The Depsipeptide Methodology for Solid Phase Peptide Synthesis: Circumventing Side Reactions and Development of an Automated Technique via

Transcription

1 Supporting Information The Depsipeptide Methodology for Solid Phase Peptide Synthesis: Circumventing Side Reactions and Development of an Automated Technique via Depsidipeptide Units. Irene Coin, Rudolf Dölling, Eberhard Krause, Michael Bienert, Michael Beyermann*, Calin Dan Sferdean, Louis A. Carpino* Table of Contents Synthesis of (VT) 10 sequence and [Asn 15 ]FBP28-WW Materials and methods S4 Synthesis of compounds: 2, 2a, 2b, 3, 3a, 3d, 3e, 4, 5b, S5 Synthesis of depsidipeptide 11 and Crambin (16-46) Materials and methods S7 Synthesis of compounds: 8a, 10, 11, 9... S8 Spectral Data 1 H-NMR: Boc-Thr-OBn..... S10 1 H-NMR: Boc-Thr(Z-Ala)-OBn S11 1 H-NMR: Boc-Thr(Z-D-Ala)-OBn... S12 IR: Boc-Thr(Z-Ala)-OBn & Boc-Thr(Z-D-Ala)-OBn... S13 1 H-NMR: Boc-Thr(Ala)-OH... S14 IR: Boc-Thr(Ala)-OH... S15 1 H-NMR: Boc-Thr(Fmoc-Ala)-OH... S16 S1

2 IR: Boc-Thr(Fmoc-Ala)-OH... S17 1 H-NMR: Boc-Thr(Fmoc-Ala)-OBn... S18 IR: Boc-Thr(Fmoc-Ala)-OBn... S19 1 H-NMR of the crude reaction mixture obtained by the O-acylation of Boc-Thr-OBn with Z-Ala OH via DCM/DMAP/ EDC A. full spectrum S20 B. Scale-expanded spectrum S21 1 H-COSY Boc-Thr(Z-D-Ala)-OBn S22 1 H- COSY Boc-Thr(Z-D-Ala)-OBn S23 Solid phase assemblies of Crambin (16-46)... S24 Fig. 1. Assembly of Crambin (16-46) via standard Fmoc chemistry (peptide 12a in the text) (A) UV monitoring trace.... S25 (B) HPLC trace S25 (C) ESI-MS trace for the major peak of the HPLC trace... S26 Fig. 2. Assembly of Crambin (16-46) via the pseudoproline methodology (positions 28-27& 39-38) (A) UV monitoring trace.... S27 (B) HPLC trace for the assembly of Crambin (16-46)...S27 (C) ESI-MS trace for the major peak of the HPLC trace s28 Fig. 3. Assembly of Crambin (16-46) via the pseudoproline methodology (positions 28-27) (A) UV monitoring trace... S29 (B) HPLC trace S29 Fig. 4. Assembly of Crambin (16-46) via the pseudoproline methodology (positions 39-38) (A) UV monitoring trace S30 (B) HPLC trace S30 Fig. 5. Assembly of Crambin (16-46) depsipeptide via the depsidipeptide methodology (positions 39-38) (A) UV monitoring trace..... S31 (B) HPLC trace S31 C) ESI-MS trace for the major of the HPLC trace.... S32 S2

3 Fig. 6. Assembly of Crambin (16-46) depsipeptide via the depsidipeptide methodology (positions 28-27), peptide 12b in the text. (A) UV monitoring trace S33 (B) HPLC trace S33 (C) HPLC trace after treatment with dilute ammonia... S34 (D) ESI-MS trace for the major peak of Crambin(16-46) obtained according to 6 C... S34 Fig. 7. Assembly of Crambin (16-46) depsipeptide via the depsidipeptide methodology (positions 28-27& 39-38) (A) UV monitoring trace S35 (B) HPLC trace S35 C) ESI-MS trace for the major peak of the HPLC trace..... S36 Proof of the lack of loss of configuration during the synthesis of derivatization agent Z-Val- Aib-Gly-OH by HPLC analysis..... S37 Determination of the lack of significant loss of configuration during O-acylation of Boc-Thr- OBn by Z-Ala-OH/EDC/DMAP/DCM via derivatization of the appropriate depsidipeptide with Z-Val-Aib-Gly-OH according to HPLC analysis of the resulting depsipentapeptide S38 S3

