A set of conformationally well-definded L/D-peptide epitopes provides a serological bar code for autoantibody subtypes
Plos One. 2018 13(8): e0201735 Andreas Schrimpf, Dörte Brödje, Petra Pfefferle, Armin Geyer.
DOI:10.1371/joural.pone.0201735
Supporting Information
Abstract Which conformational parameters lead to an antibody-affine peptide antigen? And in how many different conformations can we actually present the respective conformational epitope? To provide answers from a chemical point of view, we direct the bending and tethering of peptide backbones by the utilisation of a hydrophobic cluster, disulfides, and d-amino acids. Each mutation is employed pairwise on directly opposite sides of a β-hairpin. In combination, these synthetic modules guide the formation of complementary β-sheet-like structures, whereby the oppositely configured (l/d-)bi-disulfide pairs form with high regioselectivity. The conformational properties of the peptides are assessed by NMR spectroscopy and correlated with their antibody affinity in ELISA. From a pool of thus designed peptide antigens with distinctive complementary affinities against known rheumatoid arthritis (RA) autoantibodies, we select a set of epitopes for an immunoassay with sera of RA patients. We want to put emphasis on the idea, that the different conformational properties of the chosen antigens, containing the same epitope sequence, are mirrored in the distribution of autoantibody subtypes (or of the antibody polyclonality, respectively). Such directly comparable information can only be delivered by a set of peptides, rather than a single one. The hairpin-restriction technology of l/d-configured bi-disulfide amino acid pairs is not limited to RA but applicable to other shape-persistent hairpin motifs which are supposed to identify subgroups of protein receptors.
Hinge-Type Dimerization of Proteins by a Tetracysteine Peptide of High Pairing Specificity
Biochemistry, 2018, 57, 3658-3664. Andreas Schrimpf, Franziska Hempel, Aitao Li, Uwe Linne, Uwe G. Maier, Manfred T. Reetz, Armin Geyer. DOI: 10.1021/acs.biochem.8b00475
Supporting Information
Abstract Dimeric disulfide-linked peptides are formed by the regioselective oxidative folding of thiol precursors containing the CX3CX2CX3C tetracysteine motif. Here, we investigate the general applicability of this peptide as a dimerization motif for different proteins. By recombinant DNA technology, the peptide CHWECRGCRLVC was loaded with proteins, and functional homodimers were obtained upon oxidative folding. Attached to the N-terminus of the dodecapeptide, the prokaryotic enzyme limonene epoxide hydrolase (LEH) completely forms a covalent antiparallel dimer. In a diatom expression system, the monoclonal antibody CL4 mAb is released in its functional form when its natural CPPC central parallel hinge is exchanged for the designed tetra-Cys hinge motif. To improve our understanding of the regioselectivity of tetra-disulfide formation, we provoked the formation of heterodimeric hinge peptides by mixing two different tetra-Cys peptides and characterizing the heterodimer by mass spectrometry and nuclear magnetic resonance spectroscopy.
The stereodynamics of macrocyclic succinimide-thioethers
J. Pept. Sci. 2018, 24:e3075. Stefan Lenz, Philip Horx, Armin Geyer. DOI: 10.1002/psc.3075
Abstract Maleimide‐thiol coupling is a popular bioconjugation strategy, but little is known about the stereoselectivity and the stereodynamics of the succinimide thioether formed in a biopolymer environment. We used thiol 1,4‐addition for the macrocyclisation of 5 designed pentapeptides with the ringsize of hexapeptides because they incorporate the succinimide thioether (4‐8). Both succinimide diastereomers are observed in the constrained macrocyclic rings in each case. In spite of the low diastereoselectivity of the macrocyclisation reaction, there is a significant influence of the amino acid environment on the epimerization rate of the succinimide. Its half life can be as short as several hours at room temperature when Gly is the amino acid following the succinimide (peptide 8). On the contrary, no epimerization is detectable even after several weeks in the case of D‐Phe C‐terminal to the succinimide in peptide 4. Already the small selection of examples shows how big the differences in epimerization rates can be and that the local environment has a significant influence. The variation of amino acids in the vicinity of the ligation site points the way towards the synthesis of bioconjugates which are obtained as stable and separable diastereomers.
