Simon Ficht

Solid‐Phase Synthesis of Peptide and Glycopeptide Thioesters through Side‐Chain‐Anchoring Strategies

An efficient new strategy for the synthesis of peptide and glycopeptide thioesters is described. The method relies on the side-chain immobilization of a variety of Fmoc-amino acids, protected at their C-termini, on solid supports. Once anchored, peptides were constructed using solid-phase peptide synthesis according to the Fmoc protocol. After unmasking the C-terminal carboxylate, either thiols or amino acid thioesters were coupled to afford, after cleavage, peptide and glycopeptide thioesters in high yields. Using this method a significant proportion of the proteinogenic amino acids could be incorporated as C-terminal amino acid residues, therefore providing access to a large number of potential targets that can serve as acyl donors in subsequent ligation reactions. The utility of this methodology was exemplified in the synthesis of a 28 amino acid glycopeptide thioester, which was further elaborated to an N-terminal fragment of the glycoprotein erythropoietin (EPO) by native chemical ligation.

As Fast and Selective as Enzymatic Ligations: Unpaired Nucleobases Increase the Selectivity of DNA-Controlled Native Chemical PNA Ligation

DNA‐controlled reactions offer interesting opportunities in biological, chemical, and nanosciences. In practical applications, such as in DNA sequence analysis, the sequence fidelity of the chemical‐ligation reaction is of central importance. We present a ligation reaction that is as fast as and much more selective than enzymatic T4 ligase‐mediated oligonucleotide ligations. The selectivity was higher than 3000‐fold in discriminating matched from singly mismatched DNA templates. It is demonstrated that this enormous selectivity is the hallmark of the particular ligation architecture, which is distinct from previous ligation architectures designed as “nick ligations”. Interestingly, the fidelity of the native chemical ligation of peptide nucleic acids was increased by more than one order of magnitude when performing the ligation in such a way that an abasic‐site mimic was formed opposite an unpaired template base. It is shown that the high sequence fidelity of the abasic ligation could facilitate the MALDI‐TOF mass‐spectrometric analysis of early cancer onset by allowing the detection of as little as 0.2 % of single‐base mutant DNA in the presence of 99.8 % wild‐type DNA.

Sugar-Assisted Glycopeptide Ligation with Complex Oligosaccharides: Scope and Limitations

We have previously shown sugar-assisted ligation (SAL) to be a useful method for the convergent construction of glycopeptides. However to date SAL has only been carried out on systems where the thiol auxiliary is attached to a monosaccharide. For SAL to be truly applicable to the construction of fully elaborated glycopeptides and glycoproteins, it must be possible to carry out the reaction when the thiol auxiliary is attached to more elaborate sugars, as these are frequently what are observed in nature. Here we examine the effects of glycosylation at C-3, C-4, and C-6 of the C-2 auxiliary-containing glycan. Model glycopeptides where synthesized chemoenzymatically and reacted with peptide thioesters used in our previous work. These studies reveal that SAL is sensitive to extended glycosylation on the auxiliary-containing sugar. While it is possible to carry out SAL with extended glycosylation at C-4 and C-6, the presence of glycosylation at C-3 prevents the ligation from occurring. Additionally, with glycosylation at C-4 the ligation efficiency is affected by the identity of the N-terminal AA, while the nature of the C-terminal residue of the peptide thioester does not appear to affect ligation efficiency. These studies provide useful guidelines in deciding when it is appropriate to use SAL in the synthesis of complex glycopeptides and glycoproteins and how to choose ligation junctions for optimal yield.

DNA-templated native chemical ligation of functionalized peptide nucleic acids: a versatile tool for single base-specific detection of nucleic acids.

Single base-specific detection of DNA/RNA sequences is of importance in the diagnosis of disease-associated genetic disorders or early stage cancer. This chapter introduces DNA-templated native chemical PNA ligation as a potentially useful tool for the sequence specific detection of nucleic acids. The template-induced alignment of PNA-thioesters and 1,2-aminothiol-PNAs in close proximity leads to an increase in their effective molarities. This facilitates PNA ligation to proceed at concentrations where no reaction would be possible in absence of the template. Moreover, hybridization of the rather short PNA conjugates with non-complementary DNA/RNA is disfavored, which prevents PNA ligation to occur on single base-mismatched templates. Different readout strategies of the ligation reaction such as HPLC, MALDI-TOF-MS and fluorecence monitoring are discussed, and examples for the detection of a point mutation within single stranded and PCR-amplified double stranded DNA are provided.