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Bioconjugate Chem. 1998, 9, 108−117
Simplified Preformed Chelate Protein Radiolabeling with Technetium-99m Mercaptoacetamidoadipoylglycylglycine (N3S-Adipate) Sudhakar Kasina,* James A. Sanderson, Jeffrey N. Fitzner, Ananthachari Srinivasan, Tripuraneni N. Rao, Lori J. Hobson, John M. Reno, Donald B. Axworthy, Paul L. Beaumier, and Alan R. Fritzberg NeoRx Corporation, 410 West Harrison Street, Seattle, Washington 98119. Received April 1, 1997; Revised Manuscript Received October 7, 1997X
A simplified kiet has been developed for 99mTc protein radiolabeling using an N3S triamide mercaptide bifunctional chelating agent and the preformed chelate approach. The process combined N3S chelating agent, gluconate intermediate transfer agent, stannous reducing agent, and gentisic acid stabilizer into a lyophilized formulation. With sulfur donor atom hemithioacetal protection of the ligand, δ-2,3,5,6-tetrafluorothiophenyl R-S-(1-ethoxyethyl)mercaptoacetamido-L-adipoylglycylglycine, optimum 99m Tc chelation was achieved in a single step. Subsequent reaction with NR-LU-10 antibody Fab fragment followed by purification via QAE Sephadex anion exchange resin filter afforded 99mTc-N3SNR-LU-10 Fab conjugate with retained immunoreactivity and effective tumor targeting properties.
INTRODUCTION
Earlier investigations have shown that amide thiolate (N2S2, N3S)1 chelating agents form well-defined 99mTc complexes of high stability (1, 2). Application of N2S2 chelating agents for the bifunctional attachment of 99mTc to proteins such as antibodies was established with 4,5-dimercaptoacetamidopentanoate via chelation, in situ esterification, and protein coupling (3). The chelating agent was subsequently modified to a tetrafluorophenyl active ester and incorporated into a kit (4) that has been used clinically with various antibody fragments, including NR-ML-05 Fab fragment for melanoma (5) and NRLU-10 Fab fragment for small cell lung carcinoma and other solid tumor cancers (6-8). Additionally it has been used with annexin V, a 36 kD human placental anticoagulant protein for thrombus imaging (9). Although the pentanoate-based N2S2 chelating kit served successfully to stably attach 99mTc without negatively impacting tumor targeting ability of the Fab fragments and annexin V protein, some drawbacks were * Author to whom correspondence and reprint requests should be addressed. [Phone (206) 281-7001; Fax (206) 286-2537; email
[email protected]]. X Abstract published in Advance ACS Abstracts, December 15, 1997. 1 Abbreviations: Ab-NH , antibody fragment with lysine 2 -amino group; BSA, bovine serum albumin; BuOH, butanol; CBZ, carbobenzyloxy; DCC, 1,3-dicyclohexylcarbodiimide; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DTPA, diethylenetriaminepentaacetic acid; EtOAc, ethyl acetate; HOAc, acetic acid; HPLC, high pressure liquid chromatography; IPA, 2-propanol; ITLC, instant thin layer chromatography; NR-LU10 Fab, pancarcinoma papain fragmented murine antibody fragment; N2S2, diamide disulfur; N3S-adipate-STFP, δ-2,3,5,6tetrafluorothiophenyl-R-S-(1-ethoxyethyl)mercaptoacetamido-Ladipoylglycylglycine; N3S-adipate-OTFP, δ-2,3,5,6-tetrafluorophenyl R-S-(1-ethoxyethyl)mercaptoacetamido-L-adipoylglycylglycine; PBS, phosphate-buffered saline; PIP, P-iodophenylate; QAE, quaternary aminoethyl; TCA, trichloroacetic acid; TLC, thin layer chromatography.
