A panel of extracts was prepared under 14 different culture conditions (different media composition, changing cultivation periods, extraction procedures, UV-light exposure, and temperature) and screened by LC-MS. Two whole broth extracts of A. cruciatus 763, that were grown either in a Czapek-Dox medium supplemented with cofactors or in a Czapek-Dox medium with an altered nitrogen source, afforded a good variety of known metabolites (e.g., cyclo-phenylalanine-serine and gliovictin [9,11]) and previously unknown compounds (e.g., m/z [M + H]⁺ = 566). The HPLC profiles of these two extracts indicated that they contained almost the same metabolites but with significant differences in their relative concentrations. Thus, the two cultivation conditions were separately scaled up and processed to isolate and characterize the major metabolites present in each of these extracts.

The ethyl acetate extracts of the scaled-up fungal cultures were filtered through an RP-SPE cartridge and direct separation by RP-HPLC led to the isolation of compounds 1–6. While the identity of the known metabolites 2–6 was established by direct comparison of the [α]_D, MS, ¹H and ¹³C NMR data with literature data, for the new compound 1 a complete structure determination study was performed.

Compound 1 was assigned the molecular formula C₃₀H₅₅N₅O₅ via HR-ESI-MS data (m/z 566.4280 for [M + H]⁺), indicating a structure with six degrees of unsaturation. The IR spectrum showed absorption bands at 1633 cm⁻¹, indicating the presence of amide functionalities. The ¹H and ¹³C NMR spectra suggested that 1 was a peptide-type compound on the basis of chemical shifts and multiplicities typical for α-protons and carbons. In the ¹H NMR spectrum, signals for five α-protons (δ_H 3.49, 4.23, 4.47, 4.48, and 4.58), 4 NH protons (δ_H 6.21, 6.92, 7.27, and 7.42) and an N-methyl group (δ_H 3.29) were observed. Most other resonances were found in the upfield region, including those for both methylene and methine protons (δ_H 1.37–2.33) and ten doublet methyl resonances (δ_H 0.85–1.02). The ¹³C and DEPT135 NMR spectra indicated 30 carbon resonances, consistent with the molecular formula derived from HR-ESI-MS, including five amide carbonyl carbons, ten methine carbons (five nitrogen-bearing), four methylene groups and eleven methyl groups (one nitrogen-bearing thereof) (Table 1). Interpretation of 1D and 2D NMR spectroscopic data of 1 allowed construction of five partial structures assigned as valine (Val), N-methylleucine (N-Me-Leu), and three leucine residues (Leu-1, Leu-2 and Leu-3). Resonances of each amino acid residue were identified by starting with an α carbon resonance in the region δ_C 50–65, locating the resonance of the α proton via the HSQC (Heteronuclear Single Quantum Coherence) spectrum, and using the COSY (Correlation Spectroscopy) spectrum to delineate the five independent spin systems formed by the respective amide and side chain protons of each amino acid. The resulting substructures were extended and corroborated by HMBC (Heteronuclear Multiple Bond Correlation) correlations (See Figure 2).

The latter were key in the assignment of corresponding carbonyl groups, the γ-atoms of the leucine residues and the location of the N-methyl group. In this way, all resonances for each amino acid residue could be assigned apart from those for the Leu methyl groups, which could not be distinguished unambiguously from each other due to a heavy overlap of the methyl proton resonances. HMBC correlations from each NH/NCH₃ resonance to one carbonyl carbon established the connectivity in the core of the structure (Figure 2): the NH signal of Leu-1 (δ_H 6.21) showed a long-range correlation to δ_C 171.2 which suggested that Leu-1 is connected to N-Me-Leu via its carbonyl group; further HMBC crosspeaks, this time between the signals of NH(Leu-2)/δ_C 171.7, NH(Leu-3)/δ_C 172.5, NH(Val)/δ_C 173.1 established the primary peptide sequence to be N-Me-Leu/Leu-1/Leu-2/Leu-3/Val. The ring closure was evident from ¹H–¹³C couplings between the resonance of N-CH₃ of N-Me-Leu and δ_C 173.6. These connectivities confirmed the cyclic pentapeptidic nature of 1, thereby satisfying the required six elements of unsaturation. Based on the origin of the source microorganism and on its peptidic structure, 1 was given the name lajollamide A.

