Supporting Information. Total Synthesis and Stereochemical Revision of the Anti- Tuberculosis Peptaibol Trichoderin A

Transcription

1 Supporting Information Total Synthesis and Stereochemical Revision of the Anti- Tuberculosis Peptaibol Trichoderin A Iman Kavianinia,, Lavanya Kunalingam, Paul W. R. arris,, Gregory. M. Cook, ǁ, and Margaret A. Brimble *,,, School of Biological Sciences, The University of Auckland, 3A Symonds Street, Auckland 1010, ew Zealand Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, 3A Symonds Street, Auckland 1010, ew Zealand School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1010, ew Zealand ǁ Department of Microbiology and Immunology, School of Medical Sciences, University of tago, 720 Cumberland Street, Dunedin 9054, ew Zealand S1

2 Table of Contents General Method and Materials... S1 Synthesis of Building Blocks... S4-S10 General Procedure for Peptide Synthesis... S11-S12 Total Synthesis of Synthesis of Trichoderin A... S13-S23 Antimicrobial Screening... S24 Stereochemical Assignment... S and 13 C Spectra... S31-S42 References... S43 S2

3 General Method and Materials All reagents were purchased as reagent grade and used without further purification. Solvents for reactions were dried according to standard procedures.,-diisopropylethylamine (DIPEA), piperidine,, -diisopropylcarbodiimide (DIC), 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU),,,, - tetramethylfluoroformamidinium hexafluorophosphate (TFF) and ninhydrin were purchased from Sigma-Aldrich (St. Louis, Missouri). -(7-Azabenzotriazol-1-yl)-,,','-tetramethyluronium hexafluorophosphate (ATU), -(9-fluorenylmethoxycarbonyloxy) succinimide (Fmoc-Su), di-tert-butyl dicarbonate (Boc 2 ), Fmoc-Pro-, Fmoc-Val-, Fmoc-Ile-, Fmoc-Ala- and Fmoc-Leu- were purchased from GL Biochem (Shanghai, China). 2- Chlorotrityl chloride Polystyrene resin and ethyl 2-cyano-2-(hydroxyimino)acetate (xyma) were purchased from ovabiochem (Merck, Germany). Fmoc-α-aminoisobutyric acid (Aib) was purchased from CS Bio (Shanghai, China). 1-[(1-(Cyano-2-ethoxy-2- oxoethylideneaminooxy)-dimethylamino morpholinomethylene)] methanaminium hexafluorophosphate (CMU), (benzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBoP) ), 6-chloro-1-hydroxybenzotriazole (6-Cl-Bt) were purchased from Aapptec (Louisville, Kentucky). Analytical thin layer chromatography was performed using aluminium-backed silica plates, and compounds were visualized by ultraviolet fluorescence or by staining with potassium permanganate, ninhydrin solution, followed by heating the plate as appropriate. Infrared spectra were obtained on an FTIR spectrometer as neat samples and absorption maxima are expressed in wavenumbers (cm -1 ). Melting points were recorded on an electrothermal melting point apparatus and are uncorrected. ptical rotations were determined at the sodium D line (589 nm) at 20 C on a Perkin Elmer 341 instrument. igh resolution mass spectra (RMS) were recorded on a Bruker (Billerica, MA) micrtfq mass spectrometer. uclear magnetic resonance (MR) spectra were recorded as indicated on either a Bruker AVACE 400 spectrometer ( 1, 400 Mz; 13 C, 100 Mz), or a Bruker AVACE 600 spectrometer ( 1, 600 Mz, 13 C, 150 Mz). Chemical shifts are reported in parts per million (ppm) from tetramethylsilane (δ = 0) and were measured relative to the solvent in which the sample was analysed (CDCl 3 : δ 7.26 for 1 MR, δ 77.0 for 13 C MR; CD 3 D: δ 3.3 for 1 MR, δ 49.0 for 13 C MR) The 1 MR shift values are reported as chemical shift (δ ), the corresponding integral, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, td = triplet of doublets, qd = quartet of doublets), coupling constant (J in z) and assignments. 13 C MR values are reported as the chemical shift (δ C ), the degree of hybridisation and assignment. Semi-preparative RP-PLC was performed on a Thermo Scientific (Waltham, MA) Dionex Ultimate 3000 PLC equipped with a four channel UV Detector at 210, 225, 254 and 280 nm using either an analytical column [Waters (Milford, MA) XTerra MS C18, mm, 5 µm] at a flow rate of 1 ml min -1 or a semi-preparative column (XTerra MS C18, 10 x 250 mm 5 µm) at a flow rate of 5 ml min -1. A suitably adjusted gradient of 5% B to 95% B was used, where solvent A was 0.1% formic acid in 2 and B was 0.1% formic acid in acetonitrile. LC-MS spectra were acquired on either an Agilent Technologies (Santa Clara, CA)1120 Compact LC equipped with a ewlett-packard (Palo Alto, CA) 1100 MSD mass spectrometer or an Agilent Technologies 1260 Infinity LC equipped with an Agilent Technologies 6120 Quadrupole mass spectrometer. An analytical column (Agilent C3, 150 mm x 3.0 mm, 3.5 µm) was used at a flow rate of 0.3 ml min -1 using a linear gradient of 5% B to 95% B over 30 min, where solvent A was 0.1% formic acid in 2 and B was 0.1% formic acid in acetonitrile. S3

4 Synthesis of Building Blocks Syntheses of (R)-2-methyldecanoic acid 2 Scheme S2. Synthesis of (2R)-MDA 2 (R)-3-Decanoyl-4-phenyloxazolidin-2-one S3 To a solution of (R)-4-phenyloxazolidin-2-one S1 (1.0 g, 6.13mmol) in anhydrous TF (20 ml) cooled to -78 C, n-buli (3.15 ml, 6.25 mmol) was added dropwise followed by decanoyl chloride S2 (1.4 ml, 6.74 mmol). The solution was warmed to 0 C and stirred for another hour at this temperature. Saturated 4 Cl solution was then added slowly and the mixture was extracted with C 2 Cl 2 (3 x 30 ml). The combined organic layers were washed with saturated ac 3 solution, brine, dried over MgS 4 and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (hexanes/etac, :1) to give 1.82 g (5.74 mmol, 94%) of the desired product as a colourless oil. R f : 0.49 (hexane/etac, 6:1); [ ] α (c 1.00, C 2 Cl 2 ) = v max (neat)/cm -1 : 2794, 2339, 2118, 1449, 780; 1 -MR (CDCl 3, 400 Mz) δ (ppm): (m, 5, Ar-), 5.42 (dd, 1, J = 3.7 z, J = 8.7 z, -5), 4.68 (t, 1, J = 8.9 z,-4), 4.27 (dd, 1, J = 3.8 z, J = 8.1 z, -5), (m, 2, decanoyl -2), (m, 1, decanoyl -3), (m, 13, decanoyl 6 x C 2 ), 0.87 (t, 3, J = 6.9 z, decanoyl - 10). 13 C-MR (CDCl 3, 100 Mz) δ (ppm): (C, C), (C, C-2), (C, Ar-C), 129.2, 128.7, (C, 5x Ar- C), 69.9 (C 2, C-5), 57.6 (C, C-4), 35.5 (C, decanoyl C-2 ), 31.9, 29.4, 29.3, 29.2, 29.0, 24.1 (C 2, 6 x decanoyl C 2 ), 22.6 (C 2, decanoyl C-9), (C 3, decanoyl C-10). RMS (EI): m/z [M + a] + calculated for C a , observed: D S4

