Elucidation of enzyme mechanisms using fluorinated substrate analogues

Pongdee, Rongson; Liu, Hung Wen

doi:10.1016/j.bioorg.2004.06.012

Cited by 51 publications

(33 citation statements)

References 120 publications

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“…A survey of the recent literature data highlights 1) the need for developing new efficient and stereoselective fluorination methodologies, [31] and 2) the very broad range of applications exploiting fluorinated molecules, going from drug design [29,32] to supramolecular chemistry. Fluorinated carbohydrates themselves have found many applications from mechanistic enzymology [7,10,33] to positron emission tomography (PET). [34] The work detailed in this study required both the development of a b-stereoselective fluorophosphorylation methodology and the synthesis of a challenging [35] and its co-crystallization with the target bacterial enzyme will allow us to rationally design a new generation of inhibitors of the LPS biosynthetic pathway.…”

Section: Resultsmentioning

confidence: 99%

Stereoselective Glycal Fluorophosphorylation: Synthesis of ADP‐2‐fluoroheptose, an Inhibitor of the LPS Biosynthesis

Dohi

Périon

Durka

et al. 2008

Chemistry A European J

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Heptosides are found in important bacterial glycolipids such as lipopolysaccharide (LPS), the biosynthesis of which is targeted for the development of novel antibacterial agents. This work describes the synthesis of a fluorinated analogue of ADP‐L‐glycero‐β‐D‐manno‐heptopyranose, the donor substrate of the heptosyl transferase WaaC, which catalyzes the incorporation of this carbohydrate into LPS. Synthetically, the key step for the preparation of ADP‐2F‐heptose is the simultaneous and stereoselective installation of both the fluorine atom at C‐2 and the phosphoryl group at C‐1 through a selectfluor‐mediated (selectfluor=1‐chloromethyl‐4‐fluorodiazoniabicyclo[2.2.2]octane bis(triflate)) electrophilic addition/nucleophilic substitution involving a heptosylglycal. Therefore, we detail in this article 1) the stereoselective preparation of the key intermediates heptosylglycals, 2) the development of a new fluorophosphorylation procedure allowing an excellent β‐gluco stereoselectivity with “all‐equatorial” glycals, 3) the synthesis of the target ADP‐2F‐heptose, and 4) some comments on the contacts observed between the fluorine atom of the final molecule and the protein in the crystallographic structure of heptosyltransferase WaaC.

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Section: Resultsmentioning

confidence: 99%

Stereoselective Glycal Fluorophosphorylation: Synthesis of ADP‐2‐fluoroheptose, an Inhibitor of the LPS Biosynthesis

Dohi

Périon

Durka

et al. 2008

Chemistry A European J

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“…[5,7,18] The use of fluorinated substrates as mechanistic probes and inhibitors has proven to be a powerful method for the study of enzymatic transformations. [26] In pharmaceutical applications, the CÀF bond is usually considered an effective bioisosteric group of either the CÀH or CÀOH bond. The resemblance between fluorine and oxygen in terms of electronegativity and van der Waals radii (4.0 and 1.47 vs. 3.5 and 1.57 ) makes fluorine a possible hydroxyl group mimic.…”

Section: Discussionmentioning

confidence: 99%

Probing the Hydrophobic Pocket of the Active Site in the Particulate Methane Monooxygenase (pMMO) from Methylococcus capsulatus (Bath) by Variable Stereoselective Alkane Hydroxylation and Olefin Epoxidation

