The base catalyzed elimination of the suffhydryl protecting group affords dehydroalanine and the subsequent addition of piperidine yields the C-terminally modified peptide . This side reaction is minimized (but not avoided!) when trityl is used for the protection of the C-terminal Cys.
This reaction occurs when Trp is used unprotected. The addition of Pmc (or Pbf) to the indole ring will occur and the amount of by-product is related to the distance between the Trp and the Arg residues .
The extent of diketopiperazine formation depends on the nature of the C-terminus as well as on the penultimate amino acid, especially on the bulk of the side-chains.
Good accessibility to the ester bond facilitates this side-reaction as does a cis-amide bond making sequences containing a C-terminal Pro prone to this side-reaction. Nearly quantitative loss may occur.
During the base-induced deprotection of the penultimate amino acid, the liberated amino function may cleave the resin ester linkage whilst forming a piperazinedione, i.e. the dipeptide is split off.
In peptide synthesis diketopiperazine formation is a notorious side-reaction at the dipeptide stage and is particularly prone to occur in Fmoc based SPPS because of its mechanism.
A range of cleavage reagents for peptides synthesized on 2-chlorotrityl resin has been described. TFE/AcOH/DCM (1:1:3) has been developed by Barbs . Cleavage is also rapidly attained with 0.5% TFA/DCM as well as with HFIP/DCM (1:4 or 3:7) .
Filtrates containing sulfur scavengers have to be oxidized to eliminate the pervasive obnoxious smell. For that purpose the TFA filtrate is neutralized (NaOH/ice) before addition of an oxidant (e.g. bleach or a saturated solution of iodine in ethanol).
A growing proportion of scavengers means reduction of the amount of TFA, thus, cleavage with cocktails containing a considerable amount of scavenger or many different scavengers may take longer.
Sample and bulk cleavages should be performed in a well ventilated hood and protective equipment (gloves, goggles, mask if necessary) worn by the personnel performing the operation.
In the development of the cleavage conditions it is also advisable to perform a simultaneous time course evaluation of the evolution of the cleavage. It should be complete in 2-3 hours at room temperature in order to minimize side reactions arising from long exposure to strong acid environment.
It is strongly advised to perform sample cleavages using different cocktail compositions and to compare the qualitative and quantitative recovery before choosing the final one. The monitoring of these sample cleavage is best done by analytical HPLC.
The latter system is especially suitable for the cleavage of fully protected fragments to be coupled to another fragment in solution, as it eliminates the use of a carboxylic acid in the cleavage step.
Also silane derivatives (TES, TIS) can successfully replace the malodorous EDT [59,60,61]. They show good efficacy in quenching carbocations in sequences containing Arg and Trp residues.
The use of sulfur containing scavengers is recommended in the cleavage of sequences containing Met, Cys and Arg. Care must be taken in the use of thioanisole as it has been suggested that it participates in the premature cleavage of Acm, StBu, or tBu from Cys residues .