Diphenyl-Furanones and Diphenyl-Oxopyrrole Derivatives: From Analytical Reagents for Amino Groups to New Fluorochromes for Cytochemical Staining of Chromatin DNA and Chromosomes: Proposal for Intercalative Binding and Fluorescence Mechanism

: Diaryl-furanones are speciﬁc analytical reagents for the biochemical detection of primary amines by ﬂuorescence techniques. Well-known reagents are ﬂuorescamine (Fluram) and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), yielding ﬂuorescent products with λ em at 480– 490 nm. Although the reaction products claim to be pyrrolinones, recent studies show that they are really 3-oxopyrrole (pyrrolone) derivatives. Both reagents have been used for the cytochemical demonstration of primary amines. In this work, we have applied the ﬂuorescent products of MDPF with amines (n-butylamine, BA; glucosamine, GA; and spermine, Sp), which showed interesting ﬂuorescence reactions with chromatin DNA. 2,4-diphenyl-3-oxopyrrole products (diPOPy) can be easily synthesized according to well-known procedures, by mixing solutions of MDPF in acetone with water at pH 9 containing the amino compounds. DiPOPy derivatives of BA, GA, and Sp were used for spectroscopic, microscopic, and molecular modeling studies, showing a bright and selective blue–green ﬂuorescence on DNA substrates, mainly chromatin, kinetoplast DNA, and stretched chromatin ﬁbers. The cationic diPOPy ﬂuorophore is planar, with a high partial positive charge in the N atom, and suitable for intercalative binding to DNA. A mechanism of ﬂuorescamine ﬂuorescence due to an inner-salt isomeric form is proposed, and an astonishing correlation between adenine– thymine-rich centromeric heterochromatin in mouse metaphase chromosomes after reaction of the ﬂuorescamine reagent with protein amino groups is also discussed.

At present, previously formed fluorescent fluorescamine and MDPF products in vitro have been scarcely used as direct fluorochromes [36]. However, considering that diPOPy and the fluorescamine derivative (2-benzoic-4-phenyl-3-oxopyrrole, BzPOPy) are strongly fluorescent [13,28,29], and presuming an expected affinity of the cationic MDPF derivatives for polyanionic substrates, these derivatives could be used for staining fixed cells. Indeed, when these products were applied to fixed cells, a striking fluorescence of chromatin DNA was observed. Although a similar fluorescence is also expected to occur with other polyanionic substrates (e.g., RNA, acid glycosaminoglycans, etc.), only the selective From these early studies and throughout the literature, 2-hydroxy-pyrrolinones have been customarily accepted as the fluorescent products of fluorescamine and MDPF with primary amines. However, for non-planar molecules, such as 2-hydroxy-pyrrolinones, the long absorption (370-380 nm) and emission (480-490 nm) wavelengths they actually show are quite unexpected features [36]. In fact, the occurrence of a tetrahedral C2 atom disrupts the conjugation of the three ring systems, thus avoiding the formation of a coplanar fluorophore, necessary for such long absorption and emission wavelengths. Therefore, the 2-hydroxy-pyrrolinone ( Figure 1B,B ) just corresponds to the pseudobase of the true fluorophore, which accordingly is a coplanar, full conjugated and cationic 2,4-diphenyl-3oxopyrrole (diPOPy, Figure 1C,C ) [37][38][39].
At present, previously formed fluorescent fluorescamine and MDPF products in vitro have been scarcely used as direct fluorochromes [36]. However, considering that diPOPy and the fluorescamine derivative (2-benzoic-4-phenyl-3-oxopyrrole, BzPOPy) are strongly fluorescent [13,28,29], and presuming an expected affinity of the cationic MDPF derivatives for polyanionic substrates, these derivatives could be used for staining fixed cells. Indeed, when these products were applied to fixed cells, a striking fluorescence of chromatin DNA was observed. Although a similar fluorescence is also expected to occur with other polyanionic substrates (e.g., RNA, acid glycosaminoglycans, etc.), only the selective reaction of diaryl-POPy dyes with DNA will be emphasized in the present work. An intercalative binding mode and a fluorescence mechanism for fluorescamine reaction products are also proposed and discussed here.
Colorants 2023, 2, FOR PEER REVIEW 3 reaction of diaryl-POPy dyes with DNA will be emphasized in the present work. An intercalative binding mode and a fluorescence mechanism for fluorescamine reaction products are also proposed and discussed here.

