Synthesis of isothiocyanate-derived mercapturic acids

Vermeulen, Martijn; Zwanenburg, B.; Chittenden, G. J. F.; Verhagen, Hans

doi:10.1016/s0223-5234(03)00141-7

Cited by 50 publications

(37 citation statements)

References 33 publications

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“…To test this possibility, we used calcium imaging to determine whether two model compounds, allyl isothiocyanate complexed to N-acetyl cysteine or N-Boc-diamino hexane (Fig. 1c), could activate TRPA1 in transfected HEK293 cells (9,10). Neither compound possessed agonist activity, and because a prospective isothiocyanate conjugate must contain similar structural features, a prodrug scenario seems unlikely.…”

Section: Resultsmentioning

confidence: 99%

TRP channel activation by reversible covalent modification

Hinman

Chuang²,

Bautista

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

797

950

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Allyl isothiocyanate, the pungent principle of wasabi and other mustard oils, produces pain by activating TRPA1, an excitatory ion channel on sensory nerve endings. Isothiocyanates are membranepermeable electrophiles that form adducts with thiols and primary amines, suggesting that covalent modification, rather than classical lock-and-key binding, accounts for their agonist properties. Indeed, we show that thiol reactive compounds of diverse structure activate TRPA1 in a manner that relies on covalent modification of cysteine residues within the cytoplasmic N terminus of the channel. These findings suggest an unusual paradigm whereby natural products activate a receptor through direct, reversible, and covalent protein modification.chemical modification ͉ irritants ͉ natural products ͉ pain P lants have evolved ingenious defensive strategies to ward off herbivorous predators. Such protective mechanisms often involve the production of chemical irritants that activate sensory nerve fibers of offending creatures to produce discomfort or pain (1, 2). For example, plants of the genus Brassica (mustard), Allium (onion), and Cinnamomum (cinnamon) produce pungent isothiocyanate, thiosulfinate, and ␣,␤-unsaturated aldehyde compounds, respectively, which elicit acute pain and neurogenic inflammation by activating TRPA1, a nonselective cation channel expressed by sensory neurons of the pain pathway (3-7). Each of these compounds is capable of forming covalent adducts with thiols, primary amines, and to a lesser extent, hydroxyl groups (8), raising questions as to how electrophiles with such indiscriminate reactivity function as reversible and specific ionchannel agonists.Here we provide evidence to support a model whereby a variety of structurally distinct environmental irritants activate TRPA1 through a mechanism primarily involving covalent modification of specific cysteine side chains located within the putative cytoplasmic N-terminal domain of the channel. Pharmacological analysis of various sulfhydryl reactive agents provides a biochemical rationale for the reversible properties of mustard oil and other naturally occurring TRPA1 agonists. Importantly, TRPA1 mutants that are insensitive to these electrophilic agonists retain their ability to function as ''receptor operated'' channels, indicating that distinct biochemical pathways can contribute to TRPA1 activation by environmental or endogenous stimuli. These studies provide a mechanistic framework to further elucidate biochemical and structural parameters underlying activation of this member of the TRP ion channel family. ResultsThe diverse chemical nature of TRPA1-activating irritants (Fig. 1a) suggests that reactivity, rather than structure per se, constitutes the critical determinant of TRPA1 agonist activity. To clarify this issue, we first asked whether benzyl thiocyanate (BTC) is able to activate TRPA1. BTC is isosteric with the TRPA1 agonist benzyl isothiocyanate (BITC), and, thus, whereas both compounds possess thiocyanate functional groups of similar size, th...

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Section: Resultsmentioning

confidence: 99%

TRP channel activation by reversible covalent modification

Hinman

Chuang²,

Bautista

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

797

950

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“…[ 53] Importantly, we found that elimination of the acidic workup step and distillation of the oil 7 from the residual solid reaction byproduct greatly reduced the net loss of product. Treatment of 7 with an excess of thiophosgene under basic conditions yielded the isothiocyanate 8.…”

Section: Synthesis Of Dl-sulforaphanementioning

confidence: 96%

“…This overall procedure was modified from previously reported work by Vermeulen et al, both to increase yields and in order to obtain erysolin (10). [53] Specifically, an excess of 1,4-dibromobutane was used to form the single-displacement S N 2 product 5 predominantly upon addition of potassium phthalimide. The disubstituted product was the only significant side-product and 5 could readily be isolated by flash chromatography.…”

