Fluorescent Biaryl Uracils with C5-Dihydro- and Quinazolinone Heterocyclic Appendages in PNA.

There has been much effort to exploit fluorescence techniques in the detection of nucleic acids. Canonical nucleic acids are essentially nonfluorescent; however, the modification of the nucleobase has proved to be a fruitful way to engender fluorescence. Much of the chemistry used to prepare modified nucleobases relies on expensive transition metal catalysts. In this work, we describe the synthesis of biaryl quinazolinone-uracil nucleobase analogs prepared by the condensation of anthranilamide derivatives and 5-formyluracil using inexpensive copper salts. A selection of modified nucleobases were prepared, and the effect of methoxy- or nitro- group substitution on the photophysical properties was examined. Both the dihydroquinazolinone and quinazolinone modified uracils have much larger molar absorptivity (~4–8×) than natural uracil and produce modest blue fluorescence. The quinazolinone-modified uracils display higher quantum yields than the corresponding dihydroquinazolinones and also show temperature and viscosity dependent emission consistent with molecular rotor behavior. Peptide nucleic acid (PNA) monomers possessing quinazolinone modified uracils were prepared and incorporated into oligomers. In the sequence context examined, the nitro-substituted, methoxy-substituted and unmodified quinazolinone inserts resulted in a stabilization (∆Tm = +4.0/insert; +2.0/insert; +1.0/insert, respectively) relative to control PNA sequence upon hybridization to complementary DNA. All three derivatives responded to hybridization by the “turn-on” of fluorescence intensity by ca. 3-to-4 fold and may find use as probes for complementary DNA sequences.


Introduction
Since the 1980s, much effort has been devoted to exploiting fluorescence techniques in the detection and study of nucleic acids. While aromatic-based amino acids (phenylalanine, tyrosine and tryptophan) possess intrinsic fluorescence, natural nucleic acids are essentially nonfluorescent [1,2]. In order to facilitate studies with nucleic acids, structurally modified oligonucleotides that incorporate a luminophore have been pursued aggressively. There is a wide range of methods used to produce fluorescent oligonucleotides, for example, by the substitution of a structural feature-such as the nucleobase-with a fluorophore, or by appending a chromophore to the nucleobase or sugar-phosphate backbone [3]. An especially fruitful approach has been to expand the nucleobase by ring fusion or Peptide nucleic acid (PNA) is an oligonucleotide analog capable of forming highly stable complexes with its target nucleic acid [8]. One of the attractive features of PNA is its hybridization properties-a combination of high affinity and high selectivity [9] that enable its use as a probe molecule [10]. Numerous modified nucleobases have been incorporated into PNA [11]; two notable brightly fluorescent cytosine analogs are 3,5-diaza-4-oxophenothiazine (tC) [12] and phenylpyrrolocytosine (PhpC) [13], each involving multi-step ring construction syntheses ( Figure 2). With interest in simplifying the synthesis of a uracil with a biaryl-type bond to a heterocycle, we turned to the single-pot condensation of 5-formyluracil derivatives [14] with substituted 2aminobenzamides [15]. This route has the advantage of ease of synthesis that permitted the rapid preparation and evaluation of a selection of substituted quinazolinone-uracil derivatives.