Materials and Methods
2-Methoxy 2,4-diphenyl 3(2H)-furanone (MDPF, >98% purity) was purchased from Fluka (Buchs, Switzerland). Amino compounds (n-butylamine (BA), dopamine HCl (DA), benzylamine, α-D-glucosamine HCl (GA), and spermine 4HCl (Sp)), as well as remaining reagents were purchased from Merck-Sigma-Aldrich (Darmstadt, Germany). Amino derivatives from MDPF were prepared from the amino compounds BA, GA, and Sp using well-known synthetic procedures [2,7,13,29,38], yielding the corresponding 2,4-diphenyl-3-oxopyrrole products diPOPy-B, diPOPy-G, and bis-diPOPy-Sp, respectively ( Figure 2). Other amines were found difficult to use: benzylamine was practically insoluble in water, and blackening of dopamine by autopolymerization [40] hindered the fluorescence of the reaction product, so no further studies were performed with these amines. The fluorescent reaction products were formed directly by mixing 1:4 (v/v) a solution of MDPF (2 mg/mL in acetone) with distilled water adjusted to pH 9 containing the solid amino compounds (GA, Sp, 1 mg/mL) or a 10% solution of BA. After standing at room temperature (RT) for 5 min, the final solution was adjusted to pH 7 and either diluted 1:10 (v/v) with distilled water or allowed to dry at 40 °C. Values of pH were adjusted to 9 or 7 using sodium bicarbonate or 2.5% acetic acid, respectively. Only primary amines react with furanone reagents, giving stable fluorescent products [2][3][4]7,21,38]. In addition, as The fluorescent reaction products were formed directly by mixing 1:4 (v/v) a solution of MDPF (2 mg/mL in acetone) with distilled water adjusted to pH 9 containing the solid amino compounds (GA, Sp, 1 mg/mL) or a 10% solution of BA. After standing at room temperature (RT) for 5 min, the final solution was adjusted to pH 7 and either diluted 1:10 (v/v) with distilled water or allowed to dry at 40 • C. Values of pH were adjusted to 9 or 7 using sodium bicarbonate or 2.5% acetic acid, respectively. Only primary amines react with furanone reagents, giving stable fluorescent products [2][3][4]7,21,38]. In addition, as rapid degradation products of the reagents occur in aqueous solutions, yielding nonfluorescent 2-hydroxy-furanones [1, 13,35], no other compounds are formed during the simple synthetic procedure described here.
For staining procedures, samples of chicken blood and mouse spleen smears, as well as smears of T. cruzi epimastigotes (12 × 10 6 cells/mL) growing at 27 • C, were fixed in methanol for 2 min and stained with a 20 µg/mL solution of diPOPy-B or bis-diPOPy-Sp in distilled water for 5 min, washed in distilled water, air-dried, and mounted in DePeX. Before staining, some smears were subjected to DNA extraction by using DNase I (Sigma, 0.5 mg/mL in 1 mM MgCl 2 at 37 • C for 2 h) or 5% trichloroacetic acid (TCA, at boiling temperature for 20 min). After staining, some chicken blood and mouse spleen smears were also washed for 30 min with either aqueous 2 M NaCl solution, 4 M urea, or a saturated aqueous solution of caffeine or ortho-phenanthroline, which are known competitive assays for revealing dye binding by electrostatic forces, H-bonding, or hydrophobic interactions, respectively [36,48].
On the other hand, from a historical perspective and for comparative purposes, the microscopic visualization of amino groups from basic proteins is also presented here using the pristine analytical reagent fluorescamine. As model microscopic structures, horse eosinophils containing basic proteins are very suitable cells on account of the large size of their highly acidophilic granules, allowing a precise analysis of fluorescence reactions [29,49,50]. In this case, a solution of fluorescamine (0.5 mL of a 0.5 mg/mL solution in acetone) was added to methanol-fixed smears of horse blood previously covered with 0.5 mL of borate buffer at pH 9 [28]. After treatment for 5 min at RT, smears were washed in distilled water, air-dried, and mounted in DePeX.
Microscopic observation and photography were performed using an Olympus BX-61 epifluorescence photomicroscope (Hamburg, Germany) equipped with an HBO 100-W mercury lamp and the ultraviolet (UV, 365 nm) exciting filter. Images were captured with an Olympus DP50 CCD camera and processed using Adobe Photoshop 6.0 (San José, CA, USA) and PowerPoint 2000 (Microsoft, Redmont, WA, USA) software. Unstained smears were previously analyzed for autofluorescence, which in all cases was negligible.
