1H-1,2,3-triazolyl-1,6-naphthyridin-7(6H)-ones as Potential Fluorescent Nucleoside Analogues: Synthesis and Optical Properties

In this article, we present the synthesis and the optical properties of three original molecules as potential fluorescent ribonucleoside analogues incorporating a 1,6-naphthyridin-7(6H)-one scaffold as a fluorescent nucleobase and a 1,2,3-triazole as a linkage. The nucleosides were prepared via a Cu alkyne-azide cycloaddition (CuAAC) reaction between a ribofuranosyl azide and a 4-ethynylpyridine partner. Construction of substituted 1,6-naphthyridin-7(6H)-ones was achieved through two additional steps. Optical property studies were investigated on nucleoside analogues. Powerful fluorescence properties have been evidenced with a remarkable change of emissivity depending on the polarity of the solvent, making these molecules suitable as a new class of artificial fluorescent nucleosides for investigating enzyme binding sites as well as probing nucleic acids. In addition, we are convinced that such analogues could be of great interest in the search for new antiviral or antitumoral drugs based on nucleosides.


Introduction
Nucleic acid structure and dynamics are of fundamental importance in understanding biological processes in cells [1][2][3].In this context, nucleoside derivatives and analogues have been designed to interfere with cell metabolism and are being developed as antiviral, anticancer, and antibacterial agents [4][5][6] or as tools for several purposes, such as investigating enzyme binding sites, protein interactions, DNA features, and perturbations [7,8].
DNA-based fluorescent structures capable of labeling nucleic acids are powerful tools to investigate DNA interactions and have been used in a variety of applications in chemical biology [9][10][11][12].
In this context, numerous environmentally sensitive unnatural fluorescent nucleosides have been developed to fluorescently label nucleic acids via binding, intercalation, or covalent bonds [13,14].The design of fluorescent nucleosides is a great challenge and has stimulated much research in various fields of photophysics, synthetic chemistry, and computational studies.The main fluorescence properties should include a change in fluorescence intensity or a shift in the emission maximum when interacting with the DNA environment while maintaining the other key optical properties, such as large Stokes Shift, high brightness, and high quantum yield.
In this field, unnatural fluorescent nucleosides possessing heterocyclic nucleobases are widely represented.EthenoA [15], Coumarin nucleobase analogues [16], Nile red, imidazophenanthrene, and other types of heterocyclic nucleobases have been developed [17][18][19][20].In particular, 1,2,3-triazolyl nucleoside analogues, which consist in connecting the appropriate aromatic ring or purine residue to a 1,2,3-triazole moiety, have found a growing interest due to their powerful antiviral activities [21][22][23].4-substituted-1,2,3 triazolo nucleotide analogues are also well known for antitumoral activities as inhibitors of human ecto-5 ′ -nucleosidase CD73, a cell-surface protein associated with adenosine metabolism that promotes tumor progression [24].Interestingly, a nucleoside analogue incorporating a diaminopyrimidine linked to a 1,2,3-triazole [25], also called click fleximer [26,27], was found to be luminescent and a promising tool to investigate enzyme binding sites and to characterize protein and nucleic acid interactions (Figure 1).Indeed, unlike the classic bicyclic fused system of the parent purine nucleobase, the two heterocyclic components of the click fleximers are attached by a C-C bond that permits conformational mobility.As a consequence, the relative flexibility of nucleobase enables it to adapt rapidly to the spatial requirement of an enzyme binding site.The design of flexible bioprobes will clearly provide a better understanding of the conformational effects of enzymes, and elucidate the structure of ligand binding sites in biologically important enzyme systems [28].Other synthetic fleximer derivatives whose heterocyclic bases contain one or more than one planar ring capable of interacting with their environment, via π stacking and/or H-bonding, have been discovered.For example, a fluorescent triazolyl containing a pyrene ring has shown interesting fluorescence properties and capacity to interact with BSA via hydrophobic and electrostatic bindings [29] (Figure 1).Interestingly, an environment-sensitive fluorescent nucleoside analogue bearing a thienyl ring linked to a 3-hydroxychromone (3HCnt) as ESIPT dye has been successfully incorporated into a DNA sequence with minimal perturbation.In addition, the authors demonstrated that 3HCnt can monitor local conformation changes of oligonucleotides upon interaction with HIV-1 nucleocapsid protein [30].
We have recently reported the short synthesis of 1,6-naphthyridin-7(6H)-ones 1, a fluorescent heteroaromatic scaffold with adequate photophysical properties to be incorporated as the nucleobase into a nucleoside (Scheme 1).Indeed, 1,6-naphthyridin-7(6H)-one scaffold shows a visible absorption, a solvatochromism, an acidochromism, large Stokes shifts, and high quantum yields depending on the solvent and media.These napthyridinones are also able to generate dual fluorescence in polar solvent from an intramolecular proton transfer at the excited state mechanism (ESIPT) coming from their lactim-lactam forms [41] (Scheme 1).
