Cocrystals versus Salts of Fluorescein

: A series of nitrogen-containing organic molecules (4,4’-bipyridyl; trans -1,2-bis(4-pyridyl) ethylene; 1,2-bis(4-pyridyl)ethane; 4-aminopyridine and trans -1,4-diaminocyclohexane) was envisaged for cocrystallization experiments together with ﬂuorescein. These compounds, containing pyridyl or/and amino nitrogen atoms, can act either as hydrogen bond acceptors for the phenol groups of ﬂuorescein-generating cocrystals or as proton acceptors forming organic salts. Five cocrystals were obtained with the partners containing only pyridyl groups: {(H 2 Fl) 2 (bipy)} ( 1 ); {(H 2 Fl) 2 (bipy)(MeOH) 2 } ( 2 ); {(H 2 Fl) 2 (bpete)(EtOH) 2 } ( 3 ); {(H 2 Fl)(bpete)} ( 4 ); {(H 2 Fl)(bpeta)} ( 5 ). The compounds bearing amino groups deprotonate ﬂuorescein producing salts: [(HFl)(Hampy)] · 2H 2 O ( 6 ); [(HFl)(Hampy)] ( 7 ); [(Fl)(H 2 diach)] · 3H 2 O ( 8 ); [(HFl) 2 (H 2 diach)] · 2H 2 O · EtOH ( 9 ); and [(HFl) 2 (Fl) 2 (H 2 diach) 3 ] · 4H 2 O ( 10 ). Optical properties of the cocrystals and salts were investigated.

The chromogenic mechanism of fluorescein is based on protonation-deprotonation reactions. Due to the biological applications of the fluorescein dyes, solution studies have attracted particular attention. The ionization equilibria of fluorescein are presented in Scheme 1. Depending on pH, in solution can be identified cationic (H 3 Fl + ), neutral (H 2 Fl) or anionic species (HFl − and Fl 2− ). The neutral form (H 2 Fl) presents in solution three tautomers: zwitterion (H 2 Flz), quinonoid (H 2 Flq) and lactone (H 2 Fll). For the monoanionic form (HFl − ), the phenolate tautomer appears in small quantities only in pure solvents such as DMSO, acetonitrile or acetone [13].
In solid state, the three tautomers of the neutral form are characterized by different colors: the zwitterionic form is yellow, the quinonoid form is red and the lactonoid form of fluorescein is colorless. The crystal structures of H 2 Flq and H 2 Flz have been determined by powder X-ray diffraction [14,15]. The crystal structure of the pure lactonoid form of fluorescein has not been reported. The lactonoid form crystallizes with solvent molecules, and the methanol [16], acetone [15,17] and 1,4-dioxane [15,18] solvates were structurally characterized by X-ray diffraction on a single crystal.

Synthesis
The chemicals used as well as all the solvents were of reagent grade and were purchased from commercial sources.
The X-ray powder diffraction measurements (XRPD) were carried out on a Proto AXRD Benchtop using Cu-Kα radiation with a wavelength of 1.54059 Å in the 2θ range of 5-35 • .

Spectroscopy
IR spectra were recorded on an FT-IR Bruker Vertex 70 spectrometer in the 4500-400 cm −1 range using the ATR technique. The following abbreviations were used: w = weak, m = medium, s = strong, v = very, br = broad. Absorption spectra on powder (diffuse reflectance technique) were measured with a JASCO V-670 spectrophotometer. The fluorescence spectra were collected on powder using a JASCO FP-6500 spectrofluorometer.

