The process of developing new drug candidates has changed significantly over time, as a result of the Human Genome Project and other technological advances in computational modeling and bioformatics.1 For example, high-throughput screening methods provide unparalleled capacity to screen millions of chemical structures for potential drug efficacy,2,3 as opposed to simply developing a target candidate and anticipating relevant biochemical action. Regardless of the desired approach towards production of novel drug candidate molecules by either reverse pharmacology, the classical approach, or natural product discovery,4,5,6 most drugs take several years to develop and millions of dollars to become marketable as a requirement of validation through in-depth clinical trials, evaluation of safety risks and FDA approval.7 As part of this development process, the analytical need to study the structural characteristics of these small molecules is imperative.
Structural characterization of potential drug candidates, either derived from natural sources or synthesized in the laboratory, is a complex and time consuming process, and for rapid analyses, pharmaceutical companies value the high degree of analytical selectivity and sensitivity afforded by modern mass spectrometry (MS) methods towards overall quality control of synthesized products and characterization of new drug targets.8,9 Although mass spectrometers are highly selective, often able to assign a molecular formula for a target analyte based solely on molecular mass measurement, isomeric species are difficult to differentiate by traditional MS methods, even
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with the addition of tandem MS/MS approaches.10,11 Because the biological function of chemical compounds can change with their structural variation, isomeric species are a highly researched area of the pharmaceutical field.12 Condensed phase separation techniques such as gas or liquid chromatography are often utilized to separate complex mixtures prior to mass analysis and provide the ability to separate isomers by differences in chemical properties, such as polarity or boiling point. These methods, while effective, are often highly selective to narrow classes of isomers and are not inherently high-throughput techniques. In this chapter, we describe the application of IM- MS, an emerging analytical technique for structurally characterizing small molecule isomer systems, with a particular focus on the role of IM-MS in characterizing biological systems and relevant pharmaceutical applications.
2.1.1. Isomers
Isomers are defined as compounds having the same molecular formula, but differing in their overall chemical structure.13 Isomeric species are further sub-divided into categories that reflect their structural variations, which may include covalent bond rearrangements (constitutional isomers), stereochemical variations (stereoisomers), or rotational isomers, commonly referred to as rotamers. As constitutional isomers vary in skeletal structure between constituent atoms, these isomers possess a broad scope of biological activity based upon their particular structural arrangements. For example, the molecular formula C8H9NO2 is reported to have 33 isomers by the PubChem database,14 and many of these isomers have unique chemical behavior and physiological function (Figure 2.1). In one case, paracetamol (more commonly known as acetaminophen) is a well-known analgesic, yet its constitutional isomer, methyl anthranilate functions as a bird repellent and a flavor additive in drinks.15 The structural makeup of constitutional isomers can also
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Figure 2.1. Structures related to four constitutional isomers of chemical formula C8H9NO2 and corresponding function or typical use.
Acetaminophen (analgesic)
3-Methyoxybenzamide (polymerase inhibitor)
Methyl Anthranilate (flavoring agent)
4-Ethylnitrobenzene (chemical sensor)
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vary widely depending on their biological class. For example, lipid isomers typically vary in alkyl chain position and cis/trans double bond positioning,16 while peptides tend to have sequence order variation or even amino acid substitutions comprised of the same chemical formula (e.g.
leucine/isoleucine).17
In addition to constitutional isomers, compounds with the same molecular formula can differ in stereochemistry, (i.e. diastereomers and enantiomers) resulting in varying chemical and physical properties. For example, ethambutol is a 204 Da molecule (C10H24N2O2) possessing two stereocenters. In the (+) form (S,S) ethambutol is frequently used to treat tuberculosis.18 However, with inversion of chirality at its two stereocenters to form (-) or (R,R) ethambutol, the molecule is known to cause blindness.19 Isomers also exist for two compounds possessing the same chemical scaffold and chirality. For example, rotamers are small molecule conformers where multiple three- dimensional molecular structures can arise as a result of rotation around a single bond. In some cases this bond rotation gives rise to atropisomerism, which is the restriction of rotation around a single covalent bond which results in distinct optical isomers. A commonly cited rotamer example which exhibits freedom of rotation around a single bond are the Newman projections of butane.20 In other cases where rotation is not restricted, different stable conformations are still possible, especially in protein analysis. Small molecules in particular are noted for producing a variety of conformations as a result of their flexibility.21 Because of the diverse chemical activity that can exist within constitutional and conformational isomers, finding useful and efficient ways to explore the structure of these molecules can provide insight into their specific chemical properties.
For the past 15 years IM-MS has made large contributions in the analysis of constitutional and conformational isomers.22,23,52 As result of the commercialization and rapid adoption of IM- MS instrumentation,24,33,37 the Web of Science database cites over 3,500 articles in the last decade
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related to IM-MS studies.25 While ion mobility has been traditionally utilized to study large biological systems, more recently it has been applied to the study of smaller (< 400 Daltons) drug and drug-like molecules.26,27 In this chapter, we describe the technique and theory of IM-MS, provide examples of the use of IM-MS to characterize various small drug and drug-like molecules, and provide some basic methodology towards collecting and analyzing IM-MS data using a commercially available IM-MS platform (Agilent 6560) as an example.37