Spectroscopic studies on diPOPy derivatives (10-20 µg/mL in distilled water at pH 7) were performed using a spectrophotometer Shimadzu UV-VIS 1604, Columbia, MD, USA, and a spectrofluorometer Perkin-Elmer 650-10S (Chicago, IL, USA) equipped with a 150-W xenon lamp, two grating monochromators, the Hamamatsu R372F detector (wavelength range: 220-730 nm), and excitation at 360 nm. When used in the normal mode, emission spectra that are corrected for variation in the excitation intensity are obtained. Because of its negligible contribution, the Raman scattering of solvents was not subtracted from emission spectra. Molecular parameters were studied by simple inspection of chemical structures such as the conjugated bond number (CBN, for the overall size of the conjugated system), and calculated or measured molecular size (width/length, W/L values) [51]. Molecular modeling studies on protonated diPOPy (diPOPy-H), diPOPy-G, and bis-diPOPy-Sp intercalated into an AT-DNA tetramer, as well as fluorescamine and its reaction products with amino groups (BzPOPy), were carried out using the HyperChem v7 and v8.0.10 software (Hypercube, Inc., Gainesville, USA). As performed in previous molecular modeling studies [52,53], geometry optimization was achieved with the MM+ routine, followed by energy minimization by either the semi-empirical PM3 method (Polak-Ribière conjugate gradient) converged at 1, 0.5, 0.2, or 0.1 kcal/(Å mol), or using the extended Hückel method.

DiPOPy Fluorochromes from MDPF
The aspects of freshly made GA and diPOPy-G solutions (20 µg/mL in distilled water adjusted to pH 7) either under day light illumination or 420 nm excitation are shown in Figure 3A,B, which clearly reveal a light yellow absorption color and the bright blue-green fluorescence of diPOPy-G.

DiPOPy Fluorochromes from MDPF
The aspects of freshly made GA and diPOPy-G solutions (20 µg/mL in distilled water adjusted to pH 7) either under day light illumination or 420 nm excitation are shown in Figure 3A,B, which clearly reveal a light yellow absorption color and the bright bluegreen fluorescence of diPOPy-G. Identical absorption and fluorescence colors are also found in diPOPy-B and bis-diPOPy-Sp. Spectroscopic studies of diPOPy-B, diPOPy-G, and bis-diPOPy-Sp revealed absorption and emission maxima at 385 and 487 nm, respectively. Absorption and emission spectra of diPOPy-B (10 µg/mL in distilled water at pH 7) are shown in Figure 4. At neutral pH, all solutions were stable, and no spectral changes were found at mild alkaline or acid pH values. In the presence of 45% glycerol, 60% sucrose, and 40% dextran 60 in water, samples showed an emission peak about twice higher than that of aqueous solutions (not shown), which indicates the dependence of the fluorescence intensity on the viscosity of the solvent. Another solvent feature that modulates fluorescence wavelength red-shift is its dielectric constant. The dielectric constant is a measure of how efficiently the solvent responds to the altered electronic distribution of the fluorophore in the excited state. A solvent with a high dielectric constant (e.g., water) quickly reorients its dipoles to shield the altered electric field surrounding the excited molecule. This reorientation implies the use of energy that comes from the excited state. Therefore, once equilibrium is reached, which takes less than a picosecond, the available energy for photon emission is less (red-shifted) as compared with the initial energy absorbed. Solvents with high dielectric constant (polar solvents) show a more pronounced reorientation effect than solvents with a small dielectric constant (non-polar solvents). In the solid state, microcrystals of POPy derivatives also exhibited strong fluorescence under 365 nm excitation. Identical absorption and fluorescence colors are also found in diPOPy-B and bis-diPOPy-Sp. Spectroscopic studies of diPOPy-B, diPOPy-G, and bis-diPOPy-Sp revealed absorption and emission maxima at 385 and 487 nm, respectively. Absorption and emission spectra of diPOPy-B (10 µg/mL in distilled water at pH 7) are shown in Figure 4. At neutral pH, all solutions were stable, and no spectral changes were found at mild alkaline or acid pH values. In the presence of 45% glycerol, 60% sucrose, and 40% dextran 60 in water, samples showed an emission peak about twice higher than that of aqueous solutions (not shown), which indicates the dependence of the fluorescence intensity on the viscosity of the solvent. Another solvent feature that modulates fluorescence wavelength red-shift is its dielectric constant. The dielectric constant is a measure of how efficiently the solvent responds to the altered electronic distribution of the fluorophore in the excited state. A solvent with a high dielectric constant (e.g., water) quickly reorients its dipoles to shield the altered electric field surrounding the excited molecule. This reorientation implies the use of energy that comes from the excited state. Therefore, once equilibrium is reached, which takes less than a picosecond, the available energy for photon emission is less (red-shifted) as compared with the initial energy absorbed. Solvents with high dielectric constant (polar solvents) show a more pronounced reorientation effect than solvents with a small dielectric constant (non-polar solvents). In the solid state, microcrystals of POPy derivatives also exhibited strong fluorescence under 365 nm excitation.
