Efficient Nitrogen-13 Radiochemistry catalyzed by a Highly Stable Immobilized Biocatalyst

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Introduction
Recent advances in Positron Emission Tomography (PET) have encouraged chemists to synthesize novel radiotracers to enable the non-invasive diagnosis of a larger variety of diseases and the investigation of their molecular basis 1 . Due to the short half-life (T 1/2 ) of the most commonly used positron emitters, i.e. Fluorine-18 ( 18 F, T 1/2 = 109.8 min), Carbon-11 ( 11 C, T1/2 = 20.4 min) and Nitrogen-13 ( 13 N, T 1/2 = 9.97 min), the radiosynthesis of PET tracers requires the development of efficient chemical schemes in order to synthesize and purify the radioactive species in a short period of time 2 . Indeed, radiolabelling methods must be fast and highly efficient to perform chemical reactions and downstream processing in an appropriate time scale, preventing thus excessive radioactivity loss while improving the specific activity of the resulting radiotracers.
Biocatalysis can offer attractive solutions in the area of radiochemistry because enzymes present an exquisite chemical selectivity and high turnover numbers. Enzymes enable fast chemical conversions, and yield highly pure products under extremely mild conditions. However, whereas enzymes have been naturally evolved as soluble and labile catalysts, stability and solubility issues often limit their industrial applications. These issues may be circumvented by enzyme immobilization, that turns enzymes into stable and heterogeneous biocatalysts 3 . In this context, appropriate selection of the solid carrier and immobilization chemistry is crucial to achieve highly active and stable heterogeneous enzymes that can be readily integrated to continuous processes.
There are a handful of examples where immobilized enzymes have been utilized for the synthesis of different 18 F-and 11 C-labelled radiotracers [3][4][5][6][7] . Nevertheless, the enzyme-assisted preparation of radiotracers labelled with shorter-lived positron emitters such as 13 N is more challenging and remains rather unexplored 8 . In fact, there are only a few examples where immobilized enzymes have been applied to the catalytic incorporation of 13 N into L-amino acids 9-11 . Usually, 13 N is generated via the 16 O(p,α) 13 N nuclear reaction by proton irradiation of pure water. Nitrogen-13 is formed as a mixture of 13  . Immobilization of this enzyme is rather challenging since restrictions in the conformational flexibility required for the catalysis may compromise the reducing capacity.
This structural complexity may explain the low recovered activity of immobilized eNRs reported elsewhere 19 .
In the current work, we present the unprecedented use of eNR as an efficient heterogeneous biocatalysts for the reduction

Immobilization of eNR on functionalized porous agarose beads
Eukaryotic Nitrate reductase (eNR) from Aspergillus niger was immobilized through different immobilization chemistries on a survey of agarose-based matrixes (see Table 1) with the same physical parameters (surface area, pore size and particle diameter) but having different functional groups on their surfaces. We screened several immobilization chemistries (reversible and irreversible) in order to find the optimal one in terms of both activity and stability of the resulting heterogeneous biocatalyst. The initial offered activity was always 1000mU per gram. a Ai=The activity immobilized on 1 gram of carrier after the immobilization process. This activity was calculated as the difference between the offered activity and the activity in the supernatant after incubation with the carrier for 1h. b Immobilization yield (ψ) = (immobilized activity/offered activity) x 100. c The expressed activity (Ae) is defined as the measured activity of the immobilized enzyme after washing. d Relative expressed activity (Ae) = (expressed activity/immobilized activity) x 100. Table 1 shows that all carriers were able to efficiently immobilize eNR (immobilization yields >75% in all cases), although low values of expressed activity were achieved after immobilization when compared to the free enzyme. Irreversible immobilization and cationic exchange on Ag-CB and Ag-DS led to a dramatic decrease in the expressed activity (26±4 and 58 mU/g, respectively). On the other hand, ionic immobilization of eNR on positively charged carriers yielded values of expressed activity in the range 155-210 mU/g, resulting in up to 23% of relative expressed activity. Noteworthy, although the chemistry that drove the immobilization on Ag-MANAE, Ag-DEAE and Ag-PEI was the same, Ag-MANAE offered lower immobilization yields (82±2%) than Ag-DEAE (99±1%) and Ag-PEI (98±1%). This fact may be explained because Ag-MANAE surface presents a lower density of positively charged groups than Ag-DEAE and Ag-PEI surfaces.
