Fluorogenic derivatization of peptides with naphthalene-2,3-dicarboxaldehyde/cyanide: optimization of yield and application in the determination of leucine-enkephalin spiked human plasma samples.
Initial attempts to derivatize the alpha-amino site of several tripeptides with naphthalene-2,3-dicarboxaldehyde/cyanide (NDA/CN) resulted in poor yields of the expected N-substituted 1-cyanobenz[f]isoindole (CBI) products. Examination of the CBI-formation mechanism, in conjunction with knowledge of the general structure-reactivity properties of the tripeptides, led to the recognition of a competing non-productive reaction pathway. Through the use of model reactions and the isolation and structural elucidation of a predicted side-product the viability of the competing pathway was confirmed. From an understanding of the key features of both the productive and non-productive reaction pathways, a rational approach for the optimization of CBI-derivative yield was proposed and confirmed experimentally. This information led, in turn, to the development of HPLC methodology suitable for the determination of leu-enkephalin spiked into human plasma; fluorescence detection was used in conjunction with leu-enkephalin amide as the internal standard. The method enabled leu-enkephalin to be determined at a concentration of 0.31 nmol ml-1 with an error of less than 4% for 25 pmol injected.
de Montigny
P
P
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence 66045.
Riley
C M
CM
Sternson
L A
LA
Stobaugh
J F
JF
D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003486:Cyanides; D004743:Enkephalin, Leucine / Q000097:blood*; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D009281:Naphthalenes; D010455:Peptides
D003486:Cyanides; D007202:Indicators and Reagents; D009281:Naphthalenes; D010455:Peptides; D004743:Enkephalin, Leucine; C027717:2,3-naphthalenedicarboxaldehyde