Asperglaucide, a metabolite of Aspergillus glaucus, is shown by spectroscopic methods, chemical degradation, and synthesis to be N-benzoyl-L-phenylalanyl-L-phenylalaninol acetate (II). A new metabolite, asperglaucide, C₂₇H₂₈N₂O₄, is produced in relatively large amounts by the fungus Aspergillus glaucus, strains I.M.I. 53242 and 53243. Since the i.r. spectrum of the metabolite contained strong bands at 1730 and 1650 cm⁻¹, suggestive of ester and amide functions, respectively, it was decided to investigate the structure by hydrolytic procedures. The presence of an acetate residue was demonstrated by treatment with sodium methoxide in methanol to give deacetylasperglaucide, C₂₅H₂₆N₂O₃, which lacked both ester absorption in the i.r. spectrum and the acetyl ¹H n.m.r. signal of the parent at τ 8.02. After acid-catalysed hydrolysis of asperglaucide, benzoic acid and L-phenylalaninol (I) were isolated. The latter was identical with a sample prepared from the ethyl ester of L-phenylalanine hydrochloride by reduction with sodium borohydride. A similar hydrolysate was subjected to amino-acid analysis, and an equivalent of phenylalanine was also detected. Since all the carbon atoms of asperglaucide are thus accounted for, the material is identified as N-benzoylphenylalanyl-L-phenylalaninol acetate (II), the only undetermined feature being the absolute stereochemistry of the phenylalanine residue. The individual assignments of the ¹H n.m.r. signals to the non-aromatic protons of asperglaucide followed from chemical shift values, magnitudes of couplings, and decoupling experiments. The benzyl and O-methylene protons of the phenylalaninol residue appeared as doublets, τ 7.26 (J 7 Hz) and 6.10 (J 5 Hz), respectively, owing to vicinal couplings with the methine proton (τ 5.65). Irradiation at the frequency of this proton caused both methylene doublets and the NH doublet at τ 3.59 (J 8 Hz) to collapse to singlets. The CH₂·CH portion of the phenylalanine residue forms an ABX pattern (τ_A 6.93, τ_B 6.81, τ_X 5.17; J_AB 13.6, J_AX 8.1, J_BX 5.9 Hz), again confirmed by decoupling. Although all three methylene groups of asperglaucide contain diastereotopic hydrogen atoms, only in the phenylalanine residue are the methylene protons magnetically non-equivalent. The fifteen aromatic protons of asperglaucide and the NH of the phenylalanine residue show a complex pattern between τ 2.22 and 3.08, dominated by a five-proton singlet at τ 2.78. The synthesis of deacetylasperglaucide could in principle be effected by coupling N-benzoylphenylalanine with phenylalaninol by, for example, the dicyclohexylcarbodiimide-N-hydroxysuccinimide method. However, it is known that methods of this type produce racemisation of the N-benzoylphenylalanine residue via oxazolone formation. Since N-benzyloxycarbonyl-L-phenylalanine (IV) does not suffer from this disadvantage it was coupled with L-phenylalaninol by the above method to give the derivative (V). Acetylation of this gave the acetate (VI), from which the N-protecting group was removed by hydrogenolysis in the presence of toluene-p-sulphonic acid, and the product was isolated as the tosylate salt (VII). Benzoylation of this gave a product identical with asperglaucide, thus confirming the structure (II).