In order to explain the functional discrepancies obtained with the different immobilized preparations, we further studied the eNR surface to understand how the protein orientation on the carrier surface may affect its functionality. Unfortunately, X-ray structure of eNR from A. niger has not been solved yet, and hence its 3D-structural homology model containing the NADPH-domain, the heme-domain and the MOCO-domain was built ( Figure 1A) and the electrostatic surface potential was calculated in order to determine the spatial charge distribution across the enzyme surface ( Figure 1B and 1C). The analysis of this structural model reveals an acidic belt on the MOCOdomain located at the opposite face regarding to the active site. The model also shows that heme, FAD + , NAD + and molybdenum binding sites are surrounded by basic amino acids ( Figure 1C). These structural observations suggest that eNR immobilized on Ag-DS is oriented through its active site (basic region), hampering the accessibility of both NADPH and nitrate to the catalytic pocket with consequent decrease in catalytic efficiency. Nevertheless, the eNR orientation onto positively charged carriers seems to occur through one acidic region located far away from the binding pockets. As a consequence, active centres remain fairly accessible for the substrates, resulting in significantly higher relative expressed activity values for such immobilized preparations ( Figure 1C). Moreover, such orientation seems to negligibly affect dimmer stability because the dimerization domain is not involved in the protein-carrier interaction. In fact, when eNR immobilized on Ag-DEAE was incubated under high ionic strength conditions, enzymatic activity was quantitatively eluted to the supernatant, demonstrating the ionic character and the reversibility of this immobilization chemistry. surface. The surface electrostatic potential is depicted with a red-blue gradient that correspond to the acid-basic gradient. All the images were prepared by The values of expressed activity shown in Table 1 have been determined by using NADPH as cofactor; such values are relatively high when compared to previous results obtained using immobilization protocols based on porous hydrogels. For example, eNR from Aspergillus niger immobilized on a porous vinyl polymeric matrix expressed less than 2% of its expressed activity by using NADPH as cofactor 19 . The high structural complexity of eNR might be the main reason for the low expressed activity achieved in this previous work.
This enzyme requires enough structural flexibility for efficient electron transfer between both the cytochrome and the molybdenum domains.
Hence, immobilization chemistries that highly rigidify the 3D structure of eNR (covalent immobilization), may hinder the catalytic conformational changes or promote wrong protein orientations that limit either the electron transfer or the substrate accessibility, resulting in a low expressed activity of the immobilized preparation.

Effects of the temperature on both activity and stability of eNR immobilized on positively charged agarose beads
The thermal stabilities of immobilized and soluble eNR were tested at different temperatures. Figure 2A shows the inactivation courses of the three different immobilized preparations incubated at pH=7.5 and 25°C. The half-life time of eNR immobilized on Ag-DEAE was estimated 30-fold and 6-fold higher than eNR immobilized on Ag-MANAE and Ag-PEI, respectively, suggesting a dependence of the thermal stability of the immobilized enzymes with the chemical nature of the amine groups located at the surface of the carrier. The surface of Ag-DEAE containing tertiary amine groups seems to establish more favourable interactions with the acidic protein regions than the Ag-MANAE surface, which presents low density of primary amine groups, and the Ag-PEI surface coated by a polymeric bed with a high density of primary, secondary and tertiary amine groups. Ag-PEI has been suggested as an excellent carrier to stabilize multimeric enzymes, because the basic polymeric bed establishes 3D-protein-polymer interactions 20  Ag-DEAE surface formed by a 2D-monolayer of tertiary amine groups. Hence, our results suggest that the interaction between eNR and the positively charged carriers must be strong enough to guarantee the stabilizing immobilization effect and flexible enough to enable the conformation changes required for the catalysis.