Results
A series of nitrogen-containing organic molecules, namely 4,4'-bipyridyl; trans-1,2-bis(4pyridyl)ethylene; 1,2-bis(4-pyridyl)ethane; 4-aminopyridine and trans-1,4-diaminocyclohexane, was envisaged for cocrystallization experiments together with fluorescein. These compounds contain pyridyl or/and amino nitrogen atoms and, theoretically, these groups can act either as hydrogen bond acceptors for the phenol groups of fluorescein or as proton acceptors. The following ten compounds were obtained using solution-based crystallization methods (slow evaporation at room temperature) and different stoichiometric ratios between  (1)(2)(3)(4)(5) whereas the salts are indicated by square brackets (6-10). All ten systems were structurally characterized by X-ray diffraction on a single crystal.  The (O1-)H1···N1 distance for the intermolecular hydrogen interaction is 1.828 Å and the corresponding O1-H1···N1 angle is 165.0 • . Crystal 1 contains only fluorescein and bipy in a 2:1 molar ratio. The asymmetric unit consists of one fluorescein molecule and half a bipy molecule ( Figure 1). The fluorescein molecules are in the lactonoid form and the C-O bond lengths for the phenol groups are C3-O1 = 1.350(3) and C11-O3 = 1.355(3) Å. The bipy molecules are hydrogen bonded through the nitrogen atoms to two OH groups belonging to two different fluorescein molecules. The (O1-)H1•••N1 distance for the intermolecular hydrogen interaction is 1.828 Å and the corresponding O1-H1•••N1 angle is 165.0°. The O1 oxygen atom also acts as a hydrogen bond acceptor for an O3-H3 phenol group of neighboring fluorescein molecules. The (O3"-)H3"•••O1 distance for this hydrogen interaction is 1.984 Å and the corresponding O3"-H3"•••O1 angle is 144.8° (symmetry code: " = 0.5−x, −0.5 + y, −z). The interplay between the two types of hydrogen interactions generates a supramolecular 2D architecture ( Figure 2). The supramolecular interactions are further extended to the third dimension through π−π interactions established between the layers. Both organic molecules contain aromatic systems and two patterns of π−π interactions can be described ( Figure 3). The first one involves the xanthene cores of the fluorescein molecules with a separation of 3.51-3.62 Å between the fragments. The mean planes of interacting xanthene fragments are parallel. The second type of π−π interaction implicates one pyridine ring of a bipy molecule and one phenol moiety of a fluorescein molecule. The dihedral angle between the mean planes The O1 oxygen atom also acts as a hydrogen bond acceptor for an O3-H3 phenol group of neighboring fluorescein molecules. The (O3"-)H3"···O1 distance for this hydrogen interaction is 1.984 Å and the corresponding O3"-H3"···O1 angle is 144.8 • (symmetry code: " = 0.5−x, −0.5 + y, −z). The interplay between the two types of hydrogen interactions generates a supramolecular 2D architecture ( Figure 2).