A synthetic marine sponge collagen by late-stage dihydroxylation
Org. Lett. 2018 20, 162 -165 Christoph Priem, Armin Geyer. DOI: 10.1021/acs.orglett.7b03525
Supporting Information
Abstract Based on the observation that an increased substrate size is paralleled by an enhanced diastereoselectivity, a late-stage dihydroxylation protocol toward the 21mer CMP (collagen model peptide) Ac-(Pro-Hyp-Gly)3-Pro-Dyp-Gly-(Pro-Hyp-Gly)3-NH2 is presented. C3 and C4 hydroxylation have a converse effect on the triple-helical stability of collagen. Their combined influence on the melting temperature was studied by NMR spectroscopy.
Scaling the amphiphilic character and antimicrobial activity of Gramicidin S by dihydroxylation or ketal formation
Christoph Priem, André Wuttke, Maryna Berditsch, Anne Ulrich, Armin Geyer.
J. Org. Chem. 2017, 82, 12366-121376. DOI: 10.1021/acs.joc.7b02177
Supporting Information
Abstract The acid lability of aliphatic ketals, which often serve as protection groups for 1,2-diols, is influenced by their local structural environment. The acetonide of the protected amino acid cis-dihydroxyproline (Dyp) is a typical protecting group cleavable by traces of TFA. The tricyclic acetonide of the dipeptide d-Hot═Tap is resistant to TFA and thus can serve as a bioorthogonal modification of bioactive peptides. With the aim of improving antimicrobial activity and hemolytic properties, we use these reactivity differences to scale the membrane affinity of the decapeptide Gramicidin S cyclo(d-Phe-Pro-Val-Orn-Leu-)2 (GS). The cis-dihydroxylated amino acids are used to increase the polarity of GS or obversely decrease the polarity by stereoselective ketal formation with an aliphatic ketone. While Dyp (GS mimetic 15) has only minimal influence on the biological properties of GS, d-Hot═Tap at the position of d-Phe1-Pro2 eradicates the biological activity (GS mimetic 16). The acid-stable ketals 17–19 are bioorthogonal modifications which reconstitute the biological activity of GS. We describe an improved synthesis of orthogonally protected Fmoc-Dyp-acetonide (9) and of several Fmoc-d-Hot═Tap-ketals for solid-phase peptide synthesis.
Reversible folding of a -hairpin peptide by a metal-chelating amino acid
Jan Reutzel, Tim M. Diogo, Armin Geyer.
Chem. Eur. J. 2017, 23, 8450-8456. DOI: 10.1002/chem.201700698
Supporting Information
Abstract Reversible peptide folding: 5-(1-Hydroxy-pyridin-2(1H)-onyl)-l-alanine (Hop), a redox stable N-hydroxy-1,2-pyridone-functionalized α-amino acid with the desired metal-chelating properties of DOPA (3,4-dihydroxy phenylalanine), was synthesized in eight steps with 96 % ee and used as Fmoc-protected amino acid for the assembly of a 14mer β-hairpin peptide. The secondary structure of the peptide, which was destabilized by the introduction l-Hop, could be reversibly reconstituted by subsequent additions of Ga3+ and a competitive metal chelator.
One-pot synthesis and antimicrobial activity of new 4,6-disubstituted-3,4-dihydropyrimidine-2(1H)-thiones
Mahmoud Al-Refai, Mohammad Ibrahim, Abdullah Al-Fawwaz, Armin Geyer
Eur. J. Chem. 2017, 8, 96-100. DOI: http://dx.doi.org/10.5155/eurjchem.8.1.96-100.154
Supplementary files
Abstract A series of 3,4-dihydropyrimidine-2(1H)-thiones (3a-i) were synthesized in moderate yields via a one-pot reaction of 3-acetyl-2,5-diclorothiophene (1), aryl aldehydes (2a-i) and thiourea in methanolic solution of potassium hydroxide under reflux conditions. All newly synthesized compounds were characterized by extensive NMR analysis, including 1D NMR experiments (1H and 13C) and 2D NMR experiments (COSY, HMBC and HSQC), as well as ESI-MS and HRESI-MS data. The antimicrobial activity of all new compounds (3a-f) was tested against bacteria and fungi. Thione derivative (3c) only showed activity against Staphylococcus aureus, Bacillus subtilis and Aspergillus niger.
Self-assembly of peptide boroxoles on cis-dihydroxylated oligoamide templates in water
André Wuttke, Armin Geyer.