Therefore, the recent development in the field of triazole-linked fluorescent nucleoside conjugates and the photophysical properties of 1,6-naphthyridin-7(6H)-ones encouraged us to design and synthetize fused 1,6-naphthyridin-7(6H)-ones with triazole and to study their photophysical properties.We thus expect to develop a new class of fluorescent nucleoside analogues with potentially powerful biological properties as well as to use them as molecular probes to investigate enzyme binding sites or the structural Scheme 1.Our reported 8-substituted 1,6-naphthyridin-7(6H)-ones 1 for Ar = Ph and their emission spectra in various solvents, adapted with permission from Ref. [41].2023, Royal Chemical Society (RCS).
Therefore, the recent development in the field of triazole-linked fluorescent nucleoside conjugates and the photophysical properties of 1,6-naphthyridin-7(6H)-ones encouraged us to design and synthetize fused 1,6-naphthyridin-7(6H)-ones with triazole and to study their photophysical properties.We thus expect to develop a new class of fluorescent nucleoside analogues with potentially powerful biological properties as well as to use them as molecular probes to investigate enzyme binding sites or the structural characteristics of nucleic acids.
naphtyridinone-ribose conjugate TzNat, we chose to use a CuAAC click reaction.
Synthesis of 8-(4-ethynylphenyl)-1,6-naphthyridin-7(6H)-one 3 was achieved following our previously reported strategy from commercially available 2-chloro-3-formylpyridine. The azide partner 2 was prepared from 1,2,3,4-tetra-O acetyl β-D-ribofuranose following a reported procedure [49].However, attempts to realize the CuAAC click reaction between the two partners failed.The classical procedure using a combination of CuSO 4 and sodium ascorbate in tertbutanol/water at various temperatures failed.The use of CuI or more activated Cu(iMes)Cl in organic solvent with a base remained unsuccessful (Scheme 3 and Table S1 in Supplementary Materials).We hypothesized that the naphtyridinone bicycle 3 was not compatible with the click conditions because of the high reactivity of the pyridone moiety acting as a ligand with the copper atom.To overcome this issue, the click reaction was performed on the opened form 5 [41], the synthetic precursor of the 8-(4-ethynylphenyl)-1,6-naphthyridin-7(6H)-one 3 [41], and the azide 2 using standard conditions.Under these conditions, the click compound 6 was obtained in 40% yield.The last step consisted of an acidic treatment to promote the concomitant deprotection of the ribose, the acetal removal, and the nitrile function hydrolysis-cyclization.Unfortunately, this step did not give satisfactory results and led to an inseparable mixture containing a majority of the starting product, a very small amount of the expected product 4, and a portion containing the mono-deacetylated sugar unit (Scheme 3).Synthesis of 8-(4-ethynylphenyl)-1,6-naphthyridin-7(6H)-one 3 was achieved following our previously reported strategy from commercially available 2-chloro-3-formylpyridine. The azide partner 2 was prepared from 1,2,3,4-tetra-O acetyl β-D-ribofuranose following a reported procedure [49].However, attempts to realize the CuAAC click reaction between the two partners failed.The classical procedure using a combination of CuSO4 and sodium ascorbate in tertbutanol/water at various temperatures failed.The use of CuI or more activated Cu(iMes)Cl in organic solvent with a base remained unsuccessful (Scheme 3 and Table S1 in Supplementary Materials).We hypothesized that the naphtyridinone bicycle 3 was not compatible with the click conditions because of the high reactivity of the pyridone moiety acting as a ligand with the copper atom.To overcome this issue, the click reaction was performed on the opened form 5 [41], the synthetic precursor of the 8-(4-ethynylphenyl)-1,6-naphthyridin-7(6H)-one 3 [41], and the azide 2 using standard conditions.Under these conditions, the click compound 6 was obtained in 40% yield.The last step consisted of an acidic treatment to promote the concomitant deprotection of the ribose, the acetal removal, and the nitrile function hydrolysis-cyclization.Unfortunately, this step did not give satisfactory results and led to an inseparable mixture containing a majority of the starting product, a very small amount of the expected product 4, and a portion containing the mono-deacetylated sugar unit (Scheme 3).Encouraged by this last result, we slightly modified the strategy to target triazole conjugate A or TzNat A, which has the advantage of being easily modified at the 8-position of the naphthyridinone ring, and to allow further photophysical modification.Therefore, we have envisioned performing the click reaction with the 2-chloro-3-(dimethoxymethyl)-4-ethynylpyridine 9 as the alkyne partner and the azide ribose tetraacetate 2 in the first intention (Scheme 4).Encouraged by this last result, we slightly modified the strategy to target triazole conjugate A or TzNat A, which has the