot minimized. The crude reaction mixture was diluted with 15.25 mL (198 mmol, 19.8 equivalents) of trifluoroacetic acid (or alternatively formic acid), forming a white precipitate (spermine tetra TFA salt or tetra-formate salt). Solvent was removed with a rotary evaporator and 50 mL of ethanol was added, forming a white precipitate (spermine-4TFA). The pH 3 solution was filtered twice to remove spermine acid salt, and ethanol was removed with a rotary evaporator. 50 mL of water was added to the yellow viscous oil, yielding a precipitate (dexamethasone and dexamethasone mesylate), and the solution was filtered through 0.2 μM filter to yield a clear yellow liquid. Water was removed with a lyophilizer over several days to yield dexamethasone-spermine and disubstituted spermine tetra-acid salts. Total yield ˜30% for combined products). A 1-L round-bottom flask equipped with a magnetic stir bar rubber septum was purged with nitrogen The crude reaction mixture

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The 21-hydroxy group of dexamethasone is not required for anti-inflammatory activity, and therefore is an ideal choice for conjugation to a polycation (FIG. 1A) (Schimmer et al., 1996, in The Pharmacological Basis of Therapeutics, 1459-1485, eds. Hardman and Limbird, McGraw-Hill, New York). A one-pot reaction between spermine, 2-iminothiolane (Traut's reagent) and dexamethasone mesylate yielded the dexamethasone-spermine and disubstituted conjugates, (DS and D2S, respectively) (FIG. 1B). Traut's reagent is selectively ring-opened by the primary amines (Hermanson, 1996, Bioconjugate Techniques, Academic Press) on spermine, forming a hydrolytically sensitive amidimide bond (Hermanson, 1996, Bioconjugate Techniques, Academic Press) between spermine and iminothiolane and a reactive thiolate anion that reacts with the α-keto mesylate on the 21 position of dexamethasone mesylate (Simmons et al., 1980, J. Org. Chem. 45:084-3088), yielding an α-keto thioether linkage between the dexamethasone and iminothiolane. Dexamethasone mesylate (prepared or received from Steraloids, N.H.) or dexamethasone (Steraloids, N.H.), 2-iminothiolane (Traut's reagent) (Pierce), spermine (Sigma), and ethanol or DMSO (Aldrich) were used as received. A total of 105 mg (223 μmol) of dexamethasone-mesylate, and 28.4 mg (206 μmol) of Trout's reagent were dissolved together in either DMSO or ethanol prior to addition of 31.9 μl (145 μmol of spermine at room temperature. The conjugation reaction was complete in 45 minutes by TLC. HPLC purification (60/40 0.1% TFA/acetonitrile, Hamilton PRP-1 column), evaporative solvent removal, and freeze-drying yielded both DS and D2S as white powders, 1H NMR of DS confirmed the presence of the 3-bis-enone and 11- and 17-hydroxy groups required for glucocorticoid activity (FIG. 1A) as well as 1H signals from the conjugated spermine and the 21-α-keto thioether group. Hydrolysis of DS in 1M NaOH for 20 minutes resulted in the cleavage of the amidimide linkage between spermine and iminothiolane, forming a dexamethasone-amide (DA) (FIG. 1B), which has a 21-substituted butyl thioether amide side-chain on dexamethasone. For DA, FIG. 1B: Calc: C, 63.26; H, 7.35; N, 2.84. Found: C, 63.44; H, 7.27; N, 2.83. Water solubility: DS>100,000 mg/l; DA 60 mg/l; (dexamethasone 100 mg/l).

