Heterobifunctional Cross-Linkers Containing 4,9-Dioxa-1,12

Apr 15, 1997 - Organic Chemistry Group, Bayer Corporation, P.O. Box 70, Elkhart, Indiana 46515, and Bayer AG ZF-FDM,. Krefeld-Uerdingen, Germany...
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Bioconjugate Chem. 1997, 8, 447−452

447

Heterobifunctional Cross-Linkers Containing 4,9-Dioxa-1,12-dodecanediamine Spacers Gary M. Johnson,*,† James P. Albarella,† and Christoph Petry‡ Organic Chemistry Group, Bayer Corporation, P.O. Box 70, Elkhart, Indiana 46515, and Bayer AG ZF-FDM, Krefeld-Uerdingen, Germany. Received August 31, 1996X

A series of heterobifunctional linker arms has been prepared by functionalization of (tert-butoxycarbonyl)-4,9-dioxa-1,12-dodecanediamine [tBOC-HN(CH2)3O(CH2)4O(CH2)3NH2] with anhydrides or acid chloride.

Heterobifunctional cross-linkers are commonly used in bioconjugation technologies to prepare antibody-enzyme conjugates for use in enzyme immunoassays. Possible drawbacks can include instability and incomplete, oligomeric, and polymeric reactions, which frequently occur with commercially available cross-linkers. As part of a program to develop new reagents with enhanced stability and reactivity, 4,9-dioxa-1,12-dodecanediamine [H2N(CH2)3O(CH2)4O(CH2)3NH2] heterobifunctional crosslinkers were synthesized. This (3,4,3) extended spacer was readily available and inexpensive and had good water solubility. It has been postulated that extended length cross-linkers would reduce steric crowding between the enzyme and antibody and increase stability and may provide enhanced biochemical properties relative to those presently being used.

in Scheme 2 where intermediate 5 was converted into cross-linker 18 in 17% yield5 via amino acid 9. The linkers were activated as N-hydroxysuccinimidyl (NHS) esters before coupling to alkaline phosphatase, which in turn was then coupled to 2-iminothiolane (2IT)activated antibody.4 Activation was performed in situ with NHS in DMF/TEA as the resultant esters were unstable. In the case of linkers 10 and 14, intramolecular cyclizations to imides were determined to be significant side reactions. To overcome these problems, heterobifunctional linker 17 was modified to the very stable hydrazide 19, shown in Scheme 3. This modification used (tert-butoxycarbonyl)hydrazine (tBOCNHNH2) and dicyclohexylcarbodiimide (DCC) in DMF (61%), followed by trifluoroacetic acid (TFA) for 2 h (31% yield).5 This stable, crystalline cross-linkers binds covalently to aldehyde sites on the antibody.

CHEMISTRY

RESULTS AND DISCUSSION

Synthesis of the cross-linkers began by coupling the known1 (tert-butoxycarbonyl)-4,9-dioxa-1,12-dodecanediamine [tBOC(343)NH2] to anhydrides2 1-3 or acid chloride 4, providing carboxy-functionalized intermediates 5-8 in near quantitative yields (see Scheme 1 and Table 1). Treatment with trimethylsilyl iodide (TMSI)3 in CHCl3 followed by methanol quench furnished quantitative crude yields of amino acids 9 and 11-13. In the case of intermediate 6, the cyclic imide analog 10 was obtained via intramolecular cyclization. Compound 13 was obtained in 92% yield by treatment of 8 with NaOH in aqueous ethanol. Coupling of amino acids 9 and 11-13 with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate (SMCC)4 in dimethylformamide/triethylamine (DMF/TEA) provided the heterobifunctional crosslinkers (14-17) in 67, 7, 32, and 30% yields,5 respectively. N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) analogs4 were also developed as thiol-functionalized cross-linkers. These were prepared by treating an amino acid with SPDP in DMF/TEA, as shown by the example

