1348_C69.fm Page 1 Monday, October 13, 2003 3:22 PM 69 Photoremovable Protecting Groups 69.1 Introduction ..................................................................... 69-1 69.2 Historical Review.............................................................. 69-2 o-Nitrobenzyl • Benzoin • Phenacyl • Coumaryl and Arylmethyl 69.3 Carboxylic Acids............................................................. 69-17 o-Nitrobenzyl • Coumaryl • Phenacyl • Benzoin • Other Richard S. Givens 69.4 Phosphates and Phosphites ........................................... 69-23 o-Nitrobenzyl • Coumaryl • Phenacyl • Benzoin University of Kansas 69.5 Sulfates and Other Acids................................................ 69-26 Peter G. Conrad, II 69.6 Alcohols, Thiols, and N-Oxides .................................... 69-27 University of Kansas o-Nitrobenzyl • Thiopixyl and Coumaryl • Benzoin • Other Abraham L. Yousef 69.7 Phenols and Other Weak Acids..................................... 69-36 o-Nitrobenzyl • Benzoin University of Kansas 69.8 Amines ............................................................................ 69-37 Jong-Ill Lee o-Nitrobenzyl • Benzoin Derivatives • Arylsulfonamides University of Kansas 69.9 Conclusion...................................................................... 69-40 69.1 Introduction Photoremovable protecting groups are enjoying a resurgence of interest since their introduction by Kaplan1a and Engels1b in the late 1970s. A review of published work since 19932 is timely and will provide information about several new groups that have been recently developed. The scope of this review is, therefore, limited to recent developments in the field and will cover only the applications with major functional groups that have been “protected” by a photoremovable chromophore. The review is not intended to be comprehensive but focuses instead on a series of well-chosen examples of chromophores that were deployed as protecting groups with a select group of representative functional groups. Because the focus of this review is the application of photoremovable protecting groups, emphasis is placed on synthesis of the protected functionality and on the procedures employed for deprotection, including the protection and photodeprotection yields, the deprotection reaction rates, and the quantum efficiencies, when available. An attempt has been made to list the advantages and disadvantages of each photoremov- able protecting group as well as a brief discussion of the mechanism for the photodeprotection. 0-8493-1348-1/04/$0.00+$1.50 © 2004 by CRC Press LLC 69-1 1348_C69.fm Page 2 Monday, October 13, 2003 3:22 PM 69-2 CRC Handbook of Organic Photochemistry and Photobiology, 2nd Edition When the literature is insufficient for providing a comprehensive treatment of applications of a photoprotecting group, then only a brief discussion is provided. An exhaustive list of applications for any of the chromophores is not included; these may be found by consulting other reviews or the original literature on a topic. Several good reviews on photoremovable protecting groups have appeared since this topic was reviewed in 1993 (e.g., Adams and Tsien3 and Corrie and Trentham4). Notable among the more recent reviews are those by Wirz,5 Bochet,6 and Givens.7 A volume of Methods in Enzymology devoted entirely to the chemistry and applications of photoremovable protecting groups, also termed “caged” compounds, that are employed in biochemistry and other biological studies has also appeared. In general, photolysis reactions present a noteworthy and often ideal alternative to all other methods for introducing reagents or substrates into reactions or biological media. The ability to control the spatial, temporal, and concentration variables by using light to photochemically release a substrate provides the researcher with the ability to design more precisely the experimental applications in synthesis, physiology, and molecular biology. Among the many possible examples is the recently reported inhibition–reactiva- tion of protein kinase A by photolysis of the dormant enzyme.8–10 In this demonstration, it is necessary that the deprotection process be initiated by photolysis of the dominant chromophore of the protecting group. Covalent blocking of the functional groups at the active site of an enzyme essentially suspends its mode of action and virtually shuts down the catalytic cycle. It is this feature that has attracted biochemists to the use of protecting groups for the investigation of biological mechanisms. In synthesis, the protecting group serves as a mask that renders a functional group inert to subsequent synthetic reaction conditions,11 except, of course, conditions that are required for the removal of the protecting