Amino acid A is activated (in this example with an uronium salt) to give activated species B, which has in this case two potential reaction pathways. In the presence of a potent nucleophile such as amino acid C, the racemization-free coupling to the peptide with the desired stereochemistry E can take place.
In the absence of a potent nucleophile, B can undergo an intramolecular ring closing to the oxazolone F, which can tautomerize to the achiral enol form G. In this step, the stereochemical information of the molecule gets lost, since G can tautomerize back to F or with a comparable probability to the oxazolone H, which has the opposite stereochemistry than the starting material. The further coupling of F with a nucleophile such as the amino acid C still gives the desired peptide E, but the same coupling of H results in peptide I, which has the undesired stereochemistry
The probability of racemization strongly depends on the acidity of the proton on the chiral center CD, which is also influenced by the electron withdrawing effect of residue R1. In the case that amino acid A carries a carbamate protecting group (i.e. Boc or Fmoc), no racemization takes place under appropriate conditions. In the case that amino acid A is the terminus of a peptide chain, the probability of racemization increases notably. This is also one of the reasons
why peptides are synthesized from C- to N-terminus. Approaches of inverse peptide synthesis (the peptide chain grows from N- to C-terminus), as well as fragment condensations oftentimes suffer from racemization.
2.3.3 Protecting Groups
The examples shown so far were very simplified since the reactants were monofunctional so that the carboxylic acid of one amino acid can only react with the amine of another. In reality, amino acids are at least bifunctional, necessitating the use of protecting groups. Protecting groups are blocking reactive centers of the amino acid in order to avoid their undesired reaction during the coupling step. Protecting groups have to be easily removable. The deprotection has to proceed in high yields and ideally without the necessity of a subsequent purification step. In the synthesis of a peptide, at least two different protecting groups have to be used. It is necessary that those two protecting groups are orthogonal to each other. This means that each protecting group can be cleaved in the presence of the other one, without (partial) deprotection of the latter. The more functional groups are incorporated in the peptide (side chain functionality), the more complex the protecting group strategy. The protecting group strategy also depends on the synthesis as such. Peptide chemistry in solution needs one protecting group more than peptide synthesis of the same peptide on polymeric support, since in the latter the C-terminus is bound to the support and cannot undergo undesired side reactions. In a linear peptide synthesis, protecting groups of the backbone functional groups are so called temporary protecting groups, since they are cleaved after each coupling step, whereas protecting groups of side chain functionalities are so called permanent protecting groups, since they are cleaved at the end of the synthesis. Therefore, permanent protecting groups have to be stable enough to undergo several coupling/deprotection-cycles.
Figure 10: Examples for protecting group strategies.
Some of the most commonly used protecting groups are depicted in Figure 10.
The protected amino acid A carries a Boc group at the N-terminus (temporary protecting group), a methyl ester at the C-terminus (temporary protecting group) and a 2Cl-Z group at the amine functionality of the side chain (permanent protecting group). The Boc group can be cleaved with dilute or concentrated TFA in CH2Cl2. Under these cleaving conditions, the other two protecting groups are stable. All formed byproducts and excess TFA can easily be removed by washing (synthesis on support) or under vacuum (synthesis in solution). The methyl ester is cleaved under very mild basic conditions with LiOH in water:THF-mixtures. Under these conditions, the other two protecting groups are stable. All byproducts can easily be removed by aqueous work-up (synthesis in solution). The 2Cl-Z group is cleaved under super-acidic conditions with TFA:TFMSA. These conditions are very harsh and cleave the Boc group quantitatively and the methyl ester partially, but since the cleavage of the permanent protecting groups is usually the last step of the synthesis, this is no real drawback. The purification of the resulting peptide proceeds via precipitation procedures (and preparative HPLC). Boc and methyl ester are so called orthogonal protecting groups, whereas the 2Cl-Z group is in this strategy quasi-orthogonal to them. In peptide synthesis on support, no C-terminal
protecting group is needed and the resulting strategy is the so called Boc/Bzl-strategy.
Amino acid B (Figure 10) carries Z group at the N-terminus (temporary protecting group), a methyl ester at the C-terminus (temporary protecting group) and a Boc group at the amine functionality of the side chain (permanent protecting group). The Z group can i.e. be cleaved under hydrogenation with Pd/C/H2 in organic solvents like ethyl acetate or alcohols. Under these conditions, the other two protecting groups are stable. Boc group and methyl ester can be cleaved as described above. In this case, all three protecting groups are orthogonal.