Description of the Crystal Structures
Crystal 1 contains only fluorescein and bipy in a 2:1 molar ratio. The asymmetric unit consists of one fluorescein molecule and half a bipy molecule ( Figure 1). The fluorescein molecules are in the lactonoid form and the C-O bond lengths for the phenol groups are C3-O1 = 1.350(3) and C11-O3 = 1.355(3) Å. The bipy molecules are hydrogen bonded through the nitrogen atoms to two OH groups belonging to two different fluorescein molecules. The (O1-)H1•••N1 distance for the intermolecular hydrogen interaction is 1.828 Å and the corresponding O1-H1•••N1 angle is 165.0°. The O1 oxygen atom also acts as a hydrogen bond acceptor for an O3-H3 phenol group of neighboring fluorescein molecules. The (O3"-)H3"•••O1 distance for this hydrogen interaction is 1.984 Å and the corresponding O3"-H3"•••O1 angle is 144.8° (symmetry code: " = 0.5−x, −0.5 + y, −z). The interplay between the two types of hydrogen interactions generates a supramolecular 2D architecture ( Figure 2). The supramolecular interactions are further extended to the third dimension through π−π interactions established between the layers. Both organic molecules contain aromatic systems and two patterns of π−π interactions can be described ( Figure 3). The first one involves the xanthene cores of the fluorescein molecules with a separation of 3.51-3.62 Å between the fragments. The mean planes of interacting xanthene fragments are parallel. The second type of π−π interaction implicates one pyridine ring of a bipy molecule and one phenol moiety of a fluorescein molecule. The dihedral angle between the mean planes The supramolecular interactions are further extended to the third dimension through π-π interactions established between the layers. Both organic molecules contain aromatic systems and two patterns of π-π interactions can be described ( Figure 3). The first one involves the xanthene cores of the fluorescein molecules with a separation of 3.51-3.62 Å between the fragments. The mean planes of interacting xanthene fragments are parallel. The second type of π-π interaction implicates one pyridine ring of a bipy molecule and one phenol moiety of a fluorescein molecule. The dihedral angle between the mean planes of pyridine and xanthene fragments is 31.1 • and the shortest contacts between these fragments range between 3.32 and 3.71 Å. of pyridine and xanthene fragments is 31.1° and the shortest contacts between these fragments range between 3.32 and 3.71 Å. Cocrystal 2 is a pseudopolymorph of 1 and contains fluorescein, bipy and methanol in a 2:1:2 stoichiometry. It crystalizes in the triclinic P-1 space group and the asymmetric unit comprises one fluorescein molecule, half a bipy molecule and one methanol molecule ( Figure 4). The methanol molecule is also a hydrogen donor for another phenol group of a different fluorescein molecule generating supramolecular units formed by two fluorescein molecules and two methanol molecules ( Figure 5). The (O6-)H6•••O1" distance is 2.264 Å, while the O6-H6•••O1 angle is 135.5°. These supramolecular units are connected by the bipy molecules in a 1D array. In crystal 2, the presence of the solvent molecules prevents the extension of the hydrogen networking to a 2D system. Within the supramolecular dimers the xanthene fragments also establish π−π interactions (3.38-3.55 Å). Cocrystal 2 is a pseudopolymorph of 1 and contains fluorescein, bipy and methanol in a 2:1:2 stoichiometry. It crystalizes in the triclinic P-1 space group and the asymmetric unit comprises one fluorescein molecule, half a bipy molecule and one methanol molecule ( Figure 4). of pyridine and xanthene fragments is 31.1° and the shortest contacts between these fragments range between 3.32 and 3.71 Å.  The methanol molecule is also a hydrogen donor for another phenol group of a different fluorescein molecule generating supramolecular units formed by two fluorescein molecules and two methanol molecules ( Figure 5). The (O6-)H6•••O1" distance is 2.264 Å, while the O6-H6•••O1 angle is 135.5°. These supramolecular units are connected by the bipy molecules in a 1D array. In crystal 2, the presence of the solvent molecules prevents the extension of the hydrogen networking to a 2D system. Within the supramolecular dimers the xanthene fragments also establish π−π interactions (3.38-3.55 Å). The methanol molecule is also a hydrogen donor for another phenol group of a different fluorescein molecule generating supramolecular units formed by two fluorescein molecules and two methanol molecules ( Figure 5). The (O6-)H6···O1" distance is 2.264 Å, while the O6-H6···O1 angle is 135.5 • . These supramolecular units are connected by the bipy molecules in a 1D array. In crystal 2, the presence of the solvent molecules prevents the extension of the hydrogen networking to a 2D system. Within the supramolecular dimers the xanthene fragments also establish π-π interactions (3.38-3.55 Å).
For the cocrystallization experiments of fluorescein with trans-1,2-bis(4-pyridyl)ethylene two different molar ratios were used: 2:1 and 1:1. The corresponding cocrystals obtained are {(H 2 Fl) 2 (bpete)(EtOH) 2 } (3) and {(H 2 Fl)(bpete)} (4). Cocrystal 3 contains, similarly with crystal 2, fluorescein, bpete and solvent in a 2:1:2 stoichiometry ( Figure 6). In this case, the solvent is ethanol and the CH 3 -CH 2 -fragment is disordered on two independent crystallographic positions with site occupancy factors of 0.5 each. Similar to compound 2, the two phenol groups of fluorescein act as hydrogen bond donors towards one bpete molecule and one ethanol molecule. The (O1-)H1···N1 distance is 1.823 Å and the corresponding O1-H1···N1 angle is 161.0 • .          The supramolecular chains formed by hydrogen interactions intercalate and the bpete molecules from neighboring chains establish π−π interactions ranging between 3.27 and 3.64 Å (Figure 9). The supramolecular chains formed by hydrogen interactions intercalate and the bpete molecules from neighboring chains establish π-π interactions ranging between 3.27 and 3.64 Å (Figure 9).  We have to mention here that compound 4 is not crystallizing as a pure phase and the bulk sample probably contains a small amount of compound 3 (Figures S3-S5).
