S1

SUPPORTING INFORMATION FOR:

Synthesis and evaluation of a (3R, 6S, 9S)-2-oxo-1-azabicyclo[4.3.0]nonane scaffold as a mimic of Xaa-transPro in poly-L-proline type II helix conformation.

Boris Aillarda, Jeremy D. Kilburnb, Jeremy P. Blaydesc, Graham J. Tizzarda, Stuart Findlowd,

Jörn M. Wernerd and Sally Bloodwortha*.

aChemistry, Faculty of Natural and Environmental Sciences, University of Southampton,

Southampton, SO17 1BJ, UK.

bSchool of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London, E1 4NS, UK.

cCancer Sciences, Faculty of Medicine, University of Southampton, Southampton, SO16

6YD, UK.

dCentre for Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK.

*Correspondence author: S.Bloodworth@soton.ac.uk

CONTENTS

1 Expression and purification of 13C/15N labelled Fyn SH3 S2

1.1 Expression S2 1.2 Purification S2

2 NMR data S2

2.1 Fyn SH3 assignment S2 2.2 1H-15N HSQC titration spectra S4 2.3 Binding curves S7

3 Synthesis and characterisation of compounds S9

3.1 General methods S9

3.1.1 General techniques of SPPS S9 3.1.2 General techniques for synthesis of oligomers 9-11 S11

3.2 Experimental information S12

4 Structural data S26 5 References S26

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2015

S2

1 Expression and purification of 13C/15N labelled Fyn SH3 Methods were adapted from those of Campbell et al.1

1.1 Expression The Fyn-SH3 domain (residues 73-145) was cloned into the bacterial expression vector pGEX-2T and the derived plasmid was transformed into BL21 CodonPlus(DE3) RIPL bacteria (Agilent Technologies). The bacteria were grown in M9 minimal medium in the presence of 1 g/L of 15N NH4Cl alone or in combination with 1 g/L of 13C glucose.

1.2 Purification The protein expression was induced with 0.3 mM isopropyl-B-D-thiogalactopyranoside at 30 °C overnight, then the cells were harvested by centrifugation at 4 oC, washed once with PBS then stored at -20 oC until processed. Cells were re-suspended in TTBS (50 mM Tris-HCl, pH 8.5 +150 mM NaCl and 0.1 % (v/v) Triton X-100) (10 mL per g of cells) in the presence of lysozyme, 10 ug/mL DNase I, 10 mM MgCl2 and a cocktail of protease inhibitors (Complete from Roche). The suspension was then sonicated (4 x 20 s) to effect lysis of the cells. Samples were maintained on ice during sonication. Cell debris was then pelleted by centrifugation (30 min, 20 000 g) at 4 oC and the pellet discarded. The supernatant was applied onto a column of glutathione-Sepharose beads (GE Healthcare) in TTBS using an AktaPrime FPLC system. After extensive washing of the resin with TBS (without Triton X-100) the bound GST-SH3 fusion was eluted from the column with 15 mM glutathione in TBS. Fractions containing the fusion protein were pooled and applied to a desalting column equilibrated in 50 mM Tris-HCl pH 8.5, 150 mM NaCl to remove the glutathione, prior to thrombin cleavage of the fusion protein. Thrombin (Novagen) was added at a ratio of 1 unit of protease/mg of protein in the presence of 2.5 mM CaCl2. Cleavage was allowed to proceed at 16 °C overnight and the extent of cleavage of the fusion protein determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Cleaved material was applied to glutathione-Sepharose beads to capture free GST and the flow through was passed onto a HiLoad 26/60 Superdex 200 gel filtration column (GE Healthcare) equilibrated in buffer A (20 mM Tris-HCl, pH 8.5 containing 20 mM NaCl). Fyn-SH3 was further purified on a 1 mL Q-trap column (GE Healthcare) equilibrated in buffer A. The protein was eluted with a linear gradient from 0 to 100 % buffer B (20 mM Tris-HCl, pH 8.5 containing 500 mM) over 4 mL. Fractions containing Fyn SH3 domain were pooled and dialysed against 10 mM potassium phosphate pH 6.0, prior to be concentrated in centrifugal concentrator to 0.1 mM for NMR. 2 NMR data 2.1 Fyn SH3 assignment NMR spectra of 13C/15N labelled SH3 domain were collected at 25 oC with a 600 MHz four-channel Varian INOVA NMR spectrometer equipped with a room temperature 5 mm triple resonance z-gradient probe. Resonance assignments of the free and peptide bound form of the backbone 15N and HN nuclei were obtained by a combination of 2D 1H-15N HSQC, 3D 15N-edited NOESY-HSQC, as well as HNCA, HNCACB and CBCA(CO)NH spectra. All experiments used water flip-back and gradient-enhancement.

S3

Residue number δ H (ppm) δ N (ppm) δ Cα (ppm) δ Cβ (ppm)

Gly Thr Gly Val Thr Leu Phe Val Ala Leu Tyr Asp Tyr Glu Ala Arg Thr Glu Asp Asp Leu Ser Phe His Lys Gly Glu Lys Phe Gln Ile

Leu Asn Ser Ser Glu Gly Asp Trp Trp Glu Ala Arg Ser Leu

81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99

100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125

8.28 8.15 8.52 7.99 8.4

8.25 8.91 9.77 9.1 9.5

7.15 8.31 8.34 7.26 8.21 9.61 8.34 8.81 8.02 7.98 8.3

8.04 8.8

8.67 9.24 8.96 8.2

8.13 9.17 8.68 9.39 8.94 7.61 8.8

8.02 8.71 8.32 8.55 7.63 9.18 8.71 9.41 8.91 8.8

9.01

108.63 112.90 111.22 119.55 119.52 125.57 122.13 119.35 126.72 125.96 111.96 117.51 120.39 128.05 125.99 121.15 111.99 118.61 116.39 119.95 120.14 114.42 116.35 116.44 121.76 114.66 124.66 121.01 115.68 119.74 126.59 128.78 115.24 121.11 117.16 121.5

107.86 119.11 120.61 124.26 123.9

131.76 118.63 119.86 130.79

45.2 45.28 62.11 45.44 62.13 54.4

56.59 58.24 52.41 55.56 53.88 54.49 58.39 53.85 53.05 56.27 59.67 58.14 54.52 52.95 54.21 58.3

56.38 53.76 58.63 45.04 58.33 54.81 56.87 54.3

59.44 55.59 53.68 57.01 60.46 56.08 46.29 55.53 55.88 52.81 56.07 51.06 53.7

57.64 56.68

69.71

32.96 70.02 43.94 43.61 35.62 21.27 42.97 42.83 42.13 42.41 30.84 20.25 32.39 71.63 45.41 41.93 42.6