Synthesis of Dihydroquinazolinone-Based Uracil Scaffold
Herein, we describe a simple and convenient route for the synthesis of dihydroquinazolinoneuracil and quinazolinone-uracil scaffolds (Scheme 1), as well as an evaluation of their basic photophysical properties. The synthesis of the both the dihydroquinazolinone-and quinazolinoneuracils started with 5-formyluracil (1), accessed from 5-hydroxymethyluracil as previously described [14]. Next, 5-formyluracil (1) was alkylated under standard conditions with tert-butyl bromoacetate to give tert-butyl (5-formyluracil-1-yl)acetate (2) with high yield (Scheme 1). Peptide nucleic acid (PNA) is an oligonucleotide analog capable of forming highly stable complexes with its target nucleic acid [8]. One of the attractive features of PNA is its hybridization properties-a combination of high affinity and high selectivity [9] that enable its use as a probe molecule [10]. Numerous modified nucleobases have been incorporated into PNA [11]; two notable brightly fluorescent cytosine analogs are 3,5-diaza-4-oxophenothiazine (tC) [12] and phenylpyrrolocytosine (PhpC) [13], each involving multi-step ring construction syntheses ( Figure 2).
Molecules 2020, 25, x FOR PEER REVIEW 2 of 21 such as the nucleobase-with a fluorophore, or by appending a chromophore to the nucleobase or sugar-phosphate backbone [3]. An especially fruitful approach has been to expand the nucleobase by ring fusion or to extend the conjugation through an ethylene or ethyne bridge and/or by direct coupling to another aromatic moiety via a biaryl bond [4,5]; these routes overwhelmingly employ precious metal catalysts. The 5-heteroaromatic uracils are attractive targets as modified nucleobases since they have been shown to retain their hydrogen bonding ability to the complementary nucleobases but have the potential to be fluorescent and possess molecular rotor properties that enable them to be "turn on" probes of nucleic acid structure [6,7] (Figure 1). Peptide nucleic acid (PNA) is an oligonucleotide analog capable of forming highly stable complexes with its target nucleic acid [8]. One of the attractive features of PNA is its hybridization properties-a combination of high affinity and high selectivity [9] that enable its use as a probe molecule [10]. Numerous modified nucleobases have been incorporated into PNA [11]; two notable brightly fluorescent cytosine analogs are 3,5-diaza-4-oxophenothiazine (tC) [12] and phenylpyrrolocytosine (PhpC) [13], each involving multi-step ring construction syntheses ( Figure 2). With interest in simplifying the synthesis of a uracil with a biaryl-type bond to a heterocycle, we turned to the single-pot condensation of 5-formyluracil derivatives [14] with substituted 2aminobenzamides [15]. This route has the advantage of ease of synthesis that permitted the rapid preparation and evaluation of a selection of substituted quinazolinone-uracil derivatives.

Synthesis of Dihydroquinazolinone-Based Uracil Scaffold
Herein, we describe a simple and convenient route for the synthesis of dihydroquinazolinoneuracil and quinazolinone-uracil scaffolds (Scheme 1), as well as an evaluation of their basic photophysical properties. The synthesis of the both the dihydroquinazolinone-and quinazolinoneuracils started with 5-formyluracil (1), accessed from 5-hydroxymethyluracil as previously described [14]. Next, 5-formyluracil (1) was alkylated under standard conditions with tert-butyl bromoacetate to give tert-butyl (5-formyluracil-1-yl)acetate (2) with high yield (Scheme 1). With interest in simplifying the synthesis of a uracil with a biaryl-type bond to a heterocycle, we turned to the single-pot condensation of 5-formyluracil derivatives [14] with substituted 2-aminobenzamides [15]. This route has the advantage of ease of synthesis that permitted the rapid preparation and evaluation of a selection of substituted quinazolinone-uracil derivatives.

Synthesis of Quinazolinone-Based Uracil Scaffolds
Substituted quinazolines have been synthesized by a number of methods, including the condensation of 2-aminobenzylamines with aldehydes using copper (II) nitrate in refluxing ethanol followed by subsequent treatment with a stoichiometric oxidant [16]. It has been observed in cases that the Lewis acid catalyst for the condensation may also promote oxidation. Catalytic aerobic oxidation (CuCl/DABCO/4-HO-TEMPO) has been developed for the preparation of substituted quinazolines [17], and we used this as a starting point to determine the optimal conditions for the quinazolinone-uracils of interest (Scheme 2).

Synthesis of Quinazolinone-Based Uracil Scaffolds
Substituted quinazolines have been synthesized by a number of methods, including the condensation of 2-aminobenzylamines with aldehydes using copper (II) nitrate in refluxing ethanol followed by subsequent treatment with a stoichiometric oxidant [16]. It has been observed in cases that the Lewis acid catalyst for the condensation may also promote oxidation. Catalytic aerobic oxidation (CuCl/DABCO/4-HO-TEMPO) has been developed for the preparation of substituted quinazolines [17], and we used this as a starting point to determine the optimal conditions for the quinazolinone-uracils of interest (Scheme 2).

