Applying Molecular Networking for the Detection of

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1 Supporting Information Applying Molecular Networking for the Detection of Natural Sources and Analogues of the Selective Gq Protein Inhibitor FR Raphael Reher, Markus Kuschak, Nina Heycke, Suvi Annala, Stefan Kehraus, Hao-Fu Dai, Christa E. Müller, Evi Kostenis, Gabriele M. König, and Max Crüsemann, * Institute of Pharmaceutical Biology, Molecular, Cellular, and Pharmacobiology Section, Institute of Pharmaceutical Biology, Pharmaceutical Institute, Institute of Pharmaceutical Chemistry I, University of Bonn, Bonn 53113, Germany. Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou , Hainan, China.

2 Table of Contents LCMS² and MS n analysis of FR and FR analogues Figure S1: Figure S2: LCMS² analysis of FR, FR-1, FR-2 LCMS² analysis of FR-SC, FR-SC-1, FR-SC-2, FR-SC-3, and FR-SC-4 Figure S3: HPLC-UV chromatogram of the FR-3/FR-4 mixture (1:1) Figure S4: LCMS² analysis of FR, FR-3, and FR-4 Structures of FR and FR analogues Scheme S1: Scheme S2: Scheme S3: Structures of FR, FR-1, FR-2, FR-3, and FR-4 Putative structures of FR-SC, FR-SC-1, FR-SC-2, FR-SC-3, and FR-SC-4 Structures and numbering of FR, FR-1, FR-2, FR-3, and FR-4 NMR spectroscopic data of FR-3 (3) and FR-4 (4) Figure S5: Figure S6: Figure S7: Figure S8: Figure S9: Figure S10: Figure S11: 1 H NMR (600 MHz CDCl 3 ) of FR-3 (3) and FR-4 (4), 1:1 mixture 13 C NMR (150 MHz CDCl 3 ) of FR-3 (3) and FR-4 (4), 1:1 mixture COSY NMR (600 MHz CDCl 3 ) of FR-3 (3) and FR-4 (4), 1:1 mixture HSQC NMR (600 MHz CDCl 3 ) of FR-3 (3) and FR-4 (4), 1:1 mixture HMBC NMR (600 MHz CDCl 3 ) of FR-3 (3) and FR-4 (4), 1:1 mixture 1 H NMR (600 MHz CDCl 3 ) of FR-3 (3) and FR-4 (4), 3:1 mixture 13 C NMR (150 MHz CDCl 3 ) of FR-3 (3) and FR-4 (4), 3:1 mixture Investigation of other plants from the genus Ardisia for FR and FR analogues Figure S12: Picture of modified stomata from Ardisia lucida. Figure S13: Extracted ion chromatograms m/z ± 0.05 Da of extracts/fractions from different Ardisia species.

3 Figure S1: (A) Schematic structure of FR (FR), FR-1 (1), FR-2 (2). Peptide fragment ions are labeled according to a nomenclature system developed by Ngoka et al. 1 based on Biemanns modifications 2 of Roepstorffs nomenclature 3 in one-letter amino acid code. b = b-ion with loss of water. L = N-acetylhydroxyleucine1, L * = N-3- hydroxypropionyl-hydroxyleucine, F = phenyllactic acid, A = N-methyldehydroalanine, A = alanine, A = N- methylalanine, L = hydroxyleucine, T = N,O-dimethylthreonine, L = N-propionylhydroxyleucine2, L = N- acetylhydroxyleucine2. (B) Comparison of first-generation, product-ion spectra of FR, 1, and 2; [M+H] +. 1 has a different acylation residue at L ; instead of an acetate residue (L in FR), 1 contains a 3-hydroxypropionate residue (L*). 2 has a different acylation residue at L ; instead of a propionate residue (L in FR), 2 contains an acetate residue (L ).

4 Figure S2: Schematic structure of FR-SC (5), and derivatives thereof 6, 7, 8, and 9 on the right side. FR derivatives 5-9 are all lacking the side chain L = N-propionylhydroxyleucine2. Peptide fragment ions are labeled according to a nomenclature system developed by Ngoka et al. 1 based on Biemanns modifications 2 of Roepstorffs nomenclature 3 in one-letter amino acid code. b = b-ion with loss of water. L = N-acetylhydroxyleucine, F = phenyllactic acid, A = N-methyldehydroalanine, A = alanine, A = N-methylalanine, L = hydroxyleucine, T = N,O-dimethylthreonine, L^ = N-propionylhydroxyleucine1, L = N-3HPr-Hle = N-3hydroxypropionylhydroxyleucine1. Left side: Comparison of first-generation, product-ion spectra of 5-9; [M+H] +.5, 6 and 9 have different acylation residues at L ; instead of an acetate residue (L in 5), 6 contains a propionate residue (L^) and 8 contains a 3-hydroxypropionate residue (L ). 7 differs from 5 in the amino acid N-methylalanine (A ). A is substituted in 8 by alanine or N- Methylglycine (A). 9 contains an additional alanine (A).

