custom peptide synthesis
"Peptides" redirects here. For the journal, see Peptides (journal).
A tetrapeptide (example Val-Gly-Ser-Ala) with green marked amino end (L-Valine) and
blue marked carboxyl end (L-Alanine).
Peptides (from Greek language πεπτ??, peptós "digested"; derived from π?σσειν, péssein "to digest") are short chains of amino acid monomers linked by peptide (amide) bonds.
The covalent chemical bonds are formed when the carboxyl group of one amino acid reacts with the amino group of another. The shortest peptides are dipeptides, consisting of 2 amino acids joined by a single peptide bond, followed by tripeptides, tetrapeptides, etc. A polypeptide is a long, continuous, and unbranched peptide chain. Hence, peptides fall under the broad chemical classes of biological oligomers and polymers, alongside nucleic acids, oligosaccharides and polysaccharides, etc.
Peptides are distinguished from proteins on the basis of size, and as an arbitrary benchmark can be understood to contain approximately 50 or fewer amino acids. Proteins consist of one or more polypeptides arranged in a biologically functional way, often bound to ligands such as coenzymes and cofactors, or to another protein or other macromolecule (DNA, RNA, etc.), or to complex macromolecular assemblies. Finally, while aspects of the lab techniques applied to peptides versus polypeptides and proteins differ (e.g., the specifics of electrophoresis, chromatography, etc.), the size boundaries that distinguish peptides from polypeptides and proteins are not absolute: long peptides such as amyloid beta have been referred to as proteins, and smaller proteins like insulin have been considered peptides.
Amino acids that have been incorporated into peptides are termed "residues" due to the release of either a hydrogen ion from the amine end or a hydroxyl ion (OH?) from the carboxyl (COOH) end, or both, as a water molecule is released during formation of each amide bond. All peptides except cyclic peptides have an N-terminal and C-terminal residue at the end of the peptide (as shown for the tetrapeptide in the image).
custom peptide synthesis introduce Peptides Classes
Many kinds of peptides are known. They have been classified or categorized according to their sources and function. According to the Handbook of Biologically Active Peptides, some groups of peptides include plant peptides, bacterial/antibiotic peptides, fungal peptides, invertebrate peptides, amphibian/skin peptides, venom peptides, cancer/anticancer peptides, vaccine peptides , immune/inflammatory peptides, brain peptides, endocrine peptides, ingestive peptides, gastrointestinal peptides, cardiovascular peptides, renal peptides, respiratory peptides, opiate peptides, neurotrophic peptides, and blood–brain peptides.
Some ribosomal peptides are subject to proteolysis. These function, typically in higher organisms, as hormones and signaling molecules. Some organisms produce peptides as antibiotics, such as microcins.
Peptides frequently have posttranslational modifications such as phosphorylation, hydroxylation, sulfonation, palmitoylation, glycosylation and disulfide formation. In general, peptides are linear, although lariat structures have been observed. More exotic manipulations do occur, such as racemization of L-amino acids to D-amino acids in platypus venom.
Nonribosomal peptides are assembled by enzymes, not the ribosome. A common non-ribosomal peptide is glutathione, a component of the antioxidant defenses of most aerobic organisms. Other nonribosomal peptides are most common in unicellular organisms, plants, and fungi and are synthesized by modular enzyme complexes called nonribosomal peptide synthetases.
These complexes are often laid out in a similar fashion, and they can contain many different modules to perform a diverse set of chemical manipulations on the developing product. These peptides are often cyclic and can have highly complex cyclic structures, although linear nonribosomal peptides are also common. Since the system is closely related to the machinery for building fatty acids and polyketides, hybrid compounds are often found. The presence of oxazoles or thiazoles often indicates that the compound was synthesized in this fashion.
Peptide fragments refer to fragments of proteins that are used to identify or quantify the source protein. Often these are the products of enzymatic degradation performed in the laboratory on a controlled sample, but can also be forensic or paleontological samples that have been degraded by natural effects.
custom peptide synthesis introduce Peptides Uses in molecular biology
Use of peptides received prominence in molecular biology for several reasons. The first is that peptides allow the creation of peptide antibodies in animals without the need of purifying the protein of interest. This involves synthesizing antigenic peptides of sections of the protein of interest. These will then be used to make antibodies in a rabbit or mouse against the protein.
Another reason is that techniques such as mass spectrometry enable the identification of proteins based on the peptide masses and sequence that result from their fragmentation.