Photophysical Properties
UV-Vis spectra and fluorescence measurements of compounds 7-16 have been made in different solvents in order to characterize their basic photophysical properties (Tables 2 and 3). Determinations of UV-vis absorption molar extinction coefficients (Ɛ) were made only in DMSO due to limited solubility. Overall, the values generally indicate that the quantum yield in ethanol is greater than that in other solvents for dihydroquinazolinone compounds (Table 2). Additionally, the emission is bathochromically shifted in less polar solvents (THF) versus polar solvents (EtOH). However, since dihydroquinazolinone compounds (7)(8)(9)(10)(11) were only very weakly emissive, they were not pursued further for use in PNA monomers or oligomers. We investigated the optimal conditions for the reaction of commercially available anthranilamide (R 1 = H, R 2 = H) (1 equiv) with N1-alkylated-5-formyluracil 2 (1 equiv). Firstly, the two starting materials were allowed to condense in situ, and then 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-HO-TEMPO) (0.2 equiv) was added as the catalyst, and the reaction system was charged with an oxygen atmosphere (balloon) [18]. The target molecule 12 was obtained, but with poor yield due to presumed decomposition of the dihydroquinazolinone intermediate (Table 1, entry 1). To our delight, the reaction was dramatically improved when CuCl was used in combination with a monodentate N-containing ligand such as Et 3 N, DABCO (1,octane), or DMAP (4-dimethylaminopyridine) ( Table 1, entries 4-8). Entry seven demonstrates the need for an oxygen atmosphere for high conversions. The optimized conditions, (Table 1, entry 8) were also used for the oxidation of dihydroquinazolinones 7-11 to quinazolinones 12-16, respectively (Scheme 1).

Photophysical Properties
UV-Vis spectra and fluorescence measurements of compounds 7-16 have been made in different solvents in order to characterize their basic photophysical properties (Tables 2 and 3). Determinations of UV-vis absorption molar extinction coefficients (ε) were made only in DMSO due to limited solubility. Overall, the values generally indicate that the quantum yield in ethanol is greater than that in other solvents for dihydroquinazolinone compounds (Table 2). Additionally, the emission is bathochromically shifted in less polar solvents (THF) versus polar solvents (EtOH). However, since dihydroquinazolinone compounds (7)(8)(9)(10)(11) were only very weakly emissive, they were not pursued further for use in PNA monomers or oligomers.  (455) a The quantum yields were determined using emission wavelengths between 340 and 640 nm, with an excitation wavelength of 325 nm. b Excitation wavelength at 310 nm. c λ max emission, in nanometers, in parentheses. Table 3. Photophysical properties a of quinazolinone compounds 12-16. The planarity and rigidity of the molecular structure plays a major role in determining the fluorescence properties of aromatic compounds [19].
Thus, the dehydrogenation of dihydroquinazolinone compounds 7-11 (Schemes 1 and 2) was done to increase conjugation and molecular rigidity. It was envisioned that the quinazolinone structure would favor being coplanar with the uracil by an intramolecular hydrogen bond (Figure 3a,b) and this would potentially benefit the fluorescence quantum yield. Computational studies of the two structures (Figure 3a,b) was done at the Hartree-Foch 6-31+G* level to determine the most favorable tautomer and the minimum energy conformation. The calculated structures show that the planes defined by the dihydroquinazolinone and uracil have a severe twist (~70 • ) about the biaryl bond (shown in red, Figure 3a), whereas the quinazolinone and uracil exists in a coplanar geometry with a hydrogen bond (predicted length 1.99 Å) between the amide proton and C4-oxygen.
at the Hartree-Foch 6-31+G* level to determine the most favorable tautomer and the minimum energy conformation. The calculated structures show that the planes defined by the dihydroquinazolinone and uracil have a severe twist (~70°) about the biaryl bond (shown in red, Figure 3a), whereas the quinazolinone and uracil exists in a coplanar geometry with a hydrogen bond (predicted length 1.99 Å) between the amide proton and C4-oxygen.  The trends in the fluorescence quantum yields largely support this notion, i.e., that the quinazolinone-uracils (12)(13)(14)(15)(16) are more emissive than the dihydroquinzolinone congeners (7)(8)(9)(10)(11) (Table 3). However, the nitro-substituted quinazolinone compounds also tended to be less soluble, and measurements could not be made in ethanol. We also investigated the effects of temperature and environmental viscosity (ethanol versus glycerol) on the fluorescent properties ( Table 3). The increase in fluorescence quantum yield when moving from ethanol (or other less viscous solvents) to the viscous glycerol is consistent with a reduced loss of excited state energy through intramolecular rotations, particularly with respect to the biaryl bond. Further evidence of the molecular rotor behavior is shown by the increase in quantum yield as the temperature is lowered.