advantage of being easily modified at the 8-position of the naphthyridinone ring, and to allow further photophysical modification.Therefore, we have envisioned performing the click reaction with the 2-chloro-3-(dimethoxymethyl)-4-ethynylpyridine 9 as the alkyne partner and the azide ribose tetraacetate 2 in the first intention (Scheme 4).The synthesis of alkyne partner 9 was achieved in two steps.The mono-alkynylated product 7 was successfully prepared by a regioselective Sonogashira cross-coupling reaction performed in 5 min at 150 °C using a monowave apparatus.The aldehyde function of 7 was then protected as dimethoxyacetal under the mild conditions developed by Luche using methanol, cerium chloride, and trimethylorthoformate as the water scavenger [50].Deprotection of the alkyne function appeared very sensitive due to the high reactivity of the triple bond.Two minutes were necessary to cleave the C-Si bond with K2CO3 in methanol at room temperature and obtain compound 9.The ribofuranosyl azide partner 2 was reacted with 9 by a CuAAC click reaction with CuI, diisopropylethylene diamine in dichloromethane for 24 h at reflux, affording the desired triazole 10 in good yield.The triazole 10 was subsequently engaged in a nucleophilic aromatic substitution with phenylacetonitrile and sodium hydride.However, the reaction was unsuccessful.The presence of acetate protecting groups of the ribose part was suspected to be part of the observed lack of reactivity, due to its electron-withdrawing character and its propensity to be saponified under basic conditions (Scheme 5).The synthesis of alkyne partner 9 was achieved in two steps.The mono-alkynylated product 7 was successfully prepared by a regioselective Sonogashira cross-coupling reaction performed in 5 min at 150 • C using a monowave apparatus.The aldehyde function of 7 was then protected as dimethoxyacetal under the mild conditions developed by Luche using methanol, cerium chloride, and trimethylorthoformate as the water scavenger [50].Deprotection of the alkyne function appeared very sensitive due to the high reactivity of the triple bond.Two minutes were necessary to cleave the C-Si bond with K 2 CO 3 in methanol at room temperature and obtain compound 9.The ribofuranosyl azide partner 2 was reacted with 9 by a CuAAC click reaction with CuI, diisopropylethylene diamine in dichloromethane for 24 h at reflux, affording the desired triazole 10 in good yield.The triazole 10 was subsequently engaged in a nucleophilic aromatic substitution with phenylacetonitrile and sodium hydride.However, the reaction was unsuccessful.The presence of acetate protecting groups of the ribose part was suspected to be part of the observed lack of reactivity, due to its electron-withdrawing character and its propensity to be saponified under basic conditions (Scheme 5).
Therefore, we changed the ribose acetate protecting groups for the more stable tertbutyldimethylsilylether protecting groups.Starting from 2,3,5-triacetate-β-D-ribofuranosyl azide 2, the protected tertbutyldimethylsilylether ribose azide 13 was obtained in two steps using sodium methoxide, followed by the addition of tertbutyldimethylsilylchloride and imidazole, with excellent yield (Scheme 6).Next, the CuAAC click reaction between the azide 13 and the alkyne 9 proceeded smoothly, affording the desired triazole 14 in good yield.The reaction of 14 with phenylacetonitrile in the presence of sodium hydride gave the substituted product 15 in 30% yield.Finally, the nucleoside napthyridinone TzNat A was obtained as its chlorohydrate salt in quantitative yield after acidic treatment of compound 15.This strategy was employed to successfully prepare TzNAt B possessing a 4-methoxyphenyl substituent and TzNAt C bearing a 4-bromophenyl, starting from the same precursor 14 and using 4-methoxyphenylacetonitrile and 4-bromophenylacetonitrile respectively for the nucleophilic aromatic substitution step (Scheme 6).Therefore, we changed the ribose acetate protecting groups for the more stable tertbutyldimethylsilylether protecting groups.Starting from 2,3,5-triacetate-β-D-ribofuranosyl azide 2, the protected tertbutyldimethylsilylether ribose azide 13 was obtained in two steps using sodium methoxide, followed by the addition of tertbutyldimethylsilylchloride and imidazole, with excellent yield (Scheme 6).Next, the CuAAC click reaction between the azide 13 and the alkyne 9 proceeded smoothly, affording the desired triazole 14 in good yield.The reaction of 14 with phenylacetonitrile in the presence of sodium hydride gave the substituted product 15 in 30% yield.Finally, the nucleoside napthyridinone TzNat A was obtained as its chlorohydrate salt in quantitative yield after acidic treatment of compound 15.This strategy was employed to successfully prepare TzNAt B possessing a 4-methoxyphenyl substituent and TzNAt C bearing a 4-bromophenyl, starting from the same precursor 14 and using 4-methoxyphenylacetonitrile and 4-bromophenylacetonitrile respectively for the nucleophilic aromatic substitution step (Scheme 6).The structures of nucleosides TzNat A, B, and C were confirmed by NMR spectroscopy using 1D ( 1 H, JMOD and 13 C-DEPT) and 2D (COSY, HSQC, HMBC) experiments (See Supplementary Materials).

