Targeting Bacillus anthracis toxicity with a genetically selected inhibitor of the PA/CMG2

Supplemental Information Targeting Bacillus anthracis toxicity with a genetically selected inhibitor of the PA/CMG2 protein-protein interaction. Abigail L. Male, 1 Fedor Forafonov, 1 Francesco Cuda, 1 Gong Zhang, 2 Siqi Zheng, 3 Petra C. F. yston, 4 Peng R. Chen, 2,3 E. Diane Williamson, 4 Ali Tavassoli 1,5, * 1. Chemistry, University of Southampton, Southampton, S17 1BJ, United Kingdom. 2 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China. 3. Beijing ational Laboratory for Molecular Sciences, Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing, China. 4. Defence Science and Technology Laboratory, Porton Down, Salisbury, UK 5. Institute for Life Sciences, University of Southampton, Southampton, United Kingdom. *E-mail: a.tavassoli@soton.ac.uk 1

Supplemental Figure 1. A) The structure of PA bound to CMG2 from PDB 1T6B. The four domains of PA are highlighted in different colours for clarity. B) Drop-spotting data from the various RTS built for this study. We assessed full length PA (I-IV, top row of each plate), PA II-IV (2 nd row) and PA III-IV (bottom row). nly PA III-IV was observed to form a functional repressor with CMG2, as illustrated by the loss of growth of the PA III-IV/CMG2 RTS (loss of 4 spots = 10000 fold) in response to 50 µm IPTG. 2

Supplemental Figure 2. A) The mechanism of SICLPPS intein splicing. B) Cartoon representation of the truncated SICLPPS inteins selected in this study. C) Graphical representation of the truncated SICLPPS intein adapted from PDB ID 1ZD7. 3

Supplemental Table 1 Selected hits Rank Sequence 1 CMFPA* 1 CLRFT* 1 CLRPT* 2 CPLSLVA* 3 CPIF* 3 CITA* 3 CLIYLI 3 CSMDL 3 CAS* *=STP codon 4

Supplementary Experimental procedures and Spectra. All peptides were synthesized and purified as detailed in the methods section. CLRFT 2 S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 54 mg (34%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 12.62 (1, br. s., Thr-C) 8.51 (1, d, J=7.63 z, Leu-) 8.13 (1, d, J=8.24 z, Thr-) 8.12 (1, d, J=8.24 z, Cys-) 7.95 (1, d, J=7.93 z, Phe-) 7.51 (1, br. s., Arg-) 7.19-7.29 (4, m, Phe- Ar) 7.14-7.18 (1, m, Phe-Ar) 4.96 (1, br. s., Cys-S or Thr-) 4.69 (1, td, J=8.54, 4.27 z, Pheα) 4.28-4.38 (1, m, Leu-α) 4.20-4.27 (2, m, Thr-α and Thr-β) 4.15-4.19 (1, m, Arg-α) 3.97-4.06 (1, m, Cys-α) 3.35 (br. s., solvent- 2 ) 2.95-3.12 (4, m, Arg-β, Arg-δ or Phe-β) 2.76-2.88 (2, m, Arg-β or Phe-β) 1.62 (2, d, J=5.19 z, Cys-β) 1.35-1.51 (5, m, Leu-β, Arg-γ and Leuγ) 1.00-1.10 (3, m, Thr-γ) 0.81-0.91 (6, m, Leu-δ); Analytical PLC (220 nm) 18.1 min; IR (neat) 3270, 1631, 1525, 1188 cm -1 ; MS (ESI+) m/z (%) 639.2 ((M + ) +, 100), 320.5 ((M + 2) 2+ 41.8); RMS (ESI+) for C 28 47 8 7 S (M + ) + calcd 639.3283, found 639.3270. PLC: 5

