03 Fakultät Chemie
Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/4
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Item Open Access The effect of pooling on the detection of the nucleocapsid protein of SARS-CoV-2 with rapid antigen tests(2021) Berking, Tim; Lorenz, Sabrina; Ulrich, Alexander; Greiner, Joachim; Kervio, Eric; Bremer, Jennifer; Wege, Christina; Kleinow, Tatjana; Richert, ClemensThe COVID-19 pandemic puts significant stress on the viral testing capabilities of many countries. Rapid point-of-care (PoC) antigen tests are valuable tools but implementing frequent large scale testing is costly. We have developed an inexpensive device for pooling swabs, extracting specimens, and detecting viral antigens with a commercial lateral flow test for the nucleocapsid protein of SARS-CoV-2 as antigen. The holder of the device can be produced locally through 3D printing. The extraction and the elution can be performed with the entire set-up encapsulated in a transparent bag, minimizing the risk of infection for the operator. With 0.35 mL extraction buffer and six swabs, including a positive control swab, 43 ± 6% (n = 8) of the signal for an individual extraction of a positive control standard was obtained. Image analysis still showed a signal-to-noise ratio of approximately 2:1 at 32-fold dilution of the extract from a single positive control swab. The relative signal from the test line versus the control line was found to scale linearly upon dilution (R2 = 0.98), indicating that other pooling regimes are conceivable. A pilot project involving 14 participants and 18 pooled tests in a laboratory course at our university did not give any false positives, and an individual case study confirmed the ability to detect a SARS-CoV-2 infection with five-fold or six-fold pooling, including one swab from a PCR-confirmed COVID patient. These findings suggest that pooling can make frequent testing more affordable for schools, universities, and similar institutions, without decreasing sensitivity to an unacceptable level.Item Open Access Absolute configuration of small molecules by co‐crystallization(2020) Krupp, Felix; Frey, Wolfgang; Richert, ClemensThe most reliable method to determine the absolute configuration of chiral molecules is X‐ray crystallography, but small molecules can be difficult to crystallize. We report rapid co‐crystallization of tetraaryladamantanes with small molecules as different as n‐decane to nicotine to produce crystals for X‐ray analysis and the assignment of absolute configuration when the molecules are chiral. A screen of 52 diverse compounds gave inclusion in co‐crystals for 88 % of all cases and a high‐resolution structure in 77 % of cases. Furthermore, starting from three milligrams of analyte, a combination of NMR spectroscopy and X‐ray crystallography produced a full structure in less than three days using an adamantane crystallization chaperone that encapsulates the analyte at room temperature.Item Open Access Synthesis of a peptidoyl RNA hairpin via a combination of solid‐phase and template‐directed chain assembly(2022) Bremer, Jennifer; Richter, Christian; Schwalbe, Harald; Richert, ClemensPeptidoyl RNAs are the products of ribosome‐free, single‐nucleotide translation. They contain a peptide in the backbone of the oligoribonucleotide and are interesting from a synthetic and a bioorganic point of view. A synthesis of a stabilized version of peptidoyl RNA, with an amide bond between the C‐terminus of a peptide and a 3′‐amino‐2′,3′‐dideoxynucleoside in the RNA chain was developed. The preferred synthetic route used an N‐Teoc‐protected aminonucleoside support and involved a solution‐phase coupling of the amino‐terminal oligonucleotide to a dipeptido dinucleotide. Exploratory UV‐melting and NMR analysis of the hairpin 5′‐UUGGCGAAAGCdC‐LeuLeu‐AA‐3′ indicated that the peptide‐linked RNA segments do not fold in a cooperative fashion. The synthetic access to doubly RNA‐linked peptides on a scale sufficient for structural biology opens the door to the exploration of their structural and biochemical properties.Item Open Access Transitions in solvate crystals of a tetraaryladamantane(2023) Frey, Wolfgang; Schwenger, Alexander; Berking, Tim; Richert, ClemensObtaining high-resolution structures of liquid compounds can be difficult. Encapsulating them in the lattice of a larger organic molecule acting as crystallization chaperone is one option to overcome this difficulty. Tetraaryladamantane ethers can play the role of chaperones, accommodating a range of different guest molecules in their crystals. How well-ordered crystalline arrangements for molecules of different shape are achieved is not clear. Cases in which more than one structure is found may shed light on this phenomenon. Here, we report low-order cubic crystal structures of 1,3,5,7-tetrakis(2,4-dimethoxyphenyl)adamantane (TDA) encapsulating ortho-xylene or cyclohexane, together with better ordered structures obtained after warming the crystals to 60 °C. Evidence for cubic crystal systems was also found for limonene, hexachlorobutadiene and eucalyptol, with a transition to a triclinic system for the former two, but no transition up to 70 °C for the latter. These findings indicate that some solvate structures of TDA can readily undergo structural transitions to less solvated, better ordered systems. Crystals obtained by rapid thermal crystallization may be in kinetically trapped states, and the transition to a solvate-free crystal system appears to have a kinetic barrier that depends strongly on the structure of the liquid guest molecules encapsulated in the lattice.Item Open Access Ribosome‐free translation up to pentapeptides via template walk on RNA sequences(2024) Reußwig, Sabrina G.; Richert, ClemensThe origin of translation is one of the most difficult problems of molecular evolution. Identifying molecular systems that translate an RNA sequence into a peptide sequence in the absence of ribosomes and enzymes is a challenge. Recently, single‐nucleotide translation via coupling of 5′ phosphoramidate‐linked amino acids to 2′/3′‐aminoacyl transfer‐NMPs, as directed by the sequence of an RNA template, was demonstrated for three of the four canonical nucleotides. How single‐nucleotide translation could be expanded to include all four bases and to produce longer peptides without translocation along the template strand remained unclear. Using transfer strands of increasing length containing any of the four bases that interrogate adjacent positions along the template, we now show that pentapeptides can be produced in coupling reactions and subsequent hydrolytic release in situ. With 2′/3′‐aminoacylated mono‐, di‐, tri‐ and tetranucleotides we thus show how efficient translation can be without biomacromolecules.Item Open Access Determining the diastereoselectivity of the formation of dipeptidonucleotides by NMR spectroscopy(2021) Doppleb, Olivia; Bremer, Jennifer; Bechthold, Maren; Sánchez Rico, Carolina; Göhringer, Daniela; Griesser, Helmut; Richert, ClemensProteins are composed of l‐amino acids, but nucleic acids and most oligosaccharides contain d‐sugars as building blocks. It is interesting to ask whether this is a coincidence or a consequence of the functional interplay of these biomolecules. One reaction that provides an opportunity to study this interplay is the formation of phosphoramidate‐linked peptido RNA from amino acids and ribonucleotides in aqueous condensation buffer. Here we report how the diastereoselectivity of the first peptide coupling of the peptido RNA pathway can be determined in situ by NMR spectroscopy. When a racemic mixture of an amino acid ester was allowed to react with an 5′‐aminoacidyl nucleotide, diastereomeric ratios of up to 72 : 28 of the resulting dipeptido nucleotides were found by integration of 31P‐ or 1H‐NMR peaks. The highest diastereomeric excess was found for the homochiral coupling product d‐Ser‐d‐Trp, phosphoramidate‐linked to adenosine 5′‐monophosphate with its d‐ribose ring. When control reactions with an N‐acetyl amino acid and valine methyl ester were run in organic solvent, the diastereoselectivity was found to be lower, with diastereomeric ratios≤62 : 38. The results from the exploratory study thus indicate that the ribonucleotide residue not only facilitates the coupling of lipophilic amino acids in aqueous medium but also the formation of a homochiral dipeptide. The methodology described here may be used to search for other stereoselective reactions that shed light on the origin of homochirality.Item Open Access Synthesis of peptido RNAs from unprotected peptides and oligoribonucleotides via coupling in aqueous solution(2020) Räuchle, Maximilian; Leveau, Gabrielle; Richert, ClemensPeptido RNAs are hybrid molecules with a phosphoramidate link between the N‐terminus of a peptide and the 5'‐phosphate of an oligoribonucleotide. Such species are formed in spontaneous co‐oligomerizations of amino acids and ribonucleotides in aqueous condensation buffer. To shed light on the properties of these fascinating molecules, a synthetic method for their preparation in monodisperse form is needed. Herein, we report how peptido RNAs can be prepared via solution‐phase coupling of unprotected peptides and oligoribonucleotides in aqueous solution. The preferred protocol uses pre‐activation of the 5'‐phosphate of the RNA as an imidazolide at pH 6.5, followed by precipitation and coupling to the peptide at pH 8 with an organocatalyst. The procedure gave peptido RNAs from water‐soluble peptides and synthetic oligoribonucleotides in up to 68 % yield. The method is convenient and inexpensive and can produce NMR quantities, opening the door to the systematic exploration of the chemistry of peptido RNAs.Item