Benzyl tert‐but­oxy­carbonyl‐α‐amino­isobutyrate

The small synthetic peptide, benzyl 2‐(tert‐but­oxy­carbonyl‐amino)­isobutyrate, C₁₆H₂₃NO₄, has the α‐helical conformation [|?| = 55.8 (2)° and |ψ| = 37.9 (2)°] observed in peptide fragments of peptaibols containing the α‐amino­isobutyric acid (Aib) residue. The structure shows no intramolecular hydrogen bonding, which would disrupt the limited conformational freedom associated with this amino acid. Two weak intermolecular hydrogen contacts are observed.     

Increased carrier mobility in end-functionalized oligosilanes

We show that a class of oligosilane–arene σ, π-hybrid materials exhibits distinct and enhanced solid-state electronic properties relative to its parent components. In the single crystal structure, the σ-conjugation axis of one molecule points towards the π-face of a neighboring molecule due to an unusual gauche conformation. This organization is hypothesized to be beneficial for charge transport. We show that solution-deposited crystalline films of the hybrid materials show up to a 100-fold increase in space-charge limited current (SCLC) mobility relative to literature reports of photoinduced hole transport in oligosilane films. The discovery that σ, π-hybrids are more than the sum of their parts offers a design opportunity for new materials.     

Facet selectivity in gold binding peptides: exploiting interfacial water structure

Peptide sequences that can discriminate between gold facets under aqueous conditions offer a promising route to control the growth and organisation of biomimetically-synthesised gold nanoparticles. Knowledge of the interplay between sequence, conformations and interfacial properties is essential for predictable manipulation of these biointerfaces, but the structural connections between a given peptide sequence and its binding affinity remain unclear, impeding practical advances in the field. These structural insights, at atomic-scale resolution, are not easily accessed with experimental approaches, but can be delivered via molecular simulation. A current unmet challenge lies in forging links between predicted adsorption free energies derived from enhanced sampling simulations with the conformational ensemble of the peptide and the water structure at the surface. To meet this challenge, here we use an in situ combination of Replica Exchange with Solute Tempering with Metadynamics simulations to predict the adsorption free energy of a gold-binding peptide sequence, AuBP1, at the aqueous Au(111), Au(100)(1 × 1) and Au(100)(5 × 1) interfaces. We find adsorption to the Au(111) surface is stronger than to Au(100), irrespective of the reconstruction status of the latter. Our predicted free energies agree with experiment, and correlate with trends in interfacial water structuring. For gold, surface hydration is predicted as a chief determining factor in peptide–surface recognition. Our findings can be used to suggest how shaped seed-nanocrystals of Au, in partnership with AuBP1, could be used to control AuNP nanoparticle morphology.     

Conformational Preferences of a β‐Octapeptide as Function of Solvent and Force‐Field Parameters

The ability to design properly folded β‐peptides with specific biological activities requires detailed insight into the relationship between the amino acid sequence and the secondary and/or tertiary structure of the peptide. One of the most frequently used spectroscopic techniques for resolving the structure of a biomolecule is NMR spectroscopy. Because only signal intensities and frequencies are recorded in the experiment, a conformational interpretation of the recorded data is not straightforward, especially for flexible molecules. The occurrence of conformational and/or time averaging, and the limited amount and accuracy of experimental data hamper the precise conformational determination of a biomolecule. In addition, the relation between experimental observables with the underlying conformational ensemble is often only approximately known, thereby aggravating the difficulty of structure determination of biomolecules. The problematic aspects of structure refinement based on NMR nuclear Overhauser effect (NOE) intensities and ³J‐coupling data are illustrated by simulating a β‐octapeptide in explicit MeOH and H₂O as solvents using three different force fields. NMR Data indicated that this peptide would fold into a 3₁₄‐helix in MeOH and into a hairpin in H₂O. Our analysis focused on the conformational space visited by the peptide, on structural properties of the peptide, and on agreement of the MD trajectories with available NMR data. We conclude that 1) although the 3₁₄‐helical structure is present when the peptide is solvated in MeOH, it is not the only relevant conformation, and that 2) the NMR data set available for the peptide, when solvated in H₂O, does not provide sufficient information to derive a single secondary structure, but rather a multitude of folds that fulfill the NOE data set.     

