Lighting Up Protons with MorphFl, a Fluorescein–Morpholine Dyad

LABORATORY EXPERIMENT pubs.acs.org/jchemeduc

Lighting Up Protons with MorphFl, a Fluorescein
Morpholine Dyad: An Experiment for the Organic Laboratory Tyson A. Miller,*,# Michael Spangler, and Shawn C. Burdette*,† Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269-3060, United States

bS Supporting Information ABSTRACT: A two-period organic laboratory experiment that includes fluorescence sensing is presented. The pH-sensitive sensor MorphFl is prepared using a Mannich reaction between a fluorescein derivative and the iminium ion of morpholine. During the first laboratory, students prepare MorphFl. The second session begins with characterizing the sensor using standard techniques, followed by using MorphFl to analyze the relative acidity of several unknown aqueous solutions. MorphFl absorbs visible and near-UV light and emits a bright green fluorescence in aqueous solutions at low pH. At high pH, the emission of MorphFl is quenched. This laboratory experiment is appropriate for demonstrating condensation reactions, electrophiles, absorbance and emission of light by organic molecules, frontier molecular orbitals, and fluorescent sensors to any undergraduate audience. The visual component of the fluorescence assay provides an exciting alternative to more traditional organic laboratory experiments. KEYWORDS: Second-Year Undergraduate, Interdisciplinary/Multidisciplinary, Laboratory Instruction, Organic Chemistry, Hands-On Learning/Manipulatives, Dyes/Pigments, Fluorescence Spectroscopy, MO Theory, Photochemistry, UV
Vis Spectroscopy

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emonstrating the connection between experiments and commonplace chemical compounds remains the preferred technique to engage undergraduate organic chemistry laboratory students. The abundance of pharmaceutical connections in the standard undergraduate curriculum may lead students to underestimate the breadth of applications for organic chemistry in modern research. Classic organic reactions have been applied to preparing molecular devices,1 supramolecular constructs,2 and fluorescent sensors.3 Fluorescent sensors are small organic molecules with emissive properties that change upon binding an external analyte.4 Sensors consist of a fluorophore, typically an aromatic hydrocarbon, and a receptor capable of binding the atom or molecule of interest selectively. These molecular tools have been vital in identifying the functions of metal ions and small molecules in biology,5 detecting various environmental pollutants,6 and constructing logic gates that may be useful in making electronic devices.7 With appropriate molecular design, fluorescent sensors are capable of making sensitive, quantitative measurements of analyte concentration using signals that are easy to monitor. In addition to quantification, fluorescent sensors can provide information about the spatial distribution and changes in analyte concentration in real time. Sensor molecule construction, properties, and behavior are easily understood by applying concepts introduced in the organic chemistry classroom. Although numerous articles in this Journal have focused on topics in fluorescence,8
10 there are relatively few laboratory exercises that allow students to explore the topic of fluorescent sensors. In this laboratory, students use a Mannich reaction to synthesize a simple proton sensor from a fluorescein derivative Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.

and then use their compound to estimate the acidity and basicity of several unknown solutions. This experiment has been conducted in an introductory organic laboratory at a large university. The classic Mannich reaction involves the reaction of an iminium ion and an enol (Scheme 1). The mechanism of the Mannich reaction with phenolic substrates has a modicum similarity to electrophilic aromatic substitution with an iminium ion behaving as an electrophile (Scheme 2). The Mannich reaction has been used extensively to make metal ion sensors from fluorescein.11 For this experiment, the diamine-based metal chelator was replaced by morpholine to synthesize the protononly receptor MorphFl, the generic name for the target compound derived from the morpholine and fluorescein constituents of the sensor. The same molecule has been described as a sensor for silver.12 The iminium ion is generated by condensing morpholine with paraformaldehyde, and the fluorescein substrate contains two phenolic reaction sites. In fluorescein, proton migration between the phenolic- and keto-oxygen generates two identical tautomers. The interconversion affords both phenolic sites required for two Mannich reactions.

