Formation Mechanisms and Mitigation Strategies of Common Peptide Impurities
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Due to the relative immobilization of the SPPS synthesis process, the generation of peptide impurities has also appeared in a generalized type, and this paper makes a summary introduction to the common types of impurities in the solid-phase synthesis of peptides and the corresponding generation mechanism.
Endo-Xaan impurities
The so-called endo-Xaan impurity refers to the impurity caused by the repeated condensation of Xaan residues into the target peptide sequence during peptide synthesis. This is a very common type of peptide impurity that can often be found in crude or even final pure peptide APIs.
Table 1 Peptide synthesis services at Creative Peptides.
There are many mechanisms that produce endo-Xaan impurities, which can be classified as follows:
Fmoc-Xaa-Xaa-OH dipeptide impurity in Fmoc-Xaa-OH raw material
Fmoc-Xaa-Xaa-OH dipeptide impurities may be produced in the process of protecting amino acid synthesis with Fmoc-Cl as raw material. If this impurity is not separated by crystallization or other purification methods during the production of the raw material, then it may be condensed into the target peptide in the Fmoc-Xaa-OH raw material, resulting in the corresponding endo-Xaa impurity. In general, endo-Xaa impurities generated by this mechanism can be avoided or minimized by quality control of raw materials. Fmoc-Xaa-OH raw materials synthesized from Fmoc-Osu usually do not produce dipeptide impurities.
H-Xaa-OH impurity in Fmoc-Xaa-OH raw material
In addition to dipeptides, impurities that may be present in Fmoc-Xaa-OH also include H-Xaa-OH impurities that are de-Fmoc-protected. The source of this impurity may be caused by the incomplete protection of Nα during the synthesis of protective amino acids, or it may be the shedding of Fmoc due to temperature, humidity or other contamination during storage or transportation of Fmoc-Xaa-OH amino acid raw materials. After the H-Xaa-OH without backbone protection is condensed into the peptide chain, its N α can still condense with Fmoc-Xaa-OH in the reaction system to form the corresponding endo-Xaa impurity. It is important to note that certain amino acids that do not contain chromophores, such as Gly, have corresponding Fmoc-missing impurities in Fmoc-Xaa-OH that cannot be detected by liquid chromatography with UV detectors.
Caused by the shedding of Fmoc in peptide synthesis
During the peptide condensation process, the Nα exposure of the backbone is caused by the premature shedding of Fmoc, which condenses with the Fmoc-Xaa-OH in the reaction system, and can also produce endo-Xaa impurities. The reason for the shedding of Fmoc may be due to the presence of certain amino groups, such as Nε of the Lys side chain and Nα of Pro. It can also be caused by the presence of dimethylamine (N, N-dimethylamine) impurities in the DMF solvent. If the peptide resin coexists overnight at room temperature with DMF that has not been completely drained, there is a risk that a positive result will be obtained if the Ninhyrdin test is redone the next day, and if the recondensation is performed based on the unconventional test results, there is a high risk of endo-Xaa impurities forming.
Table 2 Peptide analysis services at Creative Peptides.
Endo-Xaa impurities caused by piperidine remain
In the peptide condensation process, a large amount of piperidine is used in the Fmoc removal reaction, and if the remaining piperidine is not completely eluted during the subsequent resin elution process, the remaining piperidine in the resin may cleave the Fmoc in the subsequent Fmoc-Xaa-OH condensation reaction, thereby exposing its Nα and producing the corresponding endo-Xaa impurity. However, based on past experience, the residual piperidine may preferentially react with the activated ester of Fmoc-Xaa-OH to form the corresponding Fmoc-Xaa-piperidide, thereby inactivating the Fmoc-Xaa-OH of this molecule. The consequence is likely to result in a decrease in the actual Fmoc-Xaa-OH equivalent, resulting in an incomplete reaction that results in the formation of the corresponding des-Xaa impurity (see next section) rather than the endo-Xaa impurity.
Endo-XaaC-terminal impurity based on CTC resin SPPS
There is also a specific -Xa impurity that is often present in solid phase synthesis based on CTC resins and is called -Xaac-terminal. The so-called -XaaC-terminal impurity refers to the endo-Xa impurity caused by repeated condensation of the amino acid residue XaC-terminal at the C-terminal of the peptide.
This impurity is very common and tends to occur in peptide synthesis such as Gly and Pro as C-terminal residues.
Interestingly, although the endo-XaaC-terminal impurity involves repeated condensation of the C-terminal amino acid residue XaaC-terminal the root of the problem is the condensation of the second-to-last amino acid, Fmoc-Xaapenultimate-OH. In the pre-activation reaction of Fmoc-Xaapenultimate-OH, DIC and the acidic condensation reagent additive HOBt or Oxyma pre-activate Fmoc-Xaapenultimate-OH before the condensation reaction. However, this pre-activation reaction is generally not 100% complete, usually 5-10 minutes, is added to the resin system, condensation with H-XaaC-terminal-CTC resin, synthesis of the corresponding dipeptide intermediate Fmoc-Xaapenultimate -XaaC-terminal-CTC resin. However, Oxyma or HOBt, which were not fully reacted, were added to the reaction system with activated esters. These acids may cause the unreacted H-XaaC-terminal-CTC resin to fracture during the condensation reaction, thus cutting H-XaaC-terminal-OH off the resin and reintroducing it into the reaction system. The H-XaaC-terminal-OH cleaved during this process may be re-activated in the presence of DIC that has not yet been fully reacted, and re-introduced into the resin to form corresponding H-XaaC-terminal-XaaC-terminal-CTC products. The naked Nα can also react with Fmoc-Xaapenultimate -OH, which exists in the system and has not been condensed into resin, to form Fmoc-Xaapenultimate-XaaC-terminal-XaaC-terminal-CTC. That's the endo-XaaC-terminal impurity. Although the endo-XaaC-terminal impurity involves the last amino acid, it is mechanically caused by the condensation process of the penultimate amino acid. This impurity often occurs in CTC resin and DIC-mediated peptide SPPS processes.