Deciphering the Non‐Equivalence of Serine and Threonine O‐Glycosylation Points: Implications for Molecular Recognition of the Tn Antigen by an anti‐MUC1 Antibody

The structural features of MUC1‐like glycopeptides bearing the Tn antigen (α‐O‐GalNAc‐Ser/Thr) in complex with an anti MUC‐1 antibody are reported at atomic resolution. For the α‐O‐GalNAc‐Ser derivative, the glycosidic linkage adopts a high‐energy conformation, barely populated in the free state. This unusual structure (also observed in an α‐S‐GalNAc‐Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α‐O‐GalNAc‐Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non‐equivalence of Ser and Thr O‐glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti‐MUC1 antibodies.     

Microdialysis SPR: diffusion-gated sensing in blood

Chemical measurements are rarely performed in crude blood due to the poor performance of sensors and devices exposed to biofluids. In particular, biosensors have been severely limited for detection in whole blood due to surface fouling from proteins, the interaction of cells with the sensor surface and potential optical interference when considering optical methods of analysis. To solve this problem, a dialysis chamber was introduced to a surface plasmon resonance (SPR) biosensor to create a diffusion gate for large molecules. This dialysis chamber relies on the faster migration of small molecules through a microporous membrane towards a sensor, located at a specified distance from the membrane. Size filtering and diffusion through a microporous membrane restricted the access of blood cells and larger biomolecules to a sensing chamber, while smaller, faster diffusing biomolecules migrated preferentially to the sensor with limited interference from blood and serum. The affinity of a small peptide (DBG178) with anti-atherosclerotic activity and targeting type B scavenger receptor CD36 was successfully monitored at micromolar concentrations in human serum and blood without any pre-treatment of the sample. This concept could be generally applied to a variety of targets for biomolecular interaction monitoring and quantification directly in whole blood, and could find potential applications in biochemical assays, pharmacokinetic drug studies, disease treatment monitoring, implantable plasmonic sensors, and point-of-care diagnostics.     

A prochelator peptide designed to use heterometallic cooperativity to enhance metal ion affinity

A peptide has been designed so that its chelating affinity for one type of metal ion regulates its affinity for a second, different type of metal ion. The prochelator peptide (PCP), which is a fusion of motifs evocative of calcium loops and zinc fingers, forms a 1 : 2 Zn : peptide complex at pH 7.4 that increases its affinity for Zn²⁺ ∼3-fold in the presence of Tb³⁺ (log β₂ from 13.8 to 14.3), while the 1 : 1 luminescent complex with Tb³⁺ is brighter, longer lived, and 20-fold tighter in the presence of Zn²⁺ (log K from 6.2 to 7.5). This unique example of cooperative, heterometallic allostery in a biologically compatible construct suggests the possibility of designing conditionally active metal-binding agents that could respond to dynamic changes in cellular metal status.     

Stereoselective synthesis of macrocyclic peptides via a dual olefin metathesis and ethenolysis approach

Macrocyclic compounds occupy an important chemical space between small molecules and biologics and are prevalent in many natural products and pharmaceuticals. The growing interest in macrocycles has been fueled, in part, by the design of novel synthetic methods to these compounds. One appealing strategy is ring-closing metathesis (RCM) that seeks to construct macrocycles from acyclic diene precursors using defined transition-metal alkylidene catalysts. Despite its broad utility, RCM generally gives rise to a mixture of E- and Z-olefin isomers that can hinder efforts for the large-scale production and isolation of such complex molecules. To address this issue, we aimed to develop methods that can selectively enrich macrocycles in E- or Z-olefin isomers using an RCM/ethenolysis strategy. The utility of this methodology was demonstrated in the stereoselective formation of macrocyclic peptides, a class of compounds that have gained prominence as therapeutics in drug discovery. Herein, we report an assessment of various factors that promote catalyst-directed RCM and ethenolysis on a variety of peptide substrates by varying the olefin type, peptide sequence, and placement of the olefin in macrocycle formation. These methods allow for control over olefin geometry in peptides, facilitating their isolation and characterization. The studies outlined in this report seek to expand the scope of stereoselective olefin metathesis in general RCM.     

Speeding up biomolecular interactions by molecular sledding

Numerous biological processes involve association of a protein with its binding partner, an event that is preceded by a diffusion-mediated search bringing the two partners together. Often hindered by crowding in biologically relevant environments, three-dimensional diffusion can be slow and result in long bimolecular association times. Similarly, the initial association step between two binding partners often represents a rate-limiting step in biotechnologically relevant reactions. We demonstrate the practical use of an 11-a.a. DNA-interacting peptide derived from adenovirus to reduce the dimensionality of diffusional search processes and speed up associations between biological macromolecules. We functionalize binding partners with the peptide and demonstrate that the ability of the peptide to one-dimensionally diffuse along DNA results in a 20-fold reduction in reaction time. We also show that modifying PCR primers with the peptide sled enables significant acceleration of standard PCR reactions.     

