Monomeric Building Blocks Research Articles

Total Chemical Synthesis of an Intra‐A‐Chain Cystathionine Human Insulin Analogue with Enhanced Thermal Stability

AbstractDespite recent advances in the treatment of diabetes mellitus, storage of insulin formulations at 4 °C is still necessary to minimize chemical degradation. This is problematic in tropical regions where reliable refrigeration is not ubiquitous. Some degradation byproducts are caused by disulfide shuffling of cystine that leads to covalently bonded oligomers. Consequently we examined the utility of the non‐reducible cystine isostere, cystathionine, within the A‐chain. Reported herein is an efficient method for forming this mimic using simple monomeric building blocks. The intra‐A‐chain cystathionine insulin analogue was obtained in good overall yield, chemically characterized and demonstrated to possess native binding affinity for the insulin receptor isoform B. It was also shown to possess significantly enhanced thermal stability indicating potential application to next‐generation insulin analogues.

Total Chemical Synthesis of an Intra-A-Chain Cystathionine Human Insulin Analogue with Enhanced Thermal Stability.

Despite recent advances in the treatment of diabetes mellitus, storage of insulin formulations at 4 °C is still necessary to minimize chemical degradation. This is problematic in tropical regions where reliable refrigeration is not ubiquitous. Some degradation byproducts are caused by disulfide shuffling of cystine that leads to covalently bonded oligomers. Consequently we examined the utility of the non-reducible cystine isostere, cystathionine, within the A-chain. Reported herein is an efficient method for forming this mimic using simple monomeric building blocks. The intra-A-chain cystathionine insulin analogue was obtained in good overall yield, chemically characterized and demonstrated to possess native binding affinity for the insulin receptor isoform B. It was also shown to possess significantly enhanced thermal stability indicating potential application to next-generation insulin analogues.

Development of a one-pot assay for screening and identification of Mur pathway inhibitors in Mycobacterium tuberculosis.

The cell wall of Mycobacterium tuberculosis (Mtb) consists of peptidoglycan, arabinogalactan and mycolic acids. The cytoplasmic steps in the peptidoglycan biosynthetic pathway, catalyzed by the Mur (A-F) enzymes, involve the synthesis of UDP-n-acetylmuramyl pentapeptide, a key precursor molecule required for the formation of the peptidoglycan monomeric building blocks. Mur enzymes are indispensable for cell integrity and their lack of counterparts in eukaryotes suggests them to be promising Mtb drug targets. However, the caveat is that most of the current assays utilize a single Mur enzyme, thereby identifying inhibitors against only one of the enzymes. Here, we report development of a one-pot assay that reconstructs the entire Mtb Mur pathway in vitro and has the advantage of eliminating the requirement for nucleotide intermediates in the pathway as substrates. The MurA-MurF enzymes were purified and a one-pot assay was developed through optimization of successive coupled enzyme assays using UDP-n-acetylglucosamine as the initial sugar substrate. The assay is biochemically characterized and optimized for high-throughput screening of molecules that could disrupt multiple targets within the pathway. Furthermore, we have validated the assay by performing it to identify D-Cycloserine and furan-based benzene-derived compounds with known Mur ligase inhibition as inhibitors of Mtb MurE and MurF.

De Novo Design of an Allosteric Metalloprotein Assembly with Strained Disulfide Bonds.

A major goal in metalloprotein design is to build protein scaffolds from scratch that allow precise control over metal coordination. A particular challenge in this regard is the construction of allosteric systems in which metal coordination equilibria are coupled to other chemical events that take place elsewhere in the protein scaffold. We previously developed a metal-templated self-assembly strategy (MeTIR) to build supramolecular protein complexes with tailorable interfaces from monomeric building blocks. Here, using this strategy, we have incorporated multiple disulfide bonds into the interfaces of a Zn-templated cytochrome cb562 assembly in order to create mechanical strain on the quaternary structural level. Structural and biophysical analyses indicate that this strain leads to an allosteric system in which Zn2+ binding and dissociation are remotely coupled to the formation and breakage of a disulfide bond over a distance of >14 Å. The breakage of this strained bond upon Zn2+ dissociation occurs in the absence of any reductants, apparently through a hydrolytic mechanism that generates a sulfenic acid/thiol pair.

