Non-technical write-up of summer research for the Department of Homeland Security

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My project at LLNL this past summer was to improve upon the available methodology for synthesis of C-terminal polypeptide {alpha}-thioesters (all of which methods suffer from certain disadvantages requiring too much detail to discuss herein). Our initial approach to synthesis of {alpha}-thioesters is outlined in Figure 2. The approach utilizes a resin containing an aryl hydrazine linker to which the growing polypeptide chain is attached. The aryl hydrazine linker can be oxidized under mild conditions to the corresponding diazene. Our objective was to use the weak N-nucleophile benzotriazole to cleave the peptide from the resin. The acyl benzotriazole formed by ... continued below

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Shoulders, M D July 29, 2005.

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My project at LLNL this past summer was to improve upon the available methodology for synthesis of C-terminal polypeptide {alpha}-thioesters (all of which methods suffer from certain disadvantages requiring too much detail to discuss herein). Our initial approach to synthesis of {alpha}-thioesters is outlined in Figure 2. The approach utilizes a resin containing an aryl hydrazine linker to which the growing polypeptide chain is attached. The aryl hydrazine linker can be oxidized under mild conditions to the corresponding diazene. Our objective was to use the weak N-nucleophile benzotriazole to cleave the peptide from the resin. The acyl benzotriazole formed by the cleavage may be thiolyzed using ethanethiol and triethylamine to form the corresponding C-terminal polypeptide {alpha}-thioester, which can then be employed in NCL. My initial experiments failed to result in formation of any {alpha}-thioester. Instead, the exclusive product of acyl diazene cleavage was the peptide hydrolysis product. A number of experiments were performed to identify the stage at which hydrolysis was occurring. It was found that hydrolysis occurred during the benzotriazole-mediated cleavage of the acyl diazene. After extensive experimentation, I discovered that C-terminal polypeptide {alpha}-thioesters could, in fact, be formed by performing the acyl diazene cleavage in the absence of diisopropylethylamine (DIEA). I performed other experiments to study the variables that could improve the ratio of the hydrolysis product to the thiolysis product and was able to obtain replicable results in which the product mixture was 30% {alpha}-thioester and 70% hydrolysis. While the yield must still be improved for this to represent a viable method of peptide {alpha}-thioester synthesis, it does represent significant progress towards development of such a method. I was able to effect a three- to five-fold improvement in the yield of {alpha}-thioester relative to the {alpha}-thioester yield when ethanethiol was used to cleave the acyl diazene, a promising result which merits further investigation. I performed some experiments utilizing alternative N-nucleophiles such as imidazole to cleave the acyl diazene, as benzotriazole appears to compete poorly with water in the cleavage reaction. Some promising results were obtained, suggesting that use of a slightly stronger N-nucleophile may increase the yield of C-terminal polypeptide {alpha}-thioester.

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  • Report No.: UCRL-TR-214224
  • Grant Number: W-7405-ENG-48
  • DOI: 10.2172/881664 | External Link
  • Office of Scientific & Technical Information Report Number: 881664
  • Archival Resource Key: ark:/67531/metadc886243

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  • July 29, 2005

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  • Sept. 21, 2016, 2:29 a.m.

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  • April 13, 2017, 6:12 p.m.

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Shoulders, M D. Non-technical write-up of summer research for the Department of Homeland Security, report, July 29, 2005; Livermore, California. (digital.library.unt.edu/ark:/67531/metadc886243/: accessed November 16, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.