The history of discovery by Rosetta Reusch of oligo- and poly-beta-hydroxybutyrates (OHBs and PHBs) consisting of less than ca. 150 HB units is described. These 'short-chain' biopolymers can be detected in all living organisms and have numerous physiological activities of fundamental importance for the chemistry of life. The largest are components of ion channels such as Ca2+-polyphosphate-PHB (Ca-PPi-PHB) in genetically competent E. coli and in mammalian mitochondria. Sequences with chain lengths < ca. 30 occur covalently attached to proteins (post-translational PHBylation), and methyl esters of the dimer and trimer are used by certain bacteria as highly efficient antioxidants. With synthetic monodisperse OHBs (up to 128mer) our group has contributed structural investigations, and we have shown that OHBs >= 16 alone make phospholipid bilayer vesicles permeable to Ca ions. An extensive biochemical analysis of the TRPM8 protein channel, responsible for the sense of heat in our skin, proved to be fully active only when PHBylated. Reasons for the difficulty of detecting OHBs and PHBs are discussed: the polyester chain is highly flexible, and there is ester cleavage by base, acid, nucleophiles, Lewis acids, and heat - in stark contrast to peptides. PHBs may be called a ubiquitous but fleeting species in the chemistry of life - worth being appreciated and studied much more intensively in the future! A speculation about PHB's possible role in prebiotic compartmentalization is presented, and recent uses of compartmentalization in organic synthesis are briefly mentioned. Portions of the figures used herein were presented in a lecture at the International Symposium on Biopolymers on September 13, 2022, in Sion (Switzerland).

Cyanophycin is a natural biopolymer produced by a wide range of bacteria, consisting of a chain of poly- l -Asp residues with l -Arg residues attached to the β-carboxylate sidechains by isopeptide bonds. Cyanophycin is synthesized from ATP, aspartic acid and arginine by a homooligomeric enzyme called cyanophycin synthetase (CphA1). CphA1 has domains that are homologous to glutathione synthetases and muramyl ligases, but no other structural information has been available. Here, we present cryo-electron microscopy and X-ray crystallography structures of cyanophycin synthetases from three different bacteria, including cocomplex structures of CphA1 with ATP and cyanophycin polymer analogs at 2.6 Å resolution. These structures reveal two distinct tetrameric architectures, show the configuration of active sites and polymer-binding regions, indicate dynamic conformational changes and afford insight into catalytic mechanism. Accompanying biochemical interrogation of substrate binding sites, catalytic centers and oligomerization interfaces combine with the structures to provide a holistic understanding of cyanophycin biosynthesis.

The transfection ofCryptosporidiumrepresents a major challenge, and current protocols are based on electroporation of freshly excysted sporozoites using a rather large amount of plasmid DNA which typically has a very poor yield. In this study, we report a fast and simple protocol for transfection ofCryptosporidium parvumthat takes advantage of the DNA condensing power of the poly cationic polymer polyethylenimine (PEI) and the gene delivery property of the short cell-penetrating peptide octaarginine. Our novel protocol requires a very low amount of plasmid DNA and does not necessitate special laboratory equipment to be performed. Transfection appears to be more efficient in oocysts just triggered for excystation than the excysted sporozoites. Altogether, the application of octaarginine with PEI allows efficient transfection. To the best of our knowledge, this is the first report on an electroporation-free protocol for transfection of sporozoites of aCryptosporidiumspecies.

Teruaki Mukaiyama, formerly Professor at Tokyo Institute of Technology, Tokyo University, and Tokyo University of Science passed away on November 17, 2018. As one of the most productive organic chemists he has enriched the field of synthetic organic chemistry in 60 years of research. His most important contributions are reviewed herein by a close friend.

There are widely unknown enantiopure building blocks and non-conventional transformations described in this old work that could become useful in today's diversity-oriented organic synthesis world. Coupling and mixed couplings of functionalized CF3-substituted chiral radicals by Kolbe electrolysis of carboxylic acids lead to hexafluoro-hexane-2,5-diol and to butyro- and valerolactone derivatives with functional-group relationships that normally require components with reactivity umpolung. Oxidative decarboxylation of amino-acid and peptide derivatives by Hofer-Moest electrolyses provide entry into the synthetic use of chiral acyliminium-ion intermediates. Chiral oxazoline and thioazoline building blocks (from serine, threonine, and cysteine) are accessible for substitutions and cycloadditions. The stereochemical course of oxidative CO2H replacement in serine by nucleophilically introduced groups with retention of configuration is discussed.

