Mass Spectrometric Study on 9-Fluorenylmethyloxycarbonyl Group Removal Assisted by 2-Methoxyacetic Acid in Gas Phase

Abstract

The detachment of 9-fluorenylmethyloxycarbonyl (Fmoc) from the amino group is widely performed in chemical synthesis reactions, involving peptides, nucleosides, polymers and some other organic compounds. Reported methods for Fmoc release mainly referred to traditional bases. Limited information about carboxylic acid-participating Fmoc detachment has been documented. This study mainly investigated the role of acetic acid (HAc), 3-methoxypropionic acid (MPA), 2-methoxyacetic acid (MAA) in the process of Fmoc deprotection in gas phase. The home-made nano emitter was used to spray the mixed solution of the three carboxylic acids and different Fmoc protected amino acids, which flew into the inlet of mass spectrometer (MS), to monitor the key adduct ions derived from the reaction of carboxylic acids and protected amino acids, such as Gly, Ala, Val, Leu, Pro, Met and Phe. Furthermore, the effects of substrate structures on the formation of the adduct in MS and its dissociation in MS/MS were discussed. The results showed that the relative abundance of MAA adducts were significantly higher than that of HAc adducts and MPA adducts for the same Fmoc protected amino acid, and only MAA related adducts can dissociate into fragments with 20% collision energy (CE). This is due to the special structure of MAA, which has a methoxy group providing a lone pair of electrons to the acidic hydrogen at the second position of the carboxylic acid. Thus, the methoxy group weakly interacted with the H at the 9th position of Fmoc ring, promoting the formation of the adducts of MAA and Fmoc protected amino acid to decrease the energy barrier of the transition state for the 9th H release process. All the [Xf+MAA-H]- adducts can generate two kinds of successor ions upon 20% CE, one is the Fmoc removal fragment, another is only MAA loss fragment. Interestingly, [Pf+MAA-H]- mainly gave fragments of the Fmoc removal products, whereas the other six compounds with relatively low abundance ratio mostly produced fragments by neutral MAA loss. This difference of the fragmentation pathways may be owing to the structure of Pf, which has no H in nitrogen atom and suppresses the formation of intermolecular hydrogen bonding. Thus, the formation of intermolecular hydrogen bond between the MAA and Pf is preferred, which benefits [Pf+MAA-H]- dissociation into Fmoc removal products. In summary, the structures of both carboxylic acids and amino acids play a vital role in formation of adducts and detachment of Fmoc group, and the research is helpful for the understanding in the mechanism of molecular ion reactions in gas phase.

Keywords

• 9-fluorenylmethyloxycarbonyl (fmoc) / deprotection / molecule/ion reaction / mass spectrometry