Optical Properties of TzNat Molecules
Optical properties of TzNat molecules as their chlorohydrate salts were studied in The structures of nucleosides TzNat A, B, and C were confirmed by NMR spectroscopy using 1D ( 1 H, JMOD and 13 C-DEPT) and 2D (COSY, HSQC, HMBC) experiments (See Supplementary Materials).

UV-Vis [b]
Fluorescence [c]   Compd Solvent [a]  λ abs (ε)  [a] .All TzNat have been pre-solubilized in DMSO at 10 −3 M and used it as stock solution to prepare 10 −5 M diluted solutions in solvents. [b] .Absorption maxima measured in solution (10 −5 M) at room temperature in nm (extinction coefficient at absorption maxima in 10 3 M −1 cm −1 ). [c] .Fluorescence wavelengths measured at room temperature at a concentration of 10 −5 M after excitation at the maximum absorption wavelength. [d] .Stokes shift in cm −1 defined as the wavelength difference between the absorption and emission peak maxima. [e] .Absolute fluorescence quantum yields were measured.
As shown in the UV-Vis spectra in Figure 2, TzNat A to C show similar absorption profiles.The wavelength of the maximum absorption depends strongly on the polarity of the solvent.In non-polar solvent and polar aprotic solvent, the maximum absorption is centered between 320 and 386 nm, assigned to a π-π* transition with molar extinction coefficients ranging from 17,200 to 3200 M −1 cm −1 .Interestingly, a bathochromic shift of this band is observed for all the TzNat from nonpolar solvent (CHCl 3 , CH 2 Cl 2 ) to polar aprotic solvent (THF, DMSO, DMF, DMSO, acetone).This phenomenon is more pronounced for TzNat B possessing a para-methoxyphenyl group, for which a red-shift of 70 nm was found for the position of the maximum absorption in EtOH or DMSO relating to nonpolar solvent CHCl 3 or CH 2 Cl 2 .This observed red-shift of the maximum absorption band from non-polar to polar solvent can be rationalized by the existence of an intramolecular charge transfer generated between the electron-donating group para-methoxy phenyl and the 1,2,3-triazole ring acting as an acceptor group [51].A less intense band (ε = 6700 to 400 M −1 •cm −1 ) centered at 459-474 nm is also observed in all solvents and is enhanced in non-polar solvent (CHCl3, CH2Cl2) as well as in protic solvent EtOH.However, in aqueous solvent (H2O and PBS), maximum absorption wavelengths of TzNat A to C ranging from 425 to 435 nm are recorded.This band is blue-shifted by 20 nm compared with that recorded in ethanol.
Overall, the UV properties of these molecules are in accordance with those recorded for 1,6-Naphthyridin-7(6H)-ones.[41] The maximum absorption ranging from 320 to 386 nm can be assigned to π→π* transition of the lactim form, whereas the absorption shift ranging from 425 to 474 nm could be related to the lactam form stabilized by intermolecular hydrogen bond with solvent.
The fluorescence properties of TzNat molecules were studied in solvents with different polarity (Table 1).Stokes shifts and fluorescence quantum yields (Φ) were determined in each solvent.The corresponding spectra are represented in Figure 3. Spectra recorded upon excitation at 320-386 nm in polar aprotic solvent acetone, THF, DMF, and DMSO show an emission band for the three molecules ranging from 458-502 nm, with modest Stokes shifts of 5300 to 6500 cm −1 and quantum yield up to Φ = 0.34 for TzNat B in THF.In particular, TzNat B shows a red-shift of emission wavelength (+10 nm) and a higher quantum yield compared to TzNat A and C when placed in a medium of increased polarity.The red-shift can be explained by dipole-dipole interactions between the excited fluorophore and the surrounding molecules decreasing the excited-state energy, which is more noticeable for molecule B possessing