Hardy Weißhoff

Test-Strip-Based Fluorometric Detection of Fluoride in Aqueous Media with a BODIPY-Linked Hydrogen-Bonding Receptor†

The measurement of biologically relevant anions, such as fluoride, is an important task in analytical chemistry, in particular, for dental health and osteoporosis. Although a large number of fluoride probes are known, the applicability under relevant conditions is limited to a few examples. To improve this situation, BODIPY-amidothiourea dyes with varying hydrogen-bond donating strengths were developed, the most H-acidic of which (1 c) could detect F(-) from an inorganic source (NaF) in 50 % aqueous solution (DMSO/water 1:1, v/v) with 0.01 ppm sensitivity through selective fluorescence quenching by a photoinduced electron-transfer (PET) process. Use of the probe and a reference dye with a test-strip assay and a portable and rapidly recording lateral-flow fluorescence reader made determination of F(-) in neat aqueous solutions, such as spiked water samples and toothpaste extracts, possible in a self-referenced manner, achieving a detection limit of 0.2 ppm.

Temperature coefficients of amide proton NMR resonance frequencies in trifluoroethanol: A monitor of intramolecular hydrogen bonds in helical peptides?

Summary2D 1H NMR spectroscopy of two α-helical peptides which differ in their amphipathicity has been used to investigate the relationships between amide-proton chemical shifts, amide-proton exchange rates, temperature, and trifluoroethanol (TFE) concentration. In 50% TFE, in which the peptides are maximally helical, the amide-proton chemical shift and temperature coefficient patterns are very similar to each other in each peptide. Temperature coefficients from −10 to −6 ppb/K, usually indicative of the lack of intramolecular hydrogen bonds, were observed even for hydrophobic amino acids in the center of the α-helices. However, slow hydrogen isotope exchange for residues from 4 to 16 in both 18-mer helices indicates intact intramolecular hydrogen bonds over most of the length of these peptides. Based on these anomalous observations, we suggest that the pattern of amide-proton shifts in α-helices in H2O/TFE solvents is dominated by bifurcated intermolecular hydrogen-bond formation between the backbone carbonyl groups and TFE. The amide-proton chemical shift changes with increasing temperature may be interpreted by a disruption of intermolecular hydrogen bonds between carbonyl groups and the TFE in TFE/water rather than by the length of intramolecular hydrogen bonds in α-helices.

Novel disulfide-constrained pentapeptides as models for β-VIa turns in proteins

The conformational behavior of cyclic peptides of the amino acid sequence Cys‐Phe/Ala‐Pro‐Ala‐Cys has been investigated through the combined use of molecular simulation methods and NMR experiments to find models for β‐VIa turns of proteins. Both oxidized (cyclic) peptides and reduced (linear) forms were investigated. At least 95% of the cyclic peptides show a cis conformation of the Xaa‐Pro bond in solution in DMSO or water, whereas all other peptide bonds are trans. Furthermore, we observed a hydrogen bond between the NH group of residue Ala4 and the C = O group of residue Cys1. Both properties are indicative of β‐VIa turns. After reduction of the disulfide bridge, the all‐trans form of the peptide bonds predominates.

On the use of N-dicyclopropylmethyl aspartyl-glycine synthone for backbone amide protection

To prevent aspartimide formation and related side products in Asp‐Xaa, particularly Asp‐Gly‐containing peptides, usually the 2‐hydroxy‐4‐methoxybenzyl (Hmb) backbone amide protection is applied for peptide synthesis according to the Fmoc‐protocols. In the present study, the usefulness of the recently proposed acid‐labile dicyclopropylmethyl (Dcpm) protectant was analyzed. Despite the significant steric hindrance of this bulky group, N‐terminal H‐(Dcpm)Gly‐peptides are quantitatively acylated by potent acylating agents, and alternatively the dipeptide Fmoc‐Asp(OtBu)‐(Dcpm)Gly‐OH derivative can be used as a building block. In contrast to the Hmb group, Dcpm is inert toward acylations, but is readily removed in the acid deprotection and resin‐cleavage step. Copyright