PNA Monomer Synthesis and Oligomerization
The quinazolinone-based uracil scaffolds were designed to retain the usual hydrogen bonding ability to a complementary nucleobase. In order to test the ability of these modified bases to respond to hybridization, i.e., the transition from a relatively unstructured single-stranded probe to the more highly structured environment of duplex, a selection of PNA monomers were prepared. A priori, it was not known how quinazolinone ring substitution would affect hybridization (stacking) interactions or fluorescence properties in the context of oligomers; thus, the unsubstituted quinazolinone-based uracil scaffold (12), the nitro-substituted quinazolinone (13) and the methoxy-substituted quinzaolinone (16) were chosen to prepare PNA monomers (Scheme 3).
Proceeding to monomer synthesis and oligomerization, the tert-butyl esters of the quinazolinone based uracil scaffolds (12, 13 and 16) were converted to the free acids (19, 20 and 21) by acidolysis with trifluoroacetic acid in the presence of triethylsilane. The free base of the tert-butyl-Fmoc-based aminoethylglycine backbone was liberated from the hydrochloride salt (18) immediately prior to use [20]. The quinazolinone-based nucleobase acetic acid was then converted to the active ester, in situ, by treatment with a solution of hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC). The active ester of the nucleobase derivative was then coupled to the tert-butyl-Fmoc-based aminoethylglycine backbone in the presence of DMAP to give the monomer esters with usual yields. Finally, the monomers (25, 26 and 27) were produced by the acidolysis of the tert-butyl esters, as previously done. The monomers were characterized using 1 H and 13 C NMR spectroscopy, as is usual for PNA monomer signals, indicating the presence of rotomers (see supplemental data). Additionally, the compounds were identified by high-resolution mass spectrometry (HRMS).
With monomers 25, 26 and 27 in hand, PNA sequences were prepared by automated peptide synthesis ( Table 1). The three PNA monomers performed well, with no significant difference between them and commercially available standard PNA monomers (Table S1 and Page S5-S9). As is commonly done, all of the oligomers were constructed with a C-terminal lysine in order to impart water solubility. Once the oligomers were in hand, thermal stability (Tm) analysis with complementary DNA and PNA was undertaken.
The unmodified PNA control sequence hybridization with complementary PNA showed excellent agreement with our previous report (67.5 °C) [20]. The unmodified quinazolinone showed a slight decrease in the thermal stability of the duplex compared to that for an unmodified PNA (~∆Tm = −1.0 °C per insert), which may be useful in pseudocomplementary PNA applications as a fluorescent reporter, although this requires further study. On the other hand, both the nitro-and methoxy-substituted quinazolinones showed an increase in Tm values when hybridized to the PNA strand. (Table 4). Similar results were observed for hybridization with complementary (underlined) DNA (5′-AGTGATCTACCT-3′): the nitro-substituted quinazolinone had the greatest stabilizing effect (∆Tm = +4.0 °C per insert); next was methoxy-substituted quinazolinone (∆Tm = +2.0 °C per insert), while the unsubstituted quinazolinone gave a slight stabilization (∆Tm = +1.0 °C per insert). Proceeding to monomer synthesis and oligomerization, the tert-butyl esters of the quinazolinone based uracil scaffolds (12, 13 and 16) were converted to the free acids (19, 20 and 21) by acidolysis with trifluoroacetic acid in the presence of triethylsilane. The free base of the tert-butyl-Fmoc-based aminoethylglycine backbone was liberated from the hydrochloride salt (18) immediately prior to use [20]. The quinazolinone-based nucleobase acetic acid was then converted to the active ester, in situ, by treatment with a solution of hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC). The active ester of the nucleobase derivative was then coupled to the tert-butyl-Fmoc-based aminoethylglycine backbone in the presence of DMAP to give the monomer esters with usual yields. Finally, the monomers (25, 26 and 27) were produced by the acidolysis of the tert-butyl esters, as previously done. The monomers were characterized using 1 H and 13 C NMR spectroscopy, as is usual for PNA monomer signals, indicating the presence of rotomers (see supplemental data). Additionally, the compounds were identified by high-resolution mass spectrometry (HRMS).
With monomers 25, 26 and 27 in hand, PNA sequences were prepared by automated peptide synthesis ( Table 1). The three PNA monomers performed well, with no significant difference between them and commercially available standard PNA monomers (Table S1 and Page S5-S9). As is commonly done, all of the oligomers were constructed with a C-terminal lysine in order to impart water solubility. Once the oligomers were in hand, thermal stability (T m ) analysis with complementary DNA and PNA was undertaken.
The unmodified PNA control sequence hybridization with complementary PNA showed excellent agreement with our previous report (67.5 • C) [20]. The unmodified quinazolinone showed a slight decrease in the thermal stability of the duplex compared to that for an unmodified PNA (~∆Tm = −1.0 • C per insert), which may be useful in pseudocomplementary PNA applications as a fluorescent reporter, although this requires further study. On the other hand, both the nitro-and methoxy-substituted quinazolinones showed an increase in T m values when hybridized to the PNA strand. (Table 4). Similar results were observed for hybridization with complementary (underlined) DNA (5 -AGTGATCTACCT-3 ): the nitro-substituted quinazolinone had the greatest stabilizing effect (∆T m = +4.0 • C per insert); next was methoxy-substituted quinazolinone (∆T m = +2.0 • C per insert), while the unsubstituted quinazolinone gave a slight stabilization (∆T m = +1.0 • C per insert).