Mass Spectrum: igh resolution mass spectrum: 6

1 MR: 7

CSY: 8

TCSY: 9

CLRPT 2 S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 21.0 mg (15%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 12.43-12.71 (1, m, Thr-C) 8.52 (1, d, J=7.93 z, Leu-) 8.25 (1, d, J=7.63 z, Arg-) 8.20 (3 br. s., Phe-) 7.86 (1, d, J=8.24 z, Pro-) 7.53 (1, t, J=5.49 z, Arg- side-chain ) 4.91 (1, br. s., Thr-) 4.52 (1, dd, J=8.39, 3.51 z, Pro-α) 4.45-4.50 (1, m, Arg-α) 4.36 (1, ddd, J=9.84, 7.86, 5.19 z, Leu-α) 4.13-4.18 (2, m, Thr-α, Thr-β) 4.02-4.07 (1, m, Cys-α) 3.61-3.68 (1, m, Proβ) 3.02-3.15 (3, m, Arg-δ) 2.99 (1, d, J=5.19 z, Cys-β) 2.88 (1, d, J=14.04 z, Cys-β) 2.00-2.12 (1, m, Pro-γ) 1.82-1.96 (3, m, Pro-γ and Pro-δ) 1.62-1.73 (2, m, Leu-β) 1.49-1.58 (3, m, Arg-γ and Leu-γ) 1.42-1.49 (2, m, Arg-β) 1.06-1.09 (3, m, Thr-γ) 0.89 (6, d, J=6.71 z, Leu-δ); Analytical PLC (220 nm) 16.1 min; IR (neat) 3278, 1655, 1182 cm -1 ; MS (ESI+) m/z (%) 589.2 ((M + ) + ), 295.2 ((M +2) 2+ ); RMS (ESI+) for C 24 45 8 7 S (M + ) + calcd 589.3126, found 589.3125. PLC: 10

1 MR: 12

CMFPA 2 S S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 56 mg (27%) of the product as a white solid. 1 MR (600Mz, DMS) δ ppm 12.57 (1, br. s., Ala-C) 8.93 (2, br. s., is- side-chain ) 8.63 (1, d, J = 8.5 z, Met-) 8.33 (2, t, J = 7.9 z, Asn- and is-) 8.09 (1, d, J = 7.3 z, Ala-) 8.03 (1, d, J = 7.3 z, Phe-) 7.46 (1, br. s., is-ind) 7.42 (1, m, Phe-Ar) 7.24-7.33 (4, m, Phe-Ar) 7.16-7.25 (1, m, is-ind) 6.97-7.06 (1, m, Cys-S) 4.57-4.64 (1, dd, is-α) 4.46-4.56 (1, m, Phe-α) 4.39-4.45 (1, m, J=8.5 z, Asn-α) 4.37 (1, d, J = 8.5 z, Met-α) 4.10-4.25 (1, dd, J=7.3 z, Ala-α) 4.03 (1, t, J=6, Cys-α) 3.94 (1, d, J = 6.1 z, Pro-α) 3.53-3.64 (1, m, Pro-β) 3.14-3.50 (, m, Pro-β and Phe-β) 3.03 (2, d, J = 15.9 z, is-β) 2.97 (2, m, Asn-β) 2.76-2.92 (3, m, Cys-β) 2.35-2.49 (4, m, Met-γ and Pro-γ) 1.97-2.07 (3, m, Met-δ) 1.81-1.95 (3, m, Met-β) 1.71-1.80 (2, m, Pro-δ) 1.28 (3, d, J = 7.3 z, Alaβ); Analytical PLC (220 nm) 17.6 min; IR (neat) 3279, 1631, 1188 cm -1 ; MS (ESI+) m/z (%) 819.2 ((M + ) +, 100.0), 410.4 ((M +2) 2+ 68.0); RMS (ESI+) for C 35 50 10 9 S 2 (M + ) + calcd 819.3276, found 819.3828. PLC: 13