The Ag-DEAE immobilized enzyme was much more stable than its soluble counterpart under a broad range of temperatures (30-55°C) ( Figure 2B). Surprisingly, when such immobilized biocatalyst was incubated for only 15 minutes in the range of 40-45°C and the enzyme activity was assayed at 25°C, a 2-fold activity enhancement (hyperactivation) was observed with respect to the non-thermally treated enzyme ( Figure 2B). This effect was not observed with the soluble enzyme, which decreased its initial activity after incubation at T>25°C due to thermal inactivation. Interestingly, both the soluble and immobilized enzymes showed similar activity/temperature profiles ( Figure   S1). Altogether, these results suggest that the aforementioned hyperactivation effect can only be attributed to the presence of the carrier and  The thermal-hyperactivation and thermal-stabilization effects observed for eNR immobilized on Ag-DEAE may be explained by some beneficial enzyme-carrier interactions that drive to some local structural re-organization in the enzyme, resulting in a more efficient electron transfer between the NADPH and the MOCO domains. Those interactions may stabilize a more beneficial conformation for eNR, which explains the 2-fold more active and 12-fold more stable enzyme. These carrier-and temperature-assisted conformational changes were further investigated by fluorescence studies that suggested a relationship between the improved functional properties and a structural reorganization (Supporting information, figure S3). Indeed, alterations in the fluorescence spectrum of the immobilized enzyme were observed with increased temperature, while no changes were observed for the soluble enzyme incubated under the same conditions. Hereby, the temperature seems to act as an external stimulus that induces an optimal fitting between the surfaces of the enzyme and the carrier, resulting in a suitable geometric congruence for the catalysis. Despite the nature of this thermally-induced more active conformation is not clear at present, it is worth mentioning that similar effects have been described for other thermostable soluble enzymes [21][22][23] . In any case, we put forth one of the few experimental demonstrations of thermal hyperactivation and stabilization assisted by the carrier surface, which is not only acting as an scaffold to stabilize eNR but also as an active surface that induces temperature-triggered positive conformational changes on the enzyme. However, before applying this biocatalyst to radiochemistry, we carried out its kinetic characterization. To this aim, Michaelis-Menten parameters were determined for both soluble and the immobilized eNRs towards both nitrate and NADPH ( Table 2).

Kinetic parameters and loading capacity of eNR immobilized on Ag-DEAE
Kinetic studies provided us interesting data that helped to understand the activity decrease resulting from the immobilization process. In a first glance, the immobilized preparation showed a 26-fold K m[NADPH] higher than the soluble enzyme. Contrarily, K m[NO3] was 4.5 times smaller for the immobilized eNR than for the soluble preparation. These results confirm that immobilization of eNR on Ag-DEAE negatively affected the enzyme binding towards NADPH, which explains the low expressed activity after the immobilization process. The high K m [NADPH] values for the immobilized preparation may due to two main reasons: i) Conformational changes induced by the carrier during the immobilization process that diminish the enzyme affinity towards NADPH, or ii) mass transfer issues that hamper NADPH diffusivity from the bulk solution to the active sites of the immobilized eNR molecules. In order to elucidate which reason contributes more to the low expressed activity, the effect of the enzyme loading on both immobilization yield and expressed activity after the immobilization was tested. Figure 3 shows that the immobilization was always quantitative regardless the enzyme load. However, the relative expressed activity presented a strong negative logarithmic correlation with the enzyme load. Under very low enzyme loads (37.5 µg eNR /g carrier ) the relative expressed activity was 85%, while the value decreased to approximately 20% at loads higher than 75 µg eNR /g carrier (Figure 3). and 25°C. Immobilization yield (full square) and relative expressed activity (open square) were determined for each enzyme load. Immobilization yield and expressed activity were calculated as was previously described in Table 1.
