Details
| Original language | English |
|---|---|
| Pages (from-to) | 1476-1480 |
| Number of pages | 5 |
| Journal | Chemistry - a European journal |
| Volume | 25 |
| Issue number | 6 |
| Early online date | 20 Dec 2018 |
| Publication status | Published - 28 Jan 2019 |
Abstract
The authors wish to provide detailed protocol that clarifies and supports their results with RvD1n−3 DPA and RvD1 in this manuscript. The following paragraph should be inserted at Line 3 on page 1479: A) Validation of n−3 DPA Resolvins The structural integrity of RvD1n−3 DPA and RvD1n−3 DPA ethyl ester were each verified using LC-MS-MS before bioassays and receptor assay. B) Activation of human ALX and GPR32: comparison of RvD1, RvD1n−3 DPA and RvD1n−3 DPA ethyl ester using GPCR-beta-arrestin based system: a step-by-step procedure (1) Ligand receptor interactions were monitored using the PathHunter® β-arrestin cell-based assays (Eurofins DiscoverX Corporation, Fremont, CA, USA) essentially as described earlier.[1] CHO-β-arrestin-GPR32 cells and HEK-β-arrestin-FPR2/ALX cells were purchased from DiscoverX, and maintained in HAM F-12 and DMEM media, respectively, supplemented with 10% FBS, 1X pen/strip, G418 (0.8 mg ml−1) and hygromycin (0.3 mg ml−1). (2) The day before each experiment, cells were plated onto 96-well plates (white with clear bottom; Costar #3903) at 20 000 cells per well. (3) The next day, the media was aspirated and cells were suspended in 90 microliters of PBS +/+ (with Ca2+ and Mg2+). Stocks of each compound (10−4 M) were used to prepare working solutions at 10−7 to 10−13 M in PBS +/+ by serial dilutions. (4) Ten microliters of each concentration was then added to each well to make final concentrations of 10−8 to 10−14 M. For vehicle controls, 10 microliters of PBS+/+ was added to the wells. Each concentration of each compound was tested in 3 to 4 replicates in 3 independent experiments. Assay plates were incubated at 37 °C and 5 % CO2 for 90 min. (5) DiscoverX PathHunter Detection Reagent (Eurofins DiscoverX Corporation, Fremont, CA, USA) was then used following the manufacturer's instruction; namely, 55 microliters of Working Detection Solution (mixture of Cell Assay Buffer: Substrate Reagent 1: Substrate Reagent 2; 19:5:1) was added to each well, and assay plates incubated in the dark at room temperature for 60 min. Chemiluminescence was then determined using a luminescence plate reader (SpectraMax M3; Molecular Device) at 0.5 s per well, collecting signals from all wavelengths as recommended by the manufacturer. Results are expressed as % increase of RLU (Relative luminescence) above vehicle controls. The 10 % increases represent 35.3±8.6 and 170.0±7.1 RLU arbitrary units for GPR32 and ALX, respectively. Note that the receptor denoted ALX/FPR2 was demonstrated bias in intracellular signaling with specific ligands and with pro-resolving mediator ligands,[2] confirmed in >175 publications in PubMed to date.
Keywords
- Karstedt's catalyst, natural products, sp–sp cross-coupling, specialized pro-resolving mediators, total synthesis
ASJC Scopus subject areas
- Chemical Engineering(all)
- Catalysis
- Chemistry(all)
- Organic Chemistry
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In: Chemistry - a European journal, Vol. 25, No. 6, 28.01.2019, p. 1476-1480.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Resolving Inflammation
T2 - Synthesis, Configurational Assignment, and Biological Evaluations of RvD1n−3 DPA
AU - Tungen, Jørn Eivind
AU - Gerstmann, Lisa
AU - Vik, Anders
AU - De Matteis, Roberta
AU - Colas, Romain Alexandre
AU - Dalli, Jesmond
AU - Chiang, Nan
AU - Serhan, Charles Nicholas
AU - Kalesse, Markus
AU - Hansen, Trond Vidar
N1 - Funding Information: The authors are grateful for collaborations and fruitful scientific interactions as well as funding for a Short Term Scientific Mission scholarship to L.G. from COST Action CM 1407. Funding to T.V.H (FRIPRO-FRINATEK 230470) from The Norwegian Research Council is gratefully appreciated. J.D. received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant no: 677542) and the Barts Charity (grant no: MGU0343). J.D. is also supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant 107613/Z/15/Z). C.N.S is supported by the National Institutes of Health GM Grant PO1GM095467 and continuous financial support is gratefully acknowledged.