Peptides have recently been used in the study of protein structure and function. For example, synthetic peptides can be used as probes to see where protein-peptide interactions occur- see the page on Protein tags.
Inhibitory peptides are also used in clinical research to examine the effects of peptides on the inhibition of cancer proteins and other diseases. For example, one of the most promising application is through peptides that target LHRH. These particular peptides act as an agonist, meaning that they bind to a cell in a way that regulates LHRH receptors. The process of inhibiting the cell receptors suggests that peptides could be beneficial in treating prostate cancer. However, additional investigations and experiments are required before the cancer-fighting attributes, exhibited by peptides, can be considered definitive.
custom peptide synthesis introduce Peptides Number of amino acids
Peptides of defined length are named using IUPAC numerical multiplier prefixes.
A monopeptide has one amino acid.
A dipeptide has two amino acids.
A tripeptide has three amino acids.
A tetrapeptide has four amino acids.
A pentapeptide has five amino acids.
A hexapeptide has six amino acids.
A heptapeptide has seven amino acids.
An octapeptide has eight amino acids (e.g., angiotensin II).
A nonapeptide has nine amino acids (e.g., oxytocin).
A decapeptide has ten amino acids (e.g., gonadotropin-releasing hormone & angiotensin I).
A neuropeptide is a peptide that is active in association with neural tissue.
A lipopeptide is a peptide that has a lipid connected to it, and pepducins are lipopeptides that interact with GPCRs.
A peptide hormone is a peptide that acts as a hormone.
A proteose is a mixture of peptides produced by the hydrolysis of proteins. The term is somewhat archaic.
A peptidergic agent (or drug) is a chemical which functions to directly modulate the peptide systems in the body or brain. An example is opioidergics, which are neuropeptidergics.
Doping in sports
The term peptide has been used to mean secretagogue peptides and peptide hormones in sports doping matters: secretagogue peptides are classified as Schedule 2 (S2) prohibited substances on the World Anti-Doping Agency (WADA) Prohibited List, and are therefore prohibited for use by professional athletes both in and out of competition. Such secretagogue peptides have been on the WADA prohibited substances list since at least 2008. The Australian Crime Commission cited the alleged misuse of secretagogue peptides in Australian sport including growth hormone releasing peptides CJC-1295, GHRP-6, and GHSR (gene) hexarelin. There is ongoing controversy on the legality of using secretagogue peptides in sports.
custom peptide synthesis introduce See also
Beefy meaty peptide
Epidermal growth factor
Journal of Peptide Science
Peptide Spectral Library
Peptidomimetics (such as peptoids and β-peptides) to peptides, but with different properties.
Protein tag, describing addition of peptide sequences to enable protein isolation or detection
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A faster solid phase custom peptide synthesis method using ultrasonic agitation
Sonication accelerates couplings in the solid phase custom peptide synthesis.
Sonical custom peptide synthesis gives products with a higher purity than the classical approach.
Sonication does not cause the racemization of sensible residues (Cys, His).
The ultrasonic bath allows the parallel synthesis of many peptides.
Solid phase-supported synthesis is a widely used strategy in peptide chemistry. A factor which limits the product purity is the individual stages yields. Here, we reported that
the use of ultrasonic agitation allows to reduce tenfold the time of synthesis in the Fmoc strategy and improve the purity of the final product. Our method is a promising
alternative to traditional synthetic methods and microwave synthesizers.
Functionalized silica shell magnetic nanoparticles for nanophase custom peptide synthesis applications
New Nanostructured Support Synthesis for peptide preparation based on core–shell magnetic nanoparticles- silica was synthesized.
Different mesoporous structures of silica shell were explored to improve solvent dispersability and magnetic separation of nanoparticles.
Preliminary successful attempts of custom peptide synthesis involving several amino acids to assess the impact of steric hindrance were performed.
