Better results will be obtained by repeating a coupling with fresh reagents (and changing coupling parameters if a low conversion was obtained) rather than by prolonging the reaction. Generally, coupling protocols may be changed in the course of a synthesis, especially when optimizing an SPPS.
As mentioned above, the generation and disappearance of Fmoc based chromophors allows the monitoring of the synthesis. Furthermore, samples may be taken to determine the load of Fmoc peptide. The completion of the deprotection reaction may be checked by cleaving samples and analyzing the obtained peptide.
Also small scale manual SPPS as well as multiple peptide synthesis and the synthesis of pep- tide libraries can be performed very rapidly and conveniently with preformed active esters. Fmoc-AA-ONp and Fmoc-AA-OSu have found only restricted application in SPPS.
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It must also be noted that during the synthesis of very long peptides the detection of the remaining free amino group will usually become difficult with increasing peptide length.
A special paragraph will be dedicated to the problems caused by peptide aggregation in the course of the synthesis. This phenomenon is a major cause of trouble as it is difficult to predict, is sequence dependent and no universal solution has been found up to now.
Minute amounts of bromophenol blue have been added to couplings in progress to monitor the conversion; the end-point is indicated by discoloration .
But, whichever cleavage reagent is preferred, it has to be washed out very carefully after Fmoc removal, the last washing must be neutral. When synthesizing large peptides the duration of Fmoc cleavage should be gradually increased. For safe removal of the deblocking reagent the resin may have to be washed more often.
Cleavage of samples may seem a rather elaborate way of monitoring an SPPS, but it is the most comprehensive method. It is especially useful when the synthesis has to be optimized or documented or when negative color tests results are obtained because of scarcely accessible coupling sites.
The facility with which the aromatic ring of phenols and phenol ethers undergoes electrophilic substitution has been noted. Two examples are shown in the following diagram. The first shows the Friedel-Crafts synthesis of the food preservative BHT from para-cresol. The second reaction is interesting in that it further demonstrates the delocalization of charge that occurs in the phenolate anion. Carbon dioxide is a weak electrophile and normally does not react with aromatic compounds; however, the negative charge concentration on the phenolate ring enables the carboxylation reaction shown in the second step. The sodium salt of salicylic acid is the major ptoduct, and the preference for ortho substitution may reflect the influence of the sodium cation. This is called the Kolbe-Schmidt reaction, and it has served in the preparation of aspirin, as the last step illustrates.
The other approach is to introduce backbone protecting groups which will prevent the formation of hydrogen bonds. Such protection is made by the introduction of the Hmb group on the αnitrogen . It has been shown that the presence of a Hmb unit every 6-7 residues is sufficient to disrupt the peptide aggregation . The Hmb protected amino acid is introduced under the form of N,O-bis-Fmoc-N-(2hydroxy-4-methoxybenzyl) derivative, the O-Fmoc protection being cleaved during the following piperidine treatment. At the end of the synthesis the Hmb group is cleaved in the final TFA cleavage.
The following problems review various aspects of aromatic chemistry. The first two questions review some simple concepts. The next two questions require you to analyze the directing influence of substituents. The fifth question asks you to draw the products of some aromatic substitution reactions. The sixth question takes you through a mutistep synthesis. The last selection leads to a large number of multiple choice questions.
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) .
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.