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A Fluorogenic Aromatic Nucleophilic Substitution Reaction for Demonstrating Normal-Phase Chromatography and Isolation of Nitrobenzoxadiazole Chromophores Jessie A. Key, Matthew D. Li, and Christopher W. Cairo* Alberta Ingenuity Centre for Carbohydrate Science, Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada *
[email protected] The spectral properties of organic dyes and fluorophores are a result of their chemical structures. In this experiment, strongly fluorescent nitrobenzoxadiazole (NBD) dyes (2-4) are generated from the commercially available nonfluorescent starting material, 4-chloro-7-nitrobenzoxadiazole (NBD-Cl, 1) by an aromatic nucleophilic substitution (SNAr) (Scheme 1) (1, 2). Students first synthesize fluorescent dyes in a microscale reaction, generating fluorophores with amine, amino alcohol, and amino acid functional groups. The second portion of this experiment develops basic thin-layer chromatography (TLC) skills by allowing students to observe the separation of the synthetic dyes with varying structure (3). Students are encouraged to explain the retention factor, Rf, of each derivative based on chemical structure. The final portion of the experiment familiarizes the student with flash-column chromatography (4, 5) as a method of separation, allowing them to isolate a synthetic NBD dye (3). The progress and elution of the product can be easily monitored visually or with the use of a long-wavelength UV lamp (365 nm). Student response has been positive, and the experiment provided an improvement in general comprehension of normal-phase chromatography.
Figure 1. Normalized absorbance and fluorescence emission (dashed) of NBDs (1) and (3) in methanol. Scheme 1. Nitrobenzoxadiazole Reactions: Three Fluorophores Generated by the Reaction of an Amine with the Starting NBD-Cl
Background Fluorescence occurs after excitation of an electron in the singlet ground state (S0) to the first excited state (S1), upon absorption of a photon. Relaxation to the ground state can occur nonradiatively or radiatively with the emission of a photon providing fluorescence emission (6-8). Molecules that can be excited and relax efficiently in a radiative pathway are known as fluorophores. Organic fluorophores are an important tool in bioconjugate chemistry where these chromophores act as specific probes for fluorescence imaging and spectroscopy. Bright fluorophores with good spectral resolution from interfering compounds provide excellent signal-to-noise ratios in complex mixtures (6, 7). Importantly, the structure of the fluorophore determines its spectra, and therefore, synthetic dyes with improved properties are an active area of investigation. The experiment described here allows students to observe the effect of changing organic structure on the visual and fluorescence spectra of these compounds. Most dramatically, students observe that the starting material, 1, is not fluorescent; however, all of the major products demonstrate fluorescent properties. Students then observe the influence of organic functional groups on normal-phase chromatography. 98
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Nitrobenzoxadiazole fluorophores are an important class of small molecule labels used in chemical biology and bioanalytical studies (8, 9). Their small size, strong fluorescence, and sensitivity to environment or substitution make them ideal for numerous applications in bioconjugation. For example, these dyes are commonly used as lipid probes and for enzymatic assays (8-10). The 4-nitro-substituted benzofurazan ring is an excellent substrate for SN Ar reactions (11), often resulting in an increase in fluorescence when the halide is replaced with an amine or other heteroatoms (Figure 1) (12). Experimental and theoretical work has shown that the key factors in control of the fluorescence of 4,7-substituted benzofurazan dyes are the dipole moment across the benzene ring (from position 4 to 7) and the electron density
