Synthesis of Novel, Dual-Targeting 68Ga-NODAGA-LacN-E[c(RGDfK)]2 Glycopeptide as a PET Imaging Agent for Cancer Diagnosis

Radiolabeled peptides possessing an Arg-Gly-Asp (RGD) motif are widely used radiopharmaceuticals for PET imaging of tumor angiogenesis due to their high affinity and selectivity to αvβ3 integrin. This receptor is overexpressed in tumor and tumor endothelial cells in the case of numerous cancer cell lines, therefore, it is an excellent biomarker for cancer diagnosis. The galectin-3 protein is also highly expressed in tumor cells and N-acetyllactosamine is a well-established ligand of this receptor. We have developed a synthetic method to prepare a lactosamine-containing radiotracer, namely 68Ga-NODAGA-LacN-E[c(RGDfK)]2, for cancer diagnosis. First, a lactosamine derivative with azido-propyl aglycone was synthetized. Then, NODAGA-NHS was attached to the amino group of this lactosamine derivative. The obtained compound was conjugated to an E[c(RGDfK)]2 peptide with a strain-promoted click reaction. We have accomplished the radiolabeling of the synthetized NODAGA-LacN-E[c(RGDfK)]2 precursor with a positron-emitting 68Ga isotope (radiochemical yield of >95%). The purification of the labeled compound with solid-phase extraction resulted in a radiochemical purity of >99%. Subsequently, the octanol–water partition coefficient (log P) of the labeled complex was determined to be −2.58. In addition, the in vitro stability of 68Ga-NODAGA-LacN-E[c(RGDfK)]2 was investigated and it was found that it was stable under the examined conditions.


Introduction
Positron emission tomography (PET) is a non-invasive, functional imaging technique that allows the visualization of physiological and pathological processes in the human body with a high sensitivity and selectivity. This imaging method requires a radiopharmaceutical that possess a positron emitter isotope attached to the targeting molecule. This vector molecule is responsible for the delivery of the radiation to a disease site. PET is capable of diagnosing diseases, monitoring disease progression, and detecting treatment response. The widespread clinical application of PET is for the visualization of glucose consumption into a precursor molecule might result in a dual-targeting radiopharmaceutical with a higher tumor uptake. Dual-targeted molecular imaging is also a promising method to enhance the binding performance of radioligands. Since the use of dual-targeting radiotracers can result in a higher tumor accumulation compared to a single-targeted probe due to the specific interaction with two different targets [17]. Successful application of the dual-targeting approach for improving imaging quality was reported in the case of peptide-based (e.g., 18 F-labeled BBN-RGD [18], 68 Ga-BBN-RGD [19,20]), antibody-based (e.g., 64 Cu-and 111 In-labeled trastuzumab fab-PEG24-EGF [21,22]), and nanoparticle-based (e.g., integrin α v β 3 /CD44-targeted nanoparticles [23]) imaging agents.
To prove this hypothesis, we have developed the synthesis and radiolabeling of a lactosamine-containing RGD derivative and investigated the in vitro stability of the labeled complex. To the best of our knowledge, this is the first report on the preparation of 68 Galabeled E[c(RGDfK)] 2 -based glycopeptide, which possesses a lactosamine moiety for the detection of α v β 3 integrin and galectin-3 expression in cancer and tumoral endothelial cells with PET imaging. The study of the in vivo kinetics and imaging properties of this novel radioligand is in progress.

Chemistry
For the design of the novel RGD-based radioligand, the following aspects were taken into consideration. The cRGDfK dimer peptide (9, E[c(RGDfK)] 2 ) was chosen as vector molecule for tumor angiogenesis imaging, since the cyclic RGD analogs have shown a higher affinity to α v β 3 integrin than linear peptides [25] and, in addition, the multimerization approach can also lead to enhanced tumor uptake [26]. We designed the application of 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA) Pharmaceutics 2021, 13, 796 7 of 13 as a chelating agent; hence, 1,4,7 triazacyclononane-1,4,7 triacetic acid (NOTA) derivatives are capable of complexation of the 68 Ga 3+ ion with a high stability [27]. A lactosamine derivative was chosen as a carbohydrate unit for the glycosylation of E[c(RGDfK)] 2 , because N-acetyllactosamine is a natural ligand of galectin-3 [11]. This receptor is also highly expressed in different cancer cells [13]; therefore, the incorporation of this carbohydrate into the RGD based radiotracer can result in dual-targeting radiopharmaceuticals with improved targeting efficacies [17].

