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Aggregation, Racemization and Side Reactions


Aggregation of the peptide-resin can cause slow or incomplete deprotection and incomplete coupling.  One indication that aggregation is occurring is that the peptide-resin fails to swell.  It has been attributed to self-association of the peptide through hydrogen bonding.Aggregation cannot be predicted reliably from sequence data, although hydrophobic sequences are more prone to aggregation and aggregation is not likely before the fifth or sixth residue or after the twenty-first residue.

If aggregation becomes troublesome, a number of different steps can be taken to disrupt the hydrogen bonding causing the aggregation.  Some actions that can be taken include:

  • If Fmoc deprotection is slow or incomplete, switch to DBU in the deprotection reagent. (DBU protocol)
  • Switch to in situ neutralization protocols if Boc/Bzl protection is employed.
  • Switch to N-methylpyrrole (NMP) or add dimethylsulfoxide (DMSO) to the solvent.
  • Sonicate the reaction mixture.
  • Couple at a higher temperature.
  • Add chaotropic salts such as CuLi, NaClO4 or KSCN.
  • Add nonionic detergents or ethylene carbonate (Magic Mixture).
  • Utilize microwave irradiation.

If these measures fail to significantly improve coupling, then resynthesizing the peptide on a low substitution resin or on a different resin such as TentaGel or SURE™ may be beneficial.

Introducing structural elements that disrupt hydrogen bonding of the peptide backbone can be effective in overcoming synthesis problems associated with difficult or long peptide sequences.  Pseudoprolines, depsipeptides and backbone protecting groups are elements that have been utilized separately or in combination to reduce aggregation during solid phase synthesis.  In reducing aggregation, these structures also increase the solubility of the cleaved peptide, thereby facilitating the purification of the crude synthetic peptide.  After the desired peptide has been purified, a simple treatment will provide the native peptide.

Esterification on serine or threonine residues efficiently disrupts aggregation.  If the peptide contains either of these residues, then it may be possible to prepare the corresponding depsipeptide and with mild base rearrange it to the desired peptide. Protocols have been recently adapted to allow for fully automated syntheses of long-chain depsipeptides, including the esterification procedures.

Utilizing backbone-protecting groups such as 2-hydroxy-4-methoxybenzyl (Hmb) or 2,4-dimethoxybenzyl (Dmb) on the alpha-nitrogen of amino acid residues will prevent hydrogen bonding.  Incorporation of a Hmb moiety every six to seven residues will effectively disrupt aggregation.Backbone protection with Hmb or Dmb also prevents aspartimide formation and resulting side products.

Since proline in a peptide sequence is known to disrupt aggregation, utilizing pseudoprolines derived from threonine and serine is another strategy for disrupting aggregation.Pseudoprolines have been shown to be quite effective in disrupting aggregation and have made the stepwise synthesis of long peptides feasible.The TFA treatment to cleave the peptide from the resin also converts the pseudoprolines to the corresponding serine or threonine residue. 


Activation of the protected amino acid can result in some degree of racemization.  The epimerization occurs through the mechanism illustrated below(Figure 1).Adding HOBt, 6-Cl-HOBt or HOAtsuppresses the racemization.Histidine and cysteine are especially prone to racemization.  Protecting the pi imidazole nitrogen in the histidine side-chain with the methoxybenzyl group greatly reduces racemization.  A number of reduced-racemization protocols  for coupling cysteine residues have been evaluated and compared.

Copper (II) chloride with HOBt has been utilized in solution phase coupling of peptide segments to suppress racemization.Recently CuCl2 has been reported to be effective in solid phase synthesis utilizing the unusual amino acid 4,4,4-trifluoro-N-Fmoc-O-tert-butyl-threonine.

Racemization Mechanism


Figure 1 - Racemization Mechanism


Side Reactions

Diketopiperadine Formation

This side reaction occurs at the dipeptide stage and is more likely in Fmoc-based syntheses.  Diketopiperazine formation is especially prevalent when proline is one of the first two residues. 

Diketopiperazine formation

Figure 2 - Diketopiperazine Formation

In Boc-based synthesis, diketopiperazine formation can be suppressed by utilizing in situ neutralization protocols.If the Fmoc/tBu protection strategy is utilized, performing the synthesis on 2-chlorotrityl chloride resin is preferred when proline, pipecolic acid or TIC is one of the first two amino acids.  The steric bulk of the 2-chlorotrityl moiety inhibits formation of diketopiperazines. 

A second alternative is to add the second and third amino acid residues as a dipeptide unit, thus avoiding the dipeptide-resin intermediate.  This strategy is limited by the availability of the appropriate dipeptide.  A third alternative is to couple an N-trityl protected amino acid in the second position.The trityl group is then removed with dilute TFA, resulting in a protonated dipeptide-resin, which can then be coupled by in situ neutralization protocols.

Aspartimide Formation

Aspartimide formation is especially prevalent in peptides containing Asp-Gly, Asp-Ala or Asp-Ser sequences.  This side reaction can occur acidic or basic conditions.  The aspartimide can reopen producing a mixture of alpha and beta coupled peptides (Figure 7).  In Fmoc-based syntheses, piperidine can open the asartimide to yield piperidides.Adding HOBt to the piperidine deprotecting solution will reduce aspartimide formation.  A special cleavage protocol has been developed that reduces aspartimide formation. In Boc synthesis, using the beta cyclohexyl ester instead of the beta benzyl ester of aspartic acid significantly lowers the amount of aspartimide formed.