Fluorescence Properties Analysis
The steady-state fluorescence excitation and emission spectra were measured for single-stranded PNA Q U, Q U (OMe) and Q U (NO2) and heteroduplexes with fully complementary DNA (Figure 4a-c). Trends similar to those in the fluorescence studies at the submonomer level were observed, that is, the nitro-substituted quinazolinone uracil scaffold shows the weakest fluorescent intensity while the methoxy-substituted has the highest. However, each of the nucleobase fluorophores displayed a "turn-on" response of approximately 3-to-4 fold to hybrid formation. Interestingly, the Q U (NO2) oligomer displayed the largest fluorescence "turn-on" response, without a notable change in the peak wavelength. The Q U (NO2) oligomer also shows the greatest increase in the stability of the complex as measured by the T m value. A plausible explanation is that the Q U (NO2) oligomer has relatively stronger stacking interactions, leading to both a more stabilized hybrid and a relatively more rigid structure, providing a slightly better fluorescence turn-on effect. The more electron-rich parent quinazolinone and 6-methoxyquinazolinone both displayed a hypsochromic shift in the emission for the hybrid, which likely reflects the change in the polarity of the environment of the fluorophore. Nonetheless, all three fluorophores showed marked increases in emission upon hybridization, which is consistent with increased rigidity in the duplex and the molecular rotor behavior of biaryl-chromophores. The degree of fluorescence intensity in quinazolinone uracil scaffolds appears to weakly correlate with the helix-stabilizing ability of the base. The most stable heteroduplex occurs with Q U (NO2) -modified PNA. PNA oligomers are listed from the pseudo 5′-terminus to the pseudo 3′-end. Lys = L-lysine, Q U (NO2) = 6-nitroquinazolinone uracil, Q U (OMe) = 6-methoxyquinazolinone uracil, Q U = quinazolinone uracil PNA residues. * The complementary DNA oligomer 5′-AGTGATCTAC-3′ was used. † The complementary PNA oligomer H-Lys-AGTGATCTAC-Lys-NH2 was used. Temperature-dependent UV spectra at 2 µM strand concentration, each in 100 mM sodium phosphate buffer, pH = 7.0.

