An Efficient Synthesis of a Heterobifunctional Coupling Agent

One-pot Synthesis of Succinimidyl-4- ( N -maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Nicholas M. Leonard , Jarmila Brunckova. Organic Preparati...
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Bioconjugate Chem. 2005, 16, 1323−1328

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An Efficient Synthesis of a Heterobifunctional Coupling Agent Rajarathnam E. Reddy,* Yon-Yih Chen, Donald D. Johnson, Gangamani S. Beligere, Sushil D. Rege, You Pan, and John K. Thottathil Core R&D Chemistry Group (Department 09MD, Building AP20), Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064-6016. Received October 18, 2004; Revised Manuscript Received June 8, 2005

An efficient synthesis of 4-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl]-N-(6-{[6-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-6-oxohexyl]amino}-6-oxohexyl)cyclohexanecarboxamide (12), a heterobifunctional coupling agent, was developed, which is critical for chemoselective conjugation of proteins and enzymes. The synthesis involved seven steps starting from 6-{[(benzyloxy)carbonyl]amino}hexanoic acid (1), and multigram quantities of coupling agent (12) were prepared using this protocol in excellent overall yield and 99.6% purity by reversed phase HPLC. The new method is suitable for the synthesis of coupling agent 12, consistently with purity >99%, and is useful for the preparation of other analogous coupling agents.

INTRODUCTION

Recent advances in the field of bioconjugate chemistry have spurred the construction of biomolecules based on chemoselective coupling for a variety of applications (13). The native physical properties of the protein and enzyme components are typically preserved in the resulting conjugate, when the coupling is carried out under the selective conditions (3-6). Use of such well-defined conjugates for applications in biology and medical fields is critical for implementation of quality control as well as for reliable interpretation of biological results (7). Heterobifunctional coupling agents containing reactive groups, such as succinimidyl ester and maleimide, are important for cross-coupling of proteins and enzymes. The succinimidyl active ester can be chemoselectively reacted with an amine on one protein, and the resulting adduct can then be conjugated to the second unit, e.g., protein or enzyme, containing a thiol via the maleimide group present in linking agent. This controlled conjugation process is quite selective when compared to other groups, e.g., iodoacetyl group, and can be carried out at physiological pH. Bieniarz and co-workers (8) have described the synthesis of four heterobifunctional reagents with varying lengths, e.g., 9, 16, 23, and 30 atoms long, and their application for the preparation of alkaline phosphatase conjugates, in a R-fetoprotein assay. They observed that the antibody-alkaline phosphatase conjugate prepared using the coupling agent with 30-atom long 12 gave 300% enhancement in the signal, when compared to the conjugate prepared with the corresponding 9-atom long analogue. The observed differences in performance of the conjugates was attributed to the improved antibody binding and lowered steric hindrance to the complementarity-determined region of the antibody, when a 30-atom long coupling agent (12) was used (8). Subsequently, the coupling agent 12 was found to be critical in the preparation of antibody-alkaline phosphatase conjugates for applications in the development of clinical assays, such * Corresponding author. Phone: 847-938-0409. Fax: 847-9385188. E-mail: [email protected].

as thyroid stimulating hormone (TSH) (9), prostate specific antigen (PSA) (10), troponin-1 (11), and progesterone (12), as well as in the preparation of thermally stabilized immunoconjugates (13). However, the synthetic protocol described for the preparation of heterobifunctional coupling agent 12 is not practical and produced the material in very low overall yield (0.16-2.5%) (8). In addition, the quality of coupling agent 12 was varied significantly from batch to batch, ranging from 89 to 98% purity, as determined by analytical reversed phased HPLC method. We needed gram quantities of a good and consistent quality of coupling agent 12, to prepare protein-enzyme conjugates, useful for diagnostic and medical applications. In this paper, we describe an efficient and convenient method for the preparation of heterobifunctional coupling agent 12. EXPERIMENTAL SECTION