A high-performance liquid chromatography (HPLC) profile of the crude reaction mixture, prepared with linker 19, of a polyclonal anti-myoglobin antibody conjugated to alkaline phosphatase (AP) is shown in Figure 1. The new conjugate (overall yield of ∼60%) was exchanged for and tested using the same procedure and conditions as the anti-myoglobin-AP conjugate normally used in the Technicon IMMUNO 1 myoglobin assay (product T013653-51) commercialized by Bayer Corp. In that assay, magnetic particles carrying antibodies against fluorescein, a fluoresceinated monoclonal antimyoglobin antibody, and an alkaline phosphatase-labeled polyclonal anti-myoglobin antibody are incubated for 15 min together with 3 µL of a sample containing an unknown amount of myoglobin. During the course of the incubation, the two different antibody conjugates form a sandwich complex with the myoglobin-antigen. This complex is captured by the magnetic particles via the fluorescein moiety on the monoclonal antibody. The supermolecule thus formed is then precipitated by an external magnetic field and washed. Any antibody-AP conjugate present in the mixture is removed unless bound to the immobilized supermolecule. The amount of antibody-AP conjugate remaining in the assay is directly proportional to the amount of myoglobin in the sample. The kinetics of p-nitrophenyl phosphate hydrolysis is

INTRODUCTION

†

Bayer Corp. Bayer AG ZF-FDM. X Abstract published in Advance ACS Abstracts, April 15, 1997. 1 Reference 1a was used to prepare tBOC(343)NH . Patent 2 references 1b-d give other preparations and some uses of this linker. 2 Anhydride 2 from ref 2a. Anhydride 3 from ref 2b. 3 As described in ref 3. 4 These reagents were purchased from Pierce Co. (P.O. Box 17, Rockford, IL, 61105). General procedures other than those listed herein, additional examples, and relevant references may be found in their catalogs. ‡

S1043-1802(97)00026-8 CCC: $14.00

5 All isolated products exhibited spectrascopic (IR, MS, 1H NMR, and 13C/D NMR) and analytical data which are fully consistent with the assigned structures. Yields are unoptimized in that good recoveries of crude product were generally obtained, but due to expediency, the preparative thin layer chromatography (TLC) plate purification method employed sacrificed quantity for purity.

© 1997 American Chemical Society

448 Bioconjugate Chem., Vol. 8, No. 3, 1997

Johnson et al.

Scheme 1

Table 1

Scheme 2

Scheme 3

measured to quantify the antibody-AP conjugate present and hence the amount of myoglobin in the sample. The new antibody-AP conjugate synthesized with linker 19 was tested in the assay after substitution for the standard antibody-AP conjugate, and all other parameters and buffers remained unchanged (Technicon IMMUNO 1, protocol 5; fluoresceinated conjugate, 2.6 µg/ mL, 65 µL of solution; antibody-AP conjugate, 6.5 µg/ mL, 65 µL of solution; magnetic particles, 10 µL of suspension; sample volume, 3 µL, 15 min of simultaneous incubation, four washes).

The activity of the enzyme in the antibody-AP conjugate was measured as follows. One tablet of p-nitrophenyl phosphate (Sigma N2640) was dissolved into 15 mL of glycine buffer (1.88 g of glycine/L, pH 9.6). The antibody-AP conjugate was diluted to a concentration of 2.5 µg/mL in AP reaction buffer (1.88 g of glycine, 0.2 g of magnesium chloride, 13 mg of zinc chloride, and 100 mL of glycerine). Five microliters of the diluted conjugate, 0.5 mL of a 0.05 M magnesium chloride solution, and 2.5 mL of a substrate solution were then mixed in a

Bioconjugate Chem., Vol. 8, No. 3, 1997 449

Technical Notes

Figure 1. HPLC profile of the crude reaction mixture, prepared with linker 19, of a polyclonal anti-myoglobin antibody conjugated to alkaline phosphatase.

Figure 2. Dose-response curve of the myoglobin assay using linker 19.

cuvette with a 1 cm path length, and the absorption increase of the solution was monitored at 25 °C and 400 nm. A typical enzyme activity for an antibody-AP conjugate was 580 units/mg, where 1 unit of alkaline phosphatase digests 1 µmol of substrate per second, and the extinction coefficient of p-nitrophenolate at 400 nm is 18.7 cm2/µmol. Conjugates prepared by procedures using shorter linkers directed against amino groups had typical enzyme activities of 400-650 units/mg, depending on the amount of NH-derivatizing agent used. Figure 2 shows a dose-response curve of the myoglobin assay using linker 19 on the Technicon IMMUNO 1

assay. The dose-response curve depicted (using a sixpoint cubic-fit-through-zero calculation) was nearly identical with the optimized standard curve obtained using the regular antibody-AP conjugate. The instrument was calibrated using six myoglobin investigational calibrators having concentrations of 0, 63, 186, 619, 1552, and 3193 ng/mL. The reaction rates measured on the Technicon IMMUNO 1 assay for the calibrators were 3.5, 29.0, 78.0, 254.0, 638.6, and 1238.2 mA/min, respectively, and the dose-response curve using a six-point cubic-fit-throughzero calculation was constructed. As controls, three serum pools with assigned concentrations of 15, 75, and 667 ng/mL were run. Using this calibration, the controls