A new nanostructured solid phase synthesis magnetic support consisting of core-shell type magnetic nanoparticles based on magnetite, para-aminobenzoic acid (PABA) and secondary
silica shell were synthesized by coprecipitation method and characterized. An newly designed organic linker with an ending hydroxyl group inspired by HMBA linker (4-hydroxy
methyl benzamide) widely employed in classical peptide solid phase synthesis was obtained from 1,4-dimethylolbenzene and gamma-isocyanatopropyltriethoxysilane and was grafted on
the surface in order to be used for the nanophase synthesis of peptides. Three reaction stages, yielding a tripeptide sequence, using n-terminal protected amino acids and the
principle of solid phase synthesis through the Fmoc strategy were performed, in order to prove the effectiveness of the nanophase custom peptide synthesis. The MS analysis
confirmed the success of the tricustom peptide synthesis, recommending the new nanostructured system as a solid support in the solid phase synthesis of peptides. The
intermediate and final materials were analysed by advanced characterization methods (SEM, TEM, BET, X-ray diffraction, magnetic properties, DLS, FT-IR, NMR for organic
intermediates characterization, TGA and LC-MS).
Effect of high hydrostatic pressure on prebiotic custom peptide synthesis
Prebiotic custom peptide synthesis is a central issue concerning life’s origins. Many studies considered that life might come from Hadean deep-sea environment, that is, under
high hydrostatic pressure conditions. However, the properties of prebiotic peptide formation under high hydrostatic pressure conditions have seldom been mentioned. Here we
report that the yields of dipeptides increase with raised pressures. Significantly, effect of pressure on the formation of dipeptide was obvious at relatively low temperature.
Considering that the deep sea is of high hydrostatic pressure, the pressure may serve as one of the key factors in prebiotic custom peptide synthesis in the Hadean deep-sea
environment. The high hydrostatic pressure should be considered as one of the significant factors in studying the origin of life.
Here we report that the high hydrostatic pressure, as a key factor of deep-sea environment conditions, promoted the peptide formation and should be considered as one of the
significant factors in studying the origin of life.
Bicyclic peptides: types, synthesis and applications
Bicyclic peptides categorized into two groups: natural and synthetic.
Bicyclic peptides form one of the promising platforms for drug development owing to their biocompatibility, similarity and chemical diversity to proteins.
Bicyclic peptides can be employed as effective alternatives to complex molecules, such as antibodies, or small chemical molecules.
Bicyclic peptides can be used as antimicrobial agents, drug targeting, imaging and diagnosis agents and therapeutics tools.
Bicyclic peptides form one of the most promising platforms for drug development owing to their biocompatibility, similarity and chemical diversity to proteins, and they are
considered as a possible practical tool in various therapeutic and diagnostic applications. Bicyclic peptides are known to have the capability of being employed as an effective
alternative to complex molecules, such as antibodies, or small molecules. This review provides a summary of the recent progress on the types, synthesis and applications of
bicyclic peptides. More specifically, natural and synthetic bicyclic peptides are introduced with their different production methods and relevant applications, including drug
targeting, imaging and diagnosis. Their uses as antimicrobial agents, as well as the therapeutic functions of different bicyclic peptides, are also discussed.
Antimicrobial alumina nanobiostructures of disulfide- and triazole-linked peptides: Synthesis, characterization, membrane interactions and biological activity
Synthesis and characterization of a new disulfide and triazole-peptide-decorated alumina nanoparticles.
Relevant biological activity of hybrid nanobiostructures containing antimicrobial peptides.
Membrane-interactions of the new synthesized nanobiostructures.
Due to the its physical-chemical properties, alumina nanoparticles have potential applications in several areas, such as nanobiomaterials for medicinal or orthodontic implants,
although the introduction of these devices poses a serious risk of microbial infection. One convenient strategy to circumvent this problem is to associate the nanomaterials to
antimicrobial peptides with broad-spectrum of activities. In this study we present two novel synthesis approaches to obtain fibrous type alumina nanoparticles covalently bound
to antimicrobial peptides. In the first strategy, thiol functionalized alumina nanoparticles were linked via disulfide bond formation to a cysteine residue of an analog of the
peptide BP100 containing a four amino acid spacer (Cys-Ala-Ala-Ala). In the second strategy, alumina nanoparticles were functionalized with azide groups and then bound to
alkyne-decorated analogs of the peptides BP100 and DD K through a triazole linkage obtained via a copper(I)-catalyzed cycloaddition reaction. The complete physical-chemical
characterization of the intermediates and final materials is presented along with in vitro biological assays and membrane interaction studies, which confirmed the activity of
the obtained nanobiostructures against both bacteria and fungi. To our knowledge, this is the first report of aluminum nanoparticles covalently bound to triazole-peptides and to
a disulfide bound antimicrobial peptide with high potential for biotechnological applications.
Iterative custom peptide synthesis in membrane cascades: Untangling operational decisions
Dynamic process model of semi-batch custom peptide synthesis in membrane cascade was developed.