Mechanisms and energetics of free radical initiated disulfide bond cleavage in model peptides and insulin by mass spectrometry

We investigate the mechanism of disulfide bond cleavage in gaseous peptide and protein ions initiated by a covalently-attached regiospecific acetyl radical using mass spectrometry (MS). Highly selective S–S bond cleavages with some minor C–S bond cleavages are observed by a single step of collisional activation. We show that even multiple disulfide bonds in intact bovine insulin are fragmented in the MS2 stage, releasing the A- and B-chains with a high yield, which has been challenging to achieve by other ion activation methods. Yet, regardless of the previous reaction mechanism studies, it has remained unclear why (1) disulfide bond cleavage is preferred to peptide backbone fragmentation, and why (2) the S–S bond that requires the higher activation energy conjectured in previously suggested mechanisms is more prone to be cleaved than the C–S bond by hydrogen-deficient radicals. To probe the mechanism of these processes, model peptides possessing deuterated β-carbon(s) at the disulfide bond are employed. It is suggested that the favored pathway of S–S bond cleavage is triggered by direct acetyl radical attack at sulfur with concomitant cleavage of the S–S bond (S_H2). The activation energy for this process is substantially lower by ∼9–10 kcal mol^–1 than those of peptide backbone cleavage processes determined by density functional quantum chemical calculations. Minor reaction pathways are initiated by hydrogen abstraction from the α-carbon or the β-carbon of a disulfide, followed by β-cleavages yielding C–S or S–S bond scissions. The current mechanistic findings should be generally applicable to other radical-driven disulfide bond cleavages with different radical species such as the benzyl and methyl pyridyl radicals.     

Convergent chemo-enzymatic synthesis of mannosylated glycopeptides; targeting of putative vaccine candidates to antigen presenting cells

The combination of solid phase peptide synthesis and endo-β-N-acetylglucosaminidase (ENGase) catalysed glycosylation is a powerful convergent synthetic method allowing access to glycopeptides bearing full-length N-glycan structures. Mannose-terminated N-glycan oligosaccharides, produced by either total or semi-synthesis, were converted into oxazoline donor substrates. A peptide from the human cytomegalovirus (CMV) tegument protein pp65 that incorporates a well-characterised T cell epitope, containing N-acetylglucosamine at specific Asn residues, was accessed by solid phase peptide synthesis, and used as an acceptor substrate. High-yielding enzymatic glycosylation afforded glycopeptides bearing defined homogeneous high-mannose N-glycan structures. These high-mannose containing glycopeptides were tested for enhanced targeting to human antigen presenting cells (APCs), putatively mediated via the mannose receptor, and for processing by the APCs for presentation to human CD8+ T cells specific for a 9-mer epitope within the peptide. Binding assays showed increased binding of glycopeptides to APCs compared to the non-glycosylated control. Glycopeptides bearing high-mannose N-glycan structures at a single site outside the T cell epitope were processed and presented by the APCs to allow activation of a T cell clone. However, the addition of a second glycan within the T cell epitope resulted in ablation of T cell activation. We conclude that chemo-enzymatic synthesis of mannosylated glycopeptides enhances uptake by human APCs while preserving the immunogenicity of peptide epitopes within the glycopeptides, provided those epitopes are not themselves glycosylated.     

Efficient Gene Transfection through Inhibition of β‐Sheet (Amyloid Fiber) Formation of a Short Amphiphilic Peptide by Gold Nanoparticles

The effect of citrate‐stabilized gold nanoparticles (AuNPs) on the secondary structure of an artificial β‐sheet‐forming cationic peptide has been studied. The AuNPs inhibited β‐sheet formation and led to fragmented fibrils and spherical oligomers with assembled AuNPs on their surface. Besides this structural change, the functional properties of the peptide are also different. Whereas the peptide was unable to act as a vector for gene delivery, formation of a complex with AuNPs allowed successful gene delivery into cells.     

Activating peptides for cellular uptake via polymerization into high density brushes

The utility of peptide therapeutics is thwarted by an inability to enter cells, preventing access to crucial intracellular targets. Herein, we describe a simple and potentially widely applicable solution involving the polymerization of a minimally modified amino acid sequence into a high density brush polymer. Specifically, non-cell penetrating peptides can be rendered competent for cell entry by first including a single Arg or Lys in their amino acid sequence, if one is not already present, along with a norbornenyl unit. This modified monomer is then polymerized by ring opening metathesis polymerization (ROMP). To demonstrate the utility of this strategy, a known therapeutic peptide, which does not penetrate cells on its own, was polymerized. The resulting polymer proficiently entered cells while maintaining its intracellular function. We anticipate that this methodology will find broad use in medicine, increasing or enabling the in vivo efficacy of promising peptide therapeutics.     