Amino acid C (Figure 10) carries a Fmoc group at the N-terminus (temporary protecting group) and a Boc group at the amine functionality of the side chain (permanent protecting group). The Fmoc group can easily be removed with dilute piperidine solutions in organic solvents. Under these mild conditions, the Boc group is stable. A byproduct in this reaction is a fluorene derivative, which has to be removed in a purification step. In synthesis on support, this purification is easily done by washing procedures, but since this washing is no option for the synthesis in solution (especially of small peptides), Fmoc develops his high potential only in synthesis on support. The Boc group can be cleaved as described above. The amino acid shown here finds its application in solid phase peptide synthesis. The protecting group strategy is the so called Fmoc/tBu-strategy.
2.3.4 Synthesis In Solution
Until the years 1960, peptide synthesis was done in solution. In a standard protocol (see Figure 11), the two amino acids with appropriate protecting groups are dissolved in a non-nucleophilic organic solvent, such as CH2Cl2, DMF or NMP and coupling reagents are added (coupling). After the reaction is complete, aqueous work-up follows. The impure peptide is then purified via column chromatography or recrystallization (work-up / purification) to give the desired, pure peptide, which reenters the cycle. In the next step, the peptide is deprotected (at the N-terminus). Most deprotection steps require a subsequent work-up procedure and if necessary also purification. The N-deprotected peptide
is then coupled again with an N-protected amino acid. Work-up and purification are as described. This procedure has some major drawbacks. The purification of the peptide has to follow after each coupling and is very time consuming. The purification as such is different for every peptide. Recrystallization works only for small peptides and column chromatography also has its limits in peptide size. The solubility of the peptide is another issue, since with increasing length, the solubility of the protected peptides usually decreases. This makes the use of solvents such as DMF or NMP obligate. These solvents and the solubility as such make a purification via the classical means of organic chemistry (column chromatography, recrystallization) impossible. With increasing length of the peptide, the differences between unreacted peptide and product vanish, rendering a purification without preparative HPLC impossible. Peptide synthesis in solution limits the maximum size of the peptide to typically less than 10, in rare cases up to 20 residues.
Figure 11: Schematic representation of a linear peptide synthesis in solution.
The only advantages of synthesis in solution are the accessibility to every organic chemist without material effort, the possibility of producing gram scale amounts of peptides and the high quality and purity of the resulting peptides.
In summary, synthesis in solution can make sense for very small peptides (up to a maximum of eight amino acids), if needed on a gram scale. For example, all peptides of the Lorenzi group (see section 2.2.2) were synthesized in solution.
Nevertheless, one should always keep in mind that each peptide is unique and
every sequence has its own properties. This makes peptides so fascinating, but also every synthesis unique and demanding.
2.3.5 Synthesis On Solid Support
The two major disadvantages of peptide synthesis in solution are its length limitation and the tedious and time consuming purification steps after each coupling. In 1963, Merrifield published the development of peptide synthesis on solid support (solid phase synthesis), which circumvented these issues and revolutionized peptide chemistry and had a large impact on organic chemistry in general.[41] For this outstanding development, Merrifield was honored with the Nobel prize in 1984.
In solid phase synthesis the growing peptide chain is anchored to a polymeric support, which is insoluble in the reaction media. The peptide as such is pseudo solvated in the solvent and can undergo chemical reactions as if in solution.
Since the peptide remains attached to the insoluble polymeric support, its purification is achieved by simple washing procedures of the polymer. At the end of the synthesis, the peptide is cleaved from the resin and purified. The advantage of this strategy is the fact than it can easily be automatized. These days, solid phase synthesis is done by peptide synthesizers, which are capable to synthesize long peptide sequences in few days. The length limitation of the resulting peptide is about 50 residues.
A schematic representation of a typical solid phase peptide synthesis is depicted in Figure 12. The initial step of the synthesis is the anchoring of and N-protected amino acid to the resin (loading). This loading also includes capping of unreacted polymeric chain ends with acetic anhydride. The next step of the protocol is the deprotection of the N-terminus of the resin-bound amino acid (deprotection), followed by a washing step to remove impurities (washing). The next step in the synthesis is the coupling of the next N-protected amino acid to give the crude, resin-bound peptide (coupling). All impurities are removed by washing the resin (washing). Unreacted peptide chain ends have to be terminated with acetic anhydride in order to avoid further reactions in the next coupling cycles, since this would lead to errors in the sequence (capping).
Removing of impurities is achieved by a further washing step (washing). This
cycle is run until the desired peptide sequence is synthesized. The peptide is in the end cleaved from the polymeric support to give the crude peptide (cleavage), which is purified i.e. via preparative HPLC (purification). Main impurities are break-off sequences.
Figure 12: Schematic representation of a linear solid phase peptide synthesis.