This experiment demonstrates that immobilization chemistry does not promote inactive enzyme conformations that intrinsically diminish its specific activity after the immobilization process. In such scenario, the expressed activity should be always low regardless of the enzyme load. Therefore, it is plausible to think that mass transfer limitations of substrate and cofactors are the main reason to explain the low expressed activity of the immobilized eNR. Contextualizing these results together with the high K m [NADPH] values for the immobilized preparation, we suggest that the cofactor undergoes severe mass transfer limitations to diffuse across the Ag-DEAE microstructure to reach the enzyme active sites. Under high enzyme loads, NADPH does not efficiently diffuse into the porous microstructure of the carrier to saturate all active sites immobilized on such carrier. On the contrary, under very low enzyme loads, the vast majority of the eNR molecules are saturated with the NADPH. These results are in good agreement with previous data 24 , which showed that eNR from A. niger covalently immobilized on non-porous particles expresses more than 90% of its specific activity after immobilization. Moreover, they strongly suggest that low specific activity of eNR immobilized on positively charged agarose beads is due to limited NADPH diffusion rather than inaccurate protein orientation, because positively charged porous carrier seem to immobilize eNR through an optimal orientation according to the protein surface analysis (Figure 1C).   We hypothesize that during the first reaction cycle some negatively charged NADPH molecules are ionically absorbed to the positively charged carrier surface. However, such molecules would present an association/dissociation equilibrium that would simultaneously provide one fraction of soluble NADPH available for the enzyme catalysis and a second fraction of NADPH trapped on the carrier surface that would be re-used in consecutive cycles. In this scenario, the effective concentration of NADPH in the second and successive cycles will be higher, explaining the higher yield observed in the second cycle compared to the first one. In order to demonstrate this ionic interaction between NADPH and Ag-DEAE, we equilibrated the eNR immobilized on Ag-DEAE with NADPH in solution before triggering the first reaction cycle. The equilibrated heterogeneous biocatalyst was able to reduce 96% of [ 13 N]NO 3 to [ 13 N]NO 2 from the first cycle (Figure 4). Moreover, we directly demonstrated that NADPH was bound to the carrier by analyzing the NADPH fluorescence inside the solid particles of the heterogeneous biocatalyst ( Figure S4). These experimental evidences confirm that a fraction of NADPH is ionically trapped into the carrier surface, increasing the internal concentration of NADPH that results in higher reduction yields. In fact, when the radiochemical reduction was carried out with the NADPH-equilibrated immobilized enzyme, the heterogeneous biocatalyst was able to catalyze the radiolabelled nitrate reduction for 5 cycles with more than 30% yield without exogenous addition of NADPH (Figure 4).
This experiment provides evidence that NADPH molecules are reversibly bound to the carrier surface providing an internal cofactor concentration that was sufficient for the immobilized eNR to catalyze the radiochemical reduction with the maximum yield during the first cycle, and proportionally lower yields with the consecutive cycles. The lower reduction yield along the cycles agrees with the fact that Cycle NADPH is oxidized to NADP + decreasing the pool of NADPH trapped into the carrier porous structure. Therefore, along the consecutive cycles, immobilized eNR suffers from a lower availability of the reduced NADPH, explaining the lower yields along the operational cycles.
Contrarily, when reduction was carried out by adding exogenous NADPH in each reaction cycle, the maximum yield was maintained during 7 cycles, demonstrating the excellent operational stability of the immobilized biocatalyst (Figure 4). As far as we know, this is the first evidence of a heterogeneous biocatalyst where the carrier plays an important role on supplying the corresponding cofactor to the enzyme.
Furthermore, this optimal heterogeneous biocatalyst, offers a clean final product without any significant protein contamination, even during the recycling process (Figure S5), confirming once again that ionic interactions between eNR and Ag-DEAE are quite strong in spite of their reversible nature.

Two step chemo-enzymatic solid-phase synthesis of S-[ 13 N]GSNO
To proof the concept that the enzymatically synthesized [ 13 N]NO 2 can be directly integrated into a radiochemical synthetic cascade, we coupled the optimal eNR immobilized on Ag-DEAE to the two-step chemo-enzymatic synthesis of S-[ 13 N]GSNO ( Figure S6). for the enzymatic reactions does not interfere with the next chemical step. Therefore, the radioactive nitrite produced by this heterogeneous biocatalyst is suitable to be used in radiochemical reactions.

Methods.
Structural modelling of nitrate reductase from Aspergillus niger. Structural homology models complexed with molybdenum, NADPH, FAD + and heme cofactors were built by using different structural templates and aided by the homology-modeling server from Expasy 28 . The modeling server selected the nitrate reductase from Pichia pastoris (2BIH) complexed with molybdopterin as template to model the molybdenum binding domain (56% identity). However, the server modeled both cytochrome and NADPH/FAD + domains by using NADHdependent cytochrome B5 reductase from rat (1IB0) complexed with both FAD + and NAD + as template (39% identity). Structures are ºC as previously described. In a different experiment, soluble and immobilized preparations were incubated at 40 ºC, and samples were withdrawn at different times and assayed at 25ºC to determine the residual activity. Reusability of eNR immobilized on Ag-DEAE was evaluated by performing consecutive catalytic cycles at 25°C using nitrate (both non-radioactive and radioactive) and NADPH as substrates.