43.93 64.59 41.24 31.99 32.91

30.83 36.01 44.91 30.79 34.82 42.27 40.33 62.03 63.88 30.13

41.14 31.47 32.13 30.85 25.95 34.17 63.33 40.38

S4

Thr Thr Gly Glu Thr Gly Tyr Ile Ser Asn Tyr Val Ala Val Asp Ser Ile Gln

126 127 128 129 130 131 132 133 135 136 137 138 139 141 142 143 144 145

8.46 8.07 7.97 8.06 8.4

8.81 8.76 9.24 7.64 8.06 7.82 7.14 8.81 8.09 8.28 8.03 7.95 7.93

116.43 108.26 111.21 120.81 113.12 111.15 118.91 112.42 121.7

115.04 119.35 108.96 121.87 121.86 121.98 115.27 122.22 128.89

65.83 61.83 45.78 56.67 60.08 45.4 58.5 57.3

60.67 53.43 58.53 58.28 49.7

63.18 54.51 58.41 61.17 57.35

69.29 71.04

31.3 71.19

40.28 40.14 29.96 36.54 39.35 36.88 21.62 32.26 41.02 64.15 38.6

30.58

2.2 1H-15N HSQC titration spectra Overlay of 1H-15N HSQC spectra from titration of 13C/15N labelled Fyn SH3 with 12:

12: Fyn SH3

S2.3 Binding Curves

0  :  1 0.25  :  1 0.5  :  1 0.75  :  1 1  :  1 2  :  1 4  :  1 8  :  1 12  :  1

S5

Overlay of 1H-15N HSQC spectra from titration of 13C/15N labelled Fyn SH3 with 13:

13: Fyn SH3

Overlay of 1H-15N HSQC spectra from titration of 13C/15N labelled Fyn SH3 with 14:

14: Fyn SH3

0  :  1 0.25  :  1 0.5  :  1 0.75  :  1 1  :  1 2  :  1 4  :  1 8  :  1 17  :  1

0  :  1 0.25  :  1 0.5  :  1 0.75  :  1 1  :  1 2  :  1 4  :  1 8  :  1 25  :  1

S6

Overlay of 1H-15N HSQC spectra from titration of 13C/15N labelled Fyn SH3 with 15:

15: Fyn SH3

0  :  1 0.25  :  1 0.5  :  1 0.75  :  1 1  :  1 2  :  1 4  :  1 8  :  1 20  :  1

S7

2.3 Binding curves The equilibrium dissociation constant Kd was obtained for residues in fast exchange by fitting Kd to the fractional shift (Δδ/Δδ max) using the formula:

Δδ/Δδ max =!!! ! ! ! ! !!! ! ! ! ^!!! ! !

! !

where Δδ represents the difference between the measured shift at any (maximal = Δδ max) ligand concentration and the shift of the free protein; and [L] and [P] are the ligand and protein concentrations respectively. Titration of 13C/15N labelled Fyn SH3 with 12:

S8

Titration of 13C/15N labelled Fyn SH3 with 13:

S9

3 Synthesis and characterisation of compounds

3.1 General methods Solution phase reactions were carried out in solvents of commercial grade unless otherwise stated, and those requiring dry conditions were conducted in flame-dried glassware under argon with freshly distilled solvents. THF was distilled under argon from benzophenone and sodium. CH2Cl2, Et3N, MeCN, MeOH, piperidine and Ac2O were distilled from calcium hydride. All chemicals were used as received from commercial suppliers. Melting points were determined in open capillary tubes using a Gallenkamp Electrothermal melting point apparatus and are uncorrected. Optical rotation was measured on an Optical Activity POLAAR 2001 polarimeter. NMR spectra recorded for structural characterisation of compounds in section 3.2 were obtained on a Bruker AV300 or DPX400 MHz spectrometer as stated. 1H chemical shifts are reported as values in ppm referenced to residual solvent. The following abbreviations are used to assign multiplicity and may be compounded: s = singlet, d = doublet, t = triplet, q = quartet and m = multiplet. Coupling constants, J, are measured in Hertz (Hz). 13C spectra are proton decoupled and referenced to solvent. 13C resonances are reported as s, d, t, q, depending on the number of directly attached protons (0, 1, 2, 3 respectively) as determined by DEPT experiments. Infrared spectra were obtained for neat samples using a PerkinElmer Spectrum One FT6IR Spectrometer. Absorptions are given in wavenumber (cm-1). [α]D values are given in 10-1 deg cm2 g-1. Low resolution ESI+ mass spectra were recorded on a Waters 2700 sample manager in methanol. Accurate mass measurements were carried out at 10 000 resolutions on a Bruker Apex III FT-ICR mass spectrometer. Values of m/z are reported in atomic mass units. Purity data was obtained for compounds 9-11 using a Dionex Ultimate® 3000 UHPLC system (Thermo Scientific, Hemel Hempstead, UK). Samples were injected directly onto a Kinetex C18 Column (50 mm × 2.1 mm, 1.7 µm particle size; Phenomenex, Torrance, CA, USA) thermostatically controlled at 40 °C. Separation was achieved using 2% MeOH and 0.1% formic acid in water, held for 2 min followed by a linear gradient to 100% MeOH over 7 min. and return to 2 % MeOH for 2 min at a flow rate of 0.3 mL min-1. Mass spectra were recorded on a Maxis™ ESI-ToF mass spectrometer (Bruker Daltonics, Bremen, Germany) using positive ion electrospray ionisation (120-1500 m/z). 3.1.1 General techniques of SPPS Solid phase synthesis of 12-15 was conducted manually in three-way filtration tubes using conventional Fmoc (fluorenylmethyloxy carbonyl) protection and supported upon Rink amide resin (Merck). All Fmoc-protected amino acids were L-confirguration and were purchased from NovaBiochem except for Fmoc-Arg(Pbf)-OH (Aldrich). 2,2,4,6,7-Pentamethyldihydrobenzofuran-sulfonyl (Pbf) L-arginine side chain protection was used. General coupling was performed with either HOBt/DIC or HBTU in the presence of iPr2NEt in DMF. Deprotection was effected with two treatments of 20% (v/v) piperidine in DMF. Deprotection and coupling steps were monitored with the Kaiser Ninhydrin test (1° L-amino acid residues) or 2,3,5,6-tetrachlorobenzoquinone (chloranil) test (2° L-amino acid residues). N-terminus acylation was carried out using 50% Ac2O (v/v) in pyridine. Cleavage of the

