Reaction Kinetics of Phenylisothiocyanate with Amino Acid Analogs Open Access

Idigo, Chinenye Abiodun (2008)

Permanent URL: https://etd.library.emory.edu/concern/etds/qj72p7427?locale=en
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Abstract

The kinetics of the chemical reactions of isothiocyanate derivative cocaine analogs with amino acids in the human dopamine transporter during labeling were investigated using small molecule models of the reactions. Analogs of the side-chains of cysteine, lysine and tyrosine were 1-octanethiol, phenethylamine (PEA), and phenol respectively. Phenylisothiocyanate (PITC) was used to represent isothiocyanate derivative cocaine analogs. A large excess (1mM) of each amino acid analog was reacted with PITC in an aqueous solution at pH 7.4. The experiments were repeated at pH 9.0. Pseudo first order rate constants were obtained for 1-octanethiol (k = 1.26 x 10-2 min-1 at pH 7.4 and k = 8.73 x 10-2 min-1 at pH 9.0), and PEA (k = 2.32 x 10-3 min-1 at pH 7.4 and k = 1.51 x 10-2 min-1 at pH 9.0). The results showed that 1-octanethiol was the most reactive analog at both pH 7.4 and pH 9.0. Phenol was the least reactive and showed no detectable reaction with PITC. At pH 9.0, 1-octanethiol and PEA showed a six to seven fold increase in reactivity relative to pH 7.4. The products of the reactions were confirmed using high resolution mass spectrometry. The product of the reaction of PITC and PEA was 1- phenethyl-3-phenylthiourea. The product of the reaction of PITC and 1-octanethiol was octyl phenylcarbamodithioate. 1-phenethyl-3-phenylthiourea is a stable compound and shows no detectable sign of degradation for 48 hours. Octyl phenylcarbamodithioate is much less stable and starts to show detectable degradation at 60 minutes at pH 7.4 and as early as 20 minutes at pH 9.0.

Table of Contents

Introduction Background on the Human Dopamine Transporter hDAT Function hDAT Structure and Topology The Role of hDAT in Cocaine Addiction Methods Used to Determine the Binding Site of Cocaine on hDAT Site-Directed Mutagenesis Photoaffinity Labeling Using Azide Derivatives Chemical Labeling Using Isothiocyanate Derivatives Project Overview and Objectives Reactivity of Amino Acid Analogs with Phenylisothiocyanate Identification of Products and Their Stability Methods

Reactivity of Amino Acid Analogs HPLC Analysis of the Reaction of PITC with Amino Acid Analogs at pH 7.4 HPLC Analysis of the Reaction of PTIC with Amino Acid Analogs at pH 9.0 Identification of Products and Their Stability Identification of Products Stability of Products

Data Analysis Experimental Data HPLC Analysis of the Reaction of PITC with Amino Acid Analogs at pH 7.4 HPLC Analysis of the Reaction of PITC with Amino Acid Analogs at pH 9.0 High Resolution Mass Spectrometry Analysis of Products Stability of Product Results HPLC Analysis of the Reaction of PITC with Amino Acid Analogs at pH 7.4 HPLC Analysis of the Reaction of PITC with Amino Acid Analogs at pH 9.0 High Resolution Mass Spectrometry Analysis of Products

Discussion

Reactivity of Amino Acid Analogs at pH 7.4

Reactivity of Amino Acid Analogs at pH 9.0

Identity and Stability of Products

Summary

References

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