LIGATION OF PEPTIDES via
RING OPENING CROSS METATHESIS (ROCM)
 

Simon Michaelis, Siegfried Blechert

Institut für Organische Chemie, Technische Universität Berlin, Germany
 
Introduction

Regarding the development of new medical therapeutics, have recently peptide mimetics increased in importance. To synthesize mimetics, it is important to have appropriate chemical methods for the cell-free synthesis of peptides to expand the repertoire of ribosomal synthesis to include non-coded amino acids and other structural compounds. To prepare biologically relevant peptide domains, consisting of 130 (±40) amino acids, one must first synthesize building blocks of about 50 amino acids by solution or by solid-phase methods according to Merrifield. These peptide units can be stitched afterwards by using several different ligation techniques, which require the presence of certain functional groups (here X and Y).[1]

 
 

Amongst other kinds of metathesis reaction, the ring opening cross metathesis (ROCM)[2] is offering an interesting possibility to connect C-C bonds. Cyclic systems which are under tension can be converted by ring opening metathesis and following cross metathesis with olefinic cross partners into ring-opened products. The driving force of this atom economic metathesis reaction is the decrease of the ring-tension. ROCM is characterized by its high efficiency providing high yields and the use of smallest amounts of metathesis catalysts.

 

Concept
The following concept of ligating the peptide units by ROCM offers the big advantage, that apart from the double bound no other functional groups are needed, which is unique amongst the other known techniques of ligation.
Cyclus 2 is characterized by a high ring tension which should allow ROCM with the metathesis partner 1 with smallest amounts of a catalyst. Furthermore, after the coupling of the two groups R and R´ a structure similar to proline is introduced at the site of ligation.
 
If the two groups R and R` are peptide units a ligation of these two segments is achieved by ROCM. Because of the high symmetry of the cyclus only one regioisomer is guaranteed.
 
It is also known, that the specific introduction of additional structure units at the site of ligation has effects on the tertiary structure of the peptide[3]. As proline is found abundantly in regions of ß-turns, the introduction of a proline-like structure could initiate the formation of a ß-turn.
 

 

Synthesis of the precursors for ROCM

The cyclic amine 5 can be synthesized by endoselective Diels-Alder reaction[4] from cyclopentadiene 7 and maleimide 8 to the imide 6 and reduction with lithium aluminium hydride.[5] The coupling of the cyclus 5 with a peptide segment can be realized by reacting the amine function of 5 and the acidic function of the peptide segment with coupling reagents (e.g. DCCI / DMAP or TBTU).[6]

 
Beneath the N- and the C-terminus of the peptide the side chains of amino acids are also available to introduce the double bond in order to guarantee a peptidic partner for metathesis. A simple method using the C-terminus, is esterification of an olefinic alcohol with the acidic function of a peptide, while allylglycine can introduce the double bond via its side chain.
 
Ring opening cross metathesis
The ROCM of Nor-Gly-Gly-(Fmoc)Cys(Trt) 10  with the coupling partner R-Pro-Ala-(Fmoc)Leu 9 shown above could be done by using 1,5 mol% of the Hoveyda catalyst of the first generation within 36h at room temperature, giving a good yield of 72% as an example.
The following table is to demonstrate the tolerance of the ruthenium catalyst against the functional groups of certain amino acids. It could be seen, that only strongly nucleophilic functional groups such as NH- or SH-groups interfered with metathesis. Yet even reactions with free SH-groups were successfully run if the Hoveyda II-calalyst was used.
Generally Hoveyda I showed to be superior to Hoveyda II and the Grubbs catalysts, as the more active catalysts lead to polymeric by-products. A modification of the Hoveyda I-catalyst, leading to the more active 2-OiPr-Hoveyda I-catalyst[7], developed within the group of S. Blechert could even outperform the established catalysts in the regarded case.
 

Currently we are examining the influence of peptide segments to the stereochemistry of the ring-opening during the ROCM. We are also working towards asymmetric ROCM with chiral metathesis catalysts[8] as well as ROCM in aqueous mediums.
 

1 P. E. Dawson, S. H. Kent, Annu. Rev. Biochem. 2000, 69, 923.

2a) A. Fürstner, Angew. Chem. Int. Ed. Engl. 2000, 39, 3013; b) M. Schuster, S. Blechert, Angew. Chem. Int. Ed. Engl. 1997, 36, 2036.  c) M. F. Schneider, S. Blechert, Angew. Chem. Int. Ed. Engl. 1996, 55, 410.
3[3  U. Nagai, K. Sato, Tetrahedron Lett. 1985, 26, 647.
4  S. Harvy, J. Amer. Chem. Soc. 1949, 71, 1121
5 Y. L. Chow, C. L. Colon, Acta Chem. Scandinavia 1982, 36, 623
6 M. Bodanszky, A. Bodanszky, The Practice of Peptide Synthesis 2nd, Springer Lab Manual, 1994
7  M. Zaja, S. J. Connon,  A. M. Dunne, M. Rivard,  N. Buschmann,  J. Jiricek,  S. Blechert, Tetrahedron 2003, 59, 6545
8  J. J. van Veldhuizen, D. G. Gilligham, S. B. Garber, O. Kataoka, A. H. Hoveyda, J. Amer. Chem. Soc. 2003, 125, 12502