www.acsprf.org

I. Narrative/Progress Report

The goal of this project was to characterize the reaction rates of ribose 5-phosphate (R5P) in Maillard reactions and to establish the cause for these high rates in comparison to other sugars such as glucose or glucose 6-phosphate. We feel we have accomplished our goals and have a better understanding of the role the phosphate plays in aiding the reaction. Even prior to the start of this PRF project in September, 2006, we had characterized the reaction of R5P with a number of amines. A paper subsequently published in Carbohydrate Research in 2007¹ gives rate constants for the reaction of R5P with a number of amines. A subsequent study identified the comparative rates of reaction for D-ribose (18-fold slower) and D-2-deoxyR5P (20-fold slower) which make statements about the intramolecular role of the phosphate in helping the reaction and the importance of the C2 carbon for the reaction. We also investigated the production of superoxide (O₂^-) during the process. In another finding, the isomerization of R5P to the ketose sugar ribulose 5-phosphate caused the Maillard reaction rate to greatly increase. This shows the tight connection of the acylic form to the reaction. These findings were published in a recent article in Carbohydrate Research.²

In order to compare the rate of Maillard reaction of the predominately cyclic R5P to that of the acyclic Ru5P we required the enzyme R5P isomerase. We purchased a spinach-derived enzyme from Sigma Chemical but we discovered this preparation to be highly contaminated with other photosynthetic enzymes. The full purification of the enzyme and then the cloning of the enzyme from E. coli became a task of a biochemistry laboratory class (CHEM 313) in Spring, 2010. The extended series of laboratory exercises are now part of the course syllabus. This exercise was viewed as being pedagogically valuable and was sent to the Journal of Chemical Education for consideration. It was recently published.³

Our explorations into the reducing nature of the R5P in the initial steps of the Maillard reaction were characterized using the proteins myoglobin and cytochrome c. We immediately realized that in both cases a loss of function accompanied the modifications caused by R5P on the protein surface. The characterization of the myoglobin-R5P reactions were published this year in Biochemical and Biophysical Research Communications.⁴ Our cytochrome c-R5P studies have been conditionally accepted (upon revision) by Biochemistry.⁵

Mechanistic Work Over the Past Year

Over the past year, the undergraduate researchers finalized some characterizations to ascertain the importance of the intramolecular phosphate's role in the catalysis of the R5P – amine reaction. The study was difficult for the undergraduates as we were attempting the chromatographic separation of relatively similar compounds from one another upon their synthesis. Specifically, we were attempting to methylate the phosphate group of R5P and determine its rate of reaction vs. an unmethylated R5P form. The results of the study would define the relative contributions of phosphate simply causing a higher level of acyclic form in solution (i.e., both the methylated and unmethylated forms would have equal rates of reaction) vs. phosphate actually participating as a general acid/base catalyst in the reaction of its attached sugar. With the low yields of methylation and the poor separation, we opted instead to produce a mixture of mono-methylated, di-methylated, and un-methylated form and then simply compare the relative rates of reaction of the three when the solution was subjected to an amine (glycine, b-alanine). The results of this study were presented in June, 2011 at the CERM meeting in Indianapolis.⁶ We concluded from these studies that the predominate factor controlling the reaction rate was the amount of acyclic form in solution. The catalytic effect of a phosphate intramolecularly was relatively small in comparison. We speculate that this is due to the thermodynamically favorable positioning of the phosphate away from the ring and distant from the reactive anomeric C1 carbon.

References:

1. Munanari, A. et al., Carbohydr Res. 342, 2575-2592, 2007

2. Gersten, R.A. et al., Carbohydr Res. 345, 2499-2506, 2010

3. Jewett, K. and Sandwick, R. J. Chem. Ed. 88, 1170-1174, 2011

4. Bokiej, M. et al., Biochem Biophy Res Commun. 407, 191-196, 2011

5. Hildick-Smith, G. et al. Biochemistry (conditionally accepted upon revision)

6. Colianni, J., Hirsch, M., and Sandwick, R. “Intramolecular Phosphate Catalysis of Ribose 5-Phosphate and Amines”, CERM, June 9, 2011

II. Intellectual Development of the Undergraduate Researchers

The project impacted two Middlebury students during the 2010 – 2011 year. Ms. Melissa Hirsch, a 2011 BA Biochemistry graduate, continued her work on the methylation project during the Fall, 2010 and was a major contributor (along with Mr. Joseph Colianni, a BA Biochemistry graduate of February, 2010) to the CERM presentation. Ms.Hirsch is currently teaching chemistry at a small private high school in Southern Vermont. Mr. Casey McGowen, a senior Biochemistry major, worked during the summer of 2011 on the glycation of proteins. Mr. McGowen is currently unsure of his plans after graduation but is leaning towards applying to medical school.

III. Professional Growth of the PI

The PI has been greatly aided by this project. Over the period of the project, the PI has had five manuscripts accepted for publication (one is accepted under condition of revision) and has given two posters at meetings (the Maillard symposium in 2007 and CERM in 2011). A presentation at the Maillard Symposium in 2012 is planned. The PRF-supported has given the PI an expertise in the Maillard/glycation field that should make him competitive in future grant applications. Most importantly, the support of the project has allowed the PI continued interaction with undergraduates. He supervised the successful completion of two senior honors thesis in 2011 and guided four other students in research in the 2010 – 2011 academic year.

IV. Financial Progress

With a small amount of shifting of the final financial resources, the PI used the remainder of the funds in the PRF grant.