“Power On” – Activity Control by Photocaging

“Power On” – Activity Control by Photocaging

Published on 30.04.2024

Discover photocages – photosensitive protecting groups that are removed by irradiation with light of a defined wavelength, thus restoring the original, “active” state.

Photocages are a class of substances bearing a photosensitive group (=photoremovable protecting group, PPG) keeping a compound in its inactive (=caged) state until being removed by irradiation with light (=photocleavage) of a defined wavelength. Light as external trigger stands out for a variety of reasons, with the main ones being its non-invasiveness, high spatiotemporal resolution, and ease of dosage control.

Photocages can either be used for synthetic purposes, e.g., during solid-phase peptide synthesis, to introduce another dimension of orthogonality besides, e.g., acid/base labile protecting groups (PGs). Furthermore, PPGs allow to temporarily mask a functional group relevant for the biological activity of a certain drug molecule or to suppress a certain protein-protein interaction. In terms of photocaged drugs, pharmacological activities can be timely and selectively induced at a desired point of action reducing the risk of undesired off-target effects.

The “cage” concept was first introduced in 1978 by Hoffmann et al., who synthesized a caged ATP bearing a o-nitrobenzyl moiety as photosensitive group.

O-nitrobenzyl residues (and derivatives thereof) absorb UV light at about 350 nm. Upon absorption of a photon, a diradical is formed, the group undergoes a Norrish type II reaction, the activated oxygen of the N=O group abstracts a proton from the benzylic position, and in a rearrangement, the bond between the nitrobenzyl residue (where the protected functional group is attached) is broken, and the functional group is released, while an o-nitroso benzaldehyde is formed. This protection/deprotection scheme works well for many functional groups present in amino acids.

Simplified scheme of the photochemical deprotection of an o-nitrobenzyl-caged cysteine residue with light. First, a biradical is formed which abstracts a benzylic proton. After a rearrangement, the side chain functional group of cysteine is restored.

To reduce photolysis time and to increase yields, the original nitrobenzyl protection has been improved by the introduction of o-nitroveratryl (Nve, oNV, also named 4,5-dimethoxy-2-nitrobenzyl [DMNB]), and nitrodibenzofuranyl (NDBF) residues.

Variations of the 2-nitrobenzyl photocage: oNB: o‑Nitrobenzyl, Nve: Nitroveratryl, MDNPE: Methyl-o-nitropiperonylethyl (R=Me), NDBF: Nitrodibenzofuranyl. R can be H or methyl; Y depicts the remainder of the protected amino acid.

Enlargement of the aromatic system allows to trigger photolysis with longer wavelengths which is especially favorable for in vitro and in vivo applications in cells and organisms due to improved penetration depth and reduced cell damaging. Nitrodibenzofuran (NDBF) has a very high quantum yield. NDBF-caged compounds are photolyzed 16-160 times more efficiently than those protected with nitrobenzoyl residues, and thus are accessible by two-photon excitation with infrared light of 800 nm.

Table of available photocaged amino acids at Iris Biotech. HAA product codes represent N‑unprotected amino acids, items beginning with FAA are N-Fmoc protected.

 

oNB

DMNB/Nve/oNV

MDNPE

NDBF

Lysine

FAA9345
FAA9365

 

 

FAA8425

Cysteine

 

HAA9320
FAA3970

HAA9270
FAA7945

FAA8420

Selenocysteine

HAA9475

HAA9255

HAA9360

HAA9230

 

Glutamate

FAA9350

 

 

 

 

Still, existing photocages have mostly centered on excitation in the ultraviolet-visible range with only single examples for deep red or NIR light within the “phototherapeutic window” (650–900 nm).

As an example for synthetic purposes, the photocaged o-nitroveratryl- (oNV-) cysteine (FAA3970) in combination with S‑pyridinesulfenyl- (Npys) cysteine (FAA1975) allows for a rapid light induced -SS- bond formation. Together with, e.g., acetamidomethyl (Acm-) cysteines (FAA8255), which may be activated by oxidation with iodine (I2), cyclic peptides with multiple disulfide bridges may easily be synthesized, by activating the orthogonally protected cysteine pairs step by step.

Selenocysteine (Sec) allows site-specific covalent protein modifications, even in the presence of native cysteines, as selenols are more reactive than thiols. Our photocaged selenocysteine may be incorporated into proteins as an unnatural amino acid in response to an amber stop codon using a mutant pyrrolysyl-tRNA/tRNA synthetase pair. Photoactivable glutamate has been incorporated into the Green Fluorescent Protein (GFP) via genetic code expansion.

Besides “single” photocages, literature reports on dual photosensitive groups to selectively control and track the activities of biomolecules and probing the dynamics of cellular systems. However, such dual systems need to have non-overlapping absorption spectra to enable independent and selective controllability.

→ You want to learn more about possibilities for subsequent disulfide-bond formation? Download our booklet Cyclic Peptides”! 

→ Interested in Photochemistry? Browse our brochure

→ Looking for a photocaged building block not yet listed in our portfolio? Get in contact for custom synthesis! 

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