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In order to prevent racemisation, Cys was introduced using Fmoc-Cys(Trt)-PFP. DOTAwas introduced using tris(tBu)-DOTA. The peptide was cleaved from the resin, and thedisulfide bridge was subsequently formed by air oxidation. During the oxidation process,the reduced and oxidized forms of DOTATOC cannot be distinguished by HPLC, and themixture of the two forms yields one sharp peak. High-resolution LC-MS clearly showspeaks with m/z values corresponding to the two species. However, MS of the final reactionproduct gave the correct molecular mass for the oxidized species and showed that noreduced peptide remained. The crude product was isolated by preparative HPLC usingwater/acetonitrile gradients containing acetic acid. In order to conclusively exclude theformation of diastereomeric side products due to racemisation of Cys, (D-Cys7)-DOTATOC and (D-Cys2)-DOTATOC, the two possible products containing one D-Cyswere synthesized using pure Fmoc-D-Cys(Trt)-OH. HPLC of a mixture containingDOTATOC and the two D-Cys diastereomers demonstrated that all three forms can beresolved with significantly different retention times (Figure 2). Finally, the purifiedDOTATOC was analyzed by 1H and 13C NMR spectroscopy at 9.4 T (400 MHz for 1H) at52 °C in D2O solution. The 1D spectra confirmed the expected non-exchangeable protonand carbon counts. Complete 1H and 13C signal assignments were made using acombination of standard 2D NMR experiments (COSY, cROESY, HSQC, HMBC), whichconfirmed the amino acid residues present, their sequence, the disulfide bridge, and theattachment of DOTA with a purity of >95 percent.100[M+2H] 2+711.3160100[M+2H] 2+711.3160UV signal at 214 nm1 2 3Relative abundance908070605040[M+3H] 3+474.5465Relative abundance908070605040302010711.8173712.3181712.8182[M+H+Na] 2+722.3065[M+H+K] 2+730.288630030 40time [min]0 10 20 30 40 50 60time [min]20100735700 705 710 715 720 725 730 740m/z[M+H] +1421.6220400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/zFig. 2. Left: HPLC analysis of purified DOTATOC using a linear gradient of 0 - 50%solvent B over 60 min, flow rate 0.5 mL/min, at 60 °C (solvent A = 0.1% TFA in water, B =0.1% TFA in acetonitrile). Insert: HPLC of the DOTATOC isomers (D-Cys7)-DOTATOC(1), DOTATOC (2) and (D-Cys2)-DOTATOC (3). Right: LC-MS analysis of DOTATOC.Positive-ion ESI-MS displays signals corresponding to [M+3H] 3+ , [M+2H] 2+ , [M+H] + aswell as sodium and potassium adducts. The most abundant peak at m/z = 711.3160corresponds to [M+2H] 2+ (calc. 711.3150) for DOTATOC with the disulfide bridge:C 65 H 92 N 14 O 18 S 2 (exact mass M = 1420.6155).ConclusionIn contrast to small-molecule drugs, peptides can exhibit complex conformationalproperties which complicate the analysis (e.g. DOTATOC in DMSO could not be analyzedby NMR). Therefore, the repertoire of methods for peptide analysis is limited, typicallyemploying a combination of RP-HPLC (to determine the homogeneity) and massspectrometry (to determine the identity). It could be shown that DOTATOC can besynthesized with high purity by solid-phase methods, provided that specific synthesisprotocols are applied.339