5 3-[(R)-2-Methyldecanoyl]-4-(R)-phenyloxazolidin-2-one S4 A solution of (R)-3-decanoyl-4-phenyloxazolidin-2-one S3 (1.8 g, 5.56 mmol) in anhydrous TF (40 ml) was added to amds (7.37 ml, 7.37 mmol, 1 M in TF) dropwise at -78 C. After 1 hour MeI (1.7 ml, 28.3 mmol) was added and the solution was stirred for 4 hours at -78 C. The reaction was quenched by addition of saturated 4 Cl solution and the aqueous layer was extracted with C 2 Cl 2 (3 x 40 ml). The organic layer was washed with 1 M a 2 S 3 solution and brine, dried over MgS 4 and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (hexanes/etac, 7:1) to give 1.2 g (3.62 mmol, 64%) of the desired product as a colourless oil R f : 0.76 (hexanes/etac 5:1); [ ] α (c 1.00, C 2 Cl 2 ); v max (neat)/cm , 2159, 1779, , 1226, 1059, 756, D 699, MR (CDCl 3, 400 Mz) δ (ppm): (m, 5, Ar-), 5.43 (dd, 1, J = 3.7 z, J = 8.7 z, -5 ), 4.67 (t, 1, J = 8.9 z, 4), 4.24 (dd, 1, J = 3.7 z, J = 8.8 z, -5), (m, 1, -2), (m, 1, decanoyl -3), (m, 13, -3b, 6 x decanoyl C 2 ), 1.09 (d, 3, J = 6.9 z, C 3 ), 0.87 (t, 3, J = 6.8 z, decanoyl -10); 13 C-MR (CDCl 3, 100 Mz) δ (ppm): (C, C), (C, C-2), (C, Ar-C), 129.3, 128.8, (C, 5x Ar-C), 69.9 (C 2, C-5), 57.9 (C, C-4), 37.9 (C, decanoyl C-2), 33.2 (C 2, decanoyl C-3), 32.0, 29.8, 29.6, 29.4, 27.4, 22.8 (C 2, 6 x decanoyl C 2 ), 17.5 (C 3 ), (C 3, 6 x decanoyl C-10). RMS (EI): m/z [M + a] + calculated for C a , observed: R-2-Methyldecanoic acid 2 To a solution of S4 (630 mg, 1.90 mmol) in TF/water (36 ml, 3:1) was added Li (155 mg, 3.80 mmol) and 2 2 (1.4 ml) at 0 C. After stirring for 4 hours at 0 C the reaction was quenched by addition of 1 M a 2 S 3 solution and the mixture was extracted with C 2 Cl 2 (2 x 20 ml). The aqueous layer was acidified with 1 M Cl until p 1 was achieved and extracted with C 2 Cl 2 (3 x 20 ml). The organic layer was dried over MgS 4 and the solvent was removed under reduced pressure to give 311 mg (1.90 mmol, 88%) of the desired product as a yellow oil [ ] α : (c 1.00, Me) (lit c 1.00, Me); v max (neat)/cm -1 : 2929, 2854, 1704, 1456, 1239, MR (CDCl 3, D 400 Mz) δ (ppm): δ (br, 1,), (m, 1, -2), (m, 1-h-3a), (m, 1-h-3a), (m, 12, 6 x C 2 ), 1.18 (d, 3, J = 7 z, C 3 ), 0.88 (t, 3, J = 6.8 z, -10). 13 C-MR (CDCl 3, 100 Mz) δ (ppm): δ (C, C), 39.3 (C, C-2), 33.5 (C2, C-3), 31.8, 29.5, 29.4, 29.2, 27.1, 22.6 (C 2, 6 x C 2 ),16.8 (C 3 ), 14.1(C 3, 6 x C-10). This data was in accordance with that reported in the literature. 1 S5

6 Synthesis of (S)-2-[(2'-aminopropyl)methylamino]ethanol 3 Scheme S3. Synthesis of AMAE 3 (S)-2-[(tert-Butoxycarbonyl)amino]propanol p-toluenesulfonate S6 To a solution of Boc-L-alaninol (5.0 g, 28.5 mmol) in C 2 Cl 2 (150 ml) at 0 C was added Et 3 (11.9 ml, 85.5 mmol), 4- toluenesulfonyl chloride (8.16 g, 42.8 mmol) and DMAP (0.70 g, 5.7 mmol), and the mixture stirred at room temperature overnight. The organic phase was then sequentially washed with aqueous citric acid, aqueous sodium bicarbonate, then brine, dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. Purification by column chromatography (petroleum ether/etac, 10:1) afforded the compound S6 as a white solid (7.0 g, 21.3 mmol, 75%) [ α] R f : 0.4 (petroleum ether/ethyl acetate, 10:1); (c 1, CCl 3 ) (lit c 1, CCl 3 ); 1 MR (400 Mz, CDCl3) δ D (ppm): 7.74 (d, 2, J = 8.4 z, Ar), 7.30 (2, d, J = 8.4 z, Ar), 4.67 (br, s, 1, ), (3, m, CC 2 Ts), 2.40 (3, s, C64-C 3 ), 1.36 (9, s, C(C 3 ) 3 ), 1.11 (3, d, J = 6.8 z, C 3 ), 13C MR (100 Mz, CDCl3) δ (ppm): (C, CC(C 3 ) 3 ), (C, Ar-C), (C, Ar-C), (C, Ar-C), (C, Ar-C), 79.8 (C, C(C 3 ) 3 ), 72.7 (C2), 45.5 (C1), 28.5 (C 3, C(C 3 ) 3 ), 21.8 (Ar-C 3 ), 17.3 (C 3 ). RMS (EI): m/z [M + a] + calculated for C₁₅₂₃a₅S, , observed: This data was in accordance with that reported in the literature. 2 (S)-2-[(2'-(tert-Butoxycarbonyl)aminopropyl)methylamino]ethanol S7 S6

