*2.2. Structure Elucidation*

Litoralimycin A (**1**) was isolated as a light-yellow oil. The molecular formula of C48H45N15O10S4 was derived from its high-resolution electrospray ionization mass spectrometry (HRESIMS) peak observed at *m*/*z* 1120.2429. 1H and heteronuclear single-quantum correlation spectroscopy (HSQC) NMR spectra indicated the presence of 5 methyls, 4 exomethylenes and 1 low-field aliphatic methylene, and 7 olefinic/aromatic as well as 3 aliphatic methines, in addition to 10 exchangeable protons bound to heteroatoms (Table 1). The number of exomethylenes in combination with the four sulfur atoms gave an early hint towards a thiopeptide. The 13C spectrum indicated the presence of 9 additional carbonyls as well as 19 further olefinic carbons bearing no hydrogens. Based on correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) and heteronuclear multiple-bond correlation spectroscopy (HMBC) correlations, the planar thiopeptide structure, containing four thiazole (Thz), a valine (Val), an oxazole (Oxa), a pyridine (Pyr) and three dehydroalanine (Dala) units, was established (Figure 2). The rotating frame nuclear Overhauser effect spectroscopy (ROESY) correlation between H3-27 and 21-NH established the Δ21,26 double bond geometry as *Z*. Since thiazole amino acids racemize very easily during acid hydrolosis, the configuration of C-15 was determined by ozonolysis of the aromatic ring for preservation of the chiral center followed by acid hydrolosis [6]. After ozonolysis, hydrolyzation and derivatization with FDAA, we detected l-Val and l-Ala according to Marfey's method [7]. Thus, both C-5 and C-15 are S-configured.


**Table 1.** NMR data (1H 700 MHz, 13C 175 MHZ) of **1** in DMSO-*d*6.

**Figure 2.** Selected 1H,1H COSY (bold lines), 1H,13C HMBC (black arrows) and 1H,1H NOESY (red arrow) correlations of **1.**

HRESIMS data of **2** gave a molecular formula of C45H42N14O9S4, implying the formal loss of a C3H3NO fragment compared to **1**. The proton and carbon NMR spectra of **2** were highly similar to those of **1**, with the key difference of the absence of a terminal dehydroalanin moiety. Therefore, the structure of **2** was established as identical to that of **1** with its side chain truncated by one dehydroalanin moiety.
