N-terminal primary amino peptides

Whereas short peptides, containing N-terminal proline, were introduced in 2003 as catalysts for Michael addition, there were no reports at that time on asymmetric catalysts based on N-terminal primary amino peptides. 

In 2004, the Tsogoeva group reported the first example of N-terminal primary amine-based unmodified dipeptide being used in a catalytic asymmetric 1,4-conjugate addition of 2-nitropropane to cyclohexenone. After examination of various N-terminal primary amino dipeptides 

Interestingly, while neither cocatalyst (H-Leu-His-OH and/or 26) was sufficiently effective independently, their combination resulted in a significant increase in yields (up to 86%) and enantioselectivities (up to 91% ee), indicating the possibility of synergistic effects.  

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Scheme 13.28 Aldol reactions catalysed by N-terminal primary amino peptides 48 and 49.

Remarkable advances in the field of peptide organocatalysis have been made in recent years. Asymmetric synthesis employing IV-allqrl imidazole-based peptides, N-terminal prolyl peptides, N-terminal primary amino peptides, supported N-terminal prolyl peptides as well as oligopeptides have become a facile tool in organic chemistry. 

Primary structure Amino acid sequencing, N-terminal Edman sequencing, peptide mapping... 

In comparison, statherin is a multi-functional protein, which inhibits the primary and secondary precipitation of hydroxyapatite in saliva, as well as serving as a boundary lubricant [37]. On the basis of the NMR T p measurements of the N-terminal 15-amino acid peptide in statherin, Drobny et al. proposed that the binding region in the N-terminus has rigid dynamics. 

His residues in the peptide sequence are fundamental for Ni11 ion interactions with peptides. When the His residue is relatively distant from the N terminus, it may compete as a primary ligation site with the N-terminal amino nitrogen. However, even the higher number of His residues inserted inside the peptide sequence may be not able to compete with the albumin-like N terminus, unless the specific peptide structure is established. 

Application of the analytical techniques discussed thus far focuses upon detection of proteinaceous impurities. A variety of additional tests are undertaken that focus upon the active substance itself. These tests aim to confirm that the presumed active substance observed by electrophoresis, HPLC, etc. is indeed the active substance, and that its primary sequence (and, to a lesser extent, higher orders of structure) conform to licensed product specification. Tests performed to verify the product identity include amino acid analysis, peptide mapping, N-terminal sequencing and spectrophotometric analyses. 

Silk fibroin contains no cystine and the content of lysine and histidine is also low (about 1% in total), but it does contain tyrosine phenolic (13%) and serine alcoholic (16%) sidechains. Since glycine accounts for 44% of the total aminoacid content, an N-terminal glycine residue is reasonably representative of most of the primary amino dyeing sites in silk fibres. Amino acid analysis of hydrolysed reactive-dyed silk indicates that the reaction between fibroin and reactive dyes takes place mainly at the e-amino group of lysine, the imino group of histidine and the N-terminal amino group of the peptide chain. In an alkaline medium, the hydroxy groups of tyrosine and serine also react [114]. 

The amino group of the N-terminal amino acid residue of a peptide will react with the FDNB reagent to form the characteristic yellow DNP derivative, which may be released from the peptide by either acid or enzymic hydrolysis of the peptide bond and subsequently identified. This is of historic interest because Dr F. Sanger first used this reaction in his work on the determination of the primary structure of the polypeptide hormone insulin and the reagent is often referred to as Sanger s reagent. 

The primary goal of peptide mapping is the verification of the amino acid sequence deduced from the genetic code of the recombinant protein. The protein backbone gets cleaved by typically two or three different endoproteinases like Lys-C, trypsin, and Glu-C to achieve maps with sequence-overlapping peptide fragments. These peptide mixtures can then be separated by LC or CE and analyzed on-line by MS to obtain sequence information. Often simple mass analysis matches the predicted primary sequence of the protein. However, sometimes mutations can lead to isobaric masses of peptides that can be overseen, if no further sequence analysis like N-terminal Edman sequencing and MS/MS is carried out. 

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