DEVELOPMENT OF A FLUORESCENT CALCIUM(Ca2+) SENSOR TO INVESTIGATE MARINE SEDIMENTARY CONDITIONS

Lili Wu, Dr. Dale G. Drueckhammer

OUTLINE

Background and Introduction

Objective

Reaction Scheme of the Amine

Experiments

Results and Discussion

Conclusion and Future Work

Acknowledgements

BACKGROUND AND INTRODUCTION

As atmospheric CO2 concentrations rise, CO2 uptake by the oceans also increases.

http://upload.wikimedia.org/wikipedia/commons/5/51/Mauna_Loa_Carbon_Dioxide-en.svg

Kleypas et al 2006

This CO2 uptake disturbs the carbonate equilibrium and causes “ocean acidification”: CO2 + H2O ↔ H2CO3

BACKGROUND AND INTRODUCTION

BACKGROUND AND INTRODUCTION

This process accelerates the dissolution of calcium carbonate and will negatively affect shelled organisms which form their supporting skeletons with CaCO3

H2CO3 + CO3-2 ↔ 2 HCO3

-

↓[CO3-2] CaCO3 → Ca2+ + CO3

-

2

Overall reaction

CaCO3 + CO2 + H2O ↔ 2 HCO3- + Ca2+

An optical sensor to study CaCO3 dissolution by monitoring Ca2+ 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 Ca2+ ion distribution.

Indicators used for intracellular Ca2+ detection do not respond to the much higher marine Ca2+

concentrations due to their high-binding affinities.

OUR OBJECTIVE

To develop fluorescence-based sensors with low-affinity binding to Ca2+ 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:

O

OHO2C

AcO

Cl

OAc

Cl

O

+ N CO2Et

O

H2N

CO2Et

O

ClCl

O-O

CO2-

C

HN

OCH3

N-O2C

-O2C

O

*Synthesized by Nuria Protopopescu (PhD Candidate, Marine Chemistry)

SYNTHETIC 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.

NH2

O Br

O

O

DMF, DIEA, NaI

O

N

O

O

O

O

NaNO2

CH3COOH

O

N

O

O

O

O

NO2

H2

Pd / C

O

N

O

O

O

O

NH2

1

1

2

2

3

ALKYLATION REACTION TO YIELD COMPOUND 1

NH2

O

+

(2.85mL, 25.3mmol)

Br

O

O

DMF, DIEA, NaI

(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.

HN CO2Et

O

N CO2Et

O

CO2Et

4 1

1H-NMR SPECTRUM OF COMPOUND 4

δ = (3.8ppm, s, 2H)

HN CO2Et

O

H H

1H-NMR SPECTRUM OF COMPOUND 1¹H-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)N CO2Et

O(d)H3C

C

H(c)

(c)H

O

OH2(b)C CH3(e)

H(a)

H(a)

H(a)

DISCUSSION

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.

NITRATION OF COMPOUND 1

O

N

O

O

O

O

O

N

O

O

O

O

NO2

(0.5g, 1.69mmol)

(0.13g, 1.89mmol)

(5.38mL)

NaNO2

CH3COOH, H2O

(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.

1 2

1H-NMR SPECTRUM OF FIRST ATTEMPT

1H-NMR SPECTRUM OF SECOND ATTEMPT

H(b)

H(a)

H(c)

H(d)

H(e)

H(f) H(g)N CO2Et

O

(f)H3CC

H(e)

(e)H

O

OH2(d)C CH3(g)

H(b)

H(a)

O2N H(c)

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.

HYDROGENATION OF COMPOUND 2

O

N

O

O

O

O

NO2

O

N

O

O

O

O

NH2

(0.2g, 0.59mmol)

(51mg)

1atm H2

10% Pd / C

(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.

1H-NMR SPECTRUM OF COMPOUND 3

O

N

O

O

O

O

NH2

A TRIAL COUPLING EXPERIMENT

O

O

AcO

Cl

OAc

Cl

OHO2C

NH2

chloroform

Ph3PCl2

+

(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)

O

O

AcO

Cl

OAc

Cl

OC

HN O

1H-NMR SPECTRUM OF COUPLING PRODUCT

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.

FUTURE WORK

Optimize the conditions in the coupling experiment

Then, couple amine to the fluorophore to produce the desired calcium ion sensor:

O

OHO2C

AcO

Cl

OAc

Cl

O

N CO2Et

O

H2N

CO2Et

O

ClCl

O-O

CO2-

C

HN

OCH3

N-O2C

-O2C

O+NaOH

ACKNOWLEDGEMENTS

Dr. Dale Drueckhammer

Dr. James F. Marecek

Dale D. James M. Nuria P. Diana M. Heng C.

Nuria Protopopescu

Diana Melo

Heng Chen