Aspartimide formation can be blocked by incorporating a blocking group on the alpha-nitrogen of the amino acid preceding aspartic acid in the peptide synthesis.  2-Hydroxy-4-methoxybenzyl (Hmb) and 2,4-dimethoxybenzyl (Dmb) groups have been utilized for this purpose in Fmoc chemistry.These groups prevent aspartimide formation during synthesis and are removed during peptide cleavage from the resin by TFA treatment.  Coupling to Dmb-protected amino acids can be difficult.  Several Fmoc-AA-(Dmb)Gly-OH dipeptides are commercially available.  

The pseudoproline formed from serine also is effective in blocking aspartimide formation in Asp-Ser sequences, as demonstrated in a semi-synthetic preparation of the R2 subunit of E. coli ribonucleotide reductase.Danishefsky has demonstrated that a pseudoproline located at n+2 to an aspartic acid residue can also prevent aspartimide formation.[30]

Aspartamide Formation

Figure 3 - Aspartimide  Formation

Pyrogluamate Formation

N-terminal glutamine residues may undergo base-catalyzed cyclization to form pyroglutamate. As with aspartic acid derivatives, adding HOBt to the deprotection solution suppresses this side reaction. 

3-(1-Piperidinyl)alanine Formation 

This side product forms when peptides containing a C-terminal cysteine are prepared by Fmoc/tBu protocols.  Base catalyzed elimination of the protected sulfhydryl group produces a dehydroalanine residue, which, in turn, adds piperidine.This side product can be confirmed by mass spectroscopy as a mass shift of +51.  Utilizing the sterically bulky trityl protecting group will minimize, but not eliminate, this side product.


3-Piperidinylalanine formation

Figure 4 - 3-(1-Piperidinyl)alanine Formation


Uronium/aminium coupling reagents will react with the unprotected N-terminal of a peptide-resin to form a guanidine moiety, which irreversibly terminates the peptide chain.  Guanidinylation can be avoided by preactivating the protected amino acids with a stoichiometric amount of the coupling reagent prior to adding the solution to the peptide-resin.  In situ neutralization suppresses guanidinylation in Boc-protection based protocols.

Guanidinylation Mechanism

Figure 5 - Guanidinylation

Transfer of Sulfonyl Protecting Groups from Arg to Trp

Sulfonyl protecting groups can transfer from arginine residues to trytophan residues during the final cleavage/deprotection of the peptide-resin.  The amount of byproduct formed depends on the protecting group and the distance between tryptophan and arginine residues.Using a cleavage cocktail containing scavengers can reduce the amount of byproducts formed.  Reagent K and Reagent R are a couple of examples of the cleavage cocktails used when arginine residues are present.

The most effective way to prevent migration of sulfonyl protecting groups to tryptophan during cleavage is to utilize indole protected tryptophan derivatives: Boc-Trp(For)-OH in Boc/Bzl based synthesis and Fmoc-Trp(Boc)-OH in Fmoc/tBu based synthesis. 

Oxidation of Methionine 

The thioether of the methionine sidechain is readily oxidized to sulfoxide under acidic conditions.  Adding dithiothreitol (DTT) to the cleavage mixture will suppress oxidation.  Alternatively, the oxidized peptide can be reduced to the desired peptide following cleavage.In some cases, methionine sulfoxide is utilized in the peptide synthesis in place of methionine.  This strategy is adopted when the methionine residue is especially prone to oxidation.  The crude peptide is purified in the oxidized form, then reduced to the native form following purification.  This results in easier purification and greater recovery of the desired peptide. 

N–O Shift

Peptides containing serine or threonine residues can undergo acid catalyzed acyl N–O shift.Treatment with base, aqueous ammonia for example, reverses the reaction.

Side Reactions during Cleavage

Homoserine Lactone Formation During HF Cleavage

Tert-butyl cations, formed in the deprotection of tert-butyl based protecting groups, can alkylate the thioether sidechain of C-terminal methionine, which subsequently cyclizes to produce homoserine lactone.This side reaction can be prevented by removing all tBu-based protecting groups prior to HF cleavage.

Homoserine lactone formation

Figure 6 - Homoserine Lactone Formation

Glutamic Acid Side Reactions

Deprotection of glutamic acid residues during HF cleavage can result in the formation of an acylium ion from the HF protonation and dehydration of the unprotected carboxyl moiety.  The acylium ion can cyclize to produce a pyroglutamine residue or it can react with scavengers such as anisole to form an aryl ketone.

Glu Side Reaction During HF Cleavage

Figure 7 - Glutamic Acid Side Reation During HF Cleavage

Asp-Pro Cleavage

Cleavage of this bond during HF cleavage has been reported.

Cys Side Reactions During Cleavage From Wang Resin

 Cysteine residues with acid labile protecting groups such as trityl (Trt), 4-methoxytrityl (Mmt), or the recently reported tetrahydropyranyl (Thp) can form S-alkylated side products arising from the fragmentation of Rink Amide or Wang resin linkers.  The 4-hydroxybenzylated side product arising from Wang linker fragmentation is especially prevalent when the Cys residue is at the C-terminal.  The popular cleavage additive triisopropylsilane (TIS) alone is unable to suppress this side reaction.  Indeed, the side product may be the major product when a TFA/TIS/H2O cleavage cocktail is utilized.  This side reaction can be suppressed by adding ethane dithiol (EDT).




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