Colletotrichum, isolated from leaves of the tropical palm species Astrocaryum sciophilum collected in pristine forests of French Guiana, was investigated. The collection was first classified based on protein fingerprints acquired by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) correlated with cytotoxicity. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) data from ethyl acetate extracts were acquired and processed to generate a massive molecular network (MN) using the MetGem software. From five Colletotrichum strains producing cytotoxic specialized metabolites, we predicted the occurrence of peptide and cytochalasin analogues in four of them by MN, including a similar ion clusters in the MN algorithm provided by MetGem software. Chemoinformatics predictions were fully confirmed after isolation of three pentacyclopeptides , cyclo(Phe-Leu-Leu-Leu-Leu) and cyclo(Phe-Leu-Leu-Leu-Ile)) and two cytochalasins  exhibiting cytotoxicity at the micromolar concentration. Finally, the chemical study of the last active cytotoxic strain BSNB-0583 led to the isolation of four colletamides bearing an identical decadienamide chain.The chemical diversity of biologically active fungal strains from 42  Microorganisms virtually occupy every living habitat on earth, and endophytes have long been thought to be a prime source of original chemical compounds2. These symbiotic microorganisms are ubiquitous in plants, living inside the tissues of a host without causing any apparent harm3. Tropical tree leaves are considered as hotspot for endophytes4. Because of their high diversity, it has been clearly established that they can serve as reservoirs of new bioactive specialized metabolites5. Indeed, endophytic strains have been shown to contribute to plant defences by preventing herbivory6 and invasion from superficial pathogens3. Therefore, a significant proportion of endophytic extracts are thought to be cytotoxic and/or antimicrobial.In the field of bioactive natural products research, the chemical diversity of microorganisms is increasingly exploredColletotrichum is represented by a large number of species widely distributed in the tropics but also in temperate regions7. Some Colletotrichum are predominant in living plant tissues as symptomless endophytes9. The presence of Colletotrichum tofieldiae in roots has been shown to enhance plant fitness10, but other species are pathogenic. Indeed, Colletotrichum spp. are also among the most important groups of plant pathogens11. Several studies have sought to advance the systematic classification of Colletotrichum and to propose better identification of the species using DNA sequencing methods13. Among them, Douanla-Meli and Unger used high-throughput sequencing on selected markers for 454 Colletotrichum spp. isolates to identify strains at the species level14. Nevertheless, there is a growing interest in developing a fast and efficient method to identify and classify strains15 to reduce the required cost and time. Over the last decade, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been implemented in hospital centres as the method of choice for microbiology diagnosis. This method compares microbial peptide/protein fingerprint against reference databases16. To improve classification when using large microorganism collections, machine learning technique, such as the Aristotle Classifier, has been introduced in the literature17. Moreover, the identification accuracy is increased by combining the data from MALDI-TOF MS and FTIR spectroscopy and hierarchical clustering analysis18. Alternatives to MALDI profiling has been recently introduced by the group of Takats demonstrating that automated laser-assisted rapid evaporative ionisation mass spectrometry (LAREIMS) platform can chemically screen over 450 yeast colonies in under 4 h, while simultaneously generating recoverable glycerol stocks of each colony in real-time19. First dedicated to the identification of human pathogenic or opportunistic yeasts and bacteria, some studies recently developed spectral databases for a more systematic identification of filamentous fungi21. Nevertheless, these databases still focus on clinical isolates. Few studies reported MALDI-TOF MS as a tool for strain identification for environmental microorganisms22 and demonstrated the correlation of MALDI classification of microorganism together with production of specialized metabolites23.Among fungal endophytes, the genus Colletotrichum strains from healthy leaves of a tropical palm species, Astrocaryum sciophilum Miq. (Pulle), Arecaceae were studied. A. sciophilum is a long-lived palm that grows in the understory of tropical forests of the Guiana Shield of Amazonia, and can be locally abundant24. A methodology was developed to acquire protein MS profile of Colletotrichum strains by MALDI-TOF MS to decipher the variability of our collection. Colletotrichum strains with similar phenotypes were recognized thereby reducing the number of strains of interest. In parallel, strains were extracted with ethyl acetate (EtOAc) and each extract was tested on cell viability bioassays for cytotoxic activity. Five unique cytotoxic Colletotrichum extracts were further investigated by the molecular network approach27 and the new t-SNE (t-distributed stochastic neighbor embedding) algorithm provided by MetGem software28. This study led to the isolation of known metabolites  and of an unprecedented series of new metabolites designated as colletamides.Forty-two endophytic and cultivable Colletotrichum extracts were produced from endophytes isolated on five Astrocaryum sciophilum palm specimens. We then quantified the cell viability of the EtOAc extract on MRC-5 cells lines . First, it confirmed that an accurate identification at the species level remained impossible based on the ITS region sequencing only due to highly similar sequences on this particular genomic region.Forty-two es Table . Among tBI Table . Second Second, there is no obvious correlation between the ITS-based phylogeny and the biological activity profile. Moreover, strains in the same clade were not necessarily morphologically similar . Given this level of variability, and in order to search for bioactive metabolites in a large culture collection, we decided to generate a classification that is more reflective of strain secondary metabolism rather than taxonomical classification.Colletotrichum isolates share the same protein profiles whereas fifteen strains are characterized by a unique one . Considering previous reports on strain identification by MALDI-TOF MS and the specificity of protein fingerprints among species22, we assumed that each protein fingerprint refers to a Operational Chemically Unit  within the genus. This expectation has been confirmed through the classification by MALDI-TOF protein profiling and hierarchical clustering data analyses of the complete collection of environmental microorganisms at CNRS-ICSN, containing more than 1000 unique strains. Thus, from the 20 different protein fingerprints drawn from the 42 Colletotrichum isolates , our collection can be realigned into 18 different chemotypes OCU. These OCUs (noted from Colletotrichum sp.1 to Colletotrichum sp.18) are classified following a hierarchical clustering based on their protein fingerprint  was generated from the liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) data acquired from the EtOAc extracts of our 42 Colletotrichum sp.2, sp.14 and sp.11 displayed noticeably specific ion clusters , m/z 72.081  and m/z 120.081  were unambiguously attributed. Structures of isolated cyclopeptides cyclo (1), cyclo(Phe-Leu-Leu-Leu-Leu) (2) and cyclo(Phe-Leu-Leu-Leu-Ile) (3) were then refined by NMR  for the first one, and one oxidized methionine, one valine and three leucines (or isoleucines) for the second one. Moreover, other peptides composed of various number of phenylalanines, leucines/isoleucines and valines were annotated based on the HRMS and the MS/MS fragmentation. Finally, nodes belonging to peptides bearing non-proteinogenic amino acids were annotated based on unusual m/z values of immonium ions and differences between b and y-ion products. These cyclopeptides have not been isolated and formally identified because of their low abundance in the strain extract.The relative configuration of cyclo(Phe-Leu-Leu-Leu-Ile) was determined by X-ray crystallography . By analysing the clusters of nodes related to these cyclopeptides Fig. , we obseColletotrichum sp.12 and sp.1 . Assignment of the two related clusters was performed after cultivation of the strain BSNB-0536 (OCU Colletotrichum sp.12) on the large scale and isolation of cytochalasin D (4), cytochalasin C (5), and the cyclohexadepsipeptide hirsutatin A (6). The spectroscopic data for these compounds were identical to those provided in the literature50. These results demonstrate the interest of the t-SNE prediction module for propagating nodes annotation.The MN approach was unable to annotate metabolites clusters specifically related to OCU p.1 Fig.  regroupsn E Fig. . After sColletotrichum sp.2 (BSNB-0583) contained specialized metabolites specific to this strain distributed into two related clusters. Targeted isolation of the strain-specific metabolites provided 4 compounds (7–10) in pure form . According to NRM data analysis (see supplementary information), compounds 7 was therefore analogous to aranorosin31. It has been referenced in a patent before as a potential inhibitor of the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase32. However, the source organism was claimed to be an unidentified fungus , the stereochemistry was not specified and the only analytical data provided was a list of 13C NMR chemical shifts. This list suggests that their compound C and our compound 7 may have been identical. We determined the relative configuration in compound 7 combining NOE NMR data DP4 approach33 (see supplementary information). A very high probability (99.5%) was obtained for the relative configuration 2′S*,4′S*,5′S*,8′S*-7 . The 2′S,4′S,5′S,8′S-7 absolute configuration of 7, was deduced by comparison of the predicted and experimental ECD spectra . Compound 7 was named colletamide A.Finally, according to the classical and t-SNE MN, the extract of OCU E,8E)-N-[(R)-1--4-hydroxy-5-oxotetrahydrofuran-2-yl)-2-phenylethyl]deca-2,8-dienamide (named colletamide B), 2--3-(2-hydroxyphenyl)propionic acid (named Colletamide C) and 4--3-hydroxy-5-phenylpentanoic acid (named colletamide D), respectively .Using the same structural elucidation process, compounds 8, 9 and 10 were identified as . This novel series of decadienamides was therefore not responsible for the activity of OCU Colletotrichum sp.2 extract, in agreement with the loss of activity of the second EtOAc extract of BSNB-0583 (OCU Colletotrichum sp.2). Indeed, second extract (BSNB-0583) exhibited 63 ± 4% of viability on MRC-5 cells (10 µg/mL), therefore showing a reduced cytotoxicity compared to the initial extract (32 ± 1%). Variation in metabolite production is a common phenomenon already described in the literature1. Comparison of the LC–MS profiles between the cytotoxic and the non-cytotoxic extract shows a difference in intensity for the ion at m/z 346.2738. This ion belongs to a cluster of nodes specific to BSNB-0583 strain in the MN and this could be responsible for the altered biological activity of the extract. Curiously, the metabolite at m/z 346.2738 has not been recovered from any fractions after the purification process suggesting that it could be sensitive to purification conditions. Nevertheless, the chemical formula C22H35NO2+ could be attributed to its protonated species [M + H]+ . Moreover, MS/MS spectrum of the precursor ion at m/z 346.2738 shows a fragment ion at m/z 107.0497 (C7H7O) and complementary fragment ion at m/z 240.2327. This typical pattern has been previously described for molecules bearing a para-substituted phenol moiety such as vitroprocine derivatives, for example34.Colletamide A-D did not exhibit any cytotoxicity  have already been isolated from bacteria of the Pantoea genus54. All of these compounds are known to have antibacterial activity but no cytotoxicity has been reported in the literature.Compound Colletotrichum strains has been chemically investigated by up-to-date analytical tools, i.e. MALDI-TOF profiling and molecular networking dereplication, to identify secondary cytotoxic metabolites. We found that Colletotrichum strains produce specific specialized metabolites depending on the Operational Chemically Unit OCU highlighted by the MALDI-TOF MS clustering. This has finally led to the isolation and complete de novo characterisation of 10 Colletotrichum specialised metabolites.The newly generated collection of 42 endophytic Cytochalasins and peptides have been isolated in three cytotoxic extracts , but chemical diversity remains unknown for the 8 remaining cytotoxic extracts. At this point, this lack of information can be due to incomplete MS/MS databases or to the fact that extracts are actually composed of currently unknown specialized metabolites.Colletotrichum sp. 4 is producing metabolites