Model validation by experimental data.
Reactions, side-reactions and diafiltration included in the process model.
Further study by readers is enabled by the downloadable simulation file (gPROMS).
Membrane enhanced custom peptide synthesis (MEPS) combines liquid-phase synthesis with membrane filtration, avoiding time-consuming separation steps such as precipitation and
drying. Although performing MEPS in a multi-stage cascade is advantageous over a single-stage configuration in terms of overall yield, this is offset by the complex combination
of operational variables such as the diavolume and recycle ratio in each diafiltration process. This research aims to tackle this problem using dynamic process simulation. The
results suggest that the two-stage membrane cascade improves the overall yield of MEPS significantly from 72.2% to 95.3%, although more washing is required to remove impurities
as the second-stage membrane retains impurities together with the anchored peptide. This clearly indicates a link between process configuration and operation. While the case
study is based on the comparison of single-stage and two-stage MEPS, the results are transferable to other biopolymers such as oligonucleotides, and more complex system
configurations (e.g. three-stage MEPS).
Coupling of two amino acids in solution. The unprotected amine of one reacts with the unprotected carboxylic acid group of the other to form a peptide bond. In this example, the second reactive group (amine/acid) in each of the starting materials bears a protecting group.
In organic chemistry, custom peptide synthesis is the production of peptides, compounds where multiple amino acids are linked via amide bonds, also known as peptide bonds. Peptides are chemically synthesized by the condensation reaction of the carboxyl group of one amino acid to the amino group of another. Protecting group strategies are usually necessary to prevent undesirable side reactions with the various amino acid side chains. Chemical custom peptide synthesis most commonly starts at the carboxyl end of the peptide (C-terminus), and proceeds toward the amino-terminus (N-terminus). Protein biosynthesis (long peptides) in living organisms occurs in the opposite direction.
The chemical synthesis of peptides can be carried out using classical solution-phase techniques, although these have been replaced in most research and development settings by solid-phase methods (see below). Solution-phase synthesis retains its usefulness in large-scale production of peptides for industrial purposes however.
Chemical synthesis facilitates the production of peptides which are difficult to express in bacteria, the incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins, which consist of D-amino acids.
The established method for the production of synthetic peptides in the lab is known as solid-phase custom peptide synthesis (SPPS). Pioneered by Robert Bruce Merrifield, SPPS allows the rapid assembly of a peptide chain through successive reactions of amino acid derivatives on an insoluble porous support.
The solid support consists of small, polymeric resin beads functionalized with reactive groups (such as amine or hydroxyl groups) that link to the nascent peptide chain.Since the peptide remains covalently attached to the support throughout the synthesis, excess reagents and side products can be removed by washing and filtration. This approach circumvents the comparatively time-consuming isolation of the product peptide from solution after each reaction step, which would be required when using conventional solution-phase synthesis.
Each amino acid to be coupled to the peptide chain N-terminus must be protected on its N-terminus and side chain using appropriate protecting groups such as Boc (acid-labile) or Fmoc (base-labile), depending on the side chain and the protection strategy used (see below).
The general SPPS procedure is one of repeated cycles of alternate N-terminal deprotection and coupling reactions. The resin can be washed between each steps. First an amino acid is coupled to the resin. Subsequently, the amine is deprotected, and then coupled with the free acid of the second amino acid. This cycle repeats until the desired sequence has been synthesized. SPPS cycles may also include capping steps which block the ends of unreacted amino acids from reacting. At the end of the synthesis, the crude peptide is cleaved from the solid support while simultaneously removing all protecting groups using a reagent strong acids like trifluoroacetic acid or a nucleophile. The crude peptide can be precipitated from a non-polar solvent like diethyl ether in order to remove organic soluble by products. The crude peptide can be purified using reversed-phase HPLC. The purification process, especially of longer peptides can be challenging, because small amounts of several byproducts, which are very similar to the product, have to be removed. For this reason so-called continuous chromatography processes such as MCSGP are increasingly being used in commercial settings to maximize the yield without sacrificing on purity levels.
SPPS is limited by reaction yields, and typically peptides and proteins in the range of 70 amino acids are pushing the limits of synthetic accessibility. Synthetic difficulty also is sequence dependent; typically aggregation-prone sequences such as amyloids are difficult to make. Longer lengths can be accessed by using ligation approaches such as native chemical ligation, where two shorter fully deprotected synthetic peptides can be joined together in solution.
