Synthesis of Perbenzoylated Carbohydrates in Preparation for

Anthocyanin Pigment Characterization

Brandon Lew

March 15, 2006

Introduction

• Purpose:

To develop a technique for carbohydrate characterization through NMR so that anthocyanin carbohydrate tails may be characterized.

Anthocyanins

http://www.mariquita.com/images/photogallery/strawberries.jpg http://www.personal.psu.edu/staff/m/h/mhl100/images/blueberries.jpg

Carbohydrate Tails

O+

OH

H

OH

OH

R2

OH

R1

35

7

3' 4'

5'

Anthocyanidin Base

O

HOH

OH

OH

H

H

OH

OH

H

Anthocyanin Characterization

• NMR of anthocyanins

Seeram et al. 2002

• Separation of tails

Takeoka et al. 2005

• Combine to perform NMR on separated tails

Carbohydrates

ChairChain

C 4

C 3

C 2

C 1

O

C 5

HOH

OH

OH

H

H

OH

H

C 6

OH

H

OH

C 2

C 3

OHH

C 4 OHH

C 5

C 6

H

OHH

OHH

HOH

C 1

OHH

Can Twist,Resulting inBeta-Anomer

In Beta-Anomer,These are Flipped

Perbenzoylation

• Levene and Meyer (1927) Glucose

• D’Accorso et al. (1981) NMR data of many monosaccharides

R

O

R

O

Perbenzoylation (Protocol 1)

O

HOH

OH

OH

H

H

OH

H OH

H

O

H

H

H

H

OBz

HOBz

BzO

BzO

OBz

O

Cl

Pyridine

100 oC

β-glucose Perbenzoylated-β-glucose

Purification

• Thin-layer chromatography (TLC)

• Column chromatography

• High-pressure liquid chromatography (HPLC)

http://www.hamline.edu/depts/chemistry/facility/images/hplc.jpg

Nuclear Magnetic Resonance Spectroscopy (NMR)

1H/13CEM radiation

Excited State

Ground StateRadio wave Detector

Magnetic Field

Protocol 1 Results: 1H NMR of perbenzoylated β glucose

Protocol 1 Results: HPLC of perbenzoylated maltose

Protocol 1 Results: 13C NMR of perbenzoylated maltose

Perbenzoylation (Protocol 2)

O

HOH

OH

OH

H

H

OH

H OH

H

O

H

H

H

H

OBz

HOBz

BzO

BzO

OBz

O

Cl

Pyridine, CHCl3

0 oC

β-glucose Perbenzoylated-β-glucose

Protocol 2 Results: 1H NMR of perbenzoylated β glucose

Protocol 2 Results: HPLC of perbenzoylated β glucose

Procedure 2 Results: 1H NMR of perbenzoylated α glucose

Conclusion• Protocol 1 – contaminated

• Some disaccharides selectively benzoylated

O

HOH

OH

OH

H

H

OH

H OH

H

O

H

H

H

H

OBz

HOBz

BzO

BzO

OBz

O

Cl

Pyridine

100 oC

β-glucose Perbenzoylated-β-glucose

Conclusion• Protocol 2 – purer, quicker

• Perbenzoylation works for characterization

O

HOH

OH

OH

H

H

OH

H OH

H

O

H

H

H

H

OBz

HOBz

BzO

BzO

OBz

O

Cl

Pyridine, CHCl3

0 oC

β-glucose Perbenzoylated-β-glucose

Future Work

• Use perbenzoylation to characterize carbohydrate tails of anthocyanins

• Anthocyanins differ by their tails

• Important for developing anti-oxidant food additives

Acknowledgements

• Dr. Matachek

• Hamline University

• Ms. Fruen

• Ms. Richter, Bill Mitchell

• Team Research

Synthesis of Perbenzoylated Carbohydrates in Preparation for

Anthocyanin Pigment Characterization

Brandon Lew

March 15, 2006