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The 21-hydroxy group of dexamethasone is not required for anti-inflammatory activity, and therefore is an ideal choice for conjugation to a polycation (FIG. 1A) (Schimmer et al., 1996, in The Pharmacological Basis of Therapeutics, 1459-1485, eds. Hardman and Limbird, McGraw-Hill, New York). A one-pot reaction between spermine, 2-iminothiolane (Traut's reagent) and dexamethasone mesylate yielded the dexamethasone-spermine and disubstituted conjugates, (DS and D2S, respectively) (FIG. 1B). Traut's reagent is selectively ring-opened by the primary amines (Hermanson, 1996, Bioconjugate Techniques, Academic Press) on spermine, forming a hydrolytically sensitive amidimide bond (Hermanson, 1996, Bioconjugate Techniques, Academic Press) between spermine and iminothiolane and a reactive thiolate anion that reacts with the α-keto mesylate on the 21 position of dexamethasone mesylate (Simmons et al., 1980, J. Org. Chem. 45:084-3088), yielding an α-keto thioether linkage between the dexamethasone and iminothiolane. Dexamethasone mesylate (prepared or received from Steraloids, N.H.) or dexamethasone (Steraloids, N.H.), 2-iminothiolane (Traut's reagent) (Pierce), spermine (Sigma), and ethanol or DMSO (Aldrich) were used as received. A total of 105 mg (223 μmol) of dexamethasone-mesylate, and 28.4 mg (206 μmol) of Trout's reagent were dissolved together in either DMSO or ethanol prior to addition of 31.9 μl (145 μmol of spermine at room temperature. The conjugation reaction was complete in 45 minutes by TLC. HPLC purification (60/40 0.1% TFA/acetonitrile, Hamilton PRP-1 column), evaporative solvent removal, and freeze-drying yielded both DS and D2S as white powders, 1H NMR of DS confirmed the presence of the 3-bis-enone and 11- and 17-hydroxy groups required for glucocorticoid activity (FIG. 1A) as well as 1H signals from the conjugated spermine and the 21-α-keto thioether group. Hydrolysis of DS in 1M NaOH for 20 minutes resulted in the cleavage of the amidimide linkage between spermine and iminothiolane, forming a dexamethasone-amide (DA) (FIG. 1B), which has a 21-substituted butyl thioether amide side-chain on dexamethasone. For DA, FIG. 1B: Calc: C, 63.26; H, 7.35; N, 2.84. Found: C, 63.44; H, 7.27; N, 2.83. Water solubility: DS>100,000 mg/l; DA 60 mg/l; (dexamethasone 100 mg/l).

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Pure cholesterol-ethylenediamine (10 mmol) was dissolved in 300 mL of DCM. 100 mmol of 2,2′-dipyridyl disulfide was dissolved in 100 mL of methanol and added to the cholesterol-ethylenediamine solution plus 20 mmol of diisopropylethylamine. 15 mmol of 2-iminothiolane was dissolved in 50 mL of methanol and then drop-wise added to the cholesterol-ethylenediamine/2,2′-dipyridyldisulfide solution over 30 minutes at R.T. Following an overnight R.T. stir, organic solvent was roto-evaporated away using medium vacuum until a minimal sample volume remained. Approximately 800 mL of acetonitrile was added to precipitate the crude product. The solid was then collected by filtration onto a Whatman #5 filter apparatus, rinsed several times with acetonitrile and then air-dried. The crude solid was dissolved in a minimal volume of chloroform and chromatographically purified using a silica column. The purified product presented a single TLC spot (4:1 chloroform/methanol), and a single peak (parent ion M+H=685) was identified by positive mode electrospray. organic solvent was roto-evaporated away Approximately 800 mL of acetonitrile was added to precipitate the crude product The solid was then collected by filtration onto a Whatman #5 filter apparatus rinsed several times with acetonitrile air-dried The crude solid was dissolved in a minimal volume of chloroform chromatographically purified

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Pure cholesterol-ethylenediamine (10 mmol) was dissolved in 300 mL of DCM. 100 mmol of 2,2′-dipyridyl disulfide was dissolved in 100 mL of methanol and added to the cholesterol-ethylenediamine solution plus 20 mmol of diisopropylethylamine. 15 mmol of 2-iminothiolane was dissolved in 50 mL of methanol and then drop-wise added to the cholesterol-ethylenediamine/2,2′-dipyridyldisulfide solution over 30 minutes at R.T. Following an overnight R.T. stir, organic solvent was roto-evaporated away using medium vacuum until a minimal sample volume remained. Approximately 800 mL of acetonitrile was added to precipitate the crude product. The solid was then collected by filtration onto a Whatman #5 filter apparatus, rinsed several times with acetonitrile and then air-dried. The crude solid was dissolved in a minimal volume of chloroform and chromatographically purified using a silica column. The purified product presented a single TLC spot (4:1 chloroform/methanol), and a single peak (parent ion M+H=685) was identified by positive mode electrospray. organic solvent was roto-evaporated away Approximately 800 mL of acetonitrile was added to precipitate the crude product The solid was then collected by filtration onto a Whatman #5 filter apparatus rinsed several times with acetonitrile air-dried The crude solid was dissolved in a minimal volume of chloroform chromatographically purified