Hydration effects on the electronic properties of eumelanin building blocks.

Theoretical results for the electronic properties of eumelanin building blocks in the gas phase and water are presented. The building blocks presently investigated include the monomeric species DHI (5,6-dihydroxyindole) or hydroquinone (HQ), DHICA (5,6-dihydroxyindole-2-carboxylic acid), indolequinone (IQ), quinone methide (MQ), two covalently bonded dimers [HM ≡ HQ + MQ and IM ≡ IQ + MQ], and two tetramers [HMIM ≡ HQ + IM, IMIM ≡ IM + IM]. The electronic properties in water were determined by carrying out sequential Monte Carlo/time dependent density functional theory calculations. The results illustrate the role played by hydrogen bonding and electrostatic interactions in the electronic properties of eumelanin building blocks in a polar environment. In water, the dipole moments of monomeric species are significantly increased ([54-79]%) relative to their gas phase values. Recently, it has been proposed that the observed enhancement of the higher-energy absorption intensity in eumelanin can be explained by excitonic coupling among eumelanin protomolecules [C.-T. Chen et al., Nat. Commun. 5, 3859 (2014)]. Here, we are providing evidence that for DHICA, IQ, and HMIM, the electronic absorption toward the higher-energy end of the spectrum ([180-220] nm) is enhanced by long-range Coulombic interactions with the water environment. It was verified that by superposing the absorption spectra of different eumelanin building blocks corresponding to the monomers, dimers, and tetramers in liquid water, the behaviour of the experimental spectrum, which is characterised by a nearly monotonic decay from the ultraviolet to the infrared, is qualitatively reproduced. This result is in keeping with a "chemical disorder model," where the broadband absorption of eumelanin pigments is determined by the superposition of the spectra associated with the monomeric and oligomeric building blocks.

Improved lactic acid productivity by simultaneous recovery during fermentation using resin exchanger

Lactic acid is a versatile organic acid that can be used in various applications. One of the promising applications of lactic acid is in bioplastic industry. Lactic acid is used as the monomer building block in polylactic acid synthesis. Unlike the existing applications in food and pharmaceutical industries in which lactic acid is used as the additive, it becomes the major raw material in polylactic acid production. This, therefore, raises the demand of lactic acid and eventually increases the market price. In order to expedite the bioplastic industry, cost competitiveness to the existing plastic is a big concern. The cost effectiveness and process robustness are the key success factors in bioplastic industry. Currently, lactic acid is produced via bacterial fermentation using the lime-based process. After fermentation, lactic acid was recovered from the fermentation broth by various techniques including solvent extraction, reactive distillation, ion exchanger, and electrodialysis. To achieve low production cost, not only high fermentation rate is necessary, effective recovery process is also required. In this study, we attempted simultaneous recovery of lactic acid coupled with fermentation to drive the productivity and long-term operation in continuous culture. Anion exchange resin, Amberlite IRA-400, was selected for recovering lactic acid from the bacterial culture. The preliminary results in batch adsorption showed that among other resins studied, Amberlite IRA-400 provided the best separation efficiency. Further study on fixed bed adsorption and simultaneous fermentation and lactic acid recovery using this resin were conducted and the results will be discussed in this presentation.

Onset of the Electronic Absorption Spectra of Isolated and π-Stacked Oligomers of 5,6-Dihydroxyindole: An Ab Initio Study of the Building Blocks of Eumelanin.