experienced. Catabolism of the radiolabeled protein following kidney and liver uptake yielded a lysine adduct of the 99mTc-chelate that was excreted via the hepatobiliary system (10). This excretion interfered in the detection of disease in the lower abdomen. Furthermore, the kit was cumbersome, involving manipulation of several components prior to the 99mTc chelation step. Thus, the opportunity existed for further simplification of the radiolabeling process to yield a product of improved biodistribution and ease of use. Previously (11), we have shown that the addition of a carboxyl group to the N2S2 ligand core decreased hepatobiliary excretion of the radioactivity. Additional carboxyl groups added to the ligand further decreased hepatobiliary excretion. We have now extended these results to the N3S ligand system and herein describe the bifunctional chelate, 2,3,5,6-tetrafluorothiophenyl-S-(1ethoxyethyl)mercaptoacetamidoadipoylglycylglycine (N3Sadipate-STFP), and its incorporation into a kit for protein radiolabeling for clinical application. This kit combined N3S-adipate-STFP, gluconate intermediate transfer agent, and stannous reducing agent into a lyophilized formulation that reduced the number of steps and time required for protein radiolabeling. Comparative mice biodistribution studies of this 99mTc-N3S Fab conjugate, noncarboxylate 99mTc-N2S2 Fab conjugate (4), and 125I-PIP Fab conjugate (2) are herein described. Comparative rat biodistribution and hepatobiliary excretion studies for the series of carboxylate-substituted amide thiolate bifunctional chelating agents will be described in a separate report. MATERIALS AND METHODS
Reagents. All reagents were obtained from Aldrich Chemical Co. (Milwaukee, WI) except BSA, transferrin, cysteine, and sodium phosphate, all obtained from Sigma Chemical Co. (St. Louis, MO), and QAE Sephadex obtained from Pharmacia Biotech, Inc. (Piscataway, NJ). All solvents were reagent or HPLC grade. Two intermediate precursor compounds, S-(1-ethoxyethyl)mercap-
S1043-1802(97)00047-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/12/1998
Chelate Protein
99mTc
Radiolabeling
toacetic acid 11 and N-hydroxysuccinimidyl S-(1-ethoxyethyl)mercaptoacetate 12, were prepared as described earlier (4). NR-LU-10 Fab antibody fragment was prepared in-house from standard papain digestion of whole antibody isolated from the supernatant of cultured murine hybridoma cells (12, 13). The 99mTc-N3S-NR-LU-10 Fab purification column was manufactured to NeoRx specifications by BioProbe, Inc. (Tustin, CA) and Sepratech, Inc. (American Canyon, CA). Lyophilization vials were obtained from Miles, Pharmaceutical Div. (Spokane, WA), and stoppers were purchased from The West Co. (Lionville, PA). General Instrumentations. Proton NMR spectra were obtained on a Varian Gemini-200 spectrometer with tetramethylsilane as an internal standard. IR data were obtained on a Perkin-Elmer 1310 infrared spectrophotometer. Mass spectral data (both low and high resolution) were obtained on a Micromass 70 SEQ tandem hybrid mass spectrometer (Micromass Ltd., Manchester, U.K.) with associated DEC PDP 11-250J Data System. Mass spectral data were obtained by fast atom bombardment (FAB+) by electron impact (EI) ionization on the sample introduced by the direct insertion probe (DIP). Melting points