Molecular Biology: Protein Structure and Functions - Stepanov V.M. 2005

Peptides
Chemical synthesis of peptides

Peptide Synthesis, whose foundations were established by E. Fischer at the beginning of the 20th century, has evolved into a vast branch of synthetic organic chemistry. The synthesis of quite lengthy peptides and several relatively small Proteins has been successfully described. Without diminishing The Significance of these recent achievements, it should be noted that for the practical production of substantial quantities of proteins, microbiological synthesis in recombinant Cells remains the preferred method, while Cell-free peptide Biosynthesis is also beginning to emerge.

Synthesizing peptides of a specific Structure, even the simplest ones, requires the preliminary temporary blocking (protection) of functional groups that should not participate in the reaction. Without this precaution, highly complex mixtures of products would be obtained.

2.2.1. Protection of the Amino Group

The α-amino group of the acylating amino acid or peptide is protected in such a way that the blocking group can be cleaved—removed under sufficiently mild conditions that guarantee the integrity of the peptide—after the peptide bond has been formed. One of the first groups proposed (which retains its importance to this day) is the benzyloxycarbonyl group (abbreviated as Z, obsolete name carbobenzoxy group), which is introduced by acylating The amino acid or peptide with benzyloxycarbonyl chloride:

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Once protection is no longer needed, the benzyloxycarbonyl group is cleaved by hydrogenation in the presence of a palladium catalyst or by the action of hydrogen bromide in glacial acetic acid:

The tert-butoxycarbonyl (BOC) group is widely used. It is introduced by acylating the amino group with mono-tert-butyl carbonate anhydride (BOC-anhydride), whereas Cleavage is achieved through the mild action of acidic agents, such as trifluoroacetic acid or hydrogen chloride in organic Solvents:

Recently, especially in Automated Peptide Synthesis, the 9-fluorenylmethyloxycarbonyl group (Fmoc group) has been utilized with increasing frequency. It is introduced by treating the amino acid or peptide with the corresponding chloroformate:

* Induces the elimination of the hydrogen atom indicated by the arrow.

A multitude of Other Protecting Groups are known, each possessing various advantages and drawbacks and applied in different synthesis schemes. The need to use A wide variety of these groups arises because specific groups are required to protect the Functional groups of side chains; moreover, the deprotection of α-amino groups must be carried out prior to each new synthetic step, whereas the side-chain protection must remain unaffected under these conditions. Side-chain groups are cleaved only at the very end of peptide synthesis.

2.2.2. Protection of the α-Carboxyl Group

For this purpose, the carboxyl group is typically converted into a methyl or tert-butyl ester using the reactions described in Chapter 1. The Cleavage of the methyl ester relies on alkaline saponification, which does not always proceed smoothly and carries the risk of racemization at the C-terminal residue. tert-Butyl esters are conveniently cleaved by mild Treatment with acidic agents, such as trifluoroacetic acid:

2.2.3. Formation of the Peptide Bond

The formation of a peptide bond—which, in chemical synthesis, is typically conducted in the absence of Water—requires the activation of the carboxyl group. Although activation of the α-amino group is theoretically possible, it is virtually never used in practice. The activated derivative of an amino group-protected amino acid or peptide can be prepared in a separate reaction, and sometimes even isolated or generated immediately prior to Condensation with the amino component (an amino acid or peptide with a protected carboxyl group).

Reaction in the presence of carbodiimides. The acylated amino acid reacts with the —N=C=N— (carbodiimide) group to form the corresponding O-acylisourea. This activated derivative then reacts with the amino component to yield the peptide and a disubstituted urea:

Activated esters. It is sometimes more convenient to convert the O-acylisourea into an activated ester (to some extent, this compound itself can be regarded as an activated ester; however, it is unstable, which can lead to undesired Side Reactions). To achieve this, pentafluorophenol, p-nitrophenol, N-hydroxysuccinimide, or similar Reagents are added to the reaction mixture containing the O-acylisourea:

The products resulting from The transfer of the aminoacyl or peptide residue to the hydroxyl group of these compounds are relatively stable and can be isolated. Their subsequent interaction with the amino component yields the desired peptide:

Symmetrical anhydrides of protected Amino Acids. o-Acyl-urea can react with the carboxyl group of a second protected amino acid molecule to form its symmetrical anhydride. The latter is an efficient acylating agent and reacts with the amino component to yield a peptide, releasing a protected amino acid:

Many other Methods for carboxyl Group activation are known and applied; their descriptions can be found in specialized manuals.