’ EXPERIMENTAL OVERVIEW This simple experiment is designed to be carried out in two, 3-h laboratory sessions by students in an introductory organic chemistry laboratory. The experiment is most appropriate after the students have had experience setting up synthetic reactions as Published: August 18, 2011 1569

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Scheme 1. Mechanism of the Mannich Reaction

Scheme 2. Synthesis of MorphFl

well as using standard isolation and characterization methodologies such as recrystallization and thin-layer chromatography. Each student in the class carried out the experiment individually, but small groups can execute the procedures as well. The first laboratory period involves the synthesis of the target sensor, MorphFl. Attaining good yields of the desired product requires student efficiency in setting up reactions to allow for adequate reflux time, so the required glassware should be set aside in the previous lab period. A final reflux time between 2
2.5 h is sufficient. Students can compare and contrast their procedure and results to several related fluorescein-based sensors reported in the literature.12
14 During the second laboratory period, the product is isolated. If implemented in a longer laboratory period, the isolation procedure can be conducted immediately after preparation without changing the yield. The purity and identity of the product can easily be verified by thinlayer chromatography (TLC) by comparing the product profile to that of 20 ,70 -dichlorofluorescein. Both compounds exhibit bright fluorescence on the TLC plate and have dramatically different Rf values. MorphFl also has distinctive 1H NMR, 13C NMR, IR, and UV
vis spectra that can be matched to the ones provided in the Supporting Information. The second component of the experiment is also completed during the second laboratory period. Rather than acquiring a detailed pH change induced emission profile, students determine the relative acidity of three unknown solutions: basic, neutral, acidic. Although the content of these aqueous solutions does not affect the fluorescence, the visual evaluation of the emission intensity precludes measuring subtle differences in pH (e.g., students cannot reliably differentiate between pH 5 and pH 4 solutions). An analogous fluorescence assay can be conducted with 20 ,70 -dichlorofluorescein, which is insensitive to changes in pH. The pH-induced emission changes of MorphFl can be quantified by

spectrophotometry between pH 2 and pH 12 using the procedures in the Supporting Information. This modification is most suitable for small groups of students in an advanced organic or analytical course.

’ EXPERIMENTAL PROCEDURE Synthesis of MorphFl

Morpholine (0.350 g, 0.350 mL, 4.00 mmol), paraformaldehyde (0.112 g, 3.74 mmol), and 10 mL of acetonitrile were combined in a 50 mL round-bottom flask and refluxed for 30 min. 20 ,70 -Dichlorofluorescein (0.500 g, 1.25 mmol) in 15 mL of acetonitrile/water (1:1) was added to the solution, and the reaction mixture was refluxed for 2.5 h with continuous stirring, and then left at room temperature until the next lab period. Adequate stirring is necessary to prevent the aqueous solvent mixture from bumping. The reaction was diluted with 20 mL of water, placed in an ice bath to induce precipitation, and the product was collected by vacuum filtration. Alternatively, the crude reaction mixture was saturated with sodium chloride and placed in an ice bath to isolate the product if no precipitate formed initially. The crude product was suspended in boiling hot ethanol (10 mL), collected by filtration, and washed with ice cold water to yield a light pink powder. The compound was characterized by TLC (silica, 9:1 dichloromethane/methanol), IR, and 1H NMR. The UV
vis and fluorescence spectrum in methanol or water can also be obtained with access to appropriate instrumentation, but is not a required analytical technique to conduct the experiment. Fluorescence Assay

Three solutions of unknown pH (5 mL each of HCl pH = 5, deionized water pH = 7, and NaOH pH = 12) were placed in test 1570

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Figure 1. Illustration of the fluorescence sensing mechanism of MorphFl. In the absence of protons, the amino lone pair quenches fluorescein emission by PeT (left). When protons are introduced, new bonds form between the nitrogen and hydrogen atoms (center). The protonation stabilizes the receptor HOMO, which prevents PeT and enhances fluorescence (right).

tubes. MorphFl was dissolved in dimethylsulfoxide (DMSO) to make a 1 mM stock solution. A drop of the DMSO stock solution was added to each of the unknown solutions and the relative emission was evaluated using a handheld UV lamp (λex = 365 nm). The students were asked to qualitatively determine the intensity of the fluorescence and determine the pH of the unknown solutions based on the described pH-sensing abilities of MorphFl. The procedure is repeated by making a 1 mM stock solution with 20 ,70 -dichlorofluorescein and comparing its fluorescence response as a control.