Eumelanin is a naturally occurring skin pigment which is responsible for developing a suntan. The complex structure of eumelanin consists of π-stacked oligomers of various indole derivatives, such as the monomeric building block 5,6-dihydroxyindole (DHI). In this work, we present an ab initio wave-function study of the absorption behavior of DHI oligomers and of doubly and triply π-stacked species of these oligomers. We have simulated the onset of the electronic absorption spectra by employing the MP2 and the linear-response CC2 methods. Our results demonstrate the effect of an increasing degree of oligomerization of DHI and of an increasing degree of π-stacking of DHI oligomers on the onset of the absorption spectra and on the degree of red-shift toward the visible region of the spectrum. We find that π-stacking of DHI and its oligomers substantially red-shifts the onset of the absorption spectra. Our results also suggest that the optical properties of biological eumelanin cannot be simulated by considering the DHI building blocks alone, but instead the building blocks indole-semiquinone and indole-quinone have to be considered as well. This study contributes to advancing the understanding of the complex photophysics of the eumelanin biopolymer.

N-containing covalent organic frameworks as supports for rhodium as transition-metal catalysts in hydroformylation reactions

Porous cross-linked polymers containing nitrogen donor functionalities are a promising class of materials for the immobilization of transition metal catalysts. These highly porous materials are built from ligand-containing building blocks using a bottom-up approach. Utilizing dinitrile containing monomers, highly porous covalent triazine frameworks (CTFs) are formed at elevated temperatures in molten zinc chloride. Containing a large number of accessible nitrogen coordination sites, metal species can be stabilized within these materials. The materials' porosity can be tuned, depending on the monomeric building block used and synthesis conditions. By immobilizing rhodium species within the framework followed by pre-activation, Rh@CTF materials are suitable as catalysts in the solvent-free hydroformylation of 1-octene. These Rh@CTF catalysts are not as active as conventional homogeneous benchmark catalysts; however, their activity and selectivity clearly outperform comparable carbon-based catalysts. A correlation between porosity and polarity of the support material and the observed catalytic activity could be established. Furthermore, CTF-based catalysts could be recycled easily with no further treatment for up to five runs with just a gradual deactivation. The leaching of active species has been studied intensively to elucidate the strength of the metal-support-interactions in this rather challenging reaction for an immobilized catalyst.

Principles Governing the Self-Assembly of Coiled-Coil Protein Nanoparticles

Self-assembly refers to the spontaneous organization of individual building blocks into higher order structures. It occurs in biological systems such as spherical viruses, which utilize icosahedral symmetry as a guiding principle for the assembly of coat proteins into a capsid shell. In this study, we characterize the self-assembling protein nanoparticle (SAPN) system, which was inspired by such viruses. To facilitate self-assembly, monomeric building blocks have been designed to contain two oligomerization domains. An N-terminal pentameric coiled-coil domain is linked to a C-terminal coiled-coil trimer by two glycine residues. By combining monomers with inherent propensity to form five- and threefold symmetries in higher order agglomerates, the supposition is that nanoparticles will form that exhibit local and global symmetry axes of order 3 and 5. This article explores the principles that govern the assembly of such a system. Specifically, we show that the system predominantly forms according to a spherical core-shell morphology using a combination of scanning transmission electron microscopy and small angle neutron scattering. We introduce a mathematical toolkit to provide a specific description of the possible SAPN morphologies, and we apply it to characterize all particles with maximal symmetry. In particular, we present schematics that define the relative positions of all individual chains in the symmetric SAPN particles, and provide a guide of how this approach can be generalized to nonspherical morphologies, hence providing unprecedented insights into their geometries that can be exploited in future applications.