Oligo‐arginines are thoroughly studied cell‐penetrating peptides ( CPP s, Figures 1 and 2 ). Previous in‐vitro investigations with the octaarginine salt of the phosphonate fosmidomycin (herbicide and anti‐malaria drug) have shown a 40‐fold parasitaemia inhibition with P. falciparum , compared to fosmidomycin alone ( Figure 3 ). We have now tested this salt, as well as the corresponding phosphinate salt of the herbicide glufosinate, for herbicidal activity with whole plants by spray application, hoping for increased activities, i.e . decreased doses. However, both salts showed low herbicidal activity, indicating poor foliar uptake ( Table 1 ). Another pronounced difference between in‐vitro and in‐vivo activity was demonstrated with various cell‐penetrating octaarginine salts of fosmidomycin: intravenous injection to mice caused exitus of the animals within minutes, even at doses as low as 1.4 μmol/kg ( Table 2 ). The results show that use of CPP s for drug delivery, for instance to cancer cells and tissues, must be considered with due care. The biopolymer cyanophycin is a poly‐aspartic acid containing argininylated side chains ( Figure 4 ); its building block is the dipeptide H‐ β Asp‐ α Arg‐ OH (H‐Adp‐ OH ). To test and compare the biological properties with those of octaarginines we synthesized Adp 8 ‐derivatives ( Figure 5 ). Intravenouse injection of H‐Adp 8 ‐ NH 2 into the tail vein of mice with doses as high as 45 μmol/kg causes no symptoms whatsoever ( Table 3 ), but H‐Adp 8 ‐ NH 2 is not cell penetrating ( HEK 293 and MCF ‐7 cells, Figure 6 ). On the other hand, the fluorescently labeled octamers FAM ‐(Adp( OM e)) 8 ‐ NH 2 and FAM ‐(Adp( NM e 2 )) 8 ‐ NH 2 with ester and amide groups in the side chains exhibit mediocre to high cell‐wall permeability ( Figure 6 ), and are toxic ( Table 3 ). Possible reasons for this behavior are discussed ( Figure 7 ) and corresponding NMR spectra are presented ( Figure 8 ).

Novel guanidinium-rich oligopeptide derivatives R-[Adp(X)](8)-NH2 are described, which consist of an octa-aspartic acid backbone with argininylated side chains that are derived from the biopolymer cyanophycin [H-(Adp)(n)-OH]. The Fmoc-Adp(X, Pbf)-OH building blocks for solidstate peptide synthesis (SSPS) of Adp octamers were prepared from Fmoc-Arg(Pbf)-OH and Fmoc-Asp-OAll. Coupling on PAL resin provided four octamers with and without N-terminal fluorescent groups (FAM) and C-terminal amide groups. Milligram quantities of Adp-octamers were isolated after preparative HPLC purification. The structure of the novel guanidinium-rich oligomers is unique insofar as the side chains of the Asp(8)-backbone include both a guanidino and a carboxylic acid group, the influence of which will be tested with the corresponding ester and amide derivatives that were synthesized in parallel. Unusual cell-penetrating properties of the Adp-octamers are expected.

We have shown for the first time that a natural protein (human insulin) can be acylated at the N ‐terminus with a β ‐amino acid (H‐ β 3 hAla‐), in a process catalyzed by the β ‐peptidyl aminopeptidase 3‐2W4‐BapA. This selective modification, which could also be applied for protein labeling and tagging, should be generally useful, also to protect peptides and proteins from attack by common aminopeptidases.

A quantitative analysis by confocal fluorescence microscopy of the entry into HEK293 and MCF‐7 cells by fluorescein‐labeled octaarginine (1) and by three octa‐Adp derivatives (2 – 4, octamers of the β‐Asp‐Arg‐dipeptide, derived from the biopolymer cyanophycin) is described, including the effects of the membrane dye R18 and of DMSO on cell penetration.