Mass Spectrum igh resolution mass spectrum: 14

1 MR: 15

FCRTL 2 S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 56 mg (35%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 8.78 (1, d, J=9.77 z, Cys-) 8.39 (1, d, J=7.32 z, Leu-) 8.09-8.17 (4, m, Phe-) 7.99 (1, d, J=7.32 z, Arg-) 7.82 (1, d, J=7.32 z, Thr-) 7.60 (1, br. s., Arg- side-chain ) 7.30-7.33 (2, m, Phe-Ar) 7.23-7.27 (3, m, Phe-Ar) 4.83 (1, br. s., Cys-S) 4.50-4.54 (1, m, Cys-α) 4.35 (1, q, J=7.32 z, Leu-α) 4.19-4.30 (2, m, Thr-α, Phe-α) 4.14 (1, br. s., Thr-) 3.90-3.97 (2, m, Thrβ, Arg-α) 3.04-3.13 (4, m, Arg-δ, Phe- β) 2.88-2.95 (1, m, Arg-γ) 2.67-2.82 (2, m, Arg-γ, Cys-β) 2.41-2.48 (1, m, Cys-β) 1.72 (1, br. s.) 1.61-1.68 (1, m, Leu-β) 1.49-1.56 (6, m, Leuγ, Leu-β) 1.06 (4, d, J=7.32 z, Thr-γ) 0.88 (5, d, J=7.32 z, Leu-δ) 0.83 (4, d, J=4.88 z, Leuδ); Analytical PLC (220 nm) 17.4 min; IR (neat) 3280, 1636, 1134 cm -1 ; MS (ESI+) m/z (%) 639.3 ((M + ) +, 61.3), 320.5 ((M +2) 2+ 100.0); RMS (ESI+) for C 28 46 8 7 S (M + ) + calcd 639.3283, found 639.3271. PLC: 16

1 MR: 18

PCAMF 2 S 2 S The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 45 mg (22%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 8.92 (1, br. s., is- side-chain ) 8.54 (1, d, J=4.39 z, Pro-) 8.23 (2, t, J=7.32 z, is-) 8.14 (1, d, J=7.32 z, Cys-) 8.04 (1, d, J=7.32 z, Asn-) 7.95-8.02 (2, m, Met- and Phe-) 7.93 (1, d, J=7.32 z, Ala-) 7.45 (2, d, J=10.25 z, is-ar) 7.23-7.28 (3, m, Phe-Ar, Asn- 2-side-chain ) 7.16-7.22 (4, m, Phe-Ar) 6.98 (1, br. s. is-ar) 4.51-4.55 (1, m, Cys-S) 4.48 (1, d, J=7.32 z, isα) 4.37-4.45 (4, m, Pro-α, Cys-α, Asn-α) 4.26-4.33 (1, m, Met-α and Phe-α) 4.21 (1, dd, J=7.32, 2.93 z, Ala-α) 3.65-3.74 (2, m, Pro-δ) 3.31-3.58 (19, m, Pro-δ) 3.17-3.23 (2, m) 3.02-3.16 (4, m, Cys-β, Asn-β) 2.86-2.93 (1, m, Asn-β) 2.77-2.86 (2, m, Cys-β) 2.70-2.77 (1, m) 2.52-2.60 (1, m, is-β) 2.46 (2, dd, J=16.11, 7.32 z, Phe-β) 2.34-2.42 (3, m) 2.13-2.25 (3, m, Pro-β, Met-β) 2.01 (3, s, Met-δ) 1.96 (2, s) 1.83-1.90 (4, m, Pro-γ) 1.74-1.81 (1, m, Phe-β) 1.67-1.73 (1, m) 1.55-1.64 (1, m, Met-β) 1.13-1.20 (5, m, Ala-β); Analytical PLC (220 nm) 17.8 min; IR (neat) 3261, 1622, 1132 cm -1 ; MS (ESI+) m/z (%) 819.4 ((M + ) +, 100.0); RMS (ESI+) for C 35 50 10 9 S 2 (M + ) + calcd 819.3276, found 819.3282. PLC: 19

1 MR: 21

CMAPA 2 S S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 56 mg (30%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 9.45 (1, s, is- side-chain ) 9.14 (1, d, J=7.81 z, Asn-) 8.82 (2, d, J=7.81 z, is-) 8.71-8.78 (3, m, Ala- 2 ) 8.59 (1, d, J=7.81 z, Ala- 1 ) 8.55 (1, d, J=7.81 z, Met-) 7.94 (1, br. s., Asn- sidechain) 7.87 (1, s, is-γ) 7.48 (1, br. s. Asn- side-chain ) 4.81-5.04 (8, m, Ala 1 -α, Met-α, Asn-α, isα) 4.66 (2, t, J=7.81 z, Ala 2 -α) 4.53 (2, br. s., Cys-α) 4.05-4.12 (1, m, Pro-α) 3.99-4.04 (1, m, Pro-α) 3.52-3.59 (1, m, Met-β) 3.42-3.49 (3, m, Cys-β, Met-β) 2.88-3.02 (8, m, Pro-γ, Met-γ, is-β) 2.50-2.52 (5, m, Asn-β) 2.32-2.46 (6, m, Pro-β, Asn-β) 2.22-2.30 (2, m, Proγ) 1.68-1.77 (9, m, Met-δ, Ala 1 -β, Ala 2 -β); Analytical PLC (220 nm) 15.9 min; IR (neat) 3273, 1652, 1133 cm -1 ; MS (ESI+) m/z (%) 743.3 ((M + ) +, 77.1), 372.5 ((M + 2) +, 100.0)); RMS (ESI+) for C 26 46 10 9 S 2 (M + ) + calcd 743.2963, found 743.2953. PLC: 22