J. Pept. Sci. 2017, 23, 549-555. DOI: 10.1002/psc.3007
Supporting Information
Abstract
We develop templates that can be used to stabilize consistent oligomers of a bioactive peptide. In the present study, we synthesize oligomers of an antibody epitope from the amyloidogenic prion protein. Dynamic covalent chemistry is the basis for the spontaneous condensation of 2, 3, 4 or 6 peptides with qualified polyol templates presenting the required number of bioorthogonal ligation sites. To study this process in aqueous solution, the N-terminal amino acid of a 13-mer peptide is first acylated with 4-carboxy-benzoboroxole (1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-5-carboxylic acid) and then mixed with the template to obtain self-assembled miniamyloids of specified degree of oligomerization. The template is assembled from bicyclic dipeptides of alternating d- and l-stereochemistry. The cis-diol group of this dipeptide hot=Tap (hot: d-hydroxythreonine, Tap: l-thiaproline) has sufficiently high affinity for boroxoles in water. A single N3-hot=Tap-OMe dipeptide template forms a 1 : 1 complex with 4-carboxy-benzoboroxole with excellent diastereoselectivity. The oligomeric template N3-(hot=Tap)n-OMe (n = 2, 3, 4 or 6) presents a regular pattern of 2, 3, 4 or 6 cis-diol groups for the spontaneous esterification with the same number of boronic acids. Nuclear magnetic resonance identifies the homogenous regioselectivity and stereoselectivity of this ligation process. The combination of electron-poor benzoboroxoles with this optimized cis-diol template allows for the complete ligation under high-dilution conditions in water with only 1.3 equivalents of peptide-boroxole per diol functionality. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Org. Biomol. Chem., 2017, 15, 2512-2521. DOI: 10.1039/C6OB02746F
Andreas Schrimpf, Uwe Linne, Armin Geyer.
Supplementary Information
Abstract We have designed a cysteine-rich β-hairpin peptide which dimerises spontaneously to the antiparallel double β-hairpin motif C1–C12′, C1′–C12, C5–C8, C5′–C8′-tricyclo-(CHWECCitGCRLVC)2. The highly regioselective oxidation of eight cysteines yields an intermolecular bi-disulfide 24mer hinge peptide from two individual 12mer β-hairpins, each rigidified by an additional intramolecular disulfide bond – all in all a tetra-disulfide. The reaction kinetics of air-oxidation were followed by HPLC and the constitutional isomer was identified by mass spectrometry. The hairpin conformation was characterised in detail by NMR spectroscopy and the opening angle of the antiparallel hinge was estimated from drift times obtained by ion-mobility spectrometry. Based on a set of investigated disulfide motifs, we are able to rationalise how the unbalanced number of bonded and non-bonded hydrogen pairs in a 12 mer hairpin causes their dimerisation. The unique dimeric bi-/tetra-disulfides provide systematic insights into β-hairpin formation. They can serve as a standalone structural element for the oligomerisation of peptide epitopes where structural diversity is generated from a minimal number of amino acids.
Bioorg. Med. Chem. 2017, 25, 603-608.
Sabrina Fischer, Matthias Lamping, Maike Gold, Yvonne Röttger, Dörte Brödje, Richard Dodel, Renate Frantz, Mobarak Abu Mraheil, Trinad Chakraborty, Armin Geyer.
Supplementary data Supplementary 3D model
Abstract The idea of privileged scaffolds – that there seem to be more bioactive compounds found around some structures than others – is well established for small drug molecules, but has little significance for standalone peptide secondary structures whose adaptable shapes escape the definition of a 3D motif in the absence of a protein scaffold. Here, we joined two independent biological functions in a single highly restricted peptide to support the hypothesis that the β-hairpin shape is the common basis of two otherwise unrelated biological recognition processes. To achieve this, the hydrophobic cluster HWX4LV from the decapeptide cyclic hairpin model peptide C1-C10 cyclo-CHWEGNKLVC was included in the bicyclic peptide 2. The designed β-hairpin peptide C4-C17, C8-C13 bicyclo-KHQCHWECTZGRCRLVCGRSGS (2, Z = citrulline), serves, on the one hand, as a specific epitope for rheumatoid autoantibodies and, on the other hand, shows a not negligible antibiotic effect against the bacterial strain E. coli AS19.
