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Bioconjugate Chem. 2000, 11, 124−130
Collagen Cross-Links. Synthesis of Immunoreagents for Development of Assays for Deoxypyridinoline, a Marker for Diagnosis of Osteoporosis Maciej Adamczyk,* Donald D. Johnson, and Rajarathnam E. Reddy Department of Chemistry (9NM), Building AP20, Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064-6016
(+)-Deoxypyridinoline (Dpd, 2) is a cross-link of bone collagen, which is released and excreted in urine during process of bone resorption. It has been shown that this bone collagen degradation product, Dpd (2) is a useful marker for diagnosis of osteoporosis and other metabolic bone diseases. In this paper, the design and synthesis of two immunogens (3, 4) via of conjugation of succinimidyl ester (14) to carrier protein, bovine serum albumin, or keyhole limphet hemocyanin, was presented. Additionally, fluorescent (5) and chemiluminescent (6) tracers were prepared from (-)-acid (13) via in situ activation and subsequent reaction with 6-Fln-CH2NH2 (17) or Acr-NH2 (18) and hydrolysis. The key hapten (-)-acid (13) was prepared by quaternization of 3-hydroxypyridine derivative (S,S)-(-)-11 with iodide (S)-(-)-10 followed by selective hydrolysis. These immunreagents (immunogens 3 and 4 and tracers 5 and 6) are useful for the development of sensitive and high throughput immunoassays, such as FPIA and CLIA for Dpd (2).
INTRODUCTION
Osteoporosis is a crippling degenerative bone disease, which affects the aged population, particularly postmenopausal women, and is responsible for 1.5 million fractured bones each year in the United States alone (1, 2). This metabolic disease is the consequence of an inbalance in the bone renewal process, which occurs when the bone resorption exceeds bone formation. Current methods for diagnosis of osteoporosis involve histomorphometry studies and densitometric measurements (2-4). Efforts for prevention of this disease as well as development of an effective antiresorptive therapy have increased the need for reliable and noninvasive biochemical markers of bone resorption (5-7). The traditional markers of bone resorption, i.e., urinary calcium (8, 9) and hydroxyproline (10, 11), lack clinical sensitivity and specificity for diagnosis of osteoporosis. Since bone is densely packed with collagen, a family of structurally related proteins, its strength is mainly governed by the physiological and pathological changes of collagen (12). It has been shown that, during the process of maturation of collagen, the cross-links, (+)pyridinoline (Pyd, 1) (13) and (+)-deoxypyridinoline (Dpd, 2) (14) (Figure 1), are formed from lysine and hydroxylysine by a lysyl oxidase-mediated enzymatic process (15-17). These pyridinium cross-links (1, 2) play an important role in maintaining the structure of the collagen fibril network. However, in the process of bone resorption, the cross-links (1, 2) are released into serum and excreted in urine either in free form or linked to different peptide fragments of collagen (5-7, 18). Therefore, markedly elevated levels of cross-links (1, 2) are present in urine of patients with osteoporosis and other metabolic bone diseases. Subsequently, it was found that the cross-link (+)-Dpd (2) shows greater specificity for * To whom correspondence should be addressed.
Figure 1.
