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I.   Research Progress

             Over the past year, the undergraduate research group made some excellent strides in advancing the study to fulfill the objectives of the project.   Previously, we had finished the study of the kinetics of R5P and 2'-deoxy-R5P (dR5P) and rate constants were obtained for steps to and from the Amadori product.  (In the case of dR5P, a pseudo-Amadori product was formed.)  These rate constants were compared to other sugars, in particular ribose and inorganic phosphate, to make statements about a) the impact of the furanose structure of R5P and b) the attached phosphate group on the rate of the Maillard sequence.  For a period of time we focused on characterizing the distribution of the downstream products of the reaction under different conditions but this proved difficult because the reaction was taking multiple post-Amadori paths. We identified carboxymethyl lysine (CML), a common product in the glycation of proteins, and 4-hydroxy-5-methyl-3(2H)-furanone (HMF), an important food aroma, as primary products.   

             Two big achievements this past year were a characterization of the Maillard reaction using 5-deoxyribose and the synthesis and characterization of the reaction using phosphate-methylated R5P.  5-deoxyribose is a similar molecule to R5P in that it assumes the furanose structure (unlike ribose which takes primarily the pyranose structure).   It therefore models R5P without the attached phosphate group.   Reactions of 5-deoxyribose with amines were approximately six-fold less rapid, showing the value of an intramolecular catalytic group on the reaction rate.  The comparative rate of reaction of 5-deoxyribose was, in fact, similar to that of ribose despite likely having a higher percent level of the acyclic form. 

             With more care to reaction conditions, we were also more successful in producing the mono- and di-methylated forms of R5P for assessing the acid/base catalysis properties of the phosphate on the reaction.  Rates of the mono-methylated form are less than with R5P, however until we successfully isolate the methylated forms from the unreactive R5P, we will not be able to satisfactorily compare rate constants. 

             The studies of the reaction of R5P and amines generated some interesting information that proved important in our protein glycation studies.  Over the past year we discovered the reactions of R5P and amines are potent producers of superoxide (O₂^-) and hence, through spontaneous dismutation, hydrogen peroxide (H₂O₂).   The generation of these reactive oxygen species has important implications in cellular systems.  A paper concerning the Maillard reaction of R5P in the presence of cytochrome c has recently been accepted for publication in the journal Carbohydrate Research.   (Acknowledgement of the support of PRF was included in the paper.)

             The studies of the Maillard reactions of R5P with amines included a comparison of the reaction rate using the ketose isomer ribulose 5-phosphate (Ru5P).  This required the purchase and use of the enzyme ribose 5-phosphate isomerase (RpiA).   The commercial product from Sigma Chemical Co. was shown to be quite impure and we ultimately had to perform a further purification on the enzyme.  The procedure proved to be an excellent undergraduate exercise and we thereupon developed this into a full semester theme for an undergraduate biochemistry methods course (CHEM 313).   We thought the exercise to be so creative that we submitted a manuscript outlining our curriculum to the Journal of Chemical Education.  We have not yet heard of their response.  (Again, acknowledgement to the support of PRF was given.)

             Future Research:  Our final efforts on the project will be to improve our procedures for synthesizing the methylated forms of R5P, to develop better methods to separate these forms from unreactive R5P, and to fully characterize their reactivity in the Maillard reaction.  We feel our present yield of synthesis of the mono- and di-methylated forms of R5P are sufficient if we can improve our purification methods.  (We are currently attempting to separate the methylated forms from R5P by DEAE Sephadex or by hydroxyapatite.)  Our procedure for quantification of the forms by ³¹P NMR is developed and ready for use, as is our procedures for characterizing the Maillard reactivity of these compounds.  These studies will be performed during the one month January term and during the summer of 2011.

II. Intellectual Development of the Undergraduate Researchers

             The project impacted two Middlebury students during the 2009 – 2010 year.  Mr. Joseph Colianni, a BA Biochemistry graduate of February, 2010, performed research on the project during the Fall semester and during the January term.  Mr. Colianni is currently in a Masters of Public Health program at the University of Minnesota.  Ms. Melissa Hirsch, a senior BA Biochemistry major, was supported in summer 2010 by these funds.  She performed the methylated R5P synthesis and characterization studies this past summer.  She is continuing research in the laboratory in Fall semester.  Ms. Hirsch is unsure of her future plans but is considering a career in chemistry teaching.

III.  Professional Growth of the PI

During the past year, the PI has submitted three manuscripts for publication.  One has been accepted and two are pending.   These publications will help establish the PI as an expert in the Maillard field.  The PI plans to attend and present some of these chemical studies at the bi-annual Maillard Symposium next year in Nancy, France.   The involvement in the project continues to offer opportunities to the PI for improving his role as an undergraduate research mentor.  He supervised the successful completion of Honors Thesis work by two students and guided five other students in research during the 2009 – 2010 academic year.  The involvement in research has kept the PI abreast of new developments in the field of biochemistry; this knowledge has been valuable in his teaching as well as in research.

IV. Financial Progress

            The PI applied for and was granted a second year extension and he gained approval for a budget reshaping.  With the remaining funds, he will be able to fund all or part of one more student stipend while maintaining enough for supplies and for partial coverage of his trip to attend the Maillard Symposium next August.