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(12) United States Patent (10) Patent No.: US 7,074,936 B2 Caprioli Et Al

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(12) United States Patent (10) Patent No.: US 7,074,936 B2 Caprioli Et Al US00707.4936 B2 (12) United States Patent (10) Patent No.: US 7,074,936 B2 Caprioli et al. (45) Date of Patent: Jul. 11, 2006 (54) CLEAVABLE SURFACTANTS AND (56) References Cited METHODS OF USE THEREOF U.S. PATENT DOCUMENTS (75) Inventors: Richard M. Caprioli, Brentwood, TN 4,713.486 A 12/1987 Buckle (US); Ned A. Porter, Franklin, TN 5,114,851 A 5/1992 Porter et al. (US); Jeremy L. Norris, Smyrna, TN 5,179,224. A 1/1993 Takaki et al. (US) 5,218,137 A 6/1993 Porter et al. 5,808,300 A 9/1998 Caprioli (73) Assignee: Vanderbilt University, Nashville, TN 6,306,249 B1 10/2001 Galante et al. US (US) FOREIGN PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this WO WOOO,70334 11 2000 patent is extended or adjusted under 35 WO WO O2/O97393 A2 12/2002 U.S.C. 154(b) by 299 days. OTHER PUBLICATIONS (21) Appl. No.: 10/479,477 Behforouz, et al.; Alkyl and Aryl Sulfenimides; J. Org. 1-1. Chem.: 34 (1): 51-55; 1969. (22) PCT Filed: May 28, 2002 Guyot, et al., Esterification of phenolic acids from green coffee with an immobilized lipase from Candida Antarctica (86). PCT No.: PCT/USO2A16640 in solvent-free medium: Biotechnology Letters; vol. 19. No. S 371 (c)(1), 6; Jun. 1997; pp. 529-532. (2), (4) Date: Dec. 1, 2003 Harpp., et al.: A New Synthesis of Unsymmetrical Disulifdes: s Tetrahedron Letters; 41; pp. 3551-3554; 1970. (87) PCT Pub. No.: WO02/0973.93 Kyte, et al.; J. Mol. Biol.: 1982: 157(1):105-32. Laemmli: Nature 227; 680-685; 1970. PCT Pub. Date: Dec. 5, 2002 Smith, et al.; March's Advanced Organic Chemistry: Reac tions, Mechanisms, and Structure; 5" Ed. pp. 1-1824, 2001. (65) Prior Publication Data Wuts, et al.; Protective Groups in Organic Synthesis; 1999; US 2004/0152913 A1 Aug. 5, 2004 3rd Ed. 1-779. Primary Examiner Paul A. Zucker Related U.S. Application Data (74) Attorney, Agent, or Firm Stites & Harbison PLLC; (60) Provisional application No. 60/294,337, filed on May Richard S. Myers, Jr. 29, 2001. (57) ABSTRACT (51) Int. Cl. C07D 2II/70 (2006.01) Cleavable compositions and methods of use especially in (52) U.S. Cl. ...................................................... 54.6/339 MALDI MS analysis of hydrophobic proteins. (58) Field of Classification Search ..................... None See application file for complete search history. 10 Claims, 7 Drawing Sheets U.S. Patent Jul. 11, 2006 Sheet 1 of 7 US 7,074,936 B2 0009!,0079),0080),00280099000€ (zyu)ssew |-61-I U.S. Patent Jul. 11, 2006 Sheet 2 of 7 US 7,074,936 B2 00000}, 00298 U.S. Patent Jul. 11, 2006 Sheet 3 of 7 US 7,074,936 B2 (zjuu)sseW 00:28 0099 U.S. Patent Jul. 11, 2006 Sheet 4 of 7 US 7,074,936 B2 09.1),009!OGZ|,000€000),091(zjuu) sse?N 7(61-) U.S. Patent Jul. 11, 2006 Sheet 5 of 7 US 7,074,936 B2 0000Z 0099}, 0029), G-61-I 0096 U.S. Patent Jul. 11, 2006 Sheet 6 of 7 US 7,074,936 B2 0000Z0079,0092),00260099000Z (zjuu)sse?N U.S. Patent Jul. 11, 2006 Sheet 7 of 7 US 7,074,936 B2 0080}, US 7,074,936 B2 1. 2 CLEAVABLE SURFACTANTS AND U.S. Pat. No. 5,114,851 to Porter et al., discloses certain METHODS OF USE THEREOF light activated acyl-enzymes. U.S. Pat. No. 5,218,187 to Porter et al., discloses certain This application is a 371 of PCT/US02/16640 filed May compounds useful as an intermediate for making light 28, 2002 which claims benefit of U.S. 60/294,337 filed May activatable acyl-enzymes. 29, 2001. Also see U.S. Pat. No. 5,808,300 to Caprioli, incorporated FIELD OF THE INVENTION herein by reference, for a discussion of MALDI MS. Additional background information may be found in the The present invention relates generally to cleavable deter 10 following publications: Kyte et al., J. Mol. Biol. (1982) gents or Surfactants and methods of use thereof including 157(1):105-32; March's Advanced Organic Chemistry sample isolation, Solubilization, emulsification, and analy Reactions, Mechanisms, and Structure, 5" Ed. by Michael sis. Furthermore, the present invention relates to cleavable B. Smith and Jerry March, John Wiley & Sons, Publishers: Surfactants which are useful for sample preparation, but Wuts et al. (1999) Protective Groups in Organic Synthesis, which can be cleaved for removal or to yield cleavage 15 products which have additional useful properties, including 3' Ed., John Wiley & Sons, Publishers; Behforouz, M.: for matrix assisted laser desorption ionization mass spec Kerwood, J. E. Alkyl and Aryl Sulfenimides. J. Org. Chem. troscopy (MALDI MS) analysis of hydrophobic molecules 34 (1), 51–55 (1969); and Harpp., D. N.; Ash, D. K. Back, including natural and synthetic polymers and polypeptides. T. G.; Gleason, J. G.; Orwig, B. A.; VanHorn, W. F. A New The cleavable detergents or surfactants of the present Synthesis of Unsymmetrical Disulifdes. Tetrahedron Letters, invention, among other things in additional embodiments, 41, 3551-3554 (1970). improve the quality of MALDI MS analyses of proteins, including high molecular weight proteins associated with SUMMARY OF THE INVENTION biological tissue. 