1 MR: 24

CLR(D-F)T 2 S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 52 mg (33%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 8.49 (1, d, J=7.81 z, Leu-) 8.29 (1, d, J=9.77 z, Thr-) 8.15 (3, d, J=7.81 z, Phe-) 8.05-8.09 (1, m, Arg-) 7.40 (1, t, J=5.86 z, Arg- side-chain ) 7.25-7.30 (2, m, Phe-Ar) 7.19-7.25 (2, m, Phe-Ar) 7.13-7.19 (1, m, Phe-Ar) 4.96 (1, br. s., Cys-S) 4.72 (1, td, J=9.77, 3.91 z, Thr-α) 4.24-4.34 (1, m, Leu-α) 4.11-4.23 (2, m, Arg-α, Phe-α) 4.02 (1, br. s., Cys-α) 3.35 (7, br. s. Pheβ) 3.13 (2, d, J=9.77 z, Thr-β) 2.83-3.01 (3, m, Cys-β, Thr-β) 2.66-2.76 (2, m, Arg-δ) 1.59-1.70 (1, m, Leu-β) 1.32-1.50 (3, m, Leu- β, Leu-γ, Arg-β) 1.22-1.30 (1, m- Arg-γ) 1.08-1.18 (1, m, Arg-γ) 1.00 (3, d, J=5.86 z, Thr-γ) 0.82-0.91 (6, m, Leu-δ); Analytical PLC (220 nm) 17.7 min; IR (neat) 3278, 1643, 1133 cm -1 ; MS (ESI+) m/z (%) 639.3 ((M + ) +, 65.8), 320.5 ((M + 2) +, 100.0)); RMS (ESI+) for C 28 46 8 7 S (M + ) + calcd 639.3283, found 639.3281. PLC: 25

1 MR: 27

CLR(hPhe)T 2 S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 63 mg (37%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 8.54 (1, d, J=7.81 z, Leu-) 8.30 (1, d, J=7.81 z, Phe-) 8.11 (2, d, J=7.81 z, Arg-) 7.82-7.89 (1, m, Thr-) 7.58 (1, br. s., Arg- side-chain ) 7.22 7.30 (2, m, Phe-Ar) 7.11 7.22 (3, m, Phe-Ar) 4.87 5.06 (1, m, Cys-S, Thr-) 4.29 4.46 (3, m, Leu-α, Arg-α) 4.11 4.24 (2, m, Thr-α) 4.00 4.05 (1, m, Cys-α) 3.25 3.40 (9, m, Thr-β) 3.09 (2, d, J=5.86 z, Arg-δ) 2.98 3.03 (1, m, Cys-β) 2.81 2.90 (1, m, Cys-β) 2.52-2.69 (3, m Arg-β) 1.90-2.01 (1, m, Arg-γ) 1.77-1.87 (1, m, Arg-γ) 1.60-1.74 (2, m, Leu-β,) 1.44-1.57 (6, m, Leu-γ, Leu-β) 1.05 (3, d, J=5.86 z, Thr-γ) 0.84-0.88 (6, m, Leu-δ); Analytical PLC (220 nm) 18.2 min; IR (neat) 3272, 1631, 1134 cm -1 ; MS (ESI+) m/z (%) 653.4 ((M + ) +, 70.8), 327.5 ((M + 2) +, 100.0)); RMS (ESI+) for C 29 48 8 7 S (M + ) + calcd 653.3439, found 653.3430. PLC: 28