Rhodium‐catalysed cyclisation by addition of unsaturated 1,4‐dicarbonyl compounds to oleic acid methyl ester

The Rh‐catalysis allows the one‐step synthesis of Diels–Alder‐like cycloadducts from mono‐unsaturated fatty acid and 1,4‐dicarbonyl components. In this article we report on the addition of maleic acid‐, fumaric acid‐ and acetylene dicarboxylic acid dimethyl ester to oleic acid methyl ester. In previous studies, Rh(OAc)2 showed the highest selectivity of the investigated catalysts towards the 3,6‐disubstituted‐1,2,3,6‐tetrahydro‐phthalic acid derivatives (THPA) for the addition of maleic anhydride to oleic acid. Thus, it was also used in this study. The relative configurations of the final products were identified by comparison with the Diels–Alder products, resulting from conjugated linoleic acid methylester (CLAMe) and the mentioned 1,4‐dicarbonyl compounds.Practical applications: The Rh‐catalysis is an appropriate method to synthesise Diels–Alder‐like cycloadducts from mono‐unsaturated fatty acids derivatives and 1,4‐dicarbonyl compounds in a one‐step process. The resulting products are considered to be used as detergents, corrosion inhibitors, plasticiser or new monomeric building blocks, e.g. for polycondensation processes. The obtained polyesters can be used for several applications, e.g. for coatings or adhesives, and lead to new products with promising properties.Diels–Alder‐like cycloadducts can be obtained in a one‐pot reaction from mono‐unsaturated fatty acids and 1,4‐dicarbonyl compounds using Rh‐catalysts. In contrast to common Diels–Alder reactions, a conjugated diene is not mandatory as educt, but will be generated in situ from the mono‐unsaturated fatty acid compound. The configurations of the resulting cyclocadducts are determined by NMR‐measurements.

Synthesis of triptycene based non-conjugated polytriazole: Temperature dependent regioselectivity and host–guest interaction with nitroaromatics

We report facile synthesis of a new triptycene based monomeric building block 2,6-diazidotriptycene (DAZT) that has been utilized to synthesize a series of triptycene based and triazole linked alternate co-polymer (TPT1-TPT4) via Cu(I)OAc catalyzed Sharpless “Click reaction”. The effect of reaction temperature on regioselective incorporation of 1,2,3-triazole motif in the polymer backbone has been studied. The newly synthesized triptycene-polytriazole (TPT) polymers (having 1,4-regioregular 1,2,3-triazole units) have been characterized using NMR, FTIR, and UV–vis spectroscopic techniques. Interestingly, TPTs possess moderately high molecular weight, are organosoluble, amorphous in nature and have reasonably high thermal stability with Tg > 400 °C. Additionally, selected polymers are fluorescent in nature. These fluorescent TPTs interact strongly with nitroaromatics (NACs), as evident from the binding constants values obtained from Benesi−Hildebrand fluorescence plots (assuming complexation 1:1). The extent of fluorescence quenching of these TPTs in presence of NACs was also estimated from Stern–Volmer (SV) plots. It was observed that polymer backbone which is richer in nitrogen, exhibited superior sensitivity towards NACs. Amongst the three NACs, PA is the most efficient quencher. KSV values of TPTs for various NACs are comparable with those of previously reported polymers.

Surface-Confined Amorphous Films from Metal-Coordinated Simple Phenolic Ligands

Coordination chemistry of natural polyphenols and transition metals allows rapid self-assembly of conformal coatings on diverse substrates. Herein, we report that this coordination-driven self-assembly process applies to simple phenolic molecules with monotopic or ditopic chelating sites (as opposed to macromolecular, multitopic polyphenols), leading to surface-confined amorphous films upon metal coordination. Films fabricated from gallic acid, pyrogallol, and pyrocatechol, which are the major monomeric building blocks of polyphenols, have been studied in detail. Pyrocatechol, with one vicinal diol group (i.e., bidentate), has been observed to be the limiting case for such assembly. This study expands the toolbox of available phenolic ligands for the formation of surface-confined amorphous films, which may find application in catalysis, energy, optoelectronics, and the biomedical sciences.

Proline-poor hydrophobic domains modulate the assembly and material properties of polymeric elastin.