25 This invention relates to the treatment of a sample, Such BACKGROUND OF THE INVENTION as a tissue section from a plant or animal, with a compound or mixture of compounds that would perform multi-func Proteomics, the study of proteins and their functions, is tional roles in the preparation of these samples for analysis, currently a focus of both university and commercial invest e.g., mass spectrometry or chromatography, at designed ment as each discovery in proteomics holds the potential to 30 times determined by treatment conditions. These com unlock yet another advance in medical Science. The extreme pounds would be able to function as a surfactant or deter variability in the chemistry of proteins in biological systems gents in helping solubilize hydrophobic or other non-soluble and especially in mammals, presents special problems. compounds. Due to built in cleavable bonds, appropriate A recurring problem with respect to proteomics involves treatment of the sample, for example, with acid, base, heat, the poor Solubility of a large percentage of proteins such as 35 light, etc., would then cause decomposition of the agent to those found in lipid membranes and other hydrophobic areas two or more Smaller parts, each of which does not materially of the cell or in the cellular environment. This is because interfere with the analysis. Further, each part may in itself many of the systems developed for the study of proteins are perform a further function; for example, one part may help geared to analysis in an aqueous environment. To isolate solubilize compounds present in the mixture and the other, hydrophobic proteins or hydrophobic protein domains, Sur 40 for MALDI mass spectrometry, may form crystals in the factants (detergents, such as sodium dodecyl sulfate (SDS) same way matrix acts in common MALDI analysis. or triton X) are commonly employed. Surfactants generally Accordingly, the present invention provides, in part, com have a polar head group and a hydrophobic tail group and positions and methods including, but not limited to: novel encapsulate hydrophobic proteins wherein the hydrophobic cleavable Surfactants and methods for preparing cleavable tail is in contact with the hydrophobic protein and the polar 45 Surfactants and using them in proteomic analysis including groups are in contact with the water. Thus, hydrophobic for matrix assisted laser desorption ionization mass spec proteins and polypeptides are sequestered in a coating of trometry (MALDI MS). Certain compositions disclosed detergent wherein the complex is soluble in an aqueous herein include the Surprising properties of being a Surfactant environment. that yields one or more analyte assisting molecules upon However, many analytical systems are sensitive to the 50 cleavage including a MALDI matrix composition and a presence of surfactants. For example, SDS and triton X volatile solvent. No aspect or embodiment of the present Suppress the analyte signal during matrix assisted laser invention including any claim is bound by theory or mecha desorption ionization mass spectrometry (MALDI MS) 1S. analysis. Signal Suppression from Surfactant contamination In embodiments of the present invention, compounds of is contemplated to result from physical and chemical block 55 the present invention may be constructed or synthesized in age of the ionization/desorption process of MALDI MS. two parts, connected by a linking group that can be cleaved What is needed are surfactant compositions and methods by the addition of another chemical agent or energy source. suitable for MALDI-MS analyses, and other analyses, of The portion of the compound that would act as a matrix after hydrophobic molecules including natural and synthetic cleavage would be polar in nature and, in certain embodi polymers and polypeptides/proteins. 60 ments, be a cinnamic acid analog or similar compound. The International Publication WO 00/70334 to Lee et al., second part of the molecule may be for example, a hydro discloses certain Surfactants and results for electrospray phobic molecule Such as hexane or octane alkyl group with mass spectroscopy (MS) analysis of myoglobin in the pres a functional group Such as thiol or alcohol. After cleavage, ence of certain of the Surfactants. this compound may act as a solvent, allowing solubilization U.S. Pat. No. 4,713,486 to Buckle discloses certain 65 of other compounds present including the other part of the arachidonic acid analogues, including certain cinnamates, agent. The linkage between the parts may comprise a bold stated to be useful in the treatment of allergic diseases.
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