450 Bioconjugate Chem., Vol. 8, No. 3, 1997

(reaction rates of 9.2, 33.7, and 284.3 mA/min) were recovered as 14.4 (-4%), 76.2 (+1.5%), and 691.6 (+3.7%), showing the versatility of the newly prepared reagent. SUMMARY

This paper describes the synthesis and preliminary testing of heterobifunctional linkers containing an extended spacer unit. A new enzyme-antibody conjugate prepared in acceptable yield with highly stable linker 19 demonstrates that new linkers of this type can be used as versatile agents to prepare fully functional antibodyAP conjugates. Linker 19 is particularly useful in that coupling can be performed in the presence of the protein’s surface amino groups. MATERIALS AND METHODS

IR spectra were recorded on a Perkin-Elmer 710B spectrophotometer. NMR spectra were recorded on a GE 300 MHz spectrometer. Mass spectra were determined by fast atom bombardment (FAB) on a HP 5985A apparatus, FAB source with direct introduction. Thin layer chromatography was carried out on Merck GF 254 silica plates. HPLC separations were performed with a Pharmacia FPLC system on Pharmacia Superdex 200 columns. Protein concentrations were determined on a HP 8452A diode array spectrophotometer by measuring the absorbance at 280 nm. Performance testing was done on a Technicon IMMUNO 1 immunoanalyzer. All isolated products exhibited spectrascopic data (IR, MS, 1H NMR, 13C NMR, and 13CD NMR) and analytical data which are fully consistent with the assigned structures. EXPERIMENTAL PROCEDURES

Preparation (1a) of tBOC-HN(CH2)3O(CH2)4O(CH2)3NH2. To tert-butyl S-(4,6-dimethylpyrimidin-2yl)thiocarbonate (35.3 g, 0.147 mmol, 0.6 equiv) in 1,4dioxane (100.0 mL) under argon was added dropwise via cannula a solution of H2N(CH2)3O(CH2)4O(CH2)3NH2 (50.0 g, 0.245 mmol) in 1,4-dioxane (200.0 mL) over 60 min. The reaction mixture was stirred for 24 h and was filtered of precipitated solid, and the crude product solution was concentrated in vacuo under high vacuum in a water bath at about 50 °C. The compound was purified via flash chromatography (7.5 × 60 cm column, 800 g of 230-400 mesh silica gel-60), eluting with 100 mL fractions of 90/20/2 CHCl3/CH3OH/NH4OH. TLC in 90/20/2 CHCl3/CH3OH/NH4OH revealed an orangecolored product spot after iodine spray solution with an Rf value of about 0.35. Product fractions were combined and concentrated in vacuo at about 45 °C and then dissolved into CHCl3/ether (1/1, 300 mL). The organics were washed with 50 mL of water, separated, and dried with granular MgSO4. The solution was filtered and concentrated in vacuo to a pale yellow product oil (∼1418 g, 40-60%). 1 H NMR (300 MHz, CD3OD, ppm): 1.42 (s, 9H), 1.62 (m, 4H), 1.72 (m, 4H), 1.75 (t, 2H), 3.12 (t, 2H), 3.46 (m, 8H). General Synthetic Method for Preparation of Intermediates 5-7. Triethylamine (TEA, 5.34 mmol, 1.3 equiv) was added in one portion to a solution of tBOC(343)NH2 (4.11 mmol, 1 equiv) in DMF (anhydrous, 10 mL, 2.5 equiv) under argon. A solution of anhydride, or acid chloride (5.0 mmol, 1.2 equiv), in dimethylformamide (DMF, anhydrous, 2.0 mL) was added to the flask dropwise via syringe over 5 min. The reaction mixture was stirred under argon for 24 h. A white TEA‚HCl precipitate was then removed by filtration, and the resultant solution was concentrated in vacuo under high

