Publication Date

Fall 2012

Document Type

Thesis

Degree Name

Master of Science

Department

Analytical Chemistry

First Advisor

Shailendra Kumar, Ph.D.

Second Advisor

Walter Henne, Jr., Ph.D.

Third Advisor

Karen D'Arcy, Ph.D.

Abstract

Photo oxidation of proteins leads to the formation of cross links of the amino acids in the proteins and leads to induction of cataracts, photo aging of the skin and in the Photodynamic therapy of tumors.

Oxidation of proteins involves significant modification of particular amino acids residues with consequent photodynamic damage. Studies show that tryptophan, tyrosine and histidine side chains gives rise to semi stable peroxides on exposure to singlet oxygen these oxidized structures are studied with low temperature NMR. These peroxides or species derived from them have been postulated as key intermediates in subsequent reactions such as cross linking of oxidized amino acids with unmodified residues. It is difficult to identify modified amino acids in photo dynamically treated proteins and to determine their positions/locations .It is tedious to isolate the cross linked amino acid moieties from proteins, hence low molecular weight histidine and imidazole derivatives have been used to study the detailed reaction mechanisms.

photo oxidation of N acetyl histidine ethyl amide derivative of histidine in which the amine group and the carboxylic acid group have been converted to amide bonds .this conversion makes this amino acid a peptide resembling amino acid so that its photo oxidation mimics the photo oxidation of histidine moiety in proteins.

Use of N-Benzoyl-L histidine as a model compound elucidates the chemical structure and mechanisms of the formation of His-His cross links .Benzoyl group protects the alpha amino group of histidine participation in the cross link reactions .Benzoyl absorbs strongly in UV hence permits the spectroscopic identification

Rose Bengal is used as a photo sensitizer which sensitizes primarily by the singlet oxygen mechanism in the aqueous solution It causes extensive damage to the imidazole ring of Histidine by forming semi stable peroxides which results in the formation of Hydrated Imidazolone with the second molecule of N acetyl histidine ethyl amide. The formation of this dimer shows the strong possibility of cross linking in the histidine residues in proteins.

Lab synthesized N acetyl L histidine ethyl amide was analyzed by TLC, Melting point and NMR and declared that it was impure.

Purification process was done through re-crystallization and Running the analyte down through silica column by gradient elution using three different solvents Hexanes, Ethyl acetate and Methanol.

Synthesis of N acetyl L histidine ethyl amide meant to synthesize in lab

Lab synthesis is done in two steps:

Step1. Synthesizing N acetyl L histidine ethyl ester from L Histidine

Step2. Synthesizing N acetyl L Histidine ethyl amide from N acetyl L histidine ethyl ester

N acetyl L histidine ethyl ester is synthesized in lab on proceeding upon second step reconversion of N acetyl L histidine ethyl ester to L histidine was observed.

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