Over the years, two protecting group strategies for solid phase synthesis turned out to be very efficient. On the one hand the Fmoc/tBu-strategy and on the other hand the Boc/Bzl-strategy. In Europe, the Fmoc/tBu-strategy is very popular. Fmoc is the temporary protecting group, which is cleaved after every coupling, tBu protecting groups (i.e. Boc) are used as permanent protecting groups. The Boc/Bzl-strategy is leading in the american region. The Boc group is the temporary protecting group and benzyl protecting groups (i.e. 2Cl-Z) are permanent. The Boc/Bzl-strategy works under very harsh reaction conditions, what corrodes the peptide synthesizers very quickly.
A very important aspect in the solid phase peptide synthesis plays the choice of the polymeric support. In general, the support consists of a polymer and a linker, which connects the peptide with the polymer. Important features of the polymer are its swelling properties in the reaction solvent, its loading capacity and its inert chemical behavior. In general, the polymer is polystyrol, which is
crosslinked with 1% m-divinylbenzene. The linker can be understood as a sort of permanent, polymer bound C-terminal protecting group, which is cleaved after the synthesis. To improve swelling properties of the resin, spacing units, such as polyethyleneglycol can be placed between the polymer and the linker.
These days, a variety of different resins with different linkers is commercially available. The choice of the resin depends on the protecting group strategy and the desired C-terminus.
Figure 13: Schematic representation of a peptide synthesis resin.
The schematic representation of a peptide synthesis resin is shown in Figure 14. The spacer unit is optional. The linkers shown are two representative examples for peptide synthesis with Fmoc/tBu-strategy. The Wang linker gives the resulting peptide after cleavage with free COOH-terminus, whereas the Rink-amide linker terminates the peptide as an amide. Not shown are linkers using the Boc/Bzl-strategy. Here, the Merrifield resin and the PAM resin are popular. The former one gives the COOH-terminated peptide after cleavage, the latter one gives the resulting amide.
In summary, solid phase peptide synthesis is the state of the art approach to peptide synthesis. It can be automatized and can produce the resulting peptide much faster as compared to synthesis in solution. With this approach, peptides to a maximum length of 50 residues are realizable, what is approximately five
times the length of a peptide accessible in solution. Its major disadvantage compared to synthesis in solution is the scale limitation to much less than a gram.
2.3.6 Synthesis On Soluble Support
Another approach to peptide synthesis is the synthesis on soluble support.[42]
Here the growing peptide chain is anchored to a polymeric support, which is in contrast to solid phase peptide synthesis soluble in the reaction media. This solubility increases the solvation of the peptide and thereby its reactivity, what makes the coupling steps more efficient.
Figure 14: Schematic representation of a linear peptide synthesis on soluble support.
The polymeric support is for example monomethylethyleneglycol with average molecular weights around 5000 g/mol (MPEG 5000). This polymer easily dissolves in all coupling reagents and precipitates quantitatively in cold diethylether. After the reaction, the polymer anchored peptide is precipitated and soluble impurities are removed by washing procedures, so that tedious purifications as in synthesis in solution are unnecessary. The schematic representation of a solution phase synthesis is shown in Figure 14. Every
reaction step requires a subsequent precipitation and washing procedure. One disadvantage of this protocol is the fact that it cannot be automatized, another, that most impurities, which occur in the synthesis are not soluble in diethylether, rendering a purification by washing very inefficient. In summary, solution phase chemistry is no state of the art approach to peptide synthesis.
2.4 Polypeptide Synthesis
Polypeptides with their interesting structure-property relations and their potential biocompatibility are very important for biomedical applications such as drug delivery or DNA-complexation and –transfection. Polypeptides cannot be synthesized by the conventional means of peptide synthesis described in paragraph 2.3, due to length limitation and small scale, but have to be synthesized via polymerization reactions. The resulting peptides should be optical pure and with a narrow polydispersity.
2.4.1 Synthesis Of Homo-L-polypeptides
The state of the art polypeptide synthesis, which meets the requirements of optical pure products with a small polydispersity index is the ring opening polymerization of D-amino acid N-carboxyanhydrides (NCA). The two major approaches to this are shown in Scheme 6.
The ring opening polymerizations of NCAs can be initiated by a nucleophile such as a primary amine. A problematic side reaction can be the deprotonation of the NCA by the basic amine initiatior. The deprotonated NCA itself can now act as an initiator, leading to an undesired broader polydispersity of the product. Schlaad circumvented this issue by using the ammonium salt of the initiator, which protonated the NCA first and thereby suppressed the side reaction via the so called “activated monomer mechanism” (see Scheme 6, top).[43] Deming used cobalt and nickel complexes for the polymerization (see Scheme 6, bottom).
[44-47] Both polymerization approaches gave the polypeptides in high optical purity with very narrow polydispersities. With the ring opening polymerization, only homo-polypeptides or co-block-polypeptides can be synthesized.
Scheme 6: Schematic representation of two efficient NCA polymerization