Johnson et al.

vacuum at about 50 °C. The crude material was then purified by flash column chromatography to provide the clean product. 5. 1H NMR (300 MHz, DMSO-d6, ppm): 1.36 (s, 9H), 1.5 (m, 4H), 1.6 (m, 4H), 2.95 (qrt, 2H), 3.15 (qrt, 2H), 3.35 (m, 8H), 3.78 (s, 2H), 3.88 (s, 2H), 6.75 (NH). 13C NMR (75 MHz, DMSO-d6, ppm): 26.1, 28.3 (tBOC), 29.4, 29.8, 35.7, 37.3, 67.7, 67.9, 69.9, 71.3, 71.5, 77.4, 155.6, 169.8, 172.9. EI-DIP/MS: 347 (M - tBOC:73, 2.7), 301 (5.5), 264 (18.5), 246 (14.1), 231 (16.2), 190 (43.3), 174 (73.4), 156 (83.6), 146 (base), 130 (17.5), 114 (25.7), 102 (37.8), 74 (65.6), 57 (34.4). IR (neat, cm-1): 3700-3000, 1710, 1690, 1650, 1580, 1530, 1450, 1420, 1270, 1170. 6. 1H NMR (300 MHz, CD3OD, ppm): 1.42 (s, 9H), 1.61 (m, 4H), 1.65-1.81 (m, 4H), 2.78 (s, 3H), 3.1 (t, 2H), 3.313.5 (m, 10H), 3.58 (s, 2H), 3.76 (s, 2H). 13CD NMR (75 MHz, CD3OD, ppm): 27.5 (methylenes), 28.8 (methyls, tBOC), 30.4 (methylene), 31.0 (methylene), 37.9 (methylene), 38.9 (methylene), 43.1 (quat, tBOC), 42.9 (Nmethyl), 58.7 (methylene), 59.6 (methylene), 69.3 (methylene), 69.5 (methylene), 71.7 (methylene), 71.8 (methylene), 167.9 (CdO), 170.9 (CdO). FAB/MS: 334 [(M + H) - tBOC]. IR (neat, cm-1): 3700-3130, 31302600, 1800-1600, 1550, 1530, 1390, 1250, 1175. 7. 1H NMR (300 MHz, CD3OD, ppm): 1.36 (brs, 9H), 1.58 (m, 4H), 1.65 (qnt, 2H), 1.75 (qnt, 2H), 3.05 (s, 2H), 3.28 (m, 2H), 3.4 (m, 8H), 3.71 (m, 4H), 3.95 (m, 4H). 13C NMR (75 MHz, CD3OD, ppm): 27.5, 28.8, 30.5, 37.9, 38.9, 69.2, 69.6, 69.9, 71.2, 71.4, 71.8, 71.9, 71.93, 79.4. FAB/ MS: 365 [(M + H) - tBOC]. IR (neat, cm-1): 3350, 2940, 1710, 1670, 1535, 1450, 1275. Preparation of Intermediate 8. Pyridine (10.0 mmol, 2.0 equiv) was added in one portion to a solution of tBOC-4,9-dioxa-1,12-dodecanediamine (1a), tBOC(343)NH2 (5.0 mmol, 1 equiv), in CH2Cl2 (20 mL, 4 equiv) under argon at 0 °C. A solution of acid chloride (5.0 mmol, 1.0 equiv) in CH2Cl2 (5 mL, 1 equiv) was added to the flask, dropwise via cannula over 5 min. The reaction mixture was allowed to warm to room temperature over 2 h and stirred under argon for 24 h. The resultant solution was diluted with CH2Cl2 (50 mL) and then washed consecutively with saturated NaHCO3, 1 M HCl, brine, and water (50 mL each). The organic phase was then dried and concentrated in vacuo under high vacuum at about 35 °C. The clean crude product was then used directly in the next step. 8. 1H NMR (300 MHz, CD3OD, ppm): 1.21 (t, 3H), 1.32 (m, 4H), 1.41 (s, 9H), 1.6 (m, 8H), 1.72 (m, 4H), 2.16 (t, 2H), 2.3 (t, 2H), 3.11 (m, 2H), 3.22 (m, 2H), 3.42 (m, 6H), 4.1 (qrt, 2H), 6.5 (NH). 13C NMR (75 MHz, CD3OD, ppm): 14.5, 25.9, 26.8, 27.5, 28.3, 29.4, 29.8, 29.9, 30.6, 31.0, 35.0, 37.1, 37.8, 38.9, 61.4, 69.5, 71.77, 71.8, 158.4, 175.5, 176.1. FAB/MS: 389 [(M + H) - tBOC:73], 242 (base). IR (neat, cm-1): 2945, 1735, 1720, 1650, 1540, 1460, 1255, 1175. General Synthetic Method for Preparation of Amino Acids 9 and 11-13. To R-(343)tBOC (5-8, 1.00 g, 2.05 mmol) in a solution of CDCl3 (15 mL, 7.5 equiv) under argon was added TMSI (7.2 mmol, ∼3.5 equiv). The reaction mixture was stirred for 20 min and then the reaction quenched with CD3OD (1.5 mL), stirring 2 min. The reaction mixture was then concentrated in vacuo. The residue in the flask was then taken up in a 4.0 mL solution of water/acetic acid/ethyl ether (1.7/0.3/ 2.0); the organic phase were separated, and the aqueous phase was extracted with ethyl ether (three times). The combined organic phase was washed once with 2.0 mL of aqueous acetic acid (1.7/0.3), dried, and concentrated in vacuo. The crude product (quantitative yield) was