were obtained on a Thomas-Hoover apparatus and are uncorrected. Elemental analyses were performed by Desert Microanalytical Laboratories, Tucson, AZ. Moisture analyses were performed on a Photo Volt Moisture Analyzer. Chromatography. High performance liquid chromatography (HPLC) of small molecule forms of the organic syntheses and radiolabeling of bifunctional chelates was carried out on Beckman-Altex systems using 5 µm ODS (Beckman Ultrasphere) C-18 columns. Beckman Models 153 and 331 spectrophotometers with fixed wavelength or 155-400 variable wavelength were used for optical detection, while a Beckman Model 170 radiometric detection in series with the former was used in identifying the radiolabeled products. 99mTc-N S-adipate-STFP analyses was performed iso3 cratically using a mobile phase of acetonitrile in 0.01 M sodium phosphate (pH 6.8) at a ratio of 24:76 at 1.5 mL/ min on a reverse phase column. Additionally the separation was performed on a gradient system going from an acetonitrile:water:glacial acetic acid ratio of 5:94:1 (pump A) to 60:39:1 (pump B) at 1.0 mL/min. The program for the gradient elution involved a 10 min pumping of 100% A followed by a 35 min pumping of 100% B. Antibody protein conjugate radiochemical purity was assessed by size-exclusion chromatography using Zorbax (DuPont de Nemours) HPLC with 0.2 M phosphate, pH 7.2 as mobile phase. Detection was performed as noted above. Separation of free and protein-bound 99mTc was also performed using silica gel impregnated glass fiber instant thin layer chromatography (ITLC) supplied by Gelman Sciences, Inc., Ann Arbor, MI, as ITLC SG. The silica gel strips were precut to 2 × 10 cm, activated at 100 °C for at least 2 h and stored according to the manufacturer’s instructions. The ITLC strips were developed with freshly prepared 12% w/v trichloroacetic acid (TCA). In this system, the 99mTc-labeled Fab fragment remained at the origin and non-protein bound 99m Tc-labeled species migrated with the solvent front. The TLC strips were cut into two halves and separately counted for final purity on a Packard MINAXI 500 series γ counter. Syntheses of N3S-Adipate-STFP (17) and N3SAdipate-OTFP (15). Scheme 1 outlines the syntheses of thio and oxo 2,3,5,6-tetrafluorophenyl S-(1-ethoxyethyl)mercaptoacetamido-L-adipoylglycylglycine bifunctional
Bioconjugate Chem., Vol. 9, No. 1, 1998 109
chelating moieties, N3S-adipate-STFP and N3S-adipateOTFP. The final compounds were purified by preparative reverse phase HPLC. The detailed procedures were performed as follows. L-R-N-(Carbobenzyloxy)aminoadipic Acid (2). 4.47 g (27.7 mmol) of L-aminoadipic acid (1) was dissolved in 14 mL of 4 N sodium hydroxide and cooled to 0 °C. To the magnetically stirred solution was added a solution of 4.35 mL (30.5 mmol) of carbobenzyloxy chloride (CBZCl) in 14 mL of dioxane dropwise over a period of 30 min. The pH of the solution was periodically monitored during the addition, maintaining a basic pH by adding portions of 8.2 mL of 4.0 N sodium hydroxide during the addition. After 2 h and 15 min, the progress of the reaction was monitored by TLC in n-BuOH:HOAc:H2O ) 3:2:1. Due to the presence of trace amounts of starting amino adipic acid, an additional 1.0 mL (0.25 equiv) of CBZ-Cl in 3.0 mL dioxane was added. Immediately following this addition, 2.0 