General Methods and Materials. 1H and 13C NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer with an Oxford AS400 magnet. The chemical shifts (δ) were reported in ppm relative to TMS, and coupling constants (J) were reported in hertz. Electrospray ionization mass spectrometry (ESI-MS) was carried out on a Perkin-Elmer (Norwalk, CT) Sciex API 100 benchtop system employing a Turbo Ionspray ion source. All chemicals were purchased either from Aldrich Chemical Co. (Milwaukee, WI) or NovaBiochem (San Diego, CA). Analytical reversed phase (RP) HPLC was performed using a Waters (Milford, MA) NovaPak (C18, 60 Å, 4 µm, 3.9 × 150 mm) reversed phase (RP) column (solvents ratio v/v reported). The final product, coupling agent 12, was analyzed using an Akzonobel (Ferry, NY) kromasil reversed phased (RP) column (5.0 µm, 100 Å, 4.6 × 250 mm) (solvents ratio v/v reported). Melting points were recorded in open capillary tubes on an Electrothermal melting point apparatus and were uncorrected. Infrared spectra (IR) and elemental analysis were carried out by Roberson Microlit Laboratories, Inc. (Madison, NJ). IUPAC names of all compounds were obtained using the ACD/Ilab Web service version 3.5 at http://www.acdlabs.com/ilab.

10.1021/bc040259x CCC: $30.25 © 2005 American Chemical Society Published on Web 08/06/2005

1324 Bioconjugate Chem., Vol. 16, No. 5, 2005

Methyl 6-[(6-{[(Benzyloxy)carbonyl]amino}hexanoyl)amino]hexanoate (3). An oven-dried 2.0 L threeneck round-bottom flask was equipped with a mechanical stirrer and nitrogen inlet/outlet. To this flask were added 6-{[(benzyloxy)carbonyl]amino}hexanoic acid (1, 75.0 g, 282.7 mmol, 1.0 equiv) and methyl 6-aminohexanoate hydrochloride (2, 51.4 g, 282.7 mmol, 1.0 equiv) followed by anhydrous dichloromethane (1.1 L) at room temperature under nitrogen atmosphere. To the resulting suspension were added sequentially 1-hydroxybenzotriazole (HOBt, 45.8 g, 339.2 mmol, 1.2 equiv), diisopropylethylamine (DIEA, 123.1 mL, 706.8 mmol, 2.5 equiv), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC, 65.0 g, 339.2 mmol, 1.2 equiv), and the mixture was stirred at room temperature (21-23 °C) for 5-6 h. After completion of the reaction, as determined by the analysis of an aliquot by using HPLC, the reaction mixture was transferred into a separatory funnel and diluted with dichloromethane (1.0 L). The mixture was washed vigorously with 1.0 N hydrochloric acid (4 × 1.0 L), followed by 1.0 N sodium hydroxide (4 × 1.0 L) and finally with 3.5 M sodium chloride solution (2 × 1.0 L). The organic layer was dried with anhydrous MgSO4 and filtered, and the solvent was removed on a rotary evaporator. The resulting solid material was dried on a vacuum pump at room temperature for 24 h to afford 105.5 g of ester 3 as a colorless (white) powder in 95% yield. Mp: 74-75 °C [Lit. (15) 73-74 °C]; Analytical reversed phase (RP) HPLC: MeCN:water:0.5% aqueous trifluoroacetic acid/35:55:10/1.0 mL/min at 215 nm, tR: 7.4 min, 98%; IR (KBr): 3347, 3310, 2949, 1730, 1684, 1635, 1533, 1271, 1175, 734, 724 cm-1; 1H NMR (CDCl3): δ 7.37-7.29 (m, 5 H), 5.58 (br s, 1 H), 5.09 (s, 2 H), 4.88 (br s, 1 H), 3.67 (s, 3 H), 3.26-3.16 (m, 4 H), 2.31 (t, 2 H, J ) 7.2 Hz), 2.15 (t, 2 H, J ) 7.6 Hz), 1.691.59 (m, 4 H), 1.55-1.46 (m, 4 H), 1.38-1.29 (m, 4 H); 13 C NMR (CDCl3): δ 174.4, 173.1, 156.8, 137.0, 128.8, 128.4, 66.9, 51.8, 41.1, 39.5, 36.9, 34.2, 30.0, 29.6, 26.7, 26.6, 25.5, 24.8; ESI-MS (m/z): 393.3 (M + H)+, 410.3 (M + NH4)+, 786.0 (2 × M + H)+, 802.5 (2 × M + NH4)+; Elemental analysis: Calcd for C21H32N2O5, C, 64.26; H 8.22; N, 7.14, Found: C, 64.20; H, 8.48; N, 7.26. Methyl 6-[(6-Aminohexanoyl)amino]hexanoate Hydrochloride (4). In a dry 2.0 L hydrogention flask equipped with a stir bar was dissolved methyl 6-[(6-{[(benzyloxy)carbonyl]amino}hexanoyl)amino]hexanoate (3, 102.2 g, 260.0 mmol, 1.0 equiv) in anhydrous methanol (675 mL). To this solution were added 1.25 M hydrochloric acid in methanol (625 mL, 780.0 mmol, 3.0 equiv) and 10% palladium on activated carbon (10.4 g, wet, Degussa type E101 NE/W), and the mixture was stirred under hydrogen atmosphere (20-30 psi) at room temperature (21-23 °C). The reaction flask was re-filled with hydrogen gas periodically, and the progress of the reaction was monitored by HPLC for consumption of starting material 3. After 5 h, an additional amount of Pd/C catalyst (2.6 g) was added, and the hydrogenation was continued for an additional 2 h. After completion of the reaction, as determined by the analysis of an aliquot by using HPLC, the mixture was filtered using a funnel containing a Celite bed (about 4-5 mm thickness). The filter cake was washed with an additional amount of methanol (50 mL), and the combined filtrates were evaporated on a rotary evaporator (bath temp: 35-40 °C). The residue was redissolved in methanol (200 mL) and toluene (400 mL), and the residual amount of water, which originated from the catalyst, was removed azeotropically on a rotary evaporator. The redissolving/azeotropic concentration was repeated two more times, and the resulting white

