Ribose 5-Phosphate: A Molecule for the AGEs? Roger K. Sandwick Department of Chemistry and Biochemistry Middlebury College.

Ribose 5-Phosphate: A Molecule for the AGEs?

Roger K. SandwickDepartment of Chemistry and Biochemistry

Middlebury College

Louis-Camille Maillard (1878-1936)

■ French researcher in late 1800s/early 1900s

■ Reported on sugar/amino acid reactions – 1912-1917

Maillard Reaction

Sugar + Amine Melanoidins(brown polymeric compounds)

Heat

Maillard “browning”

1941 – Weast and McKinney – darkening of fruits

1948 – Henry et al. – off colors and loss of nutritional value in dried milk

1951 – Patron – other sugars, SO2 inhibition

Maillard Reaction

AromasColors

Flavors

The Pop Tart® Pop-Tarts®

Apple Cinnamon

Ingredients:Apple filling (corn syrup, dextrose, high fructose corn syrup, crackermeal, partially hydrogenated soybean oil, modified wheat starch, dried apples, malic acid, natural and artificial apple flavor, sugar, pectin soy lecithin), enriched wheat flour, partially hydrogenated soybean oil, corn syrup, high fructose corn syrup, sugar, dextrose, salt, cinnamon, leavening (baking soda, sodium acid pyrophosphate, monocalcium phosphate), corn starch, niacinamide, reduced iron, vitamin A palmitate, pyridoxine hydrochloride (vitamin B6), riboflavin (vitamin B2), thiamin hydrochloride (vitamin B1), and folic acid. ®Kellogg Company

Caramelization:

“to convert into a liquid made by cooking sugar until it changes color” burns!

!

Maillard Reaction vs. Caramelization:

Maillard Reaction Caramelization:

Acrylamide in Food

CH

C

O

NH2

H2C

Acrylamide

Maillard Reaction & Acrylamide

CHO

OHH

HHO

OHH

OHH

CH2OH

H2N

HC

C

O

O-CH2

CO

NH2

+

HC

OHH

HHO

OHH

OHH

CH2OH

NH+

CH

CO

O-

H2C

C

O

NH2

H2C

OHH

HHO

OHH

OHH

CH2OH

NH+

HCH2C

C

O

NH2

H2C

O

HHO

OHH

OHH

CH2OH

NH3+

Glucose

Asparagine

CH

C

O

NH2

H2C

Acrylamide

CO2

Fructosamine

Acrylamide Levels in Foods [Acrylamide] (g/kg)*

French fries 300 - 700Potato chips 600 - 2000Boiled Potatoes < 30Pop corn ~ 400Coffee (powder) ~ 200Breakfast cereals 50 - 250Meat < 30 - 50Beer < 30

* Adapted from “Acrylamide in Food” Swedish Scientific Expert Committee

Advanced Glycation End- Products (AGEs)

1967 – Samuel Rahbar discovered HbA1C in diabetic patients

Samuel Rahbar

Advanced Glycation End- Products (AGEs)

A fast moving Hb band was the result of a glycation of the N-terminal valine of the -chain.

Production of fructosamine, a resulting AGE.

ADA recommendation - < 7% HbA1c

*Picture from Mosca et al., Clin. Chem. 44, 632 - 638

Advanced Glycation End- Products (AGEs)

N+NH

N

HN

(H2C)3

CH(CH2)4

CH

N

CHOHOH2C

CH2CH2CH2CH2

CH

CH

CH2

CH2

CH2

H2C

HNCH2

COOH

NH

NO

H2C

HC

HC

OH

H2C

OH

OH

HN (CH2)3 CH

Pentosidine

Carboxymethyl- lysine

Pyrraline

Imidazolone

Ribose 5-Phosphate (R5P)