After each cycle, the immobilized preparation was washed and the enzymatic activity towards non-radioactive nitrate was determined by spectrophotometry, while the final yield towards radioactive nitrate was determined by radio-HPLC (see section Radiochemistry).
Fluorescence Spectroscopy. Fluorescence measurements were carried out in a Varioskan Flash fluorescence spectrophotometer (Thermo Scientific), monitoring the intrinsic tryptophan fluorescence of immobilized A. niger eNR, using an excitation wavelength of 280 nm with excitation and emission bandwidths of 5 nm and recording fluorescence emission spectrum between 300 and 600 nm. All spectroscopic measurements were made in 10 mM sodium phosphate at pH 7.5 and 25ºC.

Radiochemistry.
Production of the radioactive precursor [ 13 N]NO 2 by enzyme catalysis: general procedure.
Nitrogen-13 was produced in an IBA Cyclone 18/9 cyclotron via the 16 O(p,α) 13 N nuclear reaction. The target system consisted of an aluminum insert (2 mL) covered with havar foil (thickness 25 µm, ∅ 29 mm) and with an aluminum vacuum foil (thickness 25 µm, ∅ 23 mm). The target (containing 1.75 mL of ultrapure water, Type I water, ISO 3696) was irradiated with 18MeV protons. The beam current was maintained at 20 µA (pressure in the range 5-10 bar into the target during bombardment) to reach the desired integrated currents (0. washed with distilled water (2 mL) and the QMA cartridge was dried with nitrogen gas for 15s. An acidic solution of the precursor glutathione (0.5 mL; 1 mM) was loaded into the cartridge and the nitrosation reaction was allowed to occur; the reaction mixture was finally eluted directly into a collection vial. The identification of [ 13 N]GSNO was performed by co-elution with reference standard using the same HPLC system described above; in this case, A Mediterranean SeaRP-18 column (5µm, 150 x 4.6mm, Teknokroma, Spain) was used as stationary phase and aqueous TFA solution/acetonitrile (95:5) was used as the mobile phase at a flow rate of 1 mL/min. Simultaneous UV (λ = 220 nm) and isotopic detection were used.

Conclusions
Biochemical reactions have been exploited in many different synthetic reactions by chemists; however radiochemists have not paid enough attention to biocatalysis, and there are only a handful of examples where enzymes have been utilized in radiosynthetic schemes. Such under-exploitation is even more dramatic in the case of immobilized enzymes, despite the recent advances in heterogeneous biocatalysis.
Immobilized enzymes are exquisitely selective, highly active and stable, and simplify the process work-up, potentially yielding pure radiotracers that can be directly used for biomedical purposes. Hence, they can be anticipated as ideal tools for the preparation of radiotracers labelled with short-lived radionuclides. In this work we have applied for first time one eukaryotic nitrate reductase immobilized on a solid carrier to a radiochemical process. This immobilized enzyme has been able to selectively reduce [ 13 N]NO 3 to [ 13 N]NO 2 aided by NADPH as redox cofactor. We have demonstrated that by controlling the immobilization chemistry and physico-chemical properties of the carrier, optimal heterogeneous biocatalysts with high potential in synthetic chemistry can be achieved. Noteworthy, this work is one of the few examples where an immobilized enzyme has been applied for a radiochemical process and the first report so far of a two-step chemoenzymatic route to synthesize a model radiotracers starting from a precursor ([ 13 N]NO 3 -) directly produced in the cyclotron.
We have faced the challenge to immobilize an enzyme whose catalytic mechanism depends on several cofactors and conformational changes recovering enough activity and increasing its stability to catalyze several reaction cycles. The success of this work will so far open the doors to new application of enzymes in radiochemistry and will potentiate the use of other chemo-enzymatic designs for the synthesis of novel radiotracers, even using short-lived isotopes. Successful enzyme immobilization will contribute to develop in flow radiochemical processes as well as to integrate radiochemical synthesis on-chip [31][32][33][34] .