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

Transcript

1 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. * a.tavassoli@soton.ac.uk 1

2 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 = fold) in response to 50 µm IPTG. 2

3 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

4 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

5 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 (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-) (4, m, Phe- Ar) (1, m, Phe-Ar) 4.96 (1, br. s., Cys-S or Thr-) 4.69 (1, td, J=8.54, 4.27 z, Pheα) (1, m, Leu-α) (2, m, Thr-α and Thr-β) (1, m, Arg-α) (1, m, Cys-α) 3.35 (br. s., solvent- 2 ) (4, m, Arg-β, Arg-δ or Phe-β) (2, m, Arg-β or Phe-β) 1.62 (2, d, J=5.19 z, Cys-β) (5, m, Leu-β, Arg-γ and Leuγ) (3, m, Thr-γ) (6, m, Leu-δ); Analytical PLC (220 nm) 18.1 min; IR (neat) 3270, 1631, 1525, 1188 cm -1 ; MS (ESI+) m/z (%) ((M + ) +, 100), ((M + 2) ); RMS (ESI+) for C S (M + ) + calcd , found PLC: 5

6 Mass Spectrum: igh resolution mass spectrum: 6

7 1 MR: 7

8 CSY: 8

9 TCSY: 9

10 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 (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) (1, m, Arg-α) 4.36 (1, ddd, J=9.84, 7.86, 5.19 z, Leu-α) (2, m, Thr-α, Thr-β) (1, m, Cys-α) (1, m, Proβ) (3, m, Arg-δ) 2.99 (1, d, J=5.19 z, Cys-β) 2.88 (1, d, J=14.04 z, Cys-β) (1, m, Pro-γ) (3, m, Pro-γ and Pro-δ) (2, m, Leu-β) (3, m, Arg-γ and Leu-γ) (2, m, Arg-β) (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 (%) ((M + ) + ), ((M +2) 2+ ); RMS (ESI+) for C S (M + ) + calcd , found PLC: 10

12 1 MR: 12

13 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 (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) (4, m, Phe-Ar) (1, m, is-ind) (1, m, Cys-S) (1, dd, is-α) (1, m, Phe-α) (1, m, J=8.5 z, Asn-α) 4.37 (1, d, J = 8.5 z, Met-α) (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) (1, m, Pro-β) (, m, Pro-β and Phe-β) 3.03 (2, d, J = 15.9 z, is-β) 2.97 (2, m, Asn-β) (3, m, Cys-β) (4, m, Met-γ and Pro-γ) (3, m, Met-δ) (3, m, Met-β) (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 (%) ((M + ) +, 100.0), ((M +2) ); RMS (ESI+) for C S 2 (M + ) + calcd , found PLC: 13

14 Mass Spectrum igh resolution mass spectrum: 14

15 1 MR: 15

16 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-) (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 ) (2, m, Phe-Ar) (3, m, Phe-Ar) 4.83 (1, br. s., Cys-S) (1, m, Cys-α) 4.35 (1, q, J=7.32 z, Leu-α) (2, m, Thr-α, Phe-α) 4.14 (1, br. s., Thr-) (2, m, Thrβ, Arg-α) (4, m, Arg-δ, Phe- β) (1, m, Arg-γ) (2, m, Arg-γ, Cys-β) (1, m, Cys-β) 1.72 (1, br. s.) (1, m, Leu-β) (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 (%) ((M + ) +, 61.3), ((M +2) ); RMS (ESI+) for C S (M + ) + calcd , found PLC: 16

18 1 MR: 18

19 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-) (2, m, Met- and Phe-) 7.93 (1, d, J=7.32 z, Ala-) 7.45 (2, d, J=10.25 z, is-ar) (3, m, Phe-Ar, Asn- 2-side-chain ) (4, m, Phe-Ar) 6.98 (1, br. s. is-ar) (1, m, Cys-S) 4.48 (1, d, J=7.32 z, isα) (4, m, Pro-α, Cys-α, Asn-α) (1, m, Met-α and Phe-α) 4.21 (1, dd, J=7.32, 2.93 z, Ala-α) (2, m, Pro-δ) (19, m, Pro-δ) (2, m) (4, m, Cys-β, Asn-β) (1, m, Asn-β) (2, m, Cys-β) (1, m) (1, m, is-β) 2.46 (2, dd, J=16.11, 7.32 z, Phe-β) (3, m) (3, m, Pro-β, Met-β) 2.01 (3, s, Met-δ) 1.96 (2, s) (4, m, Pro-γ) (1, m, Phe-β) (1, m) (1, m, Met-β) (5, m, Ala-β); Analytical PLC (220 nm) 17.8 min; IR (neat) 3261, 1622, 1132 cm -1 ; MS (ESI+) m/z (%) ((M + ) +, 100.0); RMS (ESI+) for C S 2 (M + ) + calcd , found PLC: 19