Technical Notes

used directly in the next step, or it was used after preparative TLC plate purification. 9. 1H NMR (300 MHz, CD3OD, ppm): 1.60 (m, 4H), 1.75 (qnt, 2H), 1.88 (qnt, 2H), 3.0 (t, 2H), 3.31 (t, 2H), 3.45 (m, 6H), 3.55 (t, 2H), 4.03 (brs, 2H), 4.22 (brs, 2H). 13 CD NMR (75 MHz, CD3OD, ppm): 27.5 (two Cs), 28.5, 30.4, 37.7, 39.4, 69.1, 69.3, 69.7, 71.5, 72.1, 71.78. EI-DIP/MS: 303 (M - OH), 261 (M - CH2CO2H), 145 (base). IR (neat, cm-1): 3700-2700, 1740, 1645, 1555, 1440, 1380, 1245. 11. 1H NMR (300 MHz, CD3OD, ppm): 1.62 (m, 4H), 1.78 (qnt, 2H), 1.9 (qnt, 2H), 3.05 (t, 2H), 3.36 (m, 2H), 3.4-3.55 (m, 6H), 3.57 (t, 2H), 3.71 (m, 2H), 3.73 (s, 2H), 3.98 (m, 2H), 4.01 (s, 2H). 13CD NMR (75 MHz, CD3OD, ppm): 27.5 (two CH2s), 28.5, 30.5, 37.8, 39.5, 69.2, 69.3, 69.6, 69.7, 71.2, 71.3, 71.8, 72.1. FAB/MS: 365 (M + H), 173 (base). IR (neat, cm-1): 3600-2700, 1710, 1640, 1450, 1375. 12. 1H NMR (300 MHz, CD3OD, ppm): 1.21 (t, 3H), 1.33 (m, 4H), 1.6 (m, 8H), 1.74 (qnt, 2H), 1.9 (qnt, 2H), 2.16 (t, 2H), 2.3 (m, 2H), 3.02 (t, 2H), 3.22 (t, 2H), 3.45 (m, 6H), 3.55 (t, 2H), 4.1 (qrt, 2H). 13C NMR (75 MHz, CD3OD, ppm): 14.5, 25.9, 26.8, 27.5, 28.5, 29.8, 29.9, 30.6, 35.0, 37.0, 37.8, 39.5, 61.4 (quat), 69.3, 69.4, 71.8, 72.1. FAB/MS: 389 (M + H, base). IR (neat, cm-1): 3300, 2940, 1735, 1640, 1555, 1465, 1280. 13. 1H NMR (300 MHz,CD3OD, ppm): 1.33 (m, 4H), 1.6 (m, 8H), 1.75 (qnt, 2H), 1.9 (qnt, 2H), 3.03 (t, 2H), 3.21 (t, 2H), 3.42 (m, 6H), 3.55 (t, 2H). 13C NMR (75 MHz, CD3OD, ppm): 25.9, 26.9, 27.5 (two Cs), 28.6, 29.9 (two Cs), 30.6, 34.7, 37.1, 37.8, 39.5, 69.3, 69.4, 71.8, 72.1, 176 (CdO). FAB/MS: 361 (M + H), 228 (base). IR (neat, cm-1): 3700-3200, 3200-3000, 1730, 1630, 1550, 1450, 1370. General Synthetic Method for Preparation of