mL of 4.0 N sodium hydroxide was added to the reaction mixture to maintain basic pH. After incubation at 0 °C for an additional 3 h, the reaction mixture was allowed to warm up to room temperature. The pH of the reaction mixture was checked, and, as necessary, the pH was adjusted to ∼10.0 with 4.0 N NaOH during the workup. The reaction mixture was washed two times with ether to remove excess CBZ-Cl, and the ether washes were discarded. The aqueous layer was then acidified with 6.0 N hydrochloric acid to ∼pH 1.0. The aqueous acidified solution was then extracted with three portions of ethyl acetate. The combined ethyl acetate extracts were then dried over anhydrous sodium sulfate and filtered. The solvent from filtrate was removed under vacuum to concentrate the product to an oil that solidified upon drying. The crude product was recrystallized from hot ethyl acetate:hexane to yield 7.4 g (90.3%) of white crystalline solid, mp 134-135 °C. 1H NMR (DMSO-d6): δ 1.40-1.80 (m, 4H), 2.10-2.30 (t, 2H), 3.85-4.05 (m, 1H), 5.05 (s, 2H), 7.37 (s, 5H, C6H5), 7.64 (d, 1H, NH), 12.36 (s, COOH). IR (KBr, cm-1): 3300, 3000, 2920, 1690, 1520, 1420, 1270, 1240, 1200, 1170, 1050, 900, 750, 700, 670. HRMS: mass calcd for C14H17NO6 (M + H)+, 296.1134; found, 296.1126. Dibenzyl L-R-N-(Carbobenzyloxy)aminoadipate (3). 6.50 g (22.0 mmol) of N-(carbobenzyloxy)aminoadipic acid was suspended in 180 mL of benzene containing 4.76 g (44.0 mmol) of benzyl alcohol. To the reaction mixture was added 0.2 g of p-toluenesulfonic acid. The reaction mixture was heated overnight under reflux with a DeanStark condenser. After 20 h, the reaction mixture turned colorless and homogeneous with about 1.6 mL of water collected. Completion of the reaction was monitored by TLC in 1:1 ethyl acetate/hexane as well as in 96:4 EtOAc/ HOAc with spots stained in potassium permanganate. Benzene from the reaction mixture was removed under vacuum. The mixture was taken up into ethyl acetate and washed twice with dilute aqueous sodium bicarbonate followed by brine solution. The organic layer was dried over anhydrous sodium sulfate and filtered. Solvent from the filtrate was removed under reduced pressure to concentrate the product. The crude product was purified by flash chromatography on a silica gel 60 column using 30% ethyl acetate in hexane as an eluting solvent. The fractions containing the product were pooled, and the solvent was removed to yield 9.09 g (87%) desired product as a viscous colorless oil: 1H NMR (DMSO-d6): δ 1.40-1.90 (m, 4H), 2.30-2.45 (t, 2H), 4.04.20 (m, 1H), 5.05 (s, 2H), 5.09 (s, 2H), 5.14 (s, 2H), 7.36 (s, 15H, C6H5), 7.84 (d, 1H, NH). IR (NaCl cells, cm-1): 3380, 3060, 3040, 2980, 1740, 1730, 1520, 1470, 1390,
110 Bioconjugate Chem., Vol. 9, No. 1, 1998
Kasina et al.
Scheme 1. Synthesis of N3S-adipate-OTFP and N3S-adipate-STFP