a donor-acceptor system.In addition, the dramatic increase in emission quantum yield (×15) in DMF and DMSO observed for TzNat B is in accordance with the presence of an intramolecular charge transfer (ICT) stabilized in a high polar solvent as mentioned above.A less intense band (ε = 6700 to 400 M −1 •cm −1 ) centered at 459-474 nm is also observed in all solvents and is enhanced in non-polar solvent (CHCl 3 , CH 2 Cl 2 ) as well as in protic solvent EtOH.However, in aqueous solvent (H 2 O and PBS), maximum absorption wavelengths of TzNat A to C ranging from 425 to 435 nm are recorded.This band is blue-shifted by 20 nm compared with that recorded in ethanol.
Overall, the UV properties of these molecules are in accordance with those recorded for 1,6-Naphthyridin-7(6H)-ones [41].The maximum absorption ranging from 320 to 386 nm can be assigned to π→π* transition of the lactim form, whereas the absorption shift ranging from 425 to 474 nm could be related to the lactam form stabilized by intermolecular hydrogen bond with solvent.
The fluorescence properties of TzNat molecules were studied in solvents with different polarity (Table 1).Stokes shifts and fluorescence quantum yields (Φ) were determined in each solvent.The corresponding spectra are represented in Figure 3. Spectra recorded upon excitation at 320-386 nm in polar aprotic solvent acetone, THF, DMF, and DMSO show an emission band for the three molecules ranging from 458-502 nm, with modest Stokes shifts of 5300 to 6500 cm −1 and quantum yield up to Φ = 0.34 for TzNat B in THF.In particular, TzNat B shows a red-shift of emission wavelength (+10 nm) and a higher quantum yield compared to TzNat A and C when placed in a medium of increased polarity.The red-shift can be explained by dipole-dipole interactions between the excited fluorophore and the surrounding molecules decreasing the excited-state energy, which is more noticeable for molecule B possessing a donor-acceptor system.In addition, the dramatic increase in emission quantum yield (×15) in DMF and DMSO observed for TzNat B is in accordance with the presence of an intramolecular charge transfer (ICT) stabilized in a high polar solvent as mentioned above.Interestingly, for TzNat A and C possessing a phenyl and a para-bromophenyl group respectively, an additional emission band is observed in DMSO and DMF at 560 nm.
Surprisingly, upon excitation of the lactim form at 320-386 nm in non-polar solvent CHCl3 and CH2Cl2 and in polar protic solvent EtOH, TzNat A, B, and C show very weak fluorescence emission centered at 470-490 nm, accompanied by a drop in quantum efficiency (Φ < 0.01).Fluorescence quenching was also observed in aqueous solvent H2O and PBS buffer after excitation at 420-430 nm corresponding to the lactam form (Φ < 0.01).
By analogy with parent 1,6-Naphthyridin-7(6H)-ones, the solvatochromic behavior of TzNat molecules, showing distinct bands in absorption and emission depending on the polarity of the solvent, could be rationalized by the presence of lactam and lactim forms in equilibrium.A lactim form would correspond to the high energy band at 458-502 nm which is favored in polar aprotic solvent, with a red-shift for TzNat B of 30 nm related to TzNat A and C.
The lactam form of TzNat generated by a proton transfer of the lactim form isomer at the excited state is thought to be responsible for the low-energy band at 560 nm in polar solvent for molecules substituted by a phenyl (TzNat A) and a para-bromo phenyl group (TzNat C).However, the lactam form is not observed for TzNat B due to the ICT.