Cross-Linkers 14-18. TEA (1-1.3 equiv) was added in one portion to a solution of amino acid (1.5 mmol, 1 equiv) in DMF (anhydrous, 10 mL, ∼7 equiv) under argon at 0 °C. SMCC or SPDP (1.5 mmol, 1.2 equiv) was added in one portion, and the reaction mixture was stirred under argon for 3-24 h, until complete via TLC monitoring. The reaction mixture was allowed to warm to room temperature if it was stirred for more than 6 h. The solution was then concentrated in vacuo under high vacuum at about 50 °C. The crude product was purified on preparative TLC plates to provide the desired crosslinker in 7-67% unoptimized5 yields. 14. 1H NMR (300 MHz, CD3OD, ppm): 1.0 (m, 4H), 1.4 (m, 4H), 1.62 (m, 4H), 1.75 (m, 6H), 2.1 (m, 2H), 3.22 (t, 2H), 3.35 (t, 2H), 3.45 (m, 4H), 3.5 (t, 2H), 4.04 (s, 2H), 4.22 (s, 2H), 6.8 (s, 2H), 7.75 (NH), 7.85 (NH). 13CD NMR (75 MHz, CD3OD, ppm): 27.5 (methylenes), 30.0 (methylenes), 30.5 (methylenes), 30.96 (methylenes), 37.8 (methylenes), 44.5 (methylene), 45.6 (methine), 46.2 (methine), 69.1 (methylene), 69.6 (methylene), 69.9 (methylene), 71.5 (methylene), 71.8 (methylene), 71.9 (methylene), 135.2 (methines). FAB/MS: 617 (MNH4+ + Na + K), 579 (MNH4+ + Na), 557 (MNH4+ base). IR (neat, cm-1): 3440, 3010, 1740, 1705, 1670, 1535, 1450, 1370, 1220. 15. 1H NMR (300 MHz, CD3OD, ppm): 1.0 (m, 4H), 1.4 (m, 4H), 1.6 (m, 4H), 1.7-1.9 (m, 10H), 2.1 (dt, 2H), 3.25 (m, 4H), 3.5 (m, 8H), 3.71 (m, 4H), 3.95 (s, 2H), 4.19 (s, 2H), 6.8 (s, 2H), 7.75 (NH), 7.88 (NH). 13CD NMR (75 MHz, CD3OD, ppm): 27.6 (methylenes), 30.1 (methylenes), 30.5 (methylenes), 31.0 (methylene), 37.7 (methylene), 37.84 (methylene), 44.5 (methylene), 46.2 (methines), 69.1 (methylene), 69.6 (methylene), 69.8 (methylene), 71.2 (methylene), 71.8 (methylene), 71.9