Peptide coupling reagents
An important feature that has enabled the broad application of SPPS is the generation of extremely high yields in the coupling step. Highly efficient amide bond-formation conditions are required. and adding an excess of each amino acid (between 2- and 10-fold). The minimization of amino acid racemization during coupling is also of vital importance to avoid epimerization in the final peptide product.
Amide bond formation between an amine and carboxylic acid is slow, and as such usually requires 'coupling reagents' or 'activators'. Activation of the carboxyl group generally involves the formation of an 'active ester' in situ. A selection of peptide coupling reagents are described below are .
Amide bond formation using DIC/HOBt.
Carbodiimides such as dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC) are frequently used for amide bond formation. The reaction proceeds via the formation of a highly reactive O-acylisourea. This reactive intermediate is attacked by the peptide N-terminal amine, forming a peptide bond. Formation of the O-acylisourea proceeds fastest in non-polar solvents such as dichloromethane.
DIC is particularly useful for SPPS since as a liquid it is easily dispensed, and the urea byproduct (N,N'-Diisopropylcarbodiimide is easily washed away. Conversely, the related carbodiimide 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is often used for solution-phase peptide couplings as its urea byproduct can be removed by washing during aqueous work-up.
Carbodiimide activation opens the possibility for racemization of the activated amino acid. Racemization can be circumvented with 'racemization suppressing' additives such as the triazoles 1-hydroxy-benzotriazole (HOBt), and 1-hydroxy-7-aza-benzotriazole (HOAt). These reagents attack the O-acylisourea intermediate to form an active ester, which subsequently reacts with the peptide to form the desired peptide bond. Ethyl cyanohydroxyiminoacetate (Oxyma), an additive for carbodiimide coupling, acts as an alternative to HOAt.
Aminium/uronium and phosphonium salts
Uronium-based peptide coupling reagents
Some coupling reagents omit the carbodiimide completely and incorporate the HOAt/HOBt moiety as an aminium/uronium or phosphonium salt of a non-nucleophilic anion (tetrafluoroborate or hexafluorophosphate). Examples of aminium/uronium reagents include HATU (HOAt), HBTU/TBTU (HOBt) and HCTU (6-ClHOBt). HBTU and TBTU differ only in the choice of anion. Phosphonium reagents include PyBOP (HOBt) and PyAOP (HOAt).
These reagents form the same active ester species as the carbodiimide activation conditions, but differ in the rate of the initial activation step, which is determined by nature of the carbon skeleton of the coupling reagent. Furthermore, aminium/uronium reagents are capable of reacting with the peptide N-terminus to form an inactive guanidino by-product, whereas as phosphonium reagents are not.
Propanephosphonic acid anhydride
Since late 2000s, propanephosphonic acid anhydride, sold commercially under various names such as "T3P", has become a useful reagent for amide bond formation in commercial applications. It converts the oxygen of the carboxylic acid into a leaving group, whose peptide-coupling byproducts are water soluble and can be easily washed away. In a performance comparison between propanephosphonic acid anhydride and other peptide coupling reagents for the preparation of a nonapeptide drug, it was found that this reagent was superior to other reagents with regards to yield and low epimerization.
Cross-linked polystyrene is the most common solid support used in SPPS.
Solid supports for custom peptide synthesis are selected for physically stability, to permit the rapid filtration of liquids. Suitable supports are inert to reagents and solvents used during SPPS, although they must swell in the solvents used to allow for penetration of the reagents, and allow for the attachment of the first amino acid.
Three primary types of solid supports are: gel-type supports, surface-type supports, and composites. Improvements to solid supports used for custom peptide synthesis enhance their ability to withstand the repeated use of TFA during the deprotection step of SPPS. Two primary resins are used, based on whether a C-terminal carboxylic acid or amide is desired. The Wang resin was, as of 1996, the most commonly used resin for peptides with C-terminal carboxylic acids.
Protecting groups schemes
As described above, the use of N-terminal and side chain protecting groups is essential during custom peptide synthesis to avoid undesirable side reactions, such as self-coupling of the activated amino acid leading to (polymerization). This would compete with the intended peptide coupling reaction, resulting in low yield or even complete failure to synthesize the desired peptide.