Elastin is a self-assembling extracellular matrix protein that provides elasticity to tissues. For entropic elastomers such as elastin, conformational disorder of the monomer building block, even in the polymeric form, is essential for elastomeric recoil. The highly hydrophobic monomer employs a range of strategies for maintaining disorder and flexibility within hydrophobic domains, particularly involving a minimum compositional threshold of proline and glycine residues. However, the native sequence of hydrophobic elastin domain 30 is uncharacteristically proline-poor and, as an isolated polypeptide, is susceptible to formation of amyloid-like structures comprised of stacked β-sheet. Here we investigated the biophysical and mechanical properties of multiple sets of elastin-like polypeptides designed with different numbers of proline-poor domain 30 from human or rat tropoelastins. We compared the contributions of these proline-poor hydrophobic sequences to self-assembly through characterization of phase separation, and to the tensile properties of cross-linked, polymeric materials. We demonstrate that length of hydrophobic domains and propensity to form β-structure, both affecting polypeptide chain flexibility and cross-link density, play key roles in modulating elastin mechanical properties. This study advances the understanding of elastin sequence-structure-function relationships, and provides new insights that will directly support rational approaches to the design of biomaterials with defined suites of mechanical properties.

Origin of life. Primordial genetics: Information transfer in a pre-RNA world based on self-replicating beta-sheet amyloid conformers

The question of the origin of life on Earth can largely be reduced to the question of what was the first molecular replicator system that was able to replicate and evolve under the presumably very harsh conditions on the early Earth. It is unlikely that a functional RNA could have existed under such conditions and it is generally assumed that some other kind of information system preceded the RNA world. Here, I present an informational molecular system that is stable, self-replicative, environmentally responsive, and evolvable under conditions characterized by high temperatures, ultraviolet and cosmic radiation. This postulated pregenetic system is based on the amyloid fold, a functionally unique polypeptide fold characterized by a cross beta-sheet structure in which the beta strands are arranged perpendicular to the fiber axis. Beside an extraordinary structural robustness, the amyloid fold possesses a unique ability to transmit information by a three-dimensional templating mechanism. In amyloidogenesis short peptide monomers are added one by one to the growing end of the fiber. From the same monomeric subunits several structural variants of amyloid may be formed. Then, in a self-replicative mode, a specific amyloid conformer can act as a template and confer its spatially encoded information to daughter molecular entities in a repetitive way. In this process, the specific conformational information, the spatially changed organization, is transmitted; the coding element is the steric zipper structure, and recognition occurs by amino acid side chain complementarity. The amyloid information system fulfills several basic requirements of a primordial evolvable replicator system: (i) it is stable under the presumed primitive Earth conditions, (ii) the monomeric building blocks of the informational polymer can be formed from available prebiotic compounds, (iii) the system is self-assembling and self-replicative and (iv) it is adaptive to changes in the environment and evolvable.

227 Views of RNA: Is RNA Unique in Its Chemical Isomer Space?

Ribonucleic acid (RNA) is one of the two nucleic acids used by extant biochemistry and plays a central role as the intermediary carrier of genetic information in transcription and translation. If RNA was involved in the origin of life, it should have a facile prebiotic synthesis. A wide variety of such syntheses have been explored. However, to date no one-pot reaction has been shown capable of yielding RNA monomers from likely prebiotically abundant starting materials, though this does not rule out the possibility that simpler, more easily prebiotically accessible nucleic acids may have preceded RNA. Given structural constraints, such as the ability to form complementary base pairs and a linear covalent polymer, a variety of structural isomers of RNA could potentially function as genetic platforms. By using structure-generation software, all the potential structural isomers of the ribosides (BC5H9O4, where B is nucleobase), as well as a set of simpler minimal analogues derived from them, that can potentially serve as monomeric building blocks of nucleic acid–like molecules are enumerated. Molecules are selected based on their likely stability under biochemically relevant conditions (e.g., moderate pH and temperature) and the presence of at least two functional groups allowing the monomers to be incorporated into linear polymers. The resulting structures are then evaluated by using molecular descriptors typically applied in quantitative structure–property relationship (QSPR) studies and predicted physicochemical properties. Several databases have been queried to determine whether any of the computed isomers had been synthesized previously. Very few of the molecules that emerge from this structure set have been previously described. We conclude that ribonucleosides may have competed with a multitude of alternative structures whose potential proto-biochemical roles and abiotic syntheses remain to be explored. Key Words: Evolution—Chemical evolution—Exobiology—Prebiotic chemistry—RNA world. Astrobiology 15, 538–558.