Cocrystal 5, {(H2Fl)(bpeta)}, was obtained by slow evaporation of a methanolic solution containing fluorescein and 1,2-bis(4-pyridyl)ethane in a 1:1 stoichiometry. In this case, the powder X-ray diffraction shows that the sample contains only one crystalline phase ( Figure S6). Structural characterization of compound 5 by X-ray diffraction on a single We have to mention here that compound 4 is not crystallizing as a pure phase and the bulk sample probably contains a small amount of compound 3 (Figures S3-S5). Cocrystal 5, {(H 2 Fl)(bpeta)}, was obtained by slow evaporation of a methanolic solution containing fluorescein and 1,2-bis(4-pyridyl)ethane in a 1:1 stoichiometry. In this case, the powder X-ray diffraction shows that the sample contains only one crystalline phase ( Figure S6). Structural characterization of compound 5 by X-ray diffraction on a single crystal reveals formation of crenel-like supramolecular chains, similarly to crystal 4, by hydrogen interactions established between phenol and pyridyl groups ( Figure 10)  We have to mention here that compound 4 is not crystallizing as a pure phase and the bulk sample probably contains a small amount of compound 3 (Figures S3-S5).
Cocrystal 5, {(H2Fl)(bpeta)}, was obtained by slow evaporation of a methanolic solution containing fluorescein and 1,2-bis(4-pyridyl)ethane in a 1:1 stoichiometry. In this case, the powder X-ray diffraction shows that the sample contains only one crystalline phase ( Figure S6). Structural characterization of compound 5 by X-ray diffraction on a single crystal reveals formation of crenel-like supramolecular chains, similarly to crystal 4, by hydrogen interactions established between phenol and pyridyl groups ( Figure 10)   The second type of nitrogen-containing organic molecules used as partners for fluorescein to obtain binary systems were the amino derivatives 4-aminopyridine and trans-1,4-diaminocyclohexane. The first notable observation is the color change induced by the amino derivatives. The solid products obtained in the presence of 4-aminopyridine and trans-1,4-diaminocyclohexane are red, while the compounds 1-5 are pale yellow or colorless. Since the chromogenic mechanism of fluorescein is based on protonation-deprotonation reactions, the color change is most probably an indication of the proton transfer between the fluorescein and the amino partner.
For a 1:1 stoichiometry between fluorescein and 4-aminopyridine, two types of crystals were obtained depending on the solvents used for crystallization: [(HFl)(Hampy)]·2H 2 O (6), obtained in small amounts as orange-red prismatic crystals on the wall of the beaker from a mixture of ethanol (96%)-acetonitrile, and [(HFl)(Hampy)] (7) as red needle-like crystals resulted from the evaporation of an ethanol-water solution.
(7) as red needle-like crystals resulted from the evaporation of an ethanol-water solution.
X-ray diffraction on a single crystal shows for compound 6 the presence of fluorescein monoanions (HFl -), Hampy + cations and crystallization water molecules (Figure 11). The lactone spirocycle of fluorescein is open and the carboxylate bond lengths are C20-O4 = 1.249(5) and C20-O5 = 1.270(6) Å. For the phenol group, the C3-O1 bond length is 1.340(5) Å, while the quinonic C11-O3 bond length is 1.271(5) Å. The ampy molecule is protonated on a pyridine nitrogen atom (N1). The quinonic O3 atom acts as a hydrogen bond acceptor for a crystallization water molecule, which is also a hydrogen bond acceptor for the pyridinium fragment ( Figure 11  The xanthene fragments establish π−π interactions (3.57-3.65 Å) forming supramolecular dimers. The fluorescein supramolecular dimers are connected into an extended supramolecular network through hydrogen interactions with crystallization water molecules and 4-aminopyridinium cations ( Figure 12). Except the xanthene O2 atom, all the other oxygen atoms of the fluorescein are involved in hydrogen interactions. The xanthene fragments establish π-π interactions (3.57-3.65 Å) forming supramolecular dimers. The fluorescein supramolecular dimers are connected into an extended supramolecular network through hydrogen interactions with crystallization water molecules and 4-aminopyridinium cations ( Figure 12). Except the xanthene O2 atom, all the other oxygen atoms of the fluorescein are involved in hydrogen interactions. In crystal 7, the lactone spirocycle of fluorescein is also open but only half of the molecules are deprotonated. The proton from one phenol group is transferred on a pyridine nitrogen atom or on a carboxylate O5 atom ( Figure 13). The ampy molecules are disordered on two crystallographic positions with site occupancy factors of 0.5. Due to the disorder of ampy molecules we could not refine the percentages of proton transfer on pyridine nitrogen atoms. We estimated that half of the protons are transferred on the carboxylate due to the short O5•••O5' distance of 2.506 Å between the carboxylate groups of neighboring fluorescein molecules, which can be explained by hydrogen interactions (the occupancy of the H5A atom was fixed at 0.5). The C3-O1 bond length is 1.336(7) Å, while the C11-O3 bond length is 1.300(6) Å. The carboxylate bond lengths are C20-O4 = 1.218 (7) and C20-O5 = 1.275(7) Å. In crystal 7, the lactone spirocycle of fluorescein is also open but only half of the molecules are deprotonated. The proton from one phenol group is transferred on a pyridine nitrogen atom or on a carboxylate O5 atom ( Figure 13). The ampy molecules are disordered on two crystallographic positions with site occupancy factors of 0.5. Due to the disorder of ampy molecules we could not refine the percentages of proton transfer on pyridine nitrogen atoms. We estimated that half of the protons are transferred on the carboxylate due to the short O5···O5' distance of 2.506 Å between the carboxylate groups of neighboring fluorescein molecules, which can be explained by hydrogen interactions (the occupancy of the H5A atom was fixed at 0.5). The C3-O1 bond length is 1.336(7) Å, while the C11-O3 bond length is 1.300(6) Å. The carboxylate bond lengths are C20-O4 = 1.218(7) and C20-O5 = 1.275(7) Å.