Two principle orthogonal protecting group schemes exist for use in solid-phase custom peptide synthesis: so-called Boc/Bzl and Fmoc/tBu approaches. The Boc/Bzl strategy utilizes TFA-labile N-terminal Boc protection alongside side chain protection that is removed using anhydrous hydrogen fluoride during the final cleavage step (with simultaneous cleavage of the peptide from the solid support). Fmoc/tBu SPPS uses base-labile Fmoc N-terminal protection, with side chain protection and a resin linkage that are acid-labile (final acidic cleavage is carried out via TFA treatment).
Both approaches, including the advantages and disadvantages of each, are outlined in more detail below.
The original method for custom peptide synthesis relied on tert-butyloxycarbonyl (or more simply 'Boc') as a temporary N-terminal α-amino protecting group. The Boc group is removed with acid, such as trifluoroacetic acid (TFA). This forms a positively charged amino group in the presence of excess TFA (note that the amino group is not protonated in the image on the right), which is neutralized and coupled to the incoming activated amino acid. Neutralization can either occur prior to coupling or in situ during the basic coupling reaction.
The Boc/Bzl approach retains its usefulness in reducing peptide aggregation during synthesis. In addition, Boc/Bzl SPPS may be preferred over the Fmoc/tBu approach when synthesizing peptides containing base-sensitive moieties (such as depsipeptides), as treatment with base is required during the Fmoc deprotection step (see below).
Permanent side-chain protecting groups used during Boc/Bzl SPPS are typically benzyl or benzyl-based groups. Final removal of the peptide from the solid support occurs simultaneously with side chain deprotection using anhydrous hydrogen fluoride via hydrolytic cleavage. The final product is a fluoride salt which is relatively easy to solubilize. Scavengers such as cresol must be added to the HF in order to prevent reactive t-butyl cations from generating undesired products. A disadvantage of this approach is the potential for degradation of the peptide by hydrogen fluoride.
The use of N-terminal Fmoc protection allows for a milder deprotection scheme than used for Boc/Bzl SPPS, and this protection scheme is truly orthogonal under SPPS conditions. Fmoc deprotection utilizes a base, typically 20–50% piperidine in DMF. The exposed amine is therefore neutral, and consequently no neutralization of the peptide-resin is required, as in the case of the Boc/Bzl approach. The lack of electrostatic repulsion between the peptide chains can lead to increased risk of aggregation with Fmoc/tBu SPPS however. Because the liberated fluorenyl group is a chromophore, Fmoc deprotection can be monitored by UV absorbance of the reaction mixture, a strategy which is employed in automated peptide synthesizers.
The ability of the Fmoc group to be cleaved under relatively mild basic conditions while being stable to acid allows the use of side chain protecting groups such as Boc and tBu that can be removed in milder acidic final cleavage conditions (TFA) than those used for final cleavage in Boc/Bzl SPPS (HF). Scavengers such as water and triisopropylsilane (TIPS) are added during the final cleavage in order to prevent side reactions with reactive cationic species released as a result of side chain deprotection. The resulting crude peptide is obtained as a TFA salt, which is potentially more difficult to solubilize than the fluoride salts generated in Boc SPPS.
Fmoc/tBu SPPS is less atom-economical, as the fluorenyl group is much larger than the Boc group. Accordingly, prices for Fmoc amino acids were high until the large-scale piloting of one of the first synthesized peptide drugs, enfuvirtide, began in the 1990s, when market demand adjusted the relative prices of Fmoc- vs Boc- amino acids.
Other protecting groups
The (Z) group is another carbamate-type amine protecting group, first used by Max Bergmann in the synthesis of oligopeptides. It is removed under harsh conditions using HBr in acetic acid, or milder conditions of catalytic hydrogenation. While it has been used periodically for α-amine protection in custom peptide synthesis, it is almost exclusively used for side chain protection.
Alloc and miscellaneous groups
The allyloxycarbonyl (alloc) protecting group is sometimes used to protect an amino group (or carboxylic acid or alcohol group) when an orthogonal deprotection scheme is required. It is also sometimes used when conducting on-resin cyclic peptide formation, where the peptide is linked to the resin by a side-chain functional group. The Alloc group can be removed using tetrakis(triphenylphosphine)palladium(0).
For special applications like synthetic steps involving protein microarrays, protecting groups sometimes termed "lithographic" are used, which are amenable to photochemistry at a particular wavelength of light, and so which can be removed during lithographic types of operations.