H

OH

H

HO OH

H H

O

H

H

OP-O

O-

O a phosphorylated aldopentose

a component of the pentose phosphate pathway

the starting material for nucleotides, some amino acids, and some co-factors

R5P Shows Enhanced Reactivity

-- 0.1 M Gly + 0.05 M sugar at 37 C and pH = 8

Reaction of R5P and Glucose with Glycine

012345678

0 10 20 30 40 50

Time (h)

A28

0

R5P

Glucose

R5P Shows Enhanced Reactivity

R5P + Glycine Glucose + Glycine

Microwaved – 35 seconds Microwaved – 35 seconds

Held at 37 C for 24 h

The Role of the Phosphate in R5P

H

OH

H

HO OH

H H

O

H

H

OP-O

O-

O

The Role of the Phosphate in R5P

H

OH

H

HO OH

H H

O

H

H

OP-O

O-

O

The Role of the Phosphate in R5P

H

OH

H

HO OH

H H

O

H

H

OPO-

O-

O

The Role of the Phosphate in R5P

H

OH

H

HO OH

H H

O

H

H

OP

O-

O-

O

The Role of the Phosphate in R5P

H

OH

H

HO OH

H H

O

H

H

OP

O-

O-

O

The Role of the Phosphate in R5P

H

OH

H

HO OH

H H

O

H

H

OP

O-

O-

O

Mechanism of Phosphate Catalysis?

H

-XO

OH

HH

OHHOH

O P O-

O

O-

H

O

OH

HH

HOH

O P O-

O

O-

H+

+O

OH

HH

HOH

OP

O-

O

O

H

OH

OH

HH

HOH

O PO-

O

-O H+

O

Encouragement of the acyclic

form

Covalent catalysis

General base catalysis

Stabilization of an

oxonium ion

NMR Study of R5P Loss

-peak-peak

DOH Gly

3.54.04.55.05.5 ppm

0 h

8.7 h

24 h

72 h

OH

H

H

HO OH

H H

O

H

H

OP-O

O-

O

H

OH

H

HO OH

H H

O

H

H

OP-O

O-

O

Reaction of R5P with glycine at pH = 8 and 37 C

R5P and the Maillard Reaction

CH

OHH

OHH

OHH

CH2OPO3H-

O

CH

OHH

OHH

OHH

CH2OPO3H-

N+ C COOHH

H

H

CH

OH

OHH

OHH

CH2OPO3H-

N C COOHH

H

H

CH

OH

H

OHH

CH2OPO3H-

N+ C COOHH

H

H

CH

O

HH

OHH

CH2OPO3H-

O

CH2

O

OHH

OHH

CH2OPO3H-

N C COOHH

H

H

CH2

OH

OH

OHH

CH2OPO3H-

N C COOHH

H

H

CH2

OH

O

OHH

CH2OPO3H-

CH3

O

O

OHH

CH2OPO3H-

Gly

Gly

Gly

OH-

OH-

OH-

3--deoxy-2-pentosulose 5-phosphate

1-deoxy-2,3-pentodiulose 5-phosphate

N-(1-deoxy-D-ribos-1-yl)-glycine 5-phosphate

Ribose 5-phosphate

Speculative

Pathway

R5P Reactivity with Proteins

66,00044,000

29,500

14,500

4 h 6 h 24 h 1 h 2 h His

Reaction of R5P with histones at

pH =7.5 and 37 C

SDS-PAGE

Some Conclusions R5P reacts with amines at a significantly

(50 – 500 x) faster rate than other common pentose and hexose sugars.

The reaction rate to form UV absorbing compounds is related to the pKa of the attacking amine and appears to be 1st order in respect to [amine] and 1.5 order in respect to [R5P].

R5P has a potential to rapidly cross-link proteins, producing both free and protein-bound melanoidins.

Acknowledgements

Funding:

- AAAS/Merck

- Vermont Genetics Network- Middlebury College

Researchers- Matthew Johanson- Elizabeth Breuer- Sarah Sandwick

- Ryan Gamble - Admire

Muranaire