1 MR: 30

CLR(Phg)T 2 S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 55 mg (35%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 8.54 (1, d, J=7.81 z, Leu-) 8.34 (2, br. s., Thr-, Phe-) 8.29 (1, d, J=7.81 z, Arg-) 7.55-7.74 (1, m, Arg- side-chain ) 7.44 (2, d, J=7.81 z, Phe-Ar) 7.25-7.34 (3, m, Phe-Ar) 5.66 (1, d, J=7.81 z, Cys-S) 4.84-5.08 (1, m Thr-α) 4.32-4.45 (2, m, Leu-α, Arg-α) 4.21 (1, d, J=5.86 z, Pheα) 4.09-4.17 (1, m, Cys-α) 3.99-4.06 (1, m Thr-β) 3.33 (8, br. s., Phe-β, Phe-γ) 3.04-3.16 (2, m, Arg-δ) 2.96-3.03 (1, m, Cys-β) 2.81-2.91 (1, m, Cys-β) 1.72 (1, br. s., Arg-β) 1.60-1.69 (1, m, Leu-β) 1.42-1.58 (5, m, Leu-γ, Leu-β, Arg-γ, Arg-β) 1.05 (3, d, J=5.86 z, Thr-γ) 0.85-0.90 (6, m, Leu-δ); Analytical PLC (220 nm) 17.4 min; IR (neat) 3270, 1630, 1137 cm -1 ; MS (ESI+) m/z (%) 625.4 ((M + ) +, 74.4), 313.5 ((M + 2) +, 100.0)); RMS (ESI+) for C 27 44 8 7 S (M + ) + calcd 625.3126, found 625.3133. PLC: 31

1 MR: 33

CLR(4-Bz-F)T 2 S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 59 mg (32%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 12.64 (1, br. s., Thr-C) 8.50 (1, d, J=7.32 z, Leu-) 8.23 (2, d, J=7.32 z, Thr-, Arg-) 8.03 (1, d, J=9.77 z, Phe-) 7.67-7.72 (2, m, Phe-Ar) 7.63 (2, d, J=7.32 z, Phe-Ar) 7.56 (2, t, J=7.32 z, Phe-Ar) 7.47-7.52 (1, m, Arg- side-chain ) 7.45 (2, d, J=7.32 z, Phe-Ar) 4.89-5.11 (1, m., Cys-S) 4.76-4.81 (1, m, Phe-α) 4.30-4.35 (1, m, Leu-α) 4.21-4.30 (2, m, Thr-α, Arg-α) 4.18 (1, d, J=4.88 z, Cys-α) 4.02 (1, br. s.,thr-) 3.18 (1, d, J=9.77 z, Phe-β) 3.02-3.08 (3, m, Thr-β, Arg-δ, Cys-β) 2.89-3.01 (2, m, Arg-δ, Phe-β) 2.79-2.88 (1, m, Arg-β) 2.54-2.65 (1, m, Arg-β) 1.55-1.69 (2, m, Leu-β) 1.34-1.52 (6, m, Leu-γ, Arg-γ) 1.04-1.08 (4, m, Thr-γ) 0.77-0.81 (5, m, Leu-δ); Analytical PLC (220 nm) 18.9 min; IR (neat) 3277, 1639, 1135 cm -1 ; MS (ESI+) m/z (%) 745.4 ((M + ) +, 45.5), 372.5 ((M + 2) +, 54.3); RMS (ESI+) for C 35 50 8 8 S (M + 2) + calcd 372.1809, found 372.1818. PLC: 34