To elucidate the configurations at the stereogenic centers of the amino acid moieties in 1, the natural product was fully hydrolyzed under acidic conditions. The hydrolysate obtained was analyzed by chiral HPLC. The stereochemical identity of the hydrolyzed amino acids observed in the HPLC chromatogram (Figure 3) was established by comparison with the respective commercial authentic standards of known absolute configuration. This led to the identification of N-Me-L-Leu, L-Val and three Leu residues, two of which are bearing L- and one D-configuration. The position of the expected D-Leu residue within the peptide backbone, however, was not evident from this, nor from the NMR spectroscopic data. To clarify this unsolved question and thus to fully stereochemically characterize 1, we embarked on the total synthesis of the three possible diastereomers of this cyclic peptide.

In order to most efficiently access the three diastereomeric forms of lajollamide feasible based on the results of the peptide hydrolysis experiments, we chose a highly convergent synthetic approach applying solution phase peptide coupling chemistry. Retrosynthetically, the cyclic pentapeptide was divided into the three diastereomeric tripeptides 10a–c and the stereochemically defined dipeptide building block 11 common to all target structures (Scheme 1). The latter can be derived of N-Boc-L-Val (12) and N-Boc-L-Leu (13). For the preparation of 10a and 10c, again a joint precursor dipeptide 14 could be defined, which is divergently transformable into the desired tripeptides by attaching N-Boc-L-Leu (13) or L-Leu-OMe (15) to its N- or C-terminus, respectively. The third fragment, 10b, is accessible from D-Leu-OMe (18) and N-Boc-L-Leu-L-Leu-OMe (17). Dipeptides 14 and 17 are easily affordable from the commercially available amino acid precursors 13, 15 and 16.

The synthesis of the tri- (10a–c) and dipeptide (11) building blocks proceeded smoothly using standard peptide deprotection and coupling chemistry (Scheme 2): all peptide coupling steps were conducted using EDC and HOBt in CH₂Cl₂ with NEt₃ as the base. Mild N-deprotection was achieved employing in-situ generation of HCl in MeOH by addition of acetylchloride. Saponification of the ester protective groups was generally carried out by applying 2 N NaOH in MeOH. Using these conditions, peptides 10a and 10c were prepared in three steps each, starting with 16 via dipeptide 14 in an overall yield of 62% and 54%, respectively. Analogously, 10b was accessible from 13 in 71% overall yield. For the preparation of the universal dipeptide precursor 11, N-Boc-L-Leu (13) was first dimethylated using MeI and NaH, with subsequent N-Boc removal to give 19. This compound was coupled to 12 by using EDC and HOBt in CH₂Cl₂ with DIPEA as the base and saponified to furnish 11 in 55% overall yield.

Having all desired building blocks in hands, we started the assembly of the three lajollamide diastereomers (Scheme 3). After removal of the N-Boc group in 10a–c using 4N HCl in dioxane, coupling to 11 was again achieved with HOBt/EDC. The resulting fully protected pentapeptides 20a–c were saponified, activated by PFP-ester formation and directly cyclized by N-deprotection followed by in-situ addition of NEt₃, leading to compounds 7–9.

With the finalization of the synthesis of all anticipated lajollamide diastereomers 7–9, we started to compare the spectroscopic data of the authentic natural product with that of the synthetic material. To our surprise, however, none of the peptides 7–9 was identical to the natural compound. This was clearly evident from substantial differences in chemical shifts in both ¹H and ¹³C NMR spectra. For example, the chemical shifts of the amino acid α protons or the N-methyl groups differed significantly, as did all carbon chemical shifts, which was particularly apparent for the amide carbonyl signals (Figure 4).