group. Construction of combinatorial platforms with photoremovable linkers is just one example of the applications in synthesis. Photorelease is sometimes termed a traceless reagent process because no reagents other than light are needed. The advantage of a process that requires no further separation of spent reagents is attractive. There are several limitations to the use of commonly employed protecting groups in synthesis and for mechanistic studies of biological processes. The reactions for incorporating and subsequently removing protecting groups often involve acid or base that may be too harsh and interfere with the normal processes or otherwise be incompatible with the chemistry or biology under investigation. In mechanistic bio- chemistry, it is often the case that the typical hydrolysis deprotection reaction is far too slow to serve as a means of investigating the initial rates of reaction for rapid biochemical processes. An ideal remedy to these limitations is a protecting group that could be removed under neutral buffered aqueous conditions, thus avoiding any alterations to the substrate or to the natural biological environ- ment.12 The release should occur on a time scale fast enough for kinetic analysis of any subsequent rapid biological processes. Such a group may be a photoremovable protecting group. 69.2 Historical Review In 1962, Barltrop et al.13 were among the first to report a photochemical deprotection reaction of a biologically significant substrate; here, glycine was released from N-benzyloxycarbonyl glycine: O ν CH3 OH h OH O N + H2N + CO2 H O O (69.1) This seminal discovery prompted the development of several additional photoremovable protecting groups. The success of many researchers in biology, particularly Kaplan,1a led to the description of the photoactivatable group as a “cage” to describe its deactivating influence on the biological substrate to which it is covalently attached.14–17 Ideally, the cage detaches only through the action of light. 1348_C69.fm Page 3 Monday, October 13, 2003 3:22 PM Photoremovable Protecting Groups 69-3 It is important that the photoremovable protecting group also possess several other desirable proper- ties. The properties were originally compiled by several researchers in the field, including Sheehan and Umezawa12 and Lester and Nerbonne,18 who provide a series of benchmarks for evaluating the efficacy of a photoremovable group in a given circumstance or for evaluating the potential of a new cage chromophore. A more useful adaptation of the Lester rules and Sheehan criteria includes the following: 1. The substrate, caged substrate, and photoproducts have good aqueous solubility for biological studies. For synthetic applications, this requirement is relaxed. 2. The photochemical release must be efficient (e.g., Φ > 0.10). 3. The departure of the substrate from the protecting group should be a primary photochemical process (i.e., occurring directly from the excited state of the cage chromophore). 4. All photoproducts should be stable to the photolysis environment. 5. Excitation wavelengths should be longer than 300 nm and must not be absorbed by the media, photoproducts, or substrate. 6. The chromophore should have a reasonable absorptivity (a) to capture the incident light efficiently. 7. The caged compounds, as well as the photoproduct from the cage portion, should be inert or at least benign with respect to the media, other reagents, and products. 8. A general, high-yielding synthetic procedure for attachment of the cage to the substrate must be available. 9. In the synthesis of a caged substrate, the separation of caged and uncaged derivatives must be quantitative. This is also necessary for the deprotection process for synthetic applications. While these are the desirable guidelines for an ideal photoremovable protecting group, a potential cage that lacks one or two of these properties may still be very useful; however, the absence of several of these features may militate against the use of that group as a photoremovable protecting group for a specific application. Some representative examples of photoremovable protecting groups that qualify as meeting the Lester and Sheehan criteria include α-substituted acetophenones, benzoins, benzyl groups, cinnamate esters, coumaryl groups, and, the most popular of them all, the o-nitrobenzyl esters and their analogs. o-Nitrobenzyl It was also Barltrop et al.19 who first reported the use of an o-nitrobenzyl group to release benzoic acid (see Eq. (69.2)). The poor yield stemmed from the subsequent conversion of 2-nitrosobenzaldehyde (3), the initial photoproduct, into azobenzene-2,2′-dicarboxylic acid (4),20 which then competed for the incident light. Yields were dramatically improved with the use of α-substituted nitrobenzyl