Dyeing Polymorphs: The MALDI Host 2,5-Dihydroxybenzoic Acid Dawn E. Cohen, Jason B. Benedict, Brian Morlan, Daniel T. Chiu, and Bart Kahr* Department of Chemistry, Box 351700, UniVersity of Washington, Seattle, Washington 98195-1700 ReceiVed December 6, 2006; ReVised Manuscript ReceiVed January 10, 2007
CRYSTAL GROWTH & DESIGN 2007 VOL. 7, NO. 3 492-495
ABSTRACT: The dye methyl red was used to distinguish concomitant polymorphs of the matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) host, 2,5-dihydroxybenzoic acid. Red and purple crystals were readily separated by inspection. They corresponded to the known Form I (P21/c, a ) 4.9110(3) Å, b ) 11.8280(7) Å, c ) 11.0580(6) Å, β ) 91.059(3)°, V ) 642.22 Å3) and Form II (P21/c, a ) 5.5610(2) Å, b ) 4.8690(1) Å, c ) 23.6880 (8) Å, β ) 100.191(1)°, V ) 631.27 Å3), respectively. X-ray structures of cocrystals of dihydroxybenzoic acids and methyl red, measurements of the linear dichroism of mixed crystals, and force-field calculations were used to investigate the mechanism of differential coloring. The relevance of the observations to investigations of the MALDI mechanism are discussed. Rapid polymorph screening has grown in recent years to become a major aspect of drug discovery.1 Here, we show how a solvatochromic dye, methyl red (MR, 2-[[4-(dimethylamino)phenyl]azo]benzoic acid, color index no. 13020) can be used to distinguish polymorphs by inspection. We illustrate the practical example of the identification by dyeing of the concomitant polymorphs of 2,5dihydroxybenzoic acid (2,5-DHB), a well-established host for biopolymers analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). MALDI-MS has revolutionized the mass analysis of biopolymers; however, the ionization mechanism is poorly understood.2 In this process, a small quantity of an analyte is precipitated with a suitable crystalline MALDI host that absorbs laser light upon irradiation. Excitation is transferred to the analyte, which is then expelled into a desorbed plume, ionized intact and readied for analysis by a timeof-flight mass spectrometer.3 Similar compounds are vastly different in their effectiveness as MALDI hosts.4 Attempts have been made to correlate matrix activity with a wide variety of crystal properties, but without success.5-8 A glaring limitation of studies of this kind is the failure to consider polymorphism. It is not clear in the many investigations of the mechanism of the MALDI ionization process using 2,5-DHB which of the two known forms9 is the active polymorph.5,10 We have extensively studied the process of dyeing crystals.11,12 Here, we show that when 2,5-DHB was recrystallized from solutions containing the solvatochromic azo dye MR, two distinctly colored solids, red and purple, precipitated concomitantly.13 X-ray analysis showed that these materials corresponded to the known polymorphs of 2,5-DHB, Form I (P21/c, a ) 4.9110(3) Å, b ) 11.8280(7) Å, c ) 11.0580(6) Å, β ) 91.059(3)°) and Form II (P21/c, a ) 5.5610(2) Å, b ) 4.8690(1) Å, c ) 23.6880(8) Å, β ) 100.191(1)°), respectively.9 Colorimetric assays might be useful in applications in which polymorph identification is important. Herein, we describe the dyed 2,5-DHB crystals and address the mechanism of the differential coloration. A saturated solution of 2,5-DHB (1 × 10-2 M) was recrystallized from 50% aqueous ethanol containing MR (1 × 10-5 M) by slow evaporation over a period of 2-3 days at room temperature by spontaneous nucleation. The habits of the crystals were wellcorrelated with the color (parts a and c of Figure 1). The red needles of I (8 × 1 × 1 mm) expressed the {011} and {102} forms; the {011} growth sectors were colored. Purple (5 × 2.5 × 0.5 mm) beveled plates of II had {001}, {101h}, and {122} forms; the {001} growth sectors were most heavily colored. Both forms were found to contain MR in proportions equal to that of the mother liquor: we found 1.1 × 10-3 and 1.4 × 10-3 moles of MR per mole of 2,5-DHB in Forms I and II, respectively. MR had little effect on * To whom correspondence should be addressed. E-mail: kahr@ chem.washington.edu. Telephone: 206-616-8195. Fax: 206-685-8665.