Regioselective disulfide bond formation
The formation of multiple native disulfides remains challenging of native custom peptide synthesis by solid-phase methods. Random chain combination typically results in several products with nonnative disulfide bonds. Stepwise formation of disulfide bonds is typically the preferred method, and performed with thiol protecting groups. Different thiol protecting groups provide multiple dimensions of orthogonal protection. These orthogonally protected cysteines are incorporated during the solid-phase synthesis of the peptide. Successive removal of these groups, to allow for selective exposure of free thiol groups, leads to disulfide formation in a stepwise manner. The order of removal of the groups must be considered so that only one group is removed at a time.
Thiol protecting groups used in custom peptide synthesis requiring later regioselective disulfide bond formation must possess multiple characteristics.[verification needed] First, they must be reversible with conditions that do not affect the unprotected side chains. Second, the protecting group must be able to withstand the conditions of solid-phase synthesis. Third, the removal of the thiol protecting group must be such that it leaves intact other thiol protecting groups, if orthogonal protection is desired. That is, the removal of PG A should not affect PG B. Some of the thiol protecting groups commonly used include the acetamidomethyl (Acm), tert-butyl (But), 3-nitro-2-pyridine sulfenyl (NPYS), 2-pyridine-sulfenyl (Pyr), and trityl (Trt) groups. Importantly, the NPYS group can replace the Acm PG to yield an activated thiol.
Using this method, Kiso and coworkers reported the first total synthesis of insulin in 1993. In this work, the A-chain of insulin was prepared with following protecting groups in place on its cysteines: CysA6(But), CysA7(Acm), and CysA11(But), leaving CysA20 unprotected.
Microwave-assisted custom peptide synthesis
Microwave-assisted custom peptide synthesis has been used to complete long peptide sequences with high degrees of yield and low degrees of racemization.
Synthesizing long peptides
Stepwise elongation, in which the amino acids are connected step-by-step in turn, is ideal for small peptides containing between 2 and 100 amino acid residues. Another method is fragment condensation, in which peptide fragments are coupled. Although the former can elongate the peptide chain without racemization, the yield drops if only it is used in the creation of long or highly polar peptides. Fragment condensation is better than stepwise elongation for synthesizing sophisticated long peptides, but its use must be restricted in order to protect against racemization. Fragment condensation is also undesirable since the coupled fragment must be in gross excess, which may be a limitation depending on the length of the fragment.
A new development for producing longer peptide chains is chemical ligation: unprotected peptide chains react chemoselectively in aqueous solution. A first kinetically controlled product rearranges to form the amide bond. The most common form of native chemical ligation uses a peptide thioester that reacts with a terminal cysteine residue.
Other methods applicable for covalently linking polypeptides in aqueous solution include the use of split inteins, spontaneous isopeptide bond formation and sortase ligation.
In order to optimize synthesis of long peptides, a method was developed in Medicon Valley for converting peptide sequences. The simple pre-sequence (e.g. Lysine (Lysn); Glutamic Acid (Glun); (LysGlu)n) that is incorporated at the C-terminus of the peptide to induce an alpha-helix-like structure. This can potentially increase biological half-life, improve peptide stability and inhibit enzymatic degradation without altering pharmacological activity or profile of action.
On resin cyclization
Peptides can be cyclized on a solid support. A variety of cylization reagents can be used such as HBTU/HOBt/DIEA, PyBop/DIEA, PyClock/DIEA. Head-to-tail peptides can be made on the solid support. The deprotection of the C-terminus at some suitable point allows on-resin cyclization by amide bond formation with the deprotected N-terminus. Once cyclization has taken place, the peptide is cleaved from resin by acidolysis and purified.
The strategy for the solid-phase synthesis of cyclic peptides in not limited to attachment through Asp, Glu or Lys side chains. Cysteine has a very reactive sulfhydryl group on its side chain. A disulfide bridge is created when a sulfur atom from one Cysteine forms a single covalent bond with another sulfur atom from a second cysteine in a different part of the protein. These bridges help to stabilize proteins, especially those secreted from cells. 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.
Off-resin cyclization is a solid-phase synthesis of key intermediates, followed by the key cyclization in solution phase, the final deprotection of any masked side chains is also carried out in solution phase. This has the disadvantages that the efficiencies of solid-phase synthesis are lost in the solution phase steps, that purification from by-products, reagents and unconverted material is required, and that undesired oligomers can be formed if macrocycle formation is involved.
The use of pentafluorophenyl esters (FDPP, PFPOH) and BOP-Cl are useful for cyclising peptides.