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1、Chinese Chemical Letters Vol. 16, No. 5, pp 643-646, 2005 http:/ 643 ESI/MS of N-(O, O-Diisopropyl) Phosphoryl Aromatic Amino Acids and their Stability Investigation Using HPLC/UV/ESI/MS Hong Xia LIU1*, Shu Sheng ZHANG1, Jian Chen ZHANG1, Xiao YANG1, Ling Bo QU1, Yu Fen ZHAO1,2 1Chemistry Department
2、, Key Laboratory of Chemical Biology and Organic Chemistry of Henan, Zhengzhou University, Zhengzhou 450052 2The Key Laboratory for Bioorganic Phosphorus Chemistry, Tsinghua University, Beijing 10084 Abstract: The full scan ESI/MS and ESI/MS2 of N-(O, O-diisopropyl) phosphoryl aromatic amino acids (
3、DIPPAAAs), N-(O, O-diisopropyl) phosphoryl phenylalanine, N-(O, O-diisopropyl) phosphoryl tryptophan and N-(O, O-diisopropyl) phosphoryl tyrosine, were obtained. The specific ions for them were found. Their stability in the LC mobile phase was investigated using developed HPLC/UV/ESI/MS and the resu
4、lts demonstrated that the DIPPAAAs were stable in the mobile phase (5 mmol/L NH4Ac-MeCN (80:20,v/v, pH7.5) within 48 h. Keywords: ESI/MS, HPLC, diisopropyl phosphorylation, amino acid. In the past work, it has been found that N-(O, O-diisopropyl) phosphoryl amino acids (DIPPAAs) took place some inte
5、resting biomimicking reactions. DIPPAA can also react with nucleosides to form nucleic acids1,2. These proved that DIPPAA as intermediate might have played an important role in prebiotic synthesis of nucleic acids and proteins during origins of life3. In the course of reaction, its stability directl
6、y influences the reaction progress and the products composition, which might lead to the different reaction mechanism. Therefore, the use of high-purity DIPPAA and keeping its high stability in the reaction system can simplify the reaction and ensure the correct information of the products. Thus, it
7、 is necessary to develop a novel approach to track its purity and stability. To date, some approaches, e.g. spectrometry4,5 and capillary electrophoresis6 have been developed for analysis of some DIPPAAs. However, HPLC technique with the advantages of rapidity, precision, accuracy and automation has
8、 not been extensively applied for their analysis. In this paper, three aromatic DIPPAAs(DIPPAAAs), N-(O, O-diisopropyl) phosphoryl phenylalanine(DIPPPhe), N-(O, O-diisopropyl) phosphoryl tryptophan(DIPPTrp) and N-(O, O-diisopropyl) phosphoryl tyrosine(DIPPTyr), were chosen as model DIPPAAs. Their ES
9、I/MS and ESI/MS2 were obtained, and a rapid HPLC/UV/ESI/MS was successfully developed to examine their stability in the mobile phase. E-mail: Hong Xia LIU et al. 644 Experimental The HPLC-MS was performed on an Agilent 1100 series HPLC (Agilent Co., Germany) and an Esquire 3000 ESI/MS with ion trap
10、mass spectrometer (Bruker Daltonik Gmbh, Germany). The separation was carried out on a Supelco LC-18 column (2504.6 mm) with mobile phase of 5 mmol/L NH4Ac-MeCN (80:20, pH7.5) at a flow-rate of 0.8 mL/min, and the detection wavelength was at 230 nm. The injection volume was 20 L. The full scan ESI/M
11、S were obtained with both negative and positive polarity. The ion source temperature was 300 C. The capillary voltage was 4.0 kV. DIPPPhe, DIPPTrp and DIPPTyr products were prepared according to our previous description7 with methanol-ether recrystallization for 3 times. Three DIPPAAAs were characte
12、rized using FTIR, NMR and MS, and the content was more than 99.0% (using the area normalization method of HPLC). All solvents and sample solutions used for HPLC/UV/ESI/MS were filtered through a 0.45 m membrane. Results and Discussion The base peak and the most abundant ions (with the relative abund
13、ance) of the ESI/MS and ESI/MS2 of DIPPPhe, DIPPTrp and DIPPTyr under both positive and negative polarity were obtained. The base peaks of DIPPPhe, DIPPTrp and DIPPTyr were their corresponding protonated or deprotonated molecule. The similar cleavage pathways were observed through successive losses
14、of one and two-molecular of propylene under the positive ESI, and with losses of one-molecular propylene and one-molecular isopropanol under the negative polarity. As an example, Figure 1 showed +ESI/MS and +ESI/MS2 for DIPPTyr. These MS and MS2 data could provide two alternative strategies to chara
15、cterize DIPPAAAs. However, the negative mode produced comparatively less fragments and a higher intensity of the signals than in the positive polarity. Therefore, the negative polarity is obviously superior and the molecular ions M-H- at m/z 328, 367 and 344 were chosen as the specific ions for iden
16、tifying DIPPPhe, DIPPTrp and DIPPTyr in the samples, respectively. Figure 1 ESI/MS (A) and ESI/MS2 of DIPPTyr under positive polarity 105 8 4 0 200 300 400 500 m/z 2 1 0 10-5 200 300 400 500 m/z ESI/MS of N-(O, O-Diisopropyl) Phosphoryl Aromatic Amino Acids 645 As DIPPAAs from the reaction with phosphorylation regent and corresponding amino acids (AAs), the possible impurities are their corresponding AAs. Thus, how
