US00879 1251B2 (12) United States Patent (10) Patent No.: US 8,791,251 B2 POWell et al. (45) Date of Patent: Jul. 29, 2014 (54) NON-IONIC ACID-LABILE SURFACTANTS F.L. Van Delft et al., A Sulfitylation-Oxidation Protocol for the Prepa AND METHODS OF USE ration of Sulfates. J. Org. Chem., 2006, 71, pp. 7473-7476. T. Takeda et al., Synthesis and Properties of Soap Types of Double (75) Inventors: Matthew Jacob Powell, Westover, WV Chain Cleavable Surfactants Derived from Pyruvate. J. Oleo Sci., (US); Trust T. RaZunguzwa, 2004, 53 (2), pp. 89-95. Morgantown, WV (US); Miaosheng Li, M. Fung et al., Hybrid Surfactants Containing Separate Hydrocarbon Morgantown, WV (US) and Fluorocarbon Chains. J. Phys. Chem., 1992, 96, pp. 6738-6742. M. Iyer et a. Synthesis of pH-Degradable Nonionic Surfactants and (73) Assignee: Protea Biosciences, Inc., Morgantown, Their Applications in Microemulsions. Langmuir 2001, 17. (22), pp. WV (US) 6816-6821. D.A. Jaeger et al., Preparation and Characterization of Glycerol (*) Notice: Subject to any disclaimer, the term of this Based Cleavable Surfactants and Derived Vesicles. J. Am. Chem. patent is extended or adjusted under 35 Soc. 1989, 111, (8), pp. 3001-3006. U.S.C. 154(b) by 112 days. D.A. Jaeger et al., Acid-Catalyzed Hydrolysis and Monolayer Prop erties of Ketal-Based Cleavable Surfactants. Langmuir 1990, 6 (3), (21) Appl. No.: 13/486,231 pp. 547-554. D.A. Jaeger et al., Cleavable Quaternary Hydrazinium Surfactants. (22) Filed: Jun. 1, 2012 Langmuir 1998, 14. (7), pp. 1940-1941. B. Guyot, et al., Esterification of phenolic acids from green coffee (65) Prior Publication Data with an immobilized lipase from Candida antarctica in solvent-free medium. Biotechnology Letters. Jun. 1997, vol. 19, (6), pp. 529-532. US 2012/0309954 A1 Dec. 6, 2012 R.M. Caprioli et al., Mass Spectrometry of Intracellular and Mem Related U.S. Application Data brane Proteins Using Cleavable Detergents. Anal. Chem. 2003, 75. pp. 6642-6647. (60) Provisional application No. 61/493,052, filed on Jun. Eckert and Liotta, “Designing Smart Surfactants' printed from www. 3, 2011. chbe.gatech.edu/eckert/pdf. Surfactant.pdf. Internet site accessed on Dec. 22, 2009, 8 pages. (51) Int. Cl. “Remove detergent from protein samples,” printed from www. C7H 3/12 (2006.01) thermo.com/eThermo/CMA/PDFs/.../articlesFile 6580.pdf, (52) U.S. Cl. Internet site accessed on Dec. 22, 2009, 2 pages. USPC ....................................................... 536/29.12 “Care and Use Manual” printed from http://www.waters.com/waters/ Support.htm? locale=en NG&lid=10008.439&cid= (58) Field of Classification Search 511442*type=USRM, Internet site accessed on Dec. 22, 2009, 4 CPC ....................................................... CO7H 13/12 pageS. USPC ....................................................... 536/29.12 ProteaseMAX Surfactant, Trypsin Enhancer, printed from www. See application file for complete search history. promega.com/tbs/lb373/tb373.pdf. Internet site accessed on Dec. 22. 2009, 20 pages. (56) References Cited PPS Silent Surfactant, Acid Cleavable Detergent, Use and Storage Instructions printed from www.proteindiscovery.com/downloads/ U.S. PATENT DOCUMENTS PPS PI UseAndStorage.pdf. Internet site accessed on Dec. 22. 2009, 1 page. 3,023,182 A 2f1962 Tanabe et al. 3,647.464 A 3, 1972 Smith et al. 4,301,044 A 11, 1981 Wentler et al. Primary Examiner — Traviss C McIntosh, III 5,093,043 A 3, 1992 Jakobson et al. (74) Attorney, Agent, or Firm — Fitch, Even, Tabin & 5,264,460 A 11/1993 Jakobson et al. Flannery LLP 6,429,200 B1 8/2002 Monahan et al. 7,022,861 B1 4/2006 McElhanon et al. (57) ABSTRACT 7,074,936 B2 7/2006 Caprioli et al. 7,091,041 B2 8/2006 Monahan et al. A compound may generally comprise the formula: 7,229,539 B1 6, 2007 Lee et al. 7,351,837 B1 4/2008 McElhanon et al. 8,013, 179 B2 9, 2011 Powell et al. RO OR 8,232,423 B2 7, 2012 Powell et al. H 8,435,756 B2 5, 2013 Powell et al. N X 8.445,223 B2 5, 2013 Powell et al. 2004/O152913 A1 8/2004 Caprioli et al. 's-X.O 2006/024.0562 A1 10/2006 Caprioli et al. 2006/0270.584 A1 11/2006 Frantz et al. 2009,0292136 A1 11/2009 Powell et al. 2011/0282096 A1 11/2011 Powell et al. wherein R is independently selected from C-C alkyl or Substituted alkyl, R is independently selected from the group FOREIGN PATENT DOCUMENTS consisting of —H and C-C alkyl or Substituted alkyl, X is selected from the group consisting of NH- and —O—, Y WO O2/O97393 A2 12/2002 is a carbohydrate, and m is an integer from 1 to 8. The WO O3/106379 A1 12/2003 compound may comprise a non-ionic acid labile Surfactant. OTHER PUBLICATIONS The compound may be used to facilitate solubilization of proteins and other molecules in an aqueous environment. Haines et al., Journal of Organic Chemistry, 1982, 47(3), pp. 474 482.