A Systematic Comparison of Different Methods to Tackle Aspartimide Formation

A Systematic Comparison of Different Methods to Tackle Aspartimide Formation

Published on 06/09/2022

Read on for detailed information on the results of our comparative study on different methods to tackle aspartimide formation. Click here!

Aspartimide formation is still a serious challenge in peptide synthesis. This side reaction is strongly sequence dependent and preferably occurs at Asp-Aaa motifs (Aaa = Gly, Asp, Asn, Gln or Arg). In a first step, the cyclic aspartimide is formed, which can re-open in a second reaction leading to (epimerized) alpha- and beta-Asp peptides and corresponding piperidides. Thus, all in all around 10 different undesired byproducts can be formed. Over the last decades, several approaches to solve this problem have been developed.

In a joint study together with Biosyntan, we systematically compared the combination of various strategies on different aspartimide-prone model peptides, namely VKDGYI-OH, VKDDYI-OH, VKDRYI-OH, which were synthesized using 50% morpholine/0.1 M formic acid.

First, the influence of various Fmoc-cleaving reagents (DMF only, 30% piperidine, 30% piperidine/0.1 M formic acid, morpholine) was studied.




The effects of the tested conditions above on the degree of aspartimide/piperidide formation can be summarized as follows:

  • Sequence dependency: DG >> DR ≥ DD
  • Cleavage reagent: 30% piperidine > 30% piperidine/0.1 M formic acid > 50% morpholine
    (pKa piperidine = 11.2; pKa morpholine = 8.4)
    If a weak cleaving reagent, e.g. morpholine, is used, there is almost no aspartimide formation but sometimes this cleavage reagent is not sufficient for complete Fmoc cleavage; thus, stronger ones have to be used
  • Addition of formic acid reduces aspartimide formation (sequence dependent)

In a next step, the influence of acidic additives (formic acid, ammonium acetate, HOBt, trifluoroethanol) as well as the steric effect of different bulky Asp side chain protecting groups (OtBu, OEpe, OBno; see related products) was investigated. These results were compared with the application of dimethoxybenzyl (Dmb) as amide backbone protection and cyanosulfurylide (CSY) as side chain protection.

Effect of Acidic Additives

Steric Effect of Bulky Protecting Groups

Effect of the CSY Protecting Group

These results show that

  • Various acidic additives show similar results:
    0.1 M formic acid ~ 0.5 M formic acid ~ 0.1 M NH4OAc ~ 0.1 M HOBt ~ 0.1 M trifluoroethanol
  • The lower the steric demand of the bulky Asp protecting group, the higher the amount of aspartimide formation: Asp(OtBu) >> Asp(OEpe) > Asp(OBno)
  • The use of Asp(CSY) allows complete suppression of aspartimide formation; but the formation of side products is observed, even when using morpholine; side reactions are the oxidation of cysteine, methionine and tryptophane; besides, chlorination of tyrosine is detected upon cleavage of the CSY group with a large excess of N-chloro-succinimide

Finally, our identified optimal conditions were tested in the synthesis of other peptide sequences prone to aspartimide formation. The results are displayed in the table shown below:



Cleavage Conditions

Crude Yield/Area [%]

Isolated Yield [%]


OtBu (3x)

30% piperidine




OtBu (3x)

30% piperidine/0.1 M FA




OBno (3x)

30% piperidine




CSY (3x)

30% piperidine




OtBu (3x)

30% piperidine




OtBu (3x)

30% piperidine/0.1 M FA




OBno (3x)

30% piperidine




CSY (3x)

30% piperidine



➔ For more information, please get in direct contact 

Interested in Asp(CSY)? Watch the recording of our free online workshop with Prof. Bode


Acid-Mediated Prevention of Aspartimide Formation in Solid Phase Peptide Synthesis; T. Michels, R. Dölling, U. Haberkorn, W. Mier; Org. Lett. 2012; 14(20): 5218-5221. https://doi.org/10.1021/ol3007925

The aspartimide problem in Fmoc-based SPPS. Part II; M. Mergler, F. Dick, B. Sax, C. Stähelin, T. Vorherr; J. Pept. Sci. 2003; 9(8): 518-526. https://doi.org/10.1002/psc.473

Prevention of aspartimide formation during peptide synthesis using cyanosulfurylides as carboxylic acid-protecting groups; K. Neumann, J. Farnung, S. Baldauf, J. W. Bode; Nat. Commun. 2020; 11: 982. https://doi.org/10.1038/s41467-020-14755-6

Preventing aspartimide formation in Fmoc SPPS of Asp-Gly containing peptides - practical aspects of new trialkylcarbinol based protecting groups; R. Behrendt, S. Huber, P. White; J. Pept. Sci. 2016; 22(2): 92-97. https://doi.org/10.1002/psc.2844

New t-butyl based aspartate protecting groups preventing aspartimide formation in Fmoc SPPS; R. Behrendt, S. Huber, R. Marti, P. White; J. Pept. Sci. 2015; 21(8): 680-687. https://doi.org/10.1002/psc.2790

2-phenyl isopropyl esters as carboxyl terminus protecting groups in the fast synthesis of peptide fragments; C. Yue, J. Thierry, P. Potier; Tetrahedron Lett. 1993; 34: 323-326. https://doi.org/10.1016/S0040-4039(00)60578-6

A new protecting group for aspartic acid that minimizes piperidine-catalyzed aspartimide formation in Fmoc solid phase peptide synthesis; A. Karlström, A. Undén; Tetrahedron Lett. 1996; 37(24): 4243-4246. https://doi.org/10.1016/0040-4039(96)00807-6

Patent EP 2 886 531 B1

Related Products
    1. Structure image for Fmoc-L-Asp(OtBu)-OH

      Product code: FAA1020

      Startet von 57,50 $

    2. Structure image for Fmoc-L-Asp(PP)-OH

      Product code: FAA1970

      Startet von 276,00 $

    3. Structure image for Fmoc-DmbGly-OH

      Product code: FAA3390

      Startet von 109,25 $

    4. Structure image for Fmoc-L-Asp(OMpe)-OH

      Product code: FAA8120

      Startet von 143,75 $

    5. Structure image for Fmoc-L-Asp-OMpe

      Product code: FAA8090

      Startet von auf Anfrage

    6. Structure image for Fmoc-L-Asp(OBno)-OH

      Product code: FAA8125

      Startet von 201,25 $

    7. Structure image for Fmoc-L-Asp(tBu)-DmbGly-OH

      Product code: FDP1380

      Startet von 908,50 $

    8. Structure image for Fmoc-L-Asp(CSY)-OH

      Product code: FAA8480

      Startet von 431,25 $

    9. Structure image for Fmoc-D-Asp(CSY)-OH

      Product code: FAA8483

      Startet von 862,50 $

    10. Fmoc-L-Asp(OEpe)-OH

      Product code: FAA8235

      Startet von 172,50 $