S10

peptide from the support and side chain protecting group removal was carried out with a mixture of CF3CO2H/iPr3SiH/H2O (96:2:2). Crude peptides were precipitated from MeOH/Et2O. Fmoc deprotection of resin Rink amide resin was pre-swollen in CH2Cl2 for 15 min. 20% v/v Piperidine in DMF (~20 mL/g of resin) was added to the resin and suspended in the vessel for 20 min. The resin was then drained and washed alternately with CH2Cl2 (20 mL/g of resin × 3), MeOH (20 mL/g of resin × 3), Et2O (20 mL/g of resin × 3) and 50% (v/v) iPr2NEt in DMF (20 mL/g of resin). Coupling of Fmoc-protected amino acids Deprotected resin (1.0 mmol of resin loading) was pre-swollen in CH2Cl2 for 20 min. To a stirred solution of Fmoc-AA-OH (3.0 mmol) in DMF (2 mL for 3.0 mmol scale) was added HOBt (3.0 mmol), DIC (3.0 mmol, and iPr2NEt (5.0 mmol), and the vessel then sealed and shaken for 5 h. All solvents were removed by suction filtration and the resin washed with DMF (20 mL/g of resin × 3), CH2Cl2 (20 mL/g of resin × 3), MeOH (20 mL/g of resin × 3) and Et2O (20 mL/g of resin × 3). General N-Fmoc deprotection The resin was treated with 20% v/v piperidine in DMF (~20 mL/g of resin) and shaken for 30 min before draining and washing alternately with DMF (20 mL/g of resin × 3), CH2Cl2 (20 mL/g of resin × 3), MeOH (20 mL/g of resin × 3) and Et2O (20 mL/g of resin × 3). N-Terminus acylation Free amine-functionalised resin (1.0 mmol of resin loading) was treated with 50% Ac2O v/v in pyridine (3.0 mmol) and shaken for 45 min. All solvents were removed by suction filtration and the resin was washed with DMF (20 mL/g of resin × 3), CH2Cl2 (20 mL/g of resin × 3), MeOH (20 mL/g of resin × 3) and Et2O (20 mL/g of resin × 3). Peptide cleavage from resin Peptide-functionalised resin (1.0 mmol of resin loading) was suspended in a mixture of CF3CO2H/iPr3SiH/H2O (96:2:2, 20 mL/g of resin). The vessel was sealed and shaken for 2 h. All solvents were removed by suction filtration. The treatment was repeated once with fresh reagents. The resin was then washed with CH2Cl2 (20 mL/g of resin × 3), MeOH (20 mL/g of resin × 3) and Et2O (20 mL/g of resin × 3). All solvents were removed by toluene azeotrope in vacuo. The peptide product was then precipitated from MeOH and Et2O. Ninhydrin test Reagent A: Solution 1: To a stirred solution of phenol (40 g) in hot ethanol (10 mL) was added amberlite mix-bed resin MB-3 (4 g) and the resulting mixture stirred for 1 h. The resin was removed by suction filtration. Solution 2: To a stirred solution of KCN (1.3 mg in 2.0 mL of H2O) was added distilled pyridine to a volume of 100 mL. Amberlite mixed bed-resin MB-3 (4 g) was added and the resulting mixture was stirred for 1 h. The resin was removed by suction filtration. Solutions 1 and 2 were mixed to afford reagent A. Reagent B: Ninhydrin (25 g) was dissolved in absolute ethanol (50 mL) and stored in the dark. To a small quantity of resin in a small test tube was added 7 drops of reagent A and 3 drops of reagent B. A control was prepared using the stains without the resin beads. Both tubes

S11

were heated at 100 oC for 5 min. Resin beads and solution became blue (positive result, free amino groups) or stayed pale yellow (negative result, complete coupling). Chloranil test A few resin beads were placed in a test tube and 2 drops of chloranil (2% v/v in DMF) were added. A control was prepared using the stains without the resin beads. Both tubes were heated at 100 oC for 5 min. The beads functionalised with free amino groups appeared as a deep blue colour (positive result) while completely coupled beads remained colourless (negative result). Purity data was obtained for compounds 12-15 using an Agilent 1200 LCMS system. Samples were directly injected onto a Zorbax SB-C18 Column (4.6 × 150 mm, 5 µm particle size). Separation was achieved following the gradient conditions in Table S1:

Time (min) % water + 0.1% formic acid % MeOH + 0.1% formic acid Flow rate (mL min-1)

0 100 0 1.0

2 98 2 1.0

8 80 20 1.0

10.5 0 100 1.0

12.5 0 100 1.0

Table S1

3.1.2 General techniques for synthesis of oligomers 9-11 N-Boc deprotection N-Boc protected substrate (0.3 mmol) was treated CF3CO2H (10 mL of a 20% v/v solution in CH2Cl2) at room temperature and the resulting mixture stirred overnight. CH2Cl2 was removed in vacuo and the residue then washed with toluene (3 × 10 mL), removal of the azeotrope with CF3CO2H was made in vacuo each time. The resulting crude oil was washed alternately with EtOAc (5 × 10 mL in total) and MeOH (5 × 10 mL in total) until a white solid of consistent weight was obtained. Saponification To a stirred solution of methyl ester substrate (0.3 mmol) in THF (1 mL) was added LiOH (1 mL of a 1M aqueous solution). The resulting solution was stirred at room temperature for 1 h and then acidified with 1M HCl (aq) (2 mL) until pH 5. The aqueous portion was extracted CH2Cl2 (5 × 15 mL), and the combined organic extracts dried over MgSO4 before concentration in vacuo. Amide coupling To a solution of carboxylic acid substrate in DMF (5 mL for a 0.3 mmol scale reaction) was added HOBt (1.2 eq.) and EDC (1.2 eq.). The resulting mixture was stirred at room temperature for 15 min. A mixture of the CF3CO2H salt of the amine substrate (1.0 eq. prepared according to the general method above) and iPr2NEt (3.0 eq.) in DMF (5 mL) was

S12

then added at 0 °C and the resulting mixture stirred overnight at room temperature. Solvents were then removed in vacuo. Purification gave compounds 9-11 as white solids in each case. Purification methods, yields and characterisation data are provided in section S3.2.