bone, and consequently, it became a marker of choice for diagnosis of osteoporosis (19-20). A number of methods were developed for quantification of cross-links (1, 2), i.e., amino acid analysis (15-17, 21), HPLC (22-26), and immunoassays (27-35). However, there is no report in the literature on preparation of immunogens and tracers (fluorescent/chemiluminescent) from a specific position of optically pure (+)-Dpd (2) with full experimental details. This fact might be attributed to the high cost ($2510.40/1.0 mg) and complex structure of Dpd (2), which requires multiple steps for its total synthesis or tedious isolation from the natural sources. The need for well-characterized reagents required for development of immunoassays is widely recognized (36-40). In this endeavor, we recently described the chiral synthesis of (+)-Dpd (2) and a variety of its analogues (41-43). In this report, we present the synthesis of immunoreagents [immunogens (3, 4) and tracers (5, 6)], which are needed for development of fluorescence polarization immunoassay (FPIA), and chemiluminescence immunoassays (CLIA) for diagnosis of osteoporosis. EXPERIMENTAL SECTION
General Methods and Materials. 1H and 13C NMR spectra were recorded on a Varian Gemini spectrometer (300 MHz), the chemical shifts (δ) were reported in parts
10.1021/bc9900892 CCC: $19.00 © 2000 American Chemical Society Published on Web 12/16/1999
Technical Notes
per million relative to TMS, and coupling constants (J) were reported in hertz. Electrospray ionization mass spectrometry (ESI/MS) was carried on a Perkin-Elmer (Norwalk, CT) Sciex API 100 Benchtop system employing Turbo Ionspray ion source, and HRMS were obtained on Nermang 3010 MS-50, JEOL SX102-A mass spectrometers. Thin-layer chromatography was performed on precoated Whatman MK6F silica gel 60 Å plates (layer thickness: 250 µm). Column chromatography was performed on silica gel, Merck grade 60 (230-400 mesh). THF was freshly distilled from a purple solution of sodium and benzophenone. All reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI) or Sigma Chemical Co. (St. Louis, MO) and used without purification, except where noted. All the solvents employed were of HPLC grade purchased from EM Science (Gibbstown, NJ) and used as received. Analytical reversed-phase (RP) HPLC was performed using a Waters, µBondapak RCM C18 10 µm (8 × 100 mm) column (solvents ratio v/v reported) unless otherwise stated. Preparative reversedphase (RP) HPLC was performed using a Waters µBondapak RCM C18 10 µm (40 × 100 mm) column (solvents ratio v/v reported) unless otherwise stated. Optical rotations were measured on Autopol III polarimeter, Rudolph Research, Flanders, NJ. (-)-tert-Butyl-(2S)-4-(4-{(2S)-3-(tert-butoxy)-2-[(tert-butoxycarbonyl)amino]-3-oxopro pyl}-5-hydroxy-3-pyridinyl)2-[(tert-butoxycarbonyl)amino]butanoate (11) was prepared from a commercially available N-t-Boc-L-glutamic acid-R-tert-butyl ester (41). (S)-(-)-Methyl-2-[(tert-butoxycarbonyl)amino]-6hydroxyhexanoate (9). (S)-(-)-6-Amino-2-[(tert-butoxycarbonyl)amino]hexanoic acid (7, 3.69 g, 15.0 mmol) was dissolved in water (50 mL) and the pH adjusted to 9.5 using 4 M NaOH(aq). The mixture was heated to 60 °C, and sodium nitroprusside (7.07 g, 23.7 mmol, 1.6 equiv) was added portionwise over 30 min period. The reaction mixture was heated for an additional 5 h, while maintaining the pH between 9 and 10 by adding 4 M NaOH(aq) during and after the addition of sodium nitroprusside. The reaction mixture was cooled to room temperature and filtered through Celite powder. The pH of the filtrate was adjusted to 3.5 using 6 M HCl and extracted with EtOAc (3 × 40 mL). The combined organic layers were washed with brine (40 mL) and dried (Na2SO4), and the solvent was removed on a rotary evaporator. The resulting crude (S)-2-[(tert-butoxycarbonyl)amino]-6-hydroxyhexanoic acid (8) (2.40 g) was dried and carried to the next reaction without purification. The above prepared crude hydroxy acid (S)-8 (1.15 g, 4.65 mmol) was dissolved in MeOH (30 mL) and cooled with an ice bath. To this mixture was added a freshly generated ethereal diazomethane solution [generated from N-nitroso-N-methylurea (4.79 g, 46.5, mmol, 10 equiv), KOH (11.6 g, 207.1 mmol, 4.4 equiv.), ether (30 mL), and water (35 mL)] at 0 °C. After 15 min, the cooling bath was removed, and stirring was continued for an additional 1.5 h. Excess diazomethane was removed by bubbling the nitrogen for 10 min and then concentrated on a rotary evaporator. The crude product was purified by silica gel column chromatography (60% EtOAc in hexanes) to afford 0.624 g of (S)-(-)-methyl-2-[(tertbutoxycarbonyl)amino]-6-hydroxyhexanoate (9) in 41% yield for two steps. Rf: 0.33 (60% EtOAc in hexane). Analytical RP HPLC: MeCN:0.1% acetic acid(aq)/40:60, 1.0 mL/min at 225 nm. Rt: 6.88 min, >99%. [R]20D: -19.4 (c 1.29, MeOH). 1H NMR (CDCl3): δ 5.07 (d, 1 H, J ) 7.8 Hz), 4.33-4.26 (m, 1 H), 3.73 (s, 3 H), 3.69-3.58 (m, 2 H), 1.85-1.55 (m, 6 H), 1.43 (s, 9 H). 13C NMR
Bioconjugate Chem., Vol. 11, No. 1, 2000 125
(CDCl3): δ 173.5, 155.6, 79.9, 62.3, 53.2, 52.2, 32.3, 31.9, 28.2, 21.4, 14.0. ESI-MS (m/z): 262 (M + H)+. (S)-(-)-Methyl-2-[(tert-butoxycarbonyl)amino]-6iodohexanoate (10). Triphenylphos phine (0.792 g, 3.02 mmol, 1.5 equiv), imidazole (0.219 g, 3.22 mmol, 1.6 equiv), and iodine (0.767 g, 3.02 mmol, 1.5 equiv) were added sequentially to a solution of (S)-(-)-9 (0.524 g, 2.01 mmol) dissolved in THF (20 mL) at room temperature under nitrogen. After stirring the mixture for 2 h, the solvent was removed on a rotary evaporator to dryness. The residue was diluted with EtOAc (40 mL) and water (40 mL). The organic layer was separated and extracted with EtOAc (2 × 40 mL). The combined organic layers were dried (Na2SO4), and the solvent was removed on a rotary evaporator. The crude product was purified by silica gel column chromatography (20% EtOAc in hexanes) to afford 0.655 g of (S)-(-)-10 in 88% yield as a colorless thick oil. Rf: 0.31 (20% EtOAc in hexanes). Analytical RP HPLC: MeCN:0.1% acetic acid(aq)/80:20, 1.0 mL/min at 225 nm. Rt: 5.91 min, 98%. [R]20D: -8.8 (c 1.51, MeOH). 1H NMR (CDCl3): δ 5.03 (d, 1 H, J ) 8.1 Hz), 4.36-4.26 (m, 1 H), 3.74 (s, 3 H), 3.16 (t, 2 H, J ) 6.9 Hz), 1.90-1.58 (m, 6 H), 1.43 (s, 9 H). 