In this blog we focus on the most frequently occurring unwanted side reactions in Fmoc peptide synthesis leading to undesired reaction products difficult or impossible to be removed resulting in lowered yields, loss of product, time-consuming purification, and increased material costs. We’ll talk about the formation of diketopiperazines, aspartimides, piperidinyl-alanine, the oxidation of methionine, incomplete Fmoc deprotection, migrating Pmc and Pbf protecting groups, N-O shifts, guanidinylation, and measures to mitigate their effects on your peptide synthesis.

Diketopiperazines (DKP): To avoid the formation of diketopiperazines is one reason why the α-amino groups of amino acids are protected in the first place; otherwise, there would be virtually no chain elongation, as all the activated building blocks would react with each other. However, to grow a peptide on the resin, the amino group of the attached amino acid needs to be deprotected to connect the next amino acid. Thus, there is an amino moiety that can act as a nucleophile and attack the penultimate amide bond of the growing peptide and form a DKP. The alkaline conditions required for Fmoc deprotection further favor the nucleophilicity of amines and thus diketopiperazine formation. Besides, C-terminal prolines are especially prone to cause DKP formation, due to the basicity of their secondary amine. With Wang resins, where, in contrast to e.g., Rink amide, the growing peptide chain is anchored by an ester (X = O), DKP formation may result in cleavage of the peptide from the resin.

Countermeasures against DKP formation include the use of dipeptide building blocks or the replacement of the base piperidine by tert. butyl ammonium fluoride (TBAF). As alternative to Fmoc for amino protection, trityl (Trt) could be used, as it may be removed with a weak acid, which then is neutralized in situ during the next coupling step with a weak base like diisopropylethylamine (DIEA/DIPEA). Further options are discussed in the book by Yang (2015) (see references).

Diketopiperazine formation in SPPS: Two unprotected amino acids may form a cyclic reaction product when their carboxyl groups are activated. The same may happen if the deprotected amino group of the last amino acid of the growing peptide chain attacks the penultimate peptide bond as nucleophile.

The formation of Aspartimides is a side reaction which affects aspartate residues. It is catalyzed by both acids and bases, so it may happen during Fmoc deprotection as well as by the action of TFA during deprotection and resin cleavage. The reaction is strongly depending on the actual sequence of the peptide and predominantly occurs when Asp is followed by Gly, Asp, Asn, Gln or Arg. The ester which is normally protecting the carboxyl side chain of the aspartate during SPPS may be attacked by the nucleophilic nitrogen of the peptide bond, forming an aspartimide while epimerization occurs. This aspartimide may be hydrolyzed into a mix of D- and L-, α- and β-peptides and with piperidine, racemic α and β may be formed.

OtBu protected aspartate may be converted into racemic aspartimides, which then are hydrolyzed into α-and β-peptides. Also, racemic adducts with the nitrogen base that is used for Fmoc-deprotection (usually piperidine) may be formed. These side reactions may be minimized by using aspartate derivatives with bulkier side chain protection groups.

To circumvent or reduce the formation of aspartimides, bulky aspartate esters may be used, where at least one methyl group in the commonly used OtBu ester protection (FAA1020) is replaced with a longer carbon chain.

Sometimes, these hydrophobic protection groups introduce solubility issues. A remedy that is completely suppressing aspartimide formation is using a cyanosulfurylide (CSY) which confers enhanced solubility. CSY can be removed by virtue of electrophilic halogen species, e.g., N-chlorosuccinimide. However, side reactions are possible, like, e.g., the oxidation of Cys, Met or Trp as well as chlorination of Tyr.

By using the cyanosulfurylylide (CSY) analog of aspartic acid as precursor, the formation of aspartimides may be suppressed completely.

→ For more information about the prevention of aspartimide formation, please see our flyer or watch the recording of our online workshop! 

The formation of 3-(1-piperidinyl)alanine is an unwanted reaction which is observed when peptides with a C-terminal Cys are synthesized. Catalyzed by a base attack on the protecting group of the thiol, dehydroalanine is formed by a β-elimination, to which then piperidine adds. This reaction can be minimized by using a trityl-protected Cys (FAA1040).

The oxidation of Methionine to the sulfoxide is frequently observed, but easily reversed by treatment with ammonium iodide and dimethyl sulfide or by adding dithiotreitol (DTT) or tris-(2-carboxyethyl)phosphine (TCEP) during workup. This side reaction is increasing the polarity and thus may improve the solubility of some peptides, so it also may be deliberately used due to its simple reversibility.

Also, incomplete Fmoc deprotection may contribute to low yields. Extending the reaction time or repeating the Fmoc deprotection step before the next amino acid is coupled helps, also adding 1 to 2% 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) as non-nucleophilic auxiliary base.

When Trp with an unprotected indol is used together with Pmc (((2,2,5,7,8-pentamethylchroman-6-yl)sulfonyl)-) or Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) protected Arg, the Pmc and Pbf protecting groups may shift to the indole ring during deprotection with TFA. The extent of this side reaction depends on the distance between the respective amino acids. Suitable protected Trp building blocks (e.g. FAA1225) are available at Iris Biotech.

In Ser- and Thr-containing peptides, rearrangements may occur during deprotection with TFA. The peptide chain migrates from the nitrogen of the amide bond to the hydroxyl group of Ser and Thr via a cyclic intermediate. Reversal of this N-O shift may be induced by bases like, e.g., aq. ammonia.

Structural representation of an N-O shift in Ser- and Thr-containing peptides via a cyclic intermediate.

If free amino groups are present during couplings with carbodiimides and uronium/aminium reagents, they may be converted into guanidium moieties. This guanidinylation can be avoided if the amino acid is pre-activated, i.e., there is no excess activation reagent present when the activated amino acid is added to the reaction. Alternatively, phosphonium salts like PyBop or Bop may be used.

References:

Side Reactions in Peptide Synthesis; Y. Yang; Academic Press 2015. ISBN: 978-0-12-801009-9

Epimerisation in Peptide Synthesis; S. Duengo, M. I. Muhajir, A. T. Hidayat, W. J. A. Musa, R. Maharani; Molecules 2023; 28(24): 8017-8055. https://doi.org/10.3390/molecules28248017

Optimized Fmoc-Removal Strategy to Suppress the Traceless and Conventional Diketopiperazine Formation in Solid-Phase Peptide Synthesis; Y. Yang, L. Hansen; ACS Omega 2022; 7: 12015-12020. https://doi.org/10.1021/acsomega.2c00214

Morpholine, a strong contender for Fmoc removal in solid-phase peptide synthesis; S. N. Mthembu, A. Chakraborty, R. Schönleber, F. Albericio, B. G. de la Torre; J. Pept. Sci. 2024; 30: e3538. https://doi.org/10.1002/psc.3538

Reduction of methionine sulfoxide with NH₄ITFA: Compatibility with peptides containing cysteine and aromatic amino acids; M. Vilaseca, E. Nicolas, F. Capdevila, E. Giralt; Tetrahedron 1998; 54(50): 15273-15286. https://doi.org/10.1016/S0040-4020(98)00954-5

Sequence-dependent modification of Trp by the Pmc protecting group of Arg during TFA deprotection; A. Stierandová, N. F. Sepetov, G. V. Nikiforovich, M. Lebl; Int J Pept Protein Res. 1994; 43(1): 31-8. https://doi.org/10.1111/j.1399-3011.1994.tb00373.x