Technical Notes

solid was dried under vacuum at room temperature (2123 °C) for 24 h to afford 81.2 g of methyl 6-[(6-aminohexanoyl)amino]hexanoate hydrochloride (4) as colorless gummy material in quantitative yield. IR (KBr): 3299, 2949, 1724, 1635, 1545, 1256, 1197, 1167, 978, 885, 732 cm-1; 1H NMR (CD3OD): δ 3.68 (s, 3 H), 3.25 (t, 2 H, J ) 7.2 Hz), 2.97 (t, 2 H, J ) 8.0 Hz), 2.37 (t, 2 H, J ) 7.2 Hz), 2.36-2.30 (m, 2 H), 1.76-1.62 (m, 6 H), 1.61-1.52 (m, 2 H), 1.50-1.42 (m, 2 H, 1.42-1.34 (m, 2 H); 13C NMR (CD3OD): δ 177.1, 176.7, 41.4, 41.3, 37.1, 35.5, 30.7, 29.1, 28.3, 27.8, 27.2, 26.5; ESI-MS (m/z): 259.2 (M + H)+, 517.8 (2 × M + H)+. Methyl 3,10,17-Trioxo-1-phenyl-2-oxa-4,11,18-triazatetracosan-24-oate (5). An oven-dried 2.0 L threeneck round-bottom flask was equipped with a mechanical stirrer and nitrogen inlet/outlet. To this flask was added 6-{[(benzyloxy)carbonyl]amino}hexanoic acid (1, Z--AhxOH, 72.0 g, 271.3 mmol, 1.0 equiv) followed by a solution of methyl 6-[(6-aminohexanoyl)amino]hexanoate hydrochloride (4, 22.1 g, 75 mmol, 1.0 equiv) in anhydrous dichloromethane (1.2 L) at room temperature under nitrogen atmosphere. To the resulting suspension were added sequentially 1-hydroxybenzotriazole (HOBt, 44.0 g, 325.6 mmol, 1.2 equiv), diisopropyl ethylamine (DIPA, 118.2 mL, 678.4 mmol, 2.5 equiv), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC, 62.4 g, 325.6 mmol, 1.2 equiv). The resulting pale yellow clear solution was stirred at room temperature (21-23 °C) for 5-6 h. After completion of the reaction as determined by the analysis of an aliquot by using HPLC, the reaction mixture was transferred into a separatory funnel and diluted with a mixture of 5% methanol in dichloromethane (1.5 L). The mixture was washed vigorously with 1 N hydrochloric acid (3 × 1.0 L), followed by 1 N sodium hydroxide (4 × 1.0 L) and finally with 3.5 M sodium chloride solution (1 × 1.0 L). The organic layer was dried with anhydrous MgSO4 and filtered, and the solvent was removed on a rotary evaporator. The resulting material was dried under vacuum for 60 h to afford 128.7 g of ester 5 as a colorless (white) powder in 94% yield. Mp: 121-24 °C [Lit. (15) 124-25 °C], IR (KBr): 3327, 2944, 2866, 1732, 1687, 1633, 1533, 1269, 733 cm1 ; Analytical reversed phase (RP) HPLC: MeCN:water: 0.5% aqueous trifluoroacetic acid/35:55:10/1.0 mL/min at 215 nm, tR: 5.85 min, 94-98.5%, 1H NMR (CDCl3): δ 7.38-7.28 (m, 5 H), 5.76 (m, 2 H), 5.08 (s, 2 H, 4.97 (m, 1 H), 3.66 (s, 3 H), 3.26-3.16 (m, 6 H), 2.31 (t, 2 H, J ) 7.2 Hz), 2.15 (t, 4 H, J ) 7.2 Hz), 1.68-1.58 (m, 6 H), 1.58-1.46 (m, 6 H), 1.38-1.28 (m, 6 H); 13C NMR (CDCl3): δ 174.4, 173.2, 156.8, 137.0, 128.8, 128.4, 128.3, 66.9, 51.8, 41.2, 39.5, 39.4, 36.8, 36.7, 34.2, 30.0, 29.6, 29.5, 26.7, 26.6, 26.5, 25.6, 25.4, 24.8; ESI-MS (m/z): 506.3 (M + H)+, 523.8 (M + NH4)+, 1029.1 (2 × M + NH4)+; Elemental analysis: Calcd for C27H43N3O6, C, 64.13, H; 8.57, N, 8.31, Found: C, 63.84, H, 8.57, N, 8.24. 3,10,17-Trioxo-1-phenyl-2-oxa-4,11,18-triazatetracosan-24-oic Acid (6). In a 3.0 L three-neck roundbottom flask equipped with mechanical stirrer was suspended the methyl 3,10,17-trioxo-1-phenyl-2-oxa-4,11,18-triazatetracosan-24-oate (5, 125.0 g, 247.2 mmol, 1.0 equiv) in tetrahydrofuran (THF, 1.5 L). In a separate 1.0 L beaker, lithium hydroxide monohydrate (20.8 g, 494.4 mmol, 2.0 equiv) was dissolved in water (0.75 L), and the solution was added to the ester (5)-THF suspension at room temperature (21-23 °C). After the addition was complete, the white suspension slowly dissolved and a clear solution formed in about 10 min. The reaction mixture was stirred for an additional 2 h, and after completion of the reaction, as determined by the analysis