13C NMR (CDCl3): δ 173.3, 155.4, 79.9, 53.0, 52.3, 32.6, 31.5, 28.2, 26.0, 6.0. ESI-MS (m/z): 372 (M + H)+. Pyridinium Compound (12). A mixture of (S,S)(-)-11 (0.369 g, 0.619 mmol) and iodide (S)-(-)-10 (0.461 g, 0.1.24 mmol, 2.0 equiv) were dissolved in anhydrous 1,4-dioxane (10 mL) and gently refluxed for 6.5 h under nitrogen. The solvent was removed on a rotary evaporator to dryness, the crude product was dissolved in MeCN0.05% acetic acid(aq) (50 mL, ratio, 80:20) and purified by preparative RP HPLC [MeCN:0.05% trifluoroacetic acid(aq)/70:30, 25 mL/min at 225 nm]. The solvent was removed on a rotary evaporator to about 300 mL volume and finally lyophilized. The resulting product (oil) was azeotroped using toluene-MeOH (1:1 ratio, 3 × 20 mL) on a rotary evaporator to afford 0.558 g of 12 as its TFA salt in 94% yield. Analytical RP HPLC: MeCN:0.05% acetic acid(aq)/80:20, 1.0 mL/min at 225 nm, Rt: 5.76 min, 96%. 1H NMR (CDCl3): δ 9.24 (s, 1 H), 7.95 (s, 1 H), 5.45-5.35 (m, 1 H), 5.23 (d, 1 H, J ) 7.8 Hz), 5.03 (d, 1 H, J ) 7.8 Hz), 4.42-4.22 (m, 4 H, 4.18-4.06 (m, 1 H), 3.75 (s, 3 H), 3.40-3.10 (m, 2 H), 3.10-2.90 (m, 2 H), 2.18-1.22 (m, 8 H), 1.47 (s, 9 H), 1.45 (s, 9 H), 1.44 (s, 9 H), 1.43 (s, 9 H), 1.34 (s, 9 H). ESI-MS (m/z): 839 (M)+. HRMS (m/z): calcd for C42H71N4O13, 839.5012; observed, 839.5010. (-)-Acid (13). Lithium hydroxide monohydrate (0.063 g, 1.5 mmol, 3.0 equiv) and water (8 mL) were added sequentially to a solution of pyridinium compound (12, 0.422 g, 0.502 mmol) dissolved in THF (24 mL) at room temperature. After stirring the mixture for 30 min, it was then concentrated on a rotary evaporator. The crude product was purified by preparative RP HPLC (MeCN: 0.05% trifluoroacetic acid(aq)/70:30, 25 mL/min at 225 nm). The solvent was removed on a rotary evaporator to about 300 mL volume and finally lyophilized to afford 0.165 g of (-)-acid (13) as its TFA salt in 40% yield. Analytical RP HPLC: MeCN:0.05% trifluoroacetic acid(aq)/70:30, 1.0 mL/min at 225 nm. Rt: 6.57 min, 98%. [R]20D: -20.0 (c 1.29, MeOH). 1H NMR (CD3OD): δ 8.37 (s, 1 H), 8.13 (s, 1 H), 4.58-4.42 (m, 2 H), 4.18-3.90 (m, 3 H), 3.40-2.82 (m, 4 H), 2.20-1.20 (m, 8 H), 1.46 (s, 9 H), 1.45 (s, 9 H), 1.44 (s, 9 H), 1.43 (s, 9 H), 1.36 (s, 9 H). ESI-MS (m/z): 839 (M)+. Succinimidyl Ester (14). N-Hydroxysuccinimide (HOSu, 0.034 g, 0.296 mmol, 2.0 equiv) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC, 0.057 g,
126 Bioconjugate Chem., Vol. 11, No. 1, 2000
0.296 mmol, 2.0 equiv) were added sequentially to solution of (-)-acid (13, 0.122 g, 0.148 mmol) and dissolved in anhydrous DMF (8 mL) at room temperature under nitrogen. The mixture was stirred for 16 h, and solvent removed on a rotary evaporator under vacuum. The crude product was dissolved in MeCN-0.1% aqueous trifluoroacetic acid (70:30 ratio, 15 mL) and purified by preparative RP HPLC [MeCN:0.05% trifluoroacetic acid(aq)/70:30, 25 mL/min at 225 nm]. The solvent was removed on a rotary evaporator (99% purity as a pale yellow powder. Similarly, the active ester (14) was treated with acridinium derivative (Acr-NH2 TFA, 18) (49) and triethylamine in DMF at room temperature to afford 20 in 72% yield after purification by preparative HPLC. Finally, hydrolysis of 20 using trifluoroacetic acid and water followed by HPLC purification afforded the chemiluminescent tracer (Acrtracer, 6) in 77% yield and >99% purity as a pale yellow powder. In summary, two immunogens [BSA-immunogen (3) and KLH-immunogen (4)] and two tracers [fluorescent (5) and chemiluminescent (6)] were prepared from succinimidyl ester (14). These immunoreagents (3,4 and 5,6)
