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Towards Auxiliary Mediated Peptide Cyclization Employing an Allylic Amination/Acyl-Transfer Cascade Reaction
Master Thesis Sjoerd Slagman HIMS, Synthetic Organic Chemistry (SOC), FNWI, UvA First supervisor Dr. Jan van Maarseveen Second supervisor Prof. Kees Elsevier Professor Prof. Henk Hiemstra
Small cyclic peptides are of high biological and chemical interest. Cyclic peptides exhibit higher bioavailability due to lower biodegradability, improved pharmacodynamic, and –kinetic properties, due to a lesser degree of flexibility when compared to their linear counterparts. To cyclize small peptides under laboratory conditions still remains a challenge. The amide bond in peptides mainly occurs in the transoid form, which enhances linearity, which, for its part, prevents both termini from coming in close proximity.
One way to overcome this is by first reacting both termini with an auxiliary by which these termini get in close proximity of one another and then peptide would cyclize spontaneously. We envisioned an auxiliary, which was based on first binding the C-terminus through standard peptide coupling and subsequent allylic amination with the N-terminus of the linear peptide. Hereafter, the peptide would then cyclize spontaneously through O-to-N acyl-transfer (figure 1, route A).
After successful development of an appropriate allylic amination procedure with the test substrate (methyl cinnamyl carbonate) and nucleophile (H-Phe-Ot-Bu) we started synthesizing the envisioned auxiliary. This auxiliary is o-OH methyl cinnamyl carbonate, through the hydroxyl group we would be able to couple the C-terminus of the peptide and the N-terminus could then attack on the allylic moiety. However, this auxiliary did not prove to be very stable at room temperature and very sensitive to traces of acid. Therefore, the product after the final step (deprotection of the hydroxyl moiety) was never isolated and follow-up chemistry (esterification) was initiated in a “one-pot” fashion. This never reached the phase of peptide coupling, only development of the test substrate, o-OAc methyl cinnamyl carbonate, was carried out.
This test substrate was then used in the allylic amination/O-to-N acyl-transfer sequence (figure 1, route B). This did not result in full conversion of the substrate. Therefore, the catalyst loading was tripled, full conversion was now achieved. However, the desired product could not be observed. Instead, several byproducts were formed in which the acetyl group was cleaved before the allylic amination could take place. Therefore, we concluded that this methodology is not suitable for small peptide cyclization, since the allylic amination reaction is far slower than the acyl-transfer.
Figure 1 graphical abstract displaying the envisioned route towards cylic peptides (in blue, A) and the failed allylic amination/acyl-transfer cascade reaction (in red, B)
List of Abbreviations
Asymmetric allylic amination
High pressure liquid chromatography
Liquid chromatography-mass spectrometry
Nuclear magnetic resonance
Thin layer chromatography
Table of Contents
Graphical abstract 3
List of Abbreviations 4
Table of Contents 6
Chapter 1, Introduction 7
Cyclic peptides 7
Peptide cyclization 10
Difficulties in cyclization 10
How to overcome these issues 12
Project objectives 16
Chapter 2 Development of a suitable Allylic Amination Methodology 20
Allylic amination 20
Chapter 3 Synthesis of the Auxiliary 28
Chapter 4 Coupling of the Auxiliary to an Amino Acid 33
Chapter 5 Testing the Allylic Amination/Acyl-transfer Sequence 35
Conclusion and Outlook 37
Experimental section 40
List of References 51
Chapter 1, Introduction Cyclic peptides
Peptides are important building blocks of nature. Peptides are build up from molecules called α-amino acids. In total there are 21 proteogenic amino acids. These amino acids are at the basis of all life on earth. A amino acid consists of a primary amine and a carboxylic acid moiety, which flank a carbon atom bearing a side chain. This side chain provides the diversity within amino acids. The side chain can be apolar, as in valine, or more polar as, for example, serine. There is also a big variety in the acidity of these side chains; from very acidic, glutamic or aspartic acid, to basic, lysine for example (figure 2).
Figure 2 variety in amino acids
By combining these amino acids through amide-bond formation, one can create peptides. The amount of peptides that can be made from these 21 proteogenic amino acids is virtually endless. Furthermore, they can differ immensely in length; from small oligopeptides to immense proteins.
The diversity in structure is also the basis for the biological activity and specificity of peptides. There are many different ‘jobs’ a peptide or protein can have. They can, for example, be either an enzyme, act as a medicine or work as cell signalling protein.
Although many examples of peptide structures have been summed up above, there is yet another very important class of peptides, these are the cyclic peptides. The class of cyclic peptides consists of a chain of amino acids where one part of the chain is connected to another part of the chain in such a way that a cyclic structure arises. These cyclic peptides are both biologically and chemically interesting targets. In 2011 Yudin and White published an elaborate review on this topic.[endnoteRef:2] There are several ways to connect both ends of the peptide. This can be either a side chain-to-side chain, head-to-side chain, side chain-to-tail or head-to-tail connection. Within this report only head-to-tail connections within small peptides (consisting of two to seven amino acids) will be discussed (figure 3). [2: C. J. White, A. K. Yudin, Nature Chemistry 2011, 3.]
Figure 3 sites for ring closure
This class of peptides has unique properties when compared to linear peptides. Where linear peptides have charged termini, (most) cyclic peptides lack these, which makes them less biodegradable and thereby their membrane permeability and bioavailability are enhanced.[endnoteRef:3],[endnoteRef:4] Furthermore, cyclic peptides are less flexible than their linear counterpart, which, in general, improves the pharmacodynamic and –kinetic properties.[endnoteRef:5] [3: T. Rezai, B. Yu, G. L. Millhauser, M. P. Jacobson, R. S. Lokey, J. Am. Chem. Soc. 2006, 128.] [4: P. Burton, R. Conradi, N. Ho, A. Hilgers, R. Borchardt, J. Pharm. Sci. 1996, 85, 1336-1340.] [5: F. Yokoyama, N. Suzuki, M. Haruki, N. Nishi, S. Oishi, N. Fujii, A. Utani, H. K. Kleinman, M. Nomizu, Biochemistry (N. Y. ) 2004,43.]
The first example of the use of such a relatively small cyclic peptide is Gramicidin S (figure 4). Gramicidin S was first isolated in 1942 by Gause and Brazhnikova and was used as an antibiotic to treat infections from superficial (gunshot) wounds, later on in the Second World War.[endnoteRef:6] [6: G. F. Gauze, M. G. Brazhni