3.2 Experimental information Pyrrolidine derivative 5 was prepared according to the method of Scolastico et al.2 as a 3:1 (Z:E) mixture of isomers. Pyrrolidine derivative 6 To a stirred solution of 5 (1.0 g, 1.96 mmol) in dry methanol (10 mL) was added (R,R)-Rh(DUPHOS-Et)OTf (10 mg, 0.014 mmol) under argon. The mixture was purged with 7 bar of H2 five times and stirred for 24 h under 7 bar of H2 before concentration in vacuo. Purification by column chromatography (SiO2 eluted with petroleum ether/Et2O (25:75)) gave the title compound 6 as a colourless oil (900 mg, 90 %). 1H NMR (DMSO-d6, 400 MHz, 80 oC) δ 7.47 (1H, broad s), 7.39-7.24 (10H, m), 5.12-5.00 (4H, m), 4.34 (1H, m), 4.04 (1H, m), 3.87 (1H, m), 3.62 (3H, s), 3.60 (3H, s), 2.21 (1H, m), 2.00-1.60 (6H, m), 1.51 (1H, m) ppm. 13C NMR (CDCl3 100 MHz) δ 173.5 (s), 172.8 (s), 156.1 (s), 154.5 (s), 136.5 (2 × s), 128.5 (2 × d), 128.4 (d), 128.0 (d), 127.9 (d), 127.6 (d), 126.5 (d), 67.2 (t), 67.0 (t), 59.7 (d), 58.6 (d), 53.9 (d), 52.2 (q), 52.1 (q), 30.4 (t), 28.9 (t), 28.5 (t), 28.0 (t) ppm. IR νmax cm-1 3324, 2952, 1699, 1501, 1409, 1349, 1203, 1173, 1111, 1045, 771, 739, 697. [α] D

24 9.9 (c 0.5, MeOH). ESI m/z 535 (M+Na)+. HRMS (ESI, m/z) Calculated for C27H32N2O8Na 535.2051, found 535.2057.

S13

ju0812ba2.014.001.1r.esp

Chemical Shift (ppm)220 200 180 160 140 120 100 80 60 40 20

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

CHLOROFORM-d17

3.45

173.

4517

3.45

173.

4517

3.45

173.

4517

3.45

173.

4517

3.45

173.

4517

3.45

173.

4517

3.45

173.

4517

3.45

173.

4517

3.45

173.

4517

3.45

173.

4517

3.45

173.

4517

2.78

172.

7817

2.78

172.

7817

2.78

172.

7817

2.78

172.

7817

2.78

172.

7817

2.78

172.

7817

2.78

172.

7817

2.78

172.

7817

2.78

172.

7817

2.78

172.

7817

2.78

172.

78

156.

1115

6.11

156.

1115

6.11

156.

1115

6.11

156.

1115

6.11

156.

1115

6.11

156.

1115

6.11

156.

1115

6.11

156.

1115

6.11

156.

1115

6.11

156.

1115

6.11

156.

1115

6.11 154.

5615

4.56

154.

5615

4.56

154.

5615

4.56

154.

5615

4.56

154.

5615

4.56

154.

5615

4.56

154.

5615

4.56

154.

5615

4.56

154.

5615

4.56

154.

5615

4.56

154.

5615

4.56 13

6.49

136.

4913

6.49

136.

4913

6.49

136.

4913

6.49

136.

4913

6.49

136.

4913

6.49

136.

4913

6.49

136.

4913

6.49

136.

4913

6.49

136.

4913

6.49

136.

4913

6.49

136.

49

128.

4612

8.46

128.

4612

8.46

128.

4612

8.46

128.

4612

8.46

128.

4612

8.46

128.

4612

8.46

128.

4612

8.46

128.

4612

8.46

128.

4612

8.46

128.

4612

8.46

128.

4612

8.46

128.

0412

8.04

128.

0412

8.04

128.

0412

8.04

128.

0412

8.04

128.

0412

8.04

128.

0412

8.04

128.

0412

8.04

128.

0412

8.04

128.

0412

8.04

128.

0412

8.04

128.

0412

8.04

127.

6112

7.61

127.

6112

7.61

127.

6112

7.61

127.

6112

7.61

127.

6112

7.61

127.

6112

7.61

127.

6112

7.61

127.

6112

7.61

127.

6112

7.61

127.

6112

7.61

127.

6112

7.61

125.

5012

5.50

125.

5012

5.50

125.

5012

5.50

125.

5012

5.50

125.

5012

5.50

125.

5012

5.50

125.

5012

5.50

125.

5012

5.50

125.

5012

5.50

125.

5012

5.50

125.