Positional Scanning Synthesis of a Peptoid Library Yields New Inducers of Apoptosis that Target Karyopherins and Tubulin.

We describe the synthesis of a library of 11, 638 N-alkylglycine peptoid trimers in a positional scanning format with adjustment of reaction conditions to account for different reactivities of the monomer building blocks. Evaluation of the library by high-content phenotypic screening for modulators of the cytoskeleton and mitosis resulted in the identification of two apoptosis-inducing peptoids, which, despite their structural similarity, target different proteins and cellular mechanisms. Whereas one peptoid binds to karyopherins, which mediate nuclear transport, the other N-alkylglycine trimer binds tubulin at the vinca alkaloid binding site.

Isolation and quantification of oligomeric and polymeric procyanidins in leaves and flowers of Hawthorn (Crataegus spp.)

Proanthocyanidins (PAs) constitute a class of polyphenols with flavan-3-ols as monomeric building blocks. These polyphenols are mostly quantified by colorimetric methods or by chromatographic determination of monomeric flavan-3-ols or low molecular oligomers as lead compounds. No reliable analytical methods are available for unambiguous identification of the homologues series of oligo- and polymeric PAs.For Hawthorn leaf and flower (Crataegi folium cum flore) from Crataegus spp. (Rosaceae) a protocol for preparative isolation of oligomeric and polymeric PAs from an acetone–water extract was developed, yielding procyanidin reference clusters with defined degree of polymerization (DP) from 2 to 10 besides a procyanidin-polymer. Identity and purity of these clusters were proven by HPLC, MS and in part NMR studies.For identification and quantification from Hawthorn an ICH-Q2 validated UHPLC method with fluorimetric detection and less than 10min runtime was developed. The method enabled quantification of procyanidin clusters with DP from 2 to 10 besides the polymer fraction. Batch analysis revealed procyanidin contents of about 20 to 45mg/g from a homologues series of oligomeric PAs and about 50% of polymer fraction.Monitoring of procyanidin distribution during seasonal growth of fresh plants of Crataegus monogyna showed more or less constant contents between 20 and 55mg/g dry weight of oligomeric procyanidins during the growing season in the different plant organs with strong accumulation in the flowers and fruits (55mg/g dry weight). From these data it can be speculated that procyanidins serve as part of the plants defense system in the reproductive organs of the plant.

Isolation and Quantification of Oligomeric and Polymeric Procyanidins in the Aerial Parts of St.John's Wort (Hypericum perforatum).