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found on the ring (13). In the reactions chosen here, students observe the effects of changing substitution at the 7-position from Cl to an amine, alkylamine, and an amino acid. The ease of the substitution reaction, availability of the starting material (1), and the strongly colored products make these compounds an excellent choice for demonstrating principles of normal-phase chromatography and spectroscopy of organic compounds. Thin-layer chromatography provides valuable information to organic chemists, allowing them to monitor reactions, support identification of a product, and to optimize conditions for isolation of desired components. As a normal-phase chromatographic technique, the ratio of the distance traveled by an analyte in relation to the solvent front (Rf) is proportional to the time spent in the mobile phase (14). Thus separation of distinct analytes depends upon the tendency of each compound to adsorb to the silica gel stationary phase. The silica gel polymer is polar, slightly acidic (pKa ∼ 2), and traditionally prepared by condensation of polysilicic acid (14, 15). Polar compounds, and those capable of hydrogen bonding, have a greater tendency to adsorb to the stationary phase, resulting in a lower Rf. Strength of adsorption typically follows the trend: alkanes < alkenes < ethers < esters < ketones and aldehydes < amines < alcohols and phenols < acids (16). The composition and polarity of the mobile phase can be adjusted to achieve the desired Rf and separation. Although TLC serves a valuable analytical role, it is cumbersome to use for purification on a preparative scale (greater than 100 mg). For this purpose, column chromatography has been developed with the capability of purifying from milligram to kilogram scales. One of the most popular chromatographies in the organic chemistry laboratory is normal-phase flash chromatography. Flash chromatography was first introduced in 1978 by Still and colleagues, and uses air pressure to drive elution, unlike TLC, which relies on capillary action (17). The process is commonly used for purification in industry and research laboratories owing to its speed and resolution of separation (ΔRf g 0.15) (17). The desired Rf for optimal separation and purification of a compound is approximately 0.35, and eluent systems should be chosen to achieve this (17). Experimental Procedures Synthesis and Analysis by TLC Three fluorophores are generated at microscale and characterized by TLC. The starting material 4-chloro-7-nitrobenzoxadiazole (1) is reacted with ammonia, ethanolamine, and glycine each dissolved in methanol. Flash Column Chromatography Purification The reaction carried out with ethanolamine can be reduced and purified by flash column chromatography. A disposable “pipet column” is prepared using a small quantity of silica (0.45 g) (18). The product is followed visually or by long-wavelength UV lamp irradiation (365 nm) by its bright yellow fluorescence (see Figures SI3 and SI4 in the supporting information). Hazards 4-Chloro-7-nitrobenzoxadiazole and glycine may be harmful if inhaled, in contact with skin, or if swallowed. Methanol and ammonia are flammable and toxic by inhalation, in contact
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with skin, or if swallowed. Ethanolamine is flammable, corrosive, and toxic by inhalation, in contact with skin, or if swallowed. Ethyl acetate and hexanes are flammable and irritants by inhalation, in contact with skin, and if swallowed. Silica gel causes respiratory tract irritation, and the column should be packed in a fume hood while wearing a mask to filter particulates. UV irradiation is harmful to the eyes. Safety glasses should be worn at all times. Results and Discussion The relative rates of the reactions proceed in the order 3 > 2 > 4. Because the substrate is identical in all three reactions, the rate determining step is expected to be attack of the nucleophile in protic solvent (2). The amino alcohol used to form 3 reacts very rapidly, and students should typically observe a yield between 40-50% from the synthesis and purification of 3 (although crude yields are ∼70-80%). The pipet-column purification of product 3 requires minimal preparation and cleanup compared to a traditional column and displays distinct separation visually and by UV illumination. However, this step is a common source of loss for students. Characterization of 3 is easily performed by IR or UV-vis and fluorescence analysis. NMR analysis may be performed, but it should be noted that some signals may be obscured by residual solvent (see the supporting information). Despite the NBD being an excellent substrate for SNAr owing to the presence of a para-nitro group on the ring, reactions to form 2 and 4 are somewhat sluggish. However, all three products should be visible by UV illumination or by eye on TLC after 