Facile Oxime Ether Synthesis: Free Carbonyl Compound Derivatization by a Brominated <i>O</i>-Benzylhydroxylamine

<div><p></p><p>The lipid peroxidation of fatty acids leads to secondary products, among which several carbonyl compounds are of concern in food toxicology. The detection of these reactive aldehydes for identification and evaluation is required. Derivatization is necessary to improve their stability and detection in liquid chromatography/high-resolution mass spectrometry (LC/HRMS) trace analyses. Therefore, a brominated <i>O</i>-benzylhydroxylamine, namely 1-((ammoniooxy)methyl)-2-bromobenzene chloride, was selected as a new probe for the mild and selective derivatization of carbonyl compounds. New oxime ethers were thus synthesized under mild reaction and workup conditions, with full analytical characterization. The relevance of the chemical reaction was assessed with nine aldehydes, especially conjugated and deuterium-labeled aldehydes, and two ketones. Virtually, the reaction should be applicable to a large set of carbonyl compounds for derivatization in complex biological samples and selective detection of the in situ–synthesized brominated oxime ethers by LC/HRMS methodology.</p></div

List of antibodies for western blot.

(DOCX)</p

Effect of nF and mF fibroblasts on the phenotype of colonocytes.

A) Effect of the different CM collected after 72 h of direct coculture on the mean area per cell for Co colonocytes growth on 8-chambered slides. *P<0.05. Data were expressed as mean ± SEM of 4 experiments. B) Western blot analysis of E-cadherin and cytokeratin 18 protein expression in Co colonocytes after 72 h of coculture with nF or mF fibroblasts. Results were normalized to Hsc70 and data were expressed as mean ± SEM of 3 experiments. *P< 0.05. C) Representative image of Lgr5 localization (green) in Co colonocytes growth on 8-chambered slides with CM collected after 72 h of direct coculture. The pictures were representative of three separate experiments. Alexa Fluor 488 goat anti-rabbit was used for secondary antibody and DAPI staining for nucleus. 1: CM-Co; 2 CM-nF/Co; 3 CM-mF/Co. Scale bar shows 50 μm.</p

Morphological characteristics of fibroblastic cell lines.

A) Phase-contrast microscope images of living fibroblasts and Co cells: (1) nF Fibroblasts, (2) mF Fibroblasts and (3) Co cells. Representative images are shown (Scale: 50 μm). B) Mean diameter of fibroblasts and epithelial cells as measured using LunaTM cell counter after trypsinizing the cells (n≥10 different cell culture passages). Values were presented as means ± SEM. **** P<0.0001.</p

Involvement of the BMP pathway.

A) Effect of BMP pathway inhibitor (K02288; 250–500 nM) on nF fibroblast-induced colonic cell growth in direct coculture. Values are mean ± SEM (n = 4 independent experiments). *P<0.05. ** P<0.01. B) Effect of SB4, a BMP4 agonist, on the number of Co cells. The graphs represented the mean ± SEM values from 4 independent experiments. *P<0.05. C) Effects of SB4 on BMP pathway activation in Co cells: Immunostaining of p-Smad1/5 was performed in Co colonocytes exposed to SB4 in 8-chambered slide and the percentage of positive cells was calculated. The graphs represented the mean ± SEM values from 4 independent experiments. * P<0.05; **** P<0.0001.</p

Cytokeratin 18 and E-cadherin expression in Co cells and in fibroblasts.

(A) Representative image of cytokeratin 18 expression (green) in Co colonocytes. No labeling was detected in nF and mF fibroblasts. The pictures were representative of three separate experiments. Alexa Fluor 488 goat anti-rabbit was used for secondary antibody and DAPI staining for nucleus. (B) Western blot analysis showing cytokeratin 18 (~48 kDa) in Co cells but not in nF and mF fibroblasts. (C) Representative image of E-cadherin expression (red) in Co colonocytes. No labeling was detected in nF and mF fibroblasts. The pictures were representative of three separate experiments. Alexa Fluor 594 goat anti-rabbit was used for secondary antibody and DAPI staining for nucleus. (TIF)</p

Effects of Co colonocytes on nF and mF fibroblasts in coculture system.

nF or mF fibroblasts were seeded into the bottom of 6-well culture plates. Epithelial cells were cultured on a membrane insert for 72 h with nF or mF fibroblasts. A) Effect on the number of nF and mF fibroblasts. nF or mF Fibroblastic cells cultured alone (without Co cells in coculture) were defined as a control. The graphs represent the mean ± SEM values from 4 independent experiments. ***PCM-nF/Co: conditioned medium resulting from the coculture between nF fibroblasts and Co cells; CM-mF/Co: conditioned medium resulting from coculture between mF fibroblasts and Co cells; CM-nF: conditioned medium of nF fibroblasts in monoculture; CM-mF: conditioned medium of mF fibroblasts in monoculture. (TIF)</p

Effects of colonic fibroblasts on Co cells in indirect coculture system.

(A) Diagram describing the indirect coculture model. Co cells were grown alone in conditioned medium from fibroblasts or Co cells in monoculture. The graphs represent the mean ± SEM values from 4 independent experiments. (B) Effect of indirect coculture on the number of Co cells. (C and D) Effect of indirect coculture on active caspase 3 and Ki67 positive Co cells. Co cells were seeded in an 8-chamber slide and allowed to settle for 6 h. The medium was then removed and replaced by the conditioned media to be tested or by fresh medium. The different media were renewed at 24 and 48 h. CM-Co: conditioned medium from Co cells in monoculture. CM-nF: conditioned medium from nF cells in monoculture. CM-mF: conditioned medium from mF cells in monoculture. M33: Fresh medium. (TIF)</p

Markers of the stem cell niche in nF and mF fibroblasts.

Representative image of CD90, Rspo1, Rspo3 and BMP4 localization in nF and mF fibroblasts. The pictures were representative of three separate experiments. Alexa Fluor 488 goat anti-rabbit or 594 goat anti-rabbit were used for secondary antibody and DAPI staining for nucleus. Scale bar shows 50 μm. (TIF)</p

Fibroblast markers determined by IF and western blot.

nF and mF fibroblasts were evaluated for the expression of vimentin (A), α-SMA (B) and FAP (C). For IF assay, the pictures were representative of four separate experiments. Alexa Fluor 488 goat anti-rabbit or 594 goat anti-rabbit were used for secondary antibody and DAPI staining for nucleus. Scale bar shows 50 μm. For western blot analysis, levels were normalized to Hsc70 and data were expressed as mean ± SEM of 3 different cell passages. *P<0.05.</p