5012

5.50

67.2

767

.27

67.2

767

.27

67.2

767

.27

67.2

767

.27

67.2

767

.27

67.2

767

.27

67.2

767

.27

67.2

767

.27

67.2

767

.27

67.2

767

.27

67.2

767

.27

67.0

467

.04

67.0

467

.04

67.0

467

.04

67.0

467

.04

67.0

467

.04

67.0

467

.04

67.0

467

.04

67.0

467

.04

67.0

467

.04

67.0

467

.04

67.0

467

.04

66.8

366

.83

66.8

366

.83

66.8

366

.83

66.8

366

.83

66.8

366

.83

66.8

366

.83

66.8

366

.83

66.8

366

.83

66.8

366

.83

66.8

366

.83

66.8

366

.83

60.0

160

.01

60.0

160

.01

60.0

160

.01

60.0

160

.01

60.0

160

.01

60.0

160

.01

60.0

160

.01

60.0

160

.01

60.0

160

.01

60.0

160

.01

60.0

160

.01

59.6

659

.66

59.6

659

.66

59.6

659

.66

59.6

659

.66

59.6

659

.66

59.6

659

.66

59.6

659

.66

59.6

659

.66

59.6

659

.66

59.6

659

.66

59.6

659

.66

58.0

058

.00

58.0

058

.00

58.0

058

.00

58.0

058

.00

58.0

058

.00

58.0

058

.00

58.0

058

.00

58.0

058

.00

58.0

058

.00

58.0

058

.00

58.0

058

.00

52.2

952

.29

52.2

952

.29

52.2

952

.29

52.2

952

.29

52.2

952

.29

52.2

952

.29

52.2

952

.29

52.2

952

.29

52.2

952

.29

52.2

952

.29

52.2

952

.29

52.1

052

.10

52.1

052

.10

52.1

052

.10

52.1

052

.10

52.1

052

.10

52.1

052

.10

52.1

052

.10

52.1

052

.10

52.1

052

.10

52.1

052

.10

52.1

052

.10

30.4

530

.45

30.4

530

.45

30.4

530

.45

30.4

530

.45

30.4

530

.45

30.4

530

.45

30.4

530

.45

30.4

530

.45

30.4

530

.45

30.4

530

.45

30.4

530

.45

30.3

330

.33

30.3

330

.33

30.3

330

.33

30.3

330

.33

30.3

330

.33

30.3

330

.33

30.3

330

.33

30.3

330

.33

30.3

330

.33

30.3

330

.33

30.3

330

.33

29.9

029

.90

29.9

029

.90

29.9

029

.90

29.9

029

.90

29.9

029

.90

29.9

029

.90

29.9

029

.90

29.9

029

.90

29.9

029

.90

29.9

029

.90

29.9

029

.90

28.8

928

.89

28.8

928

.89

28.8

928

.89

28.8

928

.89

28.8

928

.89

28.8

928

.89

28.8

928

.89

28.8

928

.89

28.8

928

.89

28.8

928

.89

28.8

928

.89

28.5

428

.54

28.5

428

.54

28.5

428

.54

28.5

428

.54

28.5

428

.54

28.5

428

.54

28.5

428

.54

28.5

428

.54

28.5

428

.54

28.5

428

.54

28.5

428

.54

28.0

228

.02

28.0

228

.02

28.0

228

.02

28.0

228

.02

28.0

228

.02

28.0

228

.02

28.0

228

.02

28.0

228

.02

28.0

228

.02

28.0

228

.02

28.0

228

.02

2-oxo-1-azabicyclo[4.3.0]nonane motif N-Boc(3R,6S,9S)-1-OMe To a solution of 6 (1.0 g, 2.0 mmol) in acetonitrile (10 mL) was added DMAP (26 mg, 0.2 mmol) followed by dropwsie addition of a solution of Boc2O (0.64 g, 3.0 mmol) in acetonitrile (5 mL) at 0 oC. The reaction mixture was stirred overnight at room temperature before concentration in vacuo. Purification by column chromatography (SiO2 eluted with petroleum ether/Et2O (4:6)) gave N-Boc-6 as a colourless oil (1.2 g, 97%). A mixture of N-Boc-6 (1.11 g, 1.81 mmol), Pd/C (10%, 30 mg) and MeOH (10 mL) under nitrogen, was purged with H2 (1 atm.). The reaction was then stirred overnight under H2 (1 atm.) before removal of solvents by filtration followed by concentration in vacuo. Purification by column chromatography (SiO2 eluted with CH2Cl2/MeOH (98:2)) gave the title compound N-Boc(3R,6S,9S)-1-OMe as a clear oil (0.55 g, 98%). 1H NMR (CDCl3, 400 MHz) δ 5.38 (1H, broad s, NH) 4.43 (1H, d, J = 9.4 Hz), 3.90 (1H, dd, J = 10.3, 5.5 Hz), 3.71 (3H, s), 3.63 (1H, tt, J = 11.3, 3.4 Hz), 2.42 (1H, m), 2.19-1.95 (4H, m), 1.79-1.57 (3H, m), 1.43 (9H, s) ppm. 13C NMR (CDCl3, 100 MHz) δ 172.1 (s), 168.6 (s), 155.9 (s), 79.7 (s), 60.6 (d), 58.2 (d), 52.2 (q), 52.1 (d), 31.5 (t), 28.6 (t), 28.4 (t), 28.3 (q), 27.7 (t) ppm. IR νmax cm-1 3303, 2977, 1748, 1700, 1653, 1532, 1401, 1368, 1255, 1175. [α] D

24 –72.6 (c 0.5, MeOH). ESI m/z 335 (M+Na)+. HRMS (ESI, m/z) Calculated for C15H24N2O5Na 335.1577, found 335.1583.

S14

dc0111ba3.011.001.1r.esp

Chemical Shift (ppm)220 200 180 160 140 120 100 80 60 40 20

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

CHLOROFORM-d

172.

0817

2.08

172.

0817

2.08

172.

0817

2.08

172.

0817

2.08

172.

0817

2.08

172.

0817

2.08

172.

0817

2.08

172.

0817

2.08

172.

0817

2.08

172.

0817

2.08

172.

0817

2.08

168.

5916

8.59

168.

5916

8.59

168.

5916

8.59

168.

5916

8.59

168.

5916

8.59

168.

5916

8.59

168.

5916

8.59

168.

5916

8.59

168.

5916

8.59

168.

5916

8.59

168.

5916

8.59

155.

9215

5.92

155.

9215

5.92

155.

9215

5.92

155.

9215

5.92

155.

9215

5.92

155.

9215

5.92

155.

9215

5.92

155.

9215

5.92

155.

9215

5.92

155.

9215

5.92

155.

9215

5.92

79.6

979

.69

79.6

979

.69

79.6

979

.69

79.6

979

.69

79.6

979

.69

79.6

979

.69

79.6

979

.69

79.6

979

.69

79.6

979

.69

79.6

979

.69

79.6

979

.69

60.6

260

.62

60.6

260

.62

60.6

260

.62

60.6

260

.62

60.6

260

.62

60.6

260

.62

60.6

260

.62

60.6

260

.62

60.6

260

.62

60.6

260

.62

60.6

260

.62

58.1

658

.16

58.1

658

.16

58.1

658

.16

58.1

658

.16

58.1

658

.16

58.1

658

.16

58.1

658

.16

58.1

658

.16

58.1

658

.16

58.1

658

.16

58.1

658

.16

52.2

552

.25

52.2

552

.25

52.2

552

.25

52.2

552

.25

52.2

552

.25

52.2

552

.25

52.2

552

.25

52.2

552

.25

52.2

552

.25

52.2

552

.25

52.2

552

.25 31

.55

31.5

531

.55

31.5

531

.55

31.5

531

.55

31.5

531

.55

31.5

531

.55

31.5

531

.55

31.5

531

.55

31.5

531

.55

31.5

531

.55

31.5

531

.55

31.5

528

.31

28.3

128

.31

28.3

128

.31

28.3

128

.31

28.3

128

.31

28.3

128

.31

28.3

128

.31

28.3

128

.31

28.3

128

.31

28.3

128

.31

28.3

128

.31

28.3

127

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

027

.70

27.7

0

S15

2-oxo-1-azabicyclo[4.3.0]nonane motif N-H(3R,6S,9S)-1-OMe A mixture of 6 (1.11 g, 2.17 mmol), Pd/C (10%, 30 mg) and MeOH (10 mL) under nitrogen, was purged with H2 (1 atm.). The reaction was stirred overnight under H2 (1 atm.) before removal of solvents by filtration followed by concentration in vacuo. Purification by column chromatography (SiO2 eluted with CH2Cl2/MeOH (93:7)) gave the title compound N-H(3R,6S,9S)-1-OMe as a yellow oil (0.33 g, 72%). 1H NMR (CDCl3, 400 MHz) δ 4.38 (1H, d, J = 9.5 Hz), 3.71 (3H, s), 3.56 (1H, tt, J = 10.1, 4.0 Hz), 3.37 (1H, dd, J = 6.3, 10.9 Hz), 2.29 (1H, m), 2.18-2.05 (2H, m), 2.04-1.91 (2H, m), 1.81-1.50 (5H, m) ppm. 13C NMR (CDCl3, 100 MHz) δ 172.3 (s), 171.8 (s), 60.8 (d), 57.9 (d), 52.2 (q), 52.0 (d), 31.6 (t), 30.6 (t), 28.5 (t), 28.0 (t) ppm. IR νmax cm-1 3365, 2952, 1736, 1703, 1629, 1514, 1439, 1365, 1328, 1167. [α] D

24 –107.4 (c 0.5, MeOH). ESI m/z 213 (M+H) +, 276 (M+Na+CH3CN) +, 447 (2M+Na)+. HRMS (ESI, m/z) Calculated for C10H16N2O3Na 235.1053, found 235.1057.

