Methods of quantifying n2-(1-carboxyethyl)-2'-deoxy-guanosine (cedg) and synthesis of oligonucleotides containing cedg

Abstract

Methods of quantifying a N2-(1-carboxyethyl)-2′-deoxyguanosine (CEdG) levels in biological samples and comparing those levels to known normal levels can diagnose a number of metabolic disorders or complications associated therewith, including diabetes, its associated complications, and cancer. Methods can also determine whether therapies for disorders are effective by measuring CEdG levels before and after treatment. Measurement of CEdG levels is achieved by using liquid chromatography electrospray ionization tandem mass spectrometry.

1 - 10 . (canceled) 
     
     
         11 . A method of synthesizing an internal standard comprising a stereochemically pure  15 N 5 -(1-carboxyethyl)-2′-deoxyguanosine ( 15 N 5 -CEdG) (R) and stereochemically pure  15 N 5 -CEdG (S), the method comprising:
 (i) generating a mixture comprising mixing DL-glyceraldehyde with  15 N 5 -labeled deoxyguanosine (dG), potassium dihydrogen phosphate, and disodium hydrogen phosphate in water; 
 (ii) heating the mixture; and 
 (iii) purifying stereochemically pure  15 N 5 -CEdG (R) and stereochemically pure  15 N 5 -CEdG (S) from the mixture using high-performance liquid chromatography (HPLC). 
 
     
     
         12 . The method of  claim 11 , wherein the HPLC comprises an (Et) 3 NH 4 OAc/CH 3 CN gradient. 
     
     
         13 . A method of synthesizing an internal standard comprising oligonucleotides comprising stereochemically pure  15 N 5 -(1-carboxyethyl)-2′-deoxyguanosine ( 15 N 5 -CEdG) (R) and oligonucleotides comprising stereochemically pure  15 N 5 -CEdG (S), the method comprising:
 (i) generating oligonucleotides containing 2-fluoro-2′-deoxyinsodine (2-FdI); 
 (ii) mixing the 2-FdI oligonucleotides with D- or L-alanine in potassium carbonate to form  15 N 5 -CEdG (R) and  15 N 5 -CEdG (S) isomers, respectively; 
 (iii) heating the mixture in concentrated ammonia; and 
 (iv) purifying oligonucleotides comprising stereochemically pure  15 N 5 -CEdG (R) and oligonucleotides comprising stereochemically pure  15 N 5 -CEdG (S) from the mixture using chromatography. 
 
     
     
         14 . The method of  claim 13 , wherein the chromatography is ion-pairing chromatography. 
     
     
         15 . The method of  claim 14 , wherein the ion-pairing chromatography comprises a gradient of acetonitrile/triethylammonium acetate. 
     
     
         16 . An internal standard comprising a stereochemically pure (R) and stereochemically pure (S)  15 N 5 -carboxyethyl-2′-deoxyguanosine ( 15 N 5 -CEdG). 
     
     
         17 . The internal standard of  claim 16 , wherein the internal standard comprises oligonucleotides containing stereochemically pure  15 N 5 -CEdG (R) and oligonucleotides containing stereochemically pure  15 N 5 -CEdG (S). 
     
     
         18 . The internal standard of  claim 16 , wherein the internal standard is for quantifying levels of N 2 -carboxyethyl-2′-deoxyguanosine (CEdG) (R) and CEdG (S) advanced glycation end products in a sample. 
     
     
         19 . The internal standard of  claim 17 , wherein the internal standard is for quantifying levels of N 2 -carboxyethyl-2′-deoxyguanosine (CEdG) (R) and CEdG (S) advanced glycation end products in a sample.