Epimerization of Peptide

2016/6/25 M1 Hirano RyoIntroduction

Fosgerau, K. Hoffman, T. Drug Discovery Today 2015, 21, 122

Introduction - chemical synthesis of peptide

Introduction - N to C synthesis
But N to C synthesis causes epimerization

Introduction - importance of N to C synthesis

Contents
0. Introduction
1. Factor of epimerization

2. To overcome epimerization
Native chemical ligation
Flow reaction
Additive

3. Summary (& Proposal)

1. Factor of the epimerization

Factor of epimerization - condensation reagent

Factor of epimerization - condensation reagent
Liquid-phase

N. L. Benoiton et al. Int. J. Peptide Protein Res. 1981, 17, 197.

・EDC tends to cause epimerization more than DCC.

Factor of epimerization - condensation reagent
Solid-phase

EDC and its derivative tends to cause more epimerization than DＩC.
L. A. Carpino, A. El-Faham Tetrahedron 1999, 55, 6813.

Factor of epimerization - condensation reagent

L. E. Conklin et al. Chem. Commun. 1967, 773.

Basicity of amine correlates with epimerization?

Positive charge of amine
inhibits the protonation of carbodiimide?

Factor of epimerization - Solvent
Liquid-phase

N. L. Benoiton et al. Int. J. Peptide Protein Res. 1981, 17, 197.

・DMF increases epimerization rate.

Factor of epimerization - Solvent
Liquid-phase

N. L. Benoiton et al. Int. J. Peptide Protein Res. 1981, 17, 197.

・Polar solvent increases epimerization rate.

Factor of epimerization - Solvent

Liquid-phase

K. Teruya et al. J. Pept. Sci. 2012, 18, 669.

・Polar solvent is likely to increase epimerization.
Accelerates hydrogen abstraction from oxazolone?
Destabilizes intermediate?

Factor of epimerization - Solvent
Solid-phase

L. A. Carpino, A. El-Faham Tetrahedron 1999, 55, 6813.

・Same tendency is observed at solid-phase synthesis.

Factor of epimerization - Solvent
So, apolar solvent is best? --- NO. Solubility problem occurs.

Liquid-phase

Chloroform : trifluoroethanol=3:1
can dissolve peptide
and suppress epimerization.
Sakakibara S. et al. Int. J. Peptide Protein Res. 1992, 40, 294.

Factor of epimerization - C-terminal residue

Sakakibara S. Biopolymers (Peptide Science) 1999, 51, 279.

Factor of epimerization - C-terminal residue

N. L. Benoiton et al. Int. J. Peptide Protein Res. 1981, 17, 197.

・Epimerization rate differs depending on C-terminal residue.
・Val or Ile tends to easily epimerize in polar solvents.

Factor of epimerization - C-terminal residue

・Hindered residue increases epimerization.
Thorpe-Ingold Effect?
N. L. Benoiton et al. Int. J. Pept. Pro. Res. 1992, 40, 559.

Factor of epimerization - N-terminal residue

Sakakibara S. Biopolymers (Peptide Science) 1999, 51, 279.

Coupling at Hindered amino acid residue should be avoided.

Factor of epimerization - temperature
Liquid-phase

N. L. Benoiton et al. Int. J. Peptide Protein Res. 1981, 17, 197.

・Low temperature suppresses epimerization.

Factor of epimerization - Summary
To supress epimerization ・・・・ Conduct reaction at low temperature
・ Use apolar solvent (but apolar solvent may cause solubility problem)
・Avoid coupling reaction using a peptidethat conteins sterically hindered residue at C-Terminal or N-terminal.
・ Aboid using a condensation reagent
that contains amino group such as EDC.
・ Epimerizagtion rate differs depend on the sequence of peptide.

2. To overcome epimerization
・NCL
・Flow reaction
・AdditiveTo overcome epimerization - NCL
NCL ・・・ Native Chemical Ligation

To overcome epimerization - NCL

E. T. Baldwin et al. Proc. Nati. Acad. Sci. USA 1991, 88, 502.
Kent, S. B. H. et al. Science 1994, 266, 776.

To overcome epimerization - NCL

P. E. Dawson et al. Proc. Nati. Acad. Sci. USA 1999, 96, 10068

・NCL enables epimerization-free coupling.

To overcome epimerization - NCLP. E. Dawson et al. Proc. Nati. Acad. Sci. USA 1999, 96, 10068.

・If X is Val, Ile, or Pro, coupling reaction doesn’t complete even in 48 hours.
One have to consider where to connect peptides.

To overcome epimerization - NCL modification
Connect peptide without using Cys

O. Seitz et al. Angew. Chem. Int. Ed. 2008, 47, 6807.

To overcome epimerization - NCL modification
Connect peptides at sterically hindered amino acid

S. J. Danishefsky et al. J. Am. Chem. Soc. 2008, 130, 15814.

To overcome epimerization - Flow reaction
Supress epimerization by mild electrophilicity.

Suppress epimerization by short reaction time.
T. Takahashi et al. Angew. Chem. Int. Ed. 2014, 53, 851.

To overcome epimerization - Flow reaction

To overcome epimerization - Flow reaction
Desired peptide is gainedin good yieldby flow reaction.

But carbamate isless epimerizable
than amide・・・.

It’s still uncertain
flow reaction can inhibit
epimerization of peptide.

To overcome epimerization - AdditiveTo overcome epimerization - AdditiveHOBt

・HOBt suppresses epimerization.
N. L. Benoiton et al. Int. J. Pept. Pro. Res. 1992, 40, 559.

To overcome epimerization - Additive
HOAt

HOAt accelerates
coupling andsuppresses epimerization
better than HOBt.

Louis A. Carpino J. Am. Chem. Soc. 1993, 115, 4397.

To overcome epimerization - Additive
HOAt

Half-times forreaction completion (min)Louis A. Carpino et al. Org. Lett., 2000, 2, 2253.

7-HOAt accelerates the reaction best.

To overcome epimerization - Additive
HOAt

Louis A. Carpino et al. Org. Lett., 2000, 2, 2253.

7-HOAt inhibits epimerization best.
Drawing of amino group by N atom
seems to exist.

To overcome epimerization - Additive

HOOBt suppresses epimerization better than HOBt.

Sakakibara S. et al. Int. J. Peptide Protein Res. 1992, 40, 294.

To overcome epimerization - Additive
HOBt

Potential energy surface scansfor carbonyl oxygen

35 kcal/mol

HOOBt

Energy barrier for cyclization

HOBt < HOOBt
HOOBt suppresses
epimerization better.
40 kcal/mol
K. Teruya et al. J. Pept. Sci. 2012, 18, 669.

To overcome epimerization - Additive
OthersF. Albericio et al. Org. Biomol. Chem., 2014, 12, 8379.

To overcome epimerization - Additive
OthersL. A. Carpino, A. El-Faham et al. J. Org. Chem. 2004, 69, 54.

It is uncertain What makes additive effective.

To overcome epimerization - Summary
・NCL realizes epimerization-suppressed fragment couplingby enabling intramolecular coupling reaction.

・Flow reaction may enable epimerization-suppressed couplingby shortening preactivation time.

・Additive can inhibit epimerization
but its reason is remained to be solved.

Summary・Epimerization rate is strongly effected by coupling condition.

・Epimerization rate depends on the sequence of amino acids,
so where to connect peptide is important to conduct ligation.
・Shortening the existing time of intermediate
can suppress the epimerization.