related to those in Colletotrichum sp.11, especially cytochalasin analogues. Indeed, the chemical study of one strain of OCU Colletotrichum sp.4 (BSNB-0536) has led to the isolation of cytochalasin metabolites.According to t-SNE representation of the MS/MS data allowing to evaluate inter-cluster distance, we demonstrate that OCU Colletotrichum sp.2 (BSNB-0583) was found to be the strain with the highest probability to isolate new and bioactive metabolites. Chemical study of the extract lead to the isolation of a new series of metabolites containing a decadienamide chain. These metabolites are named colletamide A-D and seems to have, has their known analogues, antimicrobial activities. They are the major components of the extract but minor ones also exist in this series, according to the MN.Finally, OCU Colletotrichum by MALDI-TOF MS fingerprinting of genetically close isolates and observed their chemical diversity by Molecular Networking. Application of this approach for investigating many strains exhibiting various biological activities could accelerate the discovery of novel bioactive metabolites.The methodology presented in this study targets promising bioactive strains: we distinguished different Operational Chemically Unit of 3OD or (CD3)2SO on a Bruker 500 MHz spectrometer or a Bruker 600 MHz spectrometer equipped with a 2 mm invers detection probe. Chemical shifts (δ) are reported in ppm based on TMS signal. Coupling constants (J) are in hertz. High-resolution ESI-TOF–MS measurements were performed using a Waters Acquity UPLC system with column bypass coupled to a Waters Micromass LCT Premier time-of-flight mass spectrometer equipped with an electrospray interface (ESI). Flash chromatography was performed on a Grace Reveleris system equipped with a 120 g C18 column or a 40 g C18 column55. Flow rate was 80 mL/min or 40 mL/min respectively and detection was performed with dual UV at 210 and 270 nm and ELSD. Analytical and preparative HPLCs were conducted on a Gilson system equipped with a 322 pumping device, a GX-271 fraction collector, a 171 diode array detector and a prepELSII detector electrospray nebulizer. Wavelenghts for UV absorbance were 210, 220, 254, 270 and 320 nm. Columns used for analytical and preparative experiments included a Phenomenex Luna C18 5 µm 4.6 × 250 mm and a Phenomenex Luna C18 5 µm 21.2 × 250 mm. Flow rate was 1 mL/min and 21 mL/min respectively for analytical and preparative experiments. All solvents were HPLC grade and HPLC-grade water was obtained with a Milli-Q water purification system . For UHPLC-HRMS analysis, chromatographic separation was performed on an Acquity UHPLC system interfaced to a Q-Exactive Plus mass spectrometer  with a Waters BEH C18 50 × 2.1 mm × 1.7 μm analytical column, using a heated electrospray ionization (HESI-II) source. Detection was made by an Acquity UPLC photodiode array detector from 200 to 500 nm. The optimized HESI-II parameters were as follows: source voltage, 3.5 kV (pos); sheath gas flow rate (N2), 55 units; auxiliary gas flow rate, 15 units; spare gas flow rate, 3.0; capillary temperature, 275.00 °C (pos), S-Lens RF Level, 45. The data-dependent MS/MS events were performed on the four most intense ions detected in full scan MS (Top3 experiment). The MS/MS isolation window width was 1 Da, and the normalized collision energy (NCE) was set to 35 units. In data-dependent MS/MS experiments, full scans were acquired at a resolution of 35 000 FWHM (at m/z 200) and MS/MS scans at 17 500 FWHM both with a maximum injection time of 50 ms. After being acquired in a MS/MS scan, parent ions were placed in a dynamic exclusion list for 2.0 s.NMR spectra were recorded in CDMALDI-TOF MS analyses were performed using an UltrafleXtreme mass spectrometer . Acquisitions were performed in linear positive ion mode. The laser intensity was set just above the ion generation threshold to obtain peaks with the highest possible signal-to-noise (S/N) ratio without significant peak broadening. The mass spectrometer was externally calibrated using a mixture of proteins . All data were processed using the program FlexAnalysis .Astrocaryum sciophilum palm trees were sampled in French Guiana at Piste de Saint-Elie, Sinnamary, in July 2014. The general procedures adopted for isolation of the microorganisms followed the methodology described by Casella et al.56. After collection, the plant material was washed with sterile water and surface sterilised by sequential immersion in 70% aqueous ethanol (3 min), followed by 5% aqueous sodium hypochlorite (5 min) and finally by 70% aqueous ethanol (1 min). Leaves were cut into small pieces (1–0.5 cm2) which were placed on Potato Dextrose Agar medium  in Petri dishes at 28 °C (4–5 parts per Petri dishes). Each individual hyphal tip of emerging fungi was removed and placed on a sterile PDA culture medium in 10 cm Petri dishes. The leaf fragments were cultured for a maximum of one month. All isolated endophytic strains have been deposited at the ICSN/CNRS Strain library France. Strains are maintained in triplicate in 2 ml Eppendorf tubes containing 1 ml of a solution of glycerol and water (1:1) at − 80 °C.Colletotrichum gloeosporioides (MH864569), Colletotrichum sp. strain LGMF1580 (MG976389), C. gloeosporioides (MH865232), C. gloeosporioides (MH866036), C. fragariae (MH864138), C. vietnamense (MH863700), Colletotrichum sp. strain AHGB10 (MH267867), C. gloeosporioides (KT004429), C. gloeosporioides (KR995714), Colletotrichum sp. strain AHGB15 (MH267881), C. horii (KR995727), C. horii (LC186042), C. karstii (MG602059), C. fructicola (MH865616), C. siamense (MG807423), C. queenslandicum (JX010184)). Monilochaetes infuscans (JQ005780) was used for the output group. Sequences were aligned in MAFFT version 7 57. The resulting alignment was saved into Phylip4 format. A phylogenetic tree was constructed by maximum likelihood (ML) inference on the CIPRES server (https://www.phylo.org)58. The ML analysis was performed using RaxML-HPC2 on XSEDE (8.2.10). Branch support was evaluated by a bootstrapping method with 1000 replicates. The final phylogenetic tree was visualized using FigTree version 1.4.4. The sequence data have been submitted to GenBank with an accession number for each strain . ITS sequences were blasted on NCBI GenBank (accessed 2018-03-12) to search for closest strains (See supplementary Table 20. After a culture of 72 h at 28 °C on PDA (Potato Dextrose Agar) medium, 10 mg of each isolate was suspended in 300 µL of water and 900 µL of 100% ethanol then centrifuged at 13,000 rpm. The supernatant is discarded and the pellet was resuspended in 40 µL of formic acid and vortexed. An equal volume of 100% acetonitrile was then added and the solution mixed. After centrifugation at 13,000 rpm, the supernatant was stocked at − 20 °C for maximum one week and used for MALDI measurement. For MALDI analysis, 1 µL of protein extract is spotted on the sample target, directly followed by 1 µL of α-cyano-4-hydroxycinnamic acid (CHCA) matrix (50 mg/mL in CH3CN/H2O (50/50), acidified with 0.1% trifluoroacetic acid).Protein extraction were performed in triplicate according to the acetonitrile/formic acid extraction procedure of Bruker MALDI Biotyper59. Each MALDI TOF MS spectra was preprocessed using the following parameters: intensity was square-root transformed, smoothing was done using moving average and baseline correction using TopHat. Peak detection was set to Mass SpecWavelet with a minimum peak intensity chosen in order to detect only the major peak. Peak matching on peak lists was done with a tolerance of 700 ppm for intra-sample matching. Clustering analysis was generated using Jaccard distance function and conversion values of presence.A hierarchical cluster tree was constructed using the Mass-up software2O, dried with anhydrous solid Na2SO4 and evaporated using a rotary evaporator under reduced pressure to yield a crude mixture.Each strain was cultivated at 28 °C in 10 Petri dishes (10 cm diameter) of PDA (Potato Dextrose Agar) media. Then, culture media was extracted with ethyl acetate (EtOAc) at room temperature during 24 h. The organic phase was removed via filtration, washed three times with HColletotrichum strain was profile on a UPLC-MS/MS to acquire mass data for the implementation of molecular network55. Samples were pre-treated by Solid phase extraction using Discovery® SPE 96 well plate (bed wt 100 mg/well) and plate prep vacuum Manifold. Each extract was then dissolved in 500 µL of 5% milliQ water in HPLC methanol and loaded on the cartridge bed. Elutes were injected at a concentration of 4 mg/mL for Orbitrap analysis. For UHPLC-HRMS analysis, chromatographic separation was performed on an Acquity UHPLC system interfaced to a Q- Exactive Plus mass spectrometer , using a heated electrospray ionization (HESI-II) source. Thermo Scientific Xcalibur 2.1 software was used for instrument control and data analysis.Each EtOAc extract of 3CN and H2O (0.1% formic acid modifier) was made from 5 to 100% of acetonitrile over 7 min following by an isocratic gradient of 100% CH3CN for 1 min. In positive ion mode, the di-isooctyl phthalate C24H38O4 [M + H]+ ion (m/z 391.28429) was used as an internal lock mass. The mass analyzer was calibrated using a mixture of caffeine, methionine−arginine−phenylalanine−alanine−acetate (MRFA), sodium dodecyl sulfate, sodium taurocholate, and Ultramark 1621 in an acetonitrile/methanol/water solution containing 1% formic acid by direct injection.One µL aliquot of each sample was injected and eluted at 0.6 mL/min. The linear gradient between CHThe optimized HESI-II parameters were as follows: source voltage, 3.5 kV (pos); sheath gas flow rate (N2), 55 units; auxiliary gas flow rate, 15 units; spare gas flow rate, 3.0; capillary temperature, 275.00 °C (pos), S-Lens RF Level, 45. The mass analyzer was calibrated using a mixture of caffeine, methionine−arginine−phenylalanine−alanine−acetate (MRFA), sodium dodecyl sulfate, sodium taurocholate, and Ultramark 1621 in an acetonitrile/ methanol/water solution containing 1% formic acid by direct injection. The data-dependent MS/MS events were performed on the four most intense ions detected in full scan MS (Top3 experiment). The MS/MS isolation window width was 1 Da, and the normalized collision energy (NCE) was set to 35 units. In data-dependent MS/MS experiments, full scans were acquired at a resolution of 35 000 FWHM (at m/z 200) and MS/MS scans at 17 500 FWHM both with a maximum injection time of 50 ms. After being acquired in a MS/MS scan, parent ions were placed in a dynamic exclusion list for 2.0 s.61. Mass detection was realized with centroid mass detector with the noise level set to 1.0E5 for MS level set to all. The ADAP chromatogram builder62 was achieved using a minimum group size of scans of 5, minimum group intensity threshold of 1.0E5, minimum highest intensity of 1.0E5 and m/z tolerance of 0.002 or 5 ppm. Wavelets (ADAP) algorithm was used for chromatogram deconvolution with the following settings: S/N threshold of 10, intensity window SN, minimum feature height of 1000, coefficient area threshold of 100, peak duration range between 0.01 and 0.5 and the RT wavelet range between 0.001 and 0.05. The m/z and RT range for MS/MS scan pairing was set to 0.001 Da and 0.05 min respectively. Chromatograms were deisotoped using the isotopic peaks grouper algorithm with a m/z tolerance of 0.003 (5 ppm), a RT tolerance of 0.1 (absolute), a maximum charge of 2 and the representative isotope used was the most intense. Peak alignment was performed using the join aligner method: m/z tolerance of 0.001 or 5.0 ppm, weight for m/z of 0.001, RT tolerance of 0.3 min, weight for RT of 0.1. Adduct search  was conducted on the peak list with a RT tolerance set to 1.0 min and the maximum relative peak height at 50%. Adducts found were then removed from the peak list. Peak list was gap-filled with the peak finder module: intensity tolerance of 90%, m/z tolerance of 0.001 or 5.0 ppm and RT tolerance of 0.1 min. Peak list was exported for GNPS to create a mgf file (https://doi.org/10.5281/zenodo.3862573). Row ID, row m/z, row retention time and peak area was exported in an associated CSV file. After a first analysis of the generated MN, this peak list was reduced to the ions with m/z between 200 and 900 to diminish the data size.The complete procedure has been Raw files were converted into mzXML files using MSConvert software. Then mzXML files were processed using MZmine 2.3728 to give a network containing 15,261 nodes. Molecular network was generated using the following parameters: m/z tolerance set to 0.02, Minimum Matched Peaks set to 6, topK set to 10, Minimal Cosine score Value of 0.7 and Max. Connected Component Size of 100. Associated CSV file was then loaded. For the mapping process, relative quantification of each ion was represented as pie chart-diagrams, whose proportions were based on respective areas of the corresponding extracted ion chromatograph area (EIC). The spectra in the network were then searched for analogues against the spectral libraries available. The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.7 and at least 6 matched peaks. m/z Tolerance for the search of analogues was set to 100. For t-SNE output, cosine score threshold (0.7) was similar to molecular network view. The number of iterations, perplexity, learning-rate and early exaggeration parameters were set to 1000, 6, 200 and 12, respectively. These values were previously optimized on another complex set of data28.After the preprocessing of the LC–MS/MS data with MZmine 2.37, the output mgf file was then processed with MetGem software63.For a more advanced retreatment of the network, MN was also exported on Cytoscape 3.7.0 softwareStrains were cultivated during 15 days at 28 °C in 14 cm Petri dishes of PDA (Potato Dextrose Agar) media. Each strain was extracted three times consecutively with ethyl acetate (EtOAc) at room temperature. The combined organic solution was washed as previously.Colletotrichum sp. BSNB-0580 was conducted on 226 Petri dishes (diameter 14 cm) to yield 4.7 g of a brown crude extract. 