A statistical-based approach to assessing the fidelity of combinatorial libraries encoded with electrophoric molecular tags. Development and application of tag decode-assisted single bead LC/MS analysis

Journal of Combinatorial Chemistry

Volumn 2, Issue 6, 2000, Pages 716-731

  Dolle, Roland E ^a   Guo, Joan ^a   O'Brien, Linda ^a   Jin, Ying ^a   Piznik, Mark ^a   Bowman, Kevin J ^a   Li, Wenni ^a   Egan, William J ^a   Cavallaro, Cullen L ^a   Roughton, Andrew L ^a   Zhao, Qian ^a   Reader, John C ^a   Orlowski, Marc ^a   Jacob Samuel, Biji ^a   Carroll, Carolyn Dilanni ^a  

^a PHARMACOPEIA INC   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

EID: 0001440059     PISSN: 15204766     EISSN: None     Source Type: Journal    
DOI: 10.1021/cc000052k     Document Type: Article

References (100)

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39. i = 66.7%, lb-ub = 7.9-100% (α = 0.05).
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68. The detection limit (2.5 pmol total injection) and the detector linear range (2.5∼175 pmol) used in the comparative QA analysis were established using the set of quality control compounds from library 1 (data not shown). For an example of using a set of structurally related compounds for calibration, see: Lai, C.-C.; Tsai, P.-L.; Yu, C.; Her, G.-R. Rapid Commun. Mass Spectrom. 2000, 14, 486-475 and references therein. Further details regarding the use of library QC compounds as the calibration set for the mass spectral analysis will be reported elsewhere.
69. The small quantities of cleaved compounds obtained from the beads prohibited the direct use of TIC chromatograms (ref 8) to confirm putative library compounds. This is due to the interference of bead matrix present in the reconstituted samples. The use of XIC chromatograms is standard practice in drug metabolism where biological matrix may obscure trace compounds of interest.
70. +. If the signal-to-noise (S/N) ratio of the peak defined by the molecular weight in the XIC chromatogram was less than 3:1, the targeted molecular ion was not observed and recorded as not found (NF).
71. (b) For ca. 2-3% of the F answers, the S/N of the peak in the XIC chromatogram was <3:1 and the peak height was in the lower end of the detector linear range (<4.0E+04), or the peak was a minor component of the XIC chromatogram.
72. (a) Abdel-Meguid, S. S. Med. Res. Rev. 1993, 13, 731-778.
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74. (c) Barrett, A. J. In Proteinases in Mammalian Cells and Tissues; Barrett, A. J., Ed.; Elsevier/North-Holland Biomedical Press: New York, 1977; pp 209-248.
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77. (a) Silva, A. M.; Lee, A. Y.; Gulnik, S. V.; Majer, P.; Collins, J.; Bhat, T. N.; Collins, P. J.; Cachau, R. E.; Luker, K. E.; Gluzman, I. Y.; Francis, S. E.; Oksman, A.; Goldberg, D. E.; Erickson, J. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10034-10039.
78. (b) Moon, R. P.; Tyas, L.; Certa, U.; Rupp, K.; Bur, D.; Jacquet, C.; Matile, H.; Loetscher, H.; Grueninger-Leitch, F.; Kay, J.; Dunn, B. D.; Berry, C.; Ridley, R. G. Eur. J. Biochem. 1997, 244, 552-560.
79. 2 specificity for these enzymes: (a) Carroll, C. D.; Patel, H.; Johnson, T. O.; Guo, T.; Orlowski, M.; He, Z.-M.; Cavallaro, C. L.; Guo, J.; Oksman, A.; Gluzman, I. Y.; Connelly, J.; Chelsky, D.; Goldbert, D. E.; Dolle, R. E. Bioorg. Med. Chem. Lett. 1998, 8, 2315-2320.
80. (b) Carroll, C. D.; Johnson, T. O.; Tao, Shewei; Lauri, G.; Orlowski, M.; Gluzman, I. Y.; Goldbert, D. E.; Dolle, R. E. Bioorg. Med. Chem. Lett. 1998, 8, 3203-3206.
81. Plasmepsin II has been the target for other libraries containing aspartic acid transition-state isosteres: Haque, T. S.; Skillman, G.; Lee, C. E.; Habashita, H.; Gluzman, I. Y.; Ewing, T. J. A.; Goldberg, D. E.; Kuntz, I. D.; Ellman, J. A. J. Med. Chem. 1999, 42, 1428-1440.
