Triple Helical Recognition of Pyrimidine Inversions in Polypurine Tracts of RNA by Nucleobase-modified PNA

Transcription

1 Supporting nformtion Triple Helicl Recognition of Pyrimidine nversions in Polypurine Trcts of RNA by Nucleobse-modified PNA Pnkj Gupt, Thoms Zengey nd Eriks Rozners* Deprtment of Chemistry, Binghmton University, The Stte University of New York, Binghmton, New York 1392 Tble of contents Experimentl Procedures Rw TC dt Anlyzed TC dt Rw UV melting dt Anlyzed UV melting dt Copies of NMR spectr Pg. S2 Pg. S7 Pg. S43 Pg. S46 Pg. S48 Pg. S49 S1

2 Benzyl 2-(N-(2-(((9H-fluoren-9-yl)methoxy)crbonylmino)ethyl)-2-(2-oxopyrimidin-1(2H)- yl)cetmido)cette (3). To stirring solution of 1 1 (1.57 g, 3.64 mmol), (2-oxo-1(2H)- pyrimidinyl)cetic cid 2 2 (.9 g, 5.83 mmol), nd 3,4-dihydro-3-hydroxy-4-oxo-1,2,3- benzotrizine (1.19 g, 7.29 mmol) in DMF (1 ml) t 4 C ws dded N-(3- dimethylminopropyl)-n -ethylcrbodiimide (1.5 g, 7.84 mmol). After stirring overnight t 4 C, H 2 O (15 ml) ws dded nd the product ws extrcted with EtOAc (3 3 ml). The combined orgnic lyers were wshed with 1 M queous HCl (4 ml), sturted queous NHCO 3 (4 ml), H 2 O (4 ml), nd brine (4 ml). The orgnic lyer ws dried over N 2 SO 4, evported under reduced pressure nd purified by silic gel column chromtogrphy using -7% MeOH in CH 2 Cl 2 to fford 3 s white solid mteril (1.38 g, yield 67%). R f =.36 (MeOH:CH 2 Cl 2, 7:93, v/v). Compound 3 exists in solution s pir of slowly exchnging rotmers; the signls due to the mjor (m.) nd minor (mi.) rotmers re designted: 1 H NMR (DMSO-d6, 36 MHz) δ: 8.58 (t, 1H, J = 3.6 Hz), 8.1 nd 7.96 (ddd, 1H, J = 3.6 Hz nd 3.6 Hz), 7.88 (d, 2H, J = 7.6 Hz), 7.68 (m, 1H), (m, 1H), 6.45 (t, 1H, J = 3.6 Hz), 5.22 (s,.5h, mi.), 5.13 (s, 1.5H, m.), 4.92 (s, 1.5H, m.), 4.74 (s,.5h, mi.), (m, 3H), 4.16 (s, 1H), (m, 5H). 13 C NMR (DMSO-d6, 9.5 MHz): δ: 169.3, 168.8, 167.2, 167., 166.6, 166.5, 156.3, 155.5, 15.7, 143.8, 14.7, 135.7, 128.4, 128.2, 128.1, 128., 127.9, 127.6, 127., 125.1, 12.1, 13.6, 66.6, 66., 65.5, 65.4, 5.6, 5.4, 49.2, 48., , 46.7, 4.2, ES- MS found m/z [M+H] +, clculted for C 32 H 3 N 4 O (N-(2-(((9H-fluoren-9-yl)methoxy)crbonylmino)ethyl)-2-(2-oxopyrimidin-1(2H)- yl)cetmido)cetic cid (4). Compound 3 (1.2 g, 2.11 mmol) ws dissolved in MeOH:CH 2 Cl 2 (1:1, 14 ml) nd purged with N 2. 1% Pd/C (42 mg) ws dded nd the solution ws sturted with H 2 by slow bubbling over 3 h. The rection mixture ws filtered through short pd of Celite nd the pd ws wshed with MeOH (5 ml). The solvent ws removed in vcuum to give 91 mg (91%) of 4 s white solid mteril. Compound 4 ws used for PNA synthesis without further purifiction. R f =.24 (MeOH:CH 2 Cl 2, 4:6, v/v). 1 H NMR (DMSO-d6, 36MHz) δ: 7.87 (d, 2H, J = 7.2 Hz), 7.67 (d, 2H, J = 7.2 Hz), (m, 6H), 6.25 (d, 1H, J = 3.6 Hz), (m, 4H), 3.95 (d, 2H, J = 1.8 Hz), (m, 6H), 1.76 (s, 1H). 13 C NMR (DMSO-d6, 9.5 MHz) δ: 171.1, 17.8, 169.5, 169.2, 156.3, 156.1, 155.6, 155.4, 148.6, 143.9, 14.7, 139.9, 127.6, 127.1, 125.1, 124.2, 12.1, 119.9, 65.5, 65.4, 54.9, 49.2, 48.2, 1 F. Wojciechowski, R. H. E. Hudson J. Org. Chem. 28, 73, Commercilly vilble from ChemBridge. S2

