Isomorphic Fluorescent Nucleosides

Conspectus In 1960, Weber prophesied that “There are many ways in which the properties of the excited state can be utilized to study points of ignorance of the structure and function of proteins”. This has been realized, illustrating that an intrinsic and highly responsive fluorophore such as tryptophan can alter the course of an entire scientific discipline. But what about RNA and DNA? Adapting Weber’s protein photophysics prophecy to nucleic acids requires the development of intrinsically emissive nucleoside surrogates as, unlike Trp, the canonical nucleobases display unusually low emission quantum yields, which render nucleosides, nucleotides, and oligonucleotides practically dark for most fluorescence-based applications. Over the past decades, we have developed emissive nucleoside surrogates that facilitate the monitoring of nucleoside-, nucleotide-, and nucleic acid-based transformations at a nucleobase resolution in real time. The premise underlying our approach is the identification of minimal atomic/structural perturbations that endow the synthetic analogs with favorable photophysical features while maintaining native conformations and pairing. As illuminating probes, the photophysical parameters of such isomorphic nucleosides display sensitivity to microenvironmental factors. Responsive isomorphic analogs that function similarly to their native counterparts in biochemical contexts are defined as isofunctional. Early analogs included pyrimidines substituted with five-membered aromatic heterocycles at their 5 position and have been used to assess the polarity of the major groove in duplexes. Polarized quinazolines have proven useful in assembling FRET pairs with established fluorophores and have been used to study RNA–protein and RNA–small-molecule binding. Completing a fluorescent ribonucleoside alphabet, composed of visibly emissive purine (thA, thG) and pyrimidine (thU, thC) analogs, all derived from thieno[3,4-d]pyrimidine as the heterocyclic nucleus, was a major breakthrough. To further augment functionality, a second-generation emissive RNA alphabet based on an isothiazolo[4,3-d]pyrimidine core (thA, tzG, tzU, and tzC) was fabricated. This single-atom “mutagenesis” restored the basic/coordinating nitrogen corresponding to N7 in the purine skeleton and elevated biological recognition. The isomorphic emissive nucleosides and nucleotides, particularly the purine analogs, serve as substrates for diverse enzymes. Beyond polymerases, we have challenged the emissive analogs with metabolic and catabolic enzymes, opening optical windows into the biochemistry of nucleosides and nucleotides as metabolites as well as coenzymes and second messengers. Real-time fluorescence-based assays for adenosine deaminase, guanine deaminase, and cytidine deaminase have been fabricated and used for inhibitor discovery. Emissive cofactors (e.g., SthAM), coenzymes (e.g., NtzAD+), and second messengers (e.g., c-di-tzGMP) have been enzymatically synthesized, using xyNTPs and native enzymes. Both their biosynthesis and their transformations can be fluorescently monitored in real time. Highly isomorphic and isofunctional emissive surrogates can therefore be fabricated and judiciously implemented. Beyond their utility, side-by-side comparison to established analogs, particularly to 2-aminopurine, the workhorse of nucleic acid biophysics over 5 decades, has proven prudent as they refined the scope and limitations of both the new analogs and their predecessors. Challenges, however, remain. Associated with such small heterocycles are relatively short emission wavelengths and limited brightness. Recent advances in multiphoton spectroscopy and further structural modifications have shown promise for overcoming such barriers.


