Solid-phase reaction monitoring—Chemical derivatization and off-bead analysis

Kay, Corinne; Lorthioir, Olivier; Parr, Nigel J.; Congreve, Miles; McKeown, Stephen C.; Scicinski, Jan; Ley, Steven V.

doi:10.1002/1097-0290(2000)71:2<110::aid-bit1002>3.0.co;2-2

Cited by 51 publications

(46 citation statements)

References 46 publications

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“…Further reverse phase and final normal phase HPLC purifications gave the desired products (shown in Figure 3), VBA-Asparagine (Man 8 GlcNAc 2 )-CONH 2 2 , Biotin-Asparagine (Man 8 GlcNAc 2 )-CONH 2 3 , PA-Asparagine (Man 8 GlcNAc 2 )-CONH 2 4 , 5-FAM-Asparagine (Man 8 GlcNAc 2 )-CONH 2 5 with good overall yields ranging from 65% to 85% according to initial aspartic acid loading determined by Fmoc removal quantification. 21 …”

Section: Resultsmentioning

confidence: 99%

“…Theoretical yields were calculated based on the amino acid loadings of the resins, which were determined by quantification of Fmoc removal by UV at 301 nm (ε=7800 cm −1 M −1 ). 21 400 and 500 MHz 1 H-NMR was carried on a Varian Inova NMR Spectrometer. Masses were determined either by a Bruker Autoflex III MALDI-TOF or a Waters/Micromass LCT Mass spectrometer.…”

Section: Methodsmentioning

confidence: 99%

“…The loading was determined by quantification of Fmoc removal by UV at 301 nm (ε=7800 cm −1 M −1 ) for a resin sample. 21 Capping was performed to inactivate any un-reacted amino groups by using a large excess of acetic anhydride with 3 equivalents of N-methylmorpholine in DCM. Before the on-resin glycosylation reaction, the PhiPr protecting group was selectively removed by the treatment with 94:1:5 DCM/TFA/TIS for 2 min, repeated 4 times.…”

Section: Methodsmentioning

confidence: 99%

“…Theoretical yields were calculated based on the amino acid loadings of the resins, which were determined by quantification of Fmoc removal by UV at 301 nm (ε=7800 cm −1 M −1 ). 21 …”

Section: Methodsmentioning

confidence: 99%

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Versatile on-resin synthesis of high mannose glycosylated asparagine with functional handles

Chen¹,

Pawlicki²,

Tolbert³

2014

Carbohydrate Research

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Here we present a synthetic route for solid phase synthesis of N-linked glycoconjugates containing high mannose oligosaccharides which allows the incorporation of useful functional handles on the N-terminus of asparagine. In this strategy, the C-terminus of an Fmoc protected aspartic acid residue is first attached to a solid phase support. The side chain of aspartic acid is protected by a 2-phenylisopropyl protecting group, which allows selective deprotection for the introduction of glycosylation. By using a convergent on-resin glycosylamine coupling strategy, an N-glycosidic linkage is successfully formed on the free side chain of the resin bound aspartic acid with a large high mannose oligosaccharide, Man8GlcNAc2, to yield N-linked high mannose glycosylated asparagine. The use of on-resin glycosylamine coupling provides excellent glycosylation yield, can be applied to couple other types of oligosaccharides, and also makes it possible to recover excess oligosaccharides conveniently after the on-resin coupling reaction. Useful functional handles including an alkene (p-vinylbenzoic acid), an alkyne (4-pentynoic acid), biotin, and 5-carboxyfluorescein are then conjugated onto the N-terminal amine of asparagine on-resin after the removal of the Fmoc protecting group. In this way, useful functional handles are introduced onto the glycosylated asparagine while maintaining the structural integrity of the reducing end of the oligosaccharide. The asparagine side chain also serves as a linker between the glycan and the functional group and preserves the native presentation of N-linked glycan which may aid in biochemical and structural studies. As an example of a biochemical study using functionalized high mannose glycosylated asparagine, a fluorescence polarization assay has been utilized to study the binding of the lectin Concanavalin A (ConA) using 5-carboxyfluorescein labeled high mannose glycosylated asparagine.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Theoretical yields were calculated based on the amino acid loadings of the resins, which were determined by quantification of Fmoc removal by UV at 301 nm (ε=7800 cm −1 M −1 ). 21 …”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Versatile on-resin synthesis of high mannose glycosylated asparagine with functional handles

Chen¹,

Pawlicki²,

Tolbert³

2014

Carbohydrate Research

View full text Add to dashboard Cite

show abstract

“…Fmoc protecting groups were removed using 20% piperidine in DMF (10 min × 3). The loading of the first amino acid residue was determined ( see Note 4) by UV monitoring the Fmoc removal at 301 nm (ε=7800 cm −1 M −1 )(24). …”

Section: Methodsmentioning

confidence: 99%

On-Resin Convergent Synthesis of a Glycopeptide from HIV gp120 Containing a High Mannose Type N-Linked Oligosaccharide

Chen¹,

Tolbert

2011

Methods in Molecular Biology

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Summary/Abstract This method describes a rapid and efficient approach for solid phase synthesis of N-linked glycopeptides which utilizes on-resin glycosylamine coupling to produce N-linked glycosylation sites (1). In this method, the full-length non-glycosylated peptide is first synthesized on solid phase using standard Fmoc chemistry. The glycosylation site is then introduced through an orthogonally protected 2-phenylisopropyl (PhiPr) Aspartic acid (Asp) residue. After selective deprotection of the Asp residue, a high mannose oligosaccharide glycosylamine is coupled on-resin to the free Asp side chain to form a N-glycosidic bond. Subsequent protecting group removal and peptide cleavage from the resin yield the desired glycopeptide. This strategy provides effective glycosylation reactions on the solid phase, simplifies glycopeptide purification relative to solution phase glycopeptide synthesis strategies, and enables recovery of potentially valuable, un-reacted oligosaccharides. This approach has been applied to the solid phase synthesis of the N-linked high mannose glycosylated form of peptide T (ASTTTNYT), a fragment of the HIV-1 envelope glycoprotein gp120 (2–3).

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Solid‐Phase Organic Synthesis

Fawaz

Scott

2014

Kirk-Othmer Encyclopedia of Chemical Technology

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Solid‐phase organic synthesis (SPOS), a synthetic technique in which reaction components (eg, substrates, catalysts, and reagents) are attached to insoluble polymer supports, was introduced by Prof. Robert Bruce Merrifield in the 1960s. In the decades since its introduction, SPOS has matured into a powerful technique with applications in, for example, the synthesis of small molecules, drugs and natural products, asymmetric synthesis, and organometallic chemistry. In addition, the ease with which many SPOS reactions can be automated and run in parallel means it has a pivotal role in library synthesis for combinatorial chemistry and diversity‐oriented synthesis. This article introduces the principles of SPOS and highlights key concepts including linker units, SPOS reactions, immobilized catalysts, and solid‐supported reagents through literature examples.

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Solid-phase reaction monitoring—Chemical derivatization and off-bead analysis

Cited by 51 publications

References 46 publications

Versatile on-resin synthesis of high mannose glycosylated asparagine with functional handles

Versatile on-resin synthesis of high mannose glycosylated asparagine with functional handles

On-Resin Convergent Synthesis of a Glycopeptide from HIV gp120 Containing a High Mannose Type N-Linked Oligosaccharide

Solid‐Phase Organic Synthesis

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