3 47.7, 47.4, 46.7, 46., 45.8, 41.8, 38.1, 21.9, ES-MS found m/z [M+H] +, clculted for C 25 H 24 N 4 O ). Benzyl 2-(N-(2-(((9H-fluoren-9-yl)methoxy)crbonylmino)ethyl)-3-(2-oxopyrimidin-1(2H)- yl)propnmido)cette (3b) To stirring solution of 1 1 (.81 g, 1.88 mmol), 3-(2-oxo-1,2- dihydropyrimidin-1-yl)propnoic cid 2b 3 (.61 g, 3.1 mmol), nd 3,4-dihydro-3-hydroxy-4- oxo-1,2,3-benzotrizine (.61 g, 3.76 mmol) in DMF (5 ml) t 4 C ws dded N-(3- dimethylminopropyl)-n -ethylcrbodiimide (.77 g, 4.4 mmol). After stirring overnight t 4 C, H 2 O (1 ml) ws dded nd the product ws extrcted with EtOAc (3 2 ml). The combined orgnic lyers were wshed with 1 M queous HCl (25 ml), sturted queous NHCO 3 (25 ml), H 2 O (25 ml), nd brine (25 ml). The orgnic lyer ws dried over N 2 SO 4, evported under reduced pressure nd purified by silic gel column chromtogrphy using -6% MeOH in CH 2 Cl 2 to fford 3b s white solid mteril (.71 g, yield 65%). R f =.52 (MeOH:CH 2 Cl 2, 7:93, v/v). Compound 3b exists in solution s pir of slowly exchnging rotmers; the signls due to the mjor (m.) nd minor (mi.) rotmers re designted: 1 H NMR (CDCl 3, 36 MHz, mjor rotmer) δ: 8.41 (br s, 1H), 7.82 (br s, 1H), 7.73 (d, 2H, J = 7.2 Hz), 7.57 (d, 2H, J = 7.2 Hz), (m, 11H), 6.11 (br s, 1H), 5.27 (s,.5h, mi.), 5.14 (d, 2H, J = 3.6 Hz, m.), 4.37 (d, 2H, J = 7.2 Hz), (m, 5H), 3.45 (br s, 2H), 3.28 (br s, 2H), 2.94 (br s, 2H), 2.76 (br s,.5h). 13 C NMR (CDCl 3, 9.5 MHz): δ: 171.7, 171.1, 169.6, 169.3, 165.9, 156.4, 15.2, 143.8, 141.2, 135.1, 134.7, 128.7, 128.6, 128.5, 128.4, 128.1, 127.6, 127., 125., 119.9, 13.6, 67.8, 67.1, 66.7, 66.4, 53.4, 5.6, 49., 48.8, 48.3, 48.1, 47.2, 47.1, 39.2, 39.1, 3.6, 3.5. ES-MS found m/z [M+H] +, clculted for C 33 H 32 N 4 O ). 2-(N-(2-(((9H-fluoren-9-yl)methoxy)crbonylmino)ethyl)-3-(2-oxopyrimidin-1(2H)-yl) propnmido)cetic cid (4b). Compound 3b (.67 g, 1.15 mmol) ws dissolved in MeOH (6 ml) nd purged with N 2. 1% Pd/C (26 mg) ws dded nd the solution ws sturted with H 2 by slow bubbling over 2 h. The rection mixture ws filtered through short pd of Celite nd the pd ws wshed with MeOH (5 ml). The solvent ws removed in vcuum to give 48 mg (84%) of 4b s white solid mteril. Compound 4b ws used for PNA synthesis without further purifiction. R f =.25 (MeOH:CH 2 Cl 2, 2.5:7.5, v/v). 1 H NMR (DMSO-d6, 36MHz) δ: 7.87 (m, 3H), 7.67 (br s, 1H), 7.58 (d, 1H, J = 3.6 Hz), (m, 5H), 6.11 (br s, 1H), 4.27 (m, 2H), 3 Commercilly vilble from Ukrorgsyntez Ltd. S3