1350, 1260, 1220, 1180, 1070, 1030, 1010, 980, 920, 740, 700. HRMS: mass calcd for C28H29NO6 (M + H)+, 476.2073; found, 476.2075. δ-Benzyl L-R-N-(Carbobenzyloxy)aminoadipate (4). 8.79 g (18.5 mmol) of dibenzyl N-CBZ aminoadipate was dissolved in 90 mL of dioxane and 36 mL of water. To this sollution was added a mixture containing 9.06 mL of 2 N NaOH, 43 mL of water, and 109 mL of dioxane. The reaction mixture was swirled until it became colorless and homogeneous. It was then set aside in the freezer for two days. The pH of the reaction mixture was adjusted to ∼5.5 with 6.0 N HCL and then the mixture was concentrated to a residue. To the residue was added 100 mL of 1.0 N potassium bicarbonate and the solution washed three times each with 100 mL ether to remove benzyl alcohol and any unreacted starting material. The aqueous layer was then adjusted to pH 1.0 with 6.0 N HCl and extracted three times with ethyl acetate. The combined ethyl acetate extracts were washed with brine, dried over anhydrous sodium sulfate, and filtered. Solvent from the filtrate was removed and concentrated to yield 5.51 g of viscous oil. The crude product was purified by flash chromatography on a silica gel column using an eluting solvent mixture containing 50:46:4 ratio of hexane:EtOAc:HOAc. Fractions containing the product were pooled, and the solvent was removed under vacuum to yield 1.7 g (24%) of pure compound 4, mp 69-70 °C. 1H
NMR (DMSO-d6): δ 1.50-1.80 (m, 4H), 2.30-2.45 (t, 2H), 3.85-4.05 (m, 1H), 5.05 (s, 2H), 5.10 (s, 2H), 7.37 (s, 10H, C6H5), 7.60 (d, 1H, NH). IR (KBr, cm-1): 3330, 3040, 2960, 1730, 1680, 1520, 1450, 1420, 1260, 1170, 1060, 1020, 1000, 970, 910, 830, 780, 740, 700. HRMS: mass calcd for C21H23NO6 (M + H)+, 386.16035; found, 386.1600. δ-Benzyl R-Succinimidyl L-a-N-(Carbobenzyloxy)aminoadipate (5). 0.605 g (1.57 mmol) of compound 4 was dissolved in 10.0 mL of anhydrous tetrahydrofuran. To the reaction solution was added 0.2 g (1.73 mmol) of N-hydroxysuccinimide, and the mixture was magnetically stirred at room temperature until dissolved. To the clear solution were added 0.39 g (1.88 mmol) of dicyclohexylcarbodiimide and 2.0 mL of tetrahydrofuran. The reaction mixture was stirred at room temperature overnight. The dicyclohexylurea precipitate was removed by filtration, and the solvent of the filtrate was removed under reduced pressure to yield a crude product residue. The residue was purified by flash chromatography on a silica gel column using 75% ethyl acetate/hexane as an eluting solvent. Fractions containing the product were pooled, and the solvent was removed under vacuum to yield 0.613 g (78%) of the desired product: 1H NMR (DMSO-d6): δ 1.60-2.0 (m, 4H), 2.35-2.50 (m, 4H), 2.84 (s, 4H), 4.404.55 (m, 1H), 5.09 (s, 2H), 5.11 (s, 2H), 7.38 (s, 10H, C6H5), 8.13 (d, 1H, NH). IR (NaCl cells, cm-1): 3310, 2910, 1820, 1780, 1730, 1520, 1460, 1350, 1250, 1200, 1060,
Chelate Protein
99mTc
Radiolabeling
1000, 980, 910, 860, 815, 760, 700, 650. HRMS: mass calcd for C25H26N2O8 (M + H)+, 483.17673; found 483.1767412. 2,2,2-Trichloroethyl N-(Carbobenzyloxy)glycylglycinate (7). 1.0 g (3.76 mmol) of N-(carbobenzyloxy)glycylglycine (6) was dissolved in 6.0 mL of anhydrous DMF. To this solution under magnetic stirring was added 0.56 g (3.76 mmol) of 2,2,2-trichloroethanol, followed by 0.91 mL (11.26 mmol) of gold label grade pyridine. To the stirred reaction mixture was added 0.77 g (3.76 mmol) of DCC, followed by 4.0 mL of anhydrous DMF to rinse the DCC vial. The reaction mixture was stirred at room temperature for two days. Precipitated dicyclohexylurea was removed by filtration. Solvent from the filtrate was removed under vacuum leaving a crude brown oil after five days. The crude product was purified by flash chromatography on a silica gel column using 1:1 ethyl acetate:hexane as an elution solvent. Fractions containing the product were combined. Solvent was removed under vacuum to yield 0.86 g (58%) of pure white compound 7. 