Convergent synthesis of 13N-labelled Peptidic structures using aqueous [13N]NH3

Background Nitrogen-13 has a 10-min half-life which places time constraints on the complexity of viable synthetic methods for its incorporation into PET imaging agents. In exploring ways to overcome this limitation, we have used the Ugi reaction to develop a rapid one-pot method for radiolabelling peptidic molecules using [13N]NH3 as a synthetic precursor. Methods Carrier-added [13N]NH3 (50 μL) was added to a solution of carboxylic acid, aldehyde, and isocyanide in 2,2,2-TFE (200 μL). The mixture was heated in a microwave synthesiser at 120 °C for 10 min. Reactions were analysed by radio-HPLC and radio-LCMS. Isolation of the target 13N–labelled peptidic Ugi compound was achieved via semi-preparative radio-HPLC as demonstrated for Ugi1. Results Radio-HPLC analysis of each reaction confirmed the formation of radioactive products co-eluting with their respective reference standards with radiochemical yields of the crude products ranging from 11% to 23%. Two cyclic γ-lactam structures were also achieved via intra-molecular reactions. Additional radioactive by-products observed in the radio-chromatogram were identified as 13N–labelled di-imines formed from the reaction of [13N]NH3 with two isocyanide molecules. The desired 13N–labelled Ugi product was isolated using semi-preparative HPLC. Conclusion We have developed a one-pot method that opens up new routes to radiolabel complex, peptidic molecules with 13N using aqueous [13N]NH3 as a synthetic precursor. Electronic supplementary material The online version of this article (10.1186/s41181-017-0035-7) contains supplementary material, which is available to authorized users.

Multi-component reactions are powerful tools in the efficient and rapid generation of diverse libraries of compounds, and are particularly useful for high-throughput screening. The four-component Ugi reaction combines a carboxylic acid, an amine (sometimes ammonia), an aldehyde or ketone, and an isocyanide to create α-aminoacyl amide derivatives -peptidomimetic structures with proven pharmaceutical application (Domling & Ugi, 2000). Notably, the one-pot synthesis of the local anaesthetic Xylocain and the subsequent development and marketing of at least 12 other anaesthetics is based on this scaffold (Hulme & Gore, 2003). The Ugi reaction is therefore an attractive method for the rapid radiolabelling of peptides with short-lived PET radionuclides, useful in diagnosis, drug discovery and as a research tool in understanding molecular mechanisms in vivo. Here we report the radiolabelling of α-aminoacyl amide derivatives with 13 N, via the four-component coupling (4-CC) Ugi synthesis, using aqueous [ 13 N]NH 3 as a precursor.

Reference standard synthesis
Based on methods developed by Thompson et al. (Thompson & Chen, 2009), reference standards 1-7 were successfully synthesised and isolated. LC/MS analysis of the crude product mixtures confirmed the presence of the desired 4-CC products. Full characterisation of the final isolated products can be found in the Additional file 1.

Radiochemistry
Radiosynthesis of 1-5 was achieved via the Ugi reaction by combining benzaldehyde, the respective carboxylic acid and isocyanide, and carrier-added aqueous [ 13 N]NH 3 (Fig. 1). Radiosynthesis of cyclic γ-lactams 6-7 was achieved using an intra-molecular Ugi reaction by combining the ketone and carboxylic acid in a single moleculelevulinic acid -with the respective isocyanide, and carrier-added [ 13 N]NH 3 (Fig. 1). All reaction mixtures were heated using a microwave synthesis unit. Synthesis of 1 was selected as the model reaction for optimisation (Table 1). Initially, experiments were performed in a range of solvents and heated at 100°C for 15 min. The optimum solvent was found to be TFE, affording radiochemical yields (RCY, based on radio-HPLC analysis of the crude product) of 13%, performing better than MeCN (9%), while DMF afforded no yield (Table 1, entries 1-3). Reaction time and temperature were varied, with little effect on  The established optimum conditions of 1 were applied to the radiosynthesis of a small library of compounds. 13 N-labelling of target structures 1-7 was confirmed by the co-elution of the non-radioactive standards and radio-LC/MS analysis. The radiochemical yield of these compounds ranged from 11 to 23% (Table 2). The molar activity of [ 13 N]NH 3 used in these experiments was 2.64 ± 0.12 GBq/μmol. In addition to the target Ugi compound and un-reacted [ 13 N]NH 3 , another unknown radiolabelled species was observed (Fig. 2). However, semi-preparative HPLC could be used to isolate the desired 13 N-labelled Ugi product. To demonstrate this, isolation of 1 was carried out, achieving 96% radiochemical purity (Fig. 3) and an activity yield between 4 and 6% (based on 24 min preparation time).
To identify the radioactive by-products, analogous reactions using stable isotope labelling with 14 N and 15 N were carried out, followed by LC/MS analysis to establish a molecular weight associated with the by-products. In each case, the molecular weight corresponded to the combination of one [ 14/15 N]NH 3 molecule and two isocyanide molecules, suggesting the formation of labelled di-imine structures (Fig. 4).