The quench of fluorescence in protic solvent could be explained by the nonradiative relaxation of the excited fluorophores enhanced by the polarity of the environment and hydrogen bonding with protic solvent molecules.Therefore, a twisted intramolecular charge transfer (TICT) resulting from the shift of the π-electron density on the donoracceptor system at the excited state could also be involved to explain the quenching of the fluorescence in a protic solvent.[52] The twisted conformation is usually enhanced in a highly polar solvent possessing H-bonds and leads to full charge separation and a bathochromic shift.Indeed, in H2O, the weak emission band (Φ < 0.01) of the lactam form is observed red-shifted to 560 nm, and a second band of weak intensity centered at 560 nm is also observed in DMSO and DMF, which could be attributed to the TICT emission from the lactam form (Figure 4).Interestingly, TzNat A and C possessing a phenyl and a para-bromophenyl group respectively, an additional emission band is observed in DMSO and DMF at 560 nm.
Surprisingly, upon excitation of the lactim form at 320-386 nm in non-polar solvent CHCl 3 and CH 2 Cl 2 and in polar protic solvent EtOH, TzNat A, B, and C show very weak fluorescence emission centered at 470-490 nm, accompanied by a drop in quantum efficiency (Φ < 0.01).Fluorescence quenching was also observed in aqueous solvent H 2 O and PBS buffer after excitation at 420-430 nm corresponding to the lactam form (Φ < 0.01).
By analogy with parent 1,6-Naphthyridin-7(6H)-ones, the solvatochromic behavior of TzNat molecules, showing distinct bands in absorption and emission depending on the polarity of the solvent, could be rationalized by the presence of lactam and lactim forms in equilibrium.A lactim form would correspond to the high energy band at 458-502 nm which is favored in polar aprotic solvent, with a red-shift for TzNat B of 30 nm related to TzNat A and C.
The lactam form of TzNat generated by a proton transfer of the lactim form isomer at the excited state is thought to be responsible for the low-energy band at 560 nm in polar solvent for molecules substituted by a phenyl (TzNat A) and a para-bromo phenyl group (TzNat C).However, the lactam form is not observed for TzNat B due to the ICT.
The quench of fluorescence in protic solvent could be explained by the nonradiative relaxation of the excited fluorophores enhanced by the polarity of the environment and hydrogen bonding with protic solvent molecules.Therefore, a twisted intramolecular charge transfer (TICT) resulting from the shift of the π-electron density on the donoracceptor system at the excited state could also be involved to explain the quenching of the fluorescence in a protic solvent [52].The twisted conformation is usually enhanced in a highly polar solvent possessing H-bonds and leads to full charge separation and a bathochromic shift.Indeed, in H 2 O, the weak emission band (Φ < 0.01) of the lactam form is observed red-shifted to 560 nm, and a second band of weak intensity centered at 560 nm is also observed in DMSO and DMF, which could be attributed to the TICT emission from the lactam form (Figure 4).Nucleosides TzNat A, B, and C are environmentally sensitive fluorescent molecules, which are emissive in polar aprotic solvents with an enhanced quantum yield in the presence of charge transfer (TzNat B) and much less emissive in H2O.This dramatic change of emissivity can be used to probe interactions with surrounding molecules or as a reported group to study interactions with enzymes in incorporated oligomer [25].
Conformational studies of the triazole nucleosides TzNat A-C with computational methods will yield additional information about favored conformers, and help to rationalize the optical behavior and give evidence of the TICT mechanism.In-depth photo-physical studies of these compounds is currently under investigation.

