Green Advantages of using Phacm for Cystein Protection

 

PGA (Penicillin G Amidase, Penicillin Acylase, Penicillin Amidohydrolase from E.coli on acrylic resin, Systematic name: Penicillin amidohydrolase, E.C. 3.5.1.11) has an active pocket, which is very specific for phenyl acetic acid. The prominent commercial use is hydrolysis of a phenylacetamid bond during production of the penicillin API 6-APA (De Martin et al., J. Mol. Catal. B:Enzymatic 1999; 6: 437). The high specifity of PGA towards the Phenylacetyl moiety makes the use of Phacm as new alternative for Acm very promising. The principle use already was discovered by Albericio et al in 1995 using native PGA. Today PGA is available as immobilized preparation, so by simple filtration process PGA can be removed from the reaction mixture and reused many times.

Phacm shows the same stability and orthogonality as Acm and has the additional advantage that it can be deprotected simply by water in the presence of PGA. The addition of co-solvents like acetonitrile helps to improve the solubility of hydrophobic sequences. Water in combination with DMSO (80:20) will make deprotection and cyclization a one pot reaction, which is completed (37°C, pH 7) normally within 16h to 24h. 

One disadvantage using Acm is the fact that during cleavage the Acm group tends to alkylate the electron rich aromatic rings of tyrosine and tryptophane. A synthesis of a the following model sequence (including Tyr and Trp): Ala – Cys – Phe – Trp – Lys – Tyr – Cys – Val shows clearly the three expected impurities in significant concentrations:

Ala – Cys – Phe – Trp(Acm) – Lys – Tyr – Cys – Val
Ala – Cys – Phe – Trp – Lys – Tyr(Acm) – Cys – Val
Ala – Cys – Phe – Trp(Acm) – Lys – Tyr(Acm) – Cys – Val

Through the mild and highly specific conditions removing Phacm with PGA no adduct formation occurs and the desired cyclic peptide is being formed in high yield.

Synthesis with Acm Synthesis with Phacm
   

Three impurities present in significant concentration of peptides carrying Tyr(Acm), Trp(Acm) each and both

no Phacm adducts present
Orthogonality: Fmoc, Boc same as Acm

Deprotection:
TFA, Npys, I2, Tl3+, Ph2SO, MeSiCl3

Deprotection:
same as Acm and by simple hydrolysis:
Water:DMSO (80:20), pH 7, 37°C, 16h - 24h

Forms Adducts with Trp and Tyr

No Adduct Formation !
→ clean products and high yields
References:
  • Miriam Royo, Jordi Alsina, Ernest Giralt,Urszula Slomcyznska and Fernando Albericio; S-Phenylacetamidomethyl (Phacm): an orthogonal cysteine protecting group for Boc and Fmoc solid-phase peptide synthesis strategies; J. Chem. Soc. Perkin Trans. 1995; 1095-1102.
  • M. Góngora-Benítez, A. Basso, T. Bruckdorfer, J. Tulla-Puche, F. Albericio; A green strategy for the synthesis of cysteine-containing peptides; Poster Exhibition 22nd American Peptide Symposium, San Diego 2011.

Available Reagents for Phacm Strategy

LS-1220

Imibond-PGA
Immobilised Penicillin G Amidase from E.coli on Amino Acrylic Polymer,
Particle size 150 – 300 µm, Water content: 55 – 65%,
Activity >100 U/gwet, > 280 U/gdry

 100 g
1 kg
5 kg		 230,-
1100,-
4500,-
    Order online
BAA6390 Boc-L-Cys(Phacm)-OH
CAS: 57084-73-8
C17H24N2O5S, 368,45 g/mole		 25 g
100 g		 500,-
1500,-
    Order online
FAA6910 Fmoc-L-Cys(Phacm)-OH
CAS: 159680-21-4
C27H26N2O5S, 490,57 g/mole		 25 g
100 g		 250,-
750,-
    Order online
Prices are in EUR, net, exw Germany.

Bulk Quantities Available - Prices on Request!

 
 

General literature about the synthesis of disulfide bridged cyclic peptides:

  • K. Adermann, K. Barlos; Regioselective Disulfide Formation in XXXXXXXXXXXXXXXXX
  • F. Albericio in Fmoc Solid Phase Peptide Synthesis: A Practical Approach, eds. W. C. Chan and P. White, Oxford University Press, Oxford, 2000; 81-114.
  • L. Moroder, H.-J. Musiol, N. Schaschke, L. Chen, B. Hargittai and G. Barany, Thiol group, in Houben-Weyl. Methods of Organic Chemistry. Synthesis of Peptides and Peptidomimetics, eds. M. Goodman, A. Felix, L. Moroder and C. Toniolo, Thieme, Stuttgart, 2002; 384-423.
  • K. Akaji, Y. Kiso, Synthesis of cystine peptides, in Houben-Weyl. Methods of Organic Chemistry. M. Goodman, A. Felix, L. Moroder and C. Toniolo, Synthesis of Peptides and Peptidominetics, eds. Thieme, Stuttgart, 2002; 101-141.
  • C. Boulègue, H.-J. Musiol, V. Prasad and L. Moroder, Synthesis of cystine-rich peptides, Chim. Oggi/Chemistry Today, 2006; 24: 24-36.
  • M. C.; Ferrer, M.; Barany, G. In Peptide Synthesis Protocols; Pennington, M. W., Bunn, B. M., Eds.; Humana Press: Totowa, NJ, 1994; 91-169.
  • Moroder, L.; Besse, D.; Musiol, H.-J.; Rudolph-Böhner, S.; Siedler, F. Biopolymers 1996; 40: 207-234.
  • Annis, I.; Hargittai, B.; Barany, G. Methods Enzymol. 1997; 289: 198-221.
  • trans-Dichlorotetracyanoplatinate(IV) as a Reagent for the Rapid and Quantitative Formation of Intramolecular Disulfide Bonds in Peptides; Tiesheng Shi and Dallas L. Rabenstein; J. Org. Chem. 1999; 64: 4590-4595.