A fluorescence spectroscopy primer
Figure S1 shows a basic Jablonski diagram.Excitation of a chromophore rapidly generates the Franck-Condon state (within about 10 -15 s).The efficiency of this electronic reorganization, from the ground to excited state(s), corresponds to the chromophore's absorption crosssection (σ), which is proportional to its extinction coefficient (ε).Extinction coefficients are typically listed for the peak, or maximum absorption wavelength (λmax), thus providing a measure of how capable a chromophore is in absorbing a photon of a given wavelength/energy.Vibrational relaxation (within 10 -12 -10 -10 s) rapidly populates the lowest vibronic state of the chromophore's excited state (Figure S1).This relaxation process, which, as shown, populates a lower energy emissive state, thus accounts for the lower emission energy (longer wavelength) of a chromophore compared with its higher excitation energy (shorter wavelength).The energetic difference between the absorption and the emission maxima is commonly referred to as the chromophore's Stokes shift.To calculate this value, it is advisable to convert the measured absorption and emission wavelength peaks into energy units, such as cm -1 , and subtract the two values.This is typically more meaningful than reporting the difference in wavelength (nm), as it accounts for the actual energetic difference.
Typical organic chromophores, such as the ones discussed in this Accounts of Chemical Research article, reside in their excited state for a period of (0.5-20) × 10 -9 s.Both radiative and nonradiative processes can facilitate the decay back to the ground state, and the excited state lifetime reflects the sum of the various relaxation processes (τ 0 ).The fraction responsible for emitting a photon, or the fluorescence lifetime (τ), relates to the emission quantum yield of the chromophores (Φ = τ/τ 0 ).In simplified terms, the quantum yield can be viewed as the number of photons emitted per number of photons absorbed.
For certain studies, particularly fluorescence imaging, the brightness (εΦ) of a fluorophore is reported, which is the product of the molar absorptivity (ε) and the fluorescence quantum yield (Φ).This could be useful when comparing the utility of two fluorophores with similar fluorescence quantum yields but distinct molar absorptivities.

Correlating photophysical features
When systematically studying the effect of structural modifications (or different substituents) it is useful to correlate experimental data with established quantitative empirical scales.

Substituent parameters
The Hammett substituent constants (or parameters) reflect the electronic features of a substituent (e.g., electron donating or withdrawing).They were derived from the Hammett equation, which is a linear free-energy relationship (LFER), originally used to the evaluate the effect of substituents on the equilibrium dissociation constants of benzoic acids (logK/Ko = , where K is the dissociation constant obtained for a substituted benzoic acid and Ko is the dissociation constant of benzoic acid).For example, electron withdrawing groups (e.g., nitro) facilitate dissociation and have positive  values, while electron donating groups (e.g., dimethylamino) have negative  values.Numerous scales have since been developed, reflecting the substituent position (p or m), contributions from resonance stabilization, etc. See reference 1.

Solvent polarity
Reichardt's ET( 30) is an empirical, microscopic solvent polarity scale, which is based on Reichardt's dye, a highly solvatochromic zwitterionic chromophore (see Figure 2 below and references 2 and 3).The dye is dissolved in the solvent of choice, the absorption spectrum is taken, and the maximum absorption wavelength (λmax) is extracted.The ET(30) value (in kcal/mol) is obtained as follows: ET(30) = 28591/λmax A simplified energy diagram (Figure 2) reflects the impact of solvent polarity on the ground/excited state energies and hence on the absorption maximum of the dissolved dye.As a zwitterionic molecule (Figure 2), higher polarity stabilizes the dye's ground state.Since vertical excitation (Franck-Condon principle) is an extremely fast process (Figure 1), higher polarity "destabilizes" the Franck-Condon state, as the solvation sphere remains organized in its ground state ("more polar") configuration, leading to a higher energy for its excited state (hence higher absorption energy = shorter wavelength).For photophysical studies we have always recommended using microscopic solvent polarity scales, such as the Reichardt's ET(30) scale, over the use of dielectric constants, which reflect a bulk solvent property (see reference 4 below).
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Figure S1 .
Figure S1.Left: a basic Jablonski diagram, illustrating key electronic transitions (absorption in blue and emission in ref) and the time scale associated with the excitation and relaxation processes.Right: a typical solution absorption spectrum of a chromophore (blue) and the associated normalized emission band (red).The energetic difference between the absorption and emission maxima is referred to as the Stokes shift.

Figure S2 .
Figure S2.Left: Reichardt's zwitterionic dye is the chromophore used to determine the ET(30) values of a solvents/solvent mixtures.Right: Simplified photophysical considerations associated with ground-excited state energies as solvent polarity changes from less to more polar.