Ethyl glucuronide (ethyl beta-D-6-glucosiduronic acid), a minor ethanol metabolite in serum or urine, was determined by gas chromatography-mass spectrometry. Prior to this, ethyl glucuronide was synthesized by the reaction of acetobromo-glucosiduronic acid with ethanol. For the determination of ethyl glucuronide, serum samples were precipitated with acetone, and urine specimens were analyzed after evaporation to dryness. The residues were derivatized with acetic anhydride. Capillary gas chromatography was used to find a retention index value of 1920 for the triacetyl derivative. The mass spectrum of the acetylated ethyl glucuronide was recorded. The calibration is linear in the range investigated (0.1-150 mg/L), and the detection limit is 0.1 mg/L. In individual specimens containing between 0.1 and 4 g ethanol per liter serum, ethyl glucuronide could be detected at concentrations between 3 and 14 mg/L and in the corresponding urine specimens at concentrations between 3 and 130 mg/L.

Ethyl glucuronide: an unusual ethanol metabolite in humans. Synthesis, analytical data, and determination in serum and urine

New classes of physiologically responsive magnetic resonance (MR) contrast agents are being developed that are activated by enzymes, secondary messengers, pH, and temperature. To this end, we have prepared a new class of enzyme-activated MR contrast agents using a self-immolative mechanism and investigated the properties of these agents using novel in vitro assays. We have synthesized in nine steps a Gd(III) agent 1 that is activated by the oncologically significant beta-glucuronidase. 1 consists of Gd(III)DO3A (DO3A = 1,4,7-tricarboxymethylene-1,4,7,10-tetraazacyclododecane) bearing a pendant beta-glucuronic acid moiety connected by a self-immolative linker to the macrocycle. LC-MS analysis reveals that 1 is enzymatically processed as predicted by bovine liver beta-glucuronidase, generating 2-aminoethylGdDO3A, 2. Compound 2 was prepared independently in a four-step synthetic procedure. Complex 1 displays a decrease in relaxivity upon titration with bicarbonate anion. The relaxivity increases when 1 is converted to 2 in a buffer mimicking in vivo anion concentrations (Parker, D. In Crown Compounds: Towards Future Applications; Cooper, S. R., Ed.; VCH: New York, 1992; pp 51-67) by 17%, while the relaxivity decreases by 27% for the same experiment in human blood serum. Hydrolytic kinetics catalyzed by bovine liver beta-glucuronidase at interstitial pH = 7.4 fit the Michaelis-Menten model with k cat/Km = 74.9 +/- 10.9 M(-1) s(-1). Monitoring of bulk water proton T1 during incubation with enzyme shows an increase in T1 that mirrors results obtained through the relaxivity measurements of compounds 1 and 2.

A Gadolinium Chelate for Detection of β-Glucuronidase: A Self-Immolative Approach

Resveratrol glucuronides as the metabolites of resveratrol in humans: characterization, synthesis, and anti-HIV activity.

The synthesis of O-glucuronides

Glucuronide prodrugs of 9-aminocamptothecin were synthesized. Prodrug 4, in which 9-aminocamptothecin was connected to glucuronic acid by an aromatic spacer via a carbamate linkage, was stable in both aqueous solution and human plasma. Prodrug 4 and its potassium salt 12 were 20−80-fold less toxic than 9-aminocamptothecin to human tumor cell lines. The simultaneous addition of β-glucuronidase and 4 or 12 to tumor cells resulted in a cytotoxic effect equal to that of 9-aminocamptothecin alone. Prodrugs 4 and 12 were over 80 and 4000 times more soluble than 9-aminocamptothecin in aqueous solutions at pH 4.0, respectively. Compounds 4 and 12 may be useful for prodrug monotherapy of tumors that accumulate extracellular lysosomal β-glucuronidase as well as for antibody-directed enzyme prodrug therapy (ADEPT) of cancer.

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Design and synthesis of water-soluble glucuronide derivatives of camptothecin for cancer prodrug monotherapy and antibody-directed enzyme prodrug therapy (ADEPT).

G. N. Bollenback

Papers

The Synthesis of Aryl-D-glucopyranosiduronic Acids1