|Tilley, Michael - Mike|
Submitted to: Analytical Biochemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/12/2004
Publication Date: 9/1/2004
Citation: Tilley, M., Benjamin, R.E., Tilley, K.A. 2004. Non-enzymatic preparative-scale synthesis of dityrosine and 3-bromotyrosine. Analytical Biochemistry. 334:193-195 Interpretive Summary: Amino acid side chains of proteins are very important. They can interact for crosslinking within a protein and between different proteins. Crosslinking of the side chains of two tyrosines form a compound called dityrosine (DY). DY occurs in many biological systems and has various biological functions in extensible proteins found in insect eggs, fibers such as silk and wool and may have a functional role in food processing. Excessive crosslinking of tyrosine is a sign of protein damage in diseases such as cataracts, Parkinson's and Alzheimer's diseases. Adding a bromine atom to the tyrosine side chain will form 3-bromotyrosine, a molecule that is formed in conditions such as asthma and allergic inflammation. To study the formation of these compounds, standards for analysis are needed. Several methods are available for making DY but they generally have low yields. No simple, efficient method for producing 3-bromotyrosine has been developed. In this paper we report a simple, non-enzymatic method for the simultaneous preparation of both DY and 3-bromotyrosine resulting in high (~72%) recovery of both tyrosine derivatives.
Technical Abstract: Dityrosine (DY) occurs in many biological systems with its formation catalyzed by the action of peroxidases, reactive oxygen species/free radicals or ionizing radiation. The oxidative coupling of tyrosine is of general interest in several biological processes as a marker for protein oxidation/aging as an indicator of cellular damage. DY has various biological functions in the structure of extensible proteins and polymeric structures found in insect eggs, yeast spores, fibers such as silk and wool and may have a functional role in food processing. 3-bromotyrosine serves as powerful indicator for identifying sites of eosinophil-mediated tissue injury in vivo in conditions such as asthma and allergic inflammatory disorders. A simple, rapid, non-enzymatic method for the simultaneous preparation of both DY and 3-bromotyrosine resulting in high (~72%) recovery of both tyrosine derivatives is presented in this paper. The production of these standard compounds is useful in many research areas where analysis of these compounds is performed. A solution of 10 mg tyrosine and 30 ppm potassium bromate (KBrO3) was heated at 215 C for 25 min, cooled and lyophilized. The material was suspended in 0.5 mL deionized water, filtered and analyzed by high-pressure liquid chromatography (HPLC). Three peaks were formed and collected. Compounds representing each peak were confirmed by comparing them with known standards (DY and tyrosine). The third peak, representing a new compound eluting later than DY in HPLC, was purified and subjected to mass spectral and nuclear magnetic resonance (NMR) analyses and identified as 3-bromotyrosine. Methods for synthesis and purification of DY and 3-bromotyrosine were optimized. Under the optimized conditions, nearly 100% of tyrosine was converted to either DY or 3-bromotyrosine resulting in an average of 5.7 mg DY and 7.9 mg 3-bromotyrosine from the original 10 mg of tyrosine. This represents 57% of the maximum theoretical yield for both compounds. Purification involved two rounds of preparative and analytical HPLC resulting in 72% recovery (4.1 mg DY and 5.74 mg 3-bromotyrosine).