First residue attachment

Most methods of amide bond formation involve chemical activation of the carboxy component. Those commonly employed in organic synthesis are generally regarded as too harsh to be used in peptide synthesis, leading to the formation of over-activated intermediates, which are unselective in then-reactions and consequently prone to side-reactions. Peptide chemists have therefore sought milder activating methods, mostly based on the formation of active esters, pre-formed or generated in situ. 

Those in most frequent use are listed in Table 5, together with cross-reference to the relevant protocols in later chapters. 

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The protocols have been written as they would be carried out using a manual peptide synthesis vessel. Whilst it is appreciated that most scientists preparing peptides will be using automated peptide synthesizers, it is not possible, given the wide variation in operating procedures, to describe how such methods may be applied to individual instruments. Particular emphasis has been given here to those operations which are typically carried out off-instrument, such as first residue attachment and peptide-resin cleavage. 

Figure 2. Symmetrical anhydride method for first residue attachment.

Protocol 14. Estimation of level of first residue attachment... 

AM-beads (22) leaves the majority of the peptide attachment sites in the interior uncleaved to afford (23) ( shaving methology). The first residue is attached using orthogonal FMOC-chemistry to provide (24). Coding is achieved by using standard BOC-chemistry in the interior of the bead to yield (25). Repetition of this process furnishes a surface bound peptide, which is encoded internally (26). 

The first studies indicated that the second iron atom also had a tyrosine residue attached to it. Later refinement of the structure showed that it is actually pentacoordinale, but the argument remains the same. 

Figure 2-18 Typical (3 bulges in antiparallel pleated sheets. The residues Rx, R2, and Rx identify the bulges. (A) A "classic" (3 bulge, in which < )[ and /j are nearly those of an a helix while other torsion angles are approximately those of regular (3 structures. (B) The G1 bulge in which the first residue is glycine with = 85°, /j = 0°. It is attached to a type II (3 turn of which the glycine (labeled 1) is the third residue.

Designation of the bond between two monosaccharides should specify the first residue, its anomeric configuration (a or J3), the position of attachment on both sugar residues, and the second residue. Additional locants and configurational descriptors may be present but are not required. A number of variations may be used. 

The problems involved in all of the above steps have been the subject of extensive investigations, and many significant and novel adaptations of the above basic strategy have been effected depending upon the case in hand. The use of various anchoring groups of different stability between the polymer support and the first amino acid particularly facilitated the attachment of the first residue and the final cleavage of the finished peptide U),... 

With regard to selenocysteine biosynthesis in archaea and eukarya, a tRNA has been known for some time, which accepts L-serine and possesses an anticodon complementary to UGA. It shares a number of structural featmes with tRNA from E. coli, such as extended aminoacyl acceptor and D-arms. In eukarya, the serine residue attached to this tRNA can be phosphorylated by a specific kinase and it was first assumed that this tRNA inserts phosphoserine into proteins. However, closer examination revealed that this tRNA carries selenocysteine in vivo and certainly is the pendant to tRNA from E. coli. It is still elusive whether (9-phosphoseryl-tRNA is the biosynthetic intermediate for selenocysteyl-tRNA formation in eukaryotes also, there is no evidence yet of an eukaryal and archaeal enzyme, equivalent in its function to selenocysteine synthase from bacteria. 

Trityl-based resins are highly acid-labile. The steric hindrance of the linker prevents diketopiperazine formation and the resins are recommended for Pro and Gly C-terminal peptides. Extremely mild acidolysis conditions enable the cleavage of protected peptide segments from the resin. These resins are commercially available as their chloride or alcohol precursors. The trityl chloride resin is extremely moisture-sensitive, so reagents and glassware should be carefully dried before use to avoid hydrolysis into the alcohol form. It is necessary to activate the trityl alcohol precursor and it is highly recommended to reactivate the chloride just before use see Note 4). After activation, attachment of the first residue occurs by reaction with the Fmoc amino acid derivative in the presence of a base. This reaction does not involve an activated species, so it is free from epimerization. Special precautions should be taken for Cys and His residues that are particularly sensitive to epimerization during activation (Table 2). 

To produce a carboxyamide peptide The peptide will be linked to the modified Rink linker via an amide bond. The attachment of the first residue can be carried out under conditions for peptide bond formation (e.g., with TBTU) by using the activation procedures described in Subheading 3.3.2,2, (Methods A-E) of this chapter. Do not forget to deprotect the linker before coupling of the amino acid. 

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