Introduction Since its development by Al Wolf and colleagues in the 1970s1, [11C]cyanide has been a useful synthon for a wide variety of reactions, most notably those producing [1-11C]-labeled amino acids2. However, despite its position as rote gas-phase product, the catalytic synthesis is difficult to optimize and often only perfunctorily dis-cussed in the radiochemical literature. Recently, [11C]CN– has been used in the synthesis of indole-3-[1-11C]acetic acid ([11C]IAA), the principal phytohormone responsible for a wide variety of growth and development functions in plants3. The University of Wisconsin has expertise in cyclotron production and radiochemistry of 11C and previous experience in the PET imaging of plants4,5. In this abstract, we present work on optimizing [11C]CN– production for the synthesis of [11C]IAA and the PET imaging of auxin transport in living plants. Material and Methods [11C]CH4 was produced by irradiating 270 psi of 90% N2, 10% H2 with 30 µA of 16.1 MeV protons from a GE PETtrace cyclotron. After irradiation, the [11C]CH4 was converted to [11C]CN– by passing through a quartz tube containing 3.0 g of Pt wire and powder between quartz wool frits inside a 800–1000 ˚C Carbolite tube furnace. The constituents and flow rate of the [11C]CH4 carrier gas were varied in an effort to optimize the oven\'s catalytic production of [11C]CN– from CH4 and NH3. The following conditions were investigated: i. Directly flowing irradiated target gas versus trapping, purging and releasing [11C]CH4 from a −178 ˚C HayeSep D column in He through the Pt furnace. ii. Varying the amount of anhydrous NH3 (99.995%) mixed with the [11C]CH4 carrier gas prior to the Pt furnace. Amounts varied from zero to 35 % of gas flow. iii. Varying the purity of the added NH3 gas with the addition of a hydride gas purifier (Entegris model 35KF), reducing O2 and H2O impurities to < 12 ppb. iv. Varying the flow rate of He gas carrying trapped, purged and released [11C]CH4. After flowing through the Pt furnace, the gas stream was bubbled through 300 µL of DMSO containing IAA precursor gramine (1 mg), then passed through a 60×5 cm column containing ascarite to absorb [11C]CO2, followed by a −178˚C Porapak Q column to trap [11C]CH4 and [11C]CO. After bubbling, the DMSO/gramine vial was heated to 140 ˚C to react the gramine with [11C]CN–, forming the intermediate indole-3-[1-11C]acetonitrile ([11C]IAN), which was subsequently purified by solid phase extraction (SPE). The reaction mixture was diluted into 20 mL water and loaded onto a Waters Sep-Pak light C18 cartridge, followed by rinsing with 5 mL of 0.1% HCl : acetonitrile (99 : 1) and 10 mL of the same mixture in ratio 95 : 5, and finally eluted with 0.5 mL of diethyl ether. The ether was subsequently evaporated under argon flow, followed by the hydrolysis of [11C]IAN to [11C]IAA with the addition of 300 µL 1 M NaOH and heating to 140 ˚C for 5 minutes. After hydrolysis, the solution was neutralized with 300 µL 1 M HCl and purified using preparative high-performance liquid chromatography (HPLC) using a Phenomenex Luna C18 (10μ, 250×10mm) column with a mobile phase acetonitrile : 0.1% formic acid in H2O (35 : 65) at flow rate of 3 mL/min. The [11C]IAA peak, eluting at 12 minutes, was collected and rotary evaporated to dryness, then again after the addition of 5 mL acetonitrile, followed by its reconstitution in 50 µL of water. Analytical HPLC was performed on the [11C]IAA before and after this evaporation procedure using a Phenomenex Kinetex C18 (2.6μ, 75× 4.6 mm) column with a linear gradient elution over 20 minutes of 10 : 90–30 : 70 (acetonitrile : 0.1% formic acid) at a 1 mL/min flow rate, eluting at 7.6 minutes. The transport of [11C]IAA was monitored following administration through the severed petiole of rapid cycling Brassica oleracea (rcBo) using a Siemens microPET P4 scanner. Transport was compared following administration to the first true leaf versus the final fully formed leaf in plants with and without exposure to the polar auxin transport inhibitor naphthylphthalamic acid (NPA). Results and Conclusion Optimization of the [11C]CN– gas phase chemistry was performed using two key metrics for measuring conversion yield. First is the fraction of total produced radioactivity that trapped in the DMSO/gramine solution (denoted %DMSO), and second, the fraction of DMSO/gramine-trapped activity that was able to react with gramine to form [11C]IAN (denoted %CN–). Under certain conditions, the former of these metrics experienced significant losses due to unconverted [11C]CH4 or through combustion, forming [11C]CO2 or [11C]CO. The latter metric experienced losses due to production of incomplete oxidation products of the CH4-NH3 reaction, such as methylamine. Total [11C]CH4 to [11C]CN– con-version yields is reported by the product of the two metrics. It was initially hypothesized that the irradiation of a 90% N2, 10% H2 target gas would produce sufficient in-target-hot-atom-produced NH3 to convert [11C]CH4 to [11C]CN– in the Pt furnace. However, conversion yields were found to be low and