4.5 g of this crude extract was fractionated by reverse flash chromatography on C18 column with a 5-min-step gradient of water mixed with an increasing proportion of acetonitrile . 6 fractions were generated based on the UV and ELSD detection and after spotting on TLC plates (in various mixture of acetonitrile and H2O): F1 , F2 , F3 , F4 , F5 , F6 . A step gradient of acetonitrile–methylene chloride  was conducted to generate 4 additional fractions based on the spot on TLC plates: F7 , F8 , F9  and F10 .The large-scale cultivation of 3CN/H2O 0.1% formic acid modifier for a duration of 15 min per run) to obtain 3 cyclopeptides in sufficient amount: cyclo- (1) , cyclo-(Phe-Leu-Leu-Leu-Leu) (2) , and cyclo-(Phe-Leu-Leu-Leu-Ile) (3) . The chromatogram was monitored by UV absorbance.F4 was selected for purification based on its cytotoxic activity and its HPLC profile. F4 was purified by preparative HPLC (isocratic elution 65% CHColletotrichum sp. BSNB-0536 was conducted on 220 Petri dishes (diameter 14 cm) to yield 5.4 g of a brown crude extract. 5.2 g of this crude extract was fractionated by reverse flash chromatography on C18 column with a 5-min-step gradient of water mixed with an increasing proportion of acetonitrile . 6 fractions were generated based on the UV and ELSD detection: F1 , F2 , F3 , F4 , F5 , F6 . A step gradient of acetonitrile–methylene chloride  was conducted to generate 2 additional fractions: F7  and F8 .The large-scale cultivation of 18 column with a 5-min-step gradient of water mixed with an increasing proportion of acetonitrile . 13 fractions were generated based on the UV and ELSD detection: F2-A , F2-B , F2-C , F2-D , F2-E , F2-F , F2-G , F2-H , F2-I , F2-J , F2-K , F2-L  et F2-M . A step gradient of acetonitrile–methylene chloride  was conducted to generate 2 additional fractions: F2-N  and F2-O .BSNB-0536-F2 was then again fractionated by reverse flash chromatography on C18 column with a linear gradient between water and acetonitrile (both solvent contained 0.1% formic acid): 60:40 for 5 min, 0:100 in 20 min, 0:100 for 5 min. Three molecules were generated based on the UV and ELSD detection: Cytochalasin D (4) , Cytochalasin C (5)  and Hirsutatin A (6) .138.2 mg of fraction BSNB-0536-F2-J was further purified by reverse flash chromatography on a 40 g CColletotrichum sp. BSNB-0583 was conducted on 232 Petri dishes (diameter 14 cm) to yield 1.5 g of a yellow crude extract. 1.4 g of this crude extract was fractionated by reverse flash chromatography on C18 column with a 5-min-step gradient of water mixed with an increasing proportion of acetonitrile . 12 fractions were generated based on the UV and ELSD detection: F1 , F2 , F3 , F4 , F5 , F6 , F7 , F8 , F9 , F10 , F11  and F12 . A step gradient of acetonitrile–methylene chloride  was conducted to generate 2 additional fractions: F13  and F14 .The large-scale cultivation of 3CN/H2O 0.1% formic acid for a duration of 25 min per run). Chromatograms were monitored by UV absorbance. From F6, two molecules were isolated: Colletamide A (7)  and Colletamide B (8) . From F5, two molecules were isolated: Colletamide C (9)  and Colletamide D (10) .BSNB-0583-F6 and BSNB-0583-F5 were purified by preparative HPLC  on 0.8 g was executed. 0.5 g recovered from the MeOH phase was futher fractionated by reverse flash chromatography on C18 column . For the elution, a linear gradient was performed between water and acetonitrile modified with 031% formic acid: 25/75 for 3 min, 25/75 to 0/100 in 10 min and 0/100 for 6 min. Thanks to the ELSD detection, nine zones were collected and analysed with mass spectrometry. The fraction 6 (1.7 mg), contained the targeted mass of m/z 346.275.To isolate the metabolite that give the ion of (1): White powder ; 1H-NMR 2SO) δ Phe 8.33 , 7.22 , 7.17 , 7.16 , 4.53 , 3.00 , 2.69 ; Leu1 8.48 , 4.26 , 1.41 , 1.28 , 1.23 , 0.79 , 0.76 ; Leu2 8.60 , 4.20 , 1.54 , 1.49 , 0.87 , 0.79 ; Leu3 8.40 , 4.33 , 1.49 , 1.45 , 0.87 , 0.85 ; Val 8.88 , 3.58 , 2.13 , 0.79 , 0.67 . 13C-NMR ((CD3)2SO, 600 MHz) δ Phe 170.4 (C-1), 137.8 (C-4), 129.1 (C-5), 128.0 (C-6), 126.1 (C-7), 53.4 (C-2), 37.5 (C-3); Leu1 170.9 (C-1), 51.0 (C-2), 39.4 (C-3), 24.1 (C-4), 22.6 (C-5), 22.4 (C-6); Leu2 171.5 (C-1), 52.7 (C-2), 40.6 (C-3), 24.4 (C-4), 22.9 (C-5), 21.2 (C-6); Leu3 171.1 (C-1), 52.6 (C-2), 41.1 (C-3), 24.6 (C-4), 22.4 (C-5), 22.3 (C-6); Val 171.0 (C-1), 62.5 (C-2), 29.4 (C-3), 19.2 . ESI-HRMS 586.3962 , 1171.7892 .Cyclo- (2): White powder ; 1H-NMR 2SO) δ Phe 8.34 , 7.22 , 7.17 , 7.16 , 4.48 , 3.02 , 2.70 ; Leu1 8.34 , 4.25 , 1.41 , 1.29 , 1.23 , 0.78 , 0.76 ; Leu2 8.46 , 4.21 , 1.52 , 1.44 , 0.87 , 0.84 ; Leu3 8.50 , 4.30 , 1.46 , 1.41 , 0.87 , 0.79  ; Leu4 8.94 , 4.07 , 1.47 , 1.44 , 1.36 , 0.83 , 0.77 . 13C-NMR ((CD3)2SO, 600 MHz) δ Phe 170.4 (C-1), 137.9 (C-4), 129.3 , 128.0 , 126.1 (C-7), 53.5 (C-2), 37.3 (C-3); Leu1 171.6 (C-1), 50.8 (C-2), 39.4 (C-3), 24.1 (C-4), 22.8 (C-5), 22.3 (C-6); Leu2 170.9 (C-1), 52.8 (C-2), 40.7 (C-3), 24.6 (C-4), 22.8 (C-5), 22.2 (C-6); Leu3 171.1 (C-1), 52.6 (C-2), 40.6 (C-3), 24.5 (C-4), 23.0 (C-5), 21.3 (C-6); Leu4 172.0 (C-1), 53.9 (C-2), 40.5 (C-3), 24.6 (C-4), 22.3 (C-6), 21.8 (C-5). ESI-HRMS 600.4115 , 1199.8195 .Cyclo-(Phe-Leu-Leu-Leu-Leu) (3): White powder ; 1H-NMR 2SO) δ Phe 7.99 , 7.24 , 7.18 , 7.17 , 4.65 , 2.91 , 2.72 ; Leu1 8.56 , 4.17 , 1.42 , 1.34 , 1.31 , 0.91 , 0.84 ; Leu2 7.32 , 4.10 , 1.60 , 1.52 , 0.87 , 0.78 ; Leu3 8.63 , 4.32 , 1.52 , 1.46 , 0.87 , 0.78 ; Ile 8.42 , 3.30 , 2.23 , 1.39 , 1.01 , 0.77 , 0.62  ; 13C-NMR ((CD3)2SO, 500 MHz) δ Phe 171.6 (C-1), 137.3 (C-4), 129.1 (C-5), 127.9 (C-6), 126.2 (C-7), 52.8 (C-2), 38.4 (C-3); Leu1 171.5 (C-1), 51.8 (C-2), 38.7 (C-3), 24.0 (C-4), 22.2 (C-5), 22.4 (C-6); Leu2 171.5 (C-1), 52.2 (C-2), 40.0 (C-3), 24.3 (C-4), 23.0 (C-5), 20.7 (C-6); Leu3 171.0 (C-1), 51.9 (C-2), 40.3 (C-3), 24.6 (C-4), 22.6 (C-5), 22.2 (C-6); Ile 171.1 (C-1), 62.7 (C-2), 33.2 (C-3), 25.1 (C-4), 15.1 (C-6), 9.8 (C-5); ESI-HRMS 600.4110 , 1199.8179 .Cyclo-(Phe-Leu-Leu-Leu-Ile) (4): White powder.[α]D20 –40 . 1H-NMR  δ 7.30 , 7.23 , 7.18 , 5.98 , 5.55 , 5.42 , 5.27 , 5.24 , 5.17 , 4.98 , 3.75 , 3.28 , 2.86 , 2.83 , 2.81 , 2.71 , 2.62 , 2.37 , 2.29 , 2.17 , 2.00 , 1.48 , 1.14 , 0.56 . 13C-NMR  δ 211.7 (C17), 176.5 (C1), 171.8 (C24), 150.9 (C6), 138.4 (C1′), 134.4 (C14), 133.4 (C20), 131.8 (C13), 130.9 , 129.6 , 128.9 (C19), 127.9 (C4′), 113.7 (C12), 79.3 (C9), 78.2 (C21), 72.3 (C7), 54.9 (C3), 49.7 (C4), 47.8 (C8), 44.9 (C10), 43.4 (C16), 39.4 (C15), 33.4 (C5), 24.6 (C23), 20.6 (C25), 19.7 (C22), 13.5 (C11). ESI-HRMS: 508.2717 , 530.2538 .Cytochalasin D (5): White powder.[α]D20 -16 . 1H-NMR  δ 7.31 , 7.24 , 7.23 , 5.95 , 5.87 , 5.72 , 5.28 , 5.26 , 3.72 , 3.31 , 3.03 , 2.87 , 2.83 , 2.47 , 2.40 , 2.30 , 2.01 , 1.49 , 1.60 , 1.14 , 0.4 . 13C-NMR  δ 211.6 (C17), 176.9 (C1), 172.0 (C24), 139.0 (C1′), 134.2 (C14), 134.2 (C6), 133.1 (C20), 132.1 (C13), 130.7 , 129.8 , 129.3 (C19), 127.9 (C4′), 127.9 (C5), 79.4 (C18), 76.7 (C21), 70.1 (C7), 60.2 (C3), 54.8 (C9), 50.5 (C8), 50.3 (C4), 44.9 (C10), 43.4 (C16), 39.5 (C15), 24.6 (C23), 20.7 (C25), 19.8 (C22), 17.1 (C12), 14.5 (C11). ESI-HRMS: 508.2681 , 530.2525 .Cytochalasin C (6): Yellow solid.[α]D20—63 . 1H-NMR  δ Phe 7.27 , 5.20 , 3.05 , 2.96 ; Leu 3.61 , 3.07 , 1.73 , 1.31 , 0.93 , 0.87 ; Thr 4.46 , 4.19 , 1.23 ; Ser 4.83 , 4.25 , 3.98 ; 2-hydroxyisovaleric acid 4.62 , 2.20 , 1.06 , 1.04 ; 2-hydroxyisocaproic acid 5.26 , 1.73 , 1.41 , 1.61 , 0.93 , 0.90 . 13C-NMR  δ Phe 172.7 (C-1), 137.5 (C-4), 130.8 (C-5), 129.6 , 128.1 (C-7), 52.3 (C-2), 39.8 (C-3); Leu 172.7 (C-1), 67.4 (C-2), 40.5 (N-Me), 38.1 (C-3), 26.0 (C-4), 23.5 (C-5), 22.3 (C-6); Thr 173.5 (C-1), 67.1 (C-3), 61.0 (C-2), 21.1 (C-4); Ser 171.7 (C-1), 63.8 (C-3), 55.3 (C-2); 2-hydroxyisovaleric acid 169.7 (C-1), 80.3 (C-2), 31.4 (C-3), 18.8 (C-4), 18.2 (C-5); 2-hydroxyisocaproic acid 171.9 (C-1), 74.7 (C-2), 42.6 (C-3), 25.5 (C-4), 23.4 (C-5), 21.8 (C-6). ESI-HRMS: 677.3723 , 1353.7501 .Hirsutatin A (7), -N--deca-2,8-dienamide: yellow solid; [α]D20 -3 . 1H-NMR  δ 6.82 , 5.93 , 5.42 , 4.34 , 4.15 , 4.00 , 3.66 , 3.62 , 2.83 , 2.53 , 2.22 , 2.00 , 1.63 , 1.47 , 1.39 . 13C-NMR  δ 198.9 (C-5′), 175.0 (C-1′), 168.5 (C-1), 147.5 (C-3), 132.2 (C-8), 126.0 (C-9), 123.6 (C-2), 84.6 (C-4′), 59.0 (C-7′), 57.2 (C-9′), 56.5 (C-6′), 53.8 (C-8′), 49.2 (C-2′), 34.2 (C-3′), 33.3 (C-7), 32.9 (C-4), 30.2 (C-6), 28.8 (C-5), 18.1 (C-10). ESI-HRMS: 362.1621 , 425.1692 , 745.2950 .Colletamide A (8), -N-[(R)-1--4-hydroxy-5-oxotetrahydrofuran-2-yl)-2-phenylethyl]deca-2,8-dienamide: yellow solid; [α]D20 -24 . 1H-NMR  δ 7.26 , 7.25 , 7.18 , 6.72 , 5.88 , 5.41 , 4.71 , 4.40 , 4.32 , 2.99 , 2.85 , 2.41 , 2.17 , 1.98 , 1.63 , 1.43 , 1.35 . 13C-NMR  δ 178.4 (C-1′), 169.0 (C-1), 146.8 (C-3), 139.0 (C-7′), 132.3 (C-8), 130.2 (C-8′), 129.5 (C-9′), 127.6 (C-10′), 126.0 (C-9), 124.0 (C-2), 80.7 (C-4′), 68.0 (C-2′), 54.7 (C-5′), 38.7 (C-6′), 34.6 (C-3′), 33.3 (C-7), 32.9 (C-4), 30.1 (C-6), 28.8 (C-5), 18.1 (C-10). ESI-HRMS: 372.2182 , 394.1998 , 743.4272 .Colletamide B (9), (S)-2--3-(2-hydroxyphenyl)propionic acid: yellow solid; [α]D20 -8 . 1H-NMR  δ 7.08 , 7.03 , 6.75 , 6.74 , 6.69 , 5.89 , 5.41 , 4.67 , 3.21 , 2.98 , 2.17 , 1.99 , 1.63 , 1.44 , 1.37 . 13C-NMR  δ 176.9 (C-1′), 168.4 (C-1), 156.7 (C-5′), 145.7 (C-3), 132.3 (C-8), 132.2 (C-9′), 128.9 (C-7′), 125.9 (C-9), 125.4 (C-4′), 124.7 (C-2), 120.5 (C-8′), 116.2 (C-6′), 55.6 (C-2′), 33.6 (C-3′), 33.4 (C-7), 32.9 (C-4), 30.2 (C-6), 28.9 (C-5), 18.1 (C-10). ESI-HRMS: 332.1859 , 395.1948 , 663.3648 .Colletamide C (10), -4-D20 -5 . 1H-NMR  δ 7.25 , 7.23 , 7.14 , 6.68 , 5.94 , 5.42 , 4.14 , 4.02 , 2.97 , 2.81 , 2.30 , 2.16 , 1.99 , 1.63 , 1.44 , 1.35 . 13C-NMR  δ 179.7 (C-1′), 168.6 (C-1), 145.7 (C-3), 140.2 (C-6′), 132.2 (C-8), 130.3 (C-7′), 129.3 (C-8′), 127.2 (C-9′), 126.0 (C-9), 124.7 (C-2), 70.1 (C-3′), 56.3 (C-4′), 41.6 (C-2′), 38.3 (C-5′), 33.4 (C-7), 32.9 (C-4), 30.2 (C-6), 28.9 (C-5), 18.1 (C-10). ESI-HRMS: 360.2170 , 382.1995 , 719.4293 .Colletamide D 64. The temperature of the crystal was maintained at the selected value by means of an N-Helix cooling device to within an accuracy of ± 1 K. The data were corrected for Lorentz polarization, and absorption effects. The structures were solved by direct methods using SHELXS-9765 and refined against F2 by full-matrix least-squares techniques using SHELXL-201866 with anisotropic displacement parameters for all non-hydrogen atoms. Hydrogen atoms were introduced into the calculations as a riding model with isotropic thermal parameters. All calculations were performed by using the Crystal Structure crystallographic software package WINGX67.X-ray diffraction data for cyclo-(Phe-Leu-Leu-Leu-Ile) was collected by using a VENTURE PHOTON100 CMOS Bruker diffractometer with Micro-focus IuS source Cu Kα radiation. Crystal was mounted on a CryoLoop (Hampton Research) with Paratone-N (Hampton Research) as cryoprotectant and then flashfrozen in a nitrogen-gas stream at 100 KThe crystal data collection and refinement parameters are given in Table https://www.ccdc.cam.ac.uk/Community/Requestastructure.CCDC 1,944,171 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via 2), mean average error (MAE) and DP4 probability [ \* MERGEFORMAT 33].All calculations have been performed using Gaussian 16W. Prior to DFT calculations, a conformational analysis has been performed using the GMMX plugin. Each conformer has been optimized using DFT at the B3LYP/6-31g(d) level. Frequency calculation have been performed at the same level of theory. Rotational strengths have been calculated using the B3LYP/6-311+g for 20 excited states. ECD spectra were plotted using the Gaussview 6. GIAO NMR properties were predicted using the MPW1PW91/6-311+g. Theoretical and experimental NMR chemical shift were compared using common metrics such as linear correlation  was used as positive controls. Docetaxel show 0.0005% mortality at 1 μg/mL.For each strain, a crude extract was tested to determine its cytotoxicity at 10 µg/ml using normal human lung fibroblast cells MRC-5 (ATCC CCL-171). The assay was conducted according to the procedure described by Tempête et alSupplementary Information.


