The conformational stability of the polyproline II (PPII) helix with respect to the functional groups at the C- and N-termini was examined both experimentally and theoretically. Oligoprolines AcN-[Pro](12)-CONH(2) (1), HN-[Pro](12)-CONH(2) (2), AcN-[Pro](12)-CO(2)H (3), and HN-[Pro](12)-CO(2)H (4) with charged and capped termini served as model compounds, and the relative ease with which they switch from the PPII to the polyproline I (PPI) helix was used as a measure to analyze their conformational stabilities. CD spectroscopic studies demonstrate that a positively charged N-terminus and a negatively charged C-terminus destabilize the PPII helix and favor the PPI helix, whereas capped termini favor the PPII over the PPI helix. These experimental findings are supported by the energy differences between the PPII and PPI helices of oligoprolines 1-4 computed by ab initio methods including electron-correlation effects (second-order Møller-Plesset perturbation theory, MP2). Furthermore, these quantum-chemical calculations show that differences in charge-dipole interactions are responsible for the experimentally and computationally observed relative stabilities. Although these electrostatic interactions between the terminal charges and the amide dipoles stabilize both helices, they are significantly stronger in the PPI helix where the amide bonds are oriented almost linear to the helix axis as compared to the PPII helix in which the amides are nearly perpendicular to the axis. Moreover, we demonstrate that a negative charge at the C-terminus has a more pronounced effect on the relative stability as compared to a positive charge at the N-terminus due to destabilization of the PPII helix by repulsive interaction between the C-terminal carboxylate with the neighboring amide bond. Studies at different pH values verified the electrostatic nature of the observed effects and demonstrate how changes in the protonation state can be used to deliberately stabilize the PPII helix over the PPI helix or vice versa.

The attraction of H‐bonding: Proline derivatives with a C4‐endo ring pucker and a preference for the trans amide conformer are introduced as building blocks to tune the cis/trans amide conformer ratio in Xaa‐Pro bonds within peptides. Noncovalent interactions, such as hydrogen bonding between the substituent at C4 of the proline ring and the amide backbone are key for favoring or disfavoring the trans conformer (see picture).

The trans : cis isomer ratio of Xaa–Pro bonds is significantly affected by the polarity of the environment. Computational and NMR spectroscopic studies revealed an intricate balance between polarity effects and interactions of carbonyl groups.

Phosphonate natural products possess a range of biological activities as a consequence of their ability to mimic phosphate esters or tetrahedral intermediates formed in enzymatic reactions involved in carboxyl group metabolism. The dianionic form of these compounds at pH 7 poses a drawback with respect to their ability to mimic carboxylates and tetrahedral intermediates. Microorganisms producing phosphonates have evolved two solutions to overcome this hurdle: biosynthesis of monoanionic phosphinates containing two P-C bonds or esterification of the phosphonate group. The latter solution was first discovered for the antibiotic dehydrophos that contains a methyl ester of a phosphonodehydroalanine group. We report here the expression, purification, substrate scope, and structure of the O-methyltransferase from the dehydrophos biosynthetic gene cluster. The enzyme utilizes S-adenosylmethionine to methylate a variety of phosphonates including 1-hydroxyethylphosphonate, 1,2-dihydroxyethylphosphonate, and acetyl-1-aminoethylphosphonate. Kinetic analysis showed that the best substrates are tripeptides containing as C-terminal residue a phosphonate analog of alanine suggesting the enzyme acts late in the biosynthesis of dehydrophos. These conclusions are corroborated by the X-ray structure that reveals an active site that can accommodate a tripeptide substrate. Furthermore, the structural studies demonstrate a conformational change brought about by substrate or product binding. Interestingly, the enzyme has low substrate specificity and was used to methylate the clinical antibiotic fosfomycin and the antimalaria clinical candidate fosmidomycin, showing its promise for applications in bioengineering.antibiotics | bioengineering | conformational change | X-ray crystallography | domain-swap

The conformational properties of the polyproline I (PPI) helix of oligoprolines toward heating were examined. Oligoproline H-Pro(12)-NH(2) served as a model which adopts in n-PrOH a pronounced PPI conformation with all cis amide bonds, whereas a polyproline II (PPII) conformation with all trans amide bonds is predominant in pure aqueous buffer. CD spectroscopic studies revealed that a conformational change from the PPI to the PPII helix takes place upon heating and back to the PPI helix upon cooling. This conformational transition cycle is characterized by a strong hysteresis. With a quantitative fitting of the experimentally observed hysteresis loops by a newly developed iterative integration with different starting conditions, kinetic and thermodynamic parameters for the transition from the PPI to the PPII helical conformation were determined. The transition is as expected for cis-trans isomerizations of amide bonds comparatively slow (k = 0.003 s(-1) at 80 °C) and characterized by an activation energy E(a) of 81.1 ± 3.6 kJ mol(-1). Thermodynamically, the transition from the PPI to the PPII helix is characterized by a positive standard enthalpy (ΔH(0) = 33.5 ± 2.1 kJ min(-1)) and a positive standard entropy (ΔS(0) = 102 ± 6.6 J mol(-1) K(-1)).

Attraktive H‐Brücken: Prolinderivate mit einem C4‐endo‐Ringpucker und einer Präferenz für trans‐Amidbindungen werden als Bausteine eingesetzt, um das cis/trans‐Verhältnis der Amidbindung in Xaa‐Pro‐Einheiten von Peptiden einzustellen. Nichtkovalente Wechselwirkungen wie H‐Brücken zwischen dem Substituenten am Prolinring‐C4 und dem Amidrückgrat sind ausschlaggebend für die Begünstigung oder Benachteiligung des trans‐Konformers (siehe Bild).

Synthetic derivatives of the phosphonate antibiotic dehydrophos were tested for antimicrobial activity. Both the phosphonate monomethyl ester and the vinyl phosphonate moiety proved to be important for bacteriocidal activity of the natural product.

Azidoproline (Azp) containing oligoprolines are conformationally well-defined, helical molecular scaffolds that allow for facile functionalization. Within this article we describe the synthesis of Azp-containing oligoprolines and different strategies to introduce functional moieties. In addition, the influence of factors such as substituents at the y-position of proline as well as functional groups at the termini on the conformational stability of the molecular scaffolds are briefly presented.