the habit of I, but reduced {100} faces of II to {122h}. Other additives such as cytochrome c produce the same effect.14 λmax of red I was 519 nm. Purple II absorbed at 538 and 572 nm. These values are distinct from MR in phthalic acid (PA) crystals (Figure 1b,d-g).12 MR has complex solution equilibria.15 Four species were observed: neutral (490 nm), zwitterionic (522 nm), protonated (513 nm), and deprotonated (423 nm). Form I could well be either protonated MR or the zwitterion. II is even more equivocal, absorbing far to the red of typical solution values. Cocrystal structures of MR with 2,5-DHB and PA were published previously.12 To these we add two cocrystals of MR and 2,6-DHB (1:1 and 1:2).16 From an analysis of the chromophore’s bond lengths in the X-ray structures, as well as locations of protons in difference maps, it is clear that the PA cocrystal contains neutral MR and that the 2,5-DHB cocrystal contains zwitterionic MR, whereas the two 2,6-DHB cocrystals contain protonated MR (Figure 2). This is a natural consequence of the greater acidity of 2,6-DHB (pKa1 ) 1.3) compared with 2,5-DHB (pKa1 ) 3.0)17 and phthalic acid (pKa1 ) 2.95).18 MR also forms mixed crystals with 2,6-DHB monohydrate (Pnma, a ) 6.778(1) Å, b ) 9.411(1) Å, c ) 11.890(2) Å.19 Needles elongated along [100] are homogeneously colored and show nearly complete dichroism. They are most absorbing when the incident light is polarized perpendicular to the needle axis. The spectra in Figure 1g of the mixed crystals of 2,6-monohydrate are virtually identical to the spectra of MR in II. As such, we surmise that MR in II and the 2,6-DHB mixed crystals are comparable. The greater acidity of 2,6-DHB and the two cocrystal structures suggests that MR is protonated whenever associated with 2,6-DHB, thus it is likely that the red crystals contain zwitterionic MR, whereas the purple crystals contain protonated MR. We showed that different growth sectors of potassium hydrogen phthalate will orient and overgrow 3,6-diaminoacridine in distinct protonation states.20 Therefore, it should come as little surprise that faces on distinct polymorphs can discriminate protonation states likewise. To establish how this discrimination arises, we studied the orientation of MR in I and II.21 Form I is dichroic for polarized light incident upon (011). Maximum absorbance occurs when the input polarization is 55° from a; the dichroic ratio was 3.4. (Figure 1b). The electric dipole transition moment of MR is along the long molecular axis (from the dimethylamino nitrogen to the furthest carbon on the acid ring) regardless of whether the MR is in the neutral, zwitterionic, or protonated state.12 Thus, the orientation of the long axis of MR in {011} Form I is 28° from a in the (011) projection. Form II examined through (001) showed no dichroism (Figure 1d). Conoscopy (Figure 1c) was consistent with a biaxial crystal viewed along an optic axis. A horizontal isogyre indicates that 2V ≈ 90°.22 Form II crystals cleaved easily along their long axes. Slices (200 µm) were turned 90° and laid upon their (100) surface. Vibration directions were along [001] and [010] in the (100) plane.
10.1021/cg060887w CCC: $37.00 © 2007 American Chemical Society Published on Web 02/02/2007
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Figure 1. Photograph and morphology idealization of (a) Form I 2,5-DHB and (c) Form II 2,5-DHB. Conoscopic image collected through (001). (b, d-f) Polarized absorbance spectra of 2,5-DHB growth sectors dyed by MR. Vibration directions are indicated as black and gray double-headed arrows and are correlated to the spectra. (d-f) Form II spectra. (g) Representative spectra of MR in 2,5-DHB Forms I (dashed) and II (solid) compared with the spectrum of MR in 2,6-DHB monohydrate (solid bold) for light polarized perpendicular to [100]. Morphology illustrated with WinXMorph (Kaminsky, W. J. Appl. Crystallogr. 2005, 38, 566-567).
Figure 2. Hydrogen bonding in cocrystals of (a) 1:1 MR:2,6-DHB and (b) 1:2 MR:2,6-DHB. Oxygens are red, and nitrogens are blue.
Absorbance measurements along the vibration directions gave a dichroic ratio of 1.5 with the strongest absorbance occurring for incident light in the [010] direction. The orientation of MR in Form II {001} sectors is 40° from [010] in the (100) plane. A small amount of dye in {101h} was weakly dichroic (Figure 1e). The dominant intermolecular interaction in the benzoic acid derivative:MR cocrystals is hydrogen bonding between the acid and MR carboxylic groups. We might then reasonably assume that the details of hydrogen bonding would explain MR selectivity. The lowest-energy surface of the {011} growth sectors of Form I is shown in Figure 3a. It displays one accessible H-bond donor, the disordered OH group at position 5. MR may attach to this site as an H-bond acceptor in either the zwitterionic or neutral states. These are very close in energy in the gas phase.12 The disordered hydrogen atom from the crystal structure was localized for the modeling. A geometry optimization was performed using the COMPASS forcefield23 (Forcite module of Materials Studio 4.024). The lowest-energy (011) face was cleaved, and a zwitterionic molecule was placed in close proximity. The model was then relaxed again. The long axis of MR projected in (011) (Figure 3b) was aligned 18° from [100] in the (011) projection when docked to (011), 9° from the experimental orientation. Docking to the lowest-energy (001) surface set the MR transition moment along b, in contrast to the transition moment calculated from the experiment. The volume of one MR is 329 Å3, whereas the volume of two 2,5-DHB molecules is comparable at 321 Å3 in I and 316 Å3 in II.
