Reports: B4
44216-B4 A Characterization of the Kinetics and Mechanisms of the Reaction of Ribose 5-Phosphate and Amino Acids
I. Research Progress
The advancement of the investigation of the reaction of ribose 5-phosphate (R5P) with biological amines slowed somewhat during the past year as some experimental difficulties were encountered. For the most part, the study of the kinetics of R5P and 2'-deoxy-R5P are completed and rate constants have been obtained for steps leading up to and immediately following the Amadori product. These were compared to similar reactions with the non-phosphorylated sugar ribose (with and without added inorganic phosphate). The comparison between the rates of the intramolecularly-catalyzed reaction (i.e., with R5P) vs. intermolecularly-catalyzed reaction (i.e., with ribose) is not perfect, however, due to ribose assuming a larger percentage of the pyranose form. Some attempt was made to characterize the distribution of all products formed in the R5P reaction, but this proved extremely time-consuming and we instead focused on the production of major products such as carboxymethyl lysine (CML) (an oxidized product), 4-hydroxy-5-methyl-3(2H)-furanone (HMF), and the dicarbonyl intermediates.
Most of our recent attention on the project has been focused on the synthesis of a dimethylated (on the phosphate group) R5P analogue. Once obtained, this compound will be used in Maillard reactions and the kinetics of these reactions compared to the R5P system. We have made several attempts at generating this compound using a dimethyl sulfate reaction and have obtained relatively low yields (< 40 %, as estimated by LC/MS analysis) of the fully methylated product. We are now working to isolate to high purity the dimethylated product from the monomethylated and non-methylated compounds. We attempted anion exchange chromatography using a DEAE Sephadex column and only obtained partial purification: we next will attempt to use a hydroxyapatite column for isolating the desired product.
We have also initiated a study to investigate the rate of the model compound 5-deoxyribose (5DR) in the Maillard reaction. This compound was recently obtained from a supplier (Waterstone Technologies) in high purity. Maillard reactions of 5DR with and without phosphate should help us identify the rate enhancement of phosphate (intermolecular) on the reaction for comparison purposes.
Future Research: The two investigations yet to be completed to meet the objectives of the project are centered around evaluating the role of the intramolecular phosphate on the kinetics of the reaction. As mentioned previously, we are attempting to prepare in high purity the dimethyl form of R5P in order to assess the comparative kinetics of it in the Maillard Reaction with amino acids. We hypothesize that dimethylation will decrease the rate of reaction due to the importance of this group acting as a general acid/base in the proximity of the site of reaction. Performing the reaction in the presence of inorganic phosphate will help us determine (in comparison to reactions with 5DR) the effect of a bulky phosphate group on the ring opening needed for the initial amine attack. We have spent some effort at attempting to increase our yields of the dimethyl R5P (~ 30 %) and now are working towards isolating a pure form of the compound. For the reasons just mentioned, we will characterize the kinetics of 5-DR in the Maillard reaction. These reactions are similar to what we have performed previously and should not present any difficulties. Lastly, we will continue to investigate interesting aspects of the reaction mechanism of amino acids and R5P to generate HMF and of the potential inhibitory role R5P could play in lowering acrylamide synthesis.
II. Intellectual Development of Undergraduate Researchers
The project impacted the
development of three Middlebury undergraduates during the 2008 2009
year. Risa Roppongi, a May 2009 graduate with a BA degree in
Biochemistry, performed two semesters of non-thesis research whereby, among
other things, she characterized the products of reactions between amino acids
and R5P. Ms. Roppongi
is now enrolled in a Masters in Public Health program at
III. Professional Growth of the PI
The increased research interaction with Middlebury students provided by this grant resulted in the growth of the PI as an undergraduate mentor. The experiences add to the ability of the PI to serve as an advisor to the undergraduates in their decisions towards future careers as scientists. The support has also aided him professionally to become an active scholar in this line of Maillard research. The published manuscript to Carbohydrate Research in 2008 will hopefully lead to another publication by the PI, thus strengthening his reputation in the field. Over the past five years, the PI has sponsored more thesis students and more summer researchers than any of his colleagues in the Department of Chemistry and Biochemistry.
IV. Financial Progress
Part of the requested stipend for the PI for summer, 2009 was not used as the PI used some monies from another grant to cover the maximum amount (2/9th) that one can draw in the summer. The PI used $4400 (stipend and benefits) of the 2009 budgeted $7500 amount. One student stipend (and benefits) was used. A budget request to PRF will be submitted during the upcoming year to transfer some money allocated to the PI to pay for two student stipends in summer, 2010. The amount currently available in the allocation to PI salary should cover the PI's full salary and two summer student stipends for summer, 2010. There are sufficient funds in the supply budget to handle all work to be done during the 2009 2010 academic year and during summer, 2010.