After staining suitable microscopical preparations with the fluorochromes diPOPy-B, diPOPy-G, and bis-diPOPy-Sp, a bright blue-green fluorescence was observed in DNA-containing structures. Methanol-fixed nuclei from chicken blood smears showed a strong and selective emission ( Figure 5A,B), with some fading degree like most organic fluorochromes.
Likewise, after diPOPy staining, smears of T. cruzi cultures ( Figure 6A), chromatin DNA of leucocyte nuclei from mouse spleen smears, as well as elongated chromatin fibers produced by stretching of spleen smears, showed a bright blue-green fluorescence. In this case, the observation of stretched chromatin fibers using bright-field illumination ( Figure 6B) and polarization microscopy ( Figure 6C,D) pointed out that the brighter fluorescence occurred when the analyzer filter was perpendicular to the direction of chromatin fibers, whereas a parallel orientation between them resulted in a weaker emission (⊥ > ). This agrees with an orthogonal position of the diPOPy fluorophore and the nucleosomal DNA axis, indicating an intercalative binding mode to DNA [46]. After staining suitable microscopical preparations with the fluorochromes diPOPy-B, diPOPy-G, and bis-diPOPy-Sp, a bright blue-green fluorescence was observed in DNAcontaining structures. Methanol-fixed nuclei from chicken blood smears showed a strong and selective emission ( Figure 5A,B), with some fading degree like most organic fluorochromes.    After staining suitable microscopical preparations with the fluorochromes diPOPy B, diPOPy-G, and bis-diPOPy-Sp, a bright blue-green fluorescence was observed in DNA containing structures. Methanol-fixed nuclei from chicken blood smears showed a stron and selective emission ( Figure 5A,B), with some fading degree like most organic fluoro chromes.  produced by stretching of spleen smears, showed a bright blue-green fluorescence. In this case, the observation of stretched chromatin fibers using bright-field illumination ( Figure  6B) and polarization microscopy ( Figure 6C,D) pointed out that the brighter fluorescence occurred when the analyzer filter was perpendicular to the direction of chromatin fibers, whereas a parallel orientation between them resulted in a weaker emission (⊥ > ∥). This agrees with an orthogonal position of the diPOPy fluorophore and the nucleosomal DNA axis, indicating an intercalative binding mode to DNA [46]. In control samples, after previous treatment of chicken blood, T. cruzi cultures, and mouse spleen smears with DNase and TCA, the emission induced by diPOPy-B and bis-diPOPy-Sp was negative, which indicates that DNA-containing structures are the substrate responsible for the fluorescence reaction. Washing of stained preparations with 2 M NaCl and 4 M urea solutions did not result in reduction of the fluorescence intensity, while washing in saturated aqueous solutions of caffeine or ortho-phenanthroline clearly diminished the emission, also indicating the occurrence of hydrophobic and non-ionic binding forces between fluorochromes and the DNA substrates [36,48].
Inspection of the chemical structures reveals that diPOPy is a small and planar molecule, with CBN: 20, and calculated (bonds) or measured (Å) W/L: 0.4 and 0.46, respectively, which correspond to the classical appearance of a "tea-tray" very suitable for intercalation [51]. After geometry optimization and energy minimization, molecular modeling studies showed a full conjugated aromatic structure, with a clear and differential distribution of partial charges ( Figure 7A). In control samples, after previous treatment of chicken blood, T. cruzi cultures, and mouse spleen smears with DNase and TCA, the emission induced by diPOPy-B and bis-diPOPy-Sp was negative, which indicates that DNA-containing structures are the substrate responsible for the fluorescence reaction. Washing of stained preparations with 2 M NaCl and 4 M urea solutions did not result in reduction of the fluorescence intensity, while washing in saturated aqueous solutions of caffeine or ortho-phenanthroline clearly diminished the emission, also indicating the occurrence of hydrophobic and non-ionic binding forces between fluorochromes and the DNA substrates [36,48].
Inspection of the chemical structures reveals that diPOPy is a small and planar molecule, with CBN: 20, and calculated (bonds) or measured (Å) W/L: 0.4 and 0.46, respectively, which correspond to the classical appearance of a "tea-tray" very suitable for intercalation [51]. After geometry optimization and energy minimization, molecular modeling studies showed a full conjugated aromatic structure, with a clear and differential distribution of partial charges ( Figure 7A).