21 1 MR: 21

22 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-) (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 ) (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) (1, m, Pro-α) (1, m, Pro-α) (1, m, Met-β) (3, m, Cys-β, Met-β) (8, m, Pro-γ, Met-γ, is-β) (5, m, Asn-β) (6, m, Pro-β, Asn-β) (2, m, Proγ) (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 (%) ((M + ) +, 77.1), ((M + 2) +, 100.0)); RMS (ESI+) for C S 2 (M + ) + calcd , found PLC: 22

24 1 MR: 24

25 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-) (1, m, Arg-) 7.40 (1, t, J=5.86 z, Arg- side-chain ) (2, m, Phe-Ar) (2, m, Phe-Ar) (1, m, Phe-Ar) 4.96 (1, br. s., Cys-S) 4.72 (1, td, J=9.77, 3.91 z, Thr-α) (1, m, Leu-α) (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) (3, m, Cys-β, Thr-β) (2, m, Arg-δ) (1, m, Leu-β) (3, m, Leu- β, Leu-γ, Arg-β) (1, m- Arg-γ) (1, m, Arg-γ) 1.00 (3, d, J=5.86 z, Thr-γ) (6, m, Leu-δ); Analytical PLC (220 nm) 17.7 min; IR (neat) 3278, 1643, 1133 cm -1 ; MS (ESI+) m/z (%) ((M + ) +, 65.8), ((M + 2) +, 100.0)); RMS (ESI+) for C S (M + ) + calcd , found PLC: 25

27 1 MR: 27

28 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-) (1, m, Thr-) 7.58 (1, br. s., Arg- side-chain ) (2, m, Phe-Ar) (3, m, Phe-Ar) (1, m, Cys-S, Thr-) (3, m, Leu-α, Arg-α) (2, m, Thr-α) (1, m, Cys-α) (9, m, Thr-β) 3.09 (2, d, J=5.86 z, Arg-δ) (1, m, Cys-β) (1, m, Cys-β) (3, m Arg-β) (1, m, Arg-γ) (1, m, Arg-γ) (2, m, Leu-β,) (6, m, Leu-γ, Leu-β) 1.05 (3, d, J=5.86 z, Thr-γ) (6, m, Leu-δ); Analytical PLC (220 nm) 18.2 min; IR (neat) 3272, 1631, 1134 cm -1 ; MS (ESI+) m/z (%) ((M + ) +, 70.8), ((M + 2) +, 100.0)); RMS (ESI+) for C S (M + ) + calcd , found PLC: 28

30 1 MR: 30

31 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-) (1, m, Arg- side-chain ) 7.44 (2, d, J=7.81 z, Phe-Ar) (3, m, Phe-Ar) 5.66 (1, d, J=7.81 z, Cys-S) (1, m Thr-α) (2, m, Leu-α, Arg-α) 4.21 (1, d, J=5.86 z, Pheα) (1, m, Cys-α) (1, m Thr-β) 3.33 (8, br. s., Phe-β, Phe-γ) (2, m, Arg-δ) (1, m, Cys-β) (1, m, Cys-β) 1.72 (1, br. s., Arg-β) (1, m, Leu-β) (5, m, Leu-γ, Leu-β, Arg-γ, Arg-β) 1.05 (3, d, J=5.86 z, Thr-γ) (6, m, Leu-δ); Analytical PLC (220 nm) 17.4 min; IR (neat) 3270, 1630, 1137 cm -1 ; MS (ESI+) m/z (%) ((M + ) +, 74.4), ((M + 2) +, 100.0)); RMS (ESI+) for C S (M + ) + calcd , found PLC: 31

33 1 MR: 33

34 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 (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-) (2, m, Phe-Ar) 7.63 (2, d, J=7.32 z, Phe-Ar) 7.56 (2, t, J=7.32 z, Phe-Ar) (1, m, Arg- side-chain ) 7.45 (2, d, J=7.32 z, Phe-Ar) (1, m., Cys-S) (1, m, Phe-α) (1, m, Leu-α) (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, m, Thr-β, Arg-δ, Cys-β) (2, m, Arg-δ, Phe-β) (1, m, Arg-β) (1, m, Arg-β) (2, m, Leu-β) (6, m, Leu-γ, Arg-γ) (4, m, Thr-γ) (5, m, Leu-δ); Analytical PLC (220 nm) 18.9 min; IR (neat) 3277, 1639, 1135 cm -1 ; MS (ESI+) m/z (%) ((M + ) +, 45.5), ((M + 2) +, 54.3); RMS (ESI+) for C S (M + 2) + calcd , found PLC: 34

36 1 MR: 36

37 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 ) (2, m, Tyr-Ar) 6.61 (2, d, J=7.81 z, Tyr-Ar) 4.94 (1, br. s., Cys-S) (1, m, Arg-α) (1, m, Leu-α) (1, m, Tyr-α) (1, m, Thr-) 4.16 (1, d, J=5.86 z, Thr-α) (1, m, Cys-α) (8, m, Solvent- 2 ) (3, m, Thr-β, Tyr-β) (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) (2, m, Leu-β) (7, m, Leu-γ, Arg-γ, Thr-β) 1.04 (3, d, J=7.81 z, Thr-γ) (4, m, Leu-δ ); Analytical PLC (220 nm) 18.9 min; IR (neat) 3280, 1638, 1134 cm -1 ; MS (ESI+) m/z (%) ((M + ) +, 68.1), ((M + 2) +, 100.0)); RMS (ESI+) for C S (M + ) + calcd , found PLC: 37