Chemistry
For the design of the novel RGD-based radioligand, the following aspects were taken into consideration. The cRGDfK dimer peptide (9, E[c(RGDfK)]2) was chosen as vector molecule for tumor angiogenesis imaging, since the cyclic RGD analogs have shown a higher affinity to αvβ3 integrin than linear peptides [25] and, in addition, the multimerization approach can also lead to enhanced tumor uptake [26]. We designed the application of 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA) as a chelating agent; hence, 1,4,7 triazacyclononane-1,4,7 triacetic acid (NOTA) derivatives are capable of complexation of the 68 Ga 3+ ion with a high stability [27]. A lactosamine derivative was chosen as a carbohydrate unit for the glycosylation of E[c(RGDfK)]2, because Nacetyllactosamine is a natural ligand of galectin-3 [11]. This receptor is also highly expressed in different cancer cells [13]; therefore, the incorporation of this carbohydrate into the RGD based radiotracer can result in dual-targeting radiopharmaceuticals with improved targeting efficacies [17].
First, we developed a synthetic pathway for the preparation of the functionalized lactosamine derivative, which is suitable for conjugation to both E[c(RGDfK)]2 peptide (9) and NODAGA chelator. We designed the attachment of the lactosamine unit via azidealkyne cycloaddition to the E[c(RGDfK)]2 peptide; therefore, we chose 3-azidopropyl 2phtalimido-2-deoxy-β-D-glucopyranoside (1) [24] as the starting material. For the synthesis of the glycosyl acceptor 2, the primary hydroxyl group of starting material 1 was silylated with tert-butyldiphenylsilyl chloride. Disaccharide 4 was prepared in the following way: glycosyl acceptor 2 was selectively glycosylated with the known 2,3,4,6-tetra-Oacetyl-β-D-galactopyranosyl trichloroacetimidate (3) [28] using trimethylsilyl trifluoromethanesulfonate as a catalyst in dichloromethane at −50 °C (Scheme 1). Removal of the protecting groups from the disaccharide 4 was achieved in two steps. Firstly, the phtalimido and acetyl groups were removed with ethylene diamine in ethanol to yield compound 5. Then the tert-butyl diphenylsilyl protecting group was removed with trifluoroacetic acid, which gave compound 6 (Scheme 2). In the next step, compound 6 was coupled with the commercially available NODAGA-NHS chelator (7)  Removal of the protecting groups from the disaccharide 4 was achieved in two steps. Firstly, the phtalimido and acetyl groups were removed with ethylene diamine in ethanol to yield compound 5. Then the tert-butyl diphenylsilyl protecting group was removed with trifluoroacetic acid, which gave compound 6 (Scheme 2). In the next step, compound 6 was coupled with the commercially available NODAGA-NHS chelator (7)  To avoid metal contamination, the attachment of the commercially available E[c(RGDfK)]2 peptide to chelator-bearing lactosamine 8 was designed with a copper-free, strain-promoted click reaction, which is a biorthogonal reaction of a type of azide-alkyne Huisgen cycloaddition. This method is widely used for the conjugation of biomolecules and was developed by Bertozzi et al. [29], which is based on the reaction of a cyclooctyne  To avoid metal contamination, the attachment of the commercially available E[c(RGDfK)] 2 peptide to chelator-bearing lactosamine 8 was designed with a copper-free, strain-promoted click reaction, which is a biorthogonal reaction of a type of azide-alkyne Huisgen cycloaddition. This method is widely used for the conjugation of biomolecules and was developed by Bertozzi et al. [29], which is based on the reaction of a cyclooctyne (e.g., dibenzocyclooctyne (DBCO)) moiety with an azide derivative and driven by the release of strain energy of the cyclooctyne ring. There are some examples of successful applications of this catalyst-free click reaction in the development of radiopharmaceuticals. Sapati et al. [30] used this conjugation method for the synthesis of [ 64 Cu]DOTA-ADIBON3-Ala-PEG28-A20FMDV2 and found that the introduction of the chelator-strained alkyne (DBCO) system resulted in improved pharmacokinetics for their radiotracer. Jeon et al. [31] reported the radiolabeling of a DBCO-containing cRGD peptide and gold nanoparticle with 125 I-labeled azide using a copper-free click reaction. They suggested this radiolabeling method for both in vitro and in vivo labeling of DBCO-containing molecules. Thus, the E[c(RGDfK)] 2 peptide (9) was functionalized with DBCO moiety using commercially available DBCO-NHS (10) in dry dimethyl sulfoxide and in the presence of N,N-diisopropylethylamine (Scheme 3). To avoid metal contamination, the attachment of the commercially available E[c(RGDfK)]2 peptide to chelator-bearing lactosamine 8 was designed with a copper-free, strain-promoted click reaction, which is a biorthogonal reaction of a type of azide-alkyne Huisgen cycloaddition. This method is widely used for the conjugation of biomolecules and was developed by Bertozzi et al. [29], which is based on the reaction of a cyclooctyne (e.g., dibenzocyclooctyne (DBCO)) moiety with an azide derivative and driven by the release of strain energy of the cyclooctyne ring. There are some examples of successful applications of this catalyst-free click reaction in the development of radiopharmaceuticals. Sapati et al. [30] used this conjugation method for the synthesis of [ 64 Cu]DOTA-ADIBON3-Ala-PEG28-A20FMDV2 and found that the introduction of the chelatorstrained alkyne (DBCO) system resulted in improved pharmacokinetics for their radiotracer. Jeon et al. [31] reported the radiolabeling of a DBCO-containing cRGD peptide and gold nanoparticle with 125 I-labeled azide using a copper-free click reaction. They suggested this radiolabeling method for both in vitro and in vivo labeling of DBCO-containing molecules. Thus, the E[c(RGDfK)]2 peptide (9) was functionalized with DBCO moiety using commercially available DBCO-NHS (10) in dry dimethyl sulfoxide and in the presence of N,N-diisopropylethylamine (Scheme 3). The synthetized NODAGA-LacN-E[c(RGDfK)]2 (12) was used for radiochemical investigations as a precursor molecule.