Technical Notes
are needed for generation of anti-Dpd antibodies and for development assays for diagnosis of osteoporosis. LITERATURE CITED (1) Sato, M., Grese, T. A., Dodge, J. A., Bryant, H. U., and Turner, C. H. (1999) Emerging therapies for the prevention or treatment of postmenopausal osteoporosis. J. Med. Chem. 42, 1-24. (2) Bilezikian, J. P., Raisz, L. G., and Rodan, G. A. (1996) Principles of Bone Biology, Academic Press, New York. (3) Hagiwara, S., Yang, S. O., Gluer, C. C., Bendavid, E., and Genant, H. K. (1994) Noninvasive bone mineral density measurement in the evaluation of osteoporosis. Rheum. Dis. Clin. North. Am. 20, 651-669. (4) Genant, H. K., Lang, T. F., Engelke, K., Fuerst, T., Glu¨er, C., Majumdar, S., and Jergas, M. (1996) Advances in the noninvasive assessment of bone density, quality, and structure. Calcif. Tissue Int. 59 (Suppl. 1), S10-S15. (5) Eyre, D. R. (1996) Biochemical basis of collagen metabolites as bone turnover markers. Principles of Bone Biology, pp 143-153, Academic Press, New York. (6) James, I. T., Walne, A. J., and Perrett, D. (1996) The measurement of pyridinium cross-links: A methodological overview. Ann. Clin. Biochem. 33, 397-420. (7) Knott, L., and Bailey, A. J. (1998) Collagen cross-links in mineralizing tissues: a review of their chemistry, function, and clinical relevance. Bone 22, 181-187. (8) Raisz, L. G. (1963) Stimulation of bone resorption by parathyroid hormone in tissue culture. Nature 197, 10151016. (9) Raisz, L. G. (1965) Bone resorption in tissue culture. Factors influencing the response to parathyroid hormone. J. Clin. Inv. 44, 103-116. (10) Weiss, P. H., and Klein, L. (1969) The quantitative relationship of urinary peptide hydroxyproline excretion to collagen degradation. J. Clin. Inv. 48, 1-10. (11) Lippincott, S., Chesney, R. W., Friedman, A., Pityer, R., Barden, H., and Mazess, R. B. (1989) Rapid determination of total hydroxyproline (HYP) in human urine by HPLC analysis of the phenylisothiocyonate (PITC)-derivative. Bone 10, 265-268. (12) Kadler, K. (1995) Extracellur matrix 1: Fibril-forming collagen. Protein Profile 2, 491-619. (13) Fujimoto, D., Moriguchi, T., Ishida, T., and Hayashi, H. (1978) The structure of pyridinoline, A collgen cross-link. Biochem. Biophys. Res. Commun. 84, 52-57. (14) Ogawa, T., Ono, T., Tsuda, M., and Kawanishi, Y. (1982) A novel fluor in insoluble collagen: A cross-linking moiety in collgen molecule. Biochem. Biophys. Res. Commun. 107, 1252-1257. (15) Eyre, D. R., and Oguchi, H. (1980) The hydroxypyridinium cross-links of skeletal collagens: their measurement, properties and a proposed pathway of formation. Biochem. Biophys. Res. Commun. 92, 403-410. (16) Gunja-Smith, Z., and Boucek, R. J. (1981) Collagen crosslinking compounds in urine. Biochem. J. 197, 759-762. (17) Robins, S. P. (1983) Cross-linking of Collagen. Isolation, structural Characterization and glycosylation of pyridinoline Biochem. J. 215, 167-173. (18) Hanson, D. A., and Eyre, D. R. (1996) Molecular site specificity of pyridinoline and pyrrole cross-links in type I collagen of human bone. J. Biol. Chem. 271, 26508-26516. (19) Eyre, D. R., Paz, M. A., and Gallop, P. M. (1984) Crosslinking in collagen and elastin. Annu. Rev. Biochem. 