Therefore, we propose that one MR substitutes for two 2,5-DHB molecules in the mixed crystals. Geometry optimizations of the X-ray cells were performed using the COMPASS force field.23 The relaxed cells were (I) a ) 5.429 Å, b ) 11.575 Å, c ) 9.886 Å, β ) 90.815°, V ) 621.18 Å3; (II) a ) 5.325 Å, b ) 4.950 Å, c ) 23.535 Å, β ) 99.431°, V ) 611.97 Å3. These cells are just 3% smaller than the experimental cells. Substitution of two 2,5-DHB molecules by one MR was modeled for I and II. Replacement energies (REs) were calculated for symmetry distinct substitutions of MRs for two 2,5-DHB molecules according to the formula RE ) Emc + n(EDHB) - (Epc) - n(EMR). Here, Emc is the total minimized energy of the mixed crystal, n(EDHB) is the energy of n isolated DHB molecules removed to accommodate the host, Epc is the energy of the pure 2,5-DHB crystal, and n(EMR) is the energy of n isolated MR molecules ultimately accommodated in the mixed crystal. In Form I, there are two conceivable substitutions for centric 2,5-DHB pairs with indistinguishable REs of 48 and 49 kcal/mo1. When MR substitutes for one of these pairs, the MR carboxylic acid group accepts a hydrogen bond from a nearby 2,5-DHB 5 position hydroxyl, as in the docking computations. When substituted into Form I, the long axis of MR makes an angle of 21° to a in projection, close to the experimental 27°. The Form II crystal structure has six possible substitution geometries with replacement energies of 5, 7, 18, 19, 20, and 25 kcal/mol. The arrangement corresponding to smallest RE is shown in Figure 3c. Here, MR has disrupted two R22(8) dimers, causing the network of hydrogen bonds to rearrange (Figure 3c). As intramolecular H-bonding can increase the acidity of 2,6-DHB in solution, it is conceivable that intermolecular H-bonding could likewise increase the acidity of 2,5-DHB if molecules are suitably disposed. This is inferred by changes in the interatomic distances illustrated in Figure 3. A carboxylic acid hydrogen moves toward the 2-hydroxyl oxygen of neighboring molecules by 0.9 Å. In so doing, it increases the acidity of the _OH group, which in turn activates the adjacent -COOH group. These 2,5-DHB molecules have collectively rearranged, causing the intercrystalline environment experienced by MR to be more acidic in Form II than Form
494 Crystal Growth & Design, Vol. 7, No. 3, 2007
Communications Among the numerous MALDI ionization mechanisms proposed, the “Lucky Survivor” model assumes that analytes retain their solution-state charge and exist as preformed ions within the solidstate matrix.25 Common pH indicator dyes such as MR were incorporated within MALDI matrices in an attempt to evaluate the charge state of an analyte within a MALDI matrix.8 Kru¨ger et al. examined microcrystalline samples of 2,5-DHB stained by MR during precipitation on a MALDI sample plate. They concluded, on the basis of the fact that the 2,5-DHB particles were the same color as the solution from which they were deposited, that analytes in MALDI matrices are similarly charged in the solution and crystalline states. In our work, we have observed many examples in which dyes, including MR,12 display dramatically different photophysical properties in different growth sectors of the same crystal.11,12,20 As such, no general pronouncements about the properties of dyes in crystals can be easily carried forward from expectations on the basis of solution photophysics. On the other hand, although color is not necessarily diagnostic of the state of the dye,12 we have shown here that it is diagnostic of the state of the host. Acknowledgment. B.K. thanks the NSF and D.C. thanks the Keck Foundation for financial support. Many thanks also to A. Rohl for assistance with force-field computations. Supporting Information Available: Four CIF files (two 2,5-DHB single-crystal structure redeterminations and two 2,6-DHB/MR cocrystal structures). This material is available free of charge via the Internet at http:// pubs.acs.org.
References
Figure 3. (a) Docking of zwitterionic MR (in green) to the lowest-energy (011) surface of Form I. (b) Model in (a) viewed normal to the (011) face through which the dichroism measurements were made. Orange lines indicate indistinguishable MR orientations consistent with the dichroism. Black and gray arrows indicate extinction directions. (c) Lowest replacement energy model for MR in Form II crystals. Distance indicated by d has been reduced by nearly 1 Å, likely activating the hydrogen indicated by the asterisk (*).
