Derivatization of thiol-containing compounds

Shimada, Kazutake; Mitamura, Kuniko

doi:10.1016/0378-4347(93)e0444-u

Cited by 175 publications

(86 citation statements)

References 67 publications

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“…To assess the role of -SH groups in the metabolism of NO-ASA, we pretreated the cytosolic fraction with 1 mM N-ethylmaleimide for 30 min, which reacts avidly with -SH (Shimada and Mitamura, 1994). When -SH groups were unavailable due to their reaction with maleimide, no HMP-GSH conjugate formed, but HMP did form (data not shown).…”

Section: Resultsmentioning

confidence: 99%

In Vitro Metabolism of Nitric Oxide-Donating Aspirin: The Effect of Positional Isomerism

Gao¹,

Kashfi²,

Rigas³

2004

J Pharmacol Exp Ther

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NO-donating aspirin (NO-ASA) is a potentially important chemopreventive agent against cancer. Since positional isomerism affects strongly its potency in inhibiting colon cancer cell growth, we studied the metabolic transformations of its ortho-, meta-, and para-isomers in rat liver and colon cytosolic, microsomal, and mitochondrial fractions as well as in intact HT-29 human colon cancer cells. NO-ASA and metabolites were determined by high-performance liquid chromatography and products identified by mass spectroscopy, as required. For all three isomers, the acetyl group on the ASA moiety was hydrolyzed rapidly. This was followed by hydrolysis of the ester bond linking the salicylate anion to the spacer. The ortho-and paraisomers produced salicylic acid and a putative intermediate consisting of the remainder of the molecule, which via a rapid step generated nitrate, (hydroxymethyl)phenol, and a conjugate of spacer with glutathione. The meta-isomer, in contrast, generated salicylic acid and (nitroxymethyl)phenol, the latter leading to (hydroxymethyl)phenol and the glutathione-spacer conjugate. This metabolic pathway takes place in its entirety only in the cytosolic fraction of the tissues tested and in intact human colon cancer cells, perhaps reflecting exposure to the cytosolic glutathione S-transferase, which catalyzes the formation of the spacer-glutathione conjugate. Thus, the three positional isomers of NO-ASA differ in their metabolism and these differences correlate with their differential effects on cancer cell growth, underscoring the importance of positional isomerism in modulating drug effects.NO-donating nonsteroidal anti-inflammatory drugs (NONSAIDs) are emerging as an important novel pharmacological class that has already entered the phase of clinical testing. NO-NSAIDs consist of a traditional NSAID to which the -ONO 2 group is covalently bound via a spacer (Fig. 1). Their three main features appear to be enhanced potency, efficacy, and greater safety compared with their NSAID counterparts (reviewed in Rigas et al., 2003;Rigas and Kashfi, 2004). A large body of basic work in recent years indicates that they display novel properties that set them apart from their parent traditional NSAIDs. In view of their envisioned clinical applications, it is becoming increasingly important to know their metabolic transformations by various tissues, especially those that may be targets of their pharmacological action.We have been interested in the use of NO-NSAIDs, in particularly NO-aspirin (NO-ASA), as a safe and effective chemopreventive agent against colon cancer (Williams et al., 2001;Kashfi et al., 2002). In vitro studies using cultured colon and other cell lines indicate that NO-ASA is the most potent among several NO-NSAIDs in inhibiting cell growth (preceding article), which is taken to portend its efficacy in intact organisms. Using an animal model of colon cancer, we and others have demonstrated that NO-NSAIDs, including NO-ASA, are effective in inhibiting aberrant crypt foci, which represent t...