Technical Notes

of an aliquot by using HPLC, the mixture was concentrated on a rotary evaporator to about 1.5 L final volume. The mixture was then diluted with water (750 mL), and the suspension was acidified to pH ∼3.5 with 6.0 N hydrochloric acid (85 mL), while the mixture was continuously stirred. The resulting precipitate was filtered, and the solid was washed three times with water (3 × 1.5 L). The material was transferred into crystallizing dish and dried in an oven at 45-50 °C under vacuum for 14 h. The crude acid (110.0 g) was crystallized from ethanol (700 mL) and ethyl acetate (1.6 L). The resulting white solid material was filtered and dried under vacuum at room temperature over 18-24 h to afford 100.3 g of acid 6 as a colorless (white) powder in 83% yield. Mp: 136-37 °C [Lit. (16) 133-34 °C]; Analytical reversed phase (RP) HPLC: MeCN:water:0.5% aqueous trifluoroacetic acid/25:75:10/1.0 mL/min at 215 nm, tR: 5.28 min, 99.9%; IR (KBr): 3533, 3140, 2250, 1578, 1460, 1392, 1253, 1180, 1126, 1033, 851 cm-1; 1H NMR (CD3OD): δ 7.95 (dist t, 2 H), 7.39-7.28 (m, 5 H), 7.01 (dist t, 1 H), 5.09 (s, 2 H), 3.22-3.12 (m, 6 H), 2.32 (t, 2 H, J ) 7.6 Hz), 2.20 (t, 4 H, J ) 7.6 Hz), 1.68-1.60 (m, 6 H), 1.571.48 (m, 6 H), 1.43-1.34 (m, 6 H); 13C NMR (CD3OD): δ 178.3, 176.9, 176.8, 139.4, 130.3, 129.8, 129.6, 68.2, 42.5, 41.0, 41.1, 37.9, 37.8, 35.7, 31.5, 31.0, 28.4, 28.3, 28.2, 27.6, 27.5, 26.6, ESI-MS (m/z): 492.3 (M + H)+, 509.3 (M + NH4)+; Elemental analysis: Calcd, C26H41N3O6, C, 63.52, H, 8.41, N, 8.55, Found, C, 63.54, H, 8.54, N, 8.54. 6-({6-[(6-Aminohexanoyl)amino]hexanoyl}amino)hexanoic Acid (7). In a 2.0 L dry hydrogenation flask equipped with a stir bar was suspended the 3,10,17trioxo-1-phenyl-2-oxa-4,11,18-triazatetracosan-24-oic acid (6, 50.0 g, 101.7 mmol) in methanol (900 mL). The mixture was gently heated to about 55 °C to dissolve the material, and then water (450 mL) was added. To the resulting clear solution was added 10% palladium on activated carbon (5.0 g, wet, Degussa type E101 NE/W) while the solution was still warm, and the mixture was stirred under hydrogen atmosphere (20-30 psi) for 2 h. The flask was refilled with hydrogen periodically, and the progress of the reaction was monitored by HPLC for the consumption of starting material, acid 6. An additional amount of catalyst (2.5 g) was added, and the hydrogenation was continued for an additional 