This unexpected outcome of our synthetic endeavors led us to revisit the initial analytical data of the fungal metabolite. While no ambiguities were found in the interpretation of the NMR data, a small peak with a retention time of ca. 38 min. in the HPLC chromatogram of the full hydrolysate of the natural product drew our attention (see Figure 3). In fact, based on comparison with authentic standards, this signal turned out to correspond to N-Me-D-Leu. As the planar structure of lajollamide only contains one N-Me-Leu moiety, which was assigned to be L-configured due to the large peak at ca. 27 min in our stereochemical analysis, the occurrence of a fraction of N-Me-D-Leu clearly indicated a partial epimerization of this unit during peptide hydrolysis. This led to the assumption that a similar racemization process can also be expected for the three other Leu building blocks present in the molecule. This opened the possibility of three L-Leu units initially being present in the natural product, which, assuming an epimerization rate of approx. 30% for each of these residues, would result in a 2:1 ratio of L- to D-Leu in the analysis of the peptide hydrolysate, thus potentially explaining the spectroscopic differences of the natural product 1 when compared to the synthetic analogs 7–9. With these thoughts in mind, the preparation of a lajollamide diastereomer bearing L-configuration at all amino acids residues was undertaken. The synthesis of the respective all L-tripeptide precursor 10d followed the route depicted for the preparation of 10b as shown in Scheme 2, altered only by simply attaching L-Leu-OMe to the C-terminus of 17 in the second peptide coupling step. Compound 10d was obtained in overall 67% yield and further elaborated into the desired cyclic pentapeptide via all L-configured 20d according to the sequence depicted in Scheme 3. Due to the convergent nature of our synthetic approach, the preparation of the all L-configured lajollamide-type target molecule was thus easily achieved with relatively little additional experimental work. To our great relief, this time, the analytical data for the synthetic material perfectly matched those of the natural product 1 (Figure 5). We thus unambiguously established the absolute configuration in natural 1, henceforth named lajollamide A, to be all L. The three stereochmically altered, purely synthetic analogs of 1 were consequently defined as lajollamides B–D (7–9).

No cytotoxic or enzyme inhibitory activity was observed for compounds 1–9. In antibacterial assays, metabolites 2–5 were found to be inactive. However compounds 1, 7, 8 and 9 exhibited a weak antibacterial activity against Gram-positive bacteria at a concentration of 100 μM. The growth inhibition of Bacillus subtilis was 61%, 51%, 67%, and 41%, respectively. Pathogenic bacterial strains causing infectious diseases were also inhibited. Inhibition of Staphylococcus epidermidis was shown for compound 1 (30%), 7 (43%) and 8 (32%). Only compound 7 was active against methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, in agar diffusion assays at the 50 μg level, potent antibacterial and antifungal properties towards Escherichia coli (2.5 mm total inhibition), Bacillus megaterium (7 mm total inhibition), Mycotypha microspora (13.5 mm total inhibition), Eurotium rubrum (4 mm total inhibition), and Microbotryum violaceum (13 mm total inhibition) were demonstrated by 6, which in some cases surpassed the activity of the positive controls streptomycin, benzylpenicillin and miconazole.

Optical rotations were measured on a Jasco DIP 140 polarimeter. UV and IR spectra were obtained using Perkin-Elmer Lambda and Perkin-Elmer spectrum BX instruments, respectively. All NMR spectra of the isolated material were recorded on a Bruker Avance 300 DPX spectrometer. Spectra of the synthetic compounds were acquired on Bruker AM 300 and AM 400 spectrometers. Spectra were referenced to residual protonated solvent signals with resonances at δ_H/C 3.35/49.0 (CD₃OD) or 7.26/77.0 (CDCl₃), respectively. HR-EI and HR-ESI-MS data was recorded on Kratos MS50, a Bruker Daltonics micrOTOF-Q and a Thermo Finnigan MAT 95 XL. Semipreparative HPLC was carried out using a Waters system consisting of a degasser, a 600 pump, a 996 photodiode array detector, and a 717 plus autosampler in combination with a Waters fraction collector. Chiral HPLC was performed on a Merck-Hitachi system consisting of a L-6200 A pump, a L-4500 A photodiode array detector (PDA), and a D-6000 A interface. LC-MS measurements were obtained by employing an Applied Biosystems LC/MS system consisting of an Agilent 1100 HPLC system and an MDS Sciex API 2000 mass spectrometer equipped with an API-ESI source. All solvents used were distilled under argon and dried over 3 Å molecular sieves prior to use. All amino acids, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), pentafluorophenol (PFP) were purchased from Carbolution Chemicals. 1-Hydroxybenzotriazole hydrate (HOBt∙H₂O) was purchased from Aldrich, triethylamine (NEt₃) from Grüssing GmbH and methyl iodide from Merck. Sodium hydride and 4N HCl/dioxane were delivered from Acros Organics. All chemicals were used without further purification. For thin layer chromatography TLC Silica gel 60 F254 from Merck was used. The dyeing reagent consisted of ninhydrin in ethanol. For column chromatography, silica gel was purchased from Merck.