Arabidopsis thaliana sterol profiles upon treatment with a series of azasterols reveals a specific in vivo inhibition of SMT2, a plant sterol-C-methyltransferase acting as a branch point between the campesterol and sitosterol biosynthetic segments in the pathway. Side chain azasteroids that modify sitosterol homeostasis help to refine its particular function in plant development.Inhibitors of enzymes in essential cellular pathways are potent probes to decipher intricate physiological functions of biomolecules. The analysis of  Arabidopsis thaliana  amino]-20-methyl, -;S5, C29H42NOI, Pyridinium, 4--1-methyl-iodide (1:1);S6, C29H42NOI, Pyridinium, 3--1-methyl iodide (1:1);S7, C26H43NO, 26, 27-dinorcholesta-7,22-dien-3-ol, 25-(dimethylamino), -;S8, C29H49NO2, CAS 1202777-10-3: Pregn-7-en-3-ol, 21-(butylmethylamino)-20-methyl-3-acetate, -;S9, C28H47NO2, CAS 1202777-37-4: Pregn-7-en-3-ol, 21-(butylamino)-20-methyl-3-acetate, -;S10, C28H47NO2, CAS 1202777-39-6: Pregn-7-en-3-ol, 20-methyl-21--1-methyl-iodide (1:1);S12, C29H42NOI, CAS 1001324-61-3: Pyridinium, 4--1-methyl-iodide (1:1);S13, C26H45NO, CAS 1202777-24-9: Pregn-7-en-3-ol, 20-methyl-21-(propylamino) -;S14, C26H45NO, CAS 1202777-25-0: Pregn-7-en-3-ol, 21-(butylamino)-20-methyl--;S15, C25H41NO, chola-7,22-dienol-24-(dimethylamino) ;S16, C24H41NO, CAS 1202777-23-8: Pregn-7-en-3-ol, 21-(ethylamino)-20-methyl--;S17, C27H47NO, CAS 1202777-28-3: Pregn-7-en-3-ol, 21-[amino]-20-methyl--.LDAO  Heynh ecotype Col-0 seeds were germinated on synthetic medium  containing a given concentration of LDAO or a concentration of 2 mg·L−1 of each of the azasteroids S1 to S17. Azasteroids were stored in ethanol at a concentration of 2 mg·mL−1. Control treatments consisted of 1 mL·L−1 of ethanol. The effect of the compounds on seedling growth was assessed at 15 days and 30 days after germination. Experiments were done twice independently and in a triplicate assay for each compound. One of these triplicates in each biological assay was analyzed to determine its sterol profile.2 flow rate was fixed at 2 mL.min−1 and the injector and detector temperatures were 250 ℃ and 300 ℃, respectively. The GC-MS identification of the compounds was performed using a 5973N instrument (Agilent) equipped with a primary HP5-MS column and coupled with a 6853 mass analyzer (Agilent). The temperature program of ovens (GC-FID and GC-MS) has a two-step phase: (i) a steep ramp at 30 ℃/min from 60 ℃ to 220 ℃ and (ii) a 2 ℃/min−1 increase from 220 ℃ to 300 ℃. Sterols were identified by coincidental retention time and EI-MS spectra at 70 eV [Freeze-dried seedling samples (100 mg) were saponified for one hour in a 6% potassium hydroxide methanolic solution at 80 ℃. The unsaponifiable fraction was extracted with three portions of hexane. The dried extract was acetylated with a mixture of pyridine/acetic anhydride in toluene. Steryl acetate detection, analysis, and quantification were performed as described ,52. In b


CT37 using bioassay in conjunction with mass spectrometric molecular networking (MN) driven isolation. Their complete structures were established by high-resolution electrospray ionization mass spectrometry (HR-ESIMS), and 1D and 2D nuclear magnetic resonance (NMR) data. Legonimide 1 was identified as a new alkaloid containing a rare linear imide motif in its structure, while compounds 2–4 were already known and their structures were elucidated as 1H-indole-3-carbaldehyde, actinopolymorphol B, -1-phenylbutane-2,3-diol, respectively. The biosynthetic pathways of 1–4 were proposed based on the reported biogenesis of indole alkaloids in literature. Bioactivity tests for 1 and 2 revealed moderate growth inhibition activity against Candida albicans ATCC 10231 with MIC95 values of 21.54 µg/mL and 11.47 µg/mL, respectively. Four compounds ( Streptomyces a prominent resource for natural product discovery contributing up to 75% of present day clinically used antibiotics + . 1H-indole-3-carboxaldehyde 2: pale yellow solid; UV(CH3OH) 240, 260, 280 nm; 1H, 13C NMR data, see + = 146.0603 .Actinopolymorphol B3: yellow solid; UV (MeOH) max nm 220, 280; [α]25 +27.3 ; 1H, 13C NMR data, see + = 204.1023 .-1-phenylbutane-2,3-diol4. yellow oil; [α]25D +12.1 ; UV (MeOH) max nm 250; IR νmax (cm−1) 3410, 2931, 2874, 1712, 1645, 1409, 1207, 1012, 802, 754; 1H, 13C NMR data, see + = 167.1070 .1–4 against C. albicans ATCC 10,231 were determined using a conventional broth dilution assay in accordance with standards recommended by the National Committee for Clinical Laboratory Standards (NCCLS) [600) in a SpectraMax ABS Plus (Molecular Device) plate reader. The MIC was defined as the lowest concentration of compound that inhibited ≥ 95% of the growth of C. albicans after overnight incubation.Minimum inhibitory concentrations (MIC) of  (NCCLS)  and as d (NCCLS) . The ant1-4 from the Ghanaian soil bacterium, Streptomyces sp. CT37. Their structures were deduced by analysis of the HRESIMS, 1D, and 2D NMR. Legonimide 1 was identified as a new alkaloid bearing the imide motif in its structure, while 2-4 were known and their structures were elucidated as 1H-indole-3-carbaldehyde, 3-hydroxy-4-(1H-indol-3-yl) butan-2-one, and -1-phenylbutane-2,3-diol, respectively. Alkaloids 1 and 2 exhibited moderate inhibition against C. albicans ATCC 10,231 with MIC values of 21.54 µg/mL and 11.47 µg/mL, respectively. The bioassay and molecular network-assisted isolation afforded four alkaloids


Several compounds show potential as useful selective agents that could be exploited in the recovery of several bacterial pathogens including Salmonella, Pseudomonas aeruginosa, and Listeria.In diagnostic microbiology, culture media are widely used for detection of pathogenic bacteria. Such media employ various ingredients to optimize detection of specific pathogens such as chromogenic enzyme substrates and selective inhibitors to reduce the presence of commensal bacteria. Despite this, it is rarely possible to inhibit the growth of all commensal bacteria, and thus pathogens can be overgrown and remain undetected. One approach to attempt to remedy this is the use of “suicide substrates” that can target specific bacterial enzymes and selectively inhibit unwanted bacterial species. With the purpose of identifying novel selective inhibitors, six novel phosphonopeptide derivatives based on  Antimicrobial resistance (AMR) is rapidly becoming a global crisis. According to the Centers for Disease Control and Prevention (CDC), around 2.8 million people are infected with antibiotic-resistant bacteria and approximately 35,000 deaths occur each year in the USA due to these infections ,2. Moreol-alanyl-l-fosfalin 1  were observed upon replacing the fosfalin ,12. Morearcosine .4 .A different coupling strategy was utilized where the reactive amino acid methionine, N-terminus of the tripeptide derivatives, differences in uptake may result, and different intracellular aminopeptidases may be required for the liberation of d/l-fosfalin and β-chloro-l-alanine (where incorporated), in order to act on AlaR. The rationale for varying the N-terminal amino acid was that as different species and strains of bacteria produce the transporters and enzymes involved with these processes to differing extents, differences in antibacterial activity were anticipated. The antimicrobial activity of phosphonotripeptide derivatives was evaluated as their minimum inhibitory concentration (MIC), The inhibitory action of these compounds requires efficient uptake of these molecules across the bacterial cell membrane, hydrolysis of specific peptide bonds by aminopeptidases to release the inhibitor(s), and binding of inhibitor(s) to AlaR. As different amino acids are utilized at the S. marcescens and Y. enterocolitica, with all inhibitors displaying MIC values of 2 mg/L or less. In contrast, no activity against six of the remaining Gram-negative organisms tested was observed at the highest inhibitor concentration tested, which was 8 mg/L. Turning attention to the Gram-positive organisms low or no activity was displayed at the maximum concentrations tested for L. monocytogenes, while very high activity (0.063 mg/L or less) was seen against E. faecalis for all inhibitors tested except for 21b and 25b, which still retained activity, albeit at the 4 mg/L level.Broadly speaking, all six phosphonopeptide derivatives produced similar inhibitory profiles to one another against most bacterial species and strains. However, the extent of activity for the collection of inhibitors varied markedly depending on the bacterium in question in a manner that does not appear to be specifically tied to the Gram classification of the organism. For instance, consistently high activity is seen for all inhibitors for two of the Gram-negative species 21b and 25b appear to be the least effective in terms of overall inhibitory profile. In both instances, the N-terminal amino acid is sarcosine, thus the presence of this amino acid in this position of these tripeptide derivatives appears to be detrimental to activity. However, the current data set does not definitively explain whether these poor activity profiles derive from poor uptake, or from slow liberation of the active units once inside the bacterial cell. In many instances these two poorest performing inhibitors have identical MIC values, and when differences do exist they are displayed only to the nearest increment that our tests allow. For example, for S. marcecsens the substance with a simple alanine 21b produces the greater inhibition (1 mg/L) over its β-chloro analogue 25b (2 mg/L). In contrast, for E. coli NCTC 12,241 the reverse behavior is seen with the β-chloro substance 25b exhibiting MIC = 4 mg/L, whereas in this case the simple alanine containing tripeptide 21b produces a weaker MIC of 8 mg/L. Essentially, this indicates that these two poorly performing inhibitors display very similar levels of activity over a wide range of organisms.In general, compounds 25a and 25c are the most consistently active pair of inhibitors, and contain β-Cl-Ala at the center position of each tripeptide. Thus, this indicates that the presence of the β-chloro unit in the center of the tripeptide is indeed beneficial to overall activity. While not definitive, this would indicate that the β-chloro alanine unit is liberated, and becomes active against the bacteria, acting in a synergistic manner with the fosfalin unit, which is common to all six inhibitors.With regards to the remaining four inhibitors, there is a subtle yet clear difference in activity for the central β-Cl-Ala series vs. the series of compounds with a standard alanine