53, 717748. (20) Siebel, M. J., Robins, S. P., and Bilezikian, J. P. (1992) Urinary pyridinium cross-links of collagen. Specific markers of bone resorption in metabolic bone disease. Trends Endocrinol. Metab. 3, 263-270. (21) Fugimoto, D., Suziki, M., Uchiyama, A., Miyamoto, S., and Inoue, T. (1983) Analysis of pyridinoline, a cross-linking compound of collagen fibers in human urine. J. Biochem. 94, 1133-1136. (22) Eyre, D. R., Koob, T. J., and Van Ness, K. P. (1984) Quantitation of hydroxypyridinium cross-links in collagen by
Bioconjugate Chem., Vol. 11, No. 1, 2000 129 high-performance liquid chromatography. Anal. Biochem. 137, 380-388. (23) Black, D., Duncan, A., and Robins, S. A. (1988) Quantitative analysis of the pyridinium cross-links of collagen in urine using ion-paired reversed-phase high-performance liquid chromatography. Anal. Biochem. 169, 197-203. (24) James, I. T., Perrett, D., and Thompson, P. W. (1990) Rapid assay for hard tissue collagen cross-links using isocratic ionpair reversed-phase liquid chromatography. J. Chromatogr. 525, 43-57. (25) Takahashi, M., Hoshino, H., Kushida, K., and Inoue, T. (1995) Direct measurement of cross-links, pyridinoline, deoxypyridinoline, and pentosidine, in the hydrolysate of tissues using high-performance liquid chromatography. Anal. Biochem. 232, 158-162. (26) Pratt, D. A., Daniloff, Y., Duncan, A., and Robins, S. P. (1992) Automated analysis of the pyridinium cross-links of collagen in tissue and urine using solid-phase extraction and reversed-phase high-performance liquid chromatography. Anal. Biochem. 207, 168-175. (27) Robins, S. P. (1982) An enzyme-linked immunoassay for the collagen cross-link pyridinoline. Biochem. J. 207, 617620. (28) Delmas, P. D., Gineyts, E., Bertholin, A., Garnero, P., and Marchand, F. (1993) Immunoassay of pyridinoline cross-link excretion in normal adults and in Paget’s disease. J. Bone Miner. Res. 8, 643-648. (29) Seyedin, S. M., Kung, V. T., Daniloff, Y. N., Hesley, R. P., Gomez, B., Nielsen, L. A., Rosen, H. N., and Zuk, R. F. (1993) Immunoassay for urinary pyridinoline: the new marker of bone resorption.. J. Bone Miner. Res. 8, 635-641. (30) Gomez, B., Jr., Ardakani, S., Evans, B. J., Merrell, L. D., Jenkins, D. K., and Kung, V. T. (1996) Monoclonal antibody assay for free urinary pyridinium cross-links. Clin. Chem. 42, 1168-1175. (31) Uren˜a, P., Ferreira, A., Kung, V. T., Morieux, C., Simon, P., Ang, K. S., Souberbielle, J. C., Segre, G. V., Dru¨eke, T. B., and De Vernejoul, M. C. (1995) Serum pyridinoline as a specific marker of collagen breakdown and bone metabolism in hemodialysis patients. J. Bone Miner. Res. 10, 932-939. (32) Robins, S. P., Woitge, H., Hesley, R., Ju, J., Seyedin, S., and Seibel, M. J. (1994) Direct, enzyme-linked immunoassay for urinary deoxypyridinoline as a specific marker for measuring bone resorption.. J. Bone Miner. Res. 9, 1643-1649. (33) Rosano, T. G., Peaston, R. T., Bone, H. G., Woitge, H. W., Francis, R. M., and