* 20 Claims, 6 Drawing Sheets US 8,791.251 B2 Page 2 (56) References Cited McMillen et al., “Identifying Regions of Membrane Proteins in Con tact with Phospholipid Head Groups: Covalent Attachment of a New Class of Aldehyde Lipid Labels to Cytochrome c Oxidase.” Bio OTHER PUBLICATIONS chemistry, 1986, (25) pp. 182-193. Rouhana et al., "Aggregation-Resistant Water-Soluble Gold FAQs about PPS Silent Surfactant, printed from http://www. Nanoparticles.” Langmuir, 2007 (23) pp. 12799-12801. proteindiscovery.com/pages/support/facq pps.html. Internet site Regen, Steven L.; Singh, Maninder; Samuel, N. K. P. Functionalized accessed on Dec. 22, 2009, 2 pages. liposomes. Efficient of alpha chymotrypsin, Biochemical and Bio RapiGest SF, printed from www.orbital.com.cn/up? physical Research Communications, (1984), 119(2), pp. 646-651. Waters%20Science%20Solutions.pdf. Internet site accessed on Dec. 22, 2009, 1 page. * cited by examiner U.S. Patent US 8,791.251 B2 U.S. Patent Jul. 29, 2014 Sheet 2 of 6 US 8,791.251 B2 Snoo Sunos AESue U.S. Patent Jul. 29, 2014 Sheet 4 of 6 US 8,791.251 B2 þý00372900992008?7},000),0.000$002 (zyu)ssew Asueu% U.S. Patent Jul. 29, 2014 Sheet 5 of 6 US 8,791.251 B2 US 8,791,251 B2 1. 2 NON-ONCACID-LABLE SURFACTANTS understood by considering the following description in con AND METHODS OF USE junction with the accompanying drawings. FIGS. 1A-C illustrate representative mass spectra of myo CROSS-REFERENCE TO RELATED globin according to certain embodiments. APPLICATION FIGS. 2A-C illustrate representative mass spectra of bovine serum albumin (BSA) according to certain embodi This application claims the benefit of U.S. Provisional mentS. Application No. 61/493,052, filed on Jun. 3, 2011, which is FIGS. 3A-C illustrate representative mass spectra of BSA incorporated herein by reference in its entirety. according to certain embodiments. 10 FIGS. 4A-C illustrate representative mass spectra of myo BACKGROUND globin according to certain embodiments. FIGS. 5A-C illustrate representative mass spectra of BSA The compounds and methods described herein generally according to certain embodiments. relate to non-ionic acid-labile Surfactants and methods of use. 15 FIG. 6 includes a chart listing certain properties of certain Proteomics is the study of the structure and function of embodiments. proteins and other molecules in biological systems. Some purification and identification techniques used in proteomics DESCRIPTION OF CERTAIN EMBODIMENTS require the proteins and other molecules to be solubilized in an aqueous environment. Most proteins and other hydropho A. Definitions bic molecules or molecules with significant hydrophobic regions, however, are not readily soluble in an aqueous envi As generally used herein, the term "comprising refers to ronment. A Surfactant or detergent may generally be used to various components conjointly employed in the manufacture facilitate the solubilization of proteins and other molecules in and use of the compounds and methods described herein. an aqueous environment. 25 Accordingly, the terms "consisting essentially of and “con Cleavable surfactants have been successfully used to facili sisting of are embodied in the term “comprising. tate the solubilization of proteins and other molecules in an As generally used herein, the grammatical articles includ aqueous environment. Cleavable Surfactants may generally ing “one”, “a”, “an', and “the refer to “at least one' or “one comprise a polar (hydrophilic) group joined by a cleavable or more' of what is claimed or described, unless otherwise linker to a non-polar (hydrophobic) group. Cleavable Surfac 30 indicated. Thus, the articles are used herein to refer to one or tants may be cleaved or degraded by utilizing acidic condi more than one (i.e., to at least one) of the grammatical objects tions, basic conditions, photodegradation, thermal degrada of the article. By way of example, "a component’ means one tion, or chemical reduction. The cleavage by-products may be or more components, and thus, possibly, more than one com separated from the proteins or other molecules using standard ponent is contemplated and may be employed or used. isolation techniques. Conventional non-ionic cleavable Sur 35 As generally used herein, the terms “include”, “includes’ factants, however, may generally comprise chemical struc and “including are meant to be non-limiting. tures that are complex to synthesize, require harsh conditions As generally used herein, the terms “have”, “has and (e.g., pH 1-2) or long periods of time (up to several hours) to "having are meant to be non-limiting. cleave, and/or generate cleavage by-products that interfere All numerical quantities or characteristics stated herein are with purification and identification techniques. 40 approximate unless otherwise indicated, meaning that all Accordingly, more efficient non-ionic cleavable Surfac numerical quantities are to be understood as being prefaced tants and methods of use are desirable.