Fluorescence Properties Analysis
The steady-state fluorescence excitation and emission spectra were measured for singlestranded PNA Q U, Q U (OMe) and Q U (NO2) and heteroduplexes with fully complementary DNA ( Figure  4a,b,c). Trends similar to those in the fluorescence studies at the submonomer level were observed, that is, the nitro-substituted quinazolinone uracil scaffold shows the weakest fluorescent intensity while the methoxy-substituted has the highest. However, each of the nucleobase fluorophores displayed a "turn-on" response of approximately 3-to-4 fold to hybrid formation. Interestingly, the Q U (NO2) oligomer displayed the largest fluorescence "turn-on" response, without a notable change in the peak wavelength. The Q U (NO2) oligomer also shows the greatest increase in the stability of the complex as measured by the Tm value. A plausible explanation is that the Q U (NO2) oligomer has relatively stronger stacking interactions, leading to both a more stabilized hybrid and a relatively more rigid structure, providing a slightly better fluorescence turn-on effect. The more electron-rich parent quinazolinone and 6-methoxyquinazolinone both displayed a hypsochromic shift in the emission for the hybrid, which likely reflects the change in the polarity of the environment of the fluorophore. Nonetheless, all three fluorophores showed marked increases in emission upon hybridization, which is consistent with increased rigidity in the duplex and the molecular rotor behavior of biaryl-chromophores. The degree of fluorescence intensity in quinazolinone uracil scaffolds appears to weakly correlate with the helix-stabilizing ability of the base. The most stable heteroduplex occurs with Q U (NO2) -modified PNA. (a)

2-Amino-4-methoxybenzamide (5)
60 °C, triphosgene (0.45 g, 1.5 mmol) was added and the reaction was stirred for 10 h. The reaction progress was monitored by TLC analysis. The solution was cooled to room temperature, and the isatoic anhydride intermediate was filtered and collected. Cold 1N ammonia was added to the collected intermediate, and the mixture was stirred for 24 h at 4 °C. The turbid solution was filtered and washed with water and ether to give 0.430 g (2.3 mmol, 48%) 2, 135.2, 128.0, 126.9, 116.4, 112.5.

2-Amino-4-nitrobenzamide (6)
2-Amino-4-nitrobenzoic acid (0.549 g, 3.0 mmol) was dissolved in 6 mL THF and, heated to 60 °C, triphosgene (0.3 g, 1 mmol) was added, the reaction was stirred for 5 h and the reaction progress was monitored by TLC. The solution was cooled to room temperature, and the isatoic anhydride intermediate was filtered and collected. Cold 1N ammonia was added to the collected intermediate,

2-Amino-4-nitrobenzamide (6)
2-Amino-4-nitrobenzoic acid (0.549 g, 3.0 mmol) was dissolved in 6 mL THF and, heated to 60 °C, triphosgene (0.3 g, 1 mmol) was added, the reaction was stirred for 5 h and the reaction progress was monitored by TLC. The solution was cooled to room temperature, and the isatoic anhydride intermediate was filtered and collected. Cold 1N ammonia was added to the collected intermediate,

Thermal Stability Analysis
The thermal stabilities (melting temperature, T m ) of complexes were measured in solutions of 100 mM NaCl, 10 mM sodium hydrogen phosphate, 0.1 mM EDTA, pH = 7.0 with individual PNA strand concentrations of 2 µM. Absorbance at λ = 260 nm was measured at 0.5 • C intervals while the temperature was changed at a rate of 0.7 • C/min between 15 and 90 • C. T m values were measured in triplicate and determined by the first derivative method applied through the manufacturer-supplied Varian WinUV Bio software.

Computational Studies
Structures were constructed in Spartan '14 and ground state energy was minimized using a desktop computer at the Hartree-Foch 6-31+G* level. A tautomer search was conducted at the Hartree-Foch 6-31+G* level in order to confirm the lowest energy structures that represented the most favorable tautomeric forms.

Conclusions
A facile synthesis of C5-(quinazolinone)-uracil PNA monomers has been established that is compatible with standard Fmoc-based oligomerization chemistry. The modified nucleobases bearing quinazolinone moieties were characterized by their fluorescence quantum yield in nonpolar (THF) and polar solvents (EtOH, DMSO), and their responses to changes in viscosity and temperature are consistent with molecular rotor behavior. Single modifications incorporated into a PNA decamer resulted in the stabilization of hybrids formed with complementary DNA, as judged by the UV-vis measured T m values, in the following order: Q U (NO2) > Q U (OMe) > Q U. Interestingly, this order is qualitatively reflected by the degree of fluorescence increase going from the single-stranded PNA to the PNA-DNA heteroduplex ( Q U (NO2) > Q U (OMe) > Q U). The Q U (OMe) and Q U labelled PNA oligomers dually report hybridization by an increase in fluorescence intensity and a large (~50 nm) hypsochromic shift. Thus, quinazolinone-based uracil analogs are good candidates for reporting binding events by showing emission "turn-on" properties upon hybridization to complementary DNA.