General Information
All reagents and solvents were purchased from commercial suppliers, Sigma-Aldrich, TCI (Europe), Alfa Aesar, or Fluorochem.The monowave reactor is a conventionally heated synthesis reactor from Anton Paar.Infra-red spectra were recorded on ATR VariGATR crystal Ge.All reactions were monitored by TLC on silica gel 60 F254 plates and revealed using UV lamp (l 254 nm).Flash chromatography was carried out on a Merck silica gel 60 F 254. Reactions involving air-or moisture-sensitive reagents were carried out under an inert atmosphere (argon) with oven-dried glassware.THF was distillated over sodium/benzophenone.All final compounds were analyzed by high-resolution ESI mass spectrometry (HRMS) in MeOH using a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific, San Jose, CA, USA) equipped with an electrospray ion source.NMR spectra were recorded on a Bruker Avance spectrometer at 300 or 400 MHz for 1 H and 100 MHz for 13 C. 2D NMR experiments such as 1 H-1 H COSY and 1 H- 13 C HSQC experiments were performed to enable signal attributions.UV-Vis absorption spectra were measured using a Cary 50 (Varian) spectrophotometer at 20 °C.Emission spectra were performed on a JASCO J-815 CD spectrofluorometer at 20 °C.Quantum yields were determined with a Fluoromax+ (Jobin Yvon) equipped with a quanta phi integration sphere.Data were treated with the dedicated provided by the supplier.Measurements were performed at room temperature.Synthesis and data concerning compounds 3 and compounds 5 are already described [29].Nucleosides TzNat A, B, and C are environmentally sensitive fluorescent molecules, which are emissive in polar aprotic solvents with an enhanced quantum yield in the presence of charge transfer (TzNat B) and much less emissive in H 2 O.This dramatic change of emissivity can be used to probe interactions with surrounding molecules or as a reported group to study interactions with enzymes in incorporated oligomer [25].

Attempts to
Conformational studies of the triazole nucleosides TzNat A-C with computational methods will yield additional information about favored conformers, and help to rationalize the optical behavior and give evidence of the TICT mechanism.In-depth photo-physical studies of these compounds is currently under investigation.

Scheme 3 .
Scheme 3. Attempts to use click reaction to prepare triazole conjugate 4 from acetylated ribosyl azide 2.

Scheme 3 .
Scheme 3. Attempts to use click reaction to prepare triazole conjugate 4 from acetylated ribosyl azide 2.

Scheme 4 .
Scheme 4. Retrosynthetic analysis of the synthesis of triazole conjugate TzNat A.

Scheme 5 .
Scheme 5. First attempt for the synthesis of TzNat A.

Scheme 5 . 21 Scheme 6 .
Scheme 5. First attempt for the synthesis of TzNat A. Molecules 2024, 29, x FOR PEER REVIEW 8 of 21

Scheme 6 .
Scheme 6. Successful strategy to prepare TzNat A, B, and C.

Table 1 .
UV-Vis Absorption and Fluorescence Emission Properties of Compounds TzNat A-C in various solvents.