5 Figure S3: HPLC-UV chromatogram of the FR-3/FR-4 mixture (1:1), displaying four different wavelengths. Figure S4: (A) Schematic structure of FR (FR), FR-3 (3), and FR-4 (4). Peptide fragment ions are labeled according to a nomenclature system developed by Ngoka et al. 1 based on Biemanns modifications 2 of Roepstorffs nomenclature 3 in one-letter amino acid code. b = b-ion with loss of water. L = N-acetylhydroxyleucine1, L = N- propionylhydroxyleucine1, F = phenyllactic acid, A = N-methyldehydroalanine, A = homoalanine, A = alanine, A = N-methylalanine, L = hydroxyleucine, T = N,O-dimethylthreonine, L = N-propionylhydroxyleucine2. (B) Comparison of first-generation, product-ion spectra of FR, 3, and 4; [M+H] +. Compared to FR, 3 has a different acylation residue at L ; instead of an acetate residue (L in FR), 3 contains a propionate residue (L ). 4 differs from FR in the amino acid alanine (A). A is substituted in 4 by homoalanine (A ).

6 Scheme S1: Structures of FR (FR), FR-1 (1), FR-2 (2), FR-3 (3), and FR-4 (4). Differences to FR are highlighted (blue box). For 2 complete 1D and 2D NMR data exist, but are in submission process of another manuscript.

7 Scheme S2: Putative structures of 5, 6, 7, 8 and 9. All FR derivatives shown lack the N-propionylhydroxyleucine side chain (yellow box). Putative structural differences from 5 are highlighted (blue box). Scheme S3: Structures and carbon numbering of FR (FR), FR-1 (1), FR-2 (2), FR-3 (3), and FR-4 (4).

8 Figure S5: 1 H NMR spectrum (600 MHz CDCl 3 ) of FR-3 (3) and FR-4 (4), 1:1 mixture. Figure S6: 13 C NMR spectrum (150 MHz CDCl 3 ) of FR-3 (3), and FR-4 (4), 1:1 mixture.

9 Figure S7: 1 H- 1 H-COSY NMR spectrum (600 MHz CDCl 3 ) of FR-3 (3) and FR-4 (4), 1:1 mixture. Figure S8: 1 H- 13 C HSQC NMR spectrum (600 MHz CDCl 3 ) of FR-3 (3) and FR-4 (4), 1:1 mixture.

10 Figure S9: 1 H- 13 C HMBC NMR spectrum (600 MHz CDCl 3 ) of FR-3 (3) and FR-4 (4), 1:1 mixture. Figure S10: 1 H NMR spectrum (600 MHz CDCl 3 ) of FR-3 (3) and FR-4 (4), 3:1 mixture.

11 Figure S11: 1 H- 13 C HSQC NMR spectrum (600 MHz CDCl 3 ) of FR-3 (3) and FR-4 (4), 3:1 mixture. Figure S12: (A) Ardisia lucida leaf. The arrows are pointing to potential nodule structures. (B) Modified stomata similar to the ones observed in Psychotria kirkii. 4 (Magnification 40x)

12 Figure S13: Extracted ion chromatograms m/z ± 0.05 Da of extracts/fractions from different Ardisia species. References: (1) Ngoka, L. C.; Gross, M. L. A Nomenclature System for Labeling Cyclic Peptide Fragments. J. Am. Soc. Mass Spectrom. 1999, 10 (4), (2) Biemann, K. Mass Spectrometry of Peptides and Proteins. Annu. Rev. Biochem. 1992, 61 (1), (3) Roepstorff, P.; Fohlmann, J. Proposal for a Common Nomenclature for Sequence Ions in Mass Spectra of Peptides. Biomed. Mass Spectrom. 1984, 11 (11), 601. (4) Pinto-Carbó, M.; Gademann, K.; Eberl, L.; Carlier, A. Leaf Nodule Symbiosis: Function and Transmission of Obligate Bacterial Endophytes. Curr. Opin. Plant Biol. 2018, 44,