0000000000134006
AUTHOR
Michael J. Schueller
Dynamic Precision Phenotyping Reveals Mechanism of Crop Tolerance to Root Herbivory.
The western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) is a major pest of maize (Zea mays) that is well adapted to most crop management strategies. Breeding for tolerance is a promising alternative to combat WCR but is currently constrained by a lack of physiological understanding and phenotyping tools. We developed dynamic precision phenotyping approaches using 11C with positron emission tomography, root autoradiography, and radiometabolite flux analysis to understand maize tolerance to WCR. Our results reveal that WCR attack induces specific patterns of lateral root growth that are associated with a shift in auxin biosynthesis from indole-3-pyruvic acid to indole-3-aceton…
Synthesis and PET studies of [11C-cyano]letrozole (Femara®), an aromatase inhibitor drug
Abstract Introduction Aromatase, a member of the cytochrome P 450 family, converts androgens such as androstenedione and testosterone into estrone and estradiol, respectively. Letrozole (1-[bis-(4-cyanophenyl)methyl]-1 H -1,2,4-triazole; Femara) is a high-affinity aromatase inhibitor ( K i =11.5 nM) that has Food and Drug Administration approval for breast cancer treatment. Here we report the synthesis of carbon-11-labeled letrozole and its assessment as a radiotracer for brain aromatase in the baboon. Methods Letrozole and its precursor (4-[(4-bromophenyl)-1 H -1,2,4-triazol-1-ylmethyl]benzonitrile) were prepared in a two-step synthesis from 4-cyanobenzyl bromide and 4-bromobenzyl bromide,…
Use of gaseous 13NH3 administered to intact leaves of Nicotiana tabacum to study changes in nitrogen utilization during defence induction
Nitrogen-13 (t(1/2) 9.97 m), a radioactive isotope of nitrogen, offers unique opportunities to explore plant nitrogen utilization over short time periods. Here we describe a method for administering (13)N as gaseous (13)NH(3) to intact leaves of Nicotiana tabacum L. (cv Samsun), and measuring the labelled amino acids using radio high-performance liquid chromatography (HPLC) on tissue extract. We used this method to study the effects of defence induction on plant nitrogen utilization by applying treatments of methyl jasmonate (MeJA), a potent defence elicitor. MeJA caused a significant increase relative to controls in key [(13)N]amino acids, including serine, glycine and alanine by 4 h post-…
Synthesis of l -[4-11 C]Asparagine by Ring-Opening Nucleophilic 11 C-Cyanation Reaction of a Chiral Cyclic Sulfamidate Precursor
The development of a convenient and rapid method to synthesize radiolabeled, enantiomerically pure amino acids (AAs) as potential positron emission tomography (PET) imaging agents for mapping various biochemical transformations in living organisms remains a challenge. This is especially true for the synthesis of carbon-11-labeled AAs given the short half-life of carbon-11 (11 C, t1/2 =20.4 min). A facile synthetic pathway to prepare enantiomerically pure 11 C-labeled l-asparagine was developed using a partially protected serine as a starting material with a four-step transformation providing a chiral five-membered cyclic sulfamidate as the radiolabeling precursor. Its structure and absolute…
An efficient and practical synthesis of [2-11C]indole via superfast nucleophilic [11C]cyanation and RANEY® Nickel catalyzed reductive cyclization
A rapid method for the synthesis of carbon-11 radiolabeled indole was developed using a sub-nanomolar quantity of no-carrier-added [(11)C]cyanide as radio-precursor. Based upon a reported synthesis of 2-(2-nitrophenyl)acetonitrile (), a highly reactive substrate 2-nitrobenzyl bromide () was evaluated for nucleophilic [(11)C]cyanation. Additionally, related reaction conditions were explored with the goal of obtaining of highly reactive 2-(2-nitrophenyl)-[1-(11)C]acetonitrile () while inhibiting its rapid conversion to 2,3-bis(2-nitrophenyl)-[1-(11)C]propanenitrile (). Next, a RANEY® Nickel catalyzed reductive cyclization method was utilized for synthesizing the desired [2-(11)C]indole with h…
Inhibition of trehalose breakdown increases new carbon partitioning into cellulosic biomass in Nicotiana tabacum
Abstract Validamycin A was used to inhibit in vivo trehalase activity in tobacco enabling the study of subsequent changes in new C partitioning into cellulosic biomass and lignin precursors. After 12-h exposure to treatment, plants were pulse labeled using radioactive 11 CO 2 , and the partitioning of isotope was traced into [ 11 C]cellulose and [ 11 C]hemicellulose, as well as into [ 11 C]phenylalanine, the precursor for lignin. Over this time course of treatment, new carbon partitioning into hemicellulose and cellulose was increased, while new carbon partitioning into phenylalanine was decreased. This trend was accompanied by a decrease in phenylalanine ammonia-lyase activity. After 4 d o…
Optimizing [13N]N2 radiochemistry for nitrogen-fixation in root nodules of legumes
Here we explored the conditions for synthesizing [ 13 N]N 2 in a state that is suitable for the administration to plant root nodules enabling studies of nitrogen fixation. [ 13 N]N 2 was prepared batchwise, starting with [ 13 N]NO ― 3 from the 16 O(p,α) 13 N nuclear reaction on a liquid water target. [ 13 N]NO ― 3 was first reduced to [ 13 N]NH 3 using Devarda's alloy, and then the [ 13 N]NH 3 was oxidized to [ 13 N]N 2 by hypobromite using carrier-added NH 4 CI. The amounts of carrier NH 4 CI and hypobromite were varied to determine the effects these parameters had on the radiochemical yield, and on the radiotracer specific activity. As expected, increasing the amount of carrier NH 4 CI im…
Defense Priming in Nicotiana tabacum Accelerates and Amplifies ‘New’ C/N Fluxes in Key Amino Acid Biosynthetic Pathways
: In the struggle to survive herbivory by leaf-feeding insects, plants employ multiple strategies to defend themselves. One mechanism by which plants increase resistance is by intensifying their responsiveness in the production of certain defense agents to create a rapid response. Known as defense priming, this action can accelerate and amplify responses of metabolic pathways, providing plants with long-lasting resistance, especially when faced with waves of attack. In the work presented, short-lived radiotracers of carbon administered as 11CO2 and nitrogen administered as 13NH3 were applied in Nicotiana tabacum, to examine the temporal changes in &lsquo
Radiosynthesis of 3-indolyl[1-11C]acetic acid for phyto-PET-imaging: An improved production procedure and formulation method
Abstract An improved production procedure and formulation method for the carbon-11 radiolabeled phytohormone, 3-indolyl-[l- 11 C]acetic acid ([ 11 C]IAA), was developed by modifying selected original reaction parameters. This updated procedure both doubled the yield (from 25.9±6.7% ( n =12) to 61.0±0.3% ( n =10)) and increased the concentration (0.2–0.4 GBq/0.15–0.3 mL), enabling us to provide the radiotracer [ 11 C]IAA suitable for in vivo phyto-PET-imaging studies. The specific activity was improved by more than a factor of three (26.7±5.6 GBq/µmol to 82.5±36.1 GBq/µmol). The total synthesis time for both production and formulation was 81.8±3.0 min ( n =10). In addition, a streamlined sem…
One-pot, direct incorporation of [11C]CO2 into carbamates.
Why beat about the bush? An operationally simple and mild reaction based on the direct fixation of (11)CO(2) with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) has been developed for the synthesis of (11)C-labeled carbamates at 75 degrees C within 10 minutes in radiochemical yields above 70% (see scheme). This strategy should be immediately useful for the construction of new radiotracers for positron emission tomography and other applications.