Seibel, M. J. (1998) Urinary free deoxypyridinoline by chemiluminescence immunoassay: analytical and clinical evaluation. Clin. Chem. 44, 2126-2132. (34) Sarno, M, Powel, H., Tjersland, G., Schoendorfer, D., Harris, H., Adams, K., Ogata, P., and Warnick, G. R. (1999) A collection method and high-sensitivity enzyme immunoassay for sweat pyridinoline and deoxypyridinoline cross-links. Clin. Chem. 45, 1501-1509. (35) Waelchli, R., Beerli, Ch., Meigel, H., and Revesz, L. (1997) First total synthesis of the bone resorption markers deoxypyridinoline and hydroxypyridinoline. Bioorg. Med. Chem. Lett. 7, 2831-2836. (36) Henry, C. (1996) FDA, reform, and the well characterized biologics. Anal. Chem. 68, 674A-677A. (37) Van Weemen, K. K., and Schuurs, A. H. W. M. (1975) The influence heterologous combinations of antiserum and enzyme labeled estrogen on the characteristics of estrogen enzymeimmoassays. Immunochemistry 12, 667-670. (38) Adamczyk, M., Buko, A., Chen, Y. Y., Fishpaugh, J. R., Gebler, J. C., and Johnson, D. D. (1994) Characterization of protein-hapten conjugates. 1. Matrix-assisted laser desorption ionization mass spectrometry of immuno BSA-hapten conjugates and comparison with other characterization methods. Bioconjugate Chem. 5, 631-635. (39) Adamczyk, M., Fino, L., Fishpaugh, J. R., Johnson, and D. D. Mattingly, P. G. (1994) Immunoreagents for thyroid testing. 1. Synthesis of thyroxine conjugates. Bioconjugate Chem. 5, 459-462.
130 Bioconjugate Chem., Vol. 11, No. 1, 2000 (40) Adamczyk, M., Fishpaugh, J. R., Heuser, K. J., Ramp, J. M., Reddy, R. E., and Wong, M. (1998) Synthesis of immunocomponents for the measurement of lead (Pb) by fluorescence polarization immunoassay. Tetrahedron 54, 3093-3112. (41) Adamczyk, M., Johnson, D. D., and Reddy, R. E. (1999) Collagen cross-links: Synthesis of pyridinoline, deoxypyridinoline and their analogues. Tetrahedron 55, 63-88. (42) Adamczyk, M., Johnson, D. D., and Reddy, R. E. (1999) Collagen cross-links: a convenient synthesis of tert-butyl-(2S)2-[(tert-butoxycarbonyl)amino]-4-(2-oxiranyl)butanoate. Tetrahedron: Asymmetry 10, 775-781. (43) Adamczyk, M., Akireddy, S. R, and Reddy, R. E. (1999) Versatile synthesis of (+)-deoxypyridinoline, a biochemical marker for diagnosis of osteoporosis. Tetrahedron: Asymmetry 10, 3107-3110. (44) Sokoll, L. J., and Chan, D. W. (1999) Clinical analyzers. Immunoassays. Anal. Chem. 71, 356R-362R.
Adamczyk et al. (45) Dandliker, W. B., Hsu, M.-L., and Vanderlaan, W. P. (1980) Immunoassays: Clinical laboratory Techniques for the 1980 (Nakamura, R. M., Dito, W. R., and Tucker, E. S., Eds.) pp 89-126, Alan R. Liss, New York. (46) Jandreski, M. A. (1998) Chemiluminescence Technology in immunoassays. Lab. Med. 29, 555-560. (47) Wild, D. (1994) The Immunoassay Handbook, Stocton Press, New York. (48) Kamps-Holtzapple, C., Carlin, R. J., Sheffield, C., Kubena, L., Stanker, L., and DeLoach, J. R. (1993) Analysis of haptencarrier protein conjugates by nondenaturing gel electrophoresis. J. Immunol. Methods 164, 245-253. (49) Mattingly, P. G. (1991) Chemiluminescent 10-methylacridinium-9-(N-sulfonyl carboxamide)salts. Synthesis and kinetics of light emission. J. Biolumin. Chemilumin. 6, 107-114.
BC9900892
