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The assignment of structure by tandem mass spectrometry (MS/MS) relies on the interpretation of the fragmentation behavior of gas‐phase ions. Mass spectra were acquired for a series of heterocyclic mimetics of acidic amino acids and a related series of nitrile amino acids. All amino acids were readily protonated or deprotonated by electrospray ionization (ESI), and distinctive fragmentation processes were observed when the ions were subjected to collision‐induced dissociation (CID). The deprotonated heterocycles showed bond cleavages of the 3‐hydroxyfurazan ring with formation of oxoisocyanate and the complementary deprotonated nitrile amino acid. Further fragmentation of the deprotonated nitrile amino acids was greatly dependent on the length of the alkyl nitrile side chain. Competing losses of CO2 versus HCN occurred from α‐cyanoglycinate (shortest chain), whereas water was lost from 2‐amino‐5‐cyanopentanoate (longest chain). Interestingly, loss of acrylonitrile by a McLafferty‐type fragmentation process was detected for 2‐amino‐4‐cyanobutanoate, and several competing processes were observed for β‐cyanoalanate. In one process, cyanide ion was formed either by consecutive losses of ammonia, carbon dioxide, and acetylene or by a one‐step decarboxylative elimination. In another, complementary ions were obtained from β‐cyanoalanate by loss of acetonitrile or HN=CHCO2H. Fragmentation of the protonated 3‐hydroxyfurazan and nitrile amino acids resulted in the cumulative loss (H2O + CO), a loss that is commonly observed for protonated aliphatic α‐amino acids. Overall, the distinct fragmentation behavior of the multifunctional 3‐hydroxyfurazan amino acids correlated with the charged site, whereas fragmentations of the deprotonated nitrile amino acids showed cooperative interactions between the nitrile and the carboxylate groups. [ABSTRACT FROM AUTHOR]

Over the years, human dihydroorotate dehydrogenase (hDHODH), which is a key player in the de novo pyrimidine‐biosynthesis pathway, has been targeted in the treatment of several conditions, including autoimmune disorders and acute myelogenous leukaemia, as well as in host‐targeted antiviral therapy. A molecular exploration of its inhibitor‐binding behaviours yielded promising candidates for innovative drug design. A detailed description of the enzymatic pharmacophore drove the decoration of well‐established inhibitory scaffolds, thus gaining further in vitro and in vivo efficacy. In the present work, using X‐ray crystallography, an atypical rearrangement was identified in the binding pose of a potent inhibitor characterized by a polar pyridine‐based moiety (compound 18). The crystal structure shows that upon binding compound 18 the dynamics of a protein loop involved in a gating mechanism at the cofactor‐binding site is modulated by the presence of three water molecules, thus fine‐tuning the polarity/hydrophobicity of the binding pocket. These solvent molecules are engaged in the formation of a hydrogen‐bond mesh in which one of them establishes a direct contact with the pyridine moiety of compound 18, thus paving the way for a reappraisal of the inhibition of hDHODH. Using an integrated approach, the thermodynamics of such a modulation is described by means of isothermal titration calorimetry coupled with molecular modelling. These structural insights will guide future drug design to obtain a finer Kd/logD7.4 balance and identify membrane‐permeable molecules with a drug‐like profile in terms of water solubility. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (MTB) is the etiologic agent of tuberculosis (TB), an ancient disease which causes 1.5 million deaths worldwide. Dihydroorotate dehydrogenase (DHODH) is a key enzyme of the MTB de novo pyrimidine biosynthesis pathway, and it is essential for MTB growth in vitro, hence representing a promising drug target. We present: (i) the biochemical characterization of the full‐length MTB DHODH, including the analysis of the kinetic parameters, and (ii) the previously unreleased crystal structure of the protein that allowed us to rationally screen our in‐house chemical library and identify the first selective inhibitor of mycobacterial DHODH. The inhibitor has fluorescence properties, potentially instrumental to in cellulo imaging studies, and exhibits an IC50 value of 43 μm, paving the way to hit‐to‐lead process. [ABSTRACT FROM AUTHOR]

Acidic 4‐hydroxy‐1,2,3‐triazole is a proven bioisostere of acidic functions that has recently been used to replace the acidic moieties of biologically active leads. Straightforward chemical strategies for the synthesis of the three possible N‐alkylated 4‐hydroxy‐1,2,3‐triazole regioisomers have been designed and reported herein, by identifying the optimal conditions under which the alkylation of ethyl 4‐benzyloxy‐1,2,3‐triazolecarboxylate (compound 19) can be regiodirected to the triazole N(b) position and thus produce the only isomer that cannot be obtained via the cycloaddition reaction. Furthermore, an innovative platform for parallel synthesis, called Arachno and which has been patented by the authors' group, has been used to speed up the process, and an NMR study has been carried out to better understand the reactivity of compound 19 towards the N(b) position. A library of benzyloxy protected 4‐hydroxy‐1,2,3‐triazoles has been prepared using the two strategies: regiodirection for the N(b) and N(c) isomers and cycloaddition for the N(a) isomers; the processes are described herein. The three N‐alkylated regioisomer series have been characterized spectroscopically (NMR and MS). The subsequent catalytic hydrogenation of the 4‐benzyloxy protective group on the N‐alkylated‐4‐benzyloxy‐5‐ethoxycarbonyl‐1,2,3‐triazoles provided the corresponding substituted 4‐hydroxy‐1,2,3‐triazoles. [ABSTRACT FROM AUTHOR]

Rationale: The gas‐phase fragmentation chemistry of multifunctional cations is highly influenced by the site of protonation. Possible relationships between protonation site and fragmentation processes were studied using 4‐aminoalkyl‐3‐hydroxyfurazans. For these heterocyclic amines, the starting points for competing fragmentation pathways varied with protonation at multiple sites in two tautomers. Methods: Mass spectra were acquired using electrospray ionization (positive mode) coupled to triple quadrupole and ion trap mass spectrometers; precursor–product ion relationships were studied by collision‐induced dissociation. Quantum mechanical computations were performed at the MP2/6–311++G(2d,p)//ωB97X‐D/6–311+G(d) level of theory. Results: Prominent successive losses of NO and CO and competing losses of CH2=NH or NH3 were observed as fragmentation processes. The lowest barrier computed for the initial step in a fragmentation pathway was associated with the [M + H]+ ion protonated at N5 in the heterocyclic ring, whereas an alternative ring cleavage leading to complementary product ions was initiated by protonation of the ring at N2. Side‐chain protonation led to loss of NH3 without cleavage of the 3‐hydroxyfurazan ring. Conclusions: The product ions obtained by the competing fragmentation processes varied with the site of protonation. Interestingly, the most abundant product ions observed at low collision energies were formed by cleavage of protonated molecules possessing more internal energy than other isomers. [ABSTRACT FROM AUTHOR]

A series of 4‐substituted 3‐hydroxyfurazans were subjected to electrospray ionization tandem mass spectrometry. At low collision energy, oxyisocyanate ([O=C=N–O]−, m/z 58) was formed as the predominant product ion from each deprotonated 3‐hydroxyfurazan, indicating cleavage of the heterocyclic ring. The facile energetics of this characteristic fragmentation process was confirmed by density functional computations. [ABSTRACT FROM AUTHOR]