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Section: Resultsmentioning

confidence: 99%

In Vitro Metabolism of Nitric Oxide-Donating Aspirin: The Effect of Positional Isomerism

Gao¹,

Kashfi²,

Rigas³

2004

J Pharmacol Exp Ther

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“…The SBD-F derivatization method has been reported to be superior to other precolumn dervitization methods such as o-phthaldialdehyde (OPA) (Mopper and Delmas 1984) or monobromobimane (Fahey and Newton 1987), in that SBD-F is highly specific to thiol groups, and there are no interferences from alcohols, phenols, or amino groups (Shimada and Mitamura 1994). Also, unreacted SBD-F is not fluorescent at the excitation wavelength of choice, thus eliminating any interferences attributable to the reagents (Toyooka and Imai 1983).…”

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confidence: 99%

Distribution of the thiols glutathione and 3‐mercaptopropionic acid in Connecticut lakes

Mylon

Benoit

2006

Limnology & Oceanography

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The spatiotemporal chemical characteristics of the water column in Linsley Pond, a freshwater lake in Connecticut, were studied from just before its stratification in April through the fall turnover in December. Two low-molecular-weight thiols, glutathione and 3-mercaptopropionic acid (MPA), were detected at nanomolar concentrations in the water column. GSH was detected (average concentrations were ca. 5 nmol L 21 in the particulate phase and ca. 3 nmol L 21 in the dissolved phase) in surface and near-surface waters only and covaried with chlorophyll a. Both particulate and dissolved MPA were measured in the oxic and anoxic regions. In the metalimnion, MPA concentrations were greater than in the oxic water layers above. At times, the MPA concentrations in the metalimnion were as high as those found in the anoxic hypolimnion. The MPA present at different depths in the Linsley Pond water column is most likely produced through dissimilar mechanisms. Throughout the water column, MPA may be a product of the metabolic degradation of sulfur-containing organic compounds; however, in the hypolimnetic waters, dissolved sulfide may play an important role in MPA formation through abiotic nucleophillic addition to unsaturated functionalities in dissolved organic matter. Parallel laboratory experiments were performed to assess the importance of MPA in copper speciation. The results confirmed that despite nanomolar levels in Linsley Pond, MPA does not play an important role in copper speciation. A survey of six additional lakes in Connecticut was made for detectable thiols. Only MPA was detected in four of these lakes, and its distribution was similar to that in Linsley Pond.

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“…Thiols preferentially react with maleimide groups by means of the thiolate anion. The reaction is fast and highly selective (at pH 7, the maleimide group is 1000 times more reactive towards a sulfhydryl group than towards an amine), but it is strongly influenced by pH and temperature [25,29]. The reaction progress was investigated for glutathione, a tripeptide containing one Cys residue, using flow injection analysis (FIA) with MS detection of the derivative formed.…”

Section: Resultsmentioning

confidence: 99%

Analysis of cysteine-containing proteins using precolumn derivatization with N-(2-ferroceneethyl)maleimide and liquid chromatography/electrochemistry/mass spectrometry

Seiwert

Kärst

2007

Anal Bioanal Chem

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N-(2-Ferroceneethyl)maleimide (FEM) is introduced as an electroactive derivatizing agent for thiol functionalities in proteins. Using appropriate reaction conditions, the derivatization is completed within five minutes and no unspecific labeling of free amino functions is observed. Liquid chromatography/electrochemistry/mass spectrometry was used to detect the reaction products. The reagent is a useful tool for determining the number of free thiol groups or the total number of free and disulfide-bound thiol groups in proteins. The electrochemical cell provides additional information, because the increase in mass spectrometric response upon electrochemical oxidation of the neutral ferrocene to the charged ferrocinium groups is monitored. The method was successfully applied to the analysis of native proteins and their tryptic digests.

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Derivatization of thiol-containing compounds

Cited by 175 publications

References 67 publications

In Vitro Metabolism of Nitric Oxide-Donating Aspirin: The Effect of Positional Isomerism

In Vitro Metabolism of Nitric Oxide-Donating Aspirin: The Effect of Positional Isomerism

Distribution of the thiols glutathione and 3‐mercaptopropionic acid in Connecticut lakes

Analysis of cysteine-containing proteins using precolumn derivatization with N-(2-ferroceneethyl)maleimide and liquid chromatography/electrochemistry/mass spectrometry

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