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 1-L round-bottom flask equipped with a magnetic stir bar and rubber septum was purged with nitrogen and charged with 600 mL of USP-grade dry ethanol, 60 mL of anhydrous THF, 4.95 grams (10.5 mmol; 1.05 equivalents) of dexamethasone-mesylate and 10 grams (19.5 mmol, 4.95 equivalents) of spermine. 2-Iminothiolane, 1.38 grams (10 mmol, 1.0 equivalents) in 3 mL of water, was added dropwise to the solution over 5 minutes with vigorous mixing. The solution changed color from clear to clear light yellow. The reaction was monitored by TLC and by analytical HPLC. After three hours at room temperature, the TLC spot at Rt=0 (the charged DS) (Rt=0.7 minutes DS by analytical HPLC) was maximized, and dexamethasone-mesylate (Rt 0.47, 2.8 minutes HPLC) spot minimized. The crude reaction mixture was diluted with 15.25 mL (198 mmol, 19.8 equivalents) of trifluoroacetic acid, forming a white precipitate (spermine tetra TFA salt). Solvent was removed with a rotary evaporator and 50 mL of ethanol was added, forming a white precipitate (spermine-4TFA). The pH 3 solution was filtered twice to remove spermine tetratrifluoroacetic acid salt, and ethanol was removed with a rotary evaporator. 50 mL of water was added to the yellow viscous oil, yielding a precipitate (dexamethasone and dexamethasone mesylate), and the solution was filtered through 0.2 μM filter to yield a clear yellow liquid. Water was removed with a lyophilizer over several days to yield dexamethasone-spermine tetratrifluoroacetic acid salt. Yield: 8.228 grams (7.17 mmol, 72%). A 1-L round-bottom flask equipped with a magnetic stir bar rubber septum was purged with nitrogen The crude reaction mixture

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 1-L round-bottom flask equipped with a magnetic stir bar and rubber septum was purged with nitrogen and charged with 600 ml of USP-grade dry ethanol, 60 ml of anhydrous THF, 4.95 grams (10.5 mmol, 1.05 equivalents) of dexamethasone-mesylate and 10 grams (19.5 mmol, 4.95 equivalents) of spermine. 2-Iminothiolane, 1.38 grams (10 mmol, 1.0 equivalents) in 3 ml of water, was added dropwise to the solution over 5 minutes with vigorous mixing. The solution changed color from clear to clear light yellow. The reaction was monitored by TLC and by analytical HPLC. After three hours at room temperature, the TLC spot at Rt=0 (the charged DS) (Rt=0.7 min DS by analytical HPLC) was maximized, and dexamethasone-mesylate (Rt 0.47, 2.8 min HPLC) spot minimized. The crude reaction mixture was diluted with 15.25 ml (198 mmol, 19.8 equivalents) of trifluoroacetic acid, forming a white precipitate (spermine tetra TFA salt). Solvent was removed with a rotary evaporator and 50 ml of ethanol was added, forming a white precipitate (spermine-4TFA). The pH 3 solution was filtered twice to remove spermine tetratrifluoroacetic acid salt, and ethanol was removed with a rotary evaporator. 50 ml of water was added to the yellow viscous oil, yielding a precipitate (dexamethasone and dexamethasone mesylate), and the solution was filtered through 0.2 μm filter to yield a clear yellow liquid. Water was removed with a lyophilizer over several days to yield dexamethasone-spermine tetratrifluoroacetic acid salt. Yield: 8.228 grams (7.17 mmol, 72%). A 1-L round-bottom flask equipped with a magnetic stir bar rubber septum was purged with nitrogen The crude reaction mixture

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Keyhole limpet hemocyanin (KLH, 60 mg) was reconstituted in 100 mM sodium phosphate buffer at pH 7.2. 2-Iminothiolane (2IT, 13.5 mg) was added to the protein solution as a solid, and the reaction was allowed to stir at room temperature in the dark and under argon atmosphere for 1 hour. The activated KLH—(SH)n was desalted on a Sephadex PD-10 column pre-equilibrated with 100 mM sodium phosphate buffer at pH 6.5. The SH loading was determined (Ellman's reagent) to be 886 per KLH molecule (MW 5,000,000). To 6 ml of KLH—(SH)n, 4.7 mg/mL, was added a solution of 14 mg (0.027 mmol) of MDEA-maleimide (2O) in 1 mL of DMF dropwise, and the mixture was allowed to stir at room temperature 18 hours. The resulting conjugate was placed in a dialysis tube (10,000 MW cut-off) and was dialyzed in 1 L of phosphate buffered saline (PBS) buffer (180 mM NaCl, 10 mM sodium phosphate, pH 7.2] containing 20% DMF. (3 times, at least 6 hours each). This was followed by 1 L of PBS buffer, pH 7.2 at 4° C. The protein concentration was determined to be 2.08 mg/mL using Biorad Coomassie blue protein assay (Bradford, M., Anal. Biochem. 72:248, 1976). A total of 20 mL of the conjugate was obtained. to stir at room temperature 18 hours The resulting conjugate was placed in a dialysis tube (10,000 MW cut-off) and was dialyzed in 1 L of phosphate buffered saline (PBS) buffer (180 mM NaCl, 10 mM sodium phosphate, pH 7.2] containing 20% DMF 3 times, at least 6 hours This was followed by 1 L of PBS buffer, pH 7.2 at 4° C The protein concentration A total of 20 mL of the conjugate was obtained