4 Synthesis of (VT) 10 sequence and [Asn 15 ]FBP28-WW Materials and methods. Solvents used for esterification were dried over 3Å molecular sieves for at least 24 h, the other solvents were used without any treatment. Analytical RP-HPLC was performed using a C18 column (250 x 4.6 mm, 5µm, 300Å), operated at 1mL/min. The solvent system was: buffer A = water (0.1% TFA); buffer B = 80% acetonitrile in water (0.1% TFA); linear gradient 5 to 95% B in 40 min. The temperature was 23 C, except in the case of the segment Y 19 -K 37 (Figure 3 in the text) where the analyses were carried out at 60 C to avoid splitting of the peaks due to conformational effects. Absorbance was monitored at 220 and 301nm. Product percentages are given by peak areas at 220 nm. LC-MS analysis was performed on a LC system equipped with a C18 capillary column (300µm i.d. x 15cm, 3µm, 100Å) combined with an electrospray time-of-flight (ESI-TOF) mass spectrometer. LC conditions: flow 4µl/min, eluent system: buffer C = 2% acetonitrile in water (0.1% formic acid); buffer D = 80% acetonitrile in water (0.1% formic acid), linear gradient 5 to 95% D in 40 min; room temperature. UV detection was performed at 220nm. Optical purity of peptide 3c was determined via GC-MS analysis by C.A.T. GmbH & Co, Chromatographie und Analysentechnik KG (Tübingen, Germany). The method used involves hydrolysis of the sample with 6N D 2 O/DCl and derivatization of the amino acids using deuterated reagents. Epimerization during sample preparation is accompanied by deuterium exchange in the α- position (deuterium label). The proportion of D-amino acid originally present in the peptide is thus represented by the relative amounts of the unlabeled form which is monitored by mass spectrometry. The limit of quantitation is 0.1% of the optical antipode. The standard deviation is <0.1%. S4

5 YYYNNRTLESTWEKPQELK-NH 2 (2, standard synthesis). Peptide 2 was automatically assembled on a TentaGel-SRam resin (capacity: 0.26 mmol/g), using a standard 0.25M Fmoc protocol. Cleavage was performed in standard TFA solution for 3 h. LC-MS: retention time 15.4 min, [M+H] + calc= (monoisotopic), found: YYYNNRTLESTWEKPQELK-NH 2 (2a, via the depsi analogue). The sequence TWEKPQELK- NH 2 was automatically assembled on a TentaGel-SRam resin (capacity: 0.26 mmol/g), using a standard 0.25M Fmoc protocol. Boc-Ser-OH was manually coupled and O-acylated by means of Fmoc-Glu(tBu)-OH. Acetylation in the presence of NMI followed. The rest of the sequence was assembled automatically. The depsipeptide obtained YYYNNRTL-ES-TWEKPQELK-resin was cleaved with standard TFA solution (3 h) and shifted to the native amide form. MALDI-MS: [M+H] + calc= (monoisotopic), found: YYYNNRTLESTWEKPQELK-NH 2 (2b, via pseudo-proline). The peptide YYYNNRTL-Ψ ES - TWEKPQELK-resin was automatically assembled on a TentaGel-SRam resin (capacity: 0.26 mmol/g), using a standard 0.25M Fmoc protocol, with double couplings for each step except for the pseudo-proline Fmoc-Glu(tBu)-Ser(Ψ Me, Me pro)-oh at position 27/28, which was coupled only once. Cleavage was performed in standard TFA solution for 3 h. MALDI-MS: [M+H] + calc= (monoisotopic), found: [Asn 15 ]FBP28-NH 2 (3, standard synthesis). Peptide 3 was automatically assembled on a TentaGel- SRam resin (capacity: 0.26 mmol/g), using a standard 0.25M Fmoc protocol. Cleavage was performed in standard TFA solution for 3 h. LC-MS: retention time 15.8 min, [M+H] + calc= (monoisotopic), found: Depsi-[Asn 15 ]FBP28-NH 2 GATAVSEWTEYKTANG-KT-YYYNNRTL-ES-TWEKPQELK- NH 2 (3a, two depsi units: ES 27/28, KT 17/18). The depsipeptide YYYNNRTL-ES- TWEKPQELK-NH 2 was prepared as described for peptide 2a. Boc-Thr-OH was then manually coupled and O-acylated by means of by Fmoc-Lys(Boc)-OH. Acetylation in the presence of NMI followed. The rest of the sequence was assembled automatically. Cleavage was performed in S5