Section: Synthesis Of Dl-sulforaphanementioning

confidence: 99%

Identification, Synthesis, and Enzymology of Non‐natural Glucosinolate Chemopreventive Candidates

et al. 2008

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Isothiocyanates (ITCs) are one of the many classes of breakdown products of glucosinolates found in crucifers such as broccoli and are thought to be partially responsible for the reduced risk of degenerative diseases associated with the consumption of vegetables. The production of ITCs such as L-sulforaphane is dependent on the hydrolytic bioactivities of myrosinase, localized both within vegetable tissues and within flora of the human GI tract, and is associated with important cancer chemopreventive activities. We hypothesized that novel isothiocyanates with enhanced chemopreventive properties relative to L-sulforaphane could be identified and that their glucosinolate precursors could be synthesized. From a library of 30 synthetic ITCs, we identified several with bioactivities equal or superior to those of L-sulforaphane. The corresponding non-natural glucosinolate precursors to these novel ITCs were constructed and found to be substrates for myrosinase. By utilizing a novel RP-HPLC assay to monitor myrosinase-dependent hydrolysis reactions, 2,2-diphenylethyl glucosinolate and (biphenyl-2-yl)methyl glucosinolate were shown to exhibit 26.5 and 2.8 %, respectively, of the relative activity of sinigrin and produced their corresponding ITCs in varying yields. These data support the notion that non-natural glucosinolates can act as prodrugs for novel ITCs, with a mechanism of action reliant on their hydrolytic cleavage by myrosinase. Such non-natural glucosinolates may serve as very economical chemopreventive agents for individuals at risk for cancers of and around the GI tract.

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“…4-methylthiobutyl isothiocyanate (purity > 99.9%) and 5-methylthiopentyl isothiocyanate (purity > 99.9%) were synthesized using an optimized four-step protocol based on other published literature [Schmid and Karrer, 1948;Holland et al, 1995;D'Souza et al, 2003;Vermeulen et al, 2003] The synthetic route started with the addition of the corresponding terminal dibromoalkane to phthalimide potassium salt, giving the respective N-(bromoalkyl)-phthalimide, which was treated with sodium methylmercaptide. Subsequently, the resulting N-[(methylthio)alkyl]-phthalimide was submitted to hydrazinolysis with hydrazine monohydrate, which yielded the corresponding (methylthio)alkylamine.…”

Section: Chemicalsmentioning

confidence: 99%

“…Subsequently, the resulting N-[(methylthio)alkyl]-phthalimide was submitted to hydrazinolysis with hydrazine monohydrate, which yielded the corresponding (methylthio)alkylamine. This was converted in the presence of thiophosgene to the target ITCs, which were spectroscopically identical with literature data [Holland et al, 1995;Vermeulen et al, 2003].The detailed protocol including reaction parameters, observations, and work-up procedures is reported elsewhere [Kotke, 2009].…”

Section: Chemicalsmentioning

confidence: 99%

Three structurally homologous isothiocyanates exert “Janus” characteristics in human HepG2 cells

Lamy

Crössmann

Saeed

et al. 2009

Environ and Mol Mutagen

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In this study, we used the single cell gel electrophoresis (SCGE) assay and the micronucleus (MN) test to investigate the DNA damaging effects and the antigenotoxic potencies of three structurally related ITCs in human HepG2 cells. The results show that all three ITCs possess the characteristic of a "Janus" compound, i.e., they exert both significant genotoxicity and antigenotoxicity, depending on the concentrations used in the test systems applied. Regression line analysis of the results derived by SCGE analysis showed genotoxic potency of the ITCs in the following order: 3-methylthiopropyl ITC (MTPITC) > 4-methylthiobutyl ITC (MTBITC) > 5-methylthiopentyl ITC (MTPeITC); however, this order in genotoxic potency was not confirmed by MN analysis. Additionally, the MN test showed significant mutagenicity of the test substances at higher concentrations when compared with the SCGE assay. Twenty-four hour-treatment of the cells with the ITCs, followed by a 1-hr recovery period, showed significant DNA repair in the SCGE assay at a concentration > or =10 microM MTPITC, > or =3 microM MTBITC, and > or =0.1 microM MTPeITC, respectively. In antigenotoxicity studies, the most effective concentration of MTPITC and MTPeITC toward B(a)P-induced DNA damage was 0.1 muM in both test systems. MTBITC suppressed MN formation in B(a)P-treated cells to the background level at a concentration of 1 muM. The ambivalent character of the ITCs under studymust be further clarified, especially in the possiblecontext of high dose therapeutic applications.

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Synthesis of isothiocyanate-derived mercapturic acids

Cited by 50 publications

References 33 publications

TRP channel activation by reversible covalent modification

TRP channel activation by reversible covalent modification

Identification, Synthesis, and Enzymology of Non‐natural Glucosinolate Chemopreventive Candidates

Three structurally homologous isothiocyanates exert “Janus” characteristics in human HepG2 cells

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