highly variable, with 13 ± 8 % trapping in DMSO/gramine, 9 ± 9 % of which reacted as CN– (n = 15). While in disagreement with previous reports1, this is likely as a result the batch irradiation conditions resulting ammonia losses in the target chamber and along the tubing walls. Yields and reproducibility were improved when combining the target gas with a stream of anhydrous NH3 gas flow with conversion yields reported in TABLE 1. However, these yields remained undesirably low, potentially as a result of the 10% H2 carrier gas having an adverse effect on the oxidative conversion of [11C]CH4 to [11C]CN–. To remedy this, the irradiated target gas was trapped, purged, released in He and combined with NH3 gas before flowing through the Pt furnace. Initial experiments using 99.995% anhydrous NH3 gas resulted in very poor (< 0.1%) [11C]CN– yields as a result of nearly quantitative combustion forming [11C]CO2. Installation of a hydride gas purifier to reduce O2 and H2O impurities in NH3 improved yields for CH4 in He, but did not significantly affect those from [11C]CH4 in N2/H2 target gas. In disagreement with previous reports2, conversion yields were found to be highly sensitive to overall carrier gas flow rate, with lower flow rates giving the best yields, as shown in TABLE 1. Optimization experiments are continuing. The total decay-corrected yield for the 1 hour synthesis of [11C]IAA in 50 µL of water is 2.3 ± 0.7 %, based on the total produced [11C]CH4 with a specific activity ranging from 1–100 GBq/µmol. The principal radiochemical impurity was determined to be indole-3-carboxylic acid. The SPE procedure isolating the [11C]IAN intermediate product was optimized to minimize this impurity in the final sample. After a rapid distribution of the administered [11C]IAA through the cut petiole and throughout the rcBO plant, upward vascular transport of auxin and downward polar auxin transport was visualized through time-activity curves (TACs) of regions of interest along the shoot. Comparison of these TACS with and without exposure to NPA yields insight into the fundamental physiological process of polar auxin transport in plants. In conclusion, the Pt-catalyzed oxidative conversion of [11C]CH4 and NH3 to [11C]CN– is a challenging process to optimize and highly sensitive to carrier gas composition and flow rate. Optimization for our experimental conditions yielded several results which disagreed with previous reports. [11C]IAA produced using [11C]CN– is well suited for PET imaging of polar auxin transport in living plants.

Introduction Since its development by Al Wolf and colleagues in the 1970s1, [11C]cyanide has been a useful synthon for a wide variety of reactions, most notably those producing [1-11C]-labeled amino acids2. However, despite its position as rote gas-phase product, the catalytic synthesis is difficult to optimize and often only perfunctorily dis-cussed in the radiochemical literature. Recently, [11C]CN– has been used in the synthesis of indole-3-[1-11C]acetic acid ([11C]IAA), the principal phytohormone responsible for a wide variety of growth and development functions in plants3. The University of Wisconsin has expertise in cyclotron production and radiochemistry of 11C and previous experience in the PET imaging of plants4,5. In this abstract, we present work on optimizing [11C]CN– production for the synthesis of [11C]IAA and the PET imaging of auxin transport in living plants. Material and Methods [11C]CH4 was produced by irradiating 270 psi of 90% N2, 10% H2 with 30 µA of 16.1 MeV protons from a GE PETtrace cyclotron. After irradiation, the [11C]CH4 was converted to [11C]CN– by passing through a quartz tube containing 3.0 g of Pt wire and powder between quartz wool frits inside a 800–1000 ˚C Carbolite tube furnace. The constituents and flow rate of the [11C]CH4 carrier gas were varied in an effort to optimize the oven\'s catalytic production of [11C]CN– from CH4 and NH3. The following conditions were investigated: i. Directly flowing irradiated target gas versus trapping, purging and releasing [11C]CH4 from a −178 ˚C HayeSep D column in He through the Pt furnace. ii. Varying the amount of anhydrous NH3 (99.995%) mixed with the [11C]CH4 carrier gas prior to the Pt furnace. Amounts varied from zero to 35 % of gas flow. iii. Varying the purity of the added NH3 gas with the addition of a hydride gas purifier (Entegris model 35KF), reducing O2 and H2O impurities to < 12 ppb. iv. Varying the flow rate of He gas carrying trapped, purged and released [11C]CH4. After flowing through the Pt furnace, the gas stream was bubbled through 300 µL of DMSO containing IAA precursor gramine (1 mg), then passed through a 60×5 cm column containing ascarite to absorb [11C]CO2, followed by a −178˚C Porapak Q column to trap [11C]CH4 and [11C]CO. After bubbling, the DMSO/gramine vial was heated to 140 ˚C to react the gramine with [11C]CN–, forming the intermediate indole-3-[1-11C]acetonitrile ([11C]IAN), which was subsequently purified by solid phase extraction (SPE). The reaction mixture was diluted into 20 mL water and loaded onto a