Color Test for Selective Detection of Secondary Amines on Resin and in Solution

Boas, Ulrik; Mirsharghi, Sahar

doi:10.1021/ol502936d

Cited by 8 publications

(9 citation statements)

References 20 publications

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“…The conjugation occurred by nucleophilic substitution between a primary amine group displayed by the alkyl-amine and the alkyl-bromide group displayed on the resin (Figure 1). The presence of primary amine groups on the WB resin was verified by Kaiser test [50]. The peptide ligands were subsequently immobilized to the aminated resin via iodoacetic acid (IAA) coupling chemistry as described in Section 3.2 (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

Optimization of Sequence, Display, and Mode of Operation of IgG-Binding Peptide Ligands to Develop Robust, High-Capacity Affinity Adsorbents That Afford High IgG Product Quality

Islam

Naik

Hashimoto

et al. 2019

IJMS

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This work presents the use of peptide ligand HWRGWV and its cognate sequences to develop affinity adsorbents that compete with Protein A in terms of binding capacity and quality of the eluted product. First, the peptide ligand was conjugated to crosslinked agarose resins (WorkBeads) at different densities and using different spacer arms. The optimization of ligand density and display resulted in values of static and dynamic binding capacity of 85 mg/mL and 65 mg/mL, respectively. A selected peptide-WorkBeads adsorbent was utilized for purifying Mabs from Chinese Hamster Ovary (CHO) cell culture supernatants. The peptide-WorkBeads adsorbent was found able to withstand sanitization with strong alkaline solutions (0.5 M NaOH). The purity of the eluted product was consistently higher than 95%, with logarithmic removal value (LRV) of 1.5 for host cell proteins (HCPs) and 4.0 for DNA. HCP clearance was significantly improved by adding a post-load washing step with either 0.1 M Tris HCl pH 9 or 1 M NaCl. The cognate peptide of HWRGWV, constructed by replacing arginine (R) with citrulline, further increased the HCP LRV to 2.15. The peptide-based adsorbent also showed a remarkable performance in terms of removal of Mab aggregates; unlike Protein A, in fact, HWRGWV was found to bind only monomeric IgG. Collectively, these results demonstrate the potential of peptide-based adsorbents as alternative to Protein A for the purification of therapeutic antibodies.

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Section: Resultsmentioning

confidence: 99%

Optimization of Sequence, Display, and Mode of Operation of IgG-Binding Peptide Ligands to Develop Robust, High-Capacity Affinity Adsorbents That Afford High IgG Product Quality

Islam

Naik

Hashimoto

et al. 2019

IJMS

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“…The colorimetric assays confirmed the absence of the secondary amine 3 (negative result for secondary amines test 30 ). FTIR analysis confirmed the presence of an acetyl group at 1739 cm -1 and a hydroxyl group with a broad band with a maximum at 3490 cm -1 (Figure 2, spectrum B), resulting from the deacetylation of 5 to give the resin-bound intermediate 6.…”

Section: Letter Syn Lettmentioning

confidence: 60%

“…The colorimetric assays confirmed the absence of the free aldehyde (negative result to 2-4-dinitrophenylhydrazine (DNP) test) 29 and the presence of the de-sired product 3 (positive result with chloranil/acetaldehyde in DMF, test for secondary amines). 30 FTIR analysis also confirmed the presence of a secondary amine due to a broad band with a maximum at 3411 cm -1 (Figure 1). To perform the second step, 6-acetoxy-7-methoxy-4chloro-quinazoline (4) was synthesized following a reported procedure.…”

mentioning

confidence: 73%

A Different Approach to the EGFR Inhibitor Gefitinib Involving Solid-Phase Synthesis

Sequeira

Lourenço

Ferreira

et al. 2018

Synlett

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“…This test is not reliable to determine the secondary amines. (Boas and Mirsharghi, 2014). The N-terminal amino acid such as proline, pipecolic acid or tetrahydroisoquinoline-3-carboxylic acid, we used tests such as chloranil and isatin.…”

Section: Test For Monitoring Solid-phase Reactionsmentioning

confidence: 99%

“…If the colour of the beads is blue, the secondary alkane is present. (Boas and Mirsharghi, 2014) Bromophenol Blue test Bromophenol Blue Solution: Dissolve three mg of tetrabromo-phenol sulfonephthalein in one hundred millilitres of DMF.…”

Section: Chloranil Test For Secondary Aminesmentioning

confidence: 99%

Role of capping in peptide synthesis

Verma

Pillai

Tailor

2020

ijrps

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Protecting groups like Fmoc and coupling both steps are essential to monitoring the Fmoc SPPS (Solid Phase Peptide Synthesis) reaction completion. Reliable methods are used to detect the unreacted number of amino groups for monitoring these two essential reaction steps of coupling and cleavage. The ability to detect the complete coupling, incomplete coupling or failure of coupling we use many colour tests in the laboratory and based on this the Fmoc peptide chemistry allows the control of the completion of the Fmoc cleavage. The most important test used is the Kaiser test and highly recommended to monitor the coupling and cleavage steps. If the result of colour tests is positive after coupling, then the second coupling should be performed. Then again use the colour test to detect the level of coupling. If the result is still slightly positive, repeat coupling with the smaller modification of reagents such as used PyBOP instead of HOBT AND HOAT. These colour tests help in revealing the presence of unreacted amino-functional groups. Thus, we need to block these free N-terminal of amino- acids which help in avoiding the making of deletion of sequence.

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Color Test for Selective Detection of Secondary Amines on Resin and in Solution

Cited by 8 publications

References 20 publications

Optimization of Sequence, Display, and Mode of Operation of IgG-Binding Peptide Ligands to Develop Robust, High-Capacity Affinity Adsorbents That Afford High IgG Product Quality

Optimization of Sequence, Display, and Mode of Operation of IgG-Binding Peptide Ligands to Develop Robust, High-Capacity Affinity Adsorbents That Afford High IgG Product Quality

A Different Approach to the EGFR Inhibitor Gefitinib Involving Solid-Phase Synthesis

Role of capping in peptide synthesis

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