Product Spotlight: Fmoc-Asp(OEpe)-OH |
Hindered Variant of Fmoc-Asp(OtBu)-OH
Despite the many advances in recent years, aspartimide formation still remains problematic, particularly for the synthesis of long peptides because of the repeated exposure of the growing peptide chain to piperidine leads to accumulation of aspartimide related by-products (Figure 1). Whilst in many cases aspartimides and α- and β-piperidides generated by this side reaction may be easily separated from the target peptide by HPLC, the β-aspartyl peptides and epimerized α-aspartyl peptide are almost impossible to remove as these frequently have the same retention time as the target peptide. Furthermore, as they have the same mass as the target, the presence of these side products is hard to detect.
Therefore, the implementation of synthetic strategies that minimize aspartimide formation are a prerequite to obtaining homogenous aspartyl-containing peptides in good yield. For this reason, Novabiochem has developed the novel building block, Fmoc-Asp(OEpe)-OH, for introduction of Asp residues in Fmoc SPPS.
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Learn More about Fmoc-Asp(OEpe)-OH |
Fmoc-Asp(OEpe)-OH is a hindered variant of Fmoc-Asp(OtBu)-OH in which the steric bulk of the t-butyl group is increased by linear homologation, to help shield the aspartyl β-carbonyl group and thereby reduce the formation of aspartimide derived by-products. In tests using the well established scorpion toxin II model peptide, the use of Fmoc-Asp(OEpe)-OH instead of Fmoc-Asp(OtBu)-OH or Fmoc-Asp(OMpe)-OH for introduction of the Asp residue was found to dramatically reduce the extent of aspartimide formation per 10 minute room temperature piperidine treatment from approximately 1.24% and 0.4% with Asp(tBu) and Asp(OMpe) , respectively, to only 0.13% for Asp-Arg sequences, and from 1.65% and 0.5% for Asp(OtBu) and Asp(OMpe), respectively, to 0.2% for Asp-Asn sequences (Figures 2 & 3, Table 1).
For peptide containing 40 amino acids with C-terminal Asp-Asn, this translates to an increase in yield for the product from 48% to 92%, if Asp(OEpe) is used instead of Asp(OtBu). The use of Fmoc-Asp(OEpe)-OH was found to be equally effective at 60 °C (Table 1, Figure 4).
Fig. 1: Aspartimide formation, showing potential by-products.
Asp(OR)-(X)
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Conditions |
Product [area %] |
D/L-Aspartimides [area %] |
Piperides [area %] |
Per cycle [%]a |
|---|---|---|---|---|---|
tBu
|
piperidine at RT | 28.9 | 16.3 | 54.8 | 1.24 |
| piperidine at 60°C | 30.8 | 29.1 | 40.1 | 1.19 | |
Mpe
|
piperidine at RT | 64.2 | 6.2 | 26.8 | 0.4 |
| piperidine at 60°C | 63.5 | 16.9 | 19.6 | 0.45 | |
Epe
|
piperidine at RT | 87.8 | 3.6 | 8.6 | 0.13 |
| piperidine at 60°C | 85.4 | 7.8 | 6.8 | 0.16 | |
tBu
|
piperidine at RT | 19.2 | 4.5 | 76.3 | 1.65 |
| piperidine at 60°C | 8.6 | 19.0 | 72.4 | 2.45 | |
Mpe
|
piperidine at RT | 61.3 | 3.9 | 34.8 | 0.49 |
| piperidine at 60°C | 51.4 | 11.1 | 37.5 | 0.67 | |
Epe
|
piperidine at RT | 82.7 | 3.4 | 14.9 | 0.19 |
| piperidine at 60°C | 75.8 | 5.2 | 19.0 | 0.28 |
Table 1: Composition of crude products obtained from VKDXYI peptidyl resins after treatment with 20% piperidine in DMF at room temperature and 60 °C. aCalculation of decay per cycle k: first order decay: N = N0 • e-kt → k = -ln(N)/t ; N0 = 1; t = number of cycles (100); N = area % of target peptide. Based on 10 min treatment with 20% piperidine in DMF/cycle at room temperature and 2 min treatment at 60 °C.
Figure 2: UPLC profiles of crude cleaved peptides VKDNYI after treatment with 20% piperidine in DMF for 18 h. 1: D/L aspartimides. 2: product. 3: D/L piperidides. A: made with Fmoc-Asp(OtBu)-OH; B: made with Fmoc-Asp(OMpe)-OH; C: made with Fmoc-Asp(OEpe)-OH.
Figure 3. UPLC profiles of crude cleaved peptides VKDRYI after treatment with 20% piperidine in DMF for 18 h. 1: D/L aspartimides. 2: product. 3: D/L piperidides. A: made with Fmoc-Asp(OtBu)-OH; B: made with Fmoc-Asp(OMpe)-OH; C: made with Fmoc-Asp(OEpe)-OH.
Figure 4: UPLC profiles of crude cleaved peptides VKDRYI after treatment with 20% piperidine in DMF for 2 h at 60 ̊C. 1: D/L aspartimides. 2: product. 3: D/L piperidides. A: made with Fmoc-Asp(OtBu)-OH; B: made with Fmoc-Asp(OMpe)-OH; C: made with Fmoc-Asp(OEpe)-OH.