## 3 Results and Discussion
### Synthesis of NODAGA-Glu-CFM Conjugate  
The above-described synthesis of NODAGA-Asp-CFM indicates a problem with the separation of the two isomers Fmoc-Asp-CFM(\(\alpha\)) and Fmoc-Asp-CFM(\(\beta\)). Therefore, it was decided to carry out an analogous synthesis of the NODAGA-linker-CFM conjugate using glutamic acid as a linker, which, compared to aspartic acid, contains an additional -CH\({}_{2}\) group, which should result in a better separation of the products. The stepwise synthesis of the NODAGA-Glu-CFM conjugate (Scheme 2) is analogous to that of the NODAGA-Asp-CFM, with the exception of the Boc-Glu-anh preparation step as this reagent was commercially available. For the synthesis of the Boc-Glu-CFM derivative, 13.50 mg (28 \(\upmu\)mol) of CFM and 6.42 mg (28 \(\upmu\)mol) of Boc-Glu-anh in 200 \(\upmu\)L of DMF were used. The Eppendorf tube was closed and left for 24 h on the magnetic stirrer at room temperature, and the progress of the reaction was checked by HPLC. The obtained final products with R\({}_{\rm T}\) values of 18.28 min and 18.79 min (Figure 5) were isolated from the reaction mixture by HPLC  
Figure 4: HPLC chromatogram (at 270 nm) of the reaction mixture of NODAGA-Asp-CFM (R\({}_{\rm T}\) = 19.48 min) conjugate synthesis (gradient 2). (gradient two). The results of mass spectrometry confirmed the obtainment of the Boc-Glu-CFM derivative in the form of two isomers: Boc-Glu-CFM(\(\alpha\)) (R\({}_{\rm T}\) =18.29 min) and Boc-Glu-CFM(\(\gamma\)) (R\({}_{\rm T}\) =18.79 min). MS for C\({}_{29}\)H\({}_{40}\)N\({}_{7}\)O\({}_{10}\)S\({}_{2}\)+, TOF MS ES\({}^{+}\)\(m/z\): (calculated: 710.80); found: 710.23. The chemical yield of this reaction was surprisingly low; therefore, in order to check the purity of the purchased reagent Boc-Glu-anh, mass spectrometry analysis was performed. The results of MS analysis showed that the signal from the active anhydride form (Boc-Glu-anh, \(m/z\)-228.14) was several times smaller than that of the hydrated form of the reagent (246.12 \(m/z\)). The active form of Boc-Glu-anh in the purchased reagent was present at only about 10%, which can provide an explanation for the low yield of synthesis of the Boc-Glu-CFM product obtained in the form of the two isomers. For further syntheses, only the gamma isomer obtained with a higher yield was used. The purity of the final product was higher than 98%. In the second step, the Boc-protecting group was removed. In this case, 4 mg (5.64 \(\mu\)mol) of \(\gamma\) isomer of Boc-Glu-CFM was used and dissolved in 40 \(\mu\)L of 2.5% TFA in DMF. The reaction mixture was incubated for 45 min. The final product Glu-CFM was isolated from the reaction mixture by HPLC (purity 98%) (gradient two, Figure 6, R\({}_{\rm T}\) = 13.10) and analyzed by MS.  
MS for C\({}_{24}\)H\({}_{32}\)N\({}_{7}\)O\({}_{8}\)S\({}_{2}\)+, TOF MS ES\({}^{+}\)\(m/z\): (calculated: 610.17); found: 610.32. In the last step, 1 mg (1.64 \(\mu\)mol) Glu-CFM (\(\gamma\) isomer) dissolved in 40 \(\mu\)L DMF was transferred to a tube containing 1.44 mg (1.97 \(\mu\)mol) of NODAGA-NHS, and then 0.823 \(\mu\)L (5.9 \(\mu\)mol) of Et\({}_{3}\)N was added. The vial was left for 24 h on the magnetic stirrer at room temperature, and the final product was isolated from the reaction mixture by HPLC (gradient two, purity 98%). The recorded chromatogram (Figure 7) showed a single peak with a retention time of 14.77 min. The obtained NODAGA-Glu-CFM conjugate was characterized by the methods dedicated for the analysis of radiopharmaceutical precursors recommended by the European Pharmacopoeia 11.0, monograph 07/2022:2902 (nuclear magnetic resonance spectrometry, infrared absorption spectrophotometry, mass spectrometry and chromatographic methods). The results of infrared absorption spectrophotometry and mass spectrometry are shown below. However, we do not present the results of the \({}^{1}\)H NMR analysis.