A piece of an unidentified decaying green alga, floating in the water column at a depth of 20 feet was collected by scuba in 2005 at La Jolla shore, San Diego, USA. Following transport in sterile seawater to Germany, the fungal strain #763 was isolated among other fungi from the algal sample, freed from competing microorganisms and other contaminants as previously described [23,24,25] and deposited at the fungal culture collection of the Institute for Pharmaceutical Biology, University of Bonn, Germany. The fungus was identified as Asteromyces cruciatus Moreau et Moreau ex Hennebert 1962.

The OSMAC approach was based on cultures of the fungus in media derived from the basic components of the Czapek-Dox broth [26,27]: NaNO₃ (3 g/L), FeSO₄ (0.01 g/L), KCl (0.5 g/L), MgSO₄ (0.5 g/mL), K₂HPO₄ (1.0 g/L), carbon source (30 g/L, originally sucrose, however, when not indicated, glucose was used) in artificial sea water (ASW). For the preparation of the culture medium, all components except the carbon source were mixed in 250 mL (1 L capacity, baffled Erlenmeyer flasks) of ASW and the mixtures were autoclaved (121 °C, 20 min). Subsequently, 20 mL of 40% (m/V) carbon source were added by sterile filtration to the medium, followed by inoculation with small pieces of seed culture. Small agar pieces (1 × 1 cm) of a 12–25-day-old culture of A. cruciatus grown on biomalt salt agar medium served as seed cultures. In order to find culture conditions conducive to production of new metabolites, two replicate cultures of A. cruciatus were grown in each of 14 different culture conditions: (#1) Czapek-Dox medium with shaking (#2) Czapek-Dox medium without shaking for generation of low O₂ levels [28], (#3) dixenic cultivation [29] with 10 mL of the bacterium Pseudomonas fluorescens Pf-5 [30], added on day 6, (#4) Czapek-Dox medium containing 20 g/L Amberlite XAD-16, (#5) Czapek-Dox medium supplemented with 1 mL/L cofactor solution consisting of MnCl₂·4H₂O (100 mg), CoCl₂ (20 mg), CuSO₄ (10 mg), Na₂MoO₄·2H₂O (10 mg), ZnCl₂ (20 mg), LiCl (5 mg), SnCl₂·2H₂O (5 mg), H₃BO₃ (10 mg), K₃BO₃ (10 mg), KBr (20 mg), KI (20 mg), EDTA-Na⁺ (5.8 mg) in 1 L of distilled water [28], (#6) Czapek-Dox supplemented with 4.6 mg/L CdCl₂ as polyketide inducer [31], (#7) Czapek-Dox medium, cultivated from day 10 to 13 under elevated UV-B light exposition, (#8) Czapek-Dox medium, cultivated from day 8–10 at 35 °C [28], (#9) Czapek-Dox medium, cultivated with sub-inhibitory concentrations of 0.4 mL/L cycloheximide [32,33], (#10) Czapek-Dox medium, supplemented with 8 mL/L toluene, (#11) Czapek-Dox without NaNO₃, containing 1.0 g/L Arg, 2.5 g/L Asn and 1.5 g/L Glu [28], (#12) Czapek-Dox with reduced levels of K₂HPO₄ (228.2 mg/L) for phosphate repression [28], (#13) Czapek-Dox medium without FeSO₄ for induction of siderophores, (#14) Czapek-Dox medium with sucrose instead of glucose as carbon source [28]. If not stated otherwise, the cultures were grown with shaking (120 rpm) at 24 °C. The broth was harvested at 10 days after inoculation. The culture broths of each fermentation were extracted separately with EtOAc (2 × 250 mL), and evaporated under reduced pressure to afford dark brown semisolids, ranging from 0.3 to 47.2 mg. In the case of culture condition #4, additionally the medium polarity compounds absorbed by the XAD resin material were eluted by shaking, first for 1 h in 200 mL EtOAc, and subsequently for 1 h in 200 mL acetone. All resulting three organic crude extracts of cultivation experiment #4 were combined. The resultant crude extracts of each experiment were dissolved in methanol to a final concentration of 5 mg/mL, and 10 μL was profiled by LC/MS using a 2 mM NH₄OAc buffered MeOH/H₂O gradient, increasing the MeOH portion from 10% to 100% over 20 min and holding it at 100% MeOH for additional 10 min (Macherey-Nagel C₁₈ Nucleodur 100-5 column, 2 × 125 mm, 5 μm column; 0.25 mL/min flow rate, with total ion current and photodiode array monitoring at 200–400 nm).