4 3.93 (m, 1H), 3.34 (m, 4H), 3.17 (d, 2H, J = 3.6 Hz), 3.6 (br s, 1H), 2.37 (br s, 1H), 1.73 (m, 2H), 1.46 (d, 2H, J = 7.2 Hz). 13 C NMR (DMSO-d6, 9.5 MHz) δ: 171.3, 17.8, 156.2, 155.2, 148.6, 143.9, 14.7, 139.9, 127.6, 127., 125.2, 125.1, 124.2, 12.1, 119.9, 65.5, 65.4, 47.6, 46.7, 45.5, 43.8, 41.8, 37., 31.2, 3.6, 22.1, ES-MS found m/z [M+H] +, clculted for C 26 H 26 N 4 O Benzyl 2-(N-(2-(((9H-fluoren-9-yl)methoxy)crbonylmino)ethyl)-3-(6-(benzyloxy)pyridzin -3-ylmino)propnmido)cette (6). Compound 1 1 (1.9 g, 4.41 mmol) ws dissolved in nhydrous DMF (5 ml) nd 3-(6-(benzyloxy)pyridzin-3-ylmino)propnoic cid 4 5 (1.53 g, 5.59 mmol) nd 3-hydroxy-1,2,3-benzotrizine-4(3H)-one (.921 g, 5.66 mmol) were dded. The mixture ws cooled on ice bth nd dicyclohexylcrbodiimide (1.29 g, 6.22 mmol) ws dded. After 1h the ice bth ws removed nd the mixture ws stirred overnight t room temperture. The mixture ws evported in vcuum, redissolved in dichloromethne (1 ml) nd wshed with 5% queous NHCO 3 nd 2ml of cetonitrile ws dded. The smple ws evported, dried in vcuo nd purified on silic gel column using 3% MeOH in CH 2 Cl 2 to fford 6 s white solid mteril (2.7 g, yield 89%). 1 H NMR (DMSO d6, 36MHz, TMS reference) δ (m, 15H), 6.44 (m, 1H), 5.13 (s, 2H), 4.92 (s, 1H), (m, 5H), (m, 6H). 13 C NMR (9.5 MHz, CDCl 3 ): d 169.3, 168.8, 167.2, 167., 166.6, 166.5, 156.3, 155.5, 15.7, 143.8, 14.7, 135.7, 128.4, 128.2, 128.1, 128., 127.9, 127.6, 127., 125.1, 12.1, 13.6, 66.6, 66., 65.5, 65.4, 5.6, 5.4, 49.2, 48., , 46.7, 4.2, ES-MS found m/z [M] +, clculted for C 4 H 39 N 5 O ). 2-(N-(2-(((9H-fluoren-9-yl)methoxy)crbonylmino)ethyl)-3-(6-oxo-1,6-dihydropyridzin-3- ylmino)propnmido)cetic cid (7). Compound 6 (1.2 g, 2.11 mmol) ws dissolved in bsolute ethnol (1 ml) nd purged with N 2. 1% Pd/C (96 mg) ws dded nd the solution ws sturted with H 2 by slow bubbling over 3 h. The rection mixture ws filtered through short pd of Celite nd the pd ws wshed with methnol (5 ml). The solvent ws removed in vcuum to give 7 s white solid mteril (65 mg, 73%). R f =.8 in (2% MeOH:CH 2 Cl 2, v/v). 4 A. B. Eldrup, O. Dhl, P. E. Nielsen J. Am. Chem. Soc. 1997, 119, S4

