Characterization of Nα‐Fmoc‐protected ureidopeptides by electrospray ionization tandem mass spectrometry (ESI‐MS/MS): differentiation of positional isomers

Abstract

Four pairs of positional isomers of ureidopeptides, FmocNH-CH(R(1))-ϕ(NH-CO-NH)-CH(R(2))-OY and FmocNH-CH(R(2))-ϕ(NH-CO-NH)-CH(R(1))-OY (Fmoc = [(9-fluorenyl methyl)oxy]carbonyl; R(1) = H, alkyl; R(2) = alkyl, H and Y = CH(3)/H), have been characterized and differentiated by both positive and negative ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). The major fragmentation noticed in MS/MS of all these compounds is due to --N--CH(R)--N--bond cleavage to form the characteristic N- and C-terminus fragment ions. The protonated ureidopeptide acids derived from glycine at the N-terminus form protonated (9H-fluoren-9-yl)methyl carbamate ion at m/z 240 which is absent for the corresponding esters. Another interesting fragmentation noticed in ureidopeptides derived from glycine at the N-terminus is an unusual loss of 61 units from an intermediate fragment ion FmocNH = CH(2) (+) (m/z 252). A mechanism involving an ion-neutral complex and a direct loss of NH(3) and CO(2) is proposed for this process. Whereas ureidopeptides derived from alanine, leucine and phenylalanine at the N-terminus eliminate CO(2) followed by corresponding imine to form (9H-fluoren-9-yl)methyl cation (C(14)H(11) (+)) from FmocNH = CHR(+). In addition, characteristic immonium ions are also observed. The deprotonated ureidopeptide acids dissociate differently from the protonated ureidopeptides. The [M - H](-) ions of ureidopeptide acids undergo a McLafferty-type rearrangement followed by the loss of CO(2) to form an abundant [M - H - Fmoc + H](-) which is absent for protonated ureidopeptides. Thus, the present study provides information on mass spectral characterization of ureidopeptides and distinguishes the positional isomers.