Bioconjugate Chem., Vol. 8, No. 3, 1997 451

(methylene), 135.2 (methines). FAB/MS: 602 [(M + H + HOH) or M + H, OH addition with ring opening of maleimide], 307 (base, matrix). 16. 1H NMR (300 MHz, CD3OD, ppm): 0.99 (m, 4H), 1.21 (t, 3H), 1.21-1.5 (m, 8H), 1.5-1.95 (m, 14H), 2.052.1 (dt, 2H), 2.1-2.35 (m, 4H), 3.25 (m, 4H), 3.45 (m, 8H), 4.1 (qrt, 2H), 6.8 (s, 2H), 7.75 (NH), 7.85 (NH). 13CD NMR (75 MHz, CD3OD, ppm): 14.2 (methyl), 25.9 (methylene), 26.9 (methylene), 27.6 (methylenes), 29.9 (methylenes), 30.1 (methylenes), 30.6 (methylene), 31.0 (methylene), 35.0 (methylene), 37.1 (methylene), 37.8 (methylenes), 44.5 (methylene), 46.2 (methines), 61.4 (methylenes), 69.5 (methylene), 69.6 (methylene), 71.8 (methylenes), 135.3 (methines). FAB/MS: 608 (M + H), 115 (base). IR (neat, cm-1): 3500-3300, 3020, 1710, 1645, 1520, 1450, 1370, 1280. 17. 1H NMR (300 MHz, CD3OD, ppm): 1.0 (m, 4H), 1.32 (m, 4H), 1.4 (m, 4H), 1.6 (m, 8H), 1.73 (m, 6H), 2.052.11 (dt, 2H), 2.15-2.25 (dt, 2H), 3.22 (m, 4H), 3.63 (m, 8H), 6.79 (s, 2H). 13CD NMR (75 MHz, CD3OD, ppm): 26.3 (methylene), 26.9 (methylene), 27.6 (methylenes), 29.99 (methylene), 30.1 (methylene), 30.6 (methylene), 31.0 (methylene), 35.9 (methylene), 37.1 (methylene), 37.8 (methylene), 37.9 (methylene), 44.5 (methylene), 46.2 (methines), 69.6 (methylenes), 71.8 (methylenes), 135.3 (methines). FAB/MS: 602 (M + Na), 580 (M + H), 154 (base, matrix). IR (neat, cm-1): 3300, 3100, 1725, 1640, 1550, 1450, 1370. 18. 1H NMR (300 MHz, CD3OD, ppm): 1.62 (m, 4H), 1.78 (m, 4H), 2.6 (t, 2H), 3.1 (t, 2H), 3.2-3.6 (m, 12H), 4.02 (s, 2H), 4.21 (s, 2H), 7.22 (m, 1H), 7.82 (m, 2H), 8.4 (m, 1H). 13CD NMR (75 MHz, CD3OD, ppm): 27.6 (methylenes), 30.4 (methylene), 30.5 (methylene), 35.5 (methylene), 36.1 (methylene), 37.9 (methylenes), 69.2 (methylene), 69.4 (methylene), 69.9 (methylene), 71.5 (methylene), 71.8 (methylenes), 71.9 (methylene), 121.2 (methine), 122.4 (methine), 139.1 (methine), 150.4 (methine). FAB/MS: 573 (MNH4+ + K), 557 (MNH4+ + Na), 535 (MNH4+). IR (neat, cm-1): 3310, 3080, 1750, 1665, 1540, 1420, 1370. Preparation of Hydrazide 19. Compound 17 (300 mg, 0.518 mmol) was stirred in CH2Cl2 (5.0 mL) with DCC (107 mg, 1 equiv) and tBOC hydrazide (68.4 mg, 1 equiv) at 0 °C for 1 h and then allowed to warm to room temperature, stirring for a total of ∼20 h. The precipitated urea was removed by filtration; the reaction products were concentrated in vacuo and purified on a preparative TLC plate (1 mm), eluting with 9/1 CHCl3/ MeOH, providing 220 mg (61%) of intermediate product. To this material (185 mg, 0.267 mmol) in CH2Cl2 (2.0 mL) was added TFA (2.5 mL), and the reaction mixture was stirred at room temperature, for 3 h. The reaction mixture was diluted with CH2Cl2 (50.0 mL), neutralized with NaHCO3, and the organic phase was separated, dried, and concentrated in vacuo. The crude reaction mixture was placed on a preparative TLC plate (1 mm), eluting with 90/10/1 CHCl3/MeOH/NH4OH to provide 49 mg of compound 19 in a 31% unoptimized yield. 19. 1H NMR (300 MHz, CD3OD, ppm): 1.1 (m, 4H), 1.4 (m, 8H), 1.65 (m, 8H), 1.77 (m, 6H), 2.15-2.21 (m, 4H), 3.2 (m, 4H), 3.51 (m, 8H), 6.85 (s, 2H), 7.85 (NH), 7.95 (NH). 13C NMR (75 MHz, CD3OD, ppm): 26.7, 26.9, 27.6, 29.9, 30.1, 30.6, 31.0, 34.9, 37.1, 37.8, 37.9, 44.5, 46.2, 69.5, 69.6, 71.8, 135.3. FAB/MS: 634 (M + K), 616 (M + Na), 594 (M + H), 154 (base, matrix). IR (neat, cm-1): 3700-3150, 3000-2850, 1710, 1650, 1540, 1410, 1370. Preparation of NHS-Activated Cross-Linkers. About 5 mg of maleimido carboxylic acid was dissolved in 50 µL of dry DMF. In a thoroughly dried glass flask,