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Keyhole limpet hemocyanin (KLH, 60 mg) was reconstituted in 100 mM sodium phosphate buffer at pH 7.2. 2-Iminothiolane (2IT, 13.5 mg) was added to the protein solution as a solid, and the reaction was allowed to stir at room temperature in the dark and under argon atmosphere for 1 hour. The activated KLH-(SH)n was desalted on a Sephadex PD-10 column pre-equilibrated with 100 mM sodium phosphate buffer at pH 6.5. The SH loading was determined (Ellman's reagent) to be 886 per KLH molecule (MW 5,000,000). To 6 ml of KLH-(SH)n, 4.7 mg/mL, was added a solution of 14 mg (0.027 mmol) of MDEA-maleimide (2O) in 1 mL of DMF dropwise, and the mixture was allowed to stir at room temperature 18 hours. The resulting conjugate was placed in a dialysis tube (10,000 MW cut-off) and was dialyzed in 1 L of phosphate buffered saline (PBS) buffer (180 mM NaCl, 10 mM sodium phosphate, pH 7.2] containing 20% DMF. (3 times, at least 6 hours each). This was followed by 1 L of PBS buffer, pH 7.2 at 4° C. The protein concentration was determined to be 2.08 mg/mL using Biorad Coomassie blue protein assay (Bradford, M., Anal. Biochem. 72:248, 1976). A total of 20 mL of the conjugate was obtained. to stir at room temperature 18 hours The resulting conjugate was placed in a dialysis tube (10,000 MW cut-off) and was dialyzed in 1 L of phosphate buffered saline (PBS) buffer (180 mM NaCl, 10 mM sodium phosphate, pH 7.2] containing 20% DMF 3 times, at least 6 hours This was followed by 1 L of PBS buffer, pH 7.2 at 4° C The protein concentration A total of 20 mL of the conjugate was obtained

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Pure cholesterol-ethylenediamine (10 mmol) was dissolved in 300 mL of DCM. 100 mmol of 2,2′-dipyridyl disulfide was dissolved in 100 mL of methanol and added to the cholesterol-ethylenediamine solution plus 20 mmol of diisopropylethylamine. 15 mmol of 2-iminothiolane was dissolved in 50 mL of methanol and then drop-wise added to the cholesterol-ethylenediamine/2,2′-dipyridyldisulfide solution over 30 minutes at R.T. Following an overnight R.T. stir, organic solvent was roto-evaporated away using medium vacuum until a minimal sample volume remained. Approximately 800 mL of acetonitrile was added to precipitate the crude product. The solid was then collected by filtration onto a Whatman #5 filter apparatus, rinsed several times with acetonitrile and then air-dried. The crude solid was dissolved in a minimal volume of chloroform and chromatographically purified using a silica column. The purified product presented a single TLC spot (4:1 chloroform/methanol), and a single peak (parent ion M+H=685) was identified by positive mode electrospray. organic solvent was roto-evaporated away Approximately 800 mL of acetonitrile was added to precipitate the crude product The solid was then collected by filtration onto a Whatman #5 filter apparatus rinsed several times with acetonitrile air-dried The crude solid was dissolved in a minimal volume of chloroform chromatographically purified

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 1-L round-bottom flask equipped with a magnetic stir bar and rubber septum was purged with nitrogen and charged with 600 mL of USP-grade dry ethanol, 60 mL of anhydrous THF, 4.95 grams (10.5 mmol, 1.05 equivalents) of dexamethasone-mesylate and 10 grams (19.5 mmol, 4.95 equivalents) of spermine. 2-Iminothiolane, 1.38 grams (10 mmol, 1.0 equivalents) in 3 mL of water, was added dropwise to the solution over 5 minutes with vigorous mixing. The solution changed color from clear to clear light yellow. The reaction was monitored by TLC and by analytical HPLC. After three hours at room temperature, the TLC spot at R1=0 (the charged DS) (Rt=0.7 minutes DS by analytical HPLC) was maximized, and dexamethasone-mesylate (Rt 0.47, 2.8 minutes HPLC) spot minimized. The crude reaction mixture was diluted with 15.25 mL (198 mmol, 19.8 equivalents) of trifluoroacetic acid (or alternatively formic acid), forming a white precipitate (spermine tetra TFA salt or tetra-formate salt). Solvent was removed with a rotary evaporator and 50 mL of ethanol was added, forming a white precipitate (spermine-4TFA). The pH 3 solution was filtered twice to remove spermine acid salt, and ethanol was removed with a rotary evaporator. 50 mL of water was added to the yellow viscous oil, yielding a precipitate (dexamethasone and dexamethasone mesylate), and the solution was filtered through 0.2 μM filter to yield a clear yellow liquid. Water was removed with a lyophilizer over several days to yield dexamethasone-spermine and disubstituted spermine tetra-acid salts. Total yield ˜30% for combined products). A 1-L round-bottom flask equipped with a magnetic stir bar rubber septum was purged with nitrogen The crude reaction mixture