82. Two different analysts carried out the library QA analyses over a four month period. Each QA (ca. 650 beads) required approximately 32 h total GC time (3 min per decode) and approximately 55 h total of LC/MS time (5 min per sample).
83. Between the 1902 decoded beads in the three QA runs and the 131 decodes obtained from screening, there were 16 overlapping structures identified. In each case, there was a 100% correlation between these biologically active compounds and a found (F) designation in the QA analysis adding an additional level of validation for the library QA analysis (data not shown).
84. Calculated logP (clogP) for selected synthons: 0.1, A6; -0.1, A8; 0.27, A11; 1.0, A14; -0.4, A19; 1.8, A21; 1.4, A23; -0.5, A32; 1.2, A33.
85. + was found as a major product in the respective XIC chromatograms.
86. + is well known in peptides having two or more basic residues in acidic solution (see refs 13). Thus library compounds with multiple basic synthons, if double protonated, would be recorded as NF. Cationization, solvent-adduct formation, ESI-induced oxidation, and ESI-discrimination effects (i.e., differences in ionization efficiencies for competing compounds) can also lead to NF answers.
87. The tendency to see higher overall performance of the hydrophobic synthons and a higher confirmation rate of lipophilic compounds per se may in part be an artifact of the LC/MS analysis. For example, LC is conducted employing a generic 5 min gradient on a C18 column designed to capture 95+% of the library compounds (see Experimental Section). Compounds having multiple charged groups (e.g., the putative compound A8-B14-C1-D4) may be not be retained on the column and hence register as NF.
88. A statistical sampling QA analysis (609 beads) was carried out on the tagged Fmoc-intermediate 5: p = 96.1%. UV data was also obtained for many of the intermediates, which showed >85% purity (unpublished observation). Specifically, synthon B46 gave p = 100% (n = 5), indicating chemical failure post coupling.
89. Chemical yield of resynthesized 21 was 10% versus the average 50% yields obtained for the other resynthesized compounds in Table 6.
90. 1′ residues, respectively, are reported as inhibitors of cathepsin D: Lin, T.-Y.; Williams, H. R. J. Biol. Chem. 1979, 254, 11875-11883. For other studies mapping out subsite specficity for statine-containing inhibitors of cathepsin D, see: (a) Majer, P.; Collins, J. R.; Gulnik, S. V.; Erickson, J. W. Protein Sci. 1997, 6, 1458-1466.
91. 1′ residues, respectively, are reported as inhibitors of cathepsin D: Lin, T.-Y.; Williams, H. R. J. Biol. Chem. 1979, 254, 11875-11883. For other studies mapping out subsite specficity for statine-containing inhibitors of cathepsin D, see: (a) Majer, P.; Collins, J. R.; Gulnik, S. V.; Erickson, J. W. Protein Sci. 1997, 6, 1458-1466.
92. (b) Agarwal, N. S.; Rich, D. H. J. Med. Chem. 1986, 29, 2519-2524.
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95. (e) Rao, C. M.; Scarborough, P. E.; Kay, J.; Batley, B.; Rapundalo, S.; Klutchko, S.; Taylor, M. D.; Lunney, E. A.; Humblet, C. C.; Dunn, B. M. J. Med. Chem. 1993, 36, 2614-2620.
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97. These amines are fully protonated in the assay buffer, pH = 3.5, cathepsin D assay; pH = 5.0, plm II.
98. (a) Luker, K. E.; Francis, S. E.; Gluzman, I. Y.; Goldberg, D. E. Mol. Biochem. Parasitol. 1996, 79, 71-78. Plm II was a gift from Dr. Daniel E. Goldberg, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO, 63110.
99. (b) The plasmepsin II substrate is DABCYL-γ-aminobutyric acid-Glu-Arg-Met-Phe-Leu-Ser-Phe-Pro-EDANS, a custom synthesis from AnaSpec, Inc., 2149 O'Toole Avenue, Suite F, San Jose, CA 95131.
100. 2, was purchased from AnaSpec, Inc.