7 A solution of S6 (5.0 g, 15.2 mmol) and 2-(methylamino)ethanol (6.1 ml, 76 mmol) in DMF (75 ml) was heated at 50 C for 3 d, and the solvent removed in vacuo. The residue was then taken up in brine and extracted (5x) with C 2 Cl 2, dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo. Purification by column chromatography (C 2 Cl 2 /Me, 20:1) afforded the compound S7 as a colourless oil (2.5 g, 10.8 mmol, 71%) S7: R f :0.55 (10% ( 3 :Me:EtAc, 1:9:90)); [ ] α : -6.6 (c 1.00, Me); v max (neat)/cm , 2979, 2506, 1975, 1683, D 1527, 1365, 1247, 1167, 1053, 1031, MR (CDCl 3, 400 Mz) δ (ppm): 4.60 (d, 1, J = 7.2 z, ), (m, 1, -2), 3.55 (t, 2, J = 5.3 z, -2'), 3.10 (bs, 1, ), (m, 2, -1'), (m, 1, -1a), (m, 1, -1b), 2.26 (s, 3, C 3 ), 1.40 (s, 9, C(C 3 ) 3 ), 1.10 (d, 3, J = 6.7 z, C 3 ); 13 C-MR (CDCl 3, 100 Mz) δ (ppm): ((C, CC(C 3 ) 3 ), 79.3 (C, C(C 3 ) 3 ), 63.9 (C 2, C-1), 59.6 (C 2, C-1'),, 58.7 (C 2, C-2'), 44.9 (C, C-2), 42.4 (C 3 ), (C 3, C(C 3 ) 3 ), 19.4 (C 3 ). RMS (EI): m/z [M + a] + calculated for C₁₁₂₄₂a₃, , observed: (S)-2-[(2'-Aminopropyl)methylamino]ethanol 3 A solution of S7 (2.5 g, 10.8 mmol) in TFA/ C 2 Cl 2 (50 ml, 1:1 v/v) was stirred at room temperature for 2 h, and the solvent removed in vacuo. Purification by column chromatography (C 2 Cl 2 /Me/ 4, 10:1:0.25) afforded 3 as a colourless oil (1.25 g, mmol, 72%). R f : 0.2 (C 2 Cl 2 /Me/ 4, 10:1:0.25), staining with ninhydrin; [ ] α +37 (c 0.5, Me); 1 MR (400 Mz, d 4 -Me) δ (ppm): (2, m, -2'), (m, 1, -2), (1, m, 1'a), (1, m, 1'b), 2.24 (3, s, C 3 ), 2.19 (2, d, J = 6.5 z, 1), 0.99 (3, d, J = 6.5 z, C 3 ). 13 C MR (100Mz, d 4 -Me) δ (ppm): 68.2 (C 2, C-1), 62.0 (C 2, C-1 ), 61.3 (C 2, C-2'), 46.1 (C, C-1), 44.2 (C 3 ), 21.5 (C 3 ). ). RMS (EI): m/z [M + a] + calculated for C₆₁₆₂a, , observed: D S7

8 Synthesis of (S)-(9-fluoren-9-yl)methyl-1-[methyl(2-hydroxyethylamino]propan-2-yl-carbamate 7 Scheme S1. Synthesis of Fmoc-Protected AMAE 7 2-(tert-Butyldimethylsilyloxy)--methylethanamine S9 A mixture of -methylethanolamine S8 (2.0 g, 26.6 mmol), imidazole (2.53 g, 37.3 mmol) and TBSCl (4.4 g, 29.3 mmol) in anhydrous C 2 Cl 2 (30 ml) was stirred at room temperature for 24 h. After that 2 (30 ml) was added and the aqueous layer was extracted with C 2 Cl 2 (3 x 30 ml). The combined organic layers were washed with saturated ac 3 solution and brine, dried over MgS 4, filtered and the solvent was removed under reduced pressure. The crude oil was purified by flash column chromatography using 10% ( 3 :Me:EtAc, 1:9:90) to give 3.56 g (18.8 mmol, 71%) of the desired product as colourless oil; R f : 0.36 (10% ( 3 :Me:EtAc, 1:9:90)); v max (neat)/cm -1 : 2929, 2861, 1472, 1253, 1089, 830, 774; 1 -MR (CDCl 3, 400 Mz) δ (ppm): (m, 2, -2), 2.98 (bs, 1, ), 2.65 (t, 2, J = 5.2 z, -1), 2.41 (s, 3, C 3 ), 0.85 (s, 9, C(C 3 ) 3 ), 0.20 (s, 6, Si- C 3 ). 13 C-MR (CDCl 3, 100 Mz) δ (ppm): 62.0 (C 2, C-2), 53.7 (C 2, C-1), 36.1 (C 3 ), 26.0 (C 3, C(C 3 ) 3 ), 18.4 (C, C(C 3 ) 3 ), 5.3 (Si-C 3 ). This data was in accordance with that reported in the literature. 3, 4 3-(tert-Butoxycarbonyl)-(S)-4-methyl-[1,2,3]-oxathiazolidine-2,2-dioxide S10 S8