1 MR: 36

CLRYT 2 S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 63 mg (37%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 9.16 (1, br. s., Tyr-) 8.52 (1, d, J=7.81 z, Leu-) 8.17 (1, d, J=7.81 z, Thr-) 8.04 (1, d, J=7.81 z, Tyr-) 7.86 (1, d, J=9.77 z, Arg-) 7.55 (1, br. s., Arg- side-chain ) 7.01-7.06 (2, m, Tyr-Ar) 6.61 (2, d, J=7.81 z, Tyr-Ar) 4.94 (1, br. s., Cys-S) 4.55-4.64 (1, m, Arg-α) 4.31-4.38 (1, m, Leu-α) 4.24-4.29 (1, m, Tyr-α) 4.19-4.23 (1, m, Thr-) 4.16 (1, d, J=5.86 z, Thr-α) 4.01-4.05 (1, m, Cys-α) 3.28-3.50 (8, m, Solvent- 2 ) 3.02-3.10 (3, m, Thr-β, Tyr-β) 2.91-3.01 (2, m, Cys-β) 2.86 (1, dd, J=13.67, 3.91 z, Arg-β) 2.70 (1, dd, J=13.67, 7.81 z, Arg-β) 1.57-1.69 (2, m, Leu-β) 1.37-1.52 (7, m, Leu-γ, Arg-γ, Thr-β) 1.04 (3, d, J=7.81 z, Thr-γ) 0.83-0.91 (4, m, Leu-δ ); Analytical PLC (220 nm) 18.9 min; IR (neat) 3280, 1638, 1134 cm -1 ; MS (ESI+) m/z (%) 655.3 ((M + ) +, 68.1), 328.4 ((M + 2) +, 100.0)); RMS (ESI+) for C 28 45 9 9 S (M + ) + calcd 655.3232, found 655.3225. PLC: 37

1 MR: 39

CLR(4-2 F)T 2 2 S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 65 mg (40%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 8.51 (1, d, J=7.81, Leu-) 8.26 (d, J=7.81 z, 1, Thr-) 8.15 (d, J=7.81 z, 1, Phe-) 8.10 (d, J=7.81 z, 2, Phe-Ar) 8.03-8.07 (m, 1, Arg-) 7.56 (d, J=7.81 z, 2, Phe-Ar) 5.03 (br. s., 1, Cys-S) 4.75-4.85 (m, 1, Arg-α) 4.32 (t, J=11.72 z, 1, Leu- α) 4.15-4.26 (m, 3, Phe-α, Thr-α, Thr-β) 4.03 (br. s., 1, Cys-α) 3.21 (d, J=13.67, 3.91 z, 2, Arg-β) 3.02-3.08 (m, 2, Phe-β) 2.93 (dd, J=13.67, 9.77 z, 1, Thr-β) 1.60 (d, J=5.86 z, 3, Arg-δ, Leu-β) 1.37-1.51 (m, 5, Leu-γ, Cysβ, Leu-β) 1.29-1.36 (m, 2, Arg-γ ) 1.05 (d, J=5.86 z, 3, Thr-δ) 0.82-0.85 (d, J=7.81 z, 6, Leuδ); Analytical PLC (220 nm) 19.8 min; IR (neat) 3275, 1637, 1135 cm -1 ; MS (ESI+) m/z (%) 684.3 ((M + ) +, 100.0); RMS (ESI+) for C 28 45 9 9 S (M + ) + calcd 684.3134, found 684.3133. PLC: 40

1 MR: 42

CLR(4-C-F)T C 2 S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 60 mg (36%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 12.65 (1, br. s., Thr-C) 8.51 (1, d, J=7.32 z, Leu-) 8.22 (1, d, J=9.77 z, Thr-) 8.19 (3, br. s., Arg-) 8.15 (1, d, J=7.32 z, Phe-) 8.02 (1, d, J=9.77 z, Phe-Ar) 7.68 (1, d, J=9.77 z, Phe- Ar) 7.48 (3, d, J=7.32 z, Phe-Ar, Arg- side-chain ) 5.02 (1, br. s., Cys-S) 4.73-4.80 (1, m, Pheα) 4.30-4.37 (1, m, Leu-α) 4.16-4.26 (3, m, Thr-α, Thr-β, Arg-α) 3.99-4.09 (1, m, Cys-α) 3.12-3.19 (1, m, Phe-β) 2.97-3.08 (3, m, Cys-β, Arg-δ) 2.82-2.91 (2, m, Arg-β, Phe-β) 2.58-2.66 (1, m, Arg-β) 1.55-1.68 (2, m, Leu-β) 1.31-1.50 (6, m, Leu-γ, Arg-γ) 1.05 (3, d, J=7.32 z, Thr-γ) 0.83-0.91 (6, m, Leu-δ); Analytical PLC (220 nm) 17.6 min; IR (neat) 3278, 1642, 1134 cm - 1 ; MS (ESI+) m/z (%) 664.1 ((M + ) +, 76.8), 333.0 ((M + 2) +, 100.0)); RMS (ESI+) for C 29 45 9 7 S (M + 2) + calcd 332.6654, found 332.6660. PLC: 43