S16

ju2112ba3.011.001.1r.esp

Chemical Shift (ppm)220 200 180 160 140 120 100 80 60 40 20

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

CHLOROFORM-d

172.

3417

2.34

172.

3417

2.34

172.

3417

2.34

172.

3417

2.34

172.

3417

2.34

172.

3417

2.34

172.

3417

2.34

172.

3417

2.34

172.

3417

2.34

172.

3417

2.34

172.

3417

2.34

171.

7617

1.76

171.

7617

1.76

171.

7617

1.76

171.

7617

1.76

171.

7617

1.76

171.

7617

1.76

171.

7617

1.76

171.

7617

1.76

171.

7617

1.76

171.

7617

1.76

171.

7617

1.76

60.7

860

.78

60.7

860

.78

60.7

860

.78

60.7

860

.78

60.7

860

.78

60.7

860

.78

60.7

860

.78

60.7

860

.78

60.7

860

.78

60.7

860

.78

60.7

860

.78

57.8

457

.84

57.8

457

.84

57.8

457

.84

57.8

457

.84

57.8

457

.84

57.8

457

.84

57.8

457

.84

57.8

457

.84

57.8

457

.84

57.8

457

.84

57.8

457

.84

52.1

552

.15

52.1

552

.15

52.1

552

.15

52.1

552

.15

52.1

552

.15

52.1

552

.15

52.1

552

.15

52.1

552

.15

52.1

552

.15

52.1

552

.15

52.1

552

.15

51.9

851

.98

51.9

851

.98

51.9

851

.98

51.9

851

.98

51.9

851

.98

51.9

851

.98

51.9

851

.98

51.9

851

.98

51.9

851

.98

51.9

851

.98

51.9

851

.98

31.5

731

.57

31.5

731

.57

31.5

731

.57

31.5

731

.57

31.5

731

.57

31.5

731

.57

31.5

731

.57

31.5

731

.57

31.5

731

.57

31.5

731

.57

31.5

731

.57

28.4

728

.47

28.4

728

.47

28.4

728

.47

28.4

728

.47

28.4

728

.47

28.4

728

.47

28.4

728

.47

28.4

728

.47

28.4

728

.47

28.4

728

.47

28.4

728

.47

27.9

727

.97

27.9

727

.97

27.9

727

.97

27.9

727

.97

27.9

727

.97

27.9

727

.97

27.9

727

.97

27.9

727

.97

27.9

727

.97

27.9

727

.97

27.9

727

.97

Carboxylic acid 8 To a stirred solution of N-Boc(3R,6S,9S)-1-OMe (110 mg, 0.35 mmol) in THF (1.5 mL) was added LiOH (0.1 mL of an 1M aqueous solution) at room temperature. The resulting mixture was stirred for 1 h and 1M HCl (aq) then added until pH 5. Extraction with CH2Cl2 (10 mL) was carried out and the organic extract was washed with brine (10 mL) before drying over Na2SO4 and filtration. The title compound 8 was obtained, quantitatively, without further purification. 1H NMR (D2O, 300 MHz) δ 4.18 (1H, d, J = 9.5 Hz), 3.90 (1H, m), 3.63 (1H, m), 2.35-1.50 (6H, m), 1.75-1.50 (2H, m), 1.39 (9H, s) ppm. 1 × OH and 1 × NH protons are missing from the 1H NMR spectrum. 13C NMR (CDCl3, 100 MHz) δ 174.0 (s), 169.9 (s), 155.9 (s), 79.9 (s), 60.9 (d), 58.8 (d), 52.0 (d), 31.5 (t), 28.5 (t), 28.3 (q), 28.1 (t), 27.8 (t) ppm. IR νmax cm-1 3336, 2973, 1703, 1634, 1515, 1445, 1247, 1161. [α] D

24 20.5 (c 0.8, CHCl3). ESI m/z 321 (M+Na)+, 619 (2M+Na)+. HRMS (ESI, m/z) Calculated for C28H44N4O10Na 619.2950, found 619.2955.

S17

ju2112ba4.011.001.1r.esp

Chemical Shift (ppm)160 140 120 100 80 60 40 20

0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

CHLOROFORM-d

174.

0517

4.05

174.

0517

4.05

174.

0517

4.05

174.

0517

4.05

174.

0517

4.05

174.

0517

4.05

174.

0517

4.05

174.

0517

4.05

174.

0517

4.05

174.

0517

4.05

174.

0517

4.05

169.

8916

9.89

169.

8916

9.89

169.

8916

9.89

169.

8916

9.89

169.

8916

9.89

169.

8916

9.89

169.

8916

9.89

169.

8916

9.89

169.

8916

9.89

169.

8916

9.89

169.

8916

9.89 15

5.94

155.

9415

5.94

155.

9415

5.94

155.

9415

5.94

155.

9415

5.94

155.

9415

5.94

155.

9415

5.94

155.

9415

5.94

155.

9415

5.94

155.

9415

5.94

155.

9415

5.94

155.

