This document discusses racemization in peptide synthesis. It introduces the concepts of stereoisomers, enantiomers, and chiral centers. It then discusses factors that can lead to racemization, such as the amino acid nature, activation method, solvent used, amount and character of the base, and presence of additives. Specific amino acids like histidine are highlighted as being more prone to racemization. Different racemization pathways are proposed, including those involving azlactone intermediates, enolate formation, and direct abstraction of the alpha proton. Methods to reduce racemization, such as using non-polar solvents or bulkier bases, are also covered.

1. Racemization in peptide
synthesis
Othman Al Musaimi1

2. Stereoisomers
Sequence
of bonded
atoms
Same
molecular
formula
Enantiomers
Mirror image,
non-
superimposa
ble
Two
stereoisomers
Introduction
2

3. Introduction
3
Tetrahedral
atom (Sp3)
Four
different
substituents
Chiral

4. Rules for assigning an R/S designation to a chiral center
4
S > O > N > C > H
Counter clockwise = S

5. Remember!
• R/S designation ≠ D/L designation.
• R/S designation (3D structure).
• D/L designation (biological standard format).
• In all chiral AA:
• L = S and D = R EXCEPT Cys where L = R and D = S
5
D-Glucose

6. S > O > N > C > H
Counter clockwise = R
2
1 1 1 1
2 2
3
3
3 3 2
4 4 4 4

7. Racemization
Pure form of
enantiomer
Proportions of
both
enantiomers
Racemization
7

8. Importance
8

9. Chiral integrity
• Racemization measures the fraction of racemate that is formed.
• Epimerization measures only the fraction of D-epimer.
9

10. Tolerable limit at a single amino acid residue
• Synthesis strategy.
10
• Reliability of purification procedures; the number of stages.
• Intended use of the final product.

11. Racemization in Strongly Acidic Aqueous
Solution
• Behavior of amino acids under the conditions used for protein
and peptide hydrolysis, prior to amino acid analysis.
11

12. Racemization in Acetic Acid Solution
• Several groups have established that vulnerable amino acids
racemize unusually easily if heated in acetic acid as solvent.
12

13. a) Racemization through azlactones [5(4 H)-oxazolones].
Distinct racemization pathways
13
Nα group of the activated amino acid is acylated

14. 14

15. Oxazolones tendency for racemization
15
Delocalization
Stability /
sufficient
lifetime
Endanger
chiral purity

16. b) Racemization via enolate formation
Distinct racemization pathways
16
Hydrogen
bonds
Stabilize
enols

17. Distinct racemization pathways
c) Direct abstraction of the α-proton; simple proton abstraction
mechanism.
17
*Certain cases such as the rapid racemization of derivatives of phenylglycine.
*Amino acid residues with electron-withdrawing groups on side chain C β-
position such as Cys.
Amino acid residues with relatively more acidic Hα

18. His The most racemization-prone amino acid
18

19. Factors affecting Racemization mechanism
a) Nature of AA
19
• benzoyl amino acids are more extensively racemized than acetyl
amino acids?
• The electronic forces operating in the acyl group.

20. b) Method of activation
20
Increase
activation
carboxyl
group
Increase
acidity proton
on the α-
carbon (chiral)
Racemization
/ proton
abstraction
Factors affecting Racemization mechanism

21. 21
Intramolecular base
catalysis
21
Proton
abstraction
by a basic
nitrogen
atom
O-alkyl isoureas
pronounced basic
character
Carbodiimide mediated coupling reactions
Reactive intermediates / basic center

22. 22
• Racemization is fast in highly polar solvents.
• Non-polar solvents.
• Peptide intermediates are not sufficiently soluble in non-polar
solvents.
Factors affecting Racemization mechanism
c) Solvent

23. d) Amount and chemical character of the base.
• Steric hindrance in diisopropylethylamine is insufficient to
interfere with proton abstraction from azlactone intermediates.
23
• Tribenzylamine seems to be more efficient in this respect.
• Bulkiness
• Basic strength.
Factors affecting Racemization mechanism

24. Additives
• Reducing the lifetime of overactivated,
racemization prone intermediates, such as
O-acyl-isoureas.
• Have acidic hydrogens and thus can
provide a proton which is more readily
abstracted by bases than the proton from
a chiral center.
24

25. References
• M. Bodanszky, Principles of Peptide Synthesis. Second ed. 1993:
Springer-Verlag, 10.1007/978-3-642-78056-1
• Y. Yang, 257-292 (2016), 10.1016/b978-0-12-801009-9.00011-2.
25

26. 26