Chemistry: analytical and immunological testing – Nuclear magnetic resonance – electron spin resonance or other...
Reexamination Certificate
2001-08-06
2004-05-11
Gakh, Yelena G. (Department: 1743)
Chemistry: analytical and immunological testing
Nuclear magnetic resonance, electron spin resonance or other...
C436S094000, C436S095000
Reexamination Certificate
active
06734024
ABSTRACT:
BACKGROUND OF THE INVENTION
(1) Field of the Invention
This invention is directed generally to improvements to the analysis of chemical compounds using mass spectroscopy methods. More specifically, the invention relates to ferrocenyl boronate derivatization of chemical compounds to be analyzed and to their subsequent analysis using electrospray tandem mass spectrometry methods.
(2) Description of the Related Art
Mass spectroscopy is a well-known tool used for analyzing chemical compounds. Tandem mass spectroscopy, using electrospray ionization, has been used with increasing frequency for the analysis of biological samples. Persons skilled in analytical chemistry methods are familiar with the operation of electrospray tandem mass spectrometry instruments.
Currently a need exists for improved methodologies enabling one to chemically or mechanically manipulate large numbers of compounds in order to extract subtle differences in structure. A number of complex carbohydrates and esters are biologically significant molecules in that they contain a high degree of information per molecular unit and constitute a prevalent form of protein post-translational modification. (R. A. Dwek
Biochemical Society Transactions
1995; 23: 1.) It is desirable not only to obtain amino acid sequences of a number of glycoproteins, but also to investigate the substance and microheterogeneity of certain portions of these molecules. (M. E. R. O'Brien, B. E. Souberbielle, M. E. Cowan, C. A. Allen, D. M. Luesley, J. J. Mould, G. R. P. Blackledge, G. R. B. Skinner
Cancer Letters
1991; 58: 247.) Informational properties of chemical moieties are dependent on their high degree of conformational and isomeric diversity resulting from subtle changes in unit assembly. (R. A. Laine,
Glycobiology
1994; 4: 749.) Changes in compound architecture often reflect major biological implications, including disease. (S. I. Hakormori
Cancer Research
1985; 45: 2405; A. Eiras-Segal, M. V. Croce,
Allegol. Et. Immunopathol.
1998; 25: 176; I. Brockhausen,
Biochemical Society Transactions
1997; 25: 871; C. M. Martersteck, N. L. Kedersha, D. Drapp, T. Tsui, K. J. Colley,
Glycobiology
1996; 6: 289; D. Naor, R. V. Sionov, D. Ish-Shalom,
Cancer Research
1997; 71; 241; K. O. Lloyd,
Cancer Biology
1991; 2: 421; S. Leppa, J. Heino; M. Jalkanen,
Cell Growth and Differentiation
1995; 6: 853.) As a result, the number of potential biologically significant structures is significant. (B. Fernandes, U. Sagman, M. Auger, M. Demnetrio, J. W. Dennis,
Cancer Research
1991; 51: 718.)
Structural elucidation of carbohydrates has been accomplished using a combination of techniques, usually nuclear magnetic resonance (NMR) and mass spectrometry (MS). These techniques have proven labor intensive. (J. F. Kennedy, G. Pagliuca,
Oligosaccharides
: Second Edition ed.; J. F. Kennedy, G. Pagliuca, Ed.; IRL Press at Oxford University Press: Oxford, New York, Tokyo,
1994
, pp. 43-68).
Tandem MS, particularly clectrospray quadruple ion-trap MS (ES-MS) is well suited for examining complex mixtures, particularly when coupled with a front-end separation system such as liquid chromatography (LC-MS). (N. Kawasaki,; M. Ohta; S. Hyuga; O. Hashimoto; T. Hayakawa.
Analytical Biochemistry
1999, 269, 297-303; M. Kohler; J. Leary.
Analytical Chemistry
1995, 67, 3501-3508; H. Kwon, J. Kim.
Journal of Liquid Chromatography
1995, 18, 1437-1449). ES-MS offers the possibility of working efficiently and with lower amounts of sample without the possibility of thermal losses, which can often be a requirement for the study of biological structure and function relationships. (N. H. Packer, M. J. Harrison,
Electrophoresis
1998; 19: 1872). Great potential exists for ES-MS methods eliciting fine structural and relevant details in larger chemical units. (N. Viseux, E. de Hoffmann, B. Domon,
Anal. Chem.
1998; 70: 4951.)