Wang

et al. 2008

ChemBioChem

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pMMO from M. capsulatus (Bath) oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides. According to experiments performed with cryptically chiral ethane and D,L-[2-(2)H(1),3-(2)H(1)]butane, the reactions proceed through the concerted O-atom insertion mechanism. However, when propene and but-1-ene are used as epoxidation substrates, the enantiomeric excesses (ees) of the enzymatic products are only 18 and 37 %, respectively. This relatively poor stereoselectivity in the enzymatic epoxidation presumably reflects low stereochemical differentiation between the re and si faces in the hydrophobic pocket of the active site. Further insights into the reaction mechanism are now provided by studies on trans-but-2-ene, which reveal only the D,L-2,3-dimethyloxirane products, and on cis-but-2-ene, which yield only the meso product. These observations indicate that the enzymatic epoxidation indeed proceeds through electrophilic syn addition. To achieve better facial selectivity, we have also used 3,3,3-trifluoroprop-1-ene as the substrate. The products obtained are 90 % (2S)-oxirane. When 1,1,1-trifluoropropane is the substrate, the hydroxylation at the 2-carbon exhibits an inverse chiral selectivity relative to that seen with normal butane, if we consider the size of the CF(3) group in the fluorinated propane to be comparable to one of the ethyl groups in butane. These experiments are beginning to delineate the factors that influence the orientations of various substrates in the hydrophobic cavity of the active site in the enzyme.

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“…In an approach similar to that used in the synthesis of the GalNAc-threonine conjugate 7, [13] the building block 10 was transformed into the 6-deoxy-6-fluoro-galactal 18 by zinc-mediated reductive elimination of the 1-bromo and 2-acetoxy groups (89 %). Subsequent azidonitration [25] and installation of the anomeric bromide furnished the azido bromide 19 (31 %, two steps), which was used as the galactosyl donor in the Ag 2 CO 3 / AgClO 4 -promoted coupling to Fmoc-Thr-OtBu (9). [26] The glycosylation proceeded stereoselectively at 0 8C in a mixture of toluene and CH 2 Cl 2 as the solvents and led to the formation of the desired a-configured conjugate 20 in 45 % yield.…”

Section: Resultsmentioning

confidence: 99%

“…[8] Moreover, the electron-withdrawing nature of fluorine affects the reactivity of an adjacent glycosidic bond, enabling the use of fluorinated substrate analogues as mechanistic probes for glycosyl-processing enzymes and metabolic studies. [9] The strong interest in fluorinated mono-and oligosaccharides has resulted in a number of methods for their synthesis, [10] including the syntheses of mucin-like core structures. [11] In contrast, only a few pseudo-glycopeptides containing fluorinated glycosyl amino acids are available from the literature.…”

Section: Introductionmentioning

confidence: 99%

Fluorinated Glycosyl Amino Acids for Mucin‐Like Glycopeptide Antigen Analogues

Wagner

Mersch

Hoffmann‐Röder

2010

Chemistry A European J

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The aberrant glycosylation profiles of mucin glycoproteins on epithelial tumour cells represent attractive target structures for the development of immunotherapy against cancer. Mucin-type glycopeptides have been successfully investigated as molecularly defined vaccine prototypes for triggering humoral immunity but are susceptible to rapid in vivo degradation. As a potential means to enhance the bioavailabilities of the antigenic structures, hydrolysis-resistant carbohydrate analogues with fluorine substituents at positions C6, C2' and C6' were synthesised and incorporated into the tandem repeat sequence of the mucin MUC1. The resulting pseudo-glycopeptides can be used to elucidate the effects of chemically modified antibody determinants on metabolic and immunological properties.

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Elucidation of enzyme mechanisms using fluorinated substrate analogues

Cited by 51 publications

References 120 publications

Stereoselective Glycal Fluorophosphorylation: Synthesis of ADP‐2‐fluoroheptose, an Inhibitor of the LPS Biosynthesis

Stereoselective Glycal Fluorophosphorylation: Synthesis of ADP‐2‐fluoroheptose, an Inhibitor of the LPS Biosynthesis

Probing the Hydrophobic Pocket of the Active Site in the Particulate Methane Monooxygenase (pMMO) from Methylococcus capsulatus (Bath) by Variable Stereoselective Alkane Hydroxylation and Olefin Epoxidation

Fluorinated Glycosyl Amino Acids for Mucin‐Like Glycopeptide Antigen Analogues

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