6 standard TFA solution for 3 h, giving depsipeptide 3a. MALDI-MS: [M+H] + calc= (average), found: [Asn 15 ]FBP28-NH 2 (3d, via two pseudo-prolines: ES 27/28, KT 17/18). Peptide GATAVESEWTEYKTANG-Ψ KT -YYYNNRTL-Ψ ES -TWEKPQELK-resin was automatically assembled on a TentaGel-SRam resin (capacity: 0.26 mmol/g), using a standard 0.25M Fmoc protocol, with double coupling for each step except for the pseudo-proline derivative Fmoc- Glu(tBu)-Ser(Ψ Me, Me pro)-oh at 27/28, which was coupled only once. Lys-Thr at position 17/18 was introduced as the pseudo-proline building block, Fmoc-Lys(Boc)-Thr(Ψ Me, Me pro)-oh, which was coupled according to the standard procedure. Cleavage was performed in a standard TFA solution for 3 h. MALDI-MS: [M+H] + calc= (average), found: [Asn 15 ]FBP28-NH 2 (3e, via three pseudo-prolines: ES 27/28, KT 12/13, VS 5/6). Segment ANGKTYYYNNRTL-Ψ ES -TWEKPQELK-resin was automatically assembled on a TentaGel- SRam resin (capacity: 0.26 mmol/g), using a standard 0.25M Fmoc protocol, with double coupling for each step except for pseudo-proline, Fmoc-Glu(tBu)-Ser(Ψ Me, Me pro)-oh at 27/28, which was coupled only once. Pseudo-proline Fmoc-Lys-Thr(Ψ Me, Me pro)-oh at 12/13 was coupled via N- HATU/DIEA/NMP (2 2 h) and coupling was followed by a standard capping step. The rest of the sequence was completed automatically. Lys-Thr at position 12/13 and Val-Ser at position 5/6 were introduced as pseudo-proline building blocks, Fmoc-Lys(Boc)-Thr(Ψ Me, Me pro)-oh and Fmoc-Val- Ser(Ψ Me, Me pro)-oh respectively, which were coupled according to the standard procedure. Cleavage of GATA-Ψ VS -SEWTEY-Ψ KT -ANGKTYYYNNRTL-Ψ ES -TWEKPQELK-resin was performed in a standard TFA solution for 3 h. MALDI-MS: (average), found: G-KT-YYYNNRTL-ES-TWEKPQELK-NH 2 (4, two depsi units: ES 27/28, KT 17/18). The depsi peptide YYYNNRTL-ES-TWEKPQELK-NH 2 was prepared as described for peptide 2a. Boc-Thr- OH was manually coupled. The free hydroxyl function of Thr was acylated by means of Fmoc- Lys(Boc)-OH, and acetylation in the presence of NMI followed. Fmoc was removed by the standard S6

