Lanosterol biosynthesis: push the electrons yourself

The lanosterol mechanism, step by step

Lanosterol is the four-ring sterol that animals go on to convert into cholesterol, and all of its rings come from one 30-carbon chain. An aspartic acid in the enzyme protonates the epoxide, rings A to D close one after another (ring C first closes with five atoms and then expands to six), and the 1,2-shifts walk the cation back from the side chain to C8 until a histidine removes H9 to form the C8=C9 double bond.

The 13 steps of the demo, in the exercise's own words. Try drawing each one above before you read it.

1. Protonate the epoxide oxygen

   The enzyme folds the chain so every alkene sits beside the carbon it will bond to, then switches the cascade on with one proton: Asp455 protonates the epoxide oxygen, turning it into a good leaving group.

2. Open the epoxide and close ring A

   The alkene attacks the more substituted epoxide carbon from the side opposite the breaking C–O bond, so the oxygen stays at the future 3β-hydroxyl position. Opening and ring A closure are drawn as one event, so no cation ever forms at the attacked epoxide carbon.

3. Close ring B

   Each ring closure is the same move: a π bond attacks the cation behind it and passes the positive charge one ring along. Ring B forms in the boat shape the enzyme imposes, setting its drawn ring-junction hydrogen on the β face (wedge), like the neighboring angular methyl.

4. Close ring C

   Markovnikov closure: the less substituted end of the alkene bonds to the cation, leaving the more stable tertiary cation at the other end. Ring C first forms with five atoms; the new cation hangs outside the ring.

5. Grow ring C to six atoms

   The new ring-bond electrons form a bond to the neighboring cation outside the ring. Ring C gains a sixth atom, and the positive charge appears at the ring carbon that lost the bond. That cation is only secondary, so this is uphill on its own; the enzyme stabilizes it and ring D traps it at once.

6. Expand ring C

   The migrating bond keeps its electrons as it joins the neighboring cation. Ring C now has six atoms, and the newly added ring carbon carries its methyl on the β face.

7. Close ring D

   The last alkene of the folded region closes the five-membered ring D. All four rings now exist, and the positive charge sits at the first side-chain carbon: the protosteryl cation.

8. Shift the ring hydrogen to the side chain

   Four 1,2-shifts now return the positive charge to the ring system, each group moving with its bonding pair to the neighboring cation. First, the hydrogen on the side-chain-bearing ring carbon shifts to the side chain; this sets the side-chain stereocenter of lanosterol (R, not drawn).

9. Shift the neighboring ring-junction hydrogen

   The hydrogen arrives on the α face of the carbon that just lost a hydrogen, so the side chain stays β. Each migrating group is trans to the next: the α hydrogen is followed by a β methyl, an α methyl, and finally loss of a β hydrogen. The remaining shifts alternate faces.

10. Shift the neighboring β methyl

    The β methyl next to the cation shifts to the β face of the cationic carbon, where it becomes the angular methyl at the junction of rings C and D in lanosterol.

11. Shift the neighboring α methyl

    The α methyl next to the cation shifts to its α face, giving lanosterol's 14α-methyl. The cation is now back at the junction of rings B and C, next to the drawn β hydrogen.

12. Lose the β hydrogen to form the ring alkene

    The cascade ends by losing a proton rather than capturing a nucleophile: His232 removes the drawn β hydrogen next to the cation, and the C–H electrons form the double bond at the junction of rings B and C in lanosterol.

13. Lanosterol

    Lanosterol: four fused rings (6-6-6-5) with the 3β-OH from the epoxide oxygen, methyls at C4 (two), C10, C13 and C14, the C8=C9 double bond and the C24=C25 side-chain alkene. Every carbon came from one folded chain: four ring bonds formed, one ring bond moved, and two hydrogens and two methyls each shifted one carbon. The proton donated by Asp455 remains on the product hydroxyl oxygen; His232 holds the β hydrogen it removed from C9, and regeneration of the enzyme's protonation states is not shown.

How the mechanism is drawn

(3S)-2,3-Oxidosqualene in lanosterol synthase, drawn pre-folded (chair–boat–chair) on the steroid template, steroid numbering. Asp455 is the acid and His232 the base; only their reacting groups are drawn. Each cation is drawn as a discrete intermediate. Ring C closes five-membered first and then expands. Wedges are β and hashes α. The C20 configuration (R) and C5–H (α) are not drawn.