Peptide sequencing by MS methods has rapidly developed into a mature science. (D. F. Hunt, J. E. Alexander, L. Ashley, P. A. McCormack, H. M. Martino, J. Shabanowitz, N. Sherman, M. A. Mosely, J. W. Jorgenson, K. B. Tomer,
Mass Spectromebric Methods for Protein and Peptide Sequence Analysis
; Academic Pres, Inc.: San Diego, Calif., 1991.) On the other hand, because of the large differences in fragmentation energies found in carbohydrates, the application of these types of methods in the analysis of carbohydrates has been limited. Also, due to the poor ionization in ES-MS of information-rich sugars released from their parent molecule by application of enzymatic deglycosylation, this severely affects sensitivity and consequently hinders detection.
Many analytical methods for analysis of compounds involve some form of derivatization to enhance sensitivity or fragment information in the mass spectrometer. The analysis of certain compounds has relied on derivatization chemistry to enhance the sensitivity of these important biological compounds as applied to their specific techniques. (R. A. Dwek,
Biochemical Society Transactions
1995, 23 1-25; C. M. Starr, R. I. Masada, C. Hague, E. Skop, J. C. Klock,
J. Chromatogr A
1996, 720, 295-321; M. F. Chaplin, Monosaccharides; Second Edition; M. R. Chaplin, Ed. IRL Press at Oxford University Press: Oxford, New York, Tokyo, 1994, pp 1-40.) Chemical derivatization has been employed to overcome the ionization quandry. (D. J. Harvey,
J. Am. Soc. Mass Spectrom.
2000, 11, 900-915; J. J. Pitt, J. Gorman,
Analytical Biochemistry
1997, 248, 63-75; D. Williams, T. D. Lee, N. Dinh, M. K. Young,
Rapid Commun. Mass Spectrom
2000, 14, 1530-1537; S. Susuki, K. Kakehi, S. Honda,
Anal Chem
1996, 68, 2073-2083.)
Almost all methods of MS analysis of compounds rely heavily on some form of derivatization not only to increase sensitivity, but also to augment volatility and the proclivity to form useful fragment ions. Derivatization must not only increase the propensity of the analyte to ionize, but also provide the desired quality of fragment information during successive rounds of ES-MS. Desirable fragments should provide both selective loss of chemical units and, within these fragments, cross pyranose ring fissions should reflect subtle stereochemical differences between individual molecular units. (H. Desaire, J. A. Leary,
Anal. Chem.
1999, 71, 1997-2002; Z. Zhou, S. Ogden, J. A. Leary
J. Org. Chem.
1990, 55, 5444-5446; S. P. Gaucher, J. A. Leary
Anatylical Chemistry
1998, 70, 3009-3014.) Cross pyranose ring fragmentation releases neutral fragments. The parent ion then characterizes the linkage position between molecular units. As a result, profiling, sequence identity and linkage position information may be explored. Normally, the larger molecules or compounds, particularly those that contain nitrogen in the form of N-acetylated aminohexose residues, can be electrosprayed with or without permethylation. However, the MS
2
fragmentation information is often not particularly informative due to simple water loss. This technique works even less reliably with smaller molecules found during enzymatic digestion protocols. As the molecule becomes smaller and contains fewer chargeable atoms (usually nitrogen) and a diminished propensity for cationization, the efficiency of ES-MS analysis declines. Considerable attention has been paid to various methods of mono and oligosaccharide complexation with a variety of metal ions. (H. Desaire,; J. A. Leary,
Anal. Chem.
1999; 71: 1997; S. Konig, J. A. Leary,
Journal of the American Society for Mass Spectrometry
1998; 9: 1125; G. Smith, J. A. Leary
Journal of the American Society for Mass Spectrometry
1996; 7: 953; Z. Zhou, S. Ogden, J. A. Leary,
J. Org. Chem.
1990; 44: 5444; M. Kohler, J. A. Leary,
Analytical Chemistry
1995; 67: 3501; G. F. Hofineister, Z. Zhou, J. A. Leary,
J. Am. Chem. Soc.
1991; 113: 5964.)
Furthermore, in certain cancer studies, the catechol estrogens 2-hydroxyestradiol (2-OHE) and 4-hydroxyestradiol (4-OHE) are implicated as so-called good and bad estrogens in the biogenesis of malignant cells (S. H. Safe,
Interactions Between Hormones an
Williams John Dudley
Young Mary K.
City of Hope
Gakh Yelena G.
Rothwell Figg Ernst & Manbeck
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