7 procedure and Fmoc-Gly-OH was coupled. Fmoc deprotection was carried out in the standard way leading to product 4. Samples of all intermediate peptides since the introduction of the second depsi unit were cleaved from the resin (standard cleavage solution, 3 h) and analyzed. ESI-TOF-MS of samples collected from HPLC analysis: peak at retention time min assigned to G 16 -K 37 [M+H] + calc (monoisotopic) = , found: ; peak at retention time min assigned to T 18 -K 37 [M+H] + calc (monoisotopic) = , found: Ac-LE-(+)lac-TWEKPQELK-NH 2 (peptide 5b, using Bsmoc-Leu-OH). The sequence E-lac- TWEKPQELK-NH 2 was assembled as described for peptide 5a. Bsmoc-Leu-OH was coupled via the standard procedure. The Bsmoc protected peptide was treated with 2% piperidine v/v in DMF (3 1 min), washed with DMF (1 min) and immediately acetylated using a solution of acetic anhydride 50% v/v in DMF (1 h). Peptide 5b was cleaved from the resin using a standard cleavage solution (1 h). LC-MS: retention time 16.9 min, [M+H] + calc = (monoisotopic), found: Fmoc-TLE-(+)lac-TWEKPQELK-NH 2 (peptide 6). The sequence E-lac-TWEKPQELK-NH 2 was assembled as described for peptide 5a. Bsmoc-Leu-OH was coupled via the standard procedure. The Bsmoc protected peptide was treated with piperidine 2% v/v in DMF (3 1 min) and washed with DMF (1min). Fmoc-Thr(tBu)-OH was coupled via the standard procedure. Fmoc-protected peptide 6 was cleaved from the resin using a standard cleavage solution (1 h). LC-MS: retention time 23.9 min, [M+H] + calc = (monoisotopic), found: Synthesis of depsidipeptide 11 and Crambin (16-46) Material and methods 1 H-NMR were recorded at 400 MHz. Analytical HPLC was performed using a C18 column (3.4μm, 4.6X100 mm) operated at 1mL/min. The temperature was 30 C, product percentages are given by peak areas at 220 nm. Eluent system: A = water (0.1% TFA); B = acetonitrile (0.1% TFA), the gradient system used is mentioned under each individual HPLC trace. LC-MS analysis was S7

8 performed using C18 column (3.5μm, 4.6x100cm) operated at room temperature. LC conditions: flow 1ml/min, eluent system: A = water (0.1% TFA); B = acetonitrile (0.1% TFA), linear gradient 10 to 90% B in 30 min. UV detection was performed at 220 nm. For the MS data an electrospray ion trap mass spectrometer was used. O-[(N-Benzyloxycarbonyl)]-D-alanyl-N-(tert-butyloxycarbonyl)threonine benzyl ester (8a). The same method described for 8 was followed with Z-D-Ala-OH substituted for L-form. The depsidipeptide was obtained as an oil (93% yield); 1 H-NMR (400 MHz, CDCl 3 ): δ 1.19 (d 3), 1.31 (d, 3), 1.46 (s, 9), 4.25 (m, 1), 4.49 (dd 1), (m, 6), 5.45 (m, 1), (m, 10), IR (DCM): 1749, 1721 cm -1. Anal. Calcd. for C 27 H 34 N 2 O 8 : C, 63.02; H, 6.66; N, 5.44; Found: C,63.05; H, 6.65; N, O-(Alanyl)-N-(tert-butyloxycarbonyl)threonine (10). A solution of 0.9 (1.75 mmole) g of 8 in 100 ml of absolute ethanol was treated with 90 mg of 10 % Pd/C catalyst and the mixture hydrogenated for 11 h at 40 psi. The catalyst was removed by filtration through a pad of Celite and the solvent removed with a rotary evaporator. The residual solid was suspended in 60 ml of DCM and the mixture filtered to give 0.35 g (70%) of pure depsidipetide 10, mp o C. A small sample recrystallized from water gave white crystals, m.p o C. 1 H NMR (400 MHz, DMSO-d 6 ): δ 1.13 (d, 3), 1.26 (d, 3), 1.39 (s, 9), 3.7 (q, 1) 3.93 (dd, 1), 5.27 (m, 1), 6.54 (d, 1) IR (KBr): 1748, 1712 cm -1 Anal. Calcd. for C 12 H 22 N 2 O 6 : C, 49.65; H, 7.64; N, 9.65; Found: C, 49.40; H, 7.61; N, O-[(N-9-Fluorenemethyloxycarbonyl)alanyl]-N-(tert-butyloxycarbonyl)-threonine (11) via treatment of 10 with Fmoc-OSu. According to a published procedure 23 the Fmoc derivative of 10 was prepared via FmocOSu in 85% yield in the presence of NEt 3 and obtained as a white solid, mp o C; 1 H NMR (400 MHz, CDCl 3 ): δ 1.25 (t, solvate) 1.31 (d 3), 1.37 (d, 3), 1.44 (s, 9), 2.04 (s, solvate) 4.12 (q, solvate) 4.17 (m, 1), (m, 4 ), 4.52 (dd, 1), (m, 6) 7.74 (d, 2). Anal. Calcd. for C 27 H 32 N 2 O 8 1/2 Ethyl acetate C 29 H 36 N 2 O 9 : C, 62.58; H, 6.47; N, 5.03; IR (KBr) S8