39 1 MR: 39

40 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) (m, 1, Arg-) 7.56 (d, J=7.81 z, 2, Phe-Ar) 5.03 (br. s., 1, Cys-S) (m, 1, Arg-α) 4.32 (t, J=11.72 z, 1, Leu- α) (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) (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) (m, 5, Leu-γ, Cysβ, Leu-β) (m, 2, Arg-γ ) 1.05 (d, J=5.86 z, 3, Thr-δ) (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 (%) ((M + ) +, 100.0); RMS (ESI+) for C S (M + ) + calcd , found PLC: 40

42 1 MR: 42

43 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 (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) (1, m, Pheα) (1, m, Leu-α) (3, m, Thr-α, Thr-β, Arg-α) (1, m, Cys-α) (1, m, Phe-β) (3, m, Cys-β, Arg-δ) (2, m, Arg-β, Phe-β) (1, m, Arg-β) (2, m, Leu-β) (6, m, Leu-γ, Arg-γ) 1.05 (3, d, J=7.32 z, Thr-γ) (6, m, Leu-δ); Analytical PLC (220 nm) 17.6 min; IR (neat) 3278, 1642, 1134 cm - 1 ; MS (ESI+) m/z (%) ((M + ) +, 76.8), ((M + 2) +, 100.0)); RMS (ESI+) for C S (M + 2) + calcd , found PLC: 43

45 1 MR: 45

46 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 (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) (1, m, Leu-α) (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) (2, m, Leu-β, Thr-β) (7, m, Leu-β, Leu-γ, Arg-γ) 1.04 (3, d, J=7.32 z, Thr-γ) (6, m, Leu-δ); Analytical PLC (220 nm) 19.8 min; IR (neat) 3271, 1643, 1134 cm -1 ; MS (ESI+) m/z (%) ((M + ) +, 100.0), ((M + 2) +, 78.1); RMS (ESI+) for C Br S (M + ) + calcd , found PLC: 46

48 1 MR: 48

49 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-) (2, m, Arg- and Thr-) 7.97 (1, d, J=7.93 z, Phe-) (1, m, Arg- side-chain ) (5, m, Phe-Ar) 4.99 (1, br. s., Thr- or Ser-) 4.68 (1, td, J=8.54, 4.27 z, Phe-α) (1, m, Leu-α) 4.24 (1, q, J=7.43 z, Arg-α) (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 ) (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) (3, m, Arg-β and Leu-β) (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 (%) ((M + ) +, 99.9), ((M + 2) +, 100.0)); RMS (ESI+) for C Cl 8 7 S (M + ) + calcd , found PLC: 49

51 1 MR: 51

52 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 (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-) (1, m, Phe-) 7.50 (1, br. s., Arg- side-chain ) (2, m, Phe-Ar) 7.02 (2, t, J=7.32 z, Phe-Ar) 4.99 (1, br. s., Cys-S) (1, m, Phe-α) (1, m, Leu-α) (3, m, Thr-α, Arg-α) 4.03 (1, br. s., Cys-α) 3.36 (6, br. s., Solvent- 2 ) (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) (6, m, Leu-β, Leu-γ, Arg-γ) 1.05 (4, d, J=4.88 z, Thr-γ) (6, m, Leu-δ); Analytical PLC (220 nm) 18.1 min; IR (neat) 3272, 1632, 1188 cm -1 ; MS (ESI+) m/z (%) ((M + ) +, 51.3), ((M + 2) +, 100.0)); RMS (ESI+) for C F 8 7 S (M + ) + calcd , found PLC: 52

54 1 MR: 54

55 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 (1, br. s., Thr-C) (1, m, Tyr-) 8.49 (1, d, J=7.32 z, Leu-) (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) (1, m, Arg-α) (1, m, Leu-α) (3, m, Thr-α, Tyr-α) 4.03 (1, br. s., Cys-α) (5, m, Arg-β, Leu-β, Tyr-β) (1, m Cys-β) 2.75 (1, dd, J=14.65, 9.77 z, Arg-γ) (1, m, Cys-β) 1.61 (2, br. s., Leu-δ, Leu-γ) (6, m, Arg-δ, Leu-γ, Thr-β) 1.04 (3, d, J=7.32 z, Thr-γ) (6, m, Leu-δ); Analytical PLC (220 nm) 17.6 min; IR (neat) 3284, 1627, 1182 cm -1 ; MS (ESI+) m/z (%) ((M + ) +, 91.0), ((M + 2) +, 100.0)); RMS (ESI+) for C S (M + ) + calcd , found PLC: 55

57 1 MR: 57

58 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 (%) ((M + 2) +, 35.0), ((M + 3) +, 100.0)). Mass Spectrum 58

59 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 (%) ((M + 2) +, 71.0), ((M + 3) +, 100.0)). Mass Spectrum 59