Regarding molecular orbitals of diPOPy-H ( Figure 7B), energy levels of higher occupied and lower unoccupied molecular orbitals (HOMO-0 and LUMO + 0, respectively) are well separated (LUMO + 0 subtracted from HOMO-0 = Eg = 6.01 eV), which is related to the large Stokes shift and a blue-green emission under UV excitation. As expected, the number of nodal planes is higher for LUMO + 0 than for HOMO-0 [36,40]. Regarding molecular orbitals of diPOPy-H ( Figure 7B), energy levels of higher occupied and lower unoccupied molecular orbitals (HOMO-0 and LUMO + 0, respectively) are well separated (LUMO + 0 subtracted from HOMO-0 = Eg = 6.01 eV), which is related to the large Stokes shift and a blue-green emission under UV excitation. As expected, the number of nodal planes is higher for LUMO + 0 than for HOMO-0 [36,40].
In the case of a hypothetical dye-substrate structure, molecular modeling reveals that adenine-thymine (AT)-containing nucleic acid oligomers such as tetra-(dATAT) can be bis-intercalated by bis-diPOPy-Sp, appearing as the two fluorophore rings separated by ~10.2 Å, in agreement with the "nearest neighbor exclusion principle" for intercalating agents [36,55,56]. Although there is not precise evidence for specific binding to AT base pairs, some presumptive cytological and cytogenetic data support this possibility. Figure  8 shows wire (A) and atomic volume (B) models of the bis-intercalated complex, as well as the overlapping (fused) molecular orbitals (MOs) between each dye and base pairs above and below (C), and the non-overlapping (unfused) MOs between adjacent base pairs, which abolish the in-phase π-electron conjugated stacking in these sites. In the case of a hypothetical dye-substrate structure, molecular modeling reveals that adenine-thymine (AT)-containing nucleic acid oligomers such as tetra-(dATAT) can be bis-intercalated by bis-diPOPy-Sp, appearing as the two fluorophore rings separated bỹ 10.2 Å, in agreement with the "nearest neighbor exclusion principle" for intercalating agents [36,55,56]. Although there is not precise evidence for specific binding to AT base pairs, some presumptive cytological and cytogenetic data support this possibility. Figure 8 shows wire (A) and atomic volume (B) models of the bis-intercalated complex, as well as the overlapping (fused) molecular orbitals (MOs) between each dye and base pairs above and below (C), and the non-overlapping (unfused) MOs between adjacent base pairs, which abolish the in-phase π-electron conjugated stacking in these sites. It is worth noting that all the LUMOs at low energy levels (from +0 to +12) showed full overlapping MOs between the intercalated diPOPy and its adjacent base pairs. However, only partially or non-overlapped MOs appeared between stacked base pairs, which indicates that the intercalator corresponds to an electronically isolated monomer within the intercalation cavity.

BzPOPy Fluorochromes from Fluorescamine
Although the fluorescence mechanism of the reaction product of MDPF with amino groups at neutral pH is now well known (involving the formation of planar, full conjugated and highly fluorescent diPOPy derivatives), the mechanism allowing fluorescence of fluorescamine derivatives is not so clear. As occurs for MDPF products, at alkaline pH, the pseudobase (carbinol) of the non-planar and non-fluorescent BzPOPy product is first formed, and fluorescence only appears at neutral pH [34,38,39].
In order to illustrate the fluorescamine-induced fluorescence of amino groups in situ, the reaction was carried out on horse blood smears, which revealed the basic proteins of the eosinophil leucocyte granules [29,50] generating a strong blue-green emission ( Figure 9A). It is worth noting that all the LUMOs at low energy levels (from +0 to +12) showed full overlapping MOs between the intercalated diPOPy and its adjacent base pairs. However, only partially or non-overlapped MOs appeared between stacked base pairs, which indicates that the intercalator corresponds to an electronically isolated monomer within the intercalation cavity.

BzPOPy Fluorochromes from Fluorescamine
Although the fluorescence mechanism of the reaction product of MDPF with amino groups at neutral pH is now well known (involving the formation of planar, full conjugated and highly fluorescent diPOPy derivatives), the mechanism allowing fluorescence of fluorescamine derivatives is not so clear. As occurs for MDPF products, at alkaline pH, the pseudobase (carbinol) of the non-planar and non-fluorescent BzPOPy product is first formed, and fluorescence only appears at neutral pH [34,38,39].
