Fluorine-Labeled N-Boc-l-proline as a Marker for Solid-State NMR Characterization of Biofunctionalizations on Paper Substrates

Abstract

An efficient approach employing 4-dimethylaminopyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as the coupling reagent is presented, which enables the functionalization of cotton linter paper substrates with the 19 F spin label N-boc-cis-4-fluoro-L-proline. This spin label can be easily quantified by 19 F magic angle spinning (MAS) NMR experiments to determine its loading on paper substrates. During the functionalization, the spin label stays intact, as confirmed by the 1 H− 19 F heterocorrelation ( 1 H → 19 F CP MAS FSLG HETCOR) experiments. In combination with dynamic nuclear polarization ( 19 F MAS DNP), the N-boc-cis-4-fluoro-L-proline spin label allows us to inspect 1 μmol/g and even lower molecule loadings on paper substrates, providing a highly sensitive local probe to analyze the structure of biofunctionalizations at the nanoscale on paper substrates in the future.

Peptides as Model Systems for Biofunctionalizations of Cellulose─Synthesis and Structural Characterization by Advanced Solid-State Nuclear Magnetic Resonance Techniques

“…From this spectrum, the loading of the sample is calculated (for details see Supplementary Information and ref. 5 ) to 45.8 ± 3.2 μmol/g when using 2 equiv of the peptide to 1 equiv of glucose units in MCC in the synthesis. This low value let us assume that for deeper structure analysis of the peptide on MCC, i.e., by 1D or 2D 13 C solid-state MMR techniques, a sensitivity boost is required.…”

“…It contains a linear homopolymer of d -glucose units linked by β-1,4 glycosidic bonds . In general, the hydroxyl groups of cellulose can be functionalized with bio-organic moieties via established synthesis protocols. , Cellulose-based functionalized materials are widely used in various fields of applications such as drug delivery systems, − supporting materials for catalysts, − and sensor devices. , In recent years, biosensors have gained increasing attention because of their easy handling, rapid analysis, high sensitivity, and selectivity toward detection of analytes . Especially, peptides or proteins interacting with cellulose play an important role in biosensorics.…”

“…Furthermore, the cellulose matrix generates a huge background signal in solid-state NMR making the spectral analysis challenging. A possibility to overcome these drawbacks of solid-state NMR is its combination with dynamic nuclear polarization (DNP-enhanced solid-state NMR). − This technique uses the 3 orders of magnitude higher polarization of electron spins and transfers it to nuclear spin polarization, which can be used to significantly enhance the sensitivity in solid-state NMR. − Thus, it provides a powerful technique to investigate small amounts of surface sites in solid materials − as well as to characterize biosolids. − Recently, it has also been successfully applied to characterize cellulose and cellulose-derived materials, ,,,− as well as to inspect small amounts of molecules down to the nanomolar region employing 19 F spin labels. ,, Solid-state DNP experiments typically require an appropriate sample preparation with a solvent matrix, i.e., an aqueous matrix that can mimic the conditions where biotests are performed.…”

“…50−52 Recently, it has also been successfully applied to characterize cellulose and cellulose-derived materials, 5,13,44,53−60 as well as to inspect small amounts of molecules down to the nanomolar region employing 19 F spin labels. 5,61,62 Solid-state DNP experiments typically require an appropriate sample preparation with a solvent matrix, i.e., an aqueous matrix that can mimic the conditions where biotests are performed.…”

“…The peptide functionalization on MCC is schematically illustrated in Scheme 1. The procedure refers to the approach proposed by some of the authors 5 for binding of amino acids on paper substrates. MCC (1 equiv, 0.25 mmol glucose units, 40.0 mg) was weighed in a 30 mL screw cap bottle.…”