9 3337, 1717 cm -1 Found: C, 62.58; H, 6.46; N, The amount of ethyl acetate present in the solvate was determined by NMR analysis and agreed with that determined by elemental analysis. O-[(N-9-Fluorenemethyloxycarbonyl)alanyl]-N-(tert-butyloxycarbonyl)threonine benzyl ester (9). The same method was used as described for compound 8 using the following amounts of reagents: g of (1.8 mmole) Boc-Thr-OBn, g (3.8 mmole) of Fmoc-Ala-OH, g (0.54 mmole) of DMAP, g (5.42 mmole) of EDC and 20 ml of DCM. Purification was carried out by column chromatography on silica gel using ethyl acetate/hexane as eluant (at first 1: 6 and increasing the ethyl acetate component gradually to 1: 4) to give 0.9 g (82%), of pure ester; mp o C; 1 H-NMR (400 MHz, CDCL3): δ 1.27 (d 3), 1.32 (d, 3), 1.44 (s, 9), (m, 2), 4.40 (d 2), 4.5 (dd, 1), (4, m) 5.45 (m, 1), (m 5), 7.40 (t 2), 7.60 (t 2), 7.77 (d 2); IR (DCM): 1743, 1717 cm -1 Anal. Calcd. for C 34 H 38 N 2 O 8 : C, 67.76; H, 6.36; N, 4.65; Found: C, 67.67; H, 6.50 ; N, O-[(N-9-Fluorenemethyloxycarbonyl)alanyl]-N-(tert-butyloxycarbonyl)threonine (11) via catalytic hydrogenation of 9. A solution of 0.3 g ( mmole) of 9 dissolved in 50 ml of absolute ethanol was treated with g of 5% Pd/C and the mixture shaken under a hydrogen atmosphere at 30 psi for 4 h. HPLC analysis showed 5 % of 9 left in the reaction mixture and no preliminary deblocking of the Fmoc group. The reaction time required is dependent on the degree of dilution of the reaction mixture and activity of the catalyst, factors which must be determined for each batch of catalyst. Close monitoring of the reaction by withdrawal of a small sample each hour and its analysis by HPLC or TLC techniques is advised. After catalyst removal and solvent evaporation the solid was recrystallized from ethyl acetate/hexane to give 0.15 g (58%) of pure 11. Physical constants and the 1 H-NMR spectra were identical with those described above for the sample prepared via the Fmoc-OSu/NEt 3 method. S9

