Solid‐Phase Enrichment and Analysis of Azide‐Labeled Natural Products: Fishing Downstream of Biochemical Pathways

Abstract

Many methods have been devised over the decades to trace precursors of specific molecules in cellular environments as, for example, in biosynthesis studies. The advent of click chemistry has facilitated the powerful combination of tracing and at the same time sieving the highly complex metabolome for compounds derived from simple or complex starting materials, especially when the click reaction takes place on a solid support. While the principle of solid-phase click reactions has already been successfully applied for selective protein and peptide enrichment, the successful enrichment of much smaller primary and secondary metabolites, showing great structural diversity and undergoing many different biosynthetic steps, has seen only little development. For bacterial secondary metabolism, a far broader tolerance for "clickable" precursors was observed than in ribosomal proteinogenesis, thus making this method a surprisingly valuable tool for the tracking and discovery of compounds within the cellular biochemical network. The implementation of this method has led to the identification of several new compounds from the bacterial genera Photorhabdus and Xenorhabdus, clearly proving its power.

Thiol Probes To Detect Electrophilic Natural Products Based on Their Mechanism of Action

“…8–11 In either case, only simple functional groups have been targeted, including dehydroala-nine, 12,13 ketones and aldehydes, 8,9 carboxylic acids, 8,9 amines, 7–9 thiols, 8,9 alcohols, 10 terminal alkynes, 11,14 and azides. 15 Until recently, 16 reactivity-guided isolation has not been employed for the identification of natural product pharmacophores that would lead directly to biologically active metabolites with a high potential for therapeutic development.…”

Solid-phase enrichment and analysis of electrophilic natural products

“…After disulfide-bond cleavage with a solution of tris(2-carboxyethyl)phosphine (TCEP) in a 1:5:10 mixture of phosphate-buffered saline (PBS)/chloroform/methanol at pH 7, the filtrate was analyzed by HPLC–MS. As expected the corresponding mass m/z 493.2 [M + H] + of the cleaved cycloaddition product could be directly detected in the base-peak chromatogram (BPC) showing the characteristic fragmentation pattern of the CARR adducts (Supporting Information File 1, Figure S3) [15]. Additionally, the detection limit of the model epoxide was investigated in a complex environment.…”

“…Besides the traditional chemical screening and bioactivity-guided isolation, the exploitation of the inherent properties of natural products is also feasible. Consequently, simple functional groups of natural products like dehydroalanine [6–7], ketones, aldehydes [8–9], carboxylic acids [8–9], amines [8–10], thiols [8–9], alcohols [11], epoxides [12], terminal alkynes [13–14] and azides [15] can be targeted to introduce a label. Such labels might increase the visibility in UV or MS detection in liquid chromatography coupled to UV or mass spectrometry.…”

“…Such labels might increase the visibility in UV or MS detection in liquid chromatography coupled to UV or mass spectrometry. Alternatively, natural products can be immobilized on reactive resins by making use of their chemical functionality and can be eluted after washing off all non-desired substances [8–9 11,13,15]. …”

“…The recently introduced cleavable azide-reactive resin (CARR ( 2 ), Scheme 1) is such a resin able to react with a broad range of azides [15]. Thus, the metabolic fate of azide-containing biosynthesis intermediates or building blocks can be studied and natural products containing these azides can be identified.…”