Des-Xaan impurity
The Des-Xaan impurity is the "antimatter" of the endo-Xaan impurity, which is caused by repeated condensation of Xaan, and the des-Xaan impurity, which is caused by the absence of Xaan residues when the Xaan residues are not condensed into the target peptide sequence.
Des-Xaa impurities are also one of the most easily produced impurity types during the solid phase synthesis of peptides, and there are many reasons for des-Xaa impurities:
Inadequate condensation of Fmoc-Xaan-OH
The most common cause for the formation of des-Xaan impurities is incomplete condensation of Fmoc-Xaan-OH, resulting in the deletion of Xaan residues in the peptide sequence of interest. For this obvious problem, one needs to find a corresponding solution. There are several specific solutions:
Recondensation
Due to the difficult condensation caused by some peptide sequences themselves, the common 1.5 equivalent amino acid condensation process may not ensure the complete reaction, in this case, the recondensation process can be used, that is, after the reaction is terminated, the solution of the reaction system is filtered, and 0.5-1 equivalent Fmoc-Xaa-OH and the corresponding condensation reagent are added to the resin for recondensation. In many cases, recondensation allows the target condensation reaction to be carried out completely.
Increases amino acid equivalents
Increasing amino acid equivalence can be said to be a complementary strategy to gravity condensation. This is also due to the advantage of solid-phase synthesis, where higher equivalent reactants can be used, and the remaining reactants that are not involved in the reaction are eventually eluted and excluded from the reaction system.
Use more potent condensation reagents
For the production of process peptide API, the default condensation reagent is usually DIC, which is mainly considered from the perspective of production cost control. However, if DIC is unable to achieve 100% of the target condensation reaction, a more potent condensation reagent such as PyBOP, or even a more active HATU can be considered.
Solvent
Since peptide SPPS involves many factors such as amino acid activation, condensation reaction, resin swelling, etc., the selection of organic solvent will have an important impact on the rate of condensation reaction. Although the current gold solvent for SPPS is DMF, organic solvents with different viscosities and polarities, or even binary solvents, can also be tried to achieve an increase in reaction rate.
Reaction temperature
Although increasing the temperature may lead to the formation of many side reactions, such as racemization, Fmoc early cleavage, aspartimide, hydrolysis, DKP, etc., in some specific cases, a certain amount of time and a certain degree of high temperature can accelerate the condensation of peptides without triggering significant side reactions. Of course, this process optimization process needs to be analyzed in detail, not generalized. When using high-temperature reaction conditions, it is important to be aware of other possible side reactions, including the effect on yield (premature cleavage of the peptide resin).
Catalyst
In some reaction conditions, catalysts such as DMAP can be used to convert the active intermediate into a more active DMAP amide, which is more likely to leave in the presence of nucleophiles due to the positive charge of the latter, thus accelerating the reaction. However, the introduction of DMAP also has the potential to intensify racemization, so it needs to be treated with caution.
Reduce the degree of resin substitution
Although industrial production seeks to maximize throughput, productivity, and cost efficiency, in some specific cases, these factors need to be sacrificed in exchange for product quality and process stability. Incomplete peptide condensation is usually caused by difficult amino acid condensation, and these difficult condensations, in addition to the peptide sequence itself, such as steric hindrance of amino acid side chains, and the accessibility of peptide Nα (related to the secondary structure of peptides), are largely due to the aggregation of peptide chains on solid resin, which greatly affects the accessibility and reactivity of Nα, resulting in the difficulty and incompleteness of amino acid condensation. In response to this phenomenon, a second thing is to reduce the degree of substitution of peptide resins. Although the degree of substitution of peptide resin is determined by the resin itself, the user can quantify the effective degree of substitution by controlling the equivalent amount of the first amino acid, for example, using 0.7 equivalent amino acids to replace the resin and enclosing the unreacted active site (methanol for CTC resins, Ac2O for resins such as Rink amide).
Chaotropic salt
In view of the incomplete condensation caused by the aggregation of peptide chains on the solid phase, some chaotropic salts, such as LiBr, LiCl, etc., can be added to organic solvents, which can break the H bonds between peptide chains, so as to achieve the goal of deaggregation and facilitate the condensation reaction between peptide chains and amino acids.
Dipeptides used as raw materials
In some special cases, DES-XAA may cause threatening critical impurities, i.e., impurities that cannot be removed by purification methods, in which case the quality of the impurities needs to be controlled upstream by controlling the synthesis process. Sometimes certain amino acids, such as Gly and Pro, despite their small molecular size, produce incomplete condensation, especially for target sequences such as Gly-Gly, Pro-Pro, in which case dipeptides can be synthesized using raw materials without producing key impurities that are missing a single amino acid, even if the reaction is incomplete. Blocking after condensation of amino acids can also be used to avoid the formation of key impurities of des-Xaa.
Incomplete Fmoc removal reaction
Another mechanism for the production of des-Xaa, and one that is often overlooked, is that the Fmoc protective group is not completely removed. Naturally, the sheltered Nα cannot condense with the corresponding Fmoc-Xaan-OH amino acids. However, it is possible that Fmoc will be removed during the next round of piperidine treatment, so the reexposed Nα can react with Fmoc-Xaan-1-OH, resulting in the formation of des-Xaan impurity.
This mechanism can be solved by increasing the number of Fmoc deprotection of pipridine (usually 2 times, which can be increased to 3 times), increasing the reaction temperature, or increasing the equivalent (concentration) of piperidine.