ChemBioChem 2016, 17, 2129-2132. DOI: 10.1002/cbic.201600479
Andreas Schrimpf, Armin Geyer.
Supporting Information
Abstract We have developed peptides that are able to distinguish between subgroups of polyclonal antibodies. These β-hairpin peptides act as conformational epitopes with specific shape and flexibility; they have been analyzed by NMR and CD spectroscopy, and have been shown to identify known disease markers. As a standalone mini β-sheet, a hairpin is stabilized by alternating pairs of hydrogen-bonded and non-bonded amino acids on its two opposing peptide strands. A single d mutation disrupts this secondary structure, the correlated double-d mutation of two opposing amino acids compensates for this destabilizing effect. The designed kink was introduced into both hydrogen-bonded and -non-bonded positions of an all-l hairpin that is a known conformational epitope in molecular recognition. Our peptides enabled the discrimination of different human rheumatoid arthritis autoantibodies in an ELISA assay.
RSC Adv., 2016, 6, 93343-93348. DOI: 10.1039/C6RA19624A
Maryna Abacilar, Fabian Daus, Christian Haas, Stephan Ingmar Brückner, Eike Brunner, Armin Geyer.
Abstract 15N, 13C, or both isotope labels were introduced in selected positions of several LCPAs containing 5, 11, or 13 nitrogens. The number of transformations during chemical synthesis was minimized by coupling Fmoc-glycine-15N, Fmoc-glycine-1-13C, or Fmoc-β-alanine simultaneously at both ends of the growing oligoamide chain on resin-linked norspermidine. LCPAs containing 10–20 nitrogens are the main organic constituent of diatom biosilica. Preliminary NMR studies of bioinspired silica nanocomposites obtained from double-labeled LCPA 14 and 29Si-enriched orthosilicic acid identified the close spatial arrangement of 29Si and 13C nuclei.
J. Pept. Sci. 2016, 22, 228-235. DOI: 10.1002/psc.2873
Matthias Lamping, Yvonne Grell, Armin Geyer.
Supporting Information
Abstract In this work the synthesis of a linear hexapeptide with a hydroxylamine functionality at the N-terminus and a ketone instead of the carboxylic acid at the C-terminus is described. Cyclization by ketoxime formation yields the 19-membered ring-expanded cyclic hexapeptide cyclo[Goly-Val-Ala-Pro-Leu-Kly] which adopts a main conformer with two intramolecular hydrogen bonds. The hydrolytic stability of a ketoxime lies between the inert amide and the labile imine. The substitution of an amide bond for an iminium bond transforms the irreversible macrocyclization into a reversible process, but macrocyclic imines are difficult to isolate because they are prone to hydrolysis. The enhanced chemical stability of the ketoxime justifies its application in ligation protocols. The detailed NMR analysis of a ketoxime linkage presented here identifies its local conformational preferences in a constrained peptide environment.
Org. Biomol. Chem., 2016, 14, 5032-5048. DOI: 10.1039/C6OB00565A
André Wuttke, Sebastian Nils Fischer, Annika Nebel, Michael Marsch, Armin Geyer.
Supplementary Information
Crystal structure
Front Cover 
Abstract The 6,7,8,8a-cis (all-cis) substituted δ-valerolactams of type 10, 11 and 12 are high-affinity diols for boronic ester formation, superior to the corresponding 6,7-trans analogues 1, 3 and 4. X-ray and NMR structure analysis have identified the differences of the six-membered ring conformations which cause the improved esterification properties of the all-cis stereoisomers. The homooligomeric all-cis δ-valerolactams 46–48 are used as polyol templates for the self-assembly of peptidic oligomers 49–52 by dynamic covalent chemistry. The templates have a diol spacing of approximately 5 Å, suitable for the assembly of branched peptides from the quantitative reaction between the peptide of interest, 2-formylphenylboronic acid and the respective template. According to this strategy, the tetrameric Aβ-miniamyloid 52 formed spontaneously from nine individual molecules in a three-component system. A detailed NMR analysis based on the complete sequential assignment of the trimeric Aβ(32–40)-miniamyloid 51 identified its three-dimensional structure in solution.
Synthesis, spectroscopic characterization and x-ray structure analysis of 6-(2,5-dichlorothiophen-3-yl)-2-methoxy-4-(4-methoxyphenyl)pyridine-3-carbonitrile.
