Synthesis of typical sulfonamide antibiotics with [14C]- and [13C]-labelling on phenyl ring for environmental studies

Background As a kind of widely used antibiotics, sulfonamide antibiotics (SAs) has become ubiquitous environmental contaminants that caused public concerns. The behavior of SAs in complex environmental system need to be elucidated, which is hampered by unavailability or high cost of isotope-labelled SAs. Results Using commercially available uniformly [l4C]- and [l3C]-labelled aniline as starting material, we synthesized [phenyl-ring-14C]- and [phenyl-ring-l3C]-labelled sulfamethoxazole (SMX), sulfamonomethoxine (SMM), and sulfadiazine (SDZ) using four-step (via condensation of labelled N-acetylsulfanilyl chloride and aminoheterocycles) or five-step (via condensation of labelled N-acetylsulfonamide and chloroheterocycles) reactions in good yields (5.0−22.5% and 28.1−54.1% for [14C]- and [13C]-labelled SAs, respectively) and high purities (> 98.0%). Conclusion The synthesis of [l4C]-labelled SAs could be completed on milligram-level, being feasible for preparation of labelled SAs with high specific radioactivity. This study provides efficient and maneuverable methods to obtain a variety of [14C]- or [13C]-labelled SAs for studies on their environmental behavior, such as fate, transformation, and bioaccumulation.

Targeting Metabolic Alterations in CLL Microenvironment; Inhibition of Glutamine Import Attenuates Venetoclax Resistance

Introduction: For chronic lymphocytic leukemia (CLL), especially in the lymph node (LN) setting where cells receive proliferative and pro-survival signals, in-depth studies of altered metabolism and its relationship with therapeutic responses are still lacking. Venetoclax, a BCL-2 inhibitor currently in wide clinical use for CLL, has shown high efficiency yet emerging resistance is a growing clinical problem. In cell line models, induced resistance to Venetoclax was accompanied by profound metabolic changes 1. This is in accordance with our earlier findings on metabolic and apoptotic changes that CLL cells undergo within the LN environment 2. In the current study, we performed RNA sequencing and applied fluxomics with 13C 6-glucose and 13C 5-glutamine to investigate in detail the metabolic routes in LN CLL. This led to studies to manipulate glutamine metabolism in a venetoclax resistance model. Methods: Peripheral blood (PB) samples from CLL patients were in vitro stimulated for 24 hrs by CD40 or B cell receptor (BCR), which are two potential key signals in LN. RNAseq analysis was compared with microarray data of paired PB/LN patient samples 3. For fluxomics, CLL cells were cultured for 2 hrs in medium containing either 5 mM 13C 6-glucose or 1 mM 13C 5-glutamine. Incorporation of 13C in metabolic intermediates was analyzed by LC-MS. For glutamine blockade, CLL cells were stimulated in presence of specific inhibitors of glutamine/glutamate metabolism or amino acid transporters. Cells were then treated with venetoclax, and viability was measured. Results: Gene expression profiles demonstrated that CLL cells obtained from LN tissue as well as after in vitro CD40 or BCR stimulation showed increased expression of gene sets involved in glycolysis, oxidative phosphorylation / citric acid cycle (OXPHOS/TCA) and amino acid metabolism as well as Myc activation. This confirmed that in vitro stimulation can be used to model the CLL LN setting. For unstimulated PB CLL cells, fluxomics data demonstrated low uptake of either glucose or glutamine, with 13C labelling close to zero for most metabolites. In contrast, both CD40 or BCR stimulation increased the uptake and utilization of glucose and glutamine. 13C labelling from glucose was detected in all glycolytic intermediates analyzed in both CD40- and BCR-stimulated CLL cells. Glucose was catalyzed to lactate and also partly converted to acetyl-CoA, which entered the TCA cycle. Additionally, labelling from glucose was also increased in several metabolites of the pentose phosphate pathway (PPP) suggesting it entered nucleotide synthetic routes. Compared to glucose, the contribution of glutamine was much higher in the TCA cycle in both BCR and CD40-stimulated cells. All intermediates of the TCA cycle were highly enriched with 13C from glutamine (Figure 1A). Combined, these data revealed that glutamine is the key metabolite to fuel the TCA cycle in LN CLL cells, and prompted us to study effects of glutamine blockade in conditions of Venetoclax resistance. It was found that venetoclax resistance induced by CD40 or BCR stimulation was clearly attenuated by glutamine uptake inhibition. CLL cells became re-sensitized to Venetoclax in both CD40- or BCR-stimulated samples, with an approximate 100-fold shift in IC50 (Figure 1B). Conclusions: Our study highlights the role of glutamine, in addition to glucose, in the metabolic reprogramming that CLL cells undergo in the LN (Figure 1C). These processes show potential for therapeutic targeting. Inhibition of glutamine import could contribute to dampen tumor microenvironment-induced Venetoclax resistance. References 1. Guièze, R. et al. Mitochondrial Reprogramming Underlies Resistance to BCL-2 Inhibition in Lymphoid Malignancies. Cancer Cell 36, (2019). 2. Chen, Z. et al. Effects of Ibrutinib on Metabolic Alterations and Micro-Environmental Signalling in Chronic Lymphocytic Leukaemia. Blood 136, (2020). 3. Herishanu, Y. et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-κB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood 117, 563-574 (2011). Figure 1 Figure 1. Disclosures Forconi: AbbVie: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria; Novartis: Honoraria; Gilead: Research Funding. van der Windt: Genmab: Current Employment. Kater: Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding; Abbvie: Honoraria, Other: Ad Board, Research Funding; Genmab, LAVA: Other: Ad Board, Steering Committee.