In order to illustrate the fluorescamine-induced fluorescence of amino groups in situ, the reaction was carried out on horse blood smears, which revealed the basic proteins of the eosinophil leucocyte granules [29,50] generating a strong blue-green emission ( Figure 9A). However, the relations between chemical structure, pH, and fluorescence features are intriguing, and no structures of the reaction product are expected to be fluorescent. Obviously, in the lactone reagent ( Figure 9B,B′) there is a benzoic ring perpendicular to the other two aromatic rings. The same orthogonal geometry appears after molecular modeling of the pseudobase at alkaline pH ( Figure 9C,C′), as well as in the cationic form at very low acid pH with the unionized carboxylic group ( Figure 9D,D′), and thus no fluorescence should occur for these non-planar structures. Likewise, the non-charged lactone of the BzPOPy product also appears orthogonal.
Therefore, it is tempting to assume that, at about neutral pH, the quaternary N1 and carboxylate anion could equilibrate charges themselves, forming the "inner salt" (zwitterion) with a full conjugated planar geometry, which could be the true fluorescent product from the fluorescamine reaction ( Figure 10). The same planar configuration of the fluorophore is found for the reaction products with arginine and lysine. The corresponding HOMO-0 and LUMO + 0 levels (not shown) are very similar to those of MDPF products. Examples of inner-salt colorants and fluorochromes (zwitterionic phenol blue, indophenol blue, merocyanine 540, Nile red, rhodols, Bodipy dyes, etc.) are also known [36,57,58]. However, the relations between chemical structure, pH, and fluorescence features are intriguing, and no structures of the reaction product are expected to be fluorescent. Obviously, in the lactone reagent ( Figure 9B,B ) there is a benzoic ring perpendicular to the other two aromatic rings. The same orthogonal geometry appears after molecular modeling of the pseudobase at alkaline pH ( Figure 9C,C ), as well as in the cationic form at very low acid pH with the unionized carboxylic group ( Figure 9D,D ), and thus no fluorescence should occur for these non-planar structures. Likewise, the non-charged lactone of the BzPOPy product also appears orthogonal.
Therefore, it is tempting to assume that, at about neutral pH, the quaternary N1 and carboxylate anion could equilibrate charges themselves, forming the "inner salt" (zwitterion) with a full conjugated planar geometry, which could be the true fluorescent product from the fluorescamine reaction ( Figure 10). The same planar configuration of the fluorophore is found for the reaction products with arginine and lysine. The corresponding HOMO-0 and LUMO + 0 levels (not shown) are very similar to those of MDPF products. Examples of inner-salt colorants and fluorochromes (zwitterionic phenol blue, indophenol blue, merocyanine 540, Nile red, rhodols, Bodipy dyes, etc.) are also known [36,57,58].  Figure 9. (C) The same molecule showing partial charges (eV), and 3D plotting of electrostatic potentials (from negative to positive values: red → green → blue), using the translucent isosurface rendering (total charge density contour value: 0.03), after energy minimization with HyperChem v8.0.10 and PM3 converged at E = 0.5 kcal/(Å mol). Partial charges for the N and O atoms from the inner salt are + 0.504 and −0.591 eV, respectively.
Considering the small size, planar structure, and cationic status of the POPy fluorophore, an intercalative binding mode between base pairs of a DNA duplex is expected to occur, as well as bis-intercalation in the case of bis-diPOPy-Sp. Polarized fluorescence is a suitable microscopical tool to analyze the orientation of fluorochromes in both well-orientated DNA and chromatin fibers [46,51,68]. An intercalative binding mode is expected to occur when the polarization and fiber axis orientation are perpendicular (⊥ > ).
There are still no data about the possible preference of intercalation related to AT or GC. It is known that daunomycin and berberine allow the visualization of AT-rich chromosome regions (Q banding) by fluorescence microscopy [36,55,61,69]. Likewise, quinacrine and quinacrine mustard fluoresce brighter in AT-rich regions, and it is generally accepted that Q-negative bands are produced by fluorescence quenching by guanine [70][71][72][73].
On the other hand, abundant (dA) 4-6 • (dT) [4][5][6] sequences are present in the kDNA [74]. Minicircles of kDNA contain repeated short (dA) 4-6 • (dT) 4-6 tracks alternating with normal DNA sequences, which generates the curved DNA structure typical of kDNA minicircles [75]. Likewise, the pericentromeric heterochromatin DNA regions of mouse chromosomes (C-bands) are AT-rich (approximately 69% AT), appearing composed of a highly repetitive 230-240 base pair unit [76], which contains the EcoRI GAATTC restriction site and numerous consecutive (dA) 4-6 • (dT) 4-6 tracts [77]. Therefore, due to the high AT content in kDNA and C-bands of mouse chromosomes, they can be used as a suitable test model to analyze the selectivity of fluorochrome binding at the microscopical level.