Proanthocyanidins (condensed tannins) constitute a class of oligomeric and polymeric polyphenols with flavan-3-ols as monomeric building blocks. Despite the high impact of proanthocyanidins, these polyphenols are mostly quantified by colorimetric methods or by chromatographic determination of the flavan-3-ols as cleavage products or low molecular oligomers as lead compounds. For St. John's wort (Hyperici herba) from Hypericum perforatum, a protocol for preparative isolation of oligomeric and polymeric proanthocyanidins from an acetone-water extract by chromatography on Sephadex®LH20 in combination with preparative high-performance liquid chromatography on the diol stationary phase was developed, yielding procyanidin reference clusters with a defined degree of polymerization from 3 to 10. Identity and purity of these clusters was proven by high-performance liquid chromatography (RP18 and diol phase) and mass spectrometry. For identification and quantification of proanthocyanidin clusters from St. John's wort, an ICH-Q2 (International harmonized guideline for analytical validation) validated high-performance liquid chromatography method with fluorimetric detection was developed using an acetone-water extract of the herbal material, purified by solid-phase extraction for the removal of naphthodianthrones. The method enabled the quantification of procyanidin clusters with a degree of polymerization from 2 to 10. Analysis of nine batches of Hyperici herba from different sources indicated a high variability of proanthocyanidin content in the range from 8 to 37 mg/g. In all of the batches investigated, the trimer cluster DP3 was the dominant proanthocyanidin (about 40 %), followed by DP 4 (about 15 %) and DP5 (about 12 %). Monitoring of procyanidin distribution during seasonal growth of fresh plants of H. perforatum indicated the highest proanthocyanidin content in young plants (about 50 mg/g) and a time-dependent decrease during the growing season to about 16 mg/g. The highest proanthocyanidin content was found in young leaves and flowers, while the fruits were proanthocyanidin-free; older parts of the stem and the herb had a lower proanthocyanidin content. From these data, it can be concluded that proanthocyanidins serve as part of the plant defense system in the reproductive organs.

Influence of Interface Doping on Charge-Carrier Mobilities and Sub-Bandgap Absorption in Organic Solar Cells

P3HT:PCBM (poly(3-hexylthiophene-2,5-diyl):([6,6]-phenyl-C61-butyric acid methyl ester)-based bulk heterojunctions (BHJs) were doped by using 4-toluenesulfonic acid (TSA) as dopant. This approach was inspired by the well-known interfacial doping of the active layer via the electron-blocking layer PEDOT:PSS (poly(3,4-ethylenedioxy-thiophene):poly(styrenesulfonate)) at its interface. TSA is amphiphilic, acidic, and structurally very similar to the monomeric building block of PSS. Upon TSA doping, a notable increase in the light absorption in the sub-bandgap region of pristine P3HT was observed. These features are assigned to polaron transitions within P3HT; however, the TSA impact on polaron absorption features in the BHJ is rather small. Although, for small TSA concentrations and thick active layers (∼220 nm) the fill factor of the solar cells improved dramatically with increasing TSA content in the active layer, which is discussed in terms of contact resistances at interfaces in the present paper. For 0.5% TSA concentration in the active layer solution the maximum of the power conversion efficiency was obtained. At the same time, the reproducibility of solar cell performance parameters was considerably improved.

Detection of Lipid‐Linked Peptidoglycan Precursors by Exploiting an Unexpected Transpeptidase Reaction

Penicillin-binding proteins (PBPs) are involved in the synthesis and remodeling of bacterial peptidoglycan (PG). Staphylococcus aureus expresses four PBPs. Genetic studies in S. aureus have implicated PBP4 in the formation of highly cross-linked PG, but biochemical studies have not reached a consensus on its primary enzymatic activity. Using synthetic Lipid II, we show here that PBP4 preferentially acts as a transpeptidase (TP) in vitro. Moreover, it is the PBP primarily responsible for incorporating exogenous D-amino acids into cellular PG, implying that it also has TP activity in vivo. Notably, PBP4 efficiently exchanges D-amino acids not only into PG polymer, but also into the PG monomers Lipid I and Lipid II. This is the first demonstration that any TP domain of a PBP can activate the PG monomer building blocks. Exploiting the promiscuous TP activity of PBP4, we developed a simple, highly sensitive assay to detect cellular pools of lipid-linked PG precursors, which are of notoriously low abundance. This method, which addresses a longstanding problem, is useful for assessing how genetic and pharmacological perturbations affect precursor levels, and may facilitate studies to elucidate antibiotic mechanism of action. We are currently exploring this tool to study other PG-related processes