1H NMR (DMSO-d6): δ 3.71 (d, 2H), 4.05 (d, 2H), 4.95 (s, 2H), 5.07 (s, 2H), 7.39 (s, 5H, C6H5), 7.56 (t, 1H, NH), 8.47 (t, 1H, NH). 2,2,2-Trichloroethyl Glycylglycinate (8). 0.2 g (0.503 mmol) of 2,2,2-trichloroethyl N-CBZ-glycylglycinate (7) was dissolved in 1.5 mL of glacial acetic acid. This reaction solution was added to a flask containing 2.0 mL of HBr in acetic acid (9.35 mmol, ∼30-32 wt % HBr). The reaction mixture was allowed to stand at room temperature for 30 min. The reaction solution darkened without any evidence of carbon dioxide evolution. Following addition of 40 mL of diethyl ether, the reaction mixture was cooled in the freezer for 1 h. The white precipitate that formed was collected by filtration and dried to yield 0.148 g (86%) of pure compound 8, mp 208 °C dec. 1H NMR (DMSO-d6): δ 3.67 (s, 2H), 4.17 (d, 2H), 4.97 (s, 2H), 8.06 (bs, 3H, +NH3), 8.93 (t, 1H, NH). IR (KBr, cm-1): 3280, 3200, 3000, 1740, 1660, 1530, 1410, 1370, 1250, 1160, 1100, 1070, 1040, 980, 900, 860, 800, 720, 680, 620. HRMS: mass calcd for C6H9N2O3Cl3 (M + H)+, 262.97568; found, 262.9757005. 2,2,2-Trichloroethyl δ-Benzyl L-R-N-(carbobenzyloxy)aminoadipoylglycylglycinate (9). 0.59 g (1.18 mmol) of compound 5 was dissolved in 4.0 mL of dry DMF. To the solution was added 0.406 g (1.18 mmol) of compound 8 followed by 2.0 mL of dry DMF. The reaction solution turned slightly yellow and became homogeneous. With stirring 164 µL of neat triethylamine was added dropwise over a 5 min time period. The reaction mixture was stirred at room temperature for two days. The DMF from the reaction mixture was removed under vacuum. The crude product was purified by flash chromatography on a silica gel column using 75% ethyl acetate-hexane as an eluting solvent. The fractions containing the desired product were pooled, and the solvent was removed under reduced pressure and dried to yield 0.55 g (75%) of product. 1H NMR (DMSO-d6): δ 1.45-1.80 (m, 4H), 2.37 (t, 2H), 3.78 (d, 2H), 4.04 (d, 3H), 4.93 (s, 2H), 5.04 (s, 2H), 5.09 (s, 2H), 7.37 (s, 5H, C6H5), 7.52 (d, 1H, NH), 8.27 (t, 1H, NH), 8.38 (t, 1H, NH). 2,2,2-Trichloroethyl L-R-Amino-R-adipoylglycylglycinate (10). 0.515 g (0.816 mmol) of compound 9 was dissolved in 2.0 mL of glacial acetic acid. The solution was then transferred to a Parr hydrogenation bottle containing ∼100 mg of 5% palladium on activated carbon. The reaction mixture was shaken with hydrogen at 60 psi for 24 h and then filtered through a Celite pad using additional acetic acid to rinse the flask and the Celite pad. Solvent from the filtrate was removed under
Bioconjugate Chem., Vol. 9, No. 1, 1998 111
reduced pressure yielding a thick residue. The residue was rotary evaporated from heptane three times and then triturated with ether and dried in vacuo to yield 0.39 g (100%) of hygroscopic white solid. 1H NMR (DMSO-d6): δ 1.45-1.65 (m, 2H), 1.65-1.80 (m, 2H), 2.25 (t, 2H), 3.70-3.90 (m, 4H), 4.0-4.10 (m, 2H), 4.93 (s, 2H), 8.64 (t, 1H, NH), 8.80-8.95 (m, 2H, NH). 2,2,2-Trichloroethyl L-R-S-(1-Ethoxyethyl)mercaptoacetamido-R-adipoylglycylglycinate (13). 