residue at the central position. Specifically, l-Alanine aminopeptidase is thought to be found more readily in Gram-negative bacteria undec-7-ene (DBU)  were dissolved in a round-bottom flask containing dry benzene (80 mL), followed by the addition of benzyl bromide . Caution: Benzene is a known carcinogen. The solution was stirred overnight at room temperature under nitrogen and later the solvent was removed under reduced pressure to afford an off-white residue. Ethyl acetate (100 mL) was added, the flask contents were sonicated and then washed with 1M HCl (50 mL) and brine (2 × 50 mL). The organic layer was dried over MgSO4, filtered, concentrated in vacuo and purified by column chromatography (petrol/ethyl acetate (1:1)) to give 11 as a white solid ; m.p. 61–63 °C (lit. m.p. 59–60 °C) [ῡmax/cm−1 3416 , 1756 (C=O), 1666 (C=O), 1522 (NH bend), 1200 (C-O), 1154 (C-O); 1H NMR  δH 1.36 3), 2.17 , 3.82 , 3.90 , 4.33 , 5.11 , 5.16 , 5.40 , 7.27 ; 13C NMR  δC 27.1 (C(CH3)3), 54.7 (CH-2), 62.3 (CH2-3), 66.2 (OCH2Ar), 79.1 (C(CH3)3), 127.0 (2 × CHAr), 127.3 (CHAr), 127.4 (2 × CHAr), 134.1 (CHAr quat.), 153.0 (C=O-5), 170.7 (C=O-1); m/z (ESI) calcd for (C15H21NNaO5)+, MNa+: 318.3, found 318.2. (The 1H-NMR spectrum may be found within the l-Ala-OBzl(R)-Benzyl 2-((tert-butoxycarbonyl)amino)-3-chloropropanoate or Boc-β-Cl- (12). (S)-Benzyl 2-((tert-butoxycarbonyl)amino)-3-hydroxypropanoate (11)  was dissolved in dry DCM (40 mL), followed by the addition of trichloroacetonitrile . The solution was stirred at room temperature for 2 h. To this solution, triphenylphosphine  in dry DCM (50 mL) was added slowly. The resulting solution was stirred overnight at room temperature under nitrogen; brine (100 mL) was added to quench the reaction. The organic layer was washed with brine (3 × 100 mL), dried over MgSO4, filtered and concentrated in vacuo to afford an orange residue. The residue was purified by column chromatography (petrol/ethyl acetate (7:3)) to give 12 as a white solid ; m.p. 55–58 °C; ῡmax/cm−1 3364 (NH), 1725 (C=O), 1680 (C=O), 1519 (NH bend), 1208 (C-O), 1158 (C-O); 1H NMR  δH 1.38 3), 3.78 , 3.92 , 4.67 , 5.13 , 5.18 , 5.37 , 7.29 ; 13C NMR  δC 28.3 (C(CH3)3), 45.5 (CH2-3), 54.5 (CH-2), 67.8 (OCH2Ar), 80.5 (C(CH3)3), 128.4 (CHAr), 128.6 (CHAr), 128.7 (CHAr), 134.9 (CHAr quat.), 155.0 (C=O-5), 169.0 (C=O-1); m/z (ESI) calcd for (C15H20ClNNaO4)+, MNa+: 336.1 (35Cl), 338.1 (37Cl), found 336.2 (35Cl), 338.2 (37Cl); CHN . (The mass spectrum may be found within the l-Ala-OH(R)-2-((Tert-butoxycarbonyl)amino)-3-chloropropanoic acid or Boc-β-Cl- (13). Deprotection of benzyl ester was followed, using (R)-benzyl 2-((tert-butoxycarbonyl)amino)-3-chloropropanoate (12)  to afford 13 as an off-white solid ; m.p. 125–128 °C (lit. m.p. 127–129 °C) [ῡmax/cm−1 3434 (NH), 2973 (br OH), 1752 (C=O), 1735 (C=O), 1519 (NH bend), 1159 (C-O), 1148 (C-O); 1H NMR  δH 1.40 3), 3.80 , 3.95 , 4.70 , 5.42 , 9.03 ; 13C NMR  δC 27.1 (C(CH3)3), 44.0 (CH2-3), 53.1 (CH-2), 79.8 (C(CH3)3), 154.2 (C=O-5), 172.1 (C=O-1); m/z (ESI) calcd for (C8H14ClNNaO4)+, MNa+: 246.1 (35Cl), 248.1 (37Cl), found 246.1 (35Cl), 248.1 (37Cl).–129 °C) ; ῡmax/cml-Ala-OBzl hydrochloride(R)-1-(Benzyloxy)-3-chloro-1oxopropan-2-aminium chloride or β-Cl- (14). Deprotection of tert-butoxycarbonyl was followed, using (R)-benzyl 2-((tert-butoxycarbonyl)amino)-3-chloropropanoate (12) . The white crude solid was filtered and washed by diethyl ether to give 14 as a white solid ; m.p. 145 °C (sub); ῡmax/cm−1 2841 (NH+), 1750 (C=O), 1231 (C-O); 1H NMR  δH 4.06 , 4.20 , 4.70 , 5.29 , 5.37 , 7.42-7.47 ; 13C NMR  δC 41.8 (CH2-3), 54.0 (CH-2), 69.1 (OCH2Ar), 128.6–129.1 (CHAr), 134.5 (CHAr quart.), 167.0 (C=O-1); m/z (ESI) calcd for (C10H13ClNO2), M+: 214.1 (35Cl), 216.1 (37Cl), found 214.1 (35Cl), 216.1 (37Cl); CHN .l-Nva-β-chloro-l-Ala-OBzl(R)-Benzyl 2-((S)-2-((tert-butoxycarbonyl)amino)pentanamido)-3-chloropropanoate or Boc- (22a). General peptide coupling method was followed, using Boc-l-Nva-OH (15a)  in dry THF and (R)-1-(benzyloxy)-3-chloro-1oxopropan-2-aminium chloride (14)  in dry DCM. The yellow crude liquid was purified by column chromatography (40-60 petrol/ethyl acetate (7:3)) to give 22a as a white solid ; m.p. 95–98 °C; ῡmax/cm−1 3327 (NH), 1743 (C=O), 1688 (C=O), 1653 (C=O), 1518 (NH bend), 1206 (C-O), 1169 (C-O); 1H NMR  δH 0.92 , 1.32-1.43 , 1.45 3), 1.52–1.65 , 1.75-1.82 , 3.89 , 3.99 , 4.11–4.15 , 4.96–5.00 , 5.20 , 5.25 , 6.97 , 7.33–7.37 ; 13C NMR  δC 12.7 (CH3-7″), 17.8 (CH2-7′), 27.3 (C(CH3)3), 33.4 (CH2-7), 43.8 (CH2-3), 52.2 (CH-2), 53.4 (CH-6), 67.0 (OCH2Ar), 79.3 (C(CH3)3), 127.4 (CHAr), 127.6 (CHAr), 127.7 (CHAr), 133.8 (CHAr quat.), 154.5 (C=O-9), 167.5 (C=O-1), 171.2 (C=O-5); HRMS (NSI) calcd for (C20H30ClN2O5)+, MH+: 413.1838 (35Cl), 415.1809 (37Cl), found 413.1837 (35Cl), 415.1807 (37Cl); CHN .l-Ala-OBzl(R)-Benzyl 2-(2-((tert-butoxycarbonyl)(methyl)amino) acetamido)-3-chloropropanoate or Boc-Sar-β-chloro- (22b). General peptide coupling method was followed, using Boc-Sar-OH (15b)  in dry THF and (R)-1-(benzyloxy)-3-chloro-1oxopropan-2-aminium chloride (14)  in dry DCM. The yellow crude liquid was purified by column chromatography (40–60 petrol/ethyl acetate (7:3)) to give 22b as a light yellow syrup ; ῡmax/cm−1 3302 (NH), 1747 (C=O), 1686 (br C=O), 1522 (NH bend), 1175 (C-O), 1148 (C-O); 1H NMR  δH 1.40 3), 2.87 , 3.80 , 3.82 , 3.83 , 3.94 , 4.91–4.96 , 5.13 , 5.18 , 6.97 , 7.26–7.30 ; 13C NMR  δC 28.2 (C(CH3)3), 35.6 (NCH3-8), 44.9 (CH2-3), 53.0 (CH2-6), 53.0 (CH-2), 68.0 (OCH2Ar), 81.0 (C(CH3)3), 128.4 (CHAr), 128.6 (CHAr), 128.7 (CHAr), 134.8 (CHAr quat.), 154.5 (C=O-9), 168.4 (C=O-1), 169.4 (C=O-5); HRMS (NSI) calcd for (C18H26ClN2O5)+, MH+: 385.1525 (35Cl), 387.1496 (37Cl), found 385.1527 (35Cl), 387.1498 (37Cl). LCMS purity >95% .l-Ala-d/l-Fos diethyl esterTert-butyl ((2R)-3-chloro-1-((1-(diethoxyphosphoryl)ethyl) amino)-1-oxopropan-2-yl)carbamate or Boc-β-chloro- (19e). General peptide coupling method was followed, using (R)-2-((tert-butoxycarbonyl)amino)-3-chloropropanoic acid (13)  in dry THF and diethyl 1-aminoethylphosphonate (9)  in dry THF. The light yellow crude liquid was purified by column chromatography, using 100% petrol and increasing to 100% ethyl acetate, to afford 19e as colorless syrup composed of 2 diastereoisomers, Boc-β-Cl-l-Ala-l-Fos diethyl ester and Boc-β-Cl-l-Ala-D-Fos diethyl ester ; ῡmax/cm−1 3261 (NH), 1713 (C=O), 1670 (C=O), 1517 (NH bend), 1225 (P=O), 1164 (C-O), 1020 (P-O-C), 970 (P-O-C); 1H NMR  δH 1.15–1.46 , 1.47 3), 3.74 , 4.00 , 4.06–4.22 , 4.40–4.56 , 5.46 , 5.48 , 7.01 , 7.09 ; 13C NMR  δC 15.6 (CH3-2), 15.7 (CH3-2), 16.3 , 16.4 , 16.4 , 16.4 , 28.2 (C(CH3)3), 41.2 , 41.3 , 55.2 (CH2-6), 55.3 (CH-5), 62.6 , 62.6 , 63.0 , 63.0 , 80.8 (C(CH3)3), 155.0 (C=O-8), 168.3 (C=O-4); 31P-1Hdecoup NMR  δP 24.7, 24.8; HRMS (NSI) calcd for (C14H28ClN2O6P), MH+: 409.1266 (35Cl), 411.1237 (37Cl), found 409.1258 (35Cl), 411.1231 (37Cl). LCMS purity >95% .l-Nva-β-chloro-l-Ala-OH(R)-2-((S)-2-((tert-Butoxycarbonyl)amino)pentanamido)-3-chloropropanoic acid or Boc- (23a). Deprotection of benzyl ester was followed, using (R)-benzyl 2-((S)-2-((tert-butoxycarbonyl)amino)pentanamido)-3-chloropropanoate (22a)  to afford 23a as a light yellow solid ; m.p. 60–63 °C; ῡmax/cm−1 3312 (br OH), 2963 (NH), 1655 (br C=O), 1509 (NH bend), 1161 (C-O); 1H NMR  δH 0.85 , 1.24–1.34 , 1.38 3), 1.42–1.52 , 1.54–1.59 , 3.34 , 3.84 , 3.91 , 3.95–4.02 , 4.62–4.67 , 6.92 , 8.07 ; 13C NMR  δC 14.1 (CH3-7″), 19.1 (CH2-7′), 28.6 (C(CH3)3), 34.4 (CH2-7), 45.1 (CH2-3), 53.6 (CH-2), 54.5 (CH-6), 78.5 (C(CH3)3), 155.8 (C=O-9), 170.6 (C=O-5), 173.0 (C=O-1); m/z (ESI) calcd for (C13H23ClN2NaO5)+, MNa+: 345.1 (35Cl), 347.1 (37Cl), found 345.2 (35Cl), 347.2 (37Cl); CHN .l-Ala-OH(R)-2-(2-((tert-butoxycarbonyl)(methyl)amino)acetamido)-3-chloropropanoic acid or Boc-Sar-β-chloro- (23b). Deprotection of benzyl ester was followed, using (R)-benzyl 2-(2-((tert-butoxycarbonyl)(methyl)amino)acetamido)-3-chloropropanoate (22b)  to afford 23b as an off-white solid ; m.p. 89–91 °C; ῡmax/cm−1 3342 (NH), 2982 (br OH), 1734 (C=O), 1672 (C=O), 1644 (C=O), 1524 (NH bend), 1152 (C-O); 1H NMR  δH 1.47 3), 2.99 , 3.81–4.17 , 5.01 , 7.07 , 7.45 ; 13C NMR  δC 28.3 (C(CH3)3), 36.1 (NCH3-8), 44.5 (CH2-3), 53.0 (CH2-6 and CH-2), 81.8 (C(CH3)3), 156.8 (C=O-9), 169.5 (C=O-1 and C=O-5); m/z (ESI) calcd for (C11H19ClN2NaO5)+, MNa+: 317.1 (35Cl), 319.1 (37Cl), found 317.1 (35Cl), 319.1 (37Cl); CHN .l-Ala-d/l-Fos diethyl ester hydrochloride(2R)-3-Chloro-1-((1-(diethoxyphosphoryl)ethyl)amino)-1-oxopropan-2-aminium chloride or β-Cl- (20e). Deprotection of tert-butoxycarbonyl was followed, using ((2R)-3-chloro-1-((1-(diethoxyphosphoryl)ethyl)amino)-1-oxopropan-2-yl)carbamate (19e) . The off-white hygroscopic crude solid was washed with petrol to afford 20e as a pale green solid composed of 2 diastereoisomers, β-Cl-l-Ala-l-Fos diethyl ester hydrochloride and β-Cl-l-Ala-D-Fos diethyl ester hydrochloride ; m.p. 129–133 °C (decomp.); ῡmax/cm−1 3204 (NH+), 1687 (C=O), 1562 (NH bend), 1204 (P=O), 1010 (P-O-C), 961 (P-O-C); 1H NMR  δH 1.28 , 1.29 , 1.37 , 3.92–4.04 , 4.07-4.21 , 4.38–4.48 ; 13C NMR  δC 13.7 (CH3-2), 14.0 (CH3-2), 15.7 (OCH2CH3), 15.7 (OCH2CH3), 41.7 , 42.0 , 42.4 (CH2-6), 53.7 (CH-5), 53.8 (CH-5), 64.3 , 64.5 , 165.7 (C=O-4), 165.8 (C=O-4); 31P-1Hdecoup NMR  δP 26.1, 26.2; HRMS (NSI) calcd for (C9H21ClN2O4P)+, M+: 287.0922 (35Cl), 289.0892 (37Cl), found 287.0922 (35Cl), 289.0890 (37Cl). LCMS purity >95% .l-Nva-β-chloro-l-Ala-d/l-Fos diethyl esterTert-butyl ((2S)-1-(((2R)-3-chloro-1-((1-(diethoxyphosphoryl)-ethyl)amino)-1-oxopropan-2-yl)amino)-1-oxopentan-2-yl)carbamate or Boc- (24a). General peptide coupling method was followed, using (R)-2-((S)-2-((tert-butoxycarbonyl)amino)pentanamido)-3-chloropropanoic acid (23a)  in dry THF and diethyl 1-aminoethylphosphonate (9)  in dry THF. The light yellow crude liquid was purified by column chromatography, using ethyl acetate/methanol (96:4), to afford 24a as a white solid composed of 2 diastereoisomers, Boc-l-Nva-β-Cl-l-Ala-l-Fos diethyl ester and Boc-l-Nva-β-Cl-l-Ala-D-Fos diethyl ester ; m.p. 196 °C (decomp); ῡmax/cm−1 3272 (NH), 1709 (C=O), 1680 (C=O), 1644 (C=O), 1530 (NH bend), 1229 (P=O), 1165 (C-O), 1019 (P-O-C), 972 (P-O-C); 1H NMR  δH 0.86 , 0.88 , 1.22–1.34 , 1.38 3), 1.53-1.59 , 1.70–1.77 , 3.69 , 3.78-3.81 , 3.91 , 3.97-4.13 , 4.35-4.46 , 4.73-4.79 , 4.97–5.03 , 7.01 , 7.09 , 7.25 , 7.33 ; 13C NMR  δC 12.7 (CH3-10″), 14.5 (CH3-2), 15.3 (OCH2CH3), 15.4 (OCH2CH3), 15.5 (OCH2CH3), 15.6 (OCH2CH3), 17.9 (CH2-10′), 18.0 (CH2-10′), 27.0 (C(CH3)3), 27.3 (C(CH3)3), 33.2 (CH2-10), 40.4 , 43.4 (CH2-6), 