5 Synthesis of PNA ws done on Expedite 899 synthesizer following the stndrd mnufcturers protocol (2 µmol scle) nd using NovSyn TG Sieber resin (Novbiochem) s support, HATU s n ctivtor nd Fmoc-PNA-A(Bhoc)-OH, Fmoc-PNA-C(Bhoc)-OH, Fmoc-PNA-G(Bhoc)-OH nd Fmoc-PNA-T-OH s monomers (purchsed from Link Technologies Ltd, UK). L-lysine ws coupled to N-terminus of PNA on Expedite 899 (using stndrd PNA coupling protocol) using Fmoc-L-lys(Boc)-OH nd HATU. Chin extension followed three-step cycle: (i) removl of the Fmoc-protecting group from the terminl mine with 2% piperidine in DMF, (ii) coupling of the next monomer onto the N-terminus of the growing chin with HATU, nd (iii) cpping of the unrected mines with cetic nhydride. Treting the solid resin with m-cresol/ TFA (2:8) mixture for 2h. resulted in simultneous removl of the protecting groups nd clevge of the oligomers from the resin. The crude PNA smples were precipitted from nhydrous ether. The solid ws collected, dried, dissolved in HPLC grde wter nd purified by RP-HPLC on Xbridge Prep C-18 column (5 µm, 1 mm 15 mm) t 6 C eluting with liner grdient 3%-25% of cetonitrile in wter contining.1 % of TFA over 4 min, flow rte of 5 ml/min. Absorbency ws monitored t 254 nm nd 28 nm, nd the frction contining the mjor pek ws collected, lyophilized to dryness to fford pure PNA smples. The PNA ws quntified following procedure described for DNA nd RNA. 5 The moleculr weight of the synthesized PNAs ws confirmed by ES or MALD TOF mss spectrometry: PNA1. ES-TOF (high cc.) found m/2z [M+2H] 2+, clculted for C 1 H 136 N 44 O PNA2. MALD TOF found m/z 2466 [M+H] +, clculted for C 1 H 136 N 44 O PNA3. ES found m/z [M+H] +, clculted for C 99 H 134 N 44 O PNA4. ES-TOF (high cc.) found m/2z [M+2H] 2+, clculted for C 1 H 136 N 44 O PNA5. ES found m/2z 1233 [M+2H] 2+, clculted for C 1 H 136 N 44 O PNA6. ES-TOF (high cc.) found m/2z [M+2H] 2+, clculted for C 99 H 136 N 46 O RNA ws purchsed from Dhrmcon nc. nd deprotected ccording to mnufcturers recommendtions. After deprotection RNA smples were purified using RP-HPLC on Xbridge Prep C-18 column (5 µm, 1 mm 15 mm) t 6 C eluting with liner grdient (5%-2%) of mobile phse B in mobile phse A over 4 min, flow rte 5 ml/min. Mobile phse A ws.1 M of triethylmmonium cette (ph = 7.) in HPLC wter nd mobile phse B ws mixture of.1 M of triethylmmonium cette (ph = 7.) in HPLC wter nd HPLC grde cetonitrile (6/4, v/v). Absorbency ws monitored t wvelength of 254 nm nd 28 nm, nd the frction contining 5 Puglisi, J. D.; Tinoco,., Jr., Absorbnce melting curves of RNA. Methods Enzymol. 1989, 18, S5