0.34 g (0.729 mmol) of compound 10 was dissolved in 4.0 mL of anhydrous DMF. To the solution was added 0.2 g (0.766 mmol) of compound 12 and the mixture stirred at room temperature until dissolved. Additionally 2.0 mL of anhydrous DMF was added to rinse down the sides of the flask. To the reaction mixture was added 305 µL (2.19 mmol) of triethylamine dropwise over a period of 2 min. The reaction mixture was then stirred at room temperature overnight. The DMF was removed in vacuo. Ethyl acetate was added to the residue and the precipitated solid filtered off. The filtrate was repeatedly washed with 0.1 N HCl until no NHS remained in the organic phase as indicated by TLC in a solvent mixture of Et2O:CH3OH:HOAc (92:4:4). The organic layer was then washed with brine and dried over anhydrous sodium sulfate. The solution was filtered and concentrated under vacuum to a solid. The solid was triturated with ether, collected by filtration, and dried under vacuum to yield 0.182 g (45%) of product. 1H NMR (DMSO-d6): δ 1.097 (t, 3H), 1.414 (2d, 3H), 2.200 (t, 2H), 3.225 (d, 2H), 3.253.50 (m, 4H), 3.55-3.70 (d, 1H), 3.70-3.80 (d, 2H), 4.04.05 (d, 2H), 4.20-4.35 (m, 1H), 4.70-4.85 (p, 1H), 4.924 (s, 2H), 8.10-8.225 (d, 1H), 8.25-8.45 (m, 2H). IR (KBr, cm-1): 3300, 3280, 2980, 2920, 1760, 1720, 1640, 1540, 1520, 1450, 1400, 1380, 1220, 1180, 1110, 1060, 1030, 940, 900, 850, 820, 790, 730, 660, 630. HRMS: mass calcd for C18H28Cl3N3O8S (M + H)+, 552.07409; found, 552.0724. 2,2,2-Trichloroethyl R-S-(1-Ethoxyethyl)mercaptoacetamido( L -δ-2,3,5,6-tetrafluorophenyl)adipoylglycylglycinate (14). 0.145 g (0.262 mmol) of compound 13 was dissolved in 4.0 mL methylene chloride. To the solution was added 65 mg (0.393 mmol) of tetrafluorophenol with stirring. 65 mg (0.315 mmol) of dicyclohexylcarbodiimide was added followed by 1.0 mL of methylene chloride to rinse DCC from the vial. The reaction mixture was stirred at room temperature overnight. The dicyclohexyl urea precipitate was removed by filtration, and the solvent from the filtrate was removed under vacuum to concentrate the residue. The crude product was purified by flash chromatography on a silica gel column using EtOAc:hexane:acetic acid at a ratio of 71:25:4 as an eluting solvent. Fractions containing the product were pooled. The solvent from the combined fractions was removed under reduced pressure to yield 114 mg (62%) of desired compound 14. 1H NMR (DMSO-d6): δ 1.111 (t, 3H), 1.447 (t, 3H), 1.50-1.90 (m, 3H), 2.822 (t, 2H), 3.255 (d, 2H), 3.342 (s, 2H), 3.55-3.75 (m, 1H), 3.803.90 (d, 2H), 4.0-4.10 (d, 2H), 4.30-4.45 (m, 1H), 4.754.90 (p, 1H), 4.934 (s, 2H), 7.85-8.1 (m, 1H), 8.20-8.30 (d, 1H, NH), 8.30-8.50 (m, 2H, NH). L-δ-2,3,5,6-Tetrafluorophenyl R-S-(1-Ethoxyethyl)mercaptoacetamidoadipoylglycylglycine (15). 0.107 g (0.15 mmol) of compound 14 was dissolved in 1.0 mL of THF. To the solution was added 203 µL of 1.0 M potassium dihydrogen phosphate, pH 4.26. Then 100 mg of zinc dust was added with stirring at room temperature. The progress of the reaction was monitored by TLC in a solvent mixture of ethyl acetate:acetic acid (96:4). After 30 min, an additional 203 µL of buffer and 100 mg of
112 Bioconjugate Chem., Vol. 9, No. 1, 1998