1 MR: 45

CLR(3,5-Br 2 -Y)T Br 2 S Br 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 27 mg (53%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 12.64 (1, br. s., Thr-C) 9.66 (1, br. s. Tyr-) 8.50 (1, d, J=7.32 z, Leu-) 8.27 (1, d, J=7.32 z, Thr-) 8.19 (3, d, J=7.32 z, Tyr-) 7.84 (1, d, J=7.32 z, Arg-) 7.48 (2, s, Tyr-Ar) 5.02 (1, br. s., Cys-S) 4.64 (1, br. s. Arg-α) 4.31-4.38 (1, m, Leu-α) 4.15-4.25 (3, m, Thr-α, Tyr-α) 4.02 (1, br. s., Cys-α) 3.06 (3, d, J=7.32 z, Arg-β, Cys-β) 2.98 (2, d, J=12.21 z, Arg-β, Argγ) 2.85 (1, d, J=12.21 z, Arg-δ) 2.66 (1, dd, J=14.65, 9.77 z, Arg- δ) 1.58-1.70 (2, m, Leu-β, Thr-β) 1.40-1.54 (7, m, Leu-β, Leu-γ, Arg-γ) 1.04 (3, d, J=7.32 z, Thr-γ) 0.81-0.90 (6, m, Leu-δ); Analytical PLC (220 nm) 19.8 min; IR (neat) 3271, 1643, 1134 cm -1 ; MS (ESI+) m/z (%) 813.1 ((M + ) +, 100.0), 407.1 ((M + 2) +, 78.1); RMS (ESI+) for C 28 44 Br 2 8 8 S (M + ) + calcd 811.1452, found 811.1443. PLC: 46

1 MR: 48

CLR(4-Cl-F)T Cl 2 S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 19 mg (11%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 8.51 (1, d, J=7.93 z, Leu-) 8.15-8.19 (2, m, Arg- and Thr-) 7.97 (1, d, J=7.93 z, Phe-) 7.50-7.58 (1, m, Arg- side-chain ) 7.23-7.31 (5, m, Phe-Ar) 4.99 (1, br. s., Thr- or Ser-) 4.68 (1, td, J=8.54, 4.27 z, Phe-α) 4.31-4.36 (1, m, Leu-α) 4.24 (1, q, J=7.43 z, Arg-α) 4.18-4.22 (1, m) 4.17 (1, br. s., Thr-α) 4.03 (1, t, J=5.04 z, Cys-α) 3.33 (12, br. s., Solvent- 2 ) 3.03-3.08 (4, m, Phe-β, Arg-δ) 2.99 (1, dd, J=14.34, 5.80 z, Cys-β) 2.85 (1, dd, J=14.34, 4.58 z, Cys-β) 2.79 (1, dd, J=14.04, 9.16 z, Phe-β) 1.59-1.66 (3, m, Arg-β and Leu-β) 1.35-1.51 (7, m, Leu-γ and Arg-γ) 1.04 (4, d, J=6.41 z, Thr-β and Thr-γ) 0.89 (3, d, J=6.71 z, Leu-γ) 0.85 (4, d, J=6.41 z, Leu-δ); Analytical PLC (220 nm) 18.6 min; IR (neat) 3271, 1629, 1133 cm -1 ; MS (ESI+) m/z (%) 673.3 ((M + ) +, 99.9), 337.2 ((M + 2) +, 100.0)); RMS (ESI+) for C 28 45 Cl 8 7 S (M + ) + calcd 673.2887, found 673.2888. PLC: 49

