**1. Introduction**

Natural products (NPs) are the oldest form of medicine utilized by humans. Technologies and methods improved and NPs remained one of the most important source for the development of

medicinal drugs. Today, NPs and their derivatives make up a significant percentage of approved drugs worldwide. Especially in the antibiotics sector, almost all lead structures were identified from bio-resources (~75% either unaltered or semi-synthetically modified; 1981–2014) [1,2]. Although being a traditional source for antimicrobial compounds, the pool of NP-derived structural novelty is not exhausted as exemplified by the discovery of teixobactin [3] and darobactin [4].

Besides clinical application, specialized natural products are drivers of socio-economic stability by finding application in food preservation, livestock and aquaculture treatment, as well as crop protection [5]. In all fields, humans benefit from the evolutionary shaped intrinsic antimicrobial activity of NPs. In the 1940s, the "Waksman antibiotic discovery platform" was the first systematical approach to identify antimicrobial NPs and led to the isolation of the first aminoglycosides [6].

However, high rediscovery rates make classical discovery campaigns unattractive and pose an unreasonable financial risk for the private sector. This might partly be circumvented by implementing chemo-informatics in systematic, routine processes. Data mining processes such as automatic annotation of bucket matrices [7] or MS/MS networks [8] against public databases help to identify signals and interest, even in gigantic datasets.

Besides discovery of novelty, repurposing of already known structures to different fields of application seems an encouraging approach, but high expense or no commercial availability of many natural products reduce feasibility substantially. In this context, SeaPEPR represents a methodology allowing a preliminary determination of specific bioactivity of single compounds within crude environmental extracts. Application allows us to evaluate the bio-economical value of large sets of low volume sample on the metabolite level in a standardized manner and finally facilitates decision making on downstream processes such as isolation of unknown metabolites or repurposing studies.

Here, we chose a promising and likewise challenging bio-resource as a case study to present our approach of crude environmental extract analysis. Sponges, as sessile filter feeders without physical defense, are believed to depend on chemical defense or deterrence mechanisms mediated NPs, biosynthesized either by themselves or by associated microorganisms. Due to ethical reasons, straightforward isolation and characterization of compounds by harvesting sponges from nature should be discouraged. Limited availability of material usually prohibits extensive information retrieval from a given environmental sample. Frequently, scaffolds initially discovered in environmental samples are subject to delicate chemical synthesis without a clear product application. MS/MS coupled microfractionation of environmental extracts facilitates semi-automatic dereplication and allows attributing bioactivity observed in crude extract primary screens to single compounds without the necessity of cost and time-consuming isolation or synthesis.

#### **2. Results**

## *2.1. Sample Collection and Extract Generation*

Sponges are a well-known bioresource for bioactive molecules and can be regarded as a complex environmental sample, since the holobiont (consisting of the sponge and its associated microbes) is extracted as a whole. Furthermore, the taxonomic classification of sponges, which is based on both, genetic barcoding and morphology, is time consuming and challenging. In this project, 76 sponge samples from seven different dive sites at the coastal area of Sangihe and Siau Island (Pacific Ocean, Indonesia) were obtained (Figure 1). At each diving site, around 11–15 sponge samples were collected, except sponge sample T\_5, which was the only one obtained from the site named Towo. Hence, it can be expected that the sample set represents a survey of the biodiversity around the islands. How this translates into chemical diversity was investigated in the following. As starting material, 5 mg of dried sponge, which is approximately equivalent to the size of a thumbnail, was used. From all samples, crude extracts were prepared. The extraction yield (based on dry mass) using methanol was between 12 and 86% (Figure S1).

**Figure 1.** Sampling sides of sponge specimen. Samples were retrieved by SCUBA in a depth of 4–20 m below the surface.

The generated extracts represent the material further analyzed for NP discovery. The general workflow of SeaPEPR is depicted in Figure 2.

**Figure 2.** Schematic overview of the SeaPEPR pipeline. In a first step, crude environmental extracts are subject of bioactivity assessment. At the same time, the chemical diversity of the entire set of samples is determined by cosine similarity calculation ("chemical fingerprints"). Prioritized samples are microfractionated to identify the causative agent responsible for the initially observed bioactivity. If desired, dereplicated compounds of interest can be selected for isolation.