PEPc-mediated CO2 assimilation provides carbons to gluconeogenesis and the TCA cycle in both dark-exposed and illuminated guard cells

Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO2 assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO2 assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we used 13C-HCO3 labelling of tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO2 assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination increased the 13C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the 13C-labelling into this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the 13C-enrichment in pyruvate, succinate and glutamate. Only one 13C was incorporated into malate and citrate in either dark or light conditions. Our results collectively suggest that the PEPc-mediated CO2 assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells.

Metabolic turnover and dynamics of RNA modifications by 13C labelling

RNA methylation regulates various aspects of RNA metabolism, and dynamic modulation ofRNA modifications has emerged as a major effector in cellular transitions. Yet, we lack quantitativemethods to comprehensively assess methylation dynamics, its features and regulatory inputs, acrossRNA modifications. We developed 13C-dynamods, an isotopic labelling approach using [13C-methyl]-methionine, to quantify the turnover of base modifications in newly synthesized RNA. This turnover-basedapproach resolved the contributions of mRNA vs. ncRNA modifications within polyadenylatedRNA and uncovered the distinct kinetics of N6-methyladenosine (m6A) and 7-methylguanosine (m7G)in mRNA. Moreover, we obtained converging evidence indicating presence of N6,N6-dimethyladenosine (m62A) in non-ribosomal RNA, in particular tRNA and rapidly decaying RNAs.Finally, we showed that mRNA methylation dynamics is coordinated with ribonucleotide biosynthesisduring T-cell activation, and revealed post-transcriptional lability of m6A upon metabolic stress. Thus,13C-dynamods enables studies of origin, maintenance and regulation of RNA modifications understeady-state and non-stationary conditions.

N-Heterocyclic Carbene-Catalyzed Synthesis of Ynones via C–H Alkynylation of Aldehydes with Alkynyliodonium Salts

Alkynylation of aldehydes with alkynyl(aryl)iodonium salts catalyzed by an N-heterocyclic carbene (NHC) has been developed. The application of the organocatalyst and the hypervalent iodine group-transfer reagent allowed for metal-free C–H functionalization and C–C bond formation. The reaction proceeds under exceptionally mild conditions, at –40 ⁰C and in the presence of an amine base, providing access to an array of heteroaryl-propargyl ketones containing various substituents in good to excellent yields. The mechanism of the reaction was investigated by means of both experiments and density functional theory calculations. 13C-labelling and computations determined that the key alkynyl transfer step occurs via an unusual direct SN2 substitution of iodine-based leaving group by Breslow intermediate nucleophile at an acetylenic carbon. Moreover, kinetic studies revealed that the turnover-limiting step of the catalytic cycle is the generation of the Breslow intermediate, whereas the subsequent C–C bond-formation is a fast process. These results were fully reproduced and rationalized by the computed full free energy profile of the reaction, showing that the largest energy span is located between protonated NHC and the transition state for the carbene attack on the aldehyde substrate.<br>

N-Heterocyclic Carbene-Catalyzed Synthesis of Ynones via C–H Alkynylation of Aldehydes with Alkynyliodonium Salts

Alkynylation of aldehydes with alkynyl(aryl)iodonium salts catalyzed by an N-heterocyclic carbene (NHC) has been developed. The application of the organocatalyst and the hypervalent iodine group-transfer reagent allowed for metal-free C–H functionalization and C–C bond formation. The reaction proceeds under exceptionally mild conditions, at –40 ⁰C and in the presence of an amine base, providing access to an array of heteroaryl-propargyl ketones containing various substituents in good to excellent yields. The mechanism of the reaction was investigated by means of both experiments and density functional theory calculations. 13C-labelling and computations determined that the key alkynyl transfer step occurs via an unusual direct SN2 substitution of iodine-based leaving group by Breslow intermediate nucleophile at an acetylenic carbon. Moreover, kinetic studies revealed that the turnover-limiting step of the catalytic cycle is the generation of the Breslow intermediate, whereas the subsequent C–C bond-formation is a fast process. These results were fully reproduced and rationalized by the computed full free energy profile of the reaction, showing that the largest energy span is located between protonated NHC and the transition state for the carbene attack on the aldehyde substrate.<br>