The fungus was cultivated for ten days in five 5 L-Erlenmeyer flasks, each containing 1.5 L of liquid media (=7.5 L). The liquid medium consisted of Czapek-Dox medium with glucose (30 g/L) as carbon source which was either supplemented with co-factors or contained solely the amino acids Arg, Asn and Glu as nitrogen source instead of NaNO₃. Flasks were shaken on a rotary shaker incubator at 24 °C and 120 rpm. On day 10, the cultivation medium and mycelium of each experiment was extracted with EtOAc (2 × 7.5 L). After evaporation of the organic solvent, both crude extracts (medium with trace elements: 126 mg; medium with an altered nitrogen source: 98 mg) were re-suspended separately in MeOH and filtered through reversed phase silica gel (RP-SPE cartridge, 100 mg). Separation of each filtrate by RP-HPLC analysis using a linear gradient of 40:60–100:0 MeOH-H₂O over a period of 54 min (column: Knauer Eurospher-100-C₁₈; 8 × 250 mm, 5 μm; 2 mL/min; DAD detection) yielded collectively a compound (amount from trace element medium/amount from altered nitrogen source) 1 (18.6/7.1 mg), 2 (0.9/0.7 mg), 3 (1.6/3.3 mg), 4 (19.8/19.4 mg), 5 (1.8/0.9 mg) and 6 (1.0/3.2 mg).

lajollamide A (1): yellowish glass; [α]_D²⁴ −80 (c 0.17, MeOH); UV/VIS (MeOH) λ_max (ε): 204 (12,600); IR (ATR): 3284, 2956, 1633, 1530 cm⁻¹; HR-ESI-MS m/z 566.4280 [M + H]⁺ (calc. for C₃₀H₅₆N₅O₅, 566.4276, Δ +0.7 ppm); ¹H and ¹³C NMR data in CDCl₃, see Table 1.

regiolone, syn. (−)-isosclerone (2): Colorless film; [α] _D²⁵ −16 (c 0.02, MeOH), {lit. [21] [α]_D −68 (c 0.05, MeOH)}; UV/VIS (MeOH) λ_max (ε): 212 (15,800), 262 (9900), 324 (4200); ¹H NMR (300 MHz, CDCl₃) δ_H ppm: 1.87 (1H, brs, OH-4), 2.19 (1H, m, H-3a), 2.34 (1H, m, H-3b), 2.65 (1H, m, H-2a), 3.01 (1H, m, H-2b), 4.92 (1H, m, H-4), 6.93 (1H, d, 8.5 Hz, H-7), 7.02 (1H, d, 7.4 Hz, H-5), 7.50 (1H, dd, 7.4, 8.5 Hz, H-6), 12.43 (1H, s, OH-8); ¹³C NMR (75 MHz, CDCl₃) δ_C ppm: 31.2 (CH₂, C-3), 34.6 (CH₂, C-2), 67.7 (CH, C-4), 115.2 (qC, C-8a), 117.3 (CH, C-5), 117.8 (CH, C-7), 137.0 (CH, C-6), 145.8 (qC, C-4a), 162.7 (qC, C-8), 204.2 (qC, C-1); LR-ESI-MS m/z 177.1 [M − H]⁻.