6 the mjor pek ws collected, lyophilized to dryness to fford pure RNA smples. RNA ws quntified using the extinction coefficient provided by Dhrmcon. TC Experiments were done on Nno TC G2 (TA nstruments). RNA stock solution (17.5 µl,.24 mm) ws evported to dryness nd the solid ws dissolved in 1.6 ml of cette buffer (1 mm of sodium cette, 1. mm of EDTA, ph = 5.5, 6.25 or 7.). After degssing, the RNA solution (.95 ml,.2625 mm) ws loded into TC rection cell nd the reference cell ws loded with degssed HPLC wter. PNA stock solution (7 µl,.24 mm) ws evported to dryness nd the solid ws dissolved in 35 µl of cette buffer. After degssing the PNA solution (25 µl,.48 mm) ws loded in titrtion syringe. The syringe ws inserted into rection cell nd the instrument ws equilibrted t 25 C until the bseline ws flt nd stble. The following prmeters were used: Experiment type: ncrementl titrtion Stirring rte = 25 rpm Temperture set point = 25 C Equilibrtion time = 3 sec ntervl of individul injection = 26-8 sec Number of injections = 5 Volume of individul injection = 5 µl The titrtion dt (Figures S1-S36) were nlyzed using NnoAnlyze softwre (TA nstruments) nd independent model to obtin the fitting grph nd thermodynmic dt of the experiments. UV melting of ech RNA (2.5 µm) nd PNA (12.5 µm) complexes ws done in 1 mm of sodium cette, 1. mm of EDTA, ph = Absorbnce vs. temperture profiles were mesured t 26 nm on Shimdzu 8 UV-visible spectrometers equipped with six or eight position Peltier temperture controllers, respectively. The temperture ws incresed t rte of.5 C per minute. The melting tempertures were obtined using Shimdzu LbSolutions Tm Anlysis (Version ) softwre. The experimentl bsorbnce vs. temperture curves were converted into frction of strnds remining hybridized (α) vs. temperture curves by fitting the melting profile to two-stte trnsition model, with linerly sloping lower nd upper bse lines. The melting tempertures (t m ) were obtined directly from the temperture t α =.5. S6

