Chemical structures of caged nucleobases

Most of the caging groups described in the literature employ the o-nitrobenzylic group. But other structural types have been reported as well. The caging or light-sensitive group can be synthetically incorporated into biologically active molecules such as amino acids, nucleotides, and oligonucleotides for the design of specific molecular probes. In general, for the conjugation reaction, a linkage to a hetero-atom is utilized. Common hetero atoms to which the caging moieties are linked to are oxygen (O), sulfur (S), or nitrogen (N) atoms. Functional groups used for the formation of the linkage can be ethers, thioethers, esters, including phosphate or thiophosphate esters, amines or similar groups. The structure of the caging moiety, as well as the atom to which it is linked to, affects the efficiency and the wavelength needed for the uncaging reaction. 

Figure 1: Structures of different caging groups.

Most caging groups show good water solubility and are very fast to uncage with high quantum yields. Many of them release biologically inert photolytic by-products when uncaged. Typical uncaging rates are reported to be in the microseconds.

Nomenclature for caging groups used here:

CNB      =  α-carboxy-2-nitrobenzyl;
NPE      =  1-(2-nitrophenyl)ethyl;
DMNB   =  4,5-dimethoxy-2-nitrobenzyl;
DMNPE  =  (4,5-dimethoxy-2-nitrophenyl)ethyl;
CMNB   =  5-carboxymethoxy-2-nitrobenzyl.

The addition of dithiothreitol (DTT) to caging experiments can help prevent the potential cytotoxic reaction between amines and 2-nitosobenzoyl by-products.

The following is a list of caged nucleobases that have been incorporated into oligonucleotides. Light-sensitive caging groups are shown in red.

A:  A photo-active C8 thioether-linked adenosine.

Figure 1: Photo-active C8 thioether-linked adenosine. This group has been used to cage DNAzyme to control its enzymatic activity (Liu and Deiters 2014).

Ting et al. in 2004 report the synthesis and photochemical properties of a this nucleoside: 8-(2-(4-imidazolyl)ethyl-1-thio)-2'-deoxyriboadenosine. Its light sensitivity was evaluated via an examination of the photoinduced reactivation of DNAzyme 8-17E from an inactive form that contained a single nucleotide modification. The photoinduced reversion of 8-(2-(4-imidazolyl)ethyl-1-thio)-2'-deoxyriboadenosine to unmodified deoxyadenosine restorated the activity of the DNAzyme.

B:  1-(Ortho-nitrophenyl)-ethyl (NPE) photolabile group

 The 1-(ortho-nitrophenyl)-ethyl (NPE) photolabile group has been used early onto to design and synthesize a photolabile chelator, nitrophenyl-EGTA (NPE-EGTA), that can selectively bind calcium ions (Ca²⁺) with high affinity. Photolysis of the chelator rapidly releases the calcium ions. The rapid controlled and localized increase in calcium ion concentrations has enabled studies of kinetics, regulatory and structural mechanisms of calcium based intracellular communications. More recently the NPE group has been applied to five nucleobases.

Figure 2:  Structures of NPE labeled nucleobases. The NPE group has been used for the study of biological processes since the 1980s. Nucleobases in which this caging group have been incorporated are shown (Liu and Deiters 2014).

C:   2-(Ortho-nitrophenyl)propyl (NPP)

This caging group has also been applied to nucleobases. Over the years different designs were used and tested with the goal to improve stability and quantum yields of the caging group during decaging reactions.

Figure 3:  Structures of NPP labeled nucleobases. The NPP caging group has been used for the nucleobases depicted here (Liu and Deiters 2014).

D:   6-Nitropiperonyl methyl group (NPM) and hydroxymethylene analog
       (NPOM).

This caging group was developed to improve decaging rates. The 6-nitropiperonyl-methyl group was conjugated to nucleobases to red-shift the absorption maximum and the decaging wavelength. In addition, the stability of the caging group was also improved during oligonucleotide synthesis. However, for almost all caging groups, the decaging wavelength falls typically between 360 and 366 nm. These caging groups are reported to be stable under ambient light.

Figure 4:Structures of NPM and NPOM labeled nucleobases. The NPM caging group has recently been used for cytosine and NPOM for guanine, thymine and uracil (Liu and Deiters 2014).

E:   Diethylaminocoumarin (DEACM) and nitrobenzofuran (NDBF).

Coumarin based caging groups have also been investigated for their use in nucleobases. Since they require a leaving group with a low pKa their use is limited.The diethylaminocoumarin (DEACM) group was used for the caging of guanine. The nitrobenzofuran (NDBF) group was also employed for the caging of several nucelobases. However, this group may not be as versatile as the caging groups reported earlier.

Figure 5:  Structures of DEACM and NDBF labeled nucleobases. The coumarin based caging group enables photolysis at >405 nm (Liu and Deiters 2014).

F:  o-Nitrobenzyl phosphate ester based caging

Dussy et al. in 2002 used an o-nitrobenzyl ester based caging group to enable phototriggered bond cleavage.

Figure 6: Structure of an o-nitrobenzyl ester based caging DNA building block.

The research group synthesized photocleavable nucleotides based on the photochemistry of o-nitrobenzyl esters through the introduction of o-nitrophenyl groups at the 5′C position into the oligonucleoside sugar backbone. These modified nucleosides are able to build stable DNA duplexes. Oligonucleotides modified this way can be cleaved site-specifically by irradiation with >360 nm light with high efficiency.

Reference

Adrian Dussy, Christoph Meyer, Edith Quennet, Thomas A. Bickle, Bernd Giese and Andreas Marx; New Light-Sensitive Nucleosides for Caged DNA Strand Breaks. Volume 3, Issue 1, pages 54–60, January 4, 2002.

G C Ellis-Davies, J H Kaplan; Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc Natl Acad Sci U S A. 1994 January 4; 91(1): 187–191. PMCID: PMC42911.

Ting R, Lermer L, Perrin DM. Triggering DNAzymes with light: A photoactive C8 thioetherlinked adenosine. J Am Chem Soc. 2004; 126:12720–12721. [PubMed: 15469235].