9 A solution of thionyl chloride (330 µl, 6.85 mmol) and imidazole (604 mg, 8.87 mmol) in anhydrous acetonitrile (30 ml) was cooled to -40 C under an argon atmosphere. (S)--tert-Butoxycarbonyl alaninol (400 mg, 2.84 mmol) in anhydrous acetonitrile (10 ml) was added to the mixture dropwise. The reaction was stirred for 10 minutes then triethylamine (668 µl, 4.80 mmol) was added. The mixture was allowed to warm to room temperature and was stirred for another hour. The reaction was diluted with water (20 ml) and EtAc (20 ml), the aqueous phase was extracted with EtAc (3 x 20 ml) the combined organic layers were washed with brine, dried over MgS 4, filtered and the solvent was removed under reduced pressure to afford the cyclic sulfamidite as a yellow oil. The crude intermediate was dissolved in acetonitrile (20 ml) and cooled to 0 C. To this solution was added ai 4 (630 mg, 2.97 mmol), RuCl 3 x 2 (4.8 mg, 0.02 mmol) and water (10 ml) successively. The mixture was stirred for 1 hour, diluted with water and filtrated through a pad of Celite. The filtrate was extracted with EtAc (3 x 20 ml). The combined organic layers were washed with saturated ac 3 solution and brine, dried over MgS 4, filtered and the solvent was removed under reduced pressure The crude oil was purified by flash column chromatography (hexanes/etac, 3:1) to give 492 mg (2.07 mmol, 91%) of the desired product as a colourless solid. R f : 0.68 (hexanes/etac, 2:1); m.p.: 117 C Lit C; v max (neat)/cm -1 : 2979, 2523, 2159, , 1718, 1326, 1187, 1153, 1003, 924, 796, 680, 529, 454; [ ] α (c 1.00, Me) (lit c 1.00, Me). 1 -MR (CDCl 3, 400 Mz) δ (ppm): 4.66 (dd, 1, J = 5.9 z, J = 9.1 z, -5a), (m, 1, -4), 4.19 (dd, 1, J = 2.9 z, J = 9.1 z, -5b), 1.55 (s, 9, C(C 3 ) 3 ), 1.51 (d, 3, J = 6.3 z, C 3 ). 13 C-MR (CDCl 3, 100 Mz) δ (ppm): (C, CC(C 3 ) 3 ), 85.5 (C, C(C 3 ) 3 ), 71.4 (C 2, C-5 ), 53.9 (C, C-4), 28.1 (C 3, C(C 3 ) 3 ), 18.5 (C 3 ). This data was in accordance with that reported in the literature. 5, 6 D (S)-tert-Butyl (1-((2-hydroxyethyl)(methyl)amino)propan-2-yl)carbamate S7 and (S)-tert-butyl (1-((2-((tert-butyldimethylsilyl)oxy)ethyl)(methyl)amino) propan-2-yl)carbamate S11 To a solution of amine S9 (96 mg, 0.51 mmol) in anhydrous acetonitrile (1 ml) was added the sulfamidate S10 (100 mg, 0.42 mmol) in anhydrous acetonitrile (2 ml). After stirring for 3 hours at room temperature a saturated solution of citric acid (4 ml) was added. The mixture was stirred for additional 4 hours and the p was adjusted to 12 by addition of 4 M a solution. The reaction mixture was extracted with EtAc (3 x 5 ml), the combined organic layers were washed with saturated ac 3 solution and brine, dried over MgS 4, filtered and the solvent was removed under reduced pressure. The crude oil was purified by flash column chromatography (10% ( 3 :Me:EtAc, 1:9:90)) to give 26 mg (0.08 mmol, 18%) of the TBS protected S7 product and 43 mg (0.19 mmol, 44%) of the free alcohol S11 as colourless oil S7: R f : 0.55 (10% ( 3 :Me:EtAc, 1:9:90)); [ ] α -6.6 (c 1.00, Me); v max (neat)/cm , 2979, 2506, 1975, 1683, D 1527, 1365, 1247, 1167, 1053, 1031, MR (CDCl 3, 400 Mz) δ (ppm): 4.60 (d, 1, J = 7.2 z, ), (m, 1, -2), 3.55 (t, 2, J = 5.3 z, -2'), 3.10 (bs, 1, ), (m, 2, -1'), (m, 1, -1a), (m, 1, -1b), 2.26 (s, 3, C 3 ), 1.40 (s, 9, C(C 3 ) 3 ), 1.10 (d, 3, J = 6.7 z, C 3 ); 13 C-MR (CDCl 3, 100 Mz) δ (ppm): ((C, CC(C 3 ) 3 ), 79.3 (C, C(C 3 ) 3 ), 63.9 (C 2, C-1), 59.6 (C 2, C-1'),, 58.7 (C 2, C-2'), 44.9 (C, C-2), 42.4 (C 3 ), (C 3, C(C 3 ) 3 ), 19.4 (C 3 ). RMS (EI): m/z [M + a] + calculated for C₁₁₂₄₂a₃, , observed: S11: R f : 0.91 (10% ( 3 :Me:EtAc, 1:9:90)); [ ] α (c 1.00, Me); v max (neat)/cm , 1695, 1497, 1455, 1248, D 1172, 1051, 835, 775, 667; 1 -MR (CDCl 3, 400 Mz) δ (ppm): 4.91 (bs, 1, ), 3.70 (t, 2, J = 6.1 z, -2'), (m, S9

10 1, -2), (m, 2, -1'), (m, 1, -1a), (m, 1, -1b), 2.28 (s, 3, C 3 ) 1.44 (s, 9, C(C 3 ) 3- Boc group), 1.14 (d, 3, J = 6.4 z, C 3 ), 0.89 (s, 9, C(C 3 ) 3- TBS group), 0.05 (s, 6, Si-C 3 ). 13 C-MR (CDCl 3, 100 Mz) δ (ppm): (C, CC(C 3 ) 3 ), 79.0 (C, C(C 3 ) 3 ), 63.3 (C 2, C-1), 61.7 (C 2, C-1'), 60.0 (C 2, C-2'), 44.9 (C, C-2), 43.2 (C 3 ), 28.6 (C 3, C(C 3 ) 3- BC group), 26.1 (C 3, C(C 3 ) 3- TBS group), 19.6 (C 3 ), 18.3 (C, C(C 3 ) 3- TBS group), -5.2 (Si- C 3 ). RMS (EI): m/z [M + a] + calculated for C₁₇₃₈₂a₃Si: , observed: (S)-(9-Fluoren-9-yl)methyl-1-[methyl(2-hydroxyethylamino]propan-2-yl-carbamate 7 Trifluoroacetic acid (8 ml) was added dropwise to a solution of S7 (200 mg, 0.58 mmol) or S11 (656 mg, 2.82 mmol) in C 2 Cl 2 (12 ml). After stirring for 2 hours, 2 (0.5 ml) was added and the reaction was stirred for an additional 45 minutes. The solvent was removed under reduced pressure and the residue was dissolved in 1,4-dioxane (15 ml) and 10% ac 3 solution (10 ml). To this mixture was added Fmoc-Su (128 mg, 0.38 mmol) and the reaction mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure and 2 (20 ml) and C 2 Cl 2 (20 ml) were added. The aqueous layer was extracted with C 2 Cl 2 (2 x 20 ml). The combined organic layers were washed with brine, dried over MgS 4, filtered and the solvent was removed under reduced pressure. The crude oil was purified by flash column chromatography (10% ( 3 :Me:EtAc, 1:9:90)) to give 807 mg (2.28 mmol, 67%) of the desired product as a colourless solid. R f : 0.44 (10% ( 3 :Me:EtAc, 1:9:90)); m.p.: C; [ α ] D +2.1 (c 1.00, Me); v max (neat)/cm , 2537, 2159, 1977, 1698, 1533, 1449, 1244, 1056, MR (CDCl 3, 400 Mz) δ (ppm): 7.75 (d, 2, J = 7.5 z, Ar-), 7.59 (d, 2, J = 7.5 z, 2x Ar-), 7.39 (t, 2, J = 7.4 z, 2x Ar-), 7.31 (dt, 2, J = 1.0 z, J = 7.5 z, 2x Ar-), 4.90 (bs, 1, ), 4.40 (bs, 2, C 2 -Fmoc), 4.29 (t, 1, J = 6.8 z, C-Fmoc), 3.84 (bs, 1, -2), 3.60 (t, 2, J = 5.2 z, -2), (m, 2, -1), (m, 2, -1), 2.28 (bs, 3, C 3 ) 1.14 (bs, 3, C 3 ). 13 C-MR (CDCl 3, 100 Mz) δ (ppm): (C, C), (C, 2x Ar-C), (C, 2x Ar-C), (C, 2x Ar-C), (C, 2x Ar-C), (C, 2x Ar-C), (C, 2x Ar-C), 66.6 (C 2, C 2 -Fmoc), 63.6 (C 2, C-1), 59.6 (C 2, C-1'), 58.8 (C 2, C-2'), 47.4 (C, C-Fmoc), 45.4 (C, C-2), 42.4 (C 3 ), 19.5 (C 3 ). RMS (EI): m/z [M + a] + calculated for C₂₁₂₆₂a₃: , observed: S10