Radiochemistry
For the synthesis of 68 Ga-NODAGA-LacN-E[c(RGDfK)]2, the 68 Ga isotope was tained from a 68 Ge/ 68 Ga-generator, which was eluted with 0.1 M ultra-purified (u.p.) H Ammonium acetate buffer (pH 4, 3 M) and 1 µg/µL aqueous stock solution of NODA LacN-E[c(RGDfK)]2 ligand were added to the 68 GaCl3 solution. The labeling process optimized regarding amount of peptide ligand and reaction temperature. The radiola ing efficiency was characterized by determining the radiochemical purity (RCP) usin dio-HPLC analysis of an aliquot from the crude reaction mixture. The radiolabelings w performed in triplicate for each ligand concentration and temperature (n = 3). NODAGA-LacN-E[c(RGDfK)]2 radiolabeling, the highest radiochemical purity (~9 was observed in the case of 32 µM ligand concentration for 15 min at 95 °C ( Figure 1)  The application of the lower ligand concentrations resulted in slower kinetics, but in the case of the lowest ligand concentration (10 µM) the RCY was still~85%. Details can be found in Table 1. The change in temperature from room temperature to 95 • C resulted in a significant difference in the radiochemical yield. Thus, 95 • C was found to be the optimal temperature for the synthesis of 68 Ga-NODAGA-LacN-E[c(RGDfK)] 2 using a 32-µM ligand concentration and 15 min as a reaction time. The radiolabeling of the precursor with 68 Ga isotope at 60 • C also resulted in an acceptable radiochemical yield (~92% RCP). However, no radiolabeling was observed at room temperature and the RCP was only~8.53% at 37 • C. Details can be found in Table 2. The following optimal labeling procedure was applied to further radiochemical experiments: 200 µL of NH 4 OAc buffer (3 M, pH 4) and 100 µL of aq. stock solution of NODAGA-LacN-E[c(RGDfK)] 2 (1 µg/µL, 42 nmol) were added to 1000 µL of 68 GaCl 3 eluate (approx. 60-80 MBq in 0.1 M HCl). The reaction was conducted at 95 • C for 15 min. The reaction mixture was purified with solid phase extraction using a reversed phase Oasis HLB SPE cartridge. The radiochemical purity of the labeled complex was examined with radio-HPLC and was found to be more than 99%. In addition, we developed a radio-TLC method for quality control of the 68 Ga-NODAGA-LacN-E[c(RGDfK)] 2 , applying iTLC paper and 0.5 M citrate buffer (pH 5.5) as an eluent, which gave the same RCP ( Figure 2). can be found in Table 2. The following optimal labeling procedure was applied to further radiochemical experiments: 200 µL of NH4OAc buffer (3 M, pH 4) and 100 µL of aq. stock solution of NODAGA-LacN-E[c(RGDfK)]2 (1 µg/µL, 42 nmol) were added to 1000 µL of 68 GaCl3 eluate (approx. 60-80 MBq in 0.1 M HCl). The reaction was conducted at 95 °C for 15 min. The reaction mixture was purified with solid phase extraction using a reversed phase Oasis HLB SPE cartridge. The radiochemical purity of the labeled complex was examined with radio-HPLC and was found to be more than 99%. In addition, we developed a radio-TLC method for quality control of the 68 Ga-NODAGA-LacN-E[c(RGDfK)]2, applying iTLC paper and 0.5 M citrate buffer (pH 5.5) as an eluent, which gave the same RCP ( Figure 2).  The octanol/water partition coefficient (logP) of the 68 Ga-NODAGA-LacN-E[c(RGDfK)] 2 radioligand was determined and found to be −2.58. This low logP value indicated the hydrophilic nature of the synthetized radiotracer. To assess the stability, the labeled compound was incubated with a solution of human serum, Na 2 EDTA (0.01 M) and oxalic acid (0.01 M) at room temperature, respectively. Aliquots were then taken at different time points (0, 60 and 120 min) and injected into the radio-HPLC column and the chromatograms were analyzed. Figure 3 shows the results of the in vitro stability test of the radiotracer against human serum. According to the radio-HPLC chromatograms the 68 Ga-NODAGA-LacN-E[c(RGDfK)] 2 radiotracer remained intact (>99%) for two hours. The radio-HPLC chromatograms of the Na 2 EDTA and oxalic acid challenge showed the same result and the labeled compound remained stable for 2 h. different time points (0, 60 and 120 min) and injected into the radio-HPLC column and the chromatograms were analyzed. Figure 3 shows the results of the in vitro stability test of the radiotracer against human serum. According to the radio-HPLC chromatograms the 68 Ga-NODAGA-LacN-E[c(RGDfK)]2 radiotracer remained intact (>99%) for two hours. The radio-HPLC chromatograms of the Na2EDTA and oxalic acid challenge showed the same result and the labeled compound remained stable for 2 h.