7 Figure S1. TC dt for binding of PNA1 (C) to HRP1 (G-C) ph 5.5. S7

8 Figure S2. TC dt for binding of PNA1 (C) to HRP2 (A-U) ph 5.5. S8

9 Figure S3. TC dt for binding of PNA1 (C) to HRP3 (C-G) ph 5.5. S9

10 Figure S4. TC dt for binding of PNA1 (C) to HRP4 (U-A) ph 5.5. S1

11 Figure S5. TC dt for binding of PNA2 (T) to HRP1 (G-C) ph 5.5. S11

12 Figure S6. TC dt for binding of PNA2 (T) to HRP2 (A-U) ph 5.5. S12

13 Figure S7. TC dt for binding of PNA2 (T) to HRP3 (C-G) ph 5.5. S13

14 Figure S8. TC dt for binding of PNA2 (T) to HRP4 (U-A) ph 5.5. S14

15 Figure S9. TC dt for binding of PNA3 (P) to HRP1 (G-C) ph 5.5. S15

16 Figure S1. TC dt for binding of PNA3 (P) to HRP2 (A-U) ph 5.5. S16

17 Figure S11. TC dt for binding of PNA3 (P) to HRP3 (C-G) ph 5.5. S17

18 Figure S12. TC dt for binding of PNA3 (P) to HRP4 (U-A) ph 5.5. S18

19 Figure S13. TC dt for binding of PNA4 (P ex ) to HRP1 (G-C) ph 5.5. S19

20 Figure S14. TC dt for binding of PNA4 (P ex ) to HRP2 (A-U) ph 5.5. S2

21 Figure S15. TC dt for binding of PNA4 (P ex ) to HRP3 (C-G) ph 5.5. S21

22 Figure S16. TC dt for binding of PNA4 (P ex ) to HRP4 (U-A) ph 5.5. S22

23 Figure S17. TC dt for binding of PNA5 (E) to HRP1 (G-C) ph 5.5. S23

24 Figure S18. TC dt for binding of PNA5 (E) to HRP2 (A-U) ph 5.5. S24

25 Figure S19. TC dt for binding of PNA5 (E) to HRP4 (U-A) ph 5.5. S25

26 Figure S2. TC dt for binding of PNA1 (C) to HRP1 (G-C) ph S26

27 Figure S21. TC dt for binding of PNA1 (C) to HRP2 (A-U) ph S27

28 Figure S22. TC dt for binding of PNA1 (C) to HRP3 (C-G) ph S28

29 Figure S23. TC dt for binding of PNA1 (C) to HRP4 (U-A) ph S29

30 Figure S24. TC dt for binding of PNA2 (T) to HRP1 (G-C) ph S3

31 Figure S25. TC dt for binding of PNA2 (T) to HRP2 (A-U) ph S31

32 Figure S26. TC dt for binding of PNA2 (C) to HRP3 (C-G) ph S32

33 Figure S27. TC dt for binding of PNA2 (T) to HRP4 (U-A) ph S33

34 Figure S28. TC dt for binding of PNA4 (P ex ) to HRP1 (G-C) ph S34

35 Figure S29. TC dt for binding of PNA4 (P ex ) to HRP2 (A-U) ph S35

36 Figure S3. TC dt for binding of PNA4 (P ex ) to HRP3 (C-G) ph S36

37 Figure S31. TC dt for binding of PNA4 (P ex ) to HRP4 (U-A) ph S37

38 Figure S32. TC dt for binding of PNA5 (E) to HRP1 (G-C) ph S38

39 Figure S33. TC dt for binding of PNA5 (E) to HRP2 (A-U) ph S39

40 Figure S34. TC dt for binding of PNA5 (E) to HRP3 (C-G) ph S4

41 Figure S35. TC dt for binding of PNA5 (E) to HRP4 (U-A) ph S41

42 Figure S36. TC dt for binding of PNA6 to HRP5 (bcteril A-site) t ph S42

43 Tble S1. Experimentl results of TC titrtions nlyzed dt. Sequence K ΔH ΔS ΔG order HRP1 PNA1 (5.5) 8.9E PNA2 (5.5) 7.2E PNA3 (5.5) 9.49E PNA4 (5.5) 4.71E PNA5 (5.5) 2.7E PNA1 (6.25) 6.8E E verge 8.1E stndrd dev 1.84E PNA2 (6.25) 7.5E PNA4 (6.25) <1E+4 No binding detected PNA5 (6.25) 2.9E E verge 3.5E stndrd dev 2.12E HRP2 PNA1 (5.5) 9.6E PNA2 (5.5) 8.2E PNA3 (5.5) 2.81E PNA4 (5.5) 1.53E E verge 1.72E stndrd dev 2.69E PNA5 (5.5) 2.1E PNA1 (6.25) <1E+4 No binding detected PNA2 (6.25) 3.7E E E verge 2.E stndrd dev 1.47E S43

44 PNA4 (6.25) <1E+4 No binding detected PNA5 (6.25) 4.7E HRP3 PNA1 (5.5) 7.4E PNA2 (5.5) 6.2E PNA3 (5.5) 5.4E E verge 4.64E stndrd dev 1.7E PNA4 (5.5) 2.93E PNA1 (6.25) <1E+4 No binding detected PNA2 (6.25) <1E+4 No binding detected PNA4 (6.25) 4.1E E verge 4.44E stndrd dev 4.81E PNA5 (6.25) <1E+4 No binding detected HRP4 PNA1 (5.5) 3.2E PNA2 (5.5) 8.E PNA3 (5.5) 3.16E PNA4 (5.5) 5.61E PNA5 (5.5) 3.8E PNA1 (6.25) <1E+4 No binding detected PNA2 (6.25) 8.9E PNA4 (6.25) <1E+4 No binding detected PNA5 (6.25) 2.E E verge 2.8E stndrd dev 1.13E S44

45 HRP5 PNA6 (6.25) 4.22E E verge 5.19E stndrd dev 1.37E S45

46 Absorbency 26 nm HRP1 + PNA1 HRP1 + PNA2 HRP1 + PNA4 HRP1 + PNA temperture o C Figure S37. UV therml melting of HRP1 triplexes t ph Absorbency 26 nm HRP2 + PNA1 HRP2 + PNA2 HRP2 + PNA4 HRP2 + PNA temperture o C Figure S38. UV therml melting of HRP2 triplexes t ph S46

47 Absorbency 26 nm HRP3 + PNA1 HRP3 + PNA2 HRP3 + PNA4 HRP3 + PNA temperture o C Figure S39. UV therml melting of HRP3 triplexes t ph Absorbency 26 nm HRP4 + PNA1 HRP4 + PNA2 HRP4 + PNA4 HRP4 + PNA temperture o C Figure S4. UV therml melting of HRP4 triplexes t ph S47

48 Tble S2. UV therml melting of PNA-RNA complexes t ph 6.25 Entry PNA (vrible bse) HRP1 (G-C) HRP2 (A-U) HRP3 (C-G) HRP4 (U-A) 1 PNA1 (C) >7 b NT c PNA2 (T) NT c NT c PNA4 (P ex ) PNA5(E) >6 b Melting tempertures ( C) in sodium cette buffer, ph 6.25; b Lower estimte; overlp with the duplex melting hinders precise mesurement; c n some cses we observed only the high temperture trnsition, which we interpreted s either one step triplex to single strnds melting for the highly stble HRP2-PNA1 nd HRP2-PNA2 complexes, or s no triple helix formtion for HRP2-PNA1, which hd low stbility ccording to TC results. S48