zinc dust were added. Stirring was continued for 90 min. The reaction mixture was then filtered through a Celite plug using THF to rinse material from the Celite. Solvent from the filtrate was then removed under reduced pressure and the crude product purified by flash chromatography on a silica gel column using the mixture CH2Cl2:IPA:HOAc at a ratio of 75:20:5 as eluting solvent. Fractions containing the product were pooled, and the solvent was removed. The resulting purified residue was rotatory evaporated three times from heptane to remove the residual acetic acid and dried in vacuo overnight. The dried residue was then triturated with diethyl ether and allowed to stand for a few minutes, yielding a fine white precipitate. The supernatant ether layer was carefully decanted and residual solvent was removed under reduced pressure. This dried residue was then triturated with fresh ether. The resulting combined white solid was dried in vacuo to yield 63 mg (72%) of pure product. 1H NMR (DMSO-d6): δ 1.090 (2t, 3H), 1.43 (2d, 3H), 1.501.90 (m, 4H), 2.81 (t, 2H), 3.24-3.26 (d, 2H), 3.50-3.70 (2Q, 2H), 3.69 (s, 2H), 3.76 (s, 2H), 4.25-4.45 (m, 1H), 4.70-4.85 (Q, 1H), 7.85-8.05 (m, 1H, aromatic), 8.09 (t, 1H, NH), 8.23 (d, 1H, NH), 8.34 (t, 1H, NH). MS (CI): 570 (M + 1). Anal. Calcd for C22H27N3O8SF4: C, 46.40, H, 4.78, N, 7.38, S, 5.63. Found: C, 46.29, H, 4.73, N, 7.15, S, 5.38. 2,2,2-Trichloroethyl L-δ-2,3,5,6-Tetrafluorothiophenyl R-S-(1-Ethoxyethyl)mercaptoacetamidoadipoylglycylglycinate (16). 1H NMR (DMSO-d6): δ 1.118 (t, 3H), 1.426 (t, 3H), 1.45-1.80 (m, 3H), 2.895 (t, 2H), 3.2243.238 (d, 2H), 3.324 (d, 2H), 3.55-3.75 (m, 1H), 3.703.80 (d, 2H), 4.0-4.10 (d, 2H), 4.25-4.40 (m, 1H), 4.704.85 (p, 1H), 4.914 (s, 2H), 8.05-8.25 (m, 2H, NH), 8.308.50 (m, 2H, NH). L-δ-2,3,5,6-Tetrafluorothiophenyl R-S-(1-Ethoxyethyl)mercaptoacetamidoadipoylglycylglycine (17). The synthesis of compound 17 is similar to compounds 14 and 15 except for using tetrafluorothiophenol in place of tetrafluorophenol to activate the carboxylic acid as described in 14. 1H NMR (DMSO-d6): δ 1.10 (2t, 3H), 1.45 (2d, 3H), 1.50-1.85 (m, 4H), 2.85-3.0 (t, 2H), 3.203.25 (d, 2H), 3.25-3.50 (m, 2H), 3.50-3.80 (2Q, 4H), 4.20-4.45 (m, 1H), 4.70-4.90 (P, 1H), 8.05-8.25 (m, 1H, aromatic), 8.15-8.25 (2d, 2H, NH), 8.25-8.40 (t, 1H, NH). Anal. Calcd for C22H27N3O7S2F4: C, 45.12, H, 4.65, N, 7.18, S, 10.95. Found: C, 45.16, H, 4.54, N, 6.97, S, 11.0. Formulation of N3S-Adipate-STFP Reagent. The N3S-adipate-STFP prelyophilized reagent was formulated as a 1 mL aqueous solution containing 100 µg of N3Sadipate-STFP, 2.5 mg of sodium gluconate, 0.1 mg of stannous chloride, 0.1 mg of gentisic acid, 40 mg of lactose filler, and 26% v/v tert-butyl alcohol. During preparation, N3S-adipate-STFP was dissolved at 350 µg/mL in an H2O/ tert-butyl alcohol solution (1:9) and added to an aqueous concentrate (1.4×) of the remaining ingredients adjusted to pH 1.8 with sulfuric acid. The solution was vialed in grade A borosilicate 10 mL vials fitted with 25 mm diameter gray butyl rubber lyophilization stoppers and frozen at -40 °C within 30 min of preparation. Lyophilization proceeded at 50-100 m torr vacuum with maintenance of product below a collapse temperature of -25 °C for 23 h for primary drying. Secondary drying proceeded upon 0.3 °C/min ramping to -12 °C, holding for 16 h, followed by ramping in like manner to 25 °C and holding for 20 h. Afterwards, the vials were backfilled with nitrogen to 6 in. of Hg and stoppered. Final moisture content was