11 General Procedure for Peptide Synthesis Peptides were assembled manually by Fmoc solid phase peptide synthesis (SPPS) using a fritted glass reaction vessel. Method 1. General procedure for the attachment of Fmoc-Ala- to the resin: To 2-chlorotrityl chloride polystyrene resin (70 mg, 0.1 mmol, loading: 1.42 mmol/g) pre-swollen in anhydrous C 2 Cl 2 (5 ml, 5 min), was added a solution of Fmoc-Ala- (2eq.) (62 mg, 0.2 mmol) and DIPEA (67 µl, 0.40 mmol) in anhydrous C 2 Cl 2 (2 ml). The reaction mixture was gently agitated at room temperature for 2 h. The resin was filtered and washed with C 2 Cl 2 (2 x 3 ml). A mixture of C 2 Cl 2 :Me:DIPEA (8:1.5:0.5, v/v, 2 ml) was added and the reaction agitated for 10 min, filtered and repeated once for a further 10 min. The resin was filtered and washed with C 2 Cl 2 (3 x 3 ml) and DMF (3 x 3 ml). Method 2. General procedure for the attachment of Fmoc-Aib- to the resin: To 2-chlorotrityl chloride resin (70 mg, 0.1 mmol, loading: 1.42 mmol/g) pre-swollen in anhydrous C 2 Cl 2 (5 ml, 5 min), was added a solution of Fmoc-Aib- (2eq.) (65 mg, 0.2 mmol) and DIPEA (67 µl, 0.40 mmol) in anhydrous C 2 Cl 2 (2 ml). The reaction mixture was gently agitated at room temperature for 6 h. The resin was filtered and washed with C 2 Cl 2 (2 x 3 ml). A mixture of C 2 Cl 2 :Me:DIPEA (8:1.5:0.5, v/v, 2 ml) was added and the reaction agitated for 10 min, filtered and repeated once for a further 10 min. The resin was filtered and washed with C2Cl2 (3 x 3 ml) and DMF (3 x 3 ml). Method 3. General procedure for the attachment of Fmoc-AMAE- (7) to the resin via the hydroxyl group: To 2-chlorotrityl chloride resin (70 mg, 0.1 mmol, loading: 1.42 mmol/g) pre-swollen in anhydrous C 2 Cl 2 (5 ml, 5 min), was added a solution of Fmoc-AMAE- 7 (2 eq.) (70 mg, 0.2 mmol) and DIPEA (87 µl, 0.50 mmol) in anhydrous C 2 Cl 2 /DMF (1:1, v/v, 1 ml). The reaction mixture was gently agitated at room temperature for 12 h. The resin was filtered and washed with C 2 Cl 2 (2 x 3 ml). A mixture of C 2 Cl 2 :Me:DIPEA (8:1.5:0.5, v/v, 2 ml) was added and the reaction agitated for 10 min, filtered and repeated once for a further 10 min. After washing with C 2 Cl 2 (3 x 3 ml) and DMF (3 x 3 ml) the resin was dried under high vacuum. Method 4. General procedure for the attachment of Fmoc-AMAE- (7) to the resin via the amino group: Fmoc-AMAE- 7 (2 eq.) (70 mg, 0.2 mmol) was dissolved in a solution of 2% DBU in DMF (1 ml, v/v), and the mixture was added to 2-chlorotrityl chloride resin (70 mg, 0.1 mmol, loading: 1.42 mmol/g). After agitation at room temperature for 12 h, the reaction mixture was removed by filtration and a mixture of C 2 Cl 2 :Me:DIPEA (8:1.5:0.5, v/v, 2 ml) was added and the reaction agitated for 10 min, filtered and repeated once for a further 10 min. After washing with C 2 Cl 2 (3 x 3 ml) and DMF (3 x 3 ml) the resin was dried under high vacuum. Method 5. General procedure for removal of α -Fmoc-protecting group: The peptidyl resin was treated with a solution of 20% piperidine in DMF (v/v, 2 ml) and the mixture agitated at room temperature for 5 min, filtered and repeated once for a further 15 min. The resin was filtered and washed DMF (3 x 3 ml). Method 6. General procedure for solid phase peptide synthesis (SPPS): To the peptidyl resin (0.1 mmol) was added a mixture of appropriate Fmoc-protected amino acid (5 eq, 0.5 mmol), ATU (186.4 mg, 0.49 mmol) and DIPEA (172 µl, 1 mmol) in DMF (1 ml). The reaction mixture was agitated at room temperature for 1 h, filtered and repeated once for a further 1 h with fresh reagents. The resin was washed with DMF (3 x 3 ml), and C 2 Cl 2 (3 x 3 ml). S11

12 Method 7. General Procedure for the difficult coupling reaction between resin bond Aib and Fmoc-Aib- using TFF as a coupling reagent: To the peptidyl resin (0.1 mmol) was added a mixture of Fmoc-Aib- (4 eq.) (130 mg, 0.4 mmol), TFF (110 mg, 0.40 mmol) and DIPEA (137 µl, 0.80 mmol) in DMF (2 ml). The reaction mixture was agitated at room temperature for 45 min, filtered and repeated once for a further 45 min with fresh reagents. The resin was filtered and washed with DMF (2 x 3 ml) and C 2 Cl 2 (2 x 3 ml). Method 8. General Procedure for the difficult coupling reaction between resin bond Aib and Fmoc-Aib- using CMU as a coupling reagent: To the peptidyl resin (0.1 mmol) was added a mixture of Fmoc-Aib- (4 eq.) (130 mg, 0.4 mmol), CMU (171 mg, 0.4 mmol), xyma (56 mg, 0.4 mmol) and DIPEA (137 µl, 0.8 mmol) in DMF (1 ml). The reaction mixture was agitated at room temperature for 2 h, filtered and repeated once for a further 2 h with fresh reagents. The resin was filtered and washed with DMF (2 x 3 ml) and C 2 Cl 2 (2 x 3 ml). Method 9. General procedure of the attachment of first Fmoc-Aib- to the functionalized 2-chlorotyl chloride resin in route B Fmoc-Aib- (4 eq.) (130 mg, 0.4 mmol) and DMAP (0.3 eq.) were dissolved in a mixture of C 2 Cl 2 /DMF (1/1, v/v). The resulting solution was added to functionalized resin, DIC (3 eq.) (54 µl, 0.3 mmol ) was added, and the reaction was agitated at room temperature for 12h. After washing with DMF (3 x 5 ml), Me (3 x 5 ml), C 2 Cl 2 (3 x 5 ml), the same procedure was repeated for 6 h. Method 10. General Procedure for the Attachment of Fmoc-AMD- in Trichoderin A (1) and Analogue 22: To the peptidyl resin (0.1 mmol) was added a mixture of Fmoc-protected amino acid (2 eq.) (90 mg, 0.2 mmol), ATU (72 mg, 0.19 mmol), and DIPEA (68 µl, 0.4 mmol) in DMF (1 ml). The reaction mixture was agitated at room temperature for 3 h. The resin was filtered and washed with DMF (2 x 3 ml) and C 2 Cl 2 (2 x 3 ml). Method 11. General procedure to the attachment of the -terminal dodecanoic acid: To the peptidyl resin was added a mixture of dodecanoic acid (4 eq.) (80 mg, 0.4 mmol.), CMU (171 mg, 0.4 mmol), xyma (56 mg, 0.4 mmol) and DIPEA (137 µl, 0.8 mmol) in DMF (1 ml). The reaction mixture was agitated at room temperature for 3 h, after which the resin was filtered and washed with DMF (3 x 3 ml). Method 12. General procedure to the attachment of the -terminal MDA: To the peptidyl resin (0.1 mmol) was added a mixture of MDA 2 (0.15 eq.) (28 mg, 0.15 mmol), CMU (64 mg, 0.15 mmol), xyma (21 mg, 0.15 mmol) and DIPEA (51 µl, 0.3 mmol) in DMF (1 ml). The reaction mixture was agitated at room temperature for 3 h, after which the resin was filtered and washed with DMF (3 x 3 ml). Method 13. General Procedure for Acyl Shift: To the peptidyl resin in DMF (peptide concentration 20 mg/ml) was added DBU (10 equiv.), and the reaction mixture was stirred at room temperature for 4 5 h until the completion of the acyl shift as monitored by LC-MS Method 14. General procedure for FIP-mediated resin cleavage: Resin-bound peptide was cleaved from the resin by gentle agitation in a mixture of C 2 Cl 2 /FIP (4/1, v/v, 5 ml) for 30 min. The filtrate was partially concentrated under a gentle stream of 2, diluted with 2 :C 3 C (1:1, 10 ml) and lyophilised. S12