Structure determination and by-product profile of the NK2 receptor antagonist nepadutant, a bicyclic glycopeptide

We have synthesized and fully characterized the NK2 receptor antagonist nepadutant and its by‐products using nuclear magnetic resonance (NMR) and restrained molecular dynamics. The agent consists of an active bicyclic hexapeptide combined with a sugar residue. Analysis of the high‐performance liquid chromatogram and the mass spectroscopy spectra yields traces of three by‐products with the same molecular weight as the main product. The conformation of the molecules in the bicyclic hexapeptide segment, the active region, is well defined, whereas the sugar moiety is disordered. For the peptide region of nepadutant and all of its by‐products, the NMR observables can be described by a single backbone conformation, more specifically a βI, βII‐turn arrangement. The active dipeptide unit Trp–Phe occupies the i+1 and i+2 position of a βI‐turn. The by‐product profile is characterized by different forms of sugars which are caused mainly by isomerization in the process of ring opening.

Ratiometric fluorescence detection of phosphorylated amino acids through excited‐state proton transfer using molecularly imprinted polymer (MIP) recognition nanolayers

A 2,3-diaminophenazine bis-urea fluorescent probe monomer (1) was developed. It responds to phenylphosphate and phosphorylated amino acids in a ratiometric fashion with enhanced fluorescence accompanied by the development of a redshifted emission band arising from an excited-state proton transfer (ESPT) process in the hydrogen-bonded probe/analyte complex. The two urea groups of 1 form a cleft-like binding pocket (Kb >1010  L2  mol-2 for 1:2 complex). Imprinting of 1 in presence of ethyl ester- and fluorenylmethyloxycarbonyl (Fmoc)-protected phosphorylated tyrosine (Fmoc-pTyr-OEt) as the template, methacrylamide as co-monomer, and ethyleneglycol dimethacrylate as cross-linker gave few-nanometer-thick molecularly imprinted polymer (MIP) shells on silica core microparticles with excellent selectivity for the template in a buffered biphasic assay. The supramolecular recognition features were established by spectroscopic and NMR studies. Rational screening of co-monomers and cross-linkers allowed to single out the best performing MIP components, giving significant imprinting factors (IF>3.5) while retaining ESPT emission and the ratiometric response in the thin polymer shell. Combination of the bead-based detection scheme with the phase-transfer assay dramatically improved the IF to 15.9, allowing sensitive determination of the analyte directly in aqueous media.

Elution Behavior and Structural Characterization of N- and C-functionalized DOTA Complexes for the Labelling of Biomolecules

Two types of lanthanide complexes of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for the labelling of biomolecules were investigated by HPLC, MS and NMR spectroscopy. The elution behavior of lanthanide complexes of N-functionalized DOTA [1,4,7,10-tetraazacyclododecane- 1,4,7-triacetic acid-10-maleimidoethylacetamide (nDOTA-Mal) and 1-{2-[4-(maleimido- N-propylacetamidobutyl)amino]-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid (nDOTA-Bu-Mal)] and C-functionalized DOTA [2-{4-(maleimido-N-propylacetamido)benzyl}-1,4, 7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (cDOTA-Bnz-Mal) and 2-(4-isothiocyanatobenzyl)- 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (cDOTA-Bnz-NCS)] was compared. N-functionalized lanthanide DOTA complexes coelute as required for their use as ICAT-analogous reagents. The complexation of the C-functionalized DOTA with lanthanides results in two fractions separable by HPLC. Coelution is observed for the main fractions of the lanthanide complexes. The retention times of the minor fractions show a dependence on the ionic radii of the metal ions. MALDI spectra of lanthanide-DOTA-peptide conjugates including different monoisotopic lanthanides demonstrate the advantage of the mass variations for extensive peptide and protein investigations. Graphical Abstract Elution Behavior and Structural Characterization of N- and C-functionalized DOTA Complexes for the Labelling of Biomolecules