Illuminating the chemistry of life: design, synthesis, and applications of "caged" and related photoresponsive compounds

Abstract

Biological systems are characterized by a level of spatial and temporal organization that often lies beyond the grasp of present day methods. Light-modulated bioreagents, including analogs of low molecular weight compounds, peptides, proteins, and nucleic acids, represent a compelling strategy to probe, perturb, or sample biological phenomena with the requisite control to address many of these organizational complexities. Although this technology has created considerable excitement in the chemical community, its application to biological questions has been relatively limited. We describe the challenges associated with the design, synthesis, and use of light-responsive bioreagents; the scope and limitations associated with the instrumentation required for their application; and recent chemical and biological advances in this field.

Figure 1

Caged ATP (1) and cAMP Analogs (3 and 5). These derivatives illustrate the classical caging strategy via covalent modification of an essential functional group required for biological activity. Photolysis converts the caged form of ATP (1) to its active counterpart (2) and the caged form of cAMP (3) to its active counterpart (4). However, species such as cAMP can be rapidly “deactivated” via enzyme-catalyzed hydrolysis. By contrast, the brominated cAMP analog 5 is bioorthogonal to the endogenous biochemistry of the cell. Upon photolysis, photoreleased bromo-cAMP analog displays long-lasting cAMP activity since it is resistant to phosphodiesterase-catalyzed hydrolysis.

Figure 2

Design of a Bioorthogonal Caged Protein. The actin polymerizing/depolymerizing protein cofilin exists in active dephosphorylated (6) and inactive phosphorylated (7) states. The cage moiety on 8 contains a negatively charged carboxylate, which has been introduced to mimic the phosphate in 7. Although photolysis of 8 should generate active cofilin (6), the latter can be rapidly switched off by the appropriate protein kinases. Consequently, the Cys-3 cofilin mutant 9 was prepared instead, which is impervious to kinase-catalyzed phosphorylation. Photolysis of the corresponding caged derivative 10 generates the constitutively active cofilin 9, which is resistant to intracellular down-regulatory action.

Figure 3

Single Site Regulation of Nucleic Acid Activity. Self-complementary nucleic acids form intramolecular duplexes that prevent intermolecular interactions with complementary sequences. An appropriately positioned photocleavable site (11) in a self-silenced species (12) forms, upon photolysis, duplexes with appropriate targets (13).

Figure 4

The Photoisomerizable Azobenzene Functionality and its Utility as a Biochemical Photoswitch. The azobenzene moiety is not photolytically cleaved from the biomolecule to which it is appended, but is rather reversibly interconverted between trans (14) and cis (15) isomers. This property has been used to control both peptide/protein conformation (16 ⇆ 17) and activity (18 ⇆ 19).

Figure 5

A Small Molecule Caged Protein Equivalent. A peptide-based bivalent inhibitor binds to two separate domains on the Src protein kinase (20). The high affinity inhibitor contains a photocleavable site (red). Photolysis splits the inhibitor in half, dramatically reducing inhibitory potency, and thereby liberating enzymatic activity (21). Figure reproduced from Li, H.; Hah, J. M.; Lawrence, D. S. J Am Chem Soc 2008, 130, (32), 10474-5.

Figure 6

Light-dependent Gene Activation. Caged versions of estradiol (22) and ecdysone (23) have been used to temporally and spatially control gene expression in living cells. The hydroxyl groups highlighted in yellow are required for bioactivity, which renders these sites ideal for caging (classical strategy). By contrast, some small molecules lack a caging site due to the absence of a functional group handle, such as in the case of tamoxifen (24). However, active derivatives of the latter with readily modifiable functionality (25) have been reported and covalent substitution of the latter with a photocleavable moiety renders the compound caged.

Figure 7

Coupling of Photouncaging and Fluorescence. Visualization of light-driven release of a caged compound can provide a quantitative assessment of the amount of compound liberated, information that is potentially useful when the experiment is conducted in a living cell. A protein-based system has been described that contains a fluorophore and a fluorescent quencher positioned in close proximity to one another (26). Photolysis simultaneously releases the quencher and activates the protein (27). The O-hydroxycinnamic acid caging group 29 is photolytically converted to the fluorescent coumarin derivative 30 simultaneously with active bioreagent formation.

Figure 8

Light-driven Formation or Disruption of Two Genetically Expressed Proteins. Red light-induced conversion of Pf to Pfr promotes the interaction with Pif3. Cdc42 containing bound GDP has a weak affinity for and thus is a poor activator of WASP. However, the Pf-Cdc42-GDP construct does activate Pif3-WASP in a light-dependent fashion by furnishing a dramatic enhancement in the effective concentration of the Cdc42/WASP pair.

Figure 9

The Emission Spectra of Several Common Microscope Lamps, Hg (blue), Xe (red), and Metal Halide (green) and the Absorbance Spectrum of the Common Caging Group, 4,5-Dimethoxy-2-nitrobenzyl fluorescein dextran (black). The emission spectra of the lamps are normalized by the integrated area from 250 to 700 nm. The absorbance spectrum of DMNB is in arbitrary units.

Figure 10

Caged Amino Acids with Acidic Side Chains. Peptides and proteins containing the S- and O-modified thiophosphate analogs of threonine (30), serine, and tyrosine (31) have been reported. FmocAsp analogs containing the side chain caging agents depicted in 32 and 33 have been synthesized, but only the latter has been successfully incorporated into peptides via solid phase peptide synthesis. Derivative 32 appears to be susceptible to by-product formation via an intramolecular cyclization reaction.