A Complete Set of Emissive RNA Alphabet

It is clear from the above discussion that several nucleoside analogues including the several emissive nucleosides have been reported over the years.⁴³ Describing the H-bonded/non-H-bonded RNA base analogues, though, is beyond the scope of the thesis, we found one highly inspiring and interesting report by Tor et al. who have described for the first time the isomorphic design^{43a, 44} of emissive DNA and RNA alphabets and a complete set of RNA alphabets with novel fluorescent and biophysical property.⁴⁵

Previously, Kool et al. have reported a complete set of emissive expanded RNA nucleoside analogues and studied their fluorescence photophysical properties (Figure 1.19). They have found that the benzo-expanded ribonucleosides (xRNA), analogues to A, G, C, and U RNA monomers, are efficient fluorophores with emission maxima ranging from 369-411 nm.⁴⁶

N N

O OH

HO O

OH HO

N N

O OH

HO O

OH HO rxG

1.69 rxU

1.71 rxA

1.68 rxC

1.70 N

N

NH N

N NH

NH NH NH₂

NH₂

O OH

OH OH

OH NH2

O

O O

Figure 1.19: The size expanded emissive xRNA genetic set develoed by Kool et al.

Later on, they have described the use of size-expanded versions of adenosine (rxA) and uridine (rxU) as a novel set of steric probes to investigate the RNAi mechanism. For that purpose they have incorporated these two bases into biologically active siRNAs and use in biophysical studies. Their studies demonstrate the utility of xRNA nucleobases as mechanistic tools in biologically functioning siRNAs.⁴⁷

In continuation of the isomorphic base design Tor et al. have taken very interesting and challenging task to offer a complete set of emissive RNA-alphabets derived from a single heterocyclic core that has not been addressed previously. In their design they emphasized on the structural similarity with the native counterparts which ultimately would lead to minimize structural and functional perturbation which is an inevitable consequence of replacing any native residue with a synthetic probe.

Therefore, they have reported the design and synthesis of isomorphic base analogues, a complete set of ribonucleoside alphabets, consisting of highly emissive purine (^thA,

thG) and pyrimidine (^thU, ^thC) analogues (Figure 1.20). All are derived from thieno[3,4-d]pyrimidine as the heterocyclic nucleus (1.33, Figure 1.20). The beauty of this parent heterocycle is that it can be viewed as a precursor to 5,6-modified emissive pyrimidines as well as a purine mimic with thiophene substituted for the imidazole moiety.

Figure 1.20: Isomorphic and emissive RNA alphabet designed by Tor et al. derived from a single from thieno[3,4-d]pyrimidine as the heterocyclic nucleus.

They have investigated the conformation of all the nucleoside analogues in solid state. Thus, the crystal structures of the modified ribonucleosides showed that they all display an anti orientation at their glycosidic linkages which is similar to the preference seen with the native counterparts. However, while the pyrimidine analogues ^thU and ^thC, possess partial C2′-endo conformation of sugar, the purine analogues ^thG and ^thA exhibit C2′-endo and C3′-endo ribose pucker, respectively, in the solid state, which are the predominant conformations adopted by the natural ribonucleosides. However, overlayed crystal structure of ^thG and ^thA with their native counterparts reflects minimal distortion of the ribose conformation.⁴⁵ The fundamental spectroscopic properties of the modified nucleosides ^thU, ^thC, ^thA, and

thG have also been investigated which showed good solvatochromicity along with a long wave length absorption longer than their native counterparts and highly fluorescence characteristics better that their natural counterparts. They have also incorporated ^thG into 17-mer RNA and tested the duplex stability and found the results comparable to that offered by natural G base. It is a fact that many emissive nucleoside analogues, including the classical 2-aminopurine get quenched upon incorporation into oligonucleotides.^43a,48 However, it is highly interesting to note that the oligonucleotide containing the emissive ^thG “sandwiched” between two potentially quenching G residues displayed strong visible emission with good

N N S

NH₂

O OH HO

N O OH HO

NH N S

O

O OH HO

N O OH HO thG

1.74

thU 1.76 thA

1.73

thC 1.75

OH OH

OH OH

N

NH S

S

O NH₂

O

NH₂ O

NH N S

O

NH₂ 1.72

quantum yield which is of 0.10.⁴⁵ Therefore, this set of emissive RNA nucleosides designed by Tor et al. represents a novel class of fluorescent base analogues having properties like native Watson–Crick faces, unparalleled structural isomorphicity with respect to native nucleosides, minimal perturbation in duplexes and intense visible emission.

Later on, Tor et al. have exploited the isomorphic fluorescent analogue of adenosine (^thA) as Adenosine deaminase (ADA) inhibitor, a major enzyme involved in purine metabolism. They have found that this enzyme converts ^thA into an isomorphic inosine analogue (^thI), which possesses distinct photophysical properties as compared to ^thA. They have demonstrated the utility of this sensitive fluorescence- monitored transformation for the high-throughput detection and analysis of ADA inhibitors which are of particular importance for the treatment of certain leukemias.

The conversion of ^thA (1.73, Scheme 1.1) to ^thI (1.79, Scheme 1.1) has led to an emission enhancement and is feasible even at low nM concentrations. This high- throughput method for identifying inhibitors via the detection of enhanced fluorescence signal is superior to the currently avail methods which rely on either absorption spectroscopy or chromatographic methods, which require relatively large concentrations and are not normally amenable for high-throughput formats.⁴⁹

N N N N

NH2

O OH HO

NH N N N

O

O OH HO

N N S

NH₂

O OH HO

NH N S

O

O OH

thA HO 1.73

thI 1.79

1.77 1.78

ADA

ADA (a)

(b)

OH OH

OH OH