In keeping with this, the selective fluorescence of interphase heterochromatin from chromatin DNA ( Figure 5) and kDNA ( Figure 6A) could be attributed to some preference for diPOPy to bind to AT-rich regions. According to the equation E (eV) = 1239.847/λ(nm), the energy gap (Eg) between the ground singlet S 0.0 and the first excited singlet S 1.0 of diPOPy-B corresponds to the cross (iso-absorptive/emissive) point at 435.5 nm and 2.846 eV. From these values, 2.545 eV for the 487 nm blue-green emission is obtained.
Regarding molecular modeling of POPy derivatives, simple aliphatic residues (e.g., diPOPy-B) have a rather linear structure. In the case of diPOPy-G, the C1-N1 bond is able to rotate; the hexose ring of the bulky glucosyl residue and the planar diPOPy ring are almost perpendicular. The possible intercalative binding of diPOPy-G could be accomplished with the glucosyl residue located in the minor groove, as occurs with the phenyl group of ethidium bromide and the daunosamine group of daunomycin [56,78].

Bis-Intercalative Binding
Bis-intercalation occurs when two aromatic chromophores (fluorophores) separated by a flexible or rigid linker of~10.2 Å become inserted into a DNA duplex, which is the case for some antibiotics and synthetic dyes and drugs [36,56,60]. In addition, several synthetic bis-intercalators are well-known fluorescent colorants, and they are used in microscopy and cytofluorimetry to detect and quantify DNA. Examples are 9-aminoacridine fluorophores attached by linkers with different structures and sizes [79]. The synthetic bis-intercalating fluorochromes TOTO-1 and YOYO-1 show high affinity for DNA and have wide applications for DNA imaging [64,69,80,81] due to their large increase of emission after DNA intercalation, which avoids the rotational energy decay of excited fluorophores with torsional freedom [36,82,83].
Most known bis-intercalating DNA fluorochromes (ethidium homodimer-1, TOTO-1, and YOYO-1), contain a positively charged and flexible aliphatic bridge between the intercalating cyanines. Although the use of a spermine linker has been applied for a 9-amino-acridine bis-intercalator [79], as far as we know, the spermine bridge appears as a rather overlooked linker for bis-intercalating DNA fluorochromes. The fluorogenic reaction of spermine with fluorescamine has been previously described [1,34]. Spermine is a tetracation at physiological pH and it appears as a flexible aliphatic linker that is very suitable to bridge two terminal POPy intercalators, as occurs in the case of bis-diPOPy-Sp (see Figures 2 and 8).
Apart from fluorescent labeling, bis-intercalating drugs containing suitable chromophores have biomedical applications. The biological activity and therapeutic action of DNA intercalating and bis-intercalating drugs have been reviewed [84,85]. Quinoxaline antibiotics echinomycin and triostin A bis-intercalate their quinoxaline rings into DNA and recognize GC/CG base pairs [86]. Likewise, the antitumor antibiotic carzinophilin A bis-intercalates two naphthalene rings into DNA, inducing interstrand cross-linking by means of its two alkylating aziridines [87].
Two 3-amino-naphthalimide fluorophores [42] linked by a spermine bridge can generate a bis-intercalating naphthalimide, which induces bright emission in chromatin DNA [36]. Bis-naphthalimides are also antitumor agents [96]. Regarding the possible intercalative and bis-intercalative binding mode of diPOPy fluorochromes, their physiological consequence could induce an inhibition of the replication and transcription activity of DNA, suggesting interesting pharmacological applications of these fluorescent colorants in cancer chemotherapy (e.g., [97]).

Fluorescence Mechanisms of Fluorescamine Products
As the mechanistic knowledge of histochemical reactions is a relevant issue that must be considered to avoid misleading interpretations [39], in this work we describe some microscopic and spectroscopic characteristics of MDPF fluorogenic reactions and show that, although the pseudobase is originally formed at alkaline pH, a full conjugated, cationic and planar molecule seems to be the true fluorescent product [38].
In the case of fluorescamine, a loss of fluorescence occurs at acid pH (<~4) by formation of the non-planar lactone [34], in agreement with the ionization constant of benzoic acid (pKa: 4.2). In this low-pH structure, the C2 atom becomes tetrahedral and the BzPOPy product remains non-planar. On loss of the more acidic carboxylic proton, the molecule can also exist as the free carboxylate ion, which in turn can form an inner salt (zwitterion) with the quaternary N1 atom. This polar, water-soluble form would be an equivalent of the water-insoluble lactone form, which when dissolved in organic solvents does not have color or fluorescence. It is logical to assume that the inner salt is then responsible for the emission of the fluorescamine product at pH~7, when no more pseudobase is present, and the BzPOPy inner salt becomes planar and fluorescent again (see Figure 9A).
