1. D EVELOPMENT OF A F LUORESCENT C ALCIUM (Ca 2+ ) S ENSOR TO I NVESTIGATE M ARINE S EDIMENTARY C ONDITIONS Lili Wu, Dr. Dale G. Drueckhammer

2. O UTLINE Background and Introduction Objective Reaction Scheme of the Amine Experiments Results and Discussion Conclusion and Future Work Acknowledgements

3. BACKGROUND AND INTRODUCTION As atmospheric CO 2 concentrations rise, CO 2 uptake by the oceans also increases. http://upload.wikimedia.org/wikipedia/commo ns/5/51/Mauna_Loa_Carbon_Dioxide-en.svg Kleypas et al 2006

4. This CO 2 uptake disturbs the carbonate equilibrium and causes “ocean acidification”: CO 2 + H 2 O ↔ H 2 CO 3 BACKGROUND AND INTRODUCTION

5. This process accelerates the dissolution of calcium carbonate and will negatively affect shelled organisms which form their supporting skeletons with CaCO 3 H 2 CO 3 + CO 3 -2 ↔ 2 HCO 3 - ↓ [CO 3 -2 ] CaCO 3 → Ca 2 + + CO 3 -2 Overall reaction CaCO 3 + CO 2 + H 2 O ↔ 2 HCO 3 - + Ca 2 +

6. An optical sensor to study CaCO 3 dissolution by monitoring Ca 2+ distributions in nearshore sediments would be of central importance in environmental research of the ocean ecosystem HOWEVER, No suitable sensor has yet been developed for imaging marine Ca 2+ ion distribution. Indicators used for intracellular Ca 2+ detection do not respond to the much higher marine Ca 2+ concentrations due to their high-binding affinities.

7. O UR O BJECTIVE To develop fluorescence-based sensors with low- affinity binding to Ca 2+ which will allow direct in situ, non-destructive and high-resolution 2D imaging. The experimental approach involves the coupling of a fluorophore * to an amine: * Synthesized by Nuria Protopopescu (PhD Candidate, Marine Chemistry)

8. S YNTHETIC SCHEME OF THE DESIRED AMINE My experimental work focused on the synthesis of an amine used to couple the fluorophore. The reaction pathway involves alkylation, nitration, and catalytic hydrogenation. 1 1 2 2 3

9. A LKYLATION REACTION TO YIELD COMPOUND 1 (2.85mL, 25.3mmol) (8.4mL, 75.9mmol) (30mL) (10mL, 57.5mmol) (7.73g, 51.6mmol) The reaction mixture was heated at 105 ºC overnight and monitored by TLC. Additional ethyl bromoacetate and DIEA were added in the process. Two suspected alkylation products 1 and 4 were obtained after column purification. 41

10. 1 H - NMR S PECTRUM OF COMPOUND 4 δ = (3.8ppm, s, 2H)

11. 1 H-NMR S PECTRUM OF COMPOUND 1 ¹H-NMR(300 MHz, CDCl3): δ = 6.9-6.8(m, 4H), 4.2-4.1 (q, 4H), 4.2 (s, 4H), 3.85 (s, 3H), 1.21(t, 6H) H(a) H(b) H(c) H(d) H(e)

12. D ISCUSSION The reaction did not proceed to completion. The ratio of 1 to 4 was about 1:1. Possible explanation: inadequate heating time or starting material. Then, We used sodium iodide which catalyzed the reaction. With sodium iodide added and more heating time, the reaction had a better yield(ratio of 1 : 4 ≈ 3:1) The purified product was a light brown oil after column chromatography.

13. N ITRATION OF COMPOUND 1 (0.5g, 1.69mmol) (0.13g, 1.89mmol) (5.38mL)(53.76mL) Stir overnight at room temp First attempt using compound 4 as starting material: a dark purple liquid was obtained. Second attempt using compound 1 as starting material: desired product obtained, a golden brownish oil. 12

14. 1 H - NMR S PECTRUM OF FIRST ATTEMPT

15. 1 H - NMR S PECTRUM OF SECOND ATTEMPT H(b) H(a) H(c) H(d) H(e) H(f) H(g)

16. DISCUSSION A series of trial and error identified the desired nitration product. Unexpectedly, the two suspected alkylation products were also distinguished. Column chromatography with 1:1 hexanes/ethyl acetate as a developing solvent only removed excess acetic acid from crude product. Extraction with a base would be a better choice to remove the acetic acid. Final product was a yellowish oil.

17. H YDROGENATION OF COMPOUND 2 (0.2g, 0.59mmol) (51mg) (Stir at room temp. for ~ 3 days) 2 3 The reaction was conducted on a small scale due to limited starting material. TLC verified that the product is more polar than starting material. Final product was a transparent purplish gummy solid.

18. 1 H-NMR SPECTRUM OF COMPOUND 3

19. A TRIAL COUPLING EXPERIMENT (0.023g, 0.25mmol) (0.14g, 0.26mmol) (0.3g, 0.9mmol) (10mL) (Heated at reflux for~4 hrs) The meta-isomer of the fluorophore * appeared as orange crystals. Both starting material and product possessed high polarities. TLC comparison of aniline, fluorophore and the crude product. * Synthesized by Nuria Protopopescu (PhD Candidate, Marine Chemistry)

20. 1 H - NMR SPECTRUM OF COUPLING PRODUCT

21. DISCUSSION Current results unable to confirm if the expected reaction took place. Further purification of crude product is required to separate out impurities from the product. Further spectroscopic methods are required to identify the product.

22. F UTURE WORK Optimize the conditions in the coupling experiment Then, couple amine to the fluorophore to produce the desired calcium ion sensor:

23. ACKNOWLEDGEMENTS Dr. Dale Drueckhammer Dr. James F. Marecek Dale D.James M.Nuria P.Diana M.Heng C. Nuria Protopopescu Diana Melo Heng Chen