Spectral Properties
The absorption spectra of compounds 1-5 and 7-10 have been acquired over a wavelength range from 200 to 1000 nm on solid samples (using the diffuse reflectance technique). Compound 6 was obtained only in small amounts on the wall of the beaker and we were not able to investigate the optical properties for this compound. As we already

Spectral Properties
The absorption spectra of compounds 1-5 and 7-10 have been acquired over a wavelength range from 200 to 1000 nm on solid samples (using the diffuse reflectance technique). Compound 6 was obtained only in small amounts on the wall of the beaker and we were not able to investigate the optical properties for this compound. As we already mentioned, there is significant color difference between the cocrystals of fluorescein (compounds 1-5) and the organic salts of fluorescein (compounds 6-10). The cocrystals are pale yellow or colorless and the salts are red. The cocrystals present clearly separated absorption bands in the UV (with maxima around 300-350 nm) and visible regions (with maxima around 460 and 490 nm). The salts present broad bands covering the UV and visible regions with bathochromic shifts in the absorptions. The solid-state absorption spectra of compounds 1-5 and 7-10 are presented in Figure 19a,b and in Table 1 the wavelengths for the absorption maxima are gathered. The room temperature photoluminescence of compounds 1-5 and 7-10 was investigated using different wavelengths for excitation in the 430-460 nm range. The resulting emission spectra using λex = 450 nm are presented in Figure 19c. First of all, we have to notice that no emission was observed in solid-state for the salts. The emission spectra of compounds 1-4 display symmetric bands with maxima at 550 nm. Cocrystal 5 presents a broader emission band with a maximum at 542 nm.

Conclusions
Fluorescein proved to be a versatile molecule in the binary systems formed with different nitrogen-containing organic molecules. The phenol groups of the fluorescein can act either as hydrogen bond donors generating cocrystals or as proton donors producing organic salts. For molecules containing only pyridine rings such as 4,4'-bipyridyl; trans-1,2-bis(4-pyridyl)ethylene; and 1,2-bis(4-pyridyl)ethane, we obtained cocrystals. The presence of more basic amino groups on the nitrogen-containing organic partner conducted syntheses to salts and the deprotonation degree of the fluorescein depended on the molar ratio used. With trans-1,4-diaminocyclohexane, by modifying the stoichiometry, we obtained salts containing only monoanions of fluorescein, only dianions of fluorescein or both mono-and dianions. This versatility of fluorescein allows modulation of the optical properties of the binary systems by choosing a suitable partner.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figures  S1-13: Powder X-ray diffraction patterns, Table S1: Crystallographic data, details of data collection and structure refinement parameters for compounds 1-10.   The room temperature photoluminescence of compounds 1-5 and 7-10 was investigated using different wavelengths for excitation in the 430-460 nm range. The resulting emission spectra using λ ex = 450 nm are presented in Figure 19c. First of all, we have to notice that no emission was observed in solid-state for the salts. The emission spectra of compounds 1-4 display symmetric bands with maxima at 550 nm. Cocrystal 5 presents a broader emission band with a maximum at 542 nm.

Conclusions
Fluorescein proved to be a versatile molecule in the binary systems formed with different nitrogen-containing organic molecules. The phenol groups of the fluorescein can act either as hydrogen bond donors generating cocrystals or as proton donors producing organic salts. For molecules containing only pyridine rings such as 4,4'-bipyridyl; trans-1,2bis(4-pyridyl)ethylene; and 1,2-bis(4-pyridyl)ethane, we obtained cocrystals. The presence of more basic amino groups on the nitrogen-containing organic partner conducted syntheses to salts and the deprotonation degree of the fluorescein depended on the molar ratio used. With trans-1,4-diaminocyclohexane, by modifying the stoichiometry, we obtained salts containing only monoanions of fluorescein, only dianions of fluorescein or both monoand dianions. This versatility of fluorescein allows modulation of the optical properties of the binary systems by choosing a suitable partner.