The lessons taught me by Jack Dunitz about the use of X-ray crystal structure analysis, far beyond just for determining the structure of an isolated product, are described. The direct and indirect influence Jack had on the way various topics of my synthetic group developed at ETH is demonstrated with selected examples from our various re-search areas: Li-enolates, the gem-diaryl effect and TADDOLs, self-regeneration of stereocenters, poly(hydroxybutanoates) (PHB), beta-peptides, and mechanistic investigations of organocatalytic reactions. Furthermore, the role Jack plays in the Laboratorium of Organic Chemistry at ETH Zurich is described and appreciated.

It has been suggested that the origin of regio‐ and stereoselectivity in Michael additions of pyrrolidine enamines is achieved by thermodynamic rather than kinetic control through distinct conformational preferences of the enamines. We assess this proposal by elaboration of a computational protocol that warrants sufficient accuracy. The small energy differences between the conformers necessitate a high accuracy of the electronic structure method which, in addition, must allow for computationally feasible calculations of a large number of conformers. Our protocol is based on density functional theory which we validated against explicitly correlated coupled cluster theory. The results are in agreement with the available experimental data, but illustrate that conformational preferences determined for one enamine are not readily transferable to other types of enamines. We found that an appropriate conformational sampling is inevitable to arrive at meaningful conclusions. Most prominently, s‐ cis and s‐ trans conformers are similarly stable for aldehyde‐ and ketone‐derived enamines. The regio‐ and stereoselectivity in Michael additions of pyrrolidine‐derived enamines can not be explained by pronounced stability differences of the enamine isomers and conformers in general, disproving the thermodynamic‐control hypothesis. The elucidation of the origin of regio‐ and stereoselectivity requires further theoretical investigations of the elementary steps of Michael additions.

In the formation of amyloid fibrils from small peptides, the appearance of superhelices of (P)- or (M)-helicity has been observed for the first time; high concentrations of the peptides and extended periods of incubation at physiological pH appear to be important for this phenomenon. In view of the general importance of peptide and protein aggregation, we give a brief overview with selected examples for demonstration.

β3‐Octaarginine chains were attached to the functional groups NH and CO2H of the antibacterial fluoroquinolones ciprofloxacin (→1) and enrofloxacin (→2), respectively, in order to find out whether the activity increases by attachment of the polycationic, cell‐penetrating peptide (CPP) moiety. For comparison, simple amides, 3–5, of the two antimicrobial compounds and β3‐octaarginine amide (βR8) were included in the antibacterial susceptibility tests to clarify the impact of chemical modification on the microbiological activity of either scaffold (Table).

An overview is given about our work on fluoro-organic compounds, published or described in PhD theses between 1977 and 2013. After a discussion of structural F-effects and F-tagging applications the material is ordered by the various areas of our research, in which we have used and/or prepared F-derivatives: Li- and Ti-organic compounds and reagents, polylithiated hydroxy-esters and nitroalkanes, the enantiopure trifluoro-lactic, -Roche, and -3-hydroxy-butanoic acids as toolbox for the preparation of numerous F3C-substituted compounds, including natural products and dendrimers, and fluoro-?-, -?-, and -?-amino acids, as well as peptides with back-bond-bound fluorine. The strong influence on ?-peptide folding by fluoro-substituents in the ?-position of ?-amino-acid residues is discussed in terms of the ?-fluoro-amide conformational effect. Finally, some cases of totally unexpected effects on reactivity and structure exerted by fluoro-substitution are presented and taken as examples for our use of the terms flustrate and flustration in connection with organo-fluorine chemistry.