10 Boc-Thr-OBn S10

11 Boc-Thr(Z-Ala)-OBn/CDCl3 S11

12 Boc-Thr(Z-D-Ala)-OBn/CDCl3 S12

13 Boc-Thr(Z-Ala)-OBn/ DCM S13

14 Boc-Thr(Ala)-OH/ DMSO S14

15 Boc-Thr(Ala)-OH/KBr S15

16 Boc-Thr-(Fmoc- Ala)-OH S16

17 Boc-Thr-(Fmoc-Ala)-OH/KBr S17

18 Boc-Thr(Fmoc-Ala)- -OBn /CDCl3 S18

19 Boc-Thr(Fmoc-Ala)-OBn / DCM S19

20 Crude reaction mixture Boc-Thr-OBn + Z-Ala-OH/ DCM/DMAP/EDC S20

21 Details of the crude reaction mixture (Boc-Thr-OBn + Z-Ala-OH/DCM/DMAP/EDC) S21

22 1 H-COSY Boc-Thr(Z-D-Ala)-OBn/CDCl 3 S22

23 1 H-COSY/Boc-Thr(Z-D-Ala)-OBn/CDCl 3 S23

24 Solid phase assembly of Crambin (16-46) via standard methodology and via use of depsidipeptide or pseudoproline units. All syntheses were carried out under the general conditions given in the text except that at the position indicated an appropriate depsidipeptide or pseudoproline unit was introduced in place of the threonine cartridge. Synthesized peptides, LC/MS analysis: 1. Crambin (16-46), standard synthesis (peptide 12a in the text). ESI-MS: [M+2H] 2+ calc= , found: , [M+3H] 3+ calc= , found: [M+4H] 4+ calc=786.4, found: See Fig. 1, page S Crambin (16-46) obtained via the pseudoproline methodology (positions 28-27& 39-38) ESI-MS: [M+2H] 2+ calc= , found: , [M+3H] 3+ calc= , found: See Fig. 2, page S Crambin (16-46) obtained via the pseudoproline methodology (positions 28-27) showed the same retention time as the product obtained in run 2. See Fig. 3, page S Crambin (16-46) obtained via the pseudoproline methodology (positions 39-38) showed the same retention time as the product obtained in run 2. See Fig 4, page S Depsipeptide via ester modification at positions 39-38: ESI-MS: [M+2H] 2+ calc= , found: , [M+3H] 3+ calc= , found: See Fig. 5, page S Crambin (16-46) obtained from depsipeptide made via ester modification at positions after treatment with ammonia (peptide 12b in the text): ESI-MS: reduced form [M+2H] 2+ calc= , [M+3H] 3+ calc= , oxidized form [M+2H] 2+ calc = , [M+3H] 3+ calc= , found: [M+2H] 2+ = , [M+3H] 3+ = See Fig. 6, page S Depsipeptide via ester modification at positions and 28-27: ESI-MS: [M+2H] 2+ calc= , found: , [M+3H] 3+ calc= , found: See Fig. 7, page S35. S24

25 B Fig. 1. A. UV monitoring trace for the assembly of Crambin (16-46) using standard Fmoc chemistry. Fig. 1. B. HPLC trace (220 nm) for the assembly of Crambin (16-46) under standard Fmoc conditions. LC conditions: linear gradient, solvent B 10-65% over 45 min. S25

26 Fig. 1. C. ESI-MS trace for the major peak of the product obtained by assembly of Crambin (16-46) via standard Fmoc chemistry. S26

27 FFig. 2. A. UV monitoring trace for the assembly of Crambin (16-46) via the pseudoproline methodology using standard Fmoc chemistry. The pseudoproline units were introduced at the positions indicated by the arrows (positions 28-27& 39-38). Fig. 2. B. HPLC trace for the assembly of Crambin (16-46) under the conditions mentioned in 2. A. LC conditions: linear gradient, solvent B 10-65% over 45 min. S27

28 Fig. 2. C. ESI-MS trace for the major peak obtained by assembly of Crambin (16-46) via the pseudoproline methodology under the conditions specified under 2 A. S28

29 Fig. 3. A. UV monitoring trace for the assembly of Crambin (16-46) via the pseudoproline methodology using standard Fmoc chemistry. The pseudoproline unit was introduced at the positions indicated by the arrow (positions 28-27). Fig. 3. B. HPLC trace for the assembly of Crambin (16-46) assembled under conditions mentioned in 3. A. LC conditions: linear gradient, solvent B 10-65% over 45 min. S29

30 Fig. 4. A. UV monitoring trace for the assembly of Crambin (16-46) via the pseudoproline methodology using standard Fmoc chemistry. The pseudoproline unit was introduced at the positions indicated by the arrow (positions 39-38). Fig. 4. B. HPLC trace for the assembly (220 nm) of Crambin (16-46) under conditions mentioned in 4. A. LC conditions: linear gradient: solvent B 10-65% over 45 min. S30

31 Boc-Thr(Fmoc-Ala)-OH A Fig 5. A. UV monitoring trace for the assembly of Crambin (16-46) via the depsidipeptide methodology using standard Fmoc chemistry. The depsidipeptide unit was introduced at the positions indicated by the arrow (positions 39-38). Fig. 5. B. HPLC trace for the assembly of Crambin (16-46) under conditions mentioned in 5. A.. LC conditions: linear gradient, solvent B 10-65% over 45 min. S31

32 Fig. 5. C. ESI-MS trace for the major peak of the product obtained by assembly of Crambin (16-46) via depsidipeptide methodology with ester modification found at positions For the HPLC and UV monitoring traces see page S31. S32