1 MR: 51

CLRF(4-F)T F 2 S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 48 mg (29%) of the product as a white solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 12.63 (1, br. s., Thr-C) 8.51 (1, d, J=7.32 z, Leu-) 8.19 (2, br. s., Arg-) 8.15 (1, d, J=7.32 z, Thr-) 7.92-7.98 (1, m, Phe-) 7.50 (1, br. s., Arg- side-chain ) 7.25-7.32 (2, m, Phe-Ar) 7.02 (2, t, J=7.32 z, Phe-Ar) 4.99 (1, br. s., Cys-S) 4.66-4.71 (1, m, Phe-α) 4.30-4.35 (1, m, Leu-α) 4.14-4.27 (3, m, Thr-α, Arg-α) 4.03 (1, br. s., Cys-α) 3.36 (6, br. s., Solvent- 2 ) 2.96-3.10 (5, m, Phe-β, Arg-δ, Thr-β) 2.85 (1, d, J=12.21 z, Arg-β) 2.78 (1, dd, J=14.65, 9.77 z, Arg-β) 1.62 (2, d, J=4.88 z, Leu-β, Thr-β) 1.33-1.51 (6, m, Leu-β, Leu-γ, Arg-γ) 1.05 (4, d, J=4.88 z, Thr-γ) 0.83-0.89 (6, m, Leu-δ); Analytical PLC (220 nm) 18.1 min; IR (neat) 3272, 1632, 1188 cm -1 ; MS (ESI+) m/z (%) 657.3 ((M + ) +, 51.3), 304.4 ((M + 2) +, 100.0)); RMS (ESI+) for C 28 45 F 8 7 S (M + ) + calcd 657.3199, found 657.3171. PLC: 52

1 MR: 54

CLR (3-2 -Y)T 2 2 S 2 The peptide was synthesised using standard solid phase peptide synthesis techniques, with Fmoc-protected amino acids, yielding 66 mg (38%) of the product as an orange solid. 1 MR (600 Mz, DMS-d 6 ) δ ppm 10.77 (1, br. s., Thr-C) 9.23-9.30 (1, m, Tyr-) 8.49 (1, d, J=7.32 z, Leu-) 8.17-8.25 (4, m, Thr-) 8.15 (1, d, J=7.32 z, Tyr-) 7.93 (1, d, J=7.32 z, Arg-) 7.84 (1, s, Tyr-Ar) 7.52 (1, br. s., Arg- side-chain ) 7.47 (1, d, J=9.77 z, Tyr-Ar) 7.00 (1, d, J=7.32 z, Tyr-Ar) 5.01 (1, br. s., Cys-S) 4.65-4.73 (1, m, Arg-α) 4.28-4.36 (1, m, Leu-α) 4.16-4.26 (3, m, Thr-α, Tyr-α) 4.03 (1, br. s., Cys-α) 2.94-3.10 (5, m, Arg-β, Leu-β, Tyr-β) 2.82-2.89 (1, m Cys-β) 2.75 (1, dd, J=14.65, 9.77 z, Arg-γ) 2.56-2.62 (1, m, Cys-β) 1.61 (2, br. s., Leu-δ, Leu-γ) 1.34-1.52 (6, m, Arg-δ, Leu-γ, Thr-β) 1.04 (3, d, J=7.32 z, Thr-γ) 0.82-0.88 (6, m, Leu-δ); Analytical PLC (220 nm) 17.6 min; IR (neat) 3284, 1627, 1182 cm -1 ; MS (ESI+) m/z (%) 700.2 ((M + ) +, 91.0), 350.9 ((M + 2) +, 100.0)); RMS (ESI+) for C 28 45 9 10 S (M + ) + calcd 700.3083, found 700.3072. PLC: 55

1 MR: 57

Fluorescein-tagged CLR(4-Cl-F)T CLR(4-Cl-F)T was synthesized as detailed above. The peptide (4.2 mg, 5.8 µm) was dissolved in a 1:1 solution of DMF/ 2 (1 ml). Fluorescein-5-maleimide (4.2 mg, 11.6 µm) was added, and the solution stirred overnight, with the flask protected from light. The solvent was remover in vacuo, and the product purified by column chromatography, using a DCM/Me (85:15) to remove excess fluorescein-5-maleimid, followed by 100% methanol to elute the product. The solvent was removed in vacuo to yield 2.6 mgs of the product (41% yield) as a yellow solid. MS (ESI+) m/z (%) 550.7 ((M + 2) +, 35.0), 367.7 ((M + 3) +, 100.0)). Mass Spectrum 58

Fluorescein-tagged (4-Cl-F)CRTL Fluorescein-tagged (4-Cl-F)CRTL was prepared as detailed above for fluorescein-tagged CLR(4-Cl-F)T. 2.2 mgs of the product (35% yield) was isolated as a yellow solid. MS (ESI+) m/z (%) 551.0 ((M + 2) +, 71.0), 367.6 ((M + 3) +, 100.0)). Mass Spectrum 59