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Continue to Iris Biotech GmbHSend request to US distributorPublished on 06.09.2022
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.
VKDGYI-OH
VKDRYI-OH
VKDDYI-OH
The effects of the tested conditions above on the degree of aspartimide/piperidide formation can be summarized as follows:
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
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:
Sequence |
Asp-PG |
Cleavage Conditions |
Crude Yield/Area [%] |
Isolated Yield [%] |
ASYKVTLKTPDGDNVITVPD-amide |
OtBu (3x) |
30% piperidine |
66 |
29 |
ASYKVTLKTPDGDNVITVPD-amide |
OtBu (3x) |
30% piperidine/0.1 M FA |
71 |
33 |
ASYKVTLKTPDGDNVITVPD-amide |
OBno (3x) |
30% piperidine |
76 |
42 |
ASYKVTLKTPDGDNVITVPD-amide |
CSY (3x) |
30% piperidine |
73 |
37 |
NPLGFFPDHQLDPAFRANTANPDWDy-amide |
OtBu (3x) |
30% piperidine |
53 |
23 |
NPLGFFPDHQLDPAFRANTANPDWDy-amide |
OtBu (3x) |
30% piperidine/0.1 M FA |
58 |
24 |
NPLGFFPDHQLDPAFRANTANPDWDy-amide |
OBno (3x) |
30% piperidine |
63 |
27 |
NPLGFFPDHQLDPAFRANTANPDWDy-amide |
CSY (3x) |
30% piperidine |
64 |
14 |
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References:
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