33 Boc-Thr(Fmoc-Ala)-OH Fig. 6. A. UV monitoring trace for the assembly of Crambin (16-46) depsipeptide via the depsidipeptide methodology using standard Fmoc chemistry. The depsidipeptide unit was introduced at the positions indicated by the arrow (positions 28-27). Fig. 6. B. HPLC trace for the assembly of Crambin (16-46) depsipeptide under the conditions mentioned in 6. A. LC conditions: linear gradient, solvent B 10-65% over 45 min. S33

34 Fig. 6. C. HPLC trace of Crambin (16-46) obtained under conditions mentioned in 6 A via depsipeptide methodology after treatment with dilute ammonia in order to induce the O N shift: 2 mg of depsipeptide was dissolved in 1 ml of acetonitrile/water and treated with 1 ml of 5% aqueous ammonia. After 1 h a 10μL sample was injected onto the HPLC column. Fig. 6. D. ESI-MS trace for the major peak of Crambin (16-46) according to the methodology described under 6. C. S34

35 Boc-Thr(Fmoc-Ala)-OH Fig. 7. A. UV monitoring trace for the assembly of Crambin (16-46) depsipeptide via the depsidipeptide methodology using standard Fmoc chemistry. The depsidipeptide units were introduced at the positions indicated by the arrows (positions 28-27& 39-38). Fig. 7. B. HPLC trace for the assembly of Crambin (16-46) depsipeptide under the conditions mentioned in 7. A.. LC conditions: linear gradient, solvent B 10-65% over 45 min. S35

36 Fig. 7. C. ESI-MS trace for the major peak of the product obtained by assembly of Crambin (16-46) depsipeptide via the depsidipeptide methodology under the conditions mentioned in 7. A. with ester modification found at positions 28-27& S36

37 Proof that no loss of configuration occurs during the synthesis of derivatization reagent Z-Val-Aib-Gly-OH by HPLC analysis Fig. 8. A. HPLC trace of the crude reaction mixture resulting from coupling of H-L- Val-OMe with Z-Val-Aib-Gly-OH via N-HATU/DIEA. Note the absence of any peak at min which confirms the chiral purity of the derivatization reagent. Fig. 8. B. HPLC trace of the crude reaction mixture resulting from coupling of H-D- Val-OMe with Z-Val-Aib-Gly-OH via N-HATU/DIEA in order to establish the retention time of the LD-Isomer. Fig. 8. C. HPLC trace of a mixture of the two diastereoisomeric peptides obtained under the conditions mentioned in A and B. LC conditions: eluents were A, water and B, acetonitrile containing 0.1% TFA, linear gradient: solvent B 10-90% over 50 min. S37

38 Determination of the lack of significant loss of configuration during O-acylation of Boc-Thr-OBn by Z-Ala-OH via HPLC analysis after derivatization of the Boc-deblocked depsidipeptide 8 A B C Fig. 9. A. HPLC trace of the product resulting from coupling between H-Thr(Z-Ala)- OBn and Z-Val-Aib-Gly-OH (crude reaction mixture) via N-HATU/DIEA. At the position of the LL(D)- isomer ( min) the integral shows only 0.99 % of this form. Fig. 9. B. HPLC trace of the product resulting from coupling between H-Thr(Z-D- Ala)-OBn and Z-Val-Aib-Gly-OH (crude reaction mixture) via N-HATU/DIEA. Fig. 9. C. HPLC trace of a mixture of the LL(L)- and LL(D)- depsipentapetides as made according to methods 9. A. and 9. B. above. LC conditions: eluents were A, water and B, acetonitrile containing 0.1% TFA, linear gradient: solvent B 10-90% over 50 min. S38

39 D Fig. 9. D. MALDI-MS trace for the LL(L)-depsipentapeptide prepared as indicated under 9. A, MS (m/z): LL(L)- calc: [M+Na] , found: S39

40 Fig. 9. E. MALDI-MS trace for the LL(D)-depsipentapeptide prepared as indicated under 9. B. MS (m/z): LL(D)- calc: [M+Na] , found: S40