Solid-phase enrichment and analysis of electrophilic natural products

Abstract

In search for new natural products, which may lead to the development of new drugs for all kind of applications, novel methods are needed. Here we describe the identification of electrophilic natural products in crude extracts via their reactivity against azide as a nucleophile followed by their subsequent enrichment using a cleavable azide-reactive resin (CARR). Using this approach, natural products carrying epoxides and α,β-unsaturated enones as well as several unknown compounds were identified in crude extracts from entomopathogenic Photorhabdus bacteria.

Keywords: Photorhabdus; azides; click chemistry; electrophilic natural products; enrichment; epoxides; glidobactin; stilbenes.

Scheme 1

Principle of azidation of XAD extracts from P. luminescens TT01 containing 1 and subsequent azide enrichment with CARR (2). After the vicinal azido alcohol is covalently bound to the resin through an azide–alkyne cycloaddition, compound 3 is cleaved from the resin and analyzed by HPLC–MS. Reaction conditions: i) NaN₃, NH₄Cl, 80% MeOH in H₂O, reflux overnight; ii) CARR (2), ACN, 55 °C, 1 h, then rt, overnight; iii) 5 mM TCEP in PBS/CHCl₃/MeOH 1:5:10 (v/v/v), 1 h.

Figure 1

(A) HPLC–MS base peak chromatograms of a crude XAD extract of P. luminescens TT01 and after azidation and azide enrichment. The t_R of 1 is indicated in the crude extract. After enrichment, one distinct peak at 8.1 min corresponding to 3 is visible. (B) The characteristic MS² fragmentation pattern of the derivatized azide 3 is shown highlighting the neutral loss of carbamate (−121) and dinitrogen (−28) as characteristic fragments for CARR adducts.

Scheme 2

Structures of glidobactin derivatives (glidobactin A (4), cepafungin I (5) and luminmycin D (6)) before and after azidation and azide enrichment procedure (7, 8, 9). The MS² spectrum indicates that azidation of glidobactins only took place on the reactive site that is also targeted by the proteasome (Supporting Information File 1, Figure S11) [24].