Aspartimides formation requirements

In order to prevent base-catalyzed aspartimide formation, a protecting group which provides steric hindrance in the form of both bulkiness and conformational flexibility (compared to, for example, the fert-butyl group) is required. For this purpose, the 2,4-dimethylpent-3-yl (Dmpn) ester of aspartic acid was proposed (Scheme which was shown to be very... 

Although aspartimide formation can occur during base-catalyzed acylation in Boc-based chemistry, the reaction is more pronounced during Fmoc removal with piperidine.The reaction, shown in Scheme 6, is sequence dependent, but aspartimide formation can be up to 0.3% per N-terminal deprotection cycle when the required peptide contains -Asp-Gly-. When the resultant peptide is cleaved from the support the aspartimide ring may remain intact, but can also open to form the P-branched peptide. 

With increasing use of Fmoc SPS, a number of base-mediated side reactions have been identified and reported that require careful awareness [61]. Some of these are described elsewhere in this volume (Chapters 2 and 4). The principal base-mediated side reactions are diketopiperazine formatioii caused by cyclization, particularly during A -deprotection of the residue adjacent to C-terminal resin-linked proline [62], and aspartimide formation, particularly at Asp-X residues [63,64]. The former can now be prevented by use of the substantially sterically hindered 2-chlorotrityl linker [65]. The latter side reaction is more difficult to control and appears to be largely sequence dependent. Asp-Gly, -Ser, -Thr, -Asn, and -Gin pairs are most at risk of potential imide formation, although several other Asp-X combinations have also been observed to cyclize [66,67]. For one sensitive peptide sequence, use of piperazine for A -deprotection eliminated this side reaction [68]. However, for another peptide, this base was ineffective (J Wade, unpublished). Reduction of the basicity of the piperidine solution by addition of... 

After each coupling step, the Fmoc group is removed with a solution of 20 % piperidine in DMF. Most commercially available amino-functionalized resins or preloaded hydroxy-functionalized resins require an initial M -Fmoc removal before synthesis. For longer peptide sequences, an increase in deprotection time can be beneficial further into the synthesis (after 25-30 cycles). Furthermore, for certain combinations of Asp-Xxx (in particular Asp-Gly) in the sequence, a side reaction is aspartimide formation. This side reaction can be minimized by adding 0.1 M formic acid or 1 M HOBt (1 M Oxyma) to the piperidine solutions applied in the Fmoc removal steps [24]. However, only the use of Asp(OtBu)-(Dmb) Gly-OH dipeptides completely prevent aspartimide formation. 

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