52.6 (CH-5), 52.8 (CH-5), 61.4 , 61.6 , 61.7 , 61.9 , 70.5 (CH-9), 79.4 (C(CH3)3), 154.7 (C=O-12), 166.8 (C=O-4), 171.4 (C=O-8); 31P-1Hdecoup NMR  δP 24.9, 25.0; HRMS (NSI) calcd for (C19H38ClN3O7P)+, MH+: 486.2130 (35Cl), 488.2102 (37Cl), found 486.2124 (35Cl), 488.2098 (37Cl); CHN .l-Ala-d/l-Fos diethyl esterTert-butyl (2-(((2R)-3-chloro-1-((1-(diethoxyphosphoryl)ethyl) amino)-1-oxopropan-2-yl)amino)-2-oxoethyl)(methyl)carbamate or Boc-Sar-β-chloro- (24b). General peptide coupling method was followed, using (R)-2-(2-((tert-butoxycarbonyl)(methyl)amino)acetamido)-3-chloropropanoic acid (23b)  in dry THF and diethyl 1-aminoethylphosphonate (9)  in dry THF. The light yellow crude liquid was purified by column chromatography, using DCM/methanol (95:5), to afford 24b as a light yellow syrup composed of 2 diastereoisomers, Boc-Sar-β-Cl-l-Ala-l-Fos diethyl ester and Boc-Sar-β-Cl-l-Ala-D-Fos diethyl ester ; ῡmax/cm−1 3218 (NH), 1690 (C=O), 1665 (br C=O), 1518 (NH bend), 1224 (P=O), 1148 (C-O), 1018 (P-O-C), 967 (P-O-C); 1H NMR  δH 1.11–1.35 , 1.41 3), 2.90 , 3.70–3.88 , 4.02–4.13 , 4.36–4.47 , 4.78–4.82 , 6.94 , 7.36 ; 13C NMR  δC 15.2 (CH3-2), 15.6 (CH3-2), 16.3 (OCH2CH3), 16.4 (OCH2CH3), 28.3 (C(CH3)3), 35.9 (CH3-11), 41.2 , 44.7 (CH2-6), 53.1 (CH2-9), 53.4 (CH-5), 62.7 , 63.0 , 81.0 (C(CH3)3), 152.3 (C=O-12), 167.7 (C=O-4 or C=O-8), 169.4 (C=O-4 or C=O-8); 31P-1Hdecoup NMR  δP 24.7, 24.8; HRMS (NSI) calcd for (C16H34ClN3O7P)+, MH+: 480.1637 (35Cl), 482.1608 (37Cl), found 480.1642 (35Cl), 482.1612 (37Cl). LCMS purity >92% .l-Met-β-Cl-l-Ala-d/l-Fos diethyl esterTert-butyl ((2S)-1-(((2R)-3-chloro-1-((1-(diethoxyphosphoryl) ethyl)amino)-1-oxopropan-2-yl)amino)-4-(methylthio)-1-oxobutan-2-yl) carbamate or Boc- (24c). General peptide coupling method was followed, using Boc-l-Met-OH (15c)  in dry THF and (2R)-3-chloro-1-((1-(diethoxyphosphoryl)ethyl)amino)-1-oxopropan-2-aminium chloride (20e)  in dry DCM. The yellow crude liquid was purified by column chromatography (DCM/MeOH (95:5)) and recrystallized from diethyl ether/petrol to give 24c as a white solid composed of 2 diastereoisomers, Boc-l-Met-β-Cl-l-Ala-l-Fos diethyl ester and Boc-l-Met-β-Cl-l-Ala-D-Fos diethyl ester ; m.p. 96–99 °C; ῡmax/cm−1 3278 (NH), 1709 (C=O), 1687 (C=O), 1639 (C=O), 1523 (NH bend), 1228 (P=O), 1165 (C-O), 1018 (P-O-C), 970 (P-O-C); 1H NMR  δH 1.17-1.36 , 1.38 3), 1.87–2.03 , 2.04 , 2.48–2.54 , 3.71 , 3.88 , 3.99–4.13 , 4.20 , 4.37–4.47 , 4.78–4.84 , 5.39 , 5.41 , 7.15 , 7.24 , 7.52 ; 13C NMR  δC 14.3 (CH3-2), 14.4 (CH3-2), 14.5 (CH2-10″), 15.3 (OCH2CH3), 15.4 (OCH2CH3), 15.5 (OCH2CH3), 15.6 (OCH2CH3), 27.3 (C(CH3)3), 29.2 (CH2-10′), 29.3 (CH2-10′), 30.2 (CH2-10), 30.4 (CH2-10), 40.3 , 43.5 (CH2-6), 43.7 (CH2-6), 52.7 (CH-5), 53.1 (CH-9), 61.6 , 61.7 , 62.0 , 62.1 , 79.6 (C(CH3)3), 154.8 (C=O-12), 166.7 (C=O-4), 166.8 (C=O-4), 170.7 (C=O-8), 170.8 (C=O-8); 31P-1Hdecoup NMR  δP 24.5, 24.8; HRMS (NSI) calcd for (C19H3HClN3O7PS)+, MH+: 518.1851 (35Cl), 520.1821 (37Cl), found 518.1842 (35Cl), 520.1814 (37Cl); CHN .l-Nva-β-chloro-l-Ala-d/l-Fos(1-((R)-2-((S)-2-Ammoniopentanamido)-3-chloropropanamido) ethyl)phosphonic acid or  (25a). The tert-butoxycarbonyl and diethyl ester protecting groups of tert-butyl ((2S)-1-(((2R)-3-chloro-1-((1-(diethoxyphosphoryl)-ethyl)amino)-1-oxopropan-2-yl)amino)-1-oxopentan-2-yl)carbamate (24a)  were removed. The pale green crude solid was washed with diethyl ether to give 25a as a pale green solid composed of 2 diastereoisomers, l-Nva-β-Cl-l-Ala-l-Fos and l-Nva-β-Cl-l-Ala-D-Fos ; m.p. 175 °C (sub); ῡmax/cm−1 3294 (NH+), 3000 (br OH), 1668 (C=O), 1645 (C=O), 1538 (NH bend), 1132 (P=O), 1039 (P-O-C), 921 (P-OH); 1H NMR  δH 1.01 , 1.30–1.37 , 1.44–1.54 , 1.90–1.98 , 3.91–4.15 , 4.79 ; 13C NMR  δC 12.9 (CH3-10″), 15.7 (CH3-2), 17.6 (CH2-10′), 33.0 (CH2-10), 43.3 (CH2-6), 53.1 (CH-1 and CH-9), 55.0 (CH-5), 170.4 (C=O-4 and C=O-8); 31P-1Hdecoup NMR  δP 18.5; HRMS (NSI) calcd for (C10H20ClN3O5P)-, MH-: 328.0835 (35Cl), 330.0805 (37Cl), found 328.0833 (35Cl), 330.0800 (37Cl). LCMS purity >95% . (The LCMS chromatogram and conditions may be found within the l-Ala-d/l-Fos1-((R)-3-Chloro-2-(2-(methylammonio)acetamido) propanamido)ethyl)phosphonic acid or Sar-β-chloro- (25b). The tert-butoxycarbonyl and diethyl ester protecting groups of tert-butyl (2-(((2R)-3-chloro-1-((1-(diethoxyphosphoryl)ethyl)amino)-1-oxopropan-2-yl)amino)-2-oxoethyl)(methyl)carbamate (24b)  were removed. The pale green crude solid was recrystallised from hot water/ethanol to give 25b as an off-white solid composed of 2 diastereoisomers, Sar-β-Cl-l-Ala-l-Fos and Sar-β-Cl-l-Ala-D-Fos ; m.p. 185-188 °C (decomp.); ῡmax/cm−1 3287 (NH+), 3000 (br OH), 1657 (C=O), 1634 (C=O), 1552 (NH bend), 1172 (P=O), 1054 (P-O-C), 919 (P-OH); 1H NMR  δH 1.24 , 2.74 , 3.81–3.87 , 3.93–3.94 , 3.97–4.10 , 4.79 ; 13C NMR  δC 15.4 (CH3-2), 32.9 (NCH3-11), 43.6 (CH2-6), 44.1 , 49.5 (CH2-9), 54.6 (CH-5), 166.4 (C=O-4 or C=O-9), 166.9 (C=O-4 or C=O-9); 31P-1Hdecoup NMR  δP 18.8; HRMS (NSI) calcd for (C8H18ClN3O5P)+, MH+: 302.0667 (35Cl), 304.0638 (37Cl), found 302.0670 (35Cl), 304.0640 (37Cl). LCMS purity >95% . (The LCMS chromatogram and conditions may be found within the l-Met-β-Cl-l-Ala-d/l-Fos(1-((R)-2-((S)-2-Ammonio-4-(methylthio)butanamido)-3-chloro propanamido)ethyl)phosphonic acid or  (25c). The tert-butoxycarbonyl and diethyl ester protecting groups of tert-butyl ((2S)-1-(((2R)-3-chloro-1-((1-(diethoxyphosphoryl) ethyl)amino)-1-oxopropan-2-yl)amino)-4-(methylthio)-1-oxobutan-2-yl)carbamate (24c)  were removed. The green crude solid was recrystallised from hot water/ethanol to give 25c as a pale green solid composed of 2 diastereoisomers, l-Met-β-Cl-l-Ala-l-Fos and l-Met-β-Cl-l-Ala-D-Fos ; m.p. 175–179 °C (decomp.); ῡmax/cm−1 3264 (NH+), 2829 (broad OH), 1666(C=O), 1641 (C=O), 1546 (NH bend), 1149 (P=O), 1041 (P-O-C), 921 (P-OH); 1H NMR  δH 1.31 , 2.13 , 2.18–2.29 , 2.63–2.69 , 3.89 , 3.97 , 4.01–4.13 , 4.22 , 4.75–4.79 ; 13C NMR  δC 16.9 (CH3-10″), 17.0 (CH3-10″), 18.4 (CH3-2), 31.1 (CH2-10′), 32.9 (CH2-10), 46.2 (CH2-6), 47.0 , 52.2 (CH-9), 52.3 (CH-9), 57.8 (CH-5), 58.0 (CH-5), 171.7 (C=O-4), 171.8 (C=O-4), 172.3 (C=O-8); 31P-1Hdecoup NMR  δP 18.7; HRMS (NSI) calcd for (C10H21ClN3O5PS), MNa+: 384.0520 (35Cl), 386.0489 (37Cl), found 384.0523 (35Cl), 386.0491 (37Cl). LCMS purity >95% . -glucose were purchased from Sigma Chemical Co. . l-Tyrosine, uracil, guanine, cytosine, adenine, ammonium sulphate, potassium dihydrogen phosphate, saponin, and dipotassium hydrogen phosphate were acquired from BDH Merck Ltd. . Yeast extract was supplied by bioMérieux  and bacteriological agar from Oxoid . Nicotinamide adenine dinucleotide (NAD) was obtained from Merck  and heparinized horse blood from TCS Biosciences .A. baumannii, B. cepacia, E. cloacae, E. coli (n = 2), K. pneumoniae, P. rettgeri, P. aeruginosa, S. typhimurium, S. enteritidis, S. marcescens, and Y. enterocolitica; as well as seven Gram positive bacteria: E. faecalis, E. faecium, L. monocytogenes, S. epidermidis, S. aureus, methicillin resistant S. aureus (MRSA), and S. pyogenes. These 19 bacterial strains were maintained on Columbia agar.Microbial reference strains were obtained from National Collection of Type Cultures (NCTC)  and the American Type Culture Collection (ATCC) . These included twelve Gram-negative bacteria: The methodology utilized, which was reported by Atherton et al. , was adoEach derivative was dissolved in sterile deionized water (SDW) and incorporated with AF-agar at a concentration range of 0.032–8 mg/L. A series of dilutions was performed to achieve the required concentration and then poured into the sterile Petri dishes. Plates containing AF-agar were also prepared without inhibitor. Solidified agar plates were placed in a warm cabinet (37 ± 0.5 °C) for 5 min to dry the surface of the agar and then stored at 4 °C.5 organisms was applied as a spot by this instrument on the plates containing different phosphonotripeptide derivatives (as well as inhibit-free growth control plates). Nineteen different bacterial strains were applied on a plate and incubated for 22 h at 37 ± 0.5 °C. Un-inoculated AF-agar plates from the same batch of agar were incubated as a sterility check and a sterility check was also performed on the SDW used to make bacterial suspensions by culture of aliquots on Columbia agar.Each microbial strain was isolated from the respective Columbia agar after 18 h of incubation and suspended in SDW to a density equivalent to 0.5 McFarland units using a densitometer. One hundred microliters of each suspension was transferred into the corresponded wells of a multipoint inoculation device. One microliter of the bacterial suspension, equivalent to 1.5 × 10After incubation, each plate was inspected for growth or inhibition of the inoculated spots. The minimum inhibitory concentration (MIC) was recorded as the lowest concentration of inhibitor to completely inhibit the growth of the test strain. Tests were performed in duplicate on separate occasions and in the vast majority of cases generated identical results. In a small minority of cases, results differed but were always within one double-dilution and this was always resolved by a third test. MIC values were only interpreted for any particular strain if growth on the inhibitor-free control plate was completely uninhibited and if sterility of the culture medium and diluent were validated.C-terminus. Three of the inhibitors also contain a second antimicrobial agent, β-chloroalanine, at the center of their tripeptide sequence. The identity of the N-terminal amino acid was varied in order to elicit selectivity via exploiting differences in permeability and hydrolysis rates within bacterial cells.Here we have disclosed the synthesis of six phosphonopeptide based inhibitors, all of which contain the antimicrobial agent fosfalin at their We have shown that the β-chloro containing compounds elicit lower MIC values than their alanine analogues against a wide range of clinically relevant bacteria. This is consistent with release of free β-chloroalanine which then is able to act synergistically with the fosfalin unit.25a and 25c have potential for two differing real-world clinical applications. These are the detection of lung pathogens in cystic fibrosis patients, and the detection of Salmonella within stool samples for diagnosis of food poisoning, However, further work is required with large numbers of bacterial strains and relevant clinical samples to conclusively demonstrate the utility of these compounds.Finally, we show that the MIC profiles of inhibitors Antimicrobial Compounds, 2018, WO2015140481.


