Thiol-Selective Chemo-sensor

Fall 2016

Introduction Guidelines

Thiols are important functional groups in biology, found in coenzyme A and in cysteine amino acids of peptides or proteins. The excellent nucleophilicity of thiols makes it possible to distinguish them from other nucleophiles in biological systems with mild, chemoselective electrophiles. Common examples of reagents used to selectively react with thiols are haloacetamides and maleimides (see below). Maleimides are used to prepare antibody–drug conjugates for cancer treatment (e.g. brentuximab vedotin). Some of these reagents are unstable, poorly selective, or difficult to prepare with fluorescent tags. In this experiment you will prepare a more stable and selective probe that only becomes fluorescent upon reacting with a thiol in solution. Such sensors may have a variety of uses in biology or in dyeing hair for parties.

In this experiment, you will react N-dansylfurfurylamine with dimethyl acetylenedicarboxylate at room temperature or at 37 °C with no solvent. You will then analyze and identify the structure of the product by NMR spectroscopy, and test its fluorogenic properties in selectively reacting with thiols from the amino acid N-acetylcysteine, bovine serum albumin (BSA), or your own hair!

• Synthesize a novel fluorogenic oxanorborna-diene (OND) sensor

• Develop skills for performing reactions at micro-scale, including column chromatography

• Utilize a fluorimeter to quantify the fluorescence of a for the sensor

• Apply the sensor in the detection of hair color

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Experimental Procedure Probe Synthesis: Add N-dansylfurfurylamine (16.5 mg, 0.05 mmol) to a labeled and tared microcentrifuge tube or ½-dram vial. Add dimethyl acetylenedicarboxylate (18 µL, 0.15 mmol). Cap the reaction, swirl gently to mix, and place it in a secure, dark location indicated by your instructor. Ensure that the tube or vial will remain upright and allow the mixture to react for one week at room temperature or at 37 °C.

NMR Spectroscopy: After one week, collect a 1H NMR spectrum in CDCl3 and calculate the percent conversion for the reaction. (Hint: N-dansylfurfurylamine has a doublet at δ 8.51, and the probe product has a doublet at δ 8.55). TLC Analysis: Either before or after collecting your NMR spectrum (but before running your column), compare your NMR sample to the starting material by thin-layer chromatography on silica gel plates. Use 3:1 hexanes/ethyl acetate as the eluent and visualize with short- and long-wave UV light. Purification: Purify your product by column chromatography on silica gel using a micro-column, using 3:1 hexanes/ethyl acetate as the eluent (prepare 40 mL). Dry-pack the column as instructed. Use small test tubes to collect 1-mL fractions. If there is a trace of your crude product left in your reaction vial, you may dilute it in ethyl acetate for TLC comparison with column fractions. Use TLC (3:1 hexanes/ethyl acetate) to track the progress of your separation. Your product can be visualized using UV light. The N-dansylfurfurylamine starting material fluoresces yellow-green under long-wave UV light. Your product is not visible with long-wave UV light, but will stand out as a dark spot under short-wave UV light.

If you do not think your product is completely out of the column at the end of the 40 mL of eluent, you can increase the eluent polarity to 2:1 hexanes/ethyl acetate (you should only need 15 mL more eluent: 10 mL hexanes and 5 mL ethyl acetate). Using the results of your TLC analyses, pool the fractions that contain pure product and transfer their contents to a labeled and tared vial of sufficient size using a Pasteur pipet. Rinse each test tube with about 10 drops of ethyl acetate and transfer these rinses to your collection vial with a Pasteur pipet as well. Evaporate the solvent by rotary evaporation in a tared flask to find your purified product mass.

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Analysis NMR Spectroscopy: Collect a 1H, 13C, COSY, DEPT-135 and HSQC spectrum of your purified product in CDCl3 and assign as many signals the structure as you can. Fluorescence Testing Preparation: Evaporate the CDCl3 with rotary evaporation or in a vial under a stream of nitrogen. Create a 20 mM solution of your probe in acetonitrile by adding the appropriate volume of acetonitrile to the vial of purified product. Fluorescence Testing: Before testing the effectiveness of your fluorescent probe, first shine long-wave UV light on the acetonitrile solution (you may want to hold your sample under a counter or in a cabinet to see fluorescence better). Take note of the intensity and proceed with fluorescence testing.

A) N-Acetylcysteine: Add probe solution (250 µL, 20 mM in MeCN, 0.005 mmol) to a 2-mL plastic centrifuge tube or ½-dram vial. Add N-acetylcysteine (0.73 mg, 0.0045 mmol) and swirl gently to dissolve. Place the reaction vessel under long-wave UV light and observe any changes in fluorescence. After ~1 min, add N,N-diisopropylethylamine vapors (or a drop) with a Pasteur pipet and gently swirl to mix. Observe again under UV light and note any changes in fluorescence. B) Bovine serum albumin (BSA): Add BSA (100 µL, 10 mg/mL in water, 1.5 x 10-5 mmol) to a 2-mL plastic centrifuge tube or ½-dram vial. Add probe solution (0.75 µL, 20 mM in MeCN, 1.5 x 10-5 mmol) and swirl gently to dissolve. Hold the reaction vessel under long-wave UV light and observe any changes in fluorescence. C) Hair: Cut hair into ten 1"–2" pieces, gather them together into a neat bundle, and Scotch tape one end of the bundle together. Dip the bundle of hair into 0.5 mL of 60% aqueous ammonium thioglycolate solution for 1 minute. Rinse thoroughly with deionized water and dry with a Kimwipe or paper towel. Add 0.5 mL of the 20 mM probe solution to a 2-mL plastic centrifuge tube or ½-dram vial. Dip the bundle of hair into the probe solution for 1 minute. Hold the hair under long-wave UV light and observe any changes in fluorescence. D) Determine if there is a difference in fluorescence spectra based on hair color. Here you can have fun finding volunteers and testing a hypothesis on your own. Are there any other molecules with which you would like to test your probe?

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HAZARDS

Dimethyl acetylenedicarboxylate is a lachrymator and a vesicant. Dansyl chloride is a skin and eye irritant and will cause inflammation or corneal damage. 2-Furfurylamine and ammonium thioglycolate are skin and eye irritants. Ethyl acetate, hexanes, triethylamine and N,N-diisopropylethylamine are flammable and skin and eye irritants. n-Hexane in hexanes is a neurotoxin. Triethylamine and N,N-diisopropylethylamine may cause burns on the skin. Acetonitrile is flammable and corrosive. Deuterated chloroform is a cancer suspect agent and mutagen. Silica gel is an inhalation hazard. N-Dansylfurfurylamine, the OND sensor, and the thiol adduct should be treated with care, as their hazards are unknown. Gloves and protective eyewear should be worn for this experiment.

Report

You will submit a lab report in the style of an Organic Letters communication using the provided template. The length should be 2–4 pages. Be sure to include the structure of your probe, reaction schemes, the chemical yield, and tables of relevant data (including important NMR assignments and qualitative fluorescence results). Calculate the atom economy (molecular mass of desired product / molecular masses of stoichiometric reagents) and the experimental atom economy (maximum obtainable mass of product / total mass of all reactants, not including solvents) for the probe synthesis. How is waste minimized in this experiment? What additional applications could you envision for the thiol sensor? Reference Hong, V.; Kislukhin, A. A.; Finn M. G. J. Am. Chem. Soc. 2009, 131, 9986-9994.