Development of high-performance amino acid sequensor of peptides

高性能肽氨基酸测序仪的开发

• 批准号: 10557212
• 负责人: IMAI Kazuhiro
• 金额: $ 7.49万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10557212/
• 关键词: sequensor; Edman degradation; fluorescent Edman reagent; fluorimetric HPLC; optical resolution; benzofurazan; ペプチドシークエンサー; PITC

We have developed a sensitive method for the simultaneous determination of an amino acid sequence and D/L-configuration of peptides with fluorogenic Edman reagents and BFィイD23ィエD2 as cyclization/cleavage reagent.First, New fluorogenic Edman reagents, PSBD-NCS and MSBD-NCS, were synthesized and the amino acids thiazolinone derivatives of these reagents were sensitively detected.Secondly, we tried to detect more stable amino acids derivatives of these reagents. The amino acids thiazolininone derivatives of DBD-NCS were converted to carbamoyl derivatives via thiocarbamoyl compounds and detected fluorimetrically.Thirdly, we investigated the conversion reaction of thiazolinone to thiohydantoin derivatives and found out that hydrochloric acid in methanol solution efficiently suppressed the racemization in the conversion reaction.Finally, fluorogenic Edman reagent, MTBD-NCS, was synthesized. The thiocamamoyl and carbamoyl derivatives were not fluorescent and thiohydantoin derivatives of MTBD-NCS were fluorescent, giving the superiority with regard to avoidance from the interferring peaks of the degradation products.The combined use of MTBD-NCS and BFィイD23ィエD2 gave us the sensitive determination method of amino acid sequence and D/L-configuration of peptides. This method developed in this study seems to be easily applicable to the conventional automatic sequensor.

以BFィイD23ィエD2为环化/裂解试剂，建立了一种同时测定氨基酸序列和多肽D/L构型的灵敏方法。首先，合成了新的荧光Edman试剂PSBD-NCS和MSBD-NCS，并对这两种试剂的氨基酸衍生物进行了灵敏的检测。通过硫代氨基甲酰化反应将DBD-NCS的氨基酸噻唑啉酮类化合物转化为氨基甲酰类化合物，并进行荧光检测；再次，考察了四甲基偶氮唑酮类化合物转化为硫代海因类化合物的反应，发现在甲醇溶液中盐酸能有效地抑制转化反应中的消旋反应，最终合成了荧光Edman试剂MTBD-NCS。MTBD-NCS的硫代甲酰基和氨甲酰基衍生物不是荧光的，而硫代海因衍生物是荧光的，在避免降解产物的干扰峰方面具有优势。MTBD-NCS和BFィイD23ィエD2的结合使用给出了一种灵敏的氨基酸序列和D/L构型的测定方法。本研究开发的方法似乎很容易适用于常规的自动测序仪。

项目成果

Akira Toriba: "Comparison of four fluorescence Edmam reagents with benzofurazan Structure for the detection of thiazolinone amino acid derivatives."Analyst. 124. 43-48 (1999)

Akira Toriba：“比较四种荧光 Edmam 试剂与苯并呋喃结构，用于检测噻唑啉酮氨基酸衍生物。”分析师。

T.Iida,et al.: "Amino acid sequence and D/L-configuration determination of peptides utilizing liberated N-terminus phenylthiohydantoin amino acids." J.Chromatogr.A,. 813. 267-275 (1998)

T.Iida 等人：“利用释放的 N 末端苯硫基乙内酰脲氨基酸测定肽的氨基酸序列和 D/L 构型。”

Takayuki Iida: "Semi-automatic amino acid sequencing and D/L-configuration determination of peptides with detection of liberated N-terminal phenylthiocarbamoyl amino acids."Analyst. 123. 2829-2834 (1998)

Takayuki Iida：“半自动氨基酸测序和肽的 D/L 构型测定，并检测释放的 N 末端苯硫基氨基甲酰氨基酸。”分析师。

Takayuki Iida: "Amino acid sequence and D/L-configuration determination of peptides utilizing liberated N-terminus phenylthiohydantoin amino acids."J. Chromatogr. A. 813. 267-275 (1998)

Takayuki Iida：“利用释放的 N 末端苯硫基乙内酰脲氨基酸测定肽的氨基酸序列和 D/L 构型。”

Yong Huang: "Detection of DBD-carbamoyl amino acids in amino acid sequence and D/L configuration determination of peptide width fluorogenic Edman reagent"Analytical Biochemistry. 270. 257-267 (1999)

黄勇：“氨基酸序列中DBD-氨基甲酰基氨基酸的检测及肽宽度荧光Edman试剂的D/L构型测定”分析生物化学。