AbstractThe mycosporine‐like amino acid (MAA) porphyra‐334 (1) is subjected to extensive 1H‐ and 13C‐NMR analysis as well as to density‐functional‐theory (DFT) calculations. All 1H‐ and 13C‐NMR signals of 1 are assigned, as well as the resonances of prochiral proton pairs. This is achieved by 500‐MHz standard COSY, HMQC, and HMBC experiments, as well as by one‐dimensional (DPFGSE‐NOE) and two‐dimensional (NOESY) NOE experiments. Diffusion measurements (DOSY) confirm that 1 is monomeric in D2O solution. DFT Calculations yield 13C‐NMR chemical shifts which are in good agreement for species 6 which is the imino N‐protonated form of 1. An exceptionally high proton affinity of 265.7 kcal/mol is calculated for 1, indicating that 1 may behave as a very powerful ‘proton sponge’ of comparable strength as synthetic systems studied so far. Predictions of 13C‐NMR chemical shifts by the ‘NMRPredict’ software are in agreement with the DFT data. The absolute configuration at the ring stereogenic center of 1 is concluded to be (S) from NOE data as well as from similarities with the absolute configuration (S) found in mycosporine‐glycine 16. This supports the assumption that 1 is biochemically derived from 3,3‐O‐didehydroquinic acid (17). The data obtained question the results recently published by a different research group claiming that the configuration at the imino moiety of 1 is (Z), rather than (E) as established by the here presented study.