45 min of reaction time (Figure SI1 in the supporting information). An additional concept that may be covered in this laboratory is the quantification of reaction kinetics of 2 and 4 by performing TLC at determined time intervals and comparing the product concentrations. Students also observe the striking difference in fluorescence between the starting material, 1, and the three products. This is most clearly demonstrated with purified samples of 3 obtained after flash column chromatography (see Figure SI5 in the supporting information). The amine-substituted products fulfill the expected characteristics of bright benzofurazan dyes with a 4,7-substitution pattern. The increased electron donation of the amine group relative to that of the chloride, as well as the increased dipole moment may both contribute to this effect (13). The observed order of retention of the synthetic dyes on the TLC is 1 (0.88 Rf) > 2 (0.42 Rf) > 3 (0.11 Rf) > 4 (0 Rf) with 3:2 EtOAc/hexane as the eluent. This trend follows the expected polarity of these compounds, with 1 being the least polar. All of the synthetic derivatives are more polar than the starting material, and compounds 2-4 contain multiple additional hydrogen bond donors and acceptors relative to the starting material, leading to reduced time in the mobile phase. This analysis may be combined with theoretical approaches to predict retention times (19). Summary The fluorogenic nature of the nucleophilic substitution described here allows for a simple identification of the product by TLC. As well, this experiment promotes discussion of the concepts of aromatic substitution, spectroscopy, organic structure, and polarity. Normal-phase chromatography is examined in both parts of the experiment, clearly demonstrating the effects of
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organic structure on the migration of the products. Students should observe that substitution of amine, amino alcohol, and amino acid functional groups alter the Rf of each NBD derivative in a predictable and significant way. Students gain valuable experience by performing this experiment at the microscale and are introduced to the growing field of conjugation using organic fluorophores. Acknowledgment This work was supported by the Alberta Ingenuity Centre for Carbohydrate Science. We would like to acknowledge Stuart Chambers (University of Alberta) for helpful discussions. Literature Cited 1. Zoltewicz, J. A. In Topics in Current Chemistry; Boschke, F. L., Ed.; Spinger: Berlin, 1975; pp 33-64. 2. Smith, M. B.; March, J. March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6th ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2007. 3. Calimente, D. S.; Strand, S. M.; Chang, S.-C.; Lewis, D. E. J. Chem. Educ. 1999, 76, 82–83. 4. Ponten, F.; Ellervik, U. J. Chem. Educ. 2001, 78, 363. 5. Horowitz, G. J. Chem. Educ. 2000, 77, 263–264. 6. Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006. 7. Valeur, B. Molecular Fluorescence Principles and Applications; WILEYVCH: Weinheim, 2002.
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8. Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008, 3, 142–155. 9. Bem, M.; Badea, F.; Draghici, C.; Caproiu, M. T.; Vasilescu, M.; Voicescu, M.; Beteringhe, A.; Caragheorgheopol, A.; Maganu, M.; Constantinescu, T.; Balaban, A. T. ARKIVOC 2007, 87–104. 10. Numasawa, Y.; Okabe, K.; Uchiyama, S.; Santa, T.; Imai, K. Dyes Pigm. 2005, 67, 189–195. 11. Avila, W. B.; Crow, J. L.; Utermoehlen, C. M. J. Chem. Educ. 1990, 67, 350–351. 12. Uchiyama, S.; Santa, T.; Fukushima, T.; Homma, H.; Imai, K. J. Chem. Soc., Perkin Trans. 2 1998, 2165–2173. 13. Uchiyama, S.; Santa, T.; Imai, K. J. Chem. Soc., Perkin Trans. 2 1999, 2525–2532. 14. Braithwaite, A.; Smith, F. J. Chromatographic Methods, 5th ed.; Springer: Berlin, 1996. 15. delCampo, A.; Bruce, I. J. Substrate Patterning and Activation Strategies for DNA Chip Fabrication; Springer: Berlin, 2005; Vol. 260. 16. Fifield, F. W.; Kealey, D. Principles and Practice of Analytical Chemistry, 5th ed.; Blackwell Science: Oxford, 2000. 17. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923– 2925. 18. Reynolds, R. C.; Odell, C. A. J. Chem. Educ. 1992, 69, 989–991. 19. Hessley, R. K. J. Chem. Educ. 2000, 77, 203–205.
Supporting Information Available Notes for the instructor; NMR and IR spectra. This material is available via the Internet at http://pubs.acs.org.
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