1. 4 aromatic protons Can we use 2D to assign this spectrum? Structure first determined by Woodward in 1948 (without NMR!)

2. CH 2 Identify CH, CH 2 and CH 3 peaks on HSQC (use DEPT if necessary) HSQC

3. 2223 H-22 is a good place to start COSY

4. Transfer this information to the HSQC spectrum 22 23 12 ? H-12 can be tentatively identified from C and H shifts HSQC

5. 12 11 13 We can identify 11 as a CH 2 and 13 as a CH from the HSQC H-8 can distinguished from H-14 by the doublet multiplicity and shift 13-14 correlation is weak, but is seen on one side of the diagonal 13 8 13, 14 COSY

6. 22 23 12 11 13 8 Note that have a number of “dead ends” as far as COSY is concerned 14 HSQC

7. 14 15 13 The protons at C-15 can be identified by their correlation to H-14 COSY

8. 15 16 Having identified the C-15 protons, we can now see the correlation to H-16 gem COSY

9. 22 23 12 11 13 8 14 15 This leaves 3 CH 2 s, at C-17, C-18 and C-20 16 HSQC

10. The C-20 protons can be identified as a pair of coupled (from COSY) doublets, not coupled to anything else HSQC

11. The CH 2 s at C-17 and C- 18 are left. The HSQC shows an overlapping CH 2 at 1.94 ppm. The chemical shift indicates that this must be the C-17 CH 2. The CH 2 at 2.92 and 3.35 ppm must be C-18. 17 (diagonal) 18 COSY

12. 22 23 12 11 13 8 14 15 20 17 18 16 All protonated carbons have now been assigned HSQC

13. C-10 H-12 H-11axH-11eq We can now use HMBC to assign the non-protonated carbons (and confirm other assignments, if necessary) HMBC

14. C-7 H-1 CH 2 -17 H-15 H-8 C-18 C-20 H-18 H-20 2 bond C-7 to H-16 correlation not observed? HMBC

15. COSY TOCSY 17 13 15 12118 16 15 (diagonal) 14

16. 13 15 16 NOESY Avance 300 Degassed sample Degassing removes oxygen (paramagnetic) from the sample which would otherwise provide alternate relaxation pathways. gem 15 12 8 13 15 8 12 NOESY

17. CH 2 - 17 H-15 H-13 H-11 H-8 H-18 N-9 N-19 H-18 H-16 15 N-HMBC 15 N spectroscopy is very difficult! However, we can measure 15 N shifts and determine the location of the nitrogens with HMBC (AMX 500, 10 mg sample, overnight)