49 enao N FmocHN /' yoo J"OSn N "--/ 3 5 Lbel Assigned Vlue > 8 2! ,,. 12! _.. -T---- T- 1 9 PK33B PBX-286 in DMSO-d T ,- j c--,--r-- ---, ---T-- o 2 S49 ppm

50 enao N FmocHN yoo /"-. /N '-../ OBn Jd. 14 J LU l. J ljll) U......_ o ppm PK33B PBX-286 in DMSO-d6 S5

51 en NO yoo FnlOCHr\r// /N J OH 4 Lbel Assigned Vlue ( 4 i PK34 PBXM-287 in DMSO-d6 7,,; o 2 ppm S51

52 X "" OJ (J) o -" w o o (J1 o (J) o -.J o ()O o CD o o o w o o (J1 o (J) F- ::: \ ; ;== \.. o \ J // Z.r-"? - z--< \ \, \/ \ Z ::: ("') "T -- / o ()O CD o 4 (f) $: o N o o.t>. w "" :A , ' S52

53 (l N:o FmOCHNNOBn 3b Lbel Assigned Vlue _ , t , f -; '" T ,' y-; ;_.. _ --o---j T -,.- - ; r -; -.., ; r- -T o 2 ppm PK47-PBEX-44 in CDC3 S53 - '- '

54 en o ::. OBn 3b r-., !"j " r l --:-';. -. -'!"--Y", -.,- r, ':..., y -' - T _. 11 ', 1. -,...- ', ' -- -r- ' --, , 'l (-'-r" 8 - ; ', -;--... r-' - ' - -. _ :---'1 -;.. _ r i - '..,. --l"i...., fir"- r ' "!T- r' ' r :-r""-tt' o ppm PK4 7-PBEX-44 in CDC3 S54

55 NO en oo FmOCHNNOH 4b Lbel Assigned Vlue '-..- T i , ' - 1 i-' -' , '- T -.- -, "' _. -:: -. -, 1 -, 1 o ppm PK48-PBEXM-46 in DMSO-d6 S55

56 NAO en oo FmocHNN OH 4b. ", ' 1' " ' 18.., T ' -;- ' r r.. -, l ' 15 r'. ".. ; 1-14 ".., 13 ' r "'T- rr '... '... _.-'...- T - '-r'. " r- " ',l'--r : , r ' " '-. '"' _. 1'1... " r, -' 6 1 '... '-T Tr "-- 'r; 5 1'-..-.-,... '.. r '.., n ' 1 ',.,. -,, " 1. " -rl1"-' o ppm PK48-PBEXM-46 in DMSO-d6 S56

57 H-1 in DMSO-d6 ppm Lbel Assigned Vlue , 4 2 Y.' _"., 11.,',.i..,,i.' ; 9. _' J } '."..J..J ' , i , 6.! # 9 J 1 5.,.. r ]11 d 12 13!1:1 14, : J.Y 18 1 J 'i J \t. \>j 1"",,-, JJV"tJ'*tt...,..j.,J ',.'''' r, :. 1. S57 i, '"! -1. "'...

58 w r\.j r\.j c::> w +> (]l Ol ---. co ( c::> r\.j w +> (]l Ol ---. co ( r\.j r\.j r\.j r\.j S58.: en 6. (j) s 2 3 Q. ct> - (j) : z W -6,

59 H-1 Royl in DMSO-d6 This journl is The Society of Chemistry 211 ppm Lbel Assigned Vlue i fi, i!' i f,, '! ! 9,! ' 12 ' \,3, !..,J -.l1 "".,_ ,' 5, ": S59 1.

60 f\.) w '. 2 f\.) w.po. Ul en ---J OJ (f) w U; ---J S OJ 6. (j) ::l 3 Sl (j) "" CD ::n Z,... w 3 "" "" OJ f\.) r::: f\.) f\.)