’ HAZARDS Nitrile gloves and goggles should be worn at all times. Lab coats or aprons are recommended. Reactions should be carried out in fume hoods. Acetonitrile and ethanol are flammable. Morpholine is an eye and skin irritant. Paraformaldehyde is a carcinogen. Dimethylsulfoxide readily absorbs through the skin. 20 ,70 -Dichlorofluorescein may cause irritation to skin, eyes, and respiratory tract and may be harmful if swallowed or inhaled. Limit exposure of skin and eyes to UV light. ’ PRELABORATORY LECTURE ON MOLECULAR ORBITALS, PHOTOCHEMISTRY, AND FLUORESCENT SENSORS To understand the switching behavior of MorphFl, the concepts of molecular orbitals, bonding, aromaticity, and the absorption and emission of light must be reviewed. Constructing the molecular orbital diagram for H2 provides adequate background on the types of bonding interactions involved in the sensing mechanism of MorphFl. Because only the lone pair of electrons on the morpholine nitrogen atoms and the empty s orbital of a proton are involved in the bonding, the discussion of molecular orbitals can be restricted to σ bonding until the introduction of frontier molecular orbitals involved in the photochemistry.

The fluorescein fluorophore is a (4n + 2) π electron system similar to classic examples of aromatic hydrocarbons. A simple molecular orbital diagram for the π system of benzene provides a convenient model to describe the light absorption and the emission. The promotion of an electron from π f π* upon absorption of a photon and the subsequent return to the ground state with the emission of a lower energy photon can be illustrated with the 6-electron benzene system, which parallels the conjugated π system in fluorescein. With a basic understanding of molecular orbitals and photochemistry, students can intellectually access the concepts of fluorescence sensing. The majority of the existing sensors for cationic species such as metal ions and protons utilize photo-induced electron transfer (PeT) as the signaling mechanism. In PeT systems, nonbonding electrons on receptor ligands, which are electronically decoupled from the fluorophore, quench the emission from the excited state.15,16 In MorphFl, the two tertiary nitrogen atoms derived from the morpholine groups serve as PeT quenchers. The nitrogen atoms of MorphFl serve the dual purpose of PeT quencher and analyte receptor as amines are basic and susceptible to protonation. Coordination of a metal ion or a proton to a PeT quencher, such as the morpholine nitrogen atoms, prevents these electrons from participating in quenching processes. A simple frontier molecular orbital diagram can be derived to explain the mechanism of the PeT driven fluorescence switch in MorphFl (Figure 1). Upon protonation, the previously nonbonding electrons on the nitrogen atoms reside in a stabilized σ bonding molecular orbital formed from the interaction between analyte and receptor with the concomitant formation of a σ* orbital. Although many details of the electron transfer process are beyond the scope of undergraduate organic chemistry, a comprehensive discussion of excited-state photophysics is not required to appreciate sensor behavior. With advanced students, a more detailed examination of PeT could be explored using MorphFl as an example. Amines are ideal simple receptors for protons because of built-in selectivity for cationic species. 1571

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Journal of Chemical Education Because proton concentration directly correlates with pH, a PeTbased sensor with an amine receptor will have pH-dependent emission intensity. The equilibrium between free amine and ammonium cation will result in a fluorescence profile that corresponds to the pKa of this deprotonation.