(3R,6R)-hyalodendrin, syn. A26771A (3): Yellowish film; [α] _D²⁴ −71 (c 0.15, CHCl₃) {lit. [16] [α]_D²⁷ −88 (c 0.15, MeOH)}; ¹H NMR (300 MHz, CDCl₃) δ_H ppm: 2.97 (3H, s, ⁴NCH₃), 3.21 (3H, s, ¹NCH₃), 3.61 (1H, d, 16 Hz, H-8a), 4.08 (1H, d, 16 Hz, H-8b), 4.35 (2H, m, H₂-7), 7.30 (5H, m, H-10, H-11, H-12, H-13, H-14); ¹³C NMR (75 MHz, CDCl₃) δ_C ppm: 27.5 (⁴NCH₃), 28.6 (¹NCH₃), 36.8 (CH₂, C-8), 61.2 (CH₂, C-7), 75.2 (qC, C-6), 75.7 (qC, C-3), 127.4 (CH, C-12), 128.7 (2 × CH, H-10, H-14), 129.0 (2 × CH, H-11, H-13), 134.0 (qC, C-9), 165.5 (qC, C-2), 166.9 (qC, C-5); HR-ESI-MS m/z 347.0491 [M + Na]⁺ (calc. for C₁₄H₁₆N₂NaO₃S₂, 347.0495, Δ −1.2 ppm).

(3R,6R)-gliovictin, syn. A26771E, (3R,6R)-bisdethiodi(methylthio)hyalodendrin (4): Colorless film; [α]_D²⁴ −58 (c 0.88, CHCl₃) {lit. [17] [α]_D²⁵ −65 (CHCl₃); [9] [α]_D −62 (c 1.88, CHCl₃); [16] [α]_D²⁷ −47 (c 0.13, CH₃OH); [18] [α]_D²⁴ −43 (c 0.24, CHCl₃)}; ¹H NMR (300 MHz, CDCl₃) δ_H ppm: 1.24 (1H, t, 6.9 Hz, OH-7), 2.13 (3H, s, ⁶SCH₃), 2.30 (3H, s, ³SCH₃), 3.02 (3H, s, ⁴NCH₃), 3.10 (1H, dd, 6.9 and 11.8 Hz, H-7a), 3.13 (1H, d, 13.8 Hz, H-8a), 3.31 (3H, s, ¹NCH₃), 3.71 (1H, d, 13.8 Hz, H-8b), 3.85 (1H, dd, 6.9 and 11.8 Hz, H-7b), 7.09–7.27 (5H, m, H-10, H-11, H-12, H-13, H-14). ¹³C NMR (75 MHz, CDCl₃) δ_C ppm: 13.4 (⁶SCH₃), 14.3 (³SCH₃), 29.3 (¹NCH₃), 31.0 (⁴NCH₃), 42.3 (CH₂,C-8), 64.3 (CH₂, C-7), 71.5 (qC, C-6), 73.7 (qC, C-3), 127.9 (CH-12), 128.7 (2 × CH, C-11 and C-13), 130.1 (2 × CH, C-10 and C-14), 134.1 (qC, C-9), 165.3 (qC, C-2), 165.5 (qC, C-5); HR-ESI-MS m/z 377.0965 [M + Na]⁺ (calc. for C₁₆H₂₂N₂NaO₃S₂, 377.0970, Δ −1.3 ppm).

(3R,6R)-¹N-norgliovictin, syn. bisdethiodi(methylthio)-1-demethylhyalodendrin (5): Colorless film; [α]_D²⁴ −37 (c 0.05, CH₃OH) {lit. [18] [α] _D²⁴ −63 (c 0.30, CHCl₃)}; ¹H NMR (300 MHz, CDCl₃) δ_H ppm: 1.71 (1H, brs, OH-7); 2.19 (3H, s, ³SCH₃), 2.22 (3H, s, ⁶SCH₃), 2.71 (1H, d, 11.6 Hz, H-7a), 3.15 (1H, d, 13.8 Hz, H-8a), 3.28 (3H, s, ⁴NCH₃) , 3.41 (1H, d, 11.6 Hz, H-7b), 3.53 (1H, d, 13.8 Hz, H-8b), 6.36 (1H, brs, NH-1), 7.09–7.32 (5H, m, H-10, H-11, H-12, H-13, H-14); ¹³C NMR (75 MHz, CDCl₃) δ_C ppm: 13.5 (⁶SCH₃), 14.4 (³SCH₃), 30.3 (⁴NCH₃), 42.3 (CH₂, C-8), 64.8 (qC, C-6), 65.2 (CH₂, C-7), 75.7 (qC, C-3), 128.0 (CH, C-12), 128.8 (2 × CH, C-10, C-14), 130.0 (2 × CH, C-11, C-13), 133.7 (qC, C-9), 164.9 (2 × qC, C-2, C-5); HR-ESI-MS m/z 363.0818 [M + Na]⁺ (calc. for C₁₅H₂₀N₂NaO₃S₂, 363.0813, Δ +1.4 ppm).