PY - 2019/1/28
Y1 - 2019/1/28
N2 - The authors wish to provide detailed protocol that clarifies and supports their results with RvD1n−3 DPA and RvD1 in this manuscript. The following paragraph should be inserted at Line 3 on page 1479: A) Validation of n−3 DPA Resolvins The structural integrity of RvD1n−3 DPA and RvD1n−3 DPA ethyl ester were each verified using LC-MS-MS before bioassays and receptor assay. B) Activation of human ALX and GPR32: comparison of RvD1, RvD1n−3 DPA and RvD1n−3 DPA ethyl ester using GPCR-beta-arrestin based system: a step-by-step procedure (1) Ligand receptor interactions were monitored using the PathHunter® β-arrestin cell-based assays (Eurofins DiscoverX Corporation, Fremont, CA, USA) essentially as described earlier.[1] CHO-β-arrestin-GPR32 cells and HEK-β-arrestin-FPR2/ALX cells were purchased from DiscoverX, and maintained in HAM F-12 and DMEM media, respectively, supplemented with 10% FBS, 1X pen/strip, G418 (0.8 mg ml−1) and hygromycin (0.3 mg ml−1). (2) The day before each experiment, cells were plated onto 96-well plates (white with clear bottom; Costar #3903) at 20 000 cells per well. (3) The next day, the media was aspirated and cells were suspended in 90 microliters of PBS +/+ (with Ca2+ and Mg2+). Stocks of each compound (10−4 M) were used to prepare working solutions at 10−7 to 10−13 M in PBS +/+ by serial dilutions. (4) Ten microliters of each concentration was then added to each well to make final concentrations of 10−8 to 10−14 M. For vehicle controls, 10 microliters of PBS+/+ was added to the wells. Each concentration of each compound was tested in 3 to 4 replicates in 3 independent experiments. Assay plates were incubated at 37 °C and 5 % CO2 for 90 min. (5) DiscoverX PathHunter Detection Reagent (Eurofins DiscoverX Corporation, Fremont, CA, USA) was then used following the manufacturer's instruction; namely, 55 microliters of Working Detection Solution (mixture of Cell Assay Buffer: Substrate Reagent 1: Substrate Reagent 2; 19:5:1) was added to each well, and assay plates incubated in the dark at room temperature for 60 min. Chemiluminescence was then determined using a luminescence plate reader (SpectraMax M3; Molecular Device) at 0.5 s per well, collecting signals from all wavelengths as recommended by the manufacturer. Results are expressed as % increase of RLU (Relative luminescence) above vehicle controls. The 10 % increases represent 35.3±8.6 and 170.0±7.1 RLU arbitrary units for GPR32 and ALX, respectively. Note that the receptor denoted ALX/FPR2 was demonstrated bias in intracellular signaling with specific ligands and with pro-resolving mediator ligands,[2] confirmed in >175 publications in PubMed to date.
AB - The authors wish to provide detailed protocol that clarifies and supports their results with RvD1n−3 DPA and RvD1 in this manuscript. The following paragraph should be inserted at Line 3 on page 1479: A) Validation of n−3 DPA Resolvins The structural integrity of RvD1n−3 DPA and RvD1n−3 DPA ethyl ester were each verified using LC-MS-MS before bioassays and receptor assay. B) Activation of human ALX and GPR32: comparison of RvD1, RvD1n−3 DPA and RvD1n−3 DPA ethyl ester using GPCR-beta-arrestin based system: a step-by-step procedure (1) Ligand receptor interactions were monitored using the PathHunter® β-arrestin cell-based assays (Eurofins DiscoverX Corporation, Fremont, CA, USA) essentially as described earlier.[1] CHO-β-arrestin-GPR32 cells and HEK-β-arrestin-FPR2/ALX cells were purchased from DiscoverX, and maintained in HAM F-12 and DMEM media, respectively, supplemented with 10% FBS, 1X pen/strip, G418 (0.8 mg ml−1) and hygromycin (0.3 mg ml−1). (2) The day before each experiment, cells were plated onto 96-well plates (white with clear bottom; Costar #3903) at 20 000 cells per well. (3) The next day, the media was aspirated and cells were suspended in 90 microliters of PBS +/+ (with Ca2+ and Mg2+). Stocks of each compound (10−4 M) were used to prepare working solutions at 10−7 to 10−13 M in PBS +/+ by serial dilutions. (4) Ten microliters of each concentration was then added to each well to make final concentrations of 10−8 to 10−14 M. For vehicle controls, 10 microliters of PBS+/+ was added to the wells. Each concentration of each compound was tested in 3 to 4 replicates in 3 independent experiments. Assay plates were incubated at 37 °C and 5 % CO2 for 90 min. (5) DiscoverX PathHunter Detection Reagent (Eurofins DiscoverX Corporation, Fremont, CA, USA) was then used following the manufacturer's instruction; namely, 55 microliters of Working Detection Solution (mixture of Cell Assay Buffer: Substrate Reagent 1: Substrate Reagent 2; 19:5:1) was added to each well, and assay plates incubated in the dark at room temperature for 60 min. Chemiluminescence was then determined using a luminescence plate reader (SpectraMax M3; Molecular Device) at 0.5 s per well, collecting signals from all wavelengths as recommended by the manufacturer. Results are expressed as % increase of RLU (Relative luminescence) above vehicle controls. The 10 % increases represent 35.3±8.6 and 170.0±7.1 RLU arbitrary units for GPR32 and ALX, respectively. Note that the receptor denoted ALX/FPR2 was demonstrated bias in intracellular signaling with specific ligands and with pro-resolving mediator ligands,[2] confirmed in >175 publications in PubMed to date.
KW - Karstedt's catalyst
KW - natural products
KW - sp–sp cross-coupling
KW - specialized pro-resolving mediators
KW - total synthesis
UR - http://www.scopus.com/inward/record.url?scp=85058958958&partnerID=8YFLogxK
U2 - 10.1002/chem.201806029
DO - 10.1002/chem.201806029
M3 - Article
C2 - 31774939
AN - SCOPUS:85058958958
VL - 25
SP - 1476
EP - 1480
JO - Chemistry - a European journal
JF - Chemistry - a European journal
SN - 0947-6539
IS - 6
ER -