13 Total Synthesis of Synthesis of Trichoderin A ptimization of Difficult Aib-Aib Coupling The optimal conditions for the solid phase synthesis of trichoderin A were investigated by carrying out the synthesis of an analogue of Trichoderin A wherein L-alanine, L-leucine and dodecanoic acid were replaced the original (S)-AMAE 3, (2S,4S,6S)-AMD 4 and (2R)-MDA 2 building blocks of trichoderin A, respectively. Scheme S4. Synthesis of trichoderin A analogue. The C-terminal residue Fmoc-Ala-, was attached to 2-chlorotrityl chloride resin according to Method 1. Loading was determined to be 55% by spectrophotometric analysis. The α -Fmoc-protecting group was removed using Method 5 and Fmoc-Aib- was coupled according to Method 6. The α -Fmoc-protecting group was removed using Method 5. The second Fmoc-Aib- was coupled to resin 36 using Method 7 or Method 8 to form resin 37. The α -Fmoc-protecting group was removed using Method 5 followed by coupling of Fmoc-Val-, Fmoc-Ile- and Fmoc-Aib- via Method 6, and subsequently the coupling of Fmoc- Aib- via Method 7 or Method 8 to form resin 40. Fmoc-Leu- and Fmoc-Pro- was then coupled using Method 6 followed by coupling of dodecanoic acid via Method 11 resulted in resin 42. The resin-bound peptide was cleaved from the resin using Method 14. Analysis of crude products by LC-MS showed that using mixture of CMU/xyma minimized Aib deletion (Figure S1). m/z (ESI-MS) ([M + ] + requires ). S13

14 Figure S1. LC-MS profile of crude trichoderin A analogue a) using TFF for Aib-Aib Coupling (ca. 40% as judged by peak area of RP-PLC at 210 nm), b) Using CMU/xyma for Aib-Aib Coupling (ca. 85% as judged by peak area of RP-PLC at 210 nm) b); Agilent Zorbax-C3, (150 mm x 3.0 mm, 3.5 µm, linear gradient of 5% B to 95% B (ca. 3% B/min), 0.3 ml/min. m/z [M+] + calcd: ; found: * m/z is related to fragmentation of peptide. Solid-phase synthesis of trichoderin A (1) via route A Cl 7, DIPEA, C 2 Cl 2 /DMF (1:1, v/v) Fmoc 8 SPPS (R) (S) (1) The C-terminal residue Fmoc-AMAE-, was attached to 2-chlorotrityl chloride resin according to Method 3 to form resin 8. Loading was determined to be 29% by spectrophotometric analysis. The α -Fmoc-protecting group was removed using Method 5 and Fmoc-Aib- was coupled according to Method 6. The α -Fmoc-protecting group was removed using Method 5. The second Fmoc-Aib- was coupled to resin using Method 8. The α -Fmoc-protecting group was removed using Method 5 followed by coupling of Fmoc-Val-, Fmoc-Ile- and Fmoc-Aib- via Method 6, and subsequently the coupling of Fmoc-Aib- via Method 8. Fmoc-(2S,4S,6S)-AMD 5 was then coupled using Method 10 followed by coupling of Fmoc-Pro- using Method 6 and MDA 2 via Method 12. The resin-bound peptide was cleaved from the resin using Method 14 to form peptide (1). Analysis of the crude product by LC-MS showed that the presence of a trace amount of desired product. m/z (ESI-MS) ([M+2] 2+ requires ), Figure S2. S14

15 Figure S2. LC-MS profile of crude trichoderin A synthesized using route a. Agilent Zorbax-C3, (150 mm x 3.0 mm, 3.5 µm), linear gradient of 5% B to 95% B (ca. 3% B/min), 0.3 ml/min. m/z [M+2] 2+ calcd: 582.4; found: Expected compound (1) eluted at 23 min. Solid-phase synthesis of trichoderin A (1) via route B Cl 7, 2% DBU in DMF SPPS 9 (R) (S) FIP:C 2 Cl 2 (1:4) 30 min 10 (R) (S) 2 DBU in DMF (R) (S) 11 (1) The C-terminal residue Fmoc-AMAE-, was attached to 2-chlorotrityl chloride resin according to Method 4 to form resin 9. Fmoc-Aib- was next coupled to the free hydroxyl group on resin using Method 9. The α -Fmoc-protecting group was removed using Method 5 and the loading for the resin-bound dipeptide was 37%. The second Fmoc-Aib- was coupled to resin using Method 8. The α -Fmoc-protecting group was removed using Method 5 followed by coupling of Fmoc-Val-, Fmoc-Ile- and Fmoc-Aib- via Method 6, and subsequently the coupling of Fmoc-Aib- via Method 8. Fmoc-(2S,4S,6S)-AMD 5 was then coupled using Method 10 followed by coupling of Fmoc-Pro- using Method 6 and MDA 2 via Method 12 to form resin 10. The resin-bound peptide was cleaved from the resin using Method 14 to form peptide 11. Acyl Shift was carried out using Method 13 to form Trichoderin A (1). Analysis of the crude product by LC-MS showed the presence of a trace amount of desired product. m/z (ESI-MS) ([M+2] 2+ requires ), Figure S3. S15