Oleanane and ursane pentacyclic triterpenoids are secondary metabolites of plants found in various climatic zones and regions. This group of compounds is highly attractive due to their diverse biological properties and possible use as intermediates in the synthesis of new pharmacologically promising substances. By now, their antiviral, anti-inflammatory, antimicrobial, antitumor, and other activities have been confirmed. In the last decade, methods of microbial synthesis of these compounds and their further biotransformation using microorganisms are gaining much popularity. The present review provides clear evidence that industrial microbiology can be a promising way to obtain valuable pharmacologically active compounds in environmentally friendly conditions without processing huge amounts of plant biomass and using hazardous and expensive chemicals. This review summarizes data on distribution, microbial synthesis, and biological activities of native oleanane and ursane triterpenoids. Much emphasis is put on the processes of microbial transformation of selected oleanane and ursane pentacyclic triterpenoids and on the bioactivity assessment of the obtained derivatives. SecondaCurvularia lunata ATCC 13432, Trichotecium roseum ATCC 8685, Cunninghamella sp. ATCC 3229, Mucor griseo-cyanus ATCC 1207-a, Helicostylum piriforme ATCC 8992, Fusarium lini, and Cunninghamella blakesleana dates back to about the same time [Streptomyces sp. G-20 and Chainia antibiotica IFO 12,246 were reported in the second half of the 1980s [Mucor plumbeus ATCC 4740 [Nocardia sp. NRRL 5646 [Naturally occurring triterpenoids are represented by more than 100 various types of skeletons . Native ame time ,24,25. The 1980s ,27. As fTCC 4740 ) and bacRRL 5646 ,31,32.Now, preparation of biologically active compounds based on pentacyclic triterpenoids is an actual research discussed in plenty of experimental and review publications ,32,33,341  and glycyrrhetinic acid 2 , and ursolic acid 3  is the most available ursane triterpenoid. Biosynthesis of oleanane and ursane pentacyclic triterpenoids in plants occurs by conversion of the acyclic triterpene squalene (4) to 2,3-oxidosqualene (5) and its further cyclization by specific enzyme complexes  via β-amyrin (6) or α-amyrin (7), respectively [3-29) located in oleanane and ursane derivatives at C20 and C19 of the E ring, respectively.Plant pentacyclic triterpenoids are represented by more than two dozen structural types; the most common are oleanane and ursane ones . In freeectively ,37. The Meconopsis henrici, Dracocephalum tanguticum, Comastoma pulmonaria, Corydalis impatiens, and Swertia racemosa—traditionally used in Chinese medicine—can reach 0.96 ± 0.01 mg/g and 0.64 ± 0.01 mg/g dry weight, respectively [Ocimum tenuiflorum and Syzygium aromaticum and the herbs Origanum vulgare, Rosmarus officinalis, and Salvia officinalis used as condiments contain up to 15.3 mg/g OA and up to 26.2 mg/g UA (wet weight) [Olea europaea. The acid content in olive leaves can reach 27.16 mg/g wet weight and 25.09 ± 0.72 mg/g dry weight [Glycyrrhiza [Representatives of various higher plant families are active producers of both oleanane and ursane triterpenoids . Frequenectively . Flowers weight) . The maiy weight ,40. GA icyrrhiza ,42,43; tcyrrhiza .Olea europaea) of various varieties contained OA from 0.4 ± 0.1 mg/g to 0.81 ± 0.16 mg/g dry weight and from 29.2 ± 1.8 mg/g to 34.5 ± 3.1 mg/g dry weight, respectively [Vitis vinifera) ripen [The amount of pentacyclic triterpenoids in plants is not constant and can significantly vary depending on the activity of enzyme systems and external factors . Thus, tectively ,47. The ectively . The sama) ripen . Changes4)—a common precursor of triterpenoids—by the squalene synthase (ERG9) based on two molecules of farnesyl diphosphate and its further transformation into 2,3-oxidosqualene (5) by squalene epoxidase (ERG1) [Today, pentacyclic triterpenoids and their natural derivatives are mainly obtained by extraction from plant sources. However, the extraction and separation of these compounds (often with organic solvents) are extremely labor-intensive, and energy- and time-consuming. Besides, most of pentacyclic triterpenoids are found in relatively low concentrations in plants, entailing the use of huge amounts of plant raw materials and the formation of waste biomass in large volumes . An altee (ERG1) . Subsequ6)—a precursor of oleanane pentacyclic triterpenoids—from 2,3-oxidosqualene (5) were isolated from the genomes of Glycyrrhiza glabra, Panax ginseng, Catharanthus roseus, Lotus japonicus, Artemisia annua, Chenopodium quinoa, and others [7), a precursor of ursane pentacyclic triterpenoids, this reaction involves mixed amyrin synthases (mix-AS) that catalyze the formation of both α- and β-amyrin from Eriobotrya japonica and C. roseus [Medicago truncatula is most often used to search for genes encoding CYP450 enzymes that catalyze subsequent conversion of α- and β-amyrin [Phaseolus vulgaris [Bupleurum falcatum [G. uralensis [C. roseus [Crataegus pinnatifida [Solanum lycoperum, P. ginseng [Arabidopsis thaliana [M. truncatula [L. japonicus [G. uralensis [V. vinifera [Genes encoding β-amyrin synthase (βAS) that catalyze the formation of β-amyrin  involved in the natural microbial synthesis of 2,3-oxidosqualene (TRP1 (phosphoribosylanthranilate isomerase), GAL1 , and GAL80  involved in metabolic processes that “distract” cells from biosynthesis of triterpenoids [tHMG1, ERG1, and ERG9 and inactivation of GAL1 and GAL80 in the chromosome of Saccharomyces cerevisiae JDY52 and its use in a 5-L fermenter with 40 g/L glucose resulted in 606.9 ± 9.1 mg/L OA after 144 h. To date, it is the highest yield reported [Yarrowia lipolytica ATCC 201249 with overexpressed ERG1, ERG9, ERG20, and tHMG1 and the inserted expression modules βAS and CYP716A2-linker(GSTSSG)-t46ATR1  provided the yield of 540.7 mg/L OA after 82 h in a 5-L fermenter with 100 g/L glucose [S. cerevisiae JDY52 produced relatively higher OA amounts (606.9 ± 9.1 mg/L) [Y. lipolytica was 6.59 mg/L/h and exceeded that of S. cerevisiae (4.214 mg/L/h).Overexpression of genes squalene  can enharpenoids ,66. Thusreported . In anot glucose . Despite.1 mg/L) , the proS. cerevisiae TM30 and S. cerevisiae TM44 obtained from the same parent strain by including different plasmid variations were evaluated. In the first case, the strain expressing CYP716Y1 and CYP716A12 to obtain a self-processing polyprotein with two enzymes bound via oligopeptide 2A catalyzed the formation of β-amyrin (6), erythrodiol (8), OA, oleanolic aldehyde (9), and 16α-hydroxy-oleanolic aldehyde (10), while the second strain produced two self-processing polyproteins, one consisting of CYP716Y1 and CYP716A12 and the other consisting of AtATR1 and UDP-dependent glycosyl transferase UGT73C11, and catalyzed the formation of 3-O-Glc-echinocystic acid (11) and 3-O-Glc-OA (12).Various techniques of yeast genome modification were also applied to intensify the biosynthesis of triterpenoids. It was shown that the expression of inserted plant genes from low-copy and single-copy plasmids was more effective than that from integrated, high-copy, and multicopy plasmids ,73. In RCYP93E2 orthologs from different species of the family Fabaceae. The obtained strains catalyzed the formation of 24-hydroxy-β-amyrin (13). Almost all strains (except one containing CYP93E7) simultaneously catalyzed the formation of 3β-hydroxy-olean-12-en-24-oic acid (14). The highest conversion (79.4%) of β-amyrin (6) to 24-hydroxy-β-amyrin was observed using saccharomycetes containing CYP93E9 (P. vulgaris) [Artemisia annua and Chenopodium quinoa exhibited the highest catalytic activity (10.8 ± 1.0 mg/L β-amyrin). Comparatively, CYP716AL1 (C. roseus) showed the highest activity (14.3 ± 1.6 mg/L OA) of the 16 CYP716As studied. When a combination of genes encoding these enzymes was used, the OA yield made up 8.5 ± 0.2 mg/L. At the same time, saccharomycetes containing CYP716A75 (Maesa lanceolata), CYP716A79 (C. quinoa), CYP716A110 (Aquilegia coerulea), and CYP716A1  did not catalyze the formation of OA; erythrodiol (8) but not OA was detected in all cases [CYP88D6 (C11 oxidase) for Unigene25647 (97% similarity) and ATR1  for GuCPR1  were performed, and a combination of GuCPR1 and two Unigene25647 cassettes was inserted into the genome of S. cerevisiae SGib. These manipulations resulted in the formation of 18.9 ± 2.0 mg/L GA and about 80.0 mg/L 11-oxo-β-amyrin (15) after 144 h of the fed-batch fermentation with ethanol (30 mL every 24 h) compared with the previously obtained 20.4 ± 7.7 µg/L GA and 0.5 ± 0.1 mg/L 11-oxo-β-amyrin (15) and β-amyrin (6) and trace amounts of 11α-hydroxy-β-amyrin (16), 30-hydroxy-11-oxo-β-amyrin (17), and glycyrrhetaldehyde (18) [S. cerevisiae BY-OA, substitution of CYP716C49 (C. pinnatifida) for the homologue (47.9% similarity) CaCYP716C49 (C. asiatica) allowed for obtaining 384.3 mg/L maslinic acid  after 96-h incubation in a 5-L fermenter with glucose (5 g/L). When S. cerevisiae BY-T3 containing mix-AS, VvCYP716A15, CPR, and CaCYP716C49 from different plant sources were used in the same conditions; corosolic acid  was formed after 144 h [M. truncatula was studied in Reference [21), gypsogenic acid (22), or 11-deoxo-GA (23). Importantly, saccharomycetes expressing CYP716A12 and CYP93E2 catalyzed the formation of 4-epi-hederagenin (24), and the yeasts expressing CYP716A12 and CYP72A63 catalyzed the formation of queretaroic acid (25). These compounds were not previously detected in M. truncatula tissues, indicating the great potential of combinatorial biosynthesis using microorganisms [The most promising research area in this field is combinatorial biosynthesis. Change in the natural biosynthesis pathway or combination of synthesis genes from different sources leads to enhanced efficiency of microbial biosynthesis or to the formation of new compounds. The Moses group (2014) studied the C24-oxidizing activity of ulgaris) . Dale etll cases . Substityde (18) . In straer 144 h . The acteference . The mairganisms .Betula pendula exhibited antibacterial activity against test cultures Staphylococcus aureus and Bacillus subtilis [Baccharis uncinella containing OA and UA showed antiparasitic activity by limiting the growth of promastigote and amastigote forms of Leishmania amazonensis and enhanced the immune response in infected mice [Glycyrrhiza uralensis root extract inhibited TNF-α-induced activation of NF-κB in HepG2 cells [Potentilla fulgens root extract containing ursane triterpenoids showed antioxidant activity by inhibiting the production of free radicals [Fragaria ananassa perianth containing various triterpenoids had a pronounced cytotoxic effect on B16-F10 melanoma cells and inhibited their melanogenesis by 79.1% and 80.2%, respectively [Alstonia scholaris was also due to high UA content (2.5 ± 0.6 mg/g dry weight of leaves) [Extracts obtained from plant sources using various solvents and containing the pentacyclic triterpenoids reviewed in this paper usually exhibit a wide range of biological properties. Methanolic OA-containing extracts from various parts of subtilis . Methanoted mice . The ethG2 cells . The ethradicals . An alcoectively . The all leaves) .S. aureus  1.25%) and B. subtilis (MIC 0.625%) [Pseudomonas aeruginosa cells and the level of biofilm formation. This can make a significant contribution to the development of effective antibiotic-free therapy for Pseudomonas infections [Mycobacterium tuberculosis in vitro [in silico studies [Isolation of every triterpenic acid individually allows studying their bioactive properties in detail and explaining the pharmacological properties of some plants. The antibacterial property of birch bark was determined by a high content of pentacyclic triterpenoids, in particular, OA, which exhibits a pronounced antibacterial activity against  0.625%) . Antimicfections . UA was in vitro  and the in vitro . In addi studies ,78.Fragaria ananassa perianth on B16-F10 melanoma cells was determined partly by the presence of cytotoxic UA that suppressed the melanin production by 40.2% [OA, as an antitumor agent, increased the sensitivity of sarcoma cells to chemotherapeutic drugs in human soft tissues . UA had by 40.2%  and causiNOS suppression, and COX-2 expression and by decreasing the activity of NF-κB transcription factor in HepG2 cells [The OA hepatoprotective activity was shown to be related to its inhibitory effect against carboxylesterase (therapeutic target for hypertriglyceridemia) and the hepatitis C virus (HCV) ,82. GA eG2 cells . The abiG2 cells . UA coulG2 cells . OA and G2 cells .26) and methyl 3,25-epoxy-3α-hydroxy-11-oxo-22β-senecioyloxyolean-12-en-28-oate (27) isolated from Lantana camara herb extract demonstrated antibacterial activity against a number of gram-positive and gram-negative bacteria [Along with their parent acids, the native derivatives also exhibit pronounced biological activities. Thus, the OA derivatives 22β-acetoxy-3,25-epoxy-3α-hydroxyolean-12-en-28-oic acid (bacteria .28‒30) isolated from Chrysosplenium carnosum exhibited pronounced inhibitory activities against the mouse melanoma cell lines B16F10 and SP2/0 [20, corosolic acid) had cytotoxic effects on CaSki cells  by inducing apoptosis, by arresting the cell cycle in the G2/M phase, and by inhibiting the PI3K/Akt signaling pathway [31) from Siphonodon celastrineus showed pronounced cytotoxic activity against MOLT-3 cancer cells [Oleanane 27-carboxy derivatives  and Astilbe rivularis (3β-trans-p-coumaroyloxy-olean-12-en-27-oic acid (35) and 6β-hydroxy-3-oxoolean-12-en-27-oic acid (36)) as well as oleanane and ursane polyhydroxylated derivatives (37–40) from Rosa laevigata exhibited anti-inflammatory activities [Durio zibethinus were shown to exhibit a more pronounced anti-inflammatory effect in LPS-induced NO production in RAW 264.7 cell inhibition tests