94

79.8

779

.87

79.8

779

.87

79.8

779

.87

79.8

779

.87

79.8

779

.87

79.8

779

.87

79.8

779

.87

79.8

779

.87

79.8

779

.87

79.8

779

.87

79.8

779

.87

60.9

360

.93

60.9

360

.93

60.9

360

.93

60.9

360

.93

60.9

360

.93

60.9

360

.93

60.9

360

.93

60.9

360

.93

60.9

360

.93

60.9

360

.93

60.9

360

.93

58.8

558

.85

58.8

558

.85

58.8

558

.85

58.8

558

.85

58.8

558

.85

58.8

558

.85

58.8

558

.85

58.8

558

.85

58.8

558

.85

58.8

558

.85

58.8

558

.85

52.0

752

.07

52.0

752

.07

52.0

752

.07

52.0

752

.07

52.0

752

.07

52.0

752

.07

52.0

752

.07

52.0

752

.07

52.0

752

.07

52.0

752

.07

52.0

752

.07

31.5

131

.51

31.5

131

.51

31.5

131

.51

31.5

131

.51

31.5

131

.51

31.5

131

.51

31.5

131

.51

31.5

131

.51

31.5

131

.51

31.5

131

.51

31.5

131

.51

28.3

628

.36

28.3

628

.36

28.3

628

.36

28.3

628

.36

28.3

628

.36

28.3

628

.36

28.3

628

.36

28.3

628

.36

28.3

628

.36

28.3

628

.36

28.3

628

.36

28.0

928

.09

28.0

928

.09

28.0

928

.09

28.0

928

.09

28.0

928

.09

28.0

928

.09

28.0

928

.09

28.0

928

.09

28.0

928

.09

28.0

928

.09

28.0

928

.09

27.7

727

.77

27.7

727

.77

27.7

727

.77

27.7

727

.77

27.7

727

.77

27.7

727

.77

27.7

727

.77

27.7

727

.77

27.7

727

.77

27.7

727

.77

27.7

727

.77

S18

Dimer 9 N-deprotection of N-Boc(3R,6S,9S)-1-OMe (64 mg, 0.3 mmol) and coupling of the resulting CF2CO2H salt of N-H(3R,6S,9S)-1-OMe with 8 (90 mg, 0.3 mmol), were carried out according to the general procedures of section S3.1.2 Purification by column chromatography (SiO2 eluted with CH2Cl2/MeOH/NH4OH (aq. 37%) (93:7:1)) followed by precipitation with Et2O afforded 9 (119 mg, 81% over 3 steps). m.p. 87-90 oC (Et2O). 1H NMR (CD3CN, 300 MHz) δ 6.80 (1H, broad s, NH), 5.59 (1H, broad s, NH), 4.27 (1H, d, J = 9.6 Hz), 4.19 (1H, d, J = 9.6 Hz), 4.03 (1H, m), 3.85 (1H, m), 3.63 (3H, s), 3.56-3.49 (2H, m), 2.36-1.97 (10H, m), 1.88-1.73 (2H, m), 1.72-1.50 (4H, m), 1.40 (9H, s) ppm. 13C NMR (CDCl3, 100 MHz) δ 172.3 (s), 171.1 (s), 169.0 (s), 167.7 (s), 155.8 (s), 79.7 (s), 61.3 (d), 60.3 (d), 59.4 (d), 58.1 (d), 52.4 (d), 52.2 (d), 51.8 (q), 31.8 (t), 31.6 (t), 29.0 (t), 28.4 (t), 28.3 (q), 28.1 (t), 27.8 (t), 27.7 (t), 27.6 (t) ppm. IR νmax cm-1 3295, 2949, 2350, 1740, 1631, 1518, 1440, 1364, 1248, 1200, 1166. [α] D

24 –13.8 (c 0.25, MeOH). ESI m/z 515 (M+Na)+. HRMS (ESI, m/z) Calculated for C24H36N4O7Na 515.2476, found 515.2480. UV λmax (MeOH)/nm 227 (A = 0.79, c = 0.5 mM, ε = 1580).

S19

Trimer 10 N-deprotection of 9 (80 mg, 0.16 mmol) and coupling of the resulting CF2CO2H amine salt with 8 (48 mg, 0.16 mmol), were carried out according to the general procedures of section S3.1.2. Purification by column chromatography (SiO2 eluted with CH2Cl2/MeOH/NH4OH (aq. 37%) (93:7:1)) followed by precipitation with Et2O afforded 10 (79 mg, 73% over 3 steps). m.p. 175-178 oC (CH2Cl2/Et2O). 1H NMR (CD3CN, 400 MHz) δ 7.09 (1H, broad s, NH), 6.94 (1H, d, J = 7.7 Hz, NH), 5.91 (1H, d, J = 6.6 Hz, NH), 4.30 (1H, d, J = 9.5 Hz), 4.26-4.16 (4H, m), 3.82 (1H, m), 3.62 (3H, s), 3.58-3.47 (3H, m), 2.20-1.45 (24H, m), 1.44 (9H, s) ppm. 13C NMR (CD3CN, 100 MHz) δ 173.7 (s), 173.4 (s), 173.1 (s), 170.6 (s), 169.9 (s), 157.9 (s), 80.4 (s), 61.9 (d), 61.9 (d), 61.7 (d), 61.1 (d), 61.0 (d), 59.8 (d), 53.1 (d), 52.5

S20

(q), 51.3 (d), 51.2 (d), 32.9 (t), 32.8 (t), 32.7 (t), 30.2 (t), 30.1 (t), 29.4 (t), 29.3 (t), 29.2 (t), 28.9 (q, 3C), 28.9 (t) ppm. 1 × (s) and 3 × C (t) are unobserved as a result of overlap. IR νmax cm-1 3272, 2949, 2350, 1628, 1532, 1443, 1366, 1326, 1249, 1199, 1168, 1126. [α] D

24 – 13.9 (c 0.33, MeOH). ESI m/z 695 (M+Na)+. HRMS (ESI, m/z) Calculated for C33H48N6O9Na 695.3375, found 695.3386. UV λmax (MeOH)/nm 229 (A = 1.20, c = 0.25 mM, ε = 4800), 281 (A = 0.67, c = 0.25 mM, ε = 2680).

S21

Tetramer 11 N-deprotection of 10 (48 mg, 0.07 mmol) and coupling of the resulting CF2CO2H amine salt with 8 (21 mg, 0.07 mmol), were carried out according to the general procedures of section S3.1.2. Purification by column chromatography (SiO2 eluted with CH2Cl2/MeOH/NH4OH (aq. 37%) (85:15:1)) followed by precipitation with Et2O afforded 11 (9 mg, 15% over 3 steps). m.p. 115-118 oC (CH2Cl2/Et2O). 1H NMR (CD3CN, 400 MHz) δ 6.85 (1H, d, J = 7.6 Hz, NH), 6.79 (2H, d, J = 7.6 Hz, NH), 5.35 (1H, broad s, NH), 4.29 (1H, d, J = 9.6 Hz), 4.22-4.14 (3H, m), 4.12-3.80 (4H, m), 3.64 (3H, s), 3.61-3.47 (4H, m), 2.22-1.48 (32H, m), 1.40 (9H, s) ppm. 13C NMR (CDCl3, 100 MHz) δ 172.3 (s), 171.4 (s), 171.3 (s), 169.1 (s), 168.7 (s), 168.5 (s), 167.9 (s), 155.9 (s), 79.6 (s), 61.2 (d), 60.9 (d), 60.3 (d), 59.5 (d), 59.4 (d), 59.3 (d), 58.2 (d), 52.3 (q), 51.7 (d), 51.6 (d), 51.5 (d), 31.9 (t), 31.8 (t), 31.6 (t), 30.3 (t), 28.4 (t), 28.4 (q, 3C), 28.2 (t), 28.1 (t), 28.1 (t), 27.9 (t), 27.9 (t), 27.7 (t), 27.6 (t) ppm. 1 × (s), 2 × (d) and 4 × (t) are unobserved as a result of overlap. IR νmax cm-1 3294, 2945, 2358, 1740, 1633, 1520, 1437, 1366, 1204, 1167. [α] D

24 – 17.4 (c 0.43, MeOH). ESI m/z 875 (M+Na)+ (100 %). HRMS (ESI, m/z) Calculated for C42H60N8O11Na 875.4274, found 875.4261. UV λmax (MeOH)/nm 228 (A = 1.01, c = 0.125 mM, ε = 8080), 276 (A = 0.19, c = 0.125 mM, ε = 1520).

