The principle of peptide synthesis in homogenous solution is based on the reversible blocking of the carboxylic acid function of the C-terminal amino acid and the amino group of the N-terminal amino acid. Proteinogenic amino acids contain different functional groups: amino, carboxyl, hydroxy, thio, pyrrolidinyl, imidazolyl, guanidinyl, amido and indolyl. In addition, activation of the free carboxy group of the N-terminal amino acid is necessary to obtain the peptide bond. The orthogonality is the main benefit of the Fmoc-based concept allowing a higher flexibility for complex strategies during synthesis.
In many cases, the on-resin lipidation is carried out at the lysine side chain [,]. A detailed overview of chemical approaches to obtain lipidated peptides containing examples for each strategy is given by Zhang et al. It contains a covalently attached albumin moiety and a D-amino acid at a labile position to obtain increased metabolic stability . Moreover, the Fmoc strategy does not require the use of special vessels that have to be stable towards the corrosive and toxic HF and in some cases, the repetitive TFA acidolysis for Boc deprotection could have an impact on sensitive peptide bonds and acid-catalyzed side reactions . In this study, the authors determined an increased albumin affinity of lipidated insulin variants depending on the number of carbon atoms by interaction studies with immobilized HSA. Recently, the great methodical repertoire for extending the half-lifes of biological active peptides by covalent chemical approaches has been reviewed .
In order to identify a lead compound of a relative unknown system, numerous molecules peptides have to be produced in parallel by a combination of building blocks creating a peptide library . Furthermore, possible reduced water solubility restricts their drugability. Scavengers such as cresol must be added to the HF in order to prevent reactive t-butyl cations from generating undesired products.
Scavengers such as cresol must be added to the HF in order to prevent reactive t-butyl cations from generating undesired products. Some researchers use modified cysteines using S-acetomidomethyl Acm to block the formation of the disulfide bond but preserve the cysteine and the protein's original primary structure. It has to be noted that an optimization of temperature is mandatory in order to avoid racemization and side reactions . Since peptide synthesis takes place mainly in the interior of the solid matrix, appropriate solvation, low cross linking for good accessibility and good swelling properties are very crucial.
See also: Fluorenylmethyloxycarbonyl protecting group Cleavage of the Fmoc group. First, the C-terminal amino acid is coupled to the linker. Notably, their low bioavailability owing to proteolytic degradation by enzymes of the intestine, blood and cell plasma leads to short circulating half-lives . The small resin beads can enlarge up to six times of their original volume in organic solvents. To reduce these effects, microwave-assisted instruments have been evolved . Nowadays, both protecting group strategies are used for the synthesis of peptides and both methods can be applied for automated synthesis.
Microwave energy is capable of activating any molecule containing a dipole moment, which is reflected in rapid heating on a molecular level . The linker represents the reversible connection between the solid support and the assembling peptide. Furthermore, there are linkers that enable the synthesis of partially and fully protected peptides such as the 2-chlorotrityl resin  or the Sieber amide resin . The Boc group is removed with acid, such as trifluoroacetic acid TFA. Following a coupling step, unreacted reagents and byproducts can be easily removed by washing, which makes purification of intermediates redundant.
Here, we highlight the importance of automated solid-phase peptide synthesis SPPS in the process of peptide modification. Both approaches, including the advantages and disadvantages of each, are outlined in more detail below. In order to overcome aggregate formation, a distinct short organic linker is interposed between the amino acid and the solid support, which also determines the C-terminal modification of the synthetic peptide . These properties were transferred for the first time to an important peptide hormone with high propensity to degrade in . Fragment condensation is better than stepwise elongation for synthesizing sophisticated long peptides, but its use must be restricted in order to protect against racemization.
The success story of SPPS, which has been going on for 50 years now, has shown that these molecules can be built up with a great variety of methods. In , Oxyma ethyl 2-cyano hydroxyimino acetate [59,60] was introduced as a novel additive for DIC-mediated peptide-bond formation as an alternative to the well-established HOBt. These disadvantages in the synthesis of peptides led to the revolutionary inception of a completely different strategy. Since then, many biological relevant peptides and proteins were chemically modified by fatty acid acylation . The last step of SPPS should be performed in the presence of scavengers to trap highly reactive carbocations that are formed during the cleavage procedure and that might react with the peptide to form unwanted byproducts .
All these methods are variations of the solid-phase synthesis concept . Peptide libraries can be created by directed parallel synthesis or complex peptide mixtures and identified by iterative processes or position screenings . Jump to Scheme 2 This heterogeneous synthesis technique offers great advantages.
It determines the loading of the resin, the distance between resin and peptide, chemical conditions for coupling and release and most importantly, the C-terminal functionality of the synthetic peptide.