Interception of the Bycroft-Gowland Intermediate in the Enzymatic Macrocyclization of Thiopeptides

Abstract

Thiopeptides are a broad class of macrocyclic, heavily modified peptide natural products that are unified by the presence of a substituted, nitrogen-containing heterocycle core. Early work indicated that this core might be fashioned from two dehydroalanines by an enzyme-catalyzed aza-[4 + 2] cycloaddition to give a cyclic-hemiaminal intermediate. This common intermediate could then follow a reductive path toward a dehydropiperidine, as in the thiopeptide thiostrepton, or an aromatization path to yield the pyridine groups observed in many other thiopeptides. Although several of the enzymes proposed to perform this cycloaddition have been reconstituted, only pyridine products have been isolated and any hemiaminal intermediates have yet to be observed. Here, we identify the conditions and substrates that decouple the cycloaddition from subsequent steps and allow interception and characterization of this long hypothesized intermediate. Transition state modeling indicates that the key amide-iminol tautomerization is the major hurdle in an otherwise energetically favorable cycloaddition. An anionic model suggests that deprotonation and polarization of this amide bond by TbtD removes this barrier and provides a sufficient driving force for facile (stepwise) cycloaddition. This work provides evidence for a mechanistic link between disparate cyclases in thiopeptide biosynthesis.

Figure 1. Thiopeptide structures and their proposed mechanism.

(a) Thiopeptides thiocillin and thiomuracin have pyridine cores, while thiostrepton bears a dehydropiperidine core. (b) A proposed mechanism describing the formation of diverse thiopeptide cores. Series c is not shown. (c) Biosynthetic gene clusters (BGC) for each thiopeptide shown. Highlighted in purple are the enzyme(s) that are either putatively responsible for the core formation (tsrL) or have been successfully reconstituted in vitro (tbtD, tclM).

Figure 2. Observation of a “Bycroft-Gowland” hemiaminal intermediate.

(a) The proposed structure of the bisoxazole hemiaminal intermediate observed during the course of the cyclization. (b) RP-HPLC traces (UV at 254 nm) of the MBP-TbtD-catalyzed reaction with compound (4) at 2 and 21 hours. (c) MS of linear substrate (4), hemiaminal intermediate (5) and thiopeptide product (7) in profile. (d) Time course of the MBP-TbtD-catalyzed reaction at pH 10.5. The area under each peak corresponding to (4), (5) and (7) are plotted. Similar time courses were performed across a range of basic pHs and hemiaminal intermediate (e) and thiopeptide product (f) are plotted. All time points were performed in triplicate with error bars representing the standard deviation.

Figure 3. ¹³C-¹³C COSY confirms the diketone intermediate structure.

(a) The reaction scheme of the MBP-TbtD-catalyzed cyclization of the ¹³C-labeled bisoxazole linear substrate (4’). Short exposures of (4’) to MBP-TbtD allow the buildup of hemiaminal (5’). Introducing an acidic aqueous solution to the reaction quickly hydrolyzes (5’) to the diketone intermediate (6’). Prolonged incubation of (4’) with MBP-TbtD yields the thiopeptide product (7’) as expected. (b) Profile MS of (4’), (5’) generated in situ, the isolated diketone (6’) and the thiopeptide product (7’). (c) ¹³C-¹³C COSY (DMSO-d₆) of (6’). C-C bonds formed in the formal cycloaddition are highlighted in blue.

Figure 4. Possible reaction mechanisms for the aza-[4+2] cycloaddition.

Neutral (a), cationic (b), and anionic (e, d) manifolds are investigated. (a) Cycloaddition may proceed via a concerted manner under neutral reaction system, however a high energetic amide-iminol tautomerization barrier is expected. This energy barrier may be overcome in some cationic (b) or anionic (d) systems to facilitate a stepwise cycloaddition mechanism.