S22

Parent peptide N-Ac-RPLPVAPG-NH2 (12) Solid-phase synthesis (450 mg resin loading, 0.28 mmol) and purification (to >90%) of 12 was carried out according to the general procedures described in section S3.1.1 (106 mg, 45%). m.p. 105-107 oC (MeOH/Et2O). 1H NMR (DMSO-d6, 400 MHz) δ 8.71-6.55 (11H, m, NH), 4.69-3.67 (15H, m), 3.26-2.97 (2H, m), 2.58 (1H, m), 2.40-0.70 (37H, m) ppm. IR νmax cm-1 3278, 2958, 1625, 1537, 1440, 1386, 1199, 1045. [α] D

24 –117.6 (c 0.21, MeOH). ESI m/z 847 (M+H)+. HRMS (ESI, m/z) Calculated for C39H67N12O9 847.51485, found 847.51512.

S23

Peptidomimetic analogue N-Ac-RPLPV1G-NH2 (13) Solid-phase synthesis (100 mg resin loading, 0.06 mmol) and purification (to >90%) of 13 was carried out according to the general procedures described in section S3.1.1 (21 mg, 40%). m.p. 110-112 oC (MeOH/Et2O). 1H NMR (MeOD-d4, 400 MHz) δ 4.71-4.12 (7H, m), 3.97-3.59 (7H, m), 3.25-3.15 (2H, m), 1.97 (3H, s), 2.30-1.51 (23H, m), 1.13-0.90 (12H, m) ppm. IR νmax cm-1 3284, 2954, 1628, 1536, 1443, 1200, 1173. [α] D

24 –37 (c 0.1, MeOH). ESI m/z 859 (M+H)+. HRMS (ESI, m/z) Calculated for C40H67N12O9 859.51485 found 859.51514.

S24

Peptidomimetic analogue N-Ac-RP1VAPG-NH2 (14) Solid-phase synthesis (100 mg resin loading, 0.06 mmol) and purification (to >90%) of 14 was carried out according to the general procedures described in section S3.1.1 (23 mg, 48%). m.p. 102-105 oC (MeOH/Et2O). 1H NMR (MeOD-d4, 400 MHz) δ 4.71-3.55 (14H, m), 3.26-3.12 (2H, m), 2.28-1.61 (21H, m), 1.97 (3H, s), 1.34 (3H, d, J = 6.8 Hz), 0.96 (6H, 2 × d, J = 6.6 Hz) ppm. IR νmax cm-1 3272, 2963, 1626, 1537, 1444, 1199, 1177, 1130. [α]

D24 – 46.8 (c 0.25, MeOH). ESI m/z 817 (M+H)+, 840 (M+Na)+. HRMS (ESI, m/z) Calculated

for C37H61N12O9 817.46790, found 817.46755. Purification of 14 was carried out by semi-preparative RPHPLC on a Gilson platform with Phenomenex® C18 column: 110A AXTA 50 × 21.20 mm, using 0.1% - 100% CF3CO2H in H2O and 0.1% - 100% CF3CO2H in CH3CN gradients, at 0.8 mL min-1 and detection at 220 nm. ChromatogramY axis: mAU; X axis: min.

-­‐20  0  

20  40  60  80  100  120  140  160  180  

0   5   10   15   20   25   30   35   40   45  

S25

Peptidomimetic analogue N-Ac-RP1V1G-NH2 (15) Solid-phase synthesis (100 mg resin loading, 0.06 mmol) and purification (to >90%) of 15 was carried out according to the general procedures described in section S3.1.1 (20 mg, 41%). m.p. 117-120 oC (MeOH/Et2O). 1H NMR (DMSO d-6, 400 MHz) δ 8.51-7.08 (11H, broad s, NH), 4.50-3.41 (13H, m), 3.25-3.11 (2H, m), 2.28-1.61 (25H, m), 1.97 (3H, s), 0.97 (6H, apparent t, J = 7.1 Hz) ppm. IR νmax cm-1 3287, 2948, 2358, 1665, 1625, 1553, 1446, 1182, 1131. [α] D

24 –58.5 (c 0.1, MeOH). ESI m/z m/z = 829 (M+H)+, 851 (M+Na)+. HRMS

(ESI, m/z) Calculated for C39H67N12O9 829.46790, found 829.46894.

S26

4 Structural data (3R,6S,9S)-1-OMe.HCl and 9 were co-crystallized. Formula: C34H55ClN6O11; Mr = 759.29; crystal dimensions: 0.10 x 0.05 x 0.01 mm; crystal system: Monoclinic; space group: P21; a = 13.568(15) Å, b = 7.443(7) Å, c = 19.72(2) Å, α = 90o, β = 105.441(9)o, γ = 90o; V = 1920(3) Å3; Z = 2; ρcalcd = 1.314 Mg/m3; µ = 0.159 mm-1; synchrotron radiation, λ = 0.68890 Å; T = 100 K; 2Θmax = 48.580°; 15867/5852 measured/independent reflections; Rint: 0.1905; R1(I>2σ(I)) = 0.0833, wR2(all data)= 0.2107; Δρmax = 0.348 eÅ−3, Δρmin = −0.306 eÅ−3. Colourless lath crystals were poorly diffracting with no significant data beyond 0.84Å. The data were collected on Station I19 of the Diamond Light Source, using a Crystal Logics kappa-geometry diffractometer and a Rigaku Saturn 724+ CCD detector with an Oxford Cryosystems Cryostream cooler; Rigaku CrystalClear3 was used to record images and for data integration. The structure was solved by dual space methods using SHELXD-20144 and refined on Fo

2 by full-matrix least squares refinement using SHELXL-2014. All non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were added at calculated positions. The structure was deposited with the Cambridge Structural Database with the deposition number CCDC 1014671. 5 References 1. C. J. Morton, D. J. R. Pugh, E. L. J. Brown, J. D. Kahmann, D. A. C. Renzoni and I.

D. Campbell, Structure, 1996, 4, 705. 2. M. Angiolini, S. Araneo, L. Belvisi, E. Cesarotti, A. Checchia, L. Crippa, L. Manzoni

and C. Scolastico, Eur. J. Org. Chem., 2000, 2571. 3. CrystalClear, Rigaku Corporation, The Woodlands, Texas, U.S.A., 2008-2013. 4. G. M. Sheldrick, Acta. Cryst. A, 2008, A64, 112-122.