452 Bioconjugate Chem., Vol. 8, No. 3, 1997

a 3-fold excess of dicyclohexylcarbodiimide (DCC) in 25 µL of dry DMF was added followed by N-hydroxysuccinimide (NHS, 3-fold excess). The mixture was stirred overnight and was then filtered through a piece of tissue paper (ca. 4 mm2) placed into a Pasteur pipette. The product formed was detected by TLC (CHCl3/MeOH, 9/1, visualized with iodine vapor). The Rf value of the compounds investigated is about 0.9 and can be easily distinguished from those of the starting materials (carboxylic acids, ca. 0.3-0.6, NHS near the baseline with excessive tailing). Preparation of Antibody-AP Conjugates. The antibody was activated with Traut’s reagent in triethanolamine buffer following Pierce’s Technical Bulletin 26101X (P.O. Box 117, Rockford, IL 61105). The activated antibody was purified on a Pharmacia PD 10 column in pH 7.3 buffer and stored at 4 °C prior to use. A solution of alkaline phosphatase (12 mg/mL) in glycerol/water (1/1) was incubated with the 10-fold stoichiometric excess of the crude activated cross-linker (14-17). After 25 min at 25 °C, a 1000-fold stoichiometric excess of 1 M glycine solution was added. The activated protein was purified on a Pharmacia PD 10 column with pH 7.3 buffer and stored at 4 °C prior to use. The two solutions of activated proteins were mixed in stoichiometric amounts (1/1) and kept at 25 °C for at least 3 h. The conjugate mixture obtained was fractionated by gel permeation chromatography.6 6 These reagents (Superdex resins, using standard conditions and techniques) were purchased from Pharmacia Biotech Inc. (800 Centennial Ave., P.O. Box 1327, Piscataway, NJ 088551327). General procedures other than those listed herein, additional examples, and relevant references may be found in their catalogs.

Johnson et al.

Preparation of the Antibody-AP Conjugate Using Linker 19. The enzyme was activated as described above. A solution of the antibody in 100 mM acetate buffer, (pH 5, 6 mg/mL) was incubated with a 1000-fold excess of sodium perchlorate in water (0.1 M, 1 h, 25 °C, darkness). The oxidation was then stopped by adding 0.3 M ethylene glycol solution in a 100-fold excess over perchlorate. After 5 min at room temperature, the mixture was buffer exchanged (100 mmol of acetate buffer at pH 5) and adjusted to 5 mg/mL. Cross-linker 19 was dissolved in DMF (20 mM) and added to the oxidized antibody in a 70-fold excess. The mixture was incubated for 3.5 h at room temperature. The activated antibody was buffer exchanged into the pH 7.3 conjugation buffer. Stoichiometric amounts (1/1) of the activated antibody and the activated enzyme were incubated overnight. The conjugate mixture obtained was fractionated by gel permeation chromatography.6 LITERATURE CITED (1) (a) Nagasawa, T., et al. (1973) New Agents for t-Butyloxycarbonylation and Methoxybenzyloxycarbonylation of Amino Acids. Bull. Chem. Soc. Jpn. 46, 1269-1272. (b) Sheldon, E. L., Levenson, C. H., Mullis, K. B., and Rapoport, H. (1989) N-Substituted 4,9-Dioxa-1,2-dodecane Diamines Useful for Preparing Nucleic Acid Intercalators. EP 0 309 006 A1. (c) Baer, B. W., Groves, E. S., Houston, L. L., and Levenson, C. H. (1989) Conjugates of Antisense Oligonucleotides & Therapeutic Uses Thereof. WO 91/04753. (d) Houston, L. L., Aldwin, L., and Nitecki, D. E. (1991) Thioether Linked Immunotoxin Conjugates. U.S. 5,024,834. (2) (a) Henry, D. W. (1966) Heterocycl. Chem., 503-511. (b) Voerman, M. G. L. (1904) Rec. Trav. Pays Bas, 265-282. (3) Greene, T. W. (1981) Protecting Groups in Organic Synthesis: tBOC Deprotection, John Wiley & Sons, New York.

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