Uncovered a devoted nonribosomal peptide synthase (NRPS) and various tailoring enzymes, which includes the aforementioned GetF and a second Fe/KG GetI.36 Initially proposed to catalyze -hydroxylation of 2chlorohistidine,35 GetI represented a potentially valuable biocatalyst to produce ncAAs and help in the synthesis of GE81112 B1. Surprisingly, neither 2-chlorohistidine nor histidine was converted to its corresponding hydroxylation product in reaction with GetI.37 However, the high sequence homology shared by GetI with OrfP38 and VioC,39 two previously characterized Fe/KG arginine hydroxylases, led us as an alternative to examine citrulline (55) as a substrate. To our delight, LCMS evaluation revealed a monohydroxylated product, and subsequent NMR analysis confirmed its identity as 4-hydroxycitrulline (56). At this stage, GetI was noted to exhibit comparable activity on -amino–carbamoylhydroxyvaleric acid and low levels of hydroxylation activity on arginine. Given that no committed arginine -hydroxylase was recognized at the time of this work, this discovery inspired an enzyme engineering campaign to create such enzyme. Utilizing homology models of OrfP and VioC, we performed sequential site-directed mutagenesis on GetI and obtained variant QDPYF, which was capable of converting arginine to 4-hydroxyarginine with 94 TTN. Preparative scale reaction with E. coli lysates expressing GetI QDPYF afforded almost total conversion, and we successfully implemented this course of action in a brief synthesis of a dipeptide fragment of enduracidin.37 We next targeted the chemoenzymatic synthesis of GE81112 B1 (52),40 which comprises 3hydroxypipecolic acid (AA1), 4-hydroxycitrulline (AA2), 2-aminohistidine (AA3), and hydroxy-2-chlorohistidine (AA4) (Figure 5B). Chemical construction of those monomers is nontrivial, as a Trk site earlier synthesis of GE81112 A expected 7 actions to produce each and every fragment and suffered from poor stereocontrol.41 A tactic was thus devised that would showcase the strengths of both biocatalysis and modern chemical methodology: Particularly, enzymatic hydroxylations with GetF and GetI have been proposed to allow construction of AA1 and AA2, whereas regular chemistry could grant access to AA3 and AA4. For the preparation of AA1, co-expression of GetF with GroES/GroEL8c,25 delivered comprehensive conversion of pipecolic acid (53) to 3-hydroxypipecolic acid on 500 mg scale. Upon therapy with Boc2O, protected monomer 65 was obtained in 74 yield more than two steps. Toward AA2, GetI facilitated gram-scale conversion of citrulline to 4hydroxycitrulline and provided, following subsequent safeguarding group adjustments, fragment 66 in four measures and 41 general yield. Unmasking of the main amine 67 from 66 was followed by routine coupling with 65 to provide dipeptide 68 after saponification. Preparation of AA3 proceeded by way of two-step functionalization of Boc-His-OMe (69), wherein C2 azotisation of its imidazole ring was followed by 5-HT6 Receptor Modulator Purity & Documentation saponification of the methyl ester to reveal acid 70. Building of AA4 proved extra challenging, as quite a few attempts at an asymmetric aldol reaction were met with failure. Ultimately, upon optimization in the steric environment, reaction of titanium enolate 71 and aldehyde 72 gave the desired adduct as a single diastereomer in 59 yield after methanolysis. Therapy with aqueous ammonium sulfide cleanly supplied the desired amine 73, which was coupled with acid 70 and deprotected below buffered situations to deliver dipeptide 74.Author Manuscript Author.