16 Figure S3. LC-MS profile of crude trichoderin A synthesized using route b. Agilent Zorbax-C3, (150 mm x 3.0 mm, 3.5 µm), linear gradient of 5% B to 95% B ( ca. 3% B/min), 0.3 ml/min. m/z [M+2] 2+ calcd: 582.4; found: Expected compound (1) eluted at 23 min. In order to investigate the possibility of premature cleavage of peptide, Fmoc-AMAE was anchored to the resin via hydroxyl group (route A) followed by acetylation after each coupling step using acetic anhydride ( 20%) in DMF. Analysis of the crude product by LC-MS after each coupling step showed high intensity for the mass peak corresponding to the acetylated AMAE ([M+] + : 175.2). The acetylated AMAE mass related peak intensified after each coupling step to the point that just a major peak corresponding to acetylated AMAE was observed. This finding confirmed the instability of the AMAE bond to the first Aib residue during peptide elongation, Figure S4. Figure S4. LC-MS profile shows a distinct peak representative of the acetylated AMAE ([M+] + : 175.2). Agilent Zorbax-C3, (150 mm x 3.0 mm, 3.5 µm), linear gradient of 5% B to 95% B (ca. 3% B/min), 0.3 ml/min. S16

17 When we tried the same protocol with AMAE anchored to the resin via the amino group (route B), a distinct peak was observed with a retention time of 2.8 min ([M+]+: 171.2) which also intensified after each coupling. We were unable to identify the composition of this by-product, Figure S5. Figure S5. LC-MS profile shows a distinct peak with a retention time of 2.8 min ([M+] + : 171.2). Agilent Zorbax-C3, (150 mm x 3.0 mm, 3.5 µm), linear gradient of 5% B to 95% B (ca. 3% B/min), 0.3 ml/min. Total synthesis of trichoderin A (1) Scheme S5. Synthesis of trichoderin A (1) S17

18 Solid-phase synthesis of peptide 20 (R) (S) 20 The C-terminal residue Fmoc-Aib-, was attached to 2-chlorotrityl chloride resin according to Method 2 to form resin 12. Loading was determined to be 49% by spectrophotometric analysis. The α -Fmoc-protecting group was removed using Method 5 and Fmoc-Aib- was coupled according to Method 8. The α -Fmoc-protecting group was removed using Method 5 followed by coupling of Fmoc-Val-, Fmoc-Ile- and Fmoc-Aib- via Method 6, and subsequently the coupling of Fmoc-Aib- via Method 8 to form resin 15. Fmoc-(2S,4S,6S)-AMD 5 was then coupled using Method 10 followed by coupling of Fmoc-Pro- using Method 6 and MDA 2 via Method 12 to complete the linear peptide sequence of 18. The resin-bound peptide was cleaved using Method 14, and the crude residue was purified by semi-preparative RP-PLC using conditions outlined in general method. Fractions were collected at 1 min intervals and analysed by ESI-MS and RP-PLC. Fractions identified with the correct m/z were combined and lyophilised to afford the title peptide 20 as white fluffy flakes (14.8 mg, 14.1% yield, 98% purity); R t 25.5 min; LRMS: m/z (ESI-MS) ([M+] + requires ). Figure S6. LC-MS profile of crude peptide 20 (ca. 35% as judged by peak area of RP-PLC at 210 nm); Agilent Zorbax-C3, (150 mm x 3.0 mm, 3.5 µm), linear gradient of 5% B to 95% B (ca. 3% B/min), 0.3ml/min. m/z [M+] + calcd: ; found: S18

19 Figure S7. LC-MS profile of pure peptide 20 (ca. 98% as judged by peak area of RP-PLC at 210 nm); Agilent Zorbax-C3, (150 mm x 3.0 mm, 3.5 µm), linear gradient of 5% B to 95% B (ca. 3% B/min), 0.3 ml/min. m/z [M+] + calcd: ; found: * m/z is related to fragmentation of peptide. Late-stage solution phase C-terminal coupling of AMAE 3 (R) (S) (1) The linear peptide 20 (14 mg, 14 µmol) was added to a mixture of AMAE 3 (5 mg, 42 µmol), DIC (11 µl, 72 µmol) and 6-Cl- Bt (12 mg, 72 µmol) in DMF (1 ml). The reaction mixture was agitated at room temperature for 24 h. The crude trichoderin A (1) was purified by semi-preparative RP-PLC using conditions outlined in general method. Fractions were collected at 1 min intervals and analysed by ESI-MS and RP-PLC. Fractions identified with the correct m/z were combined and lyophilised to afford the trichoderin A (1) as a white amorphous solid (7 mg, 6% yield, 97% purity); R t min; RMS (EI): m/z [M + ] + calculated for C₆₀₁₁₁₁₀₁₂: , observed: ; LRMS: m/z (ESI-MS) ([M+] + requires ), Figure S8. S19

20 Figure S8. LC-MS profile of crude trichoderin A (1) in DMF (ca. 85% as judged by peak area of RP-PLC at 210 nm); Agilent Zorbax-C3, (150 mm x 3.0 mm, 3.5 µm), linear gradient of 5% B to 95% B (ca. 3% B/min), 0.3ml/min. m/z [M+] + calcd: ; found: The earlier eluting peaks at 0-5 min and 9-12 mins are attributed to 6-Cl-Bt and DMF respectively. Figure S9. LC-MS profile of pure trichoderin A (1) (ca. 97% as judged by peak area of RP-PLC at 210 nm); Agilent Zorbax- C3, (150 mm x 3.0 mm, 3.5 µm), linear gradient of 5% B to 95% B (ca. 3% B/min), 0.3 ml/min. m/z [M+] + calcd: ; found: S20

21 Figure S10. RMS profile of trichoderin A (1). RMS (EI): m/z [M + ] + calculated for C₆₀₁₁₁₁₀₁₂: , observed: Total synthesis of trichoderin A 22 Scheme S6. Synthesis of trichoderin A 22 S21

22 Solid-phase synthesis of Peptide 21 (R) 21 The C-terminal residue Fmoc-Aib-, was attached to 2-chlorotrityl chloride resin according to Method 2 to form resin 12. Loading was determined to be 47% by spectrophotometric analysis. The α -Fmoc-protecting group was removed using Method 5 and Fmoc-Aib- was coupled according to Method 8. The α -Fmoc-protecting group was removed using Method 5 followed by coupling of Fmoc-Val-, Fmoc-Ile- and Fmoc-Aib- via Method 6, and subsequently the coupling of Fmoc-Aib- via Method 8 to form resin 15. Fmoc-(2S,4S,6R)-AMD 6 was then coupled using Method 10 followed by coupling of Fmoc-Pro- using Method 6 and MDA 2 via Method 12 to complete the linear peptide sequence of 19. The resin-bound peptide was cleaved using Method 14, and the crude residue was purified by semi-preparative RP-PLC using conditions outlined in general method. Fractions were collected at 1 min intervals and analysed by ESI-MS and RP-PLC. Fractions identified with the correct m/z were combined and lyophilised to afford the peptide 21 as white fluffy flakes (13.2 mg, 12.6% yield, 91% purity); R t min; LRMS: m/z (ESI-MS) ([M+] + requires ). Figure S11. LC-MS profile of crude peptide 21 (ca. 30% as judged by peak area of RP-PLC at 210 nm); Agilent Zorbax-C3, (150 mm x 3.0 mm, 3.5 µm), linear gradient of 5% B to 95% B (ca. 3% B/min), 0.3ml/min. m/z [M+] + calcd: ; found: S22