An example of cytochemical visualization of protein amino groups by staining mouse metaphase chromosomes with fluorescamine is reproduced in Figure 11 [28]. In this case, the strong and mild fluorescence of C and Q/G bands, respectively, are very apparent, and the close correspondence between the position of these chromosome bands and the emission of the fluorescamine-amino group product is astonishing.
As the fluorescamine reactions that reveals protein amino groups from arginine and lysine residues near to the negatively charged floor of the DNA minor groove [104] colocalize with AT-rich chromosome regions, it is tempting to speculate that DNA intercalation of BzPOPy takes place precisely within these regions. The observed more intense fluorescence can be due to the rigid intercalative binding of the planar inner-salt fluorophore. As the phenyl and benzoic rings of BzPOPy can rotate lightly, a close fitting of the high propeller-twisted (dA)n • (dT)n regions [105] with the intercalated fluorophore would occur. The flexible bridge of five and six atoms (lysine and arginine, respectively) between the fluorophore and the polypeptide chain could also provide the approach needed for suitable DNA intercalation ( Figure 12A). A schematic representation of the proposed intercalation of BzPOPy-Lys and BzPOPy-Arg from labeled proteins into DNA containing dA6dT6 sequences is shown in Figure 12B.  The absence of chromatin fluorescence after the fluorescamine reaction (see Figure  9A) deserves some comments. It is possible that, after methanol fixation, amino groups from nuclear cationic proteins could remain attached to DNA and are non-available to react with fluorescamine. The usual cytogenetic protocol using hypotonic treatment, fixation with methanol-acetic acid, and cell spreading could be relevant to disorganize the chromatin structure, improving the substrate reactivity and banding pattern. In keeping with this, fluorescamine fluorescence was found to be dependent on the fixative, chromatin, and chromosomes showing high emission after use of acetic-acid-containing fixatives [26]. A rigid intercalative binding mode will also increase the emission of tagged structures. As the fluorescamine reactions that reveals protein amino groups from arginine and lysine residues near to the negatively charged floor of the DNA minor groove [104] colocalize with AT-rich chromosome regions, it is tempting to speculate that DNA intercalation of BzPOPy takes place precisely within these regions. The observed more intense fluorescence can be due to the rigid intercalative binding of the planar inner-salt fluorophore. As the phenyl and benzoic rings of BzPOPy can rotate lightly, a close fitting of the high propeller-twisted (dA) n • (dT) n regions [105] with the intercalated fluorophore would occur. The flexible bridge of five and six atoms (lysine and arginine, respectively) between the fluorophore and the polypeptide chain could also provide the approach needed for suitable DNA intercalation ( Figure 12A). A schematic representation of the proposed intercalation of BzPOPy-Lys and BzPOPy-Arg from labeled proteins into DNA containing dA 6 dT 6 sequences is shown in Figure 12B.
The absence of chromatin fluorescence after the fluorescamine reaction (see Figure 9A) deserves some comments. It is possible that, after methanol fixation, amino groups from nuclear cationic proteins could remain attached to DNA and are non-available to react with fluorescamine. The usual cytogenetic protocol using hypotonic treatment, fixation with methanol-acetic acid, and cell spreading could be relevant to disorganize the chromatin structure, improving the substrate reactivity and banding pattern. In keeping with this, fluorescamine fluorescence was found to be dependent on the fixative, chromatin, and chromosomes showing high emission after use of acetic-acid-containing fixatives [26]. A rigid intercalative binding mode will also increase the emission of tagged structures.

Conclusions
In this work, we show that planar and cationic diPOPy or neutral BzPOPy products are strongly fluorescent dyes that can bind selectively to chromatin DNA, likely by intercalation into AT-rich chromosome regions. Likewise, a planar inner-salt BzPOPy fluorophore is proposed for the reaction product of protein amino groups with fluorescamine. Interestingly, there is no formal charge in this type of compound, and thus no binding with anionic substrates (only based on ionic forces) should occur. These fluorescence mechanisms are supported by molecular properties and microscopical evidence, which are shared with wellknown DNA intercalators. Although further studies are obviously necessary to ascertain better the affinity and selectivity for a wider range of cell and tissue substrates, it is tempting to assume that, like known intercalating and bis-intercalating antibiotics and synthetic drugs, these new diaryl-POPy derivatives could well be used in DNA cytochemistry and would also be of potential pharmacological value in cancer chemotherapy.