Conclusions
In summary, we have developed a synthetic method for the preparation of a 68 Ga-NODAGA-LacN-E[c(RGDfK)]2 radioligand containing lactosamine, which can be used for cancer diagnosis via PET imaging. NODAGA-NHS was attached to the amino group of a lactosamine derivative, which was functionalized with azido-propyl aglycone. Then, the obtained compound was conjugated with E[c(RGDfK)]2 peptide with copper-free, strain promoted click reaction. The radiolabeling of the synthetized NODAGA-LacN-E[c(RGDfK)]2 with a positron-emitting 68 Ga isotope was carried out. After purification, the octanol-water partition coefficient of the labeled compound was determined and its stability was examined against human serum, Na2EDTA (0.01 M) and oxalic acid (0.01 M). We suppose that the synthetized, novel 68 Ga-NODAGA-LacN-E[c(RGDfK)]2 radiopharmaceutical will be suitable for the detection of αvβ3 integrin and galectin-3 expression in tumor and tumor endothelial cells with PET imaging. In further studies, we will assess the in vivo kinetics and imaging properties of this radiotracer.

Conclusions
In summary, we have developed a synthetic method for the preparation of a 68 Ga-NODAGA-LacN-E[c(RGDfK)] 2 radioligand containing lactosamine, which can be used for cancer diagnosis via PET imaging. NODAGA-NHS was attached to the amino group of a lactosamine derivative, which was functionalized with azido-propyl aglycone. Then, the obtained compound was conjugated with E[c(RGDfK)] 2 peptide with copper-free, strain promoted click reaction. The radiolabeling of the synthetized NODAGA-LacN-E[c(RGDfK)] 2 with a positron-emitting 68 Ga isotope was carried out. After purification, the octanol-water partition coefficient of the labeled compound was determined and its stability was examined against human serum, Na 2 EDTA (0.01 M) and oxalic acid (0.01 M). We suppose that the synthetized, novel 68 Ga-NODAGA-LacN-E[c(RGDfK)] 2 radiopharmaceutical will be suitable for the detection of α v β 3 integrin and galectin-3 expression in tumor and tumor endothelial cells with PET imaging. In further studies, we will assess the in vivo kinetics and imaging properties of this radiotracer.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/pharmaceutics13060796/s1, Figure S1: 1 H NMR spectrum of compound 2, Figure S2: 13 C NMR spectrum of compound 2, Figure S3: 1 H NMR spectrum of compound 4, Figure S4: 13 C NMR spectrum of compound 4, Figure S5: 1 H NMR spectrum of compound 5, Figure S6: 13 C NMR spectrum of compound 5, Figure S7: 1 H NMR spectrum of compound 6, Figure S8: 13 C NMR spectrum of compound 6, Figure S9: Mass spectrum of compound 2, Figure S10: Mass spectrum of compound 4, Figure S11: Mass spectrum of compound 5, Figure S12: Mass spectrum of compound 6, Figure S13: Mass spectrum of compound 8, Figure S14: Mass spectrum of compound 11, Figure S15: Mass spectrum of compound 12. Conflicts of Interest: Adrienn Vágner, Dezső Szikra are employees of Scanomed Ltd. Scanomed provided access to mass spectrometer and did not provide financial support. The other authors declare no conflict of interest.