(3R,6R)-bis-N-norgliovictin (6): [α] _D²⁴ −8 (c 0.1, CH₃OH), {lit. [20] [α]_D −32 (c 0.1, CH₃OH); [18] [α]_D²⁵ −34 (c 0.30, dioxane)}; ¹H NMR (300 MHz, CH₃OD) δ_H ppm: 2.23 (3H, s, ⁶SCH₃), 2.37 (3H, s, ³SCH₃), 3.05 (H, d, 11.5 Hz, H-7a), 3.07 (1H, d, 13.3 Hz, H-8a), 3.40 (H, d, 11.5 Hz, H-7b), 3.62 (1H, d, 13.3 Hz, H-8b), 7.29 (5H, m, H-10, H-11, H-12, H-13, H-14); ¹³C NMR (75 MHz, CH₃OD) δ_C ppm: 13.5 (³SCH₃ or ⁶SCH₃), 13.9 (³SCH₃ or ⁶SCH₃), 46.0 (CH₂, C-8), 66.5 (CH₂, C-7), 67.8 (qC, C-3 or C-6), 68.8 (qC, C-3 or C-6), 128.4 (CH, C-12), 129.4 (2 × CH, C-11, C-13), 131.9 (2 × CH, C-10, C-14), 135.7 (qC, C-9), 167.8 (qC, C2 or C5), 168.2 (qC, C-2 or C-5); HR-ESI-MS m/z 349.0651 [M + Na]⁺ (calc. for C₁₄H₁₈N₂NaO₃S₂, 349.0657, Δ −1.7 ppm).

Lajollamide A (1.3 mg) was hydrolyzed with 6 N HCl in a sealed vial at 108 °C for 19 h. Excess aqueous HCl was removed under vacuum and the resulting hydrolysate was analyzed by chiral HPLC employing a Phenomenex Chirex 3126 (D) column (4.6 × 250 mm) eluting with 2 mM CuSO₄/MeCN (95:5) at a flow of 0.8 mL/min, monitoring at 240 nm. Comparison with commercially available standards suggested the presence of L-Val, N-Me-L-Leu, 2 × L-Leu and 1 × D-Leu. The hydrolysate was chromatographed alone and co-injected with standards to confirm assignments.

The antimicrobial activities of compounds 1, 7, 8 and 9 were determined as previously described [34] using the following indicator strains: Candida albicans (DSM 1386), Bacillus subtilis (DSM 347), E. coli K12 (DSM 498), Xanthomonas campestris (DSM 2405), Staphylococcus epidermidis (DSM 20044) and methicillin-resistant Staphylococcusaureus (MRSA) (DSM 18827). Furthermore, the compounds were tested for inhibition of phosphodiesterase (PDE4-4B2) and cytotoxicity to HepG2(human hepatocellular carcinoma) cells. The determination of the acetylcholinesterase inhibitory activity was performed according to Ohlendorf et al. (2012) [35]. Glycogen synthase kinase-3β inhibition was determined as described by Baki et al. (2007) [36]. The activity of compounds 4 and 6 was tested in agar diffusion assays against the bacteria Escherichiacoli and Bacillus megaterium, the fungi Microbotryum violoaceum, Eurotium repens and Mycothypha microsporum, and the green microalga Chlorella fusca, as previously described [37]. Benzylpenicillin (6 mm growth inhibition towards Bacillus megaterium) and streptomycin (5 mm and 10 mm total inhibition towards Escherichia coli and Bacillus megaterium, respectively) were used as positive control for the antibacterial agar diffusion assays. In antifungal and antialgal assays, miconazole served as a positive control, resulting in total inhibition zones of 6–10 mm.