Discussion
Ammonia is rarely used as the amine component in the Ugi reaction, as it reportedly leads to low yields and extensive by-product formation. A common by-product is the six-component coupling (6-CC) product formed through participation of the solvent (usually methanol) (Ackermann et al., 2012;Kazmaier & Hebach, 2003). Thompson et al. synthesised a library of structures via the Ugi reaction using ammonia, and replacing methanol with the less nucleophilic solvent TFE suppressed formation of the 6-CC product in favour of the desired 4-CC product (Thompson & Chen, 2009). Therefore, in the present study TFE was used for all reactions. Using this strategy, reference standards 1-7 were successfully synthesised. LC/MS analysis of the crude product mixtures confirmed the presence of the desired 4-CC product, with no evidence for a 6-CC product.
The radiolabelling of structures 1-7 via the Ugi reaction using aqueous carrier-added [ 13 N]NH 3 as a precursor was successful, with RCYs ranging from 11 to 23%. Under the tested conditions, the reaction did not proceed in the absence of carrier-added ammonia and the RCY varied significantly depending on the amount of carrier ammonia added: reducing the amount of ammonia from 10 μL (2.9 equivalents) to near stoichiometric amounts (5 μL, 1.4 equivalents) reduced the RCY; increasing to 20 μL (6 equivalents), also reduced the RCY. Therefore, 10 μL was selected for all subsequent reactions. We note that under these specific high temperature, closed vessel microwave conditions, the amount of ammonia in the solution versus the gas phase (and thus unavailable for reaction) has not been quantified. The necessary addition of carrier ammonia to this reaction mixture will inherently result in a tracer with low molar activity, potentially preventing the targeting of low-abundance receptors. The detrimental effect on the RCY upon increasing the volume of aqueous [ 13 N]NH 3 suggests this reaction could be affected by the presence of water and may benefit from anhydrous conditions. The use of microwave technology is often beneficial when rapid chemistry is required, particularly when using such short-lived isotopes as nitrogen-13. A limited number of experiments using conventional heating methods showed the target product could be obtained, but in lower RCY than that obtained using microwave heating. This Table 2 Radiolabelling of a small library of 13 N-labelled α-aminoacyl amide derivatives and γlactams indicates that the use of microwave technology is indeed effective in this case (see Additional file 1).
Despite the presence of un-reacted [ 13 N]NH 3 and radioactive by-products in the crude reaction mixture, the desired 13 N-labelled Ugi product could be easily isolated using semi-preparative HPLC, as demonstrated for reaction 1. The UV chromatogram of the final isolated product showed two major products co-eluting with the radioactive product. These are likely to be the non-radioactive α-aminoacyl amide Ugi product, and a structurally similar α-acyloxy amide, the product of the three-component coupling Passerini reaction between the isocyanide, carboxylic acid and aldehyde (see Additional file 1). This is consistent with our observations of the presence of the 3-component coupling Passerini product in the LC/MS analysis of the crude reaction mixture during synthesis of the non-radioactive reference standards, and in the analogous 15 N reactions. The radioactive by-products observed in the radio-chromatograms of all reactions 1-7 were identified as 13 N-labelled di-imines in which one [ 13 N]NH 3 molecule reacts with the electrophilic carbon of two isocyanide molecules to form the 13 N-labelled di-imine (Fig. 4). Identification of these by-products further contributes to expanding the breadth of known radiochemistry available for radiosynthesis with [ 13 N]NH 3. Derivatisation of R 1 and R 2 substituents via the isocyanide and carboxylic acid moieties bearing targeting groups such as peptides, sugars or other functional groups, could enable rapid access to libraries of functionalised 13 N-labelled compounds. Furthermore, it may be possible to label molecules of specific biological interest using this method, such as 1,4-benzodiazepine-2,5diones which have shown promise as anticonvulsant agents, opiate receptor antagonists  (Table 1, entry 9). a Radioactivity (counts per second); target compound 1 at R t 11.58 min; unreacted [ 13 N]NH 3 at R t 1.82 min; unknown by-product at R t 8.55 min. b UV absorption (254 nm) of reaction 1 and as inhibitors of histone deacetylases which are linked to the pathogenesis of several cancers (Hulme et al., 1998;Loudni et al., 2007).

Conclusion
The novel methodology presented here demonstrates an efficient strategy for quickly obtaining libraries of diverse 13 N-labelled peptidomimetics. The Ugi reaction has been successfully adapted to radiolabel a library of α-aminoacyl amide derivatives and γ-lactams with 13 N from cyclotron-produced aqueous [ 13 N]NH 3 precursor in a onepot method. This is also the first report of radiosynthesis with 13 N using microwave heating technology. This work demonstrates that despite its short half-life, complex molecules can be rapidly labelled with 13 N and as such, 13 N should be regarded as a viable option for labelling peptides in the future.