Synthesis of Alkyl-ACP

The acetyl-PPant unit is likely to have arisen from the excess acetyl-SNAC (20) remaining in the reaction mixture from the KS acylation reaction, acylating any free PPant thiol. It was proposed that this species may also be arising from activation of the elongated acetoacetyl-PPant, however, the MS activation conditions used in the assay were optimised using acetoacetyl-ACP ensuring that only the m/z 345.2 is ejected. The disulphide-Unked A-acetylcysteamine-PPant species is a result of a relatively minor side reaction between the free thiols of (V-acetylcysteamine and the PPant arm. 

In order to confirm that the production of the acetoacetyl-PPant was a result of PsyA KSl catalysis, a control reaction was performed under the same reaction conditions in the absence of PsyA KSl. The MS analysis of this reaction yielded no peak at m/z 345, demonstrating that the production of acetoacetyl-ACP is solely due to KS-catalysed condensation of the malonyl-ACP with the acetyl unit (Fig. 6.5). 

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The synthesis of alkyl-ACP, for use as an internal standard, was achieved by incubation of 200 pM ACP (25 mM Tris HCl, 500 mM NaCl, pH 7.4) with 4mM iodoacetamide, in the absence of fight (Scheme 2.5). Reactions were monitored by ESI-MS at regular time intervals for the presence of the alkyl-ACP adduct. Alkylation reactions were complete after 4 h at 25 C, and excess iodoacetamide was removed by spin filtration using 5 kDa MWCO columns. 

Scheme 2.5 Synthesis of alkyl-ACP from iodoacetamide and holo-ACP, in the absence of light...

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