’ STUDENT EXPERIMENTAL RESULTS AND LEARNING This experiment was piloted with 161 introductory undergraduate organic chemistry laboratory students. Several issues were examined to determine the experiment’s viability as a component of organic laboratory curriculum, including yield, ease of completion, and a superficial analysis of concept acquisition through pre- and postlab surveys and lab reports. From the execution of the experiment, the average MorphFl yield was 37.3% ( 17.2% with nearly 60% of the students able to produce a yield greater than 40%. Only 12 students were unable to successfully execute the synthesis. The outcome is typical of the success rate that students at this stage in their chemistry training have with synthetic experiments. Typical difficulties reported by the students included maintaining reaction temperature and efficiency in setting up the reactions. Although some students reported difficulty isolating the product, many of those cases correspond to low yields or no product being recovered, which suggests student error in executing the procedures during the first laboratory period. Students had no difficulty identifying the unknowns in the pH-sensing assay. Pre- and postlab anonymous surveys and the lab reports provided information about student attitudes and understanding about six specific concepts related to the lab: electrophilic aromatic substitution, fluorescence, molecular sensors, Mannich reactions and mechanism, effect of pH on structure and reactivity, and the electromagnetic spectrum. The lab’s greatest impact on student understanding was on the Mannich reaction and on the concept of molecular sensors. Students reported little confidence in explaining a Mannich reaction or its mechanism before the lab, but increased significantly postlab with reasonable answers in the lab report conclusions. Modest gains in confidence and understanding were observed regarding the concept of molecular sensors, what they are, and how they function. No significant impact from the lab was observed overall on confidence or knowledge gains in electrophilic aromatic substitution and effect of pH on structure and reactivity. Students were observed to have difficulties with the concept of fluorescence and molecular interactions with UV light. Although many students could correctly define fluorescence and state UV light
molecule interactions in lab reports, the understanding and confidence in these topics was not preserved in postlab surveys. Although this lab constitutes a novel and innovative approach to introducing photochemistry topics, the results demonstrate the lab is one component toward successfully illustrating and integrating photochemistry concepts into an organic chemistry laboratory curriculum. Overall, the students appreciated how traditional undergraduate organic chemistry can be applied to modern molecular tools involved in chemical, biological, and medicinal research. ’ CONCLUSIONS The fluorescent sensor MorphFl can be prepared by a Mannich reaction and used to identify unknown solutions with different pH in a simple fluorescence assay. This new experiment is suitable for a typical undergraduate introductory organic laboratory

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experiment and was demonstrated to be viable in pilot testing. The visual component of the fluorescence assay is effective in generating interest in the experiment because it provides an alternative to the typical curriculum. The experiment introduces students to an underrepresented application of organic transformations: the construction of molecular sensors that have applications in biology and the environment. The experimental background reinforces the concept of molecular orbitals and the interaction of organic molecules with light, which are both important topics introduced in organic chemistry.

’ ASSOCIATED CONTENT

bS

Supporting Information Notes for the instructor; extensions of the laboratory; student evaluation; 1H NMR, 13C NMR, IR, and UV
vis spectra. This material is available via the Internet at http://pubs.acs.org. Present Addresses †

Shawn C. Burdette, Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609-2280, United States. # Tyson A. Miller, Heritage University, Toppenish, Washington 98948 United States.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected], [email protected].

’ ACKNOWLEDGMENT S.C.B. acknowledges the support of this work by NSF grant CHE-0955361. We also like to thank the teaching assistants and students from the spring 2008 organic chemistry laboratory courses for their participation. ’ REFERENCES (1) Nagamani, S. A.; Norikane, Y.; Tamaoki, N. J. Org. Chem. 2005, 70 (23), 9304–9313. (2) Cantrill, S. J.; Youn, G. J.; Stoddart, J. F.; Williams, D. J. J. Org. Chem. 2001, 66 (21), 6857–6872. (3) Chi, L.; Zhao, J. Z.; James, T. D. J. Org. Chem. 2008, 73 (12), 4684–4687. (4) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515–1566. (5) Domaille, D. W.; Que, E. L.; Chang, C. J. Nat. Chem. Biol. 2008, 4 (3), 168–175. (6) Nolan, E. M.; Lippard, S. J. Chem. Rev. 2008, 108 (9), 3443–3480. (7) de Silva, A. P.; Uchiyama, S. Nat. Nanotechnol. 2007, 2 (7), 399–410. (8) Windisch, C. F.; Exarhos, G. J.; Sharma, S. K. J. Chem. Educ. 2005, 82 (6), 916–918. (9) Blitz, J. P.; Sheeran, D. J.; Becker, T. L. J. Chem. Educ. 2006, 83 (5), 758–760. (10) Cumberbatch, T.; Hanley, Q. S. J. Chem. Educ. 2007, 84 (8), 1319–1322. (11) Nolan, E. M.; Lippard, S. J. Acc. Chem. Res. 2009, 42 (1), 193–203. (12) Swamy, K. M. K.; Kim, H. N.; Soh, J. H.; Kim, Y.; Kim, S. J.; Yoon, J. Chem. Commun. 2009, 10, 1234–1236. 1572

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