Open Access Prolinyl phosphoramidates of nucleotides with increased reactivity(2024) Humboldt, Adrian; Rami, Fabian; Topp, Franka M.; Arnold, Dejana; Göhringer, Daniela; Pallan, Pradeep S.; Egli, Martin; Richert, ClemensNucleoside monophosphates (NMPs) are the subunits of RNA. They are incorporated into growing complementary strands when sequences are copied in enzyme‐free reactions using organic leaving groups at the phosphates. Amino acids are rarely considered as leaving groups, but proline can act as a leaving group when N‐linked to NMPs, so that prolinyl NMPs hydrolyze in aqueous buffer at 37 °C, with half‐life times as short as 2.4 h, and they act as monomers in enzyme‐free primer extension. Still, their level of reactivity is insufficient for practical purposes, requiring months for some extensions. Herein we report the synthesis of eight substituted prolinyl AMPs together with seven related compounds and the results of a study of their reactivity. A δ‐carboxy prolinyl NMP was found to be converted with a half‐life time of just 11 min in magnesium‐free buffer, and a δ‐isopropyl prolinyl NMP was shown to react sevenfold faster than its prolinyl counterpart in enzyme‐free genetic copying of RNA. Our results indicate that both anchimeric and steric effects can be employed to increase the reactivity of aminoacidyl nucleotides, i.e. compounds that combine two fundamental classes of biomolecules in one functional entity.Item Open Access Efficient oligomerization of aromatic amino acids induced by gaps in four‐helix bundles of DNA or RNA(2022) Doppleb, Olivia; Schwarz, Rainer Joachim; Landa, Maria; Richert, ClemensThe formation of peptides from amino acids is one of the processes associated with life. Because of the dominant role of translation in extant biology, peptide‐forming processes that are RNA induced are of particular interest. We have previously reported the formation of phosphoramidate‐linked peptido RNAs as the products of spontaneous condensation reactions between ribonucleotides and free amino acids in aqueous solution. We now asked whether four‐helix bundle (4HB) DNA or RNA folding motifs with a single‐ or double‐nucleotide gap next to a 5’‐phosphate can act as reaction sites for phosphoramidate formation. For glycine, this was found to be the case, whereas phenylalanine and tryptophan showed accelerated formation of peptides without a covalent link to the nucleic acid. Free peptides with up to 11 tryptophan or phenylalanine residues were found in precipitates forming in the presence of gap‐containing DNA or RNA 4HBs. Control experiments using motifs with just a nick or primer alone did not have the same effect. Because folded structures with a gap in a double helix are likely products of hybridization of strands formed in statistically controlled oligomerization reactions, our results are interesting in the context of prebiotic scenarios. Independent of a putative role in evolution, our findings suggest that for some aromatic amino acids an RNA‐induced pathway for oligomerization exists that does not have a discernable link to translation.Item Open Access Non-porous organic crystals and their interaction with guest molecules from the gas phase(2020) Casco, Mirian Elizabeth; Krupp, Felix; Grätz, Sven; Schwenger, Alexander; Damakoudi, Vassiliki; Richert, Clemens; Frey, Wolfgang; Borchardt, LarsSome organic molecules encapsulate solvents upon crystallization. One class of compounds that shows a high propensity to form such crystalline solvates are tetraaryladamantanes (TAAs). Recently, tetrakis(dialkoxyphenyl)-adamantanes have been shown to encapsulate a wide range of guest molecules in their crystals, and to stabilize the guest molecules against undesired reactions. The term ‘encapsulating organic crystals’ (EnOCs) has been coined for these species. In this work, we studied the behavior of three TAAs upon exposition to different guest molecules by means of sorption technique. We firstly measured the vapor adsorption/desorption isotherms with water, tetrahydrofuran and toluene, and secondly, we studied the uptake of methane on dry and wet TAAs. Uptake of methane beyond one molar equivalent was detected for wet crystals, even though the materials showed a lack of porosity. Thus far, such behavior, which we ascribe to methane hydrate formation, had been described for porous non-crystalline materials or crystals with detectable porosity, not for non-porous organic crystals. Our results show that TAA crystals have interesting properties beyond the formation of conventional solvates. Gas-containing organic crystals may find application as reservoirs for gases that are difficult to encapsulate or are slow to form crystalline hydrates in the absence of a host compound. Wet tetraaryladamantane crystals take up methane in form of methane hydrate structure I, even though they appear non-porous to argon.