compared to oleanane triterpenoids, for which the activity was reduced by C2 hydroxylation [Oleanane derivatives from tivities ,89,90 duCecropia telentida root extracts against 11β-hydroxysteroid dehydrogenase could be determined by the presence of a new, probably 2α,20β-dihydroxylated UA derivative  [42) and B (43) isolated from Potentilla fulgens exhibited antioxidant effect by inhibiting the formation of free radicals [Bupleurum chinense and B. scorzonerifolium species that are widespread in China were partially provided by the presence of bioactive oleanane saikosaponins (44–46) [The pronounced inhibitory activity of ic acid) . Ursane radicals . Antivir (44–46) .Due to the wide distribution and availability of the pentacyclic triterpenoids discussed in the review, they are often used to synthesize new semi-synthetic derivatives with antibacterial , antivir1–3. However, chemical methods often require extreme acidity and temperature values, expensive catalysts, or protective groups of molecule reactive centers [Taking into account the relative availability of the discussed triterpenic acids in natural sources and their high bioactivity, it is interesting to assess the possibility of directed transformations of these compounds to expand the range of biologically active compounds and to increase their bioavailability. Chemical methods are currently the most tested and used to transform acids  centers ,9,10,106 centers .One of the most promising ways to highlight the pharmacological potential of native pentacyclic triterpenoids is the functionalization of their molecules by polyhydroxylation. Such functionalized derivatives hydroxylated by plant P450-dependent monooxygenases  are wideAscomycota  and Mucoromycota . Fungal conversions of these compounds are accompanied by the formation of derivatives with hydroxyl groups at C1, C7, C15, C21, C24, or C30; oxo groups at C3, C7, or C21; glucopyranoside groups at C3, C28, or C30; lactone groups at C28/C13 or C3/C4, etc. as well as by the A ring fragmentation. The acid concentration used in biotransformation experiments usually ranges from 0.02 g/L to 1.0 g/L. The yield of transformation products (1.0% to 77.5%) and the duration of the processes (2 to 20 days) vary depending on the fungal catalyst characteristics  for 13 days to form of 1β,30-dihydroxy-OA (47), 7β,30-dihydroxy-OA (48), and 30-hydroxy-OA (25) in equivalent amounts (5.0‒6.0%) [25, known as queretaroic acid, was first isolated from Lemaireocereus queretaroensis and L. beneckei endemic to Mexico [25 (3.3%) was obtained by the 24-h transformation of OA (0.2 g/L) by Escherichia coli cells expressing Nonomuraea recticatena CYP450 moxA and Pseudomonas redox partner camAB. At the same time, the use of cell-free reaction systems allowed to increase the yield of compound 25 to 17.0% [R. miehei CECT 2749 during 13 days was accompanied by 7,30-dihydroxylation and, in addition, a 9(11),12-diene moiety formation in the C ring, producing 15.0% methyl 3β,7β,30-trihydroxy-oleane-9(11),12-dien-28-oate (49) [Abundantly found in nature, .0‒6.0%) . Compouno Mexico . This coo Mexico . Queretato 17.0% . Transfoate (49) .Colletotrichum lini AS 3.4486 was shown to catalyze the C15 hydroxylation of OA to form 15α-hydroxy-OA (50) [Trichothecium roseum (M 95.56) [Circinella muscae AS 3.2695 [51) on day 6 (7.5%) and day 7 (6.1%), respectively. At the same time, an intermediate (6.25%) of the dihydroxylation process—15α-hydroxy-3-oxo-olean-12-en-28-oic acid (52)—was also isolated from the culture medium of T. roseum (M 95.56) [C. muscae AS 3.2695 simultaneously catalyzed a wide variety of hydroxylation and glycosylation reactions with the formation of 7β-hydroxy-OA (53), 7β,21β-dihydroxy-OA (54), 7α,21β-dihydroxy-OA (55), 7β,15α-dihydroxy-OA (56), 7β-hydroxy-3-oxo-olean-12-en-28-oic acid (57), 7β,15α-dihydroxy-OA 28-O-β-d-glucopyranosyl ester (58), 21β-hydroxy-OA 28-O-β-d-glucopyranosyl ester (59), and OA 28-O-β-d-glucopyranosyl ester (60) ranging from 3.1% to 5.8% [-OA (50) . In turnM 95.56)  and mucoM 95.56) . C. muscC. muscae AS 3.2695 was also active against GA (approximately 0.06 g/L) and initiated oxidation, acetylation, and glycosylation reactions with the formation of metabolites (yield did not exceed 2.4%) on day 7. The metabolites included 7β-hydroxy-GA (61), 15α-hydroxy-GA (62), 7β,15α-dihydroxy-GA (63), 3,11-dioxo-7β-hydroxy-18β-olean-12-en-30-oic acid (64), 7β,15α-dihydroxy-3,11-dioxo-18β-olean-12-en-30-oic acid (65), 7β-hydroxy-11-oxo-18β -olean-12-en-30-oic acid 3-O-β-d-glucopyranoside (66), 7β-hydroxy-11-oxo-18β-olean-12-en-30-oic acid 3-O-β-d-6′-O-acetyl-glucopyranoside (67), 15α-hydroxy-11-oxo-18β-olean-12-en-30-oic acid 3-O-β-d-glucopyranoside (68), 15α-hydroxy-11-oxo-18β-olean-12-en-30-oic acid 3-O-β-d-6′-O-acetyl-glucopyranoside (69), and 7β-hydroxy-GA 30-O-β-D-glucopyranoside (70) [61 and 64 were shown to exhibit antimicrobial activity against the antibiotic-resistant strain Enterococcus faecalis [63 exhibited antioxidant and hepatoprotective properties [ide (70) . The abofaecalis , while coperties .Rhizopus arrhizus AS 3.2893 to perform oxidative transformation of GA (approximately 0.06 g/L) at C3, C7, and C15 for 7 days with the formation of 7β-hydroxy-GA (61), 15α-hydroxy-GA (62), 7β,15α-dihydroxy-GA (63), 3β-acetoxy-7β-hydroxy-11-oxo-18β -olean-12-en-30-oic acid (71), 7-oxo-GA (72), 7α-hydroxy-GA (73), and 15α-hydroxy-7-oxo-GA (74) (the yield of each compound did not exceed 2.8%). They also exhibited anti-inflammatory effects in the LPS-induced NO production inhibition test in RAW 264.7 cells [The same authors reported .7 cells .63) was also catalyzed by C. lini AS 3.4486 [Absidia pseudocylinderospora ATCC 24169 [A. pseudocylinderospora ATCC 24169, during long-term cultivation (14 days), catalyzed the formation of more than 18.0% of this compound (63) possessing antioxidant, hepatoprotective [Biological hydroxylation of GA at concentrations of 0.25 g/L (48 h) and 1.0 g/L (14 days) at C7 and C15 producing the main metabolite 7β,15α-dihydroxy-GA , 15α,24-dihydroxy-3,11-dioxo-18β -olean-12-en-30-oic acid (76), 7β,24-dihydroxy-3,11-dioxo-18β-olean-12-en-30-oic acid (77), 3,11-dioxo-7β,15α,24-trihydroxy-18β-olean-12-en-30-oic acid (78), and 7α,24-dihydroxy-3,11 -dioxo-18β-olean-12-en-30-oic acid (79). The yield of each acid did not exceed 1.3% [75, 78, and 79 were found to effectively inhibit LPS-induced NO production in mouse microglia cells with IC50 values of 0.76 mmol/L, 0.94 mmol/L, and 0.16 mmol/L, respectively [C. blakesleana AS 3.970 was used, an increase in GA concentration to 0.3 g/L led to accumulation of two main products 7β-hydroxy-GA  and 3,11-dioxo-7β-hydroxy-18β-olean-12-en-30-oic acid  with pronounced antibacterial activity after 5 days [C. elegans TSY-0865, only 2.5% of 7β-hydroxy-GA (61) was formed during 8 days [Preparative biotransformation of 0.1 g/L GA by eed 1.3% . Compounectively . Interesr 5 days . In the g 8 days .Syncephalastrum racemosum CGMCC 3.2500 transformed approximately 0.1 g/L UA to 7β,21β-dihydroxy-UA , 1β,21β-dihydroxy-UA , 1β-hydroxy-21-oxo-UA , 3β,21β-dihydroxy-urs-11-en-28,13-olide , and 3β,7β,21β-trihydroxy-urs-11-en-28,13-olide  within 10 days [S. racemosum AS 3.264 [UA biotransformation processes include oxidation and lactone formation reactions. Thus, AS 3.264 . UA deriGliocladium roseum CGMCC 3.3657 catalyzed oxidative transformation of the A ring of UA (0.1 g/L) by Baeyer–Villiger-type reaction and C21 oxidation to form 21β-hydroxy-3-oxo-urs-12-en-3,4-olide-28-oic acid , 3,21-dioxo-urs-12-en-3,4-olide-28-oic acid , 21β-hydroxy-3,4-seco-ursane-4(23),12-dien-3,28-dioic acid , and 21-oxo-3,4-seco-ursane-4(23),12-dien-3,28-dioic acid . Derivatives 86 and 88 containing a 21-oxo group showed the most pronounced anti-HCV activity [For 20 days, activity .Mucor spinosus AS 3.3450 was used to bioconvert UA, three metabolites, 7β-hydroxy-UA 28-ethanone , 7β,21β-dihydroxy-UA , and 21β-hydroxy-urs-12-en-28-oic acid 3-O-β-D-glucopyranoside , were formed within 96 h. Compound 89 was shown to exhibit pronounced (higher than that of UA) cytotoxic activity against HeLa, K562, and KB tumor cell lines [When ll lines .Bacillus, Nocardia, and Streptomyces and accompanied by the formation of C1, C2, C7, C11, C21, C24, or C29 hydroxylated derivatives, derivatives with a methyl ester group at C28; oxogroup at C3; additional carboxyl groups at C29 or C30; glucopyranoside groups at C3, C28, or C30; lactone group at C28/C13; and derivatives with a fragmented A ring. In biotransformation experiments, the compounds are usually used in concentrations ranging from 0.04 g/L to 0.3 g/L, and the yield of derivatives ranges from 5.0% to 60.0%. The duration of bioconversion is 3 to 5 days; only in the case of using Nocardia, it reached 13 days  transformation process for 13 days resulted in the formation of methyl OA (91) as the main bioconversion product (more than 60.0%), small amounts (≤5.0%) of methyl 3-oxo-olean-12-en-28-oat (92), and metabolite 93 unidentified by the authors [92) was shown to have pronounced antimelanoma [Nocardia cells in alginate carriers led to a decrease in their catalytic activity, as confirmed by a 10-fold decrease in the formation of compound 91 and only a short-term occurrence of compound 93 in the culture medium [Nocardia sp. to transform UA by methylation, by C3 oxidation, and by formation of the enone moiety in the A ring was previously shown. It was noted that the biotransformation process did not depend on the composition of the culture medium used, while the temperature increase (from 28 °C to 36 °C) for actinobacteria cultivation contributed to a 2-fold increase in the reaction rate [Actinobaceria of the genus rpenoids . The use authors . 3-oxo-Omelanoma , antileimelanoma . Despitemelanoma , the usee medium . The abiion rate .Streptomyces griseus ATCC 13273 catalyzed hydroxylation and site-selective oxidation of the C29 methyl group of OA (0.04 g/L) to the carboxyl group within 5 days to form 3β-hydroxy-olean-12-ene-28,29-dioic acid , 3β,24-dihydroxy-olean-12-ene-28,29-dioic acid , and 3β,21β,24-trihydroxy-olean-12-ene-28,29-dioic acid . Hydroxylation at C21 was shown to increase the anti-inflammatory activity of OA derivatives [94 and 96 was previously described by Y. Zhu et al. [The bacterial culture of ivatives . Using tu et al. .S. griseus ATCC 13273 cells can also transform UA (0.04 g/L) by catalyzing site-selective oxidation of the C30 methyl group to the carboxyl one and C24-hydroxylation within 3 days to produce 3β-hydroxy-urs-12-ene-28,30-dioic acid (97) and 3β,24-dihydroxy-urs-12-ene-28,30-dioic acid (98), with the product yield exceeding 30.0%. Transformation of UA derivatives 3-oxo-UK (99) and 2α-hydroxy-UA  at a concentration of 0.04 g/L by this actinobacterial strain also occurred by selective C30-oxidation and hydroxylation, that led to a mixture of 3-oxo-urs-12-ene-28,30-dioic acid  and 24-hydroxy-3-oxo-urs-12-ene-28,30-dioic acid  in the former case and a mixture of 2α,3β-dihydroxy-urs-12-ene-28,30-dioic acid  and 2α,3β,24-trihydroxy-urs-12-ene-28,30-dioic acid  in the latter case [In addition to OA, ter case .Bacillus megaterium CGMCC 1.1741 was able to transform UA (0.2 g/L), generating the main derivative 1β,11α-dihydroxy-UA  and minor derivatives (5.03–13.50%) 3-oxo-urs-12-en-28-oic acid (99), 1β,11α-dihydroxy-3-oxo-urs-12-en-28-oic acid (105), 1β-hydroxy-3-oxo-urs-12-en-28,13-olide (106), and 1β,11α-dihydroxy-3-oxo-urs-12-en -28-O-β-D-glucopyranoside (107) over 4 days. Derivatives 105 and 106 were shown to effectively inhibit LPS-induced NO release in RAW 264.7 cells  [99 was shown to inhibit cathepsin L-like rCPB2.8 protease of Leishmania mexicana [A gram-positive ctively) , and dermexicana .Rhizopus chinensis CICC 40335 and bacterial strains Bacillus subtilis ATCC 6633 and Streptomyces griseus ATCC 13273 [R. chinensis CICC 40335 occurred within 4 days by the formation of 7β,21β-dihydroxy-OA  previously obtained using the fungal strains Mucor rouxii NRRL 1894 [Circinella muscae AS 3.2695 [54) led to the formation of 7β,21β-dihydroxy-olean-12-en-28-oic acid 3-O-β-D-glucopyranoside  using B. subtilis ATCC 6633 cells and a mixture of 7β,21β,29-trihydroxy-OA  and 3β,7β,21β-trihydroxy-olean-12-ene-28,29-dioic acid  using S. griseus ATCC 13273 cells [Xu et al. (2020) have recently used the tandem biotransformation of oleanane-type pentacyclic triterpenoids using the fungal strain CC 13273 . The priRRL 1894  and CircS 3.2695 . Further73 cells .R. chinensis CICC 40335 occurred by selective oxidation with the formation of 7β-hydroxy-GA  on day 4 [C. muscae AS 3.2695, Rhizopus arrhizus AS 3.2893 [Cunninghamella [61 in the culture medium of B. subtilis ATCC 6633 led to the formation of 30-O-β-d-glucopyranoside derivatives (111 (27.5%) and 68 (44.0%), respectively) previously obtained by B. Fan et al. [Bioconversion of GA (0.2 g/L) using on day 4 . Note thS 3.2893 , and repghamella ,119. Furn et al. . Assessmn et al. .de novo DNA synthesis significantly simplified the abovementioned processes and gave a new impetus to research in this field [Triterpenoids are secondary metabolites of plants, fungi, marine invertebrates, and algae that are formed during cyclization of an acyclic triterpene squalene ,137,138.is field . With this field ,74,82,83is field ,94,141.Studies of the possibility of obtaining new OA, GA, and UA derivatives by directed biotransformations should be considered a promising area. Over 20 examples of biotransformations of these compounds using fungal and bacterial cultures most often catalyzing hydroxylation have been described since 2013. Less frequently, the literature describes processes of deeper oxidation of triterpenoids as well as their glycosylation, esterification, acetylation, or carboxylation. Biocatalytic formation of triterpenic lactones or their derivatives with fragmented C–C bond was reported only in a few cases using UA . In the