Protein (Lab) Synthesis

Question 1

What is the main problem when it comes to synthesizing peptides in the lab?

State the 2 solutions

Answer 1

• Other parts of the amino acid may react
• These reactions are prevented via protecting groups, which temporarily and reversibly convert those groups to something less reactive

Solutions:

1. Solution phase synthesis
2. Solid phase synthesis

Question 2

In solution phase synthesis, how is COOH protected?

How is the protecting group removed?

Answer 2

• Protected as an ester (OBn) using benzyl ester
  
  • Added by Fischer esterification
  • Removed by strong acid

Question 3

In solution phase synthesis, how is NH2 protected?

Answer 3

• Protected as t-butyloxycarbamate (BOC) using tert-butyloxycarbonyl anhydride
  
  • Added by nucleophilic substitution

Question 4

In solution phase synthesis, how is BOC removed?

Answer 4

• Removed by trifluoroacetic acid (TFA/CF3COOH) via E1 elimination

Question 5

In solution phase synthesis, how is the amide bond formed once the protected groups are added?

Answer 5

• Note DCC is first added to make COOH electrophilic (chlorine acid derivate may tautomerise)

Question 6

In solution phase synthesis, how do I extend my peptide once I have successfully made an amide bond?

How do I remove both protecting groups whe I am done?

Answer 6

• TFA removes the BOC group (N-terminal is to be extended!)
• Once we are done, treatment with HBr/HOAc will remove both protecting groups

Question 7

In solid phase synthesis, the NH2 group is protected just as it is in solution phase synthesis, but COOH is not. How is COOH protected?

Answer 7

• Protected as an ester connected to an insoluble, robust and reusable polymer bead

Question 8

In solid phase synthesis, how do I extend my peptide once I have successfully made an amide bond?

How do I remove both protecting groups whe I am done?

Answer 8

• To extend, use TFA and ET₃N
• To finish, use HBr/HOAc and remove the bead and BOC!

Question 9

Describe the significance of yield in peptide synthesis

Answer 9

Relatively small changes in yield at each step can have a large effect on the overall yield

• Ex., 90% yield at each step leads to a 0.003% yield after 100 runs

Anything not in the yield remains uncoupled