Synthesis of versatile chemical tools toward a structure/properties relationships study onto targeting colloids

Jolimaître, Pascale; Poirier, Cécile; Richard, Antoine; Blanpain, Annick; Delord, B.; Roux, D.; Bourel-Bonnet, Line

doi:10.1016/j.ejmech.2006.08.011

Cited by 9 publications

(10 citation statements)

References 38 publications

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“…For the preparation of probes 2–4 , Fmoc-PEGO 18 was coupled to the N-terminus of the resin-bound peptides. Following removal of the Fmoc, DTPA, 19 activated as the HOBt ester, was attached to the N-terminus of the PEGO spacer.…”

Section: Resultsmentioning

confidence: 99%

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Synthesis and Characterization of Time-resolved Fluorescence Probes for Evaluation of Competitive Binding to Melanocortin Receptors

Alleti

Vagner

Dehigaspitiya

et al. 2013

Bioorganic & Medicinal Chemistry

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Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9 nM and 4.2±0.48 nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.

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Section: Resultsmentioning

confidence: 99%

“…Attachment of the PEGO linker was performed using DIC/HOBt activation (3 equiv Fmoc-PEGO 18 , 3 equiv of HOBt, and 3 equiv of DIC). Next, the DTPA chelator was attached to the N-terminus of the resin–bound construct as follows.…”

Section: Methodsmentioning

confidence: 99%

Synthesis and Characterization of Time-resolved Fluorescence Probes for Evaluation of Competitive Binding to Melanocortin Receptors

Alleti

Vagner

Dehigaspitiya

et al. 2013

Bioorganic & Medicinal Chemistry

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“…For the preparation of probe 3 , Fmoc-PEGO 26 was coupled to the N-terminus of the resin-bound peptide. Following removal of the Fmoc, DTPA, activated as the HOBt ester, was attached to the N-terminus of the PEGO spacer.…”

Section: Resultsmentioning

confidence: 99%

Development of a time-resolved fluorescence probe for evaluation of competitive binding to the cholecystokinin 2 receptor

Elshan

Jayasundera

Weber

et al. 2015

Bioorganic & Medicinal Chemistry

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The synthesis, characterization, and use of Eu-DTPA-PEGO-Trp-Nle-Asp-Phe-NH2 (Eu-DTPA-PEGO-CCK4), a luminescent probe targeted to cholecystokinin 2 receptor (CCK2R, aka CCKBR), are described. The probe was prepared by solid phase synthesis. A Kd value of 17 ± 2 nM was determined by means of saturation binding assays using HEK-293 cells that overexpress CCK2R. The probe was then used in competitive binding assays against Ac-CCK4 and three new trivalent CCK4 compounds. Repeatable and reproducible binding assay results were obtained. Given its ease of synthesis, purification, receptor binding properties, and utility in competitive binding assays, Eu-DTPA-PEGO-CCK4 could become a standard tool for high-throughput screening of compounds in development targeted to cholecystokinin receptors.

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“…The Kaiser test was used to determine coupling completion at each attachment step. The PEGO linker [10] was then attached following the same coupling cycle, with the activation of Fmoc-PEGO-OH (2.04 mmol in 4.1 mL of DCM) with Cl-HOBt (345 mg, 2.04 mmol) and DIC (512 mg, 4.08 mmol) in DMF (10 mL).…”

Section: Methodsmentioning

confidence: 99%

Spectrophotometric determination and removal of unchelated europium ions from solutions containing Eu-diethylenetriaminepentaacetic acid chelate–peptide conjugates

Elshan

Patek

Vagner

et al. 2014

Analytical Biochemistry

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Europium chelates conjugated with peptide ligands are routinely used as probes for conducting in vitro binding experiments. The presence of unchelated Eu ions in these formulations gives high background luminescence and can lead to poor results in binding assays. In our experience, the reported methods for purification of these probes do not achieve adequate removal of unchelated metal ions in a reliable manner. In this work, a xylenol orange-based assay for the quantification of unchelated metal ions was streamlined and used to determine levels of metal ion contamination, as well as the success of metal ion removal upon attempted purification. We compared the use of Empore™ chelating disks and Chelex® 100 resin for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Both purification methods gave complete and selective removal of the contaminant metal ions. However, Empore™ chelating disks were found to give much higher recoveries of the probes under the conditions utilized. Related to the issue of probe recovery, we also describe a significantly more efficient method for the synthesis of one such probe using Rink amide AM resin in place of Tentagel S resin.

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Synthesis of versatile chemical tools toward a structure/properties relationships study onto targeting colloids

Cited by 9 publications

References 38 publications

Synthesis and Characterization of Time-resolved Fluorescence Probes for Evaluation of Competitive Binding to Melanocortin Receptors

Synthesis and Characterization of Time-resolved Fluorescence Probes for Evaluation of Competitive Binding to Melanocortin Receptors

Development of a time-resolved fluorescence probe for evaluation of competitive binding to the cholecystokinin 2 receptor

Spectrophotometric determination and removal of unchelated europium ions from solutions containing Eu-diethylenetriaminepentaacetic acid chelate–peptide conjugates

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