Many years ago, β(2) /β(3) -peptides, consisting of alternatively arranged β(2) - and β(3) h-amino-acid residues, have been found to undergo folding to a unique type of helix, the 10/12-helix, and to exhibit non-polar, lipophilic properties (Helv. Chim. Acta 1997, 80, 2033). We have now synthesized such 'mixed' hexa-, nona-, dodeca-, and octadecapeptides, consisting of Val-Ala-Leu triads, with N-terminal fluorescein (FAM) labels, i.e., 1-4, and studied their interactions with POPC (=1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) giant unilamellar vesicles (GUVs) and with human white blood cancer cells U937. The methods used were microfluidic technology, fluorescence correlation spectroscopy (FCS), a flow-cytometry assay, a membrane-toxicity assay with the dehydrogenase G6PDH as enzymatic reporter, and visual microscopy observations. All β(3) /β(2) -peptide derivatives penetrate the GUVs and/or the cells. As shown with the isomeric β(3) /β(2) -, β(3) -, and β(2) -nonamers, 2, 5, and 6, respectively, the derivatives 5 and 6 consisting exclusively of β(3) - or β(2) -amino-acid residues, respectively, interact neither with the vesicles nor with the cells. Depending on the method of investigation and on the pretreatment of the cells, the β(3) /β(2) -nonamer and/or the β(3) /β(2) -dodecamer derivative, 2 and/or 3, respectively, cause a surprising disintegration or lysis of the GUVs and cells, comparable with the action of tensides, viral fusion peptides, and host-defense antimicrobial peptides. Possible sources of the chain-length-dependent destructive potential of the β(3) /β(2) -nona- and β(3) /β(2) -dodecapeptide derivatives, and a possible relationship with the phosphate-to-phosphate and hydrocarbon thicknesses of GUVs, and eukaryotic cells are discussed. Further investigations with other types of GUVs and of eukaryotic or prokaryotic cells will be necessary to elucidate the mechanism(s) of interaction of 'mixed' β(3) /β(2) -peptides with membranes and to evaluate possible biomedical applications.

The diastereoselective Mannich reaction of functionalized aldehydes, using a phenethylamine-derived iminium precursor, by activation with prolines and prolinol derivatives have been studied. Optimized reaction conditions have been developed, allowing for scale-up and preparation of γ-amino alcohol derivatives on multi-gram scale from β-hydroxypropanal and -butanal, with diastereoselectivites of typically >73:27 and yields of >60 %. After chromatographic diastereoisomer separation, hydrogenolytic debenzylation, enantiomerically pure Fmoc-β2-Ser(tBu)-OH and Fmoc-β2-Thr(tBu)-OH were thus prepared on multi-gram scale in 6 steps and with overall yields of 24 % and 10 %, respectively, starting from commercially available starting compounds.

While the powder charge flipping (pCF) algorithm has been applied successfully to a variety of inorganic compounds, reports on its application to organic structures, in particular those consisting of light atoms only, are rare. To investigate the reason for this apparent incongruity, a series of light-atom structures were tested using the pCF algorithm implemented in the program Superflip . The data sets, which covered varying degrees of reflection overlap, had resolutions of approximately 1 Å, and the structures ranged from 40 to 136 atoms per unit cell. Both centrosymmetric and noncentrosymmetric structures were investigated. A modified pCF approach, which was developed in a separate study, was tested on several compounds whose structures could not be solved by applying the basic pCF algorithm in Superflip . The results show that organic structures with no heavy atoms and low symmetry do indeed test the limits of the pCF algorithm in Superflip . The study has allowed a few guidelines for approaching such problems to be formulated.

The roles of polyhydroxy-butyrates/alkanoates (PHB/PHA) in biology, for the preparation of chiral building blocks, and as a source of inspiration for the discovery of β- and γ-peptides are discussed. The syntheses and structures of β-peptides are outlined. The prerequisites for mimicking peptide/protein interactions with β-peptides and two examples are presented. Single terminal β-amino-acid residues can lead to stabilization of peptides (cf. NTS(8-13)) in plasma. Cell-penetrating α-L-, α-D-, mixed α-L/D- and β-oligoarginines (OAs) and -oligoprolines, as well as the mechanism(s) of internalization are compared. Recent studies show that infected erythrocytes, parasitic organisms and mycobacteria are entered by OA-derivatives, which have been employed as transporters of the antibiotic fosmidomycin. While β-peptides are generally enzymatically stable (for days in mammals), a microorganism (S. xenopeptidilytica) with an Ntn enzyme (3-2W4 BapA) was discovered that cleaves only β-peptides, and that was applied in preparations of (enantiopure) β-amino acids and β-peptides.