Application of Biocatalysis to DCC

1
Application of Biocatalysis to DCC

• Sabine Flitsch
• Agnes Zlotorowics
• Martin Dauner, Valeria Barratini
• MIB School of Chemistry
• The University of Manchester, UK

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Protease-catalysed peptide synthesis
aa3
aa3
aa1
aa1
aa2
aa2
aa1
aa2
aa3
Protease
aa1

aa3
aa2
aa3
aa1
aa1
Leu2
Leu2-8
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Protease-catalysed peptide synthesis

• On solid support in bulk aqueous solvent
• In solution organic co-solvent
• In solution aqueous buffer

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Mixtures are obtained with higher oligomers
n 5
n 4
Fiona Stefanowicz
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Enzymatic coupling of protected amino acids
Thermolysin Fmoc-Xxx-OH
H-Leu
Fmoc-Xxx-Leu
PEGA
PEGA
side chain protection
Martin Dauner
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Reversibility of reaction
1) Wash 2) FmocTyr, FmocTrp, FmocPhe
FmocMet, FmocAsp, FmocAla
FmocMetLeu-Wang-PEGA FmocAspLeu-Wang-PEGA FmocAlaL
eu-Wang-PEGA
FmocTyrLeu-Wang-PEGA FmocTrpLeu-Wang-PEGA FmocPheL
eu-Wang-PEGA
Leu-Wang-PEGA
gt FmocMet/Asp/Ala are completely removed!
Martin Dauner
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Thermolysin Fmoc-Xxx-OH
Fmoc-Xxx-Phe(His)Gly
H-Phe(His)Gly
PEGA
PEGA
-His
His
Martin Dauner
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Library formation in solution using organic
co-solvents
precipitation

• Organic solvent shifts equilibrium to peptide
  bond formation
• Poor reproducibility

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Library formation in aqueous buffer

• Only small amounts of peptides formed, analysis
  is difficult
• Reactions are fast
• Large choice of proteases
• compatible with biological systems

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LC/MS analysis of polyleucine library in buffer
UV 245 nm
UV 210 nm
TIC
Leu3
SIMS All oligomers
Leu3
Leu4
Leu5
Leu6
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Labelled peptides as internal standards
A
B
M
M1

• Mix libraries A and B
• Use library B as internal standard
• Quantification of library components
• by ms

m/z
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Synthesis of H-Leu-Leu(13C)-OH
Valeria Barratini
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(No Transcript)
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Consistency of quantification using labelled
libraries as standard

• Quantification by monitoring the ratio of MS
  signal intenisity of labelled to unlabelled
  oligomers

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Reversibility of oligoleucine library formation
in buffer
12CLeu/ 13CLeu
24hrs
24hrs
A
A2
B
B2
C
C2
If reaction is fully reversible, A2, B2 and C2
should be identical in composition
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Generation of labelled and unlabelled
tetraleucine libraries
12CLeu/ 13CLeu
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Generation of labelled and unlabelled trileucine
libraries
12CLeu/ 13CLeu
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Generation of labelled and unlabelled trileucine
libraries
12CLeu/ 13CLeu
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Labelled and unlabelled trileucine libraries
12CLeu/ 13CLeu
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Reversibility of oligoleucine library formation
in buffer
12CLeu/ 13CLeu
24hrs
24hrs
A
A2
B
B2
C
C2
If reaction is fully reversible, A2, B2 and C2
should be identical in composition
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Reversibility of oligoleucine library formation
in buffer
Leu2
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Reversibility of oligoleucine library formation
in buffer
Leu3
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Reversibility of oligoleucine library formation
in buffer
Leu4
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Application of Biocatalysis to DCC

• Sabine Flitsch
• Agnes Zlotorowics
• Martin Dauner, Valeria Barratini
• MIB School of Chemistry
• The University of Manchester, UK