I, in accordance with Form II’s purple color. The orientation displayed in Figure 3c is the same as that found in the docking simulations. The deviation between this orientation and the orientation expected from linear dichroism is likely due to the incorporation of MR in more than one way, making the measured dichroic ratio an average.
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73, 1016-1022. (d) Sadeghi, M.; Wu, X.; Vertes, A. J. Phys. Chem. B 2001, 105, 2578-2587. (e) Kinsel, G. R.; Quinchun, Z.; Narayanasamy, J.; Yassin, F.; Rasika Dias, H. V.; Niesner, B.; Prater, K.; St. Marie, C.; Ly, L.; Marynick, D. S. J. Phys. Chem. A 2004, 108, 3153-3161. (f) Horneffer, V.; Reichelt, R.; Strupat, K. Int. J. Mass Spectrom. 2003, 226, 117-131. (g) Wu, X.; Sadeghi, M.; Vertes, A. J. Phys. Chem. B 1998, 102, 4770-4778. Kahr, B.; Gurney, R. W. Chem. ReV. 2001, 101, 893-951. Benedict, J. B.; Cohen, D. E.; Lovell, S.; Rohl, A. L.; Kahr, B. J. Am. Chem. Soc. 2006, 128, 5548-5559. Bernstein, J. Polymorphism in Molecular Crystals; Clarendon Press: Oxford, U.K., 2002. Trimpin, S.; Keune, S.; Rader, H. J.; Mullen, K. J. Am. Soc. Mass Spectrom. 2006, 17, 661-671. Drummond, C. J.; Grieser, F.; Healy, T. W. J. Chem. Soc., Faraday Trans. 1 1989, 85, 561-578. Crystals of 1:1 and 2:1 2,6-DHB:MR were grown by thermal reduction of ethanol solutions containing 1.54 g of 2,6-DHB and 0.0269 g of MR, and 1.54 g of 2,6-DHB and 0.269 g of MR, respectively. Crystal data for 1:1 2,6-DHB:MR, C22H21N3O6: M ) 423.42, monoclinic, a ) 13.9970(3) Å, b ) 11.4720(3) Å, c ) 13.8990(4) Å, β ) 118.3631(12)°, V ) 1963.89(9) Å3, spacgroup P21/c, T ) 130(2) K, Z ) 4, Fcalcd ) 1.432 mg m-3, µ (Mo-KR) ) 0.106 mm-1, 7287 reflns measured, 4424 unique reflns, R1obs ) 0.0448, wR2obs ) 0.1190. Crystal data for 2:1 2,6-DHB:MR, C29H27N3O10: M ) 577.54, monoclinic, a ) 16.9900(8) Å, b ) 11.5710(9) Å, c ) 13.9390(14) Å, β ) 93.993(5)°, V ) 2733.6(4) Å3, space group P21/c, T ) 130(2) K, Z ) 4, Fcalcd ) 1.403 mg/m-3,
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(17) (18)
(19) (20)
(21)
(22) (23) (24) (25)
µ (Mo-KR) ) 0.107 mm-1, 8306 reflections measured, 4822 unique reflns, R1obs ) 0.0507, wR2obs ) 0.1138. Papadopoulos, N.; Avranas, A. J. Solid Chem. 1991, 20, 293-300. (a) Singh, A. K.; Ghosh, J. C. J. Indian Chem. Soc. 1983, 60, 702704. (b) Singh, A. K.; Ghosh, J. C. J. Indian Chem. Soc. 1985, 62, 158-160. Gdaniec, M.; Gilski, M.; Denisov, G. S. Acta Crystallogr., Sect. C 1994, 50, 1622-1626. Barbon, A.; Bellinazzi, M.; Benedict, J. B.; Brustolon, M.; Fleming, S. D.; Jang, S.-H.; Kahr, B.; Rohl, A. L. Angew. Chem., Int. Ed. 2004, 43, 5328-5331. Certainly, conformational distortions will affect the MR electronic transition energies as in the famous case of ROY (Yu, L. J. Phys. Chem. A 2002, 106, 544-550). We have no direct evidence of the MR conformations in our mixed crystals. But, of the six MR and MR cocrystal structures reported in this paper and in ref 12, the angles between aromatic planes varied between 2 and 13°, with an average value of just 6°. On the other hand, an unpublished cocrystal structure of trimesic acid and MR (CSD deposition no. 632878) was considerably nonplanar, with an angle between aromatic planes of 37°. Hartshorne, N. H.; Stuart, A. Practical Optical Crystallography, 2nd ed.; Elsevier: New York, 1969. Sun, H. J. Phys. Chem. B 1998, 102, 7338-7364. Accelrys, 4.0 ed.; Accelrys, Inc.: San Diego, 2005. Karas, M.; Gluckmann, M.; Schafer, J. J. Mass Spectrom. 2000, 35, 1-12.
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