Photochemistry has long been used as a method to induce chemical reactions in synthetic chemistry. The first report of a light induced chemical transformation was by Trommsdorff in 1834 when he described that sunlight caused a color change and
subsequent destruction of α-santonin crystals.[152] A number of different chemical moieties
that absorb light and undergo some sort of rearrangement have been used as chemical protecting groups since then. However, it was not until 1978, when Hoffman and colleagues appended an ortho-nitrobenzyl moiety to the γ-phosphate of ATP (“caged” ATP) and used
light to release free ATP and thus activate the Na+/K+ ATPase[153] that photoprotecting
groups found their way into the world of biological research.
Since the advent of caged ATP, a wide variety of photoprotected biomolecules have been developed that provide an unprecedented level of spatial and temporal control over cellular processes (Figure 4.2). The ideal caged compound is described as having: 1) a high quantum yield (φ, the number of photons required to induce the reaction), 2) high molar extinction coefficients (the measure of a molecule’s ability to absorb light of a given
wavelength) above 300 nm (the lowest cutoff for minimal interaction with biomolecules), 3) inactive photochemical byproducts, 4) a photolytic rate faster than that of the process studied, 5) biological inertness prior to photolysis, and 6) aqueous solubility.[154]
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Indeed, the majority of caged compounds fall under these guidelines. By far, the most commonly used photoprotecting group is the ortho-nitrobenzyl moiety. Cyclic
nucleotides[155], cell permeable Ca2+ chelators that release Ca2+ upon photolysis[156], steroids
for manipulating gene expression[157], and a myriad of neurotransmitters[158] have been
developed and utilized to probe activation of cellular signaling. In addition, caged
peptides[159], proteins[159], and nucleic acids[160] have been developed to study intracellular
signaling and gene expression respectively. However, the nitrobenzyl photoprotecting group has a major drawback: it is activated by ultraviolet (UV) light. UV light, while useful in
vitro and in cellulo, does not penetrate tissue and can damage biomolecules such as
DNA.[161] As in vivo whole animal studies became more common, it was necessary to
develop photoprotecting groups that responded to longer wavelengths of light, ideally within the optical window of tissue (Figure 4.3). The optical window of tissue lies between 650 and Figure 4.2: A sample of different nitrobenzyl caged biomolecules.
Figure 4.2: A sample of different nitrobenzyl caged biomolecules.
Figure 4.2: A sample of different nitrobenzyl caged biomolecules.
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1000 nm.[162] Below 650 nm, light is significantly absorbed by biomolecules and above 1000
nm, water begins to absorb light and is heated.
Some long-wavelength photoprotecting groups have been reported including the use of two-photon technology with nitrobenzyl derivatives at 700 nm[163] and single photon
photolysis of boron dipyrromethene (Bodipy) at green wavelengths (500 – 550 nm).[164]
However, two-photon imaging requires concentrating photons to the extent that a molecule absorbs two photons at the same time and thus, requires an incredibly high photon flux that
can result in sample heating. The Bodipy derivatives utilize green light which is red-shifted further than nitrobenzyl or coumarin photoprotecting groups, but is still outside the optical window of tissue.
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Work by Dr. Shell and Dr. Lawrence investigating the photochemistry of vitamin B12 (cobalamin, Figure 4.4) found that, through a yet-to-be ascertained mechanism, homolytic photolysis of an alkyl axial ligand of the central cobalt atom can be tuned by placing a long wavelength fluorophore in close proximity to the cobalamin core (Figure 4.4).[165] The
fluorophores act as molecular antennae to transmit the energy from light to cobalamin initiating photolysis. Since the initial discovery that cobalamin photochemistry is tuneable, our group has developed a number of technologies including erythrocyte mediated drug delivery[166] and long-wavelength polymerization of hydrogels.[167]
Figure 4.4: Green light (500 – 560 nm) photolyzes the cobalt-carbon bond in the axial position on cobalamin (Top). When a long wavelength fluorophore, such as Cy5, is in close proximity to cobalamin (Bottom) the long wavelength energy is transferred to cobalamin and induces photolysis. The red hydroxyl (OH) on the underside ribose can be modified.
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Cobalamin is a much larger photoprotecting group than the nitrobenzyl moiety and is not cell permeable. In addition, cobalamin photochemistry at long wavelengths only occurs when an alkyl chain is bound to cobalt. The result is that there is a limitation to the
functional groups that can be photolytically released. Given that many biologically active functional groups are heteroatoms, masking key functionality may be difficult with
cobalamin. Indeed, Lawrence and Shell found that cAMP appended to cobalamin via the 8’- amine is still capable of activating PKA before photolysis despite the steric bulk of cobalamin (personal communication). This did not spell doom of cobalamin as a photoprotecting group. Cobalamin is endosomally taken up into cells in a receptor mediated fashion and is spatially sequestered from the cytoplasm. In addition, we have appended fatty acyl chains to the reactive hydroxyl group on the ribose moiety of cobalamin thus furnishing a molecule capable of being loaded on the outer leaflet of the PM.[166] Thus, cobalamin functions as a
sequestering photoprotective group, which, upon photolysis, liberates a cell permeable small molecule of interest. We are using the sequestering and long-wavelength tuning
characteristics of cobalamin to develop drug delivery platforms.