Synthesis of 5,5,6,6-D4-L-lysine-aflatoxin B1 for use as a mass spectrometric internal standard

Human exposure to the hepatocarcinogenic mycotoxin aflatoxin 131 results in modification of serum albumin lysine epsilon-amino residues to form lysine-aflatoxin adducts. A perdeuterated reference standard is now required to quantitatively measure this adduct in epidemiologic studies of liver cancer using isotopic dilution mass spectrometry. A convenient method for the preparation of D4-(L)-lysine-AFB(1) using commercially available 5,5,6,6-D4-L-lysine is demonstrated for the first time. The application of two standard a-amino protection methods is also reported that simplifies the production of natural isotopic abundance lysine-AFB(1) over the currently used method employing N-alpha-acetyl-L-lysine. t-Boc-N-alpha-lysine was used to prepare lysine-AFB(1); however, a preferred method for directing reaction of AFB(1)-dialdehyde to the e-amino group of 5,5,6,6-D4-L-lysine utilized cupric ions that were spontaneously removed during the reverse phase HPLC purification of D4-lysine-AFB(1) using 1% HOAc. This strategy eliminates the need to otherwise synthesize and purify t-Boc-N-alpha- or N-alpha-acetyl-5,5,6,6-D4-lysine and then TFA or enzymatically deprotect overnight to obtain the target compound. Copyright (C) 2004 John Wiley Sons, Ltd.