23 Figure S12. LC-MS profile of pure peptide 21 (ca. 91% as judged by peak area of RP-PLC at 210 nm); Agilent Zorbax-C3, (150 mm x 3.0 mm, 3.5 µm), linear gradient of 5% B to 95% B (ca. 3% B/min), 0.3 ml/min. m/z [M+] + calcd: ; found: * m/z is related to fragmentation of peptide. Late-stage solution phase C-terminal coupling of AMAE 3 (R) (R) 22 The linear peptide 21 (13 mg, 12 µmol) was added to a mixture of AMAE 3 (4 mg, 36 µmol), DIC (11 µl, 72 µmol) and 6-Cl- Bt (12 mg, 72 µmol) in DMF (1 ml). The reaction mixture was agitated at room temperature for 24 h. The crude trichoderin A 22 was purified by semi-preparative RP-PLC using conditions outlined in general method. Fractions were collected at 1 min intervals and analysed by ESI-MS and RP-PLC. Fractions identified with the correct m/z were combined and lyophilised to afford the trichoderin 22 as a white amorphous solid (4 mg, 3.4% yield, 98% purity); R t min; RMS (EI): m/z [M + ] + calculated for C₆₀₁₁₁₁₀₁₂: , observed: ; LRMS: m/z (ESI-MS) ([M+] + requires ). S23

24 Figure S13. LC-MS profile of crude trichoderin A 22 in DMF (ca. 65% as judged by peak area of RP-PLC at 210 nm); Agilent Zorbax-C3, (150 mm x 3.0 mm, 3.5 µm), linear gradient of 5% B to 95% B (ca. 3% B/min), m/z [M+] + calcd: ; found: The earlier eluting peaks at 0-5 min and 9-12 mins are attributed to 6-Cl-Bt and DMF respectively. Figure S14. LC-MS profile of pure trichoderin A 22 (ca. 98% as judged by peak area of RP-PLC at 210 nm); Agilent Zorbax- C3, (150 mm x 3.0 mm, 3.5 µm), linear gradient of 5% B to 95% B (ca. 3% B/min), 0.3 ml/min. m/z [M + ] + calcd: ; found: S24

25 Figure S15. RMS profile of trichoderin A 22. RMS (EI): m/z [M + ] + calculated for C₆₀₁₁₁₁₀₁₂: , observed: Antimicrobial Screening The two C-6 AMD epimers of trichoderin A (1) and 22 were tested for their antimicrobial activity (minimum inhibitory concentration, MIC) against a range of bacteria by serial macrobroth dilution as previously described. 7 Cultures were incubated at 37 C (200 rpm) where the visual presence or absence of growth was recorded after 12 h for Staphylococcus aureus (strain BB255), Streptococcus uberis, and Escherichia coli; 24 h for Mycobacterium smegmatis, and 5 days for Mycobacterium tuberculosis (Table 1). S25

26 Table S1. 1 (δ) and 13C MR (δc) chemical shifts (ppm) of natural and synthetic trichoderin A (1) and Trichoderin A 22 atural Synthetic 22 Synthetic 1 Position δ δ C δ δ C δ δ C (R)-2-methyldecanoic acid (MDA) L-Proline (Pro) , , , (d, 6.4) (d, 6.0) (d, 6.4) 15.5 C α β 1.96, , , γ 1.92, , , δ 3.44, , , amino-6-hydroxy-4-methyl-8-oxodecanoic acid (AMD) 9.07 (br) , , , , , , , , , aminoisobutyric acid (Aib-1) 2-aminoisobutyric acid (Aib-2) C α β C α β S26

27 Table S1 continues atural Synthetic (1) Synthetic 22 Position δ δ C δ δ C δ δ C L-Isoleucine (Ile) L-valine (Val) 7.58 (d, 5.6) C α β γ 1.23, , , δ 0.94 (d, 6.7) (d, 6.7) (d, 6.7) C α 3.36 (dd, 11.4, 3.36 (dd, 3.38 (dd, ) 16.3, 5.3) , 5.4) 65.5 β γ aminoisobutyric acid (Aib-3) 2-aminoisobutyric acid (Aib-4) C α β C α β (S)-2-((2-aminopropyl(methyl)amino)ethanol (AMAE) (2 -aminopropyl) 7.71 (d, 10.0) (d, 10.28) (d, 10.3) , , , , , , , , , C (d, 4.4) (d, 4.7) (d, 4.1) 42.2 (quaternary) 8.23 (br) S27

28 Deviation from calculated and reported 1 and 13 C MR chemical shift of AMD residue for isolated trichoderin A and synthetic trichoderin A (1) (S) (R) S28

29 Deviation from calculated and reported 1 and 13 C MR chemical shift of AMD residue for isolated trichoderin A and synthetic trichoderin A 22 (R) (R) S29

30 Deviation from calculated and reported 1 MR chemical shift values for isolated trichoderin A) synthetic trichoderin A (1) and synthetic trichoderin A 22 a) Deviation from reported 1 MR chemical shift values MDA Pro AMD Aib 1 Aib2 Ile Val Aib 3 Aib 4 AMAE b) Deviation from reported 1 MR chemical shift values MDA Pro AMD Aib 1 Aib 2 Ile Val Aib 3 Aib 4 AMAE S30

31 Deviation from calculated and reported 13 CMR chemical shift values for isolated trichoderin A) synthetic trichoderin A (1) and synthetic trichoderin A 22 a) 1 Deviation from reported 13 C MR chemical shift values MDA Pro AMD Aib 1 Aib2 Ile Val Aib 3 Aib 4 AMAE b) Deviation from 13 CMR chmical shift values MDA Pro AMD Aib 1 Aib2 Ile Val Aib 3 Aib 4 AMAE S31

32 1 and 13 C MR Spectra S32

33 S33

34 S34

35 S35

36 Boc S7 Boc S7 S36

37 S37

38 S38

39 S39

40 S40

41 S41

42 S42

43 S43

44 References (1) Darley, D. J.; Butler, D. S.; Prideaux, S. J.; Thornton, T. W.; Wilson, A. D.; Woodman, T. J.; Threadgill, M. D.; Lloyd, M. D. rg. Biomol. Chem. 2009, 7, (2) Zhang, W.; Sun, T. T.; Li, Y. X. J. Pept. Sci. 2009, 15, (3) Igarashi, Y.; Yanagisawa, E.; hshima, T.; Takeda, S.; Aburada, M.; Miyamoto, K.-i. Chem. Pharm. Bull. 2007, 55, (4) Yamazaki, S.; Iwata, Y.; Fukushima, Y. rg. Biomol. Chem. 2009, 7, (5) Bower, J. F.; Szeto, P.; Gallagher, T. rg. Lett. 2007, 9, (6) Posakony, J. J.; Grierson, J. R.; Tewson, T. J. J. rg. Chem. 2002, 67, (7) Dunn, E. A.; Roxburgh, M.; Larsen, L.; Smith, R. A.; McLellan, A. D.; eikal, A.; Murphy, M. P.; Cook, G. M. Biorg. Med. Chem. 2014, 22, S44