2.2.4. Peptide Synthesis Tactics

The approaches outlined above enable the directed synthesis of highly complex peptides. The optimal synthesis pathway is chosen based on several considerations. Particular attention is paid to preventing racemization, which is most likely to occur at the C_a atom of the residue whose carboxyl group is activated. This is because activating groups cause a shift in electron density not only away from the carbonyl carbon (which is necessary to facilitate the nucleophilic attack of this atom by the amino group of the amino component) but also away from the C _a atom. The latter favors the abstraction of the hydrogen atom attached to this carbon as a proton. This abstraction converts the remaining three substituents at the a-carbon atom into a planar trigonal configuration. Upon the return of the proton, which can approach the C_a atom from either side of the plane, an equal mixture of L- and D-isomers is formed, i.e., racemization occurs. Thus, carboxyl group activation carries an inherent risk of racemization at C-terminal amino acid residues in peptide fragments:

Urethane-type amide bonds in benzyloxycarbonyl and BOC Amino acids are less prone to racemization compared to standard peptide bonds. Consequently, when planning a peptide synthesis, it is advantageous not to condense peptide fragments—since this would require activating the peptide carboxyl groups—but rather to add protected activated amino acids to the growing peptide one by one, starting from the C-terminal amino acid residue. Nevertheless, the synthesis of long peptides sometimes necessitates The Use of fragments that are subsequently joined together. In such cases, fragments are carefully chosen so that the C-terminal position of each contains either a Glycine residue, which lacks an asymmetric carbon and therefore cannot racemize, or a Proline residue, which lacks a hydrogen atom at the a-carbon atom and thus cannot undergo racemization via the mechanism described above. If this approach is unfeasible for any reason, fragment condensation is performed using peptide azides—a method where the risk of racemization is minimal, though not entirely excluded.

An exceptionally valuable advancement in stepwise peptide synthesis was the solid-phase peptide synthesis method proposed by R. Merrifield. The C-terminal amino acid is attached to a polymer matrix via an ester bond, for example, by reacting an amino acid salt with active bromomethyl groups On the surface of cross-linked polystyrene:

Following the formation of the ester bond between the C-terminal residue and the matrix, the protecting group (usually BOC or Fmoc) is cleaved by treating the polymer with an acid or an amine, respectively:

The newly liberated a-amino group is then coupled with the amino acid preceding the C-terminal one, using either a protected amino acid anhydride or another activation method, such as a reaction in the presence of a carbodiimide, a protected amino acid pentafluorophenyl ester, etc.:

The protecting group is then cleaved again, and the cycle is repeated to attach the next amino acid residue, and so on.

Such cycles can be repeated many times to yield very long peptide chains containing up to 100 residues or more. Multi-step solid-phase synthesis is particularly advantageous because it prevents the losses of the synthesized peptide that are inevitable during product isolation under conventional conditions. The yield per amino acid coupling and deprotection step typically reaches 99–99.5%, largely due to the use of a large excess of the acylating component. Importantly, the growing peptide chains, being anchored to the support, are protected from aggregation—a phenomenon that severely limits the feasibility of synthesizing long peptides in solution.

Solid-phase peptide synthesis, consisting of strictly identical repeating steps, has been successfully automated, leading to The Development of automated peptide synthesizers.

In practice, the method is still limited by incomplete coupling of the acylamino acid in individual cycles. Naturally, a peptide that fails to couple in a given cycle will be acylated in the next, resulting in a skipped amino acid residue. The formation of such sequence-deletion peptides leads to a highly complex mixture of products during the synthesis of long chains. The method has proven particularly productive when combined with High-Performance Liquid Chromatography, which allows the Isolation of the major component from the multitude of solid-phase synthesis products.

The combination of solid-phase synthesis and high-performance liquid chromatography is currently the primary approach for obtaining small quantities of 15–20-residue peptides, specifically peptide determinants used to generate specific Antibodies against a given protein.

The application of various peptide synthesis techniques, predominantly solid-phase synthesis, has made it possible to produce several generally small proteins. Initially, these efforts were purely of fundamental significance, demonstrating the feasibility of purely chemical Protein Synthesis. Recently, however, practically significant protein syntheses have been achieved. In particular, significant quantities of Insulin, A number of other Peptide Hormones such as fish Calcitonin (32 amino acid residues, produced in batches of 50–100 g), epidermal growth factor (53 residues), transforming growth factor-a (50 residues), interleukin-3 (140 residues), human leukocyte a₁- and a₂-interferons and their analogs, and pancreatic Trypsin inhibitor have been successfully produced. The proteinase of the HUMAN IMMUNODEFICIENCY VIRUS, comprising a 99-amino-acid peptide chain, has also been synthesized and used to determine its Spatial Structure. Notably, the folding of synthetically produced peptide chains into their native structure occurs spontaneously and without significant difficulty.

Although large-scale protein production in the near future will likely rely on microbiological synthesis or cell-free systems, further advances in chemical METHODS FOR PRODUCING large Peptides and Proteins remain possible. So-called protein semisynthesis also holds certain promise; in this approach, a protein polypeptide chain is assembled from peptide fragments obtained via Limited proteolysis and purely chemical methods, employing chemical and sometimes enzymatic condensation techniques. This approach has been applied, in particular, to produce human proinsulin by attaching a series of synthetic peptide units to the A- and B-chains isolated from natural insulin.