J. Chem. Crystallogr. 2016, 46, 331-340. DOI: 10.1007/s10870-016-0661-z
Mahmoud Al-Refai, Mohammad M. Ibrahim, Armin Geyer, Michael Marsch, Badem F. Ali.
Abstract The title compound was prepared by the condensation of an equimolar mixture of 1-(2,5-dichlorothiophen-3-yl)-3-(4-methoxyphenyl)prop-2-en-1-one, malononitrile and sodium hydroxide. The molecular structure was fully characterized using different spectroscopic methods. Mass ESI–HRMS measurements were performed. The HRESIMS analysis revealed the molecular formula, C18H12Cl2N2O2SNa, with [M + Na]+ and [M + Na + 2]+ and [M + Na + 4]+ isotopic clusters characteristic for a dichlorinated compound. The compound was also thoroughly characterized by 1H, 13C, NMR spectra and 2D NMR spectra (COSY, HSQC and HMBC). The molecular structure was confirmed by X-ray single crystal analysis. The new compound crystallizes in the orthorhombic, Pbcn space group with unit cell dimensions: a = 31.901(7) Å, b = 15.412(4) Å, c = 7.3655(14) Å, V = 3621.3(13) Å3 and Z = 8. In the title compound, the central pyridine ring carries four substituents, a thiophene ring, a methoxyphenyl ring, a carbonitrile group and a methoxy group. The dihedral angles between the planes of the pyridine ring, the thiophene ring and the methoxyphenyl ring are 36.66 and 40.18°, respectively. Intermolecular C–H···O/N, π···π and anion···π [Cl···π] interactions are found in the crystal structure. All interactions consolidate a three dimensional network.
Reversible boronic ester formation of ribopyranosylated glycopeptides
Chemistry Select 2016, 1, 4570-4576. DOI: 10.1002/slct.201601240
Romina Kirschner, Armin Geyer.
Supporting Information
Abstract Six natural amino acids bearing hydroxy or thiol groups are transformed into N-Fmoc and O-acetyl protected ribopyranosylated amino acids (Raa) for further use in solid-phase peptide synthesis (SPPS). Although the different O- and S-glycosidic bonds influence the 1C4/4C1 equilibrium of the ribopyranosyl side chain significantly, the oligopeptides containing Raa are capable of spontaneous boronic ester formation. Ligation of model peptides with pyrene boronic acid and competition experiments between different peptides are monitored by NMR spectroscopy. In addition to boronic ester formation, we further propose ribopyranosylated peptides with adjustable orientation of the trihydroxy ax-eq-ax disposition for other applications.
Langmuir 2016, 32, 10144-10152. DOI: 10.1021/acs.langmuir.6b02575
Oliver Gräb, Maryna Abacilar, Fabian Daus, Armin Geyer, Claudia Steinem.
Abstract Long-chain polyamines (LCPAs) are intimately involved in the biomineralization process of diatoms taking place in silica deposition vesicles being acidic compartments surrounded by a lipid bilayer. Here, we addressed the question whether and how LCPAs interact with lipid membranes composed of glycerophospholipids and glyceroglycolipids mimicking the membranes of diatoms and higher plants. Solid supported lipid bilayers and monolayers containing the three major components that are unique in diatoms and higher plants, i.e., monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG), were prepared by spreading small unilamellar vesicles. The integrity of the membranes was investigated by fluorescence microscopy and atomic force microscopy showing continuous flat bilayers and monolayers with small protrusions on top of the membrane. The addition of a synthetic polyamine composed of 13 amine groups separated by a propyl spacer (C3N13) results in flat but three-dimensional membrane stacks within minutes. The membrane stacks are connected with the underlying membrane as verified by fluorescence recovery after photobleaching experiments. Membrane stack formation was found to be independent of the lipid composition; i.e., neither glyceroglycolipids nor negatively charged lipids were required. However, the formation process was strongly dependent on the chain length of the polyamine. Whereas short polyamines such as the naturally occurring spermidine, spermine, and the synthetic polyamines C3N4 and C3N5 do not induce stack formation, those containing seven and more amine groups (C3N7, C3N13, and C3N18) do form membrane stacks. The observed stack formation might have implications for the stability and expansion of the silica deposition vesicle during valve and girdle band formation in diatoms.