Waters Sep-Pak light C18 cartridge, followed by rinsing with 5 mL of 0.1% HCl : acetonitrile (99 : 1) and 10 mL of the same mixture in ratio 95 : 5, and finally eluted with 0.5 mL of diethyl ether. The ether was subsequently evaporated under argon flow, followed by the hydrolysis of [11C]IAN to [11C]IAA with the addition of 300 µL 1 M NaOH and heating to 140 ˚C for 5 minutes. After hydrolysis, the solution was neutralized with 300 µL 1 M HCl and purified using preparative high-performance liquid chromatography (HPLC) using a Phenomenex Luna C18 (10μ, 250×10mm) column with a mobile phase acetonitrile : 0.1% formic acid in H2O (35 : 65) at flow rate of 3 mL/min. The [11C]IAA peak, eluting at 12 minutes, was collected and rotary evaporated to dryness, then again after the addition of 5 mL acetonitrile, followed by its reconstitution in 50 µL of water. Analytical HPLC was performed on the [11C]IAA before and after this evaporation procedure using a Phenomenex Kinetex C18 (2.6μ, 75× 4.6 mm) column with a linear gradient elution over 20 minutes of 10 : 90–30 : 70 (acetonitrile : 0.1% formic acid) at a 1 mL/min flow rate, eluting at 7.6 minutes. The transport of [11C]IAA was monitored following administration through the severed petiole of rapid cycling Brassica oleracea (rcBo) using a Siemens microPET P4 scanner. Transport was compared following administration to the first true leaf versus the final fully formed leaf in plants with and without exposure to the polar auxin transport inhibitor naphthylphthalamic acid (NPA). Results and Conclusion Optimization of the [11C]CN– gas phase chemistry was performed using two key metrics for measuring conversion yield. First is the fraction of total produced radioactivity that trapped in the DMSO/gramine solution (denoted %DMSO), and second, the fraction of DMSO/gramine-trapped activity that was able to react with gramine to form [11C]IAN (denoted %CN–). Under certain conditions, the former of these metrics experienced significant losses due to unconverted [11C]CH4 or through combustion, forming [11C]CO2 or [11C]CO. The latter metric experienced losses due to production of incomplete oxidation products of the CH4-NH3 reaction, such as methylamine. Total [11C]CH4 to [11C]CN– con-version yields is reported by the product of the two metrics. It was initially hypothesized that the irradiation of a 90% N2, 10% H2 target gas would produce sufficient in-target-hot-atom-produced NH3 to convert [11C]CH4 to [11C]CN– in the Pt furnace. However, conversion yields were found to be low and highly variable, with 13 ± 8 % trapping in DMSO/gramine, 9 ± 9 % of which reacted as CN– (n = 15). While in disagreement with previous reports1, this is likely as a result the batch irradiation conditions resulting ammonia losses in the target chamber and along the tubing walls. Yields and reproducibility were improved when combining the target gas with a stream of anhydrous NH3 gas flow with conversion yields reported in TABLE 1. However, these yields remained undesirably low, potentially as a result of the 10% H2 carrier gas having an adverse effect on the oxidative conversion of [11C]CH4 to [11C]CN–. To remedy this, the irradiated target gas was trapped, purged, released in He and combined with NH3 gas before flowing through the Pt furnace. Initial experiments using 99.995% anhydrous NH3 gas resulted in very poor (< 0.1%) [11C]CN– yields as a result of nearly quantitative combustion forming [11C]CO2. Installation of a hydride gas purifier to reduce O2 and H2O impurities in NH3 improved yields for CH4 in He, but did not significantly affect those from [11C]CH4 in N2/H2 target gas. In disagreement with previous reports2, conversion yields were found to be highly sensitive to overall carrier gas flow rate, with lower flow rates giving the best yields, as shown in TABLE 1. Optimization experiments are continuing. The total decay-corrected yield for the 1 hour synthesis of [11C]IAA in 50 µL of water is 2.3 ± 0.7 %, based on the total produced [11C]CH4 with a specific activity ranging from 1–100 GBq/µmol. The principal radiochemical impurity was determined to be indole-3-carboxylic acid. The SPE procedure isolating the [11C]IAN intermediate product was optimized to minimize this impurity in the final sample. After a rapid distribution of the administered [11C]IAA through the cut petiole and throughout the rcBO plant, upward vascular transport of auxin and downward polar auxin transport was visualized through time-activity curves (TACs) of regions of interest along the shoot. Comparison of these TACS with and without exposure to NPA yields insight into the fundamental physiological process of polar auxin transport in plants. In conclusion, the Pt-catalyzed oxidative conversion of [11C]CH4 and NH3 to [11C]CN– is a challenging process to optimize and highly sensitive to carrier gas composition and flow rate. Optimization for our experimental conditions yielded several results which disagreed with previous reports. [11C]IAA produced using [11C]CN– is well suited for PET imaging of polar auxin transport in living plants.