2 h. After completion of the reaction, as determined by the analysis of an aliquot by using HPLC (total time: 4 h), the mixture was then filtered over a Celite bed. The filter cake was washed with an additional methanol (100 mL), and the combined filtrates were evaporated on a rotary evaporator. The resulting solid was transferred into a crystallizing dish and dried in oven at 45-50 °C under vacuum for 36 h to afford 36.5 g of 6-({6-[(6-aminohexanoyl)amino]hexanoyl}amino)hexanoic acid (Tri-ACA-peptide, 6) as a colorless (white) powder in 99% yield. Mp: 21820 °C [Lit. (16) 203-204 °C]; IR (KBr): 3366, 3026, 2886, 1846, 1488, 1444, 1340, 1289, 1230, 1184, 1111, 985, 940 cm-1; 1H NMR (D2O + CD3OD): δ 3.19 (t, 4 H, J ) 6.8 Hz), 3.01 (t, 2 H, J ) 7.6 Hz), 2.26 (t, 2 H, J ) 7.6 Hz), 2.25 (t, 2 H, J ) 6.8 Hz), 2.20 (t, 2 H, J ) 7.2 Hz), 1.741.49 (m, 12 H), 1.45-1.29 (m, 6 H); 13C NMR (D2O + CD3OD): δ 185.0, 177.0, 177.8, 40.8, 40.7, 40.6, 40.0, 37.1, 37.0, 29.6, 29.5, 28.0, 27.6, 27.0, 26.9, 26.6, 26.5, 26.4; ESI-MS (m/z): 358.2 (M + H)+, 715.9 (2 × M + H)+; Elemental analysis: Calcd for C18H35N3O4, C, 60.48, H, 9.87, N, 11.75, Found: C, 59.89, H, 9.81, N, 11.58. Pentafluorophenyl 4-[(2,5-Dioxo-2,5-dihydro-1Hpyrrol-1-yl)methyl]cyclohexanecarboxylate (FMCC, 10). In a dry three-neck 2.0 L round-bottom flask equipped with mechanical stirrer and a nitrogen inlet,

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the powdered maleic anhydride (31.2 g, 318.1 mmol, 1.0 equiv) was dissolved in acetonitrile (500 mL) under nitrogen atmosphere. To this clear solution was added trans-4-(aminomethyl)cyclohexanecarboxylic acid (8, 50.0 g, 318.1 mmol, 1.0 equiv) at room temperature (21-23 °C). After the reaction mixture was stirred for 6 h, the resulting suspension was cooled to 2-5 °C with an ice bath. To this cold mixturewas added quickly diisopropylethylamine (DIEA, 150.0 mL, 858.9 mmol, 2.7 equiv) followed by pentafluorophenyl trifluoroacetate (9, 132.7 mL, 795.5 mmol, 2.5 equiv) slowly using an additional funnel over 20-25 min period. During this period, the temperature of the reaction mixture was maintained below 10 °C. After the addition was complete, the resulting pale brown color reaction mixture was stirred for 1.5 h at low temperature (99% by HPLC, and can be useful for preparation of other analogous coupling agents, which are critical for bioconjugation. ACKNOWLEDGMENT

We thank Dr. Phillip G. Mattingly of Abbott Laboratories for on-line literature search. LITERATURE CITED (1) Aslam, M., and Dent, A. H. (1999) Bioconjugation: Protein coupling techniques for the biomedical sciences, Macmillan Publishers, Houndsmills, England.

1328 Bioconjugate Chem., Vol. 16, No. 5, 2005 (2) Hermanson, G. T. (1996) Bioconjugate Techniques, Academic Press, New York. (3) Sasay, M. A. (2003) Monoclonal antibody conjugation via chemical modification. Biopharm. Int. December, 32-39 (4) Brinkley, M. (1992) A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents. Bioconjugate Chem. 3, 2-13. (5) Koppel, G. A. (1990) Recent advances with monoclonal antibody drug targeting for the treatment of human cancer. Bioconjugate Chem. 1, 13-23. (6) Means, G. E., and Feeney, R. E. (1990) Chemical modifications of proteins: History and applications. Bioconjugate Chem. 1, 2-12. (7) Henry, C. (1996) FDA, reform and the well-characterized biologic. Anal. Chem. 68, 674A-677A. (8) Bieniarz, C.; Husain, M., Barnes, G., King, C. A., and Welch, C. J. (1996) Extended length heterobifunctional coupling agents for protein conjugations. Bioconjugate Chem. 7, 8895. (9) Bogacz, J. P., Novotny, M., Lewis, C. A., Peters, T., Sramek, L., Chacko, M., Sequeira, A., Jacob, J., and Wilson, D. H, (1997) Development of an automated random/continues access microparticle enzyme immunoassay (MEIA) for the determination of hTSH with 3rd generation sensitivity on the Abbott AxSYM automated immunoassay system. Clin. Chem. 43, S189. (10) Dewell, B., Jacobson, L., Friese, J., Rapp, J., Miceli, C., Loewen, N., Lauren, L., Salle, J., Slota, J., Seguado, O., and Weigand, R. (1997) Development of the Abbott AxSYM free

Technical Notes PSA assay: Performance characteristics and preliminary clinical evaluation. Anticancer Res. 17, 3037-3038. (11) Qiu, K. Y., Bouma, S. R., Sumerdon, G. A., Biegalski, T. T., Gardiner, M. E., Munoz, J. J., and Peters, T. L. (2003) Development of a second generation troponin I assay for the Abbott AxSYM(R) immunoassay system. Clin. Chem. 49, A62. (12) Wilson, D. H., Groskopf, W., Hsu, S., Caplan, D., Langner, T., Baumann, M., DeManno, D., Williams, G., Paytte, D., Dagel, C., Lynch, D., and Manderino, G. (1998) Rapid, automated assay for progesterone on the Abbott AxSym analyzer. Clin. Chem. 44, 86-91. (13) Bieniarz, C., Young, D. F., and Cornwell, M. J. (1998) Thermally stabilized immunoconjugates: Conjugation of antibodies to alkaline phosphatase stabilized with polymeric cross-linkers. Bioconjugate Chem. 9, 399-402. (14) Zahn, H., and Determann, H. (1957) Nonakis-, decakis-, undecakis-, und dodecakis- -aminocapronsa¨ure. Chem. Ber. 90, 1963-1970. (15) Cairns-Smith, A. G., and Pettigrew, R. (1969) Synthesis of nylon-like oligoamides. J. Chem Soc. 1606-1609. (16) Zhan, H., and Hildebrand, D. (1957) Zur kenntnis der linearen oligamide der -amino-capronsa¨ure. Chem. Ber. 90, 320-329. (17) Adamczyk, M., and Johnson, D. (1993) Synthesis of pentafluorophenyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (FMCC). Org. Prep. Proced. Int. 25, 592-594.

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