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.
The crucial link in any polypeptide chain is the amide bond, which is formed by the condensation of an amine group of one amino acid and a carboxyl group of another.
Usually the color is developed mainly in the beads and partly in the supernatant; but for spectrometric quantitative determination of the amount of unreacted amino groups the color has to be transferred completely to the solution . The intensity of the color depends on the nature of the amino terminus to be detected; rather unspecific shades are obtained with N-terminal (side-chain protected!) Asp, Asn, Cys, Ser, and Thr. Brownish red beads result with N-terminal Pro. As the resin sample has to be heated, “hidden” NH2-groups may become more accessible and thus detectable. However, prolonged heating as well as overheating should be avoided as it may cause Lys(Boc) cleavage or Fmoc removal (by pyridine).
Reliable methods detecting minute amounts of unreacted amino groups are essential for the monitoring of the coupling reaction. On the other hand the ability of detecting small quantities of Fmoc peptide allows the control of the completion of the Fmoc cleavage.
After the cleavage the sample is analyzed by HPLC as Fmoc protected and free amino sequences are usually well separated in a standard HPLC gradient.
A recent publication has described the in situ preparation of Fmoc amino acid chlorides by reaction with bis(trichloromethyl) carbonate and their use in difficult couplings .
Fmoc amino acid chlorides can also be used but their applicability is more limited ; also acid-sensitive groups may not be present when treating Fmoc amino acids with oxalyl chloride or thionyl chloride to obtain the acid chlorides [34,35].
For that purpose a deficient amount of the C-terminal or penultimate Fmoc amino acid is coupled to the unloaded or preloaded resin. The resulting load is determined and when the desired level of substitution has been reached the remaining free amino groups are blocked by acetylation.
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.
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.
In recent years acylphosphonium (BOP, PyBOP) [24,25] and acyluronium/aminium salts (HBTU, TBTU)  have become extremely popular coupling agents in SPPS.
The same is true for the activation of Fmoc-His(Trt)-OH since racemization catalyzed by the nitrogen of the imidazole ring may occur. For the coupling of especially bulky amino acids such as Aib, Tic, ... or in the case of recognized difficult coupling we recommend the replacement of HOBt by HOAt, or the use of other activating agents.
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.
The preactivation time must be kept at a minimum when generating the Fmoc-Arg(Pmc,Pbf)-OBt derivative as the activated Arg derivatives may cyclize yielding an unreactive lactam.