Although VT-NMR provides some information on the conformational changes between different peptides, it does not provide information about their absolute spatial orientation or where actual IMHBs are formed. To gain more insight into the structures we generated 3D models of the compounds SA-A1, SA-B1, and SA-B3 based on 2D-NMR experiments. The NMR solution structures were determined for Sanguinamide A and some of its analogues (SA-A1, SA-B1 and SA-B3) in DMSO-d6 and CDCl 3 at 298 K, using a combination of 2D NMR spectra including 1 H, C-HSQC, COSY, TOCSY and 1 H, 1 H-ROESY spectra. ROE distance restraints (ranging from 49 to 64 per structure, as detailed in Table ) were combined with torsion angle restraints (2 to 3 per structure) derived from 3JH-Hα coupling constants, along with the respective proline conformations (cis for 2Phe-3Pro and trans for 4Ile-5Pro, as shown in Figures ). The structures were calculated using the CYANA software and the 20 lowest-energy structures from a total of 100 were selected and had no distance violations > 0.2 Å. The structures are in adequation with the observations made from VT NMR. Indeed, in CDCl 3 , SA-B1 and SA-B3 the Ile-6 NH is shielded by the Ile-6 side chain closing in towards Pro-5, contrary to SA-A1 (Fig. and S16-S17). The opposite occurs in DMSO-d6, where the Ile-6 side chain is turned away from the Ile-6 NH. In CDCl 3 , SA-B3 displays the most differences to SA-A1, as the Ala-1 NH is released from the hydrogen bond to be exposed to the solvent, while the Ile-4 NH undergoes the opposite variation, forming an intramolecular hydrogen bond with the Ala-1 CO. In DMSO, the same intramolecular hydrogen bond pattern is observable between Ala-1 NH -Ile-4 CO and Ala-1 CO-Ile-4 NH in all structures. The Phe-2 NH remains solvent-exposed throughout the various macrocycles. Here, VT-NMR shows shielding in DMSO which is not in accordance with the NMR-derived structure. Since the newly introduced fragments in SA-B1 and SA-B3 add an extra amide to the macrocycle, the overall conformation of their backbones is shifted in comparison to SA-A1 (Fig. ). However, this shift is only apparent in the right side of the macrocycle (Ala-1 and Phe-2), while the left side is quite similar (Ile-4 and Pro-5). In DMSO, both the SA-B1 pyridine and the SA-B3 benzene take a very identical position. However, in CDCl 3 there is a larger shift observed between the two.