The extent of protein hydration dictates the preference for heterogeneous or homogeneous nucleation generating either parallel or antiparallel β-sheet α-synuclein aggregates

α-Synuclein amyloid self-assembly is the hallmark of a number of neurodegenerative disorders, including Parkinson''s disease, although there is still very limited understanding about the factors and mechanisms that trigger this process. Primary nucleation has been observed to be initiated in vitro at hydrophobic/hydrophilic interfaces by heterogeneous nucleation generating parallel β-sheet aggregates, although no such interfaces have yet been identified in vivo. In this work, we have discovered that α-synuclein can self-assemble into amyloid aggregates by homogeneous nucleation, without the need of an active surface, and with a preference for an antiparallel β-sheet arrangement. This particular structure has been previously proposed to be distinctive of stable toxic oligomers and we here demonstrate that it indeed represents the most stable structure of the preferred amyloid pathway triggered by homogeneous nucleation under limited hydration conditions, including those encountered inside α-synuclein droplets generated by liquid–liquid phase separation. In addition, our results highlight the key role that water plays not only in modulating the transition free energy of amyloid nucleation, and thus governing the initiation of the process, but also in dictating the type of preferred primary nucleation and the type of amyloid polymorph generated depending on the extent of protein hydration. These findings are particularly relevant in the context of in vivo α-synuclein aggregation where the protein can encounter a variety of hydration conditions in different cellular microenvironments, including the vicinity of lipid membranes or the interior of membraneless compartments, which could lead to the formation of remarkably different amyloid polymorphs by either heterogeneous or homogeneous nucleation.     

Flaws in foldamers: conformational uniformity and signal decay in achiral helical peptide oligomers

Although foldamers, by definition, are extended molecular structures with a well-defined conformation, minor conformers must be populated at least to some extent in solution. We present a quantitative analysis of these minor conformers for a series of helical oligomers built from achiral but helicogenic α-amino acids. By measuring the chain length dependence or chain position dependence of NMR or CD quantities that measure screw-sense preference in a helical oligomer, we quantify values for the decay constant of a conformational signal as it passes through the molecular structure. This conformational signal is a perturbation of the racemic mixture of M and P helices that such oligomers typically adopt by the inclusion of an N or C terminal chiral inducer. We show that decay constants may be very low (<1% signal loss per residue) in non-polar solvents, and we evaluate the increase in decay constant that results in polar solvents, at higher temperatures, and with more conformationally flexible residues such as Gly. Decay constants are independent of whether the signal originates from the N or the C terminus. By interpreting the decay constant in terms of the probability with which conformations containing a screw-sense reversal are populated, we quantify the populations of these alternative minor conformers within the overall ensemble of secondary structures adopted by the foldamer. We deduce helical persistence lengths for Aib polymers that allow us to show that in a non-polar solvent a peptide helix, even in the absence of chiral residues, may continue with the same screw sense for approximately 200 residues.     

Monolayer formation of short helical turn forming peptide derivatives at the air–water and air–solid interfaces

Two tetrapeptide derivatives [peptide A (Boc–Ala–Ile–Ile–Gly–OMe) and peptide B (Boc–Ala–Ile–Leu–Ser–OMe)], that take helical turn conformation in solution, were shown to form monolayer at the air/water interface. Circular dichroism (CD) measurements indicate that peptide A has more helical turn propensity than peptide B in sodium dodecyl sulphate (SDS) micelles. Langmuir–Blodgget film study of peptides A and B suggest that both the peptides form stable monolayer at the air/water interface. Spectroscopic investigations reveal that peptide A forms helical turn assemblage on transferring the film into hydrophilic quartz and hydrophobic ZnSe surfaces. Whereas, peptide B adopts β-sheet structure on hydrophilic surface and a mixture of β-sheet and helical turn conformation on hydrophobic surface.     

A Multicomponent Conjugation Strategy to Unique N‐Steroidal Peptides: First Evidence of the Steroidal Nucleus as a β‐Turn Inducer in Acyclic Peptides

Constraining small peptides into specific secondary structures has been a major challenge in peptide ligand design. So far, the major solution for decreasing the conformational flexibility in small peptides has been cyclization. An alternative is the use of topological templates, which are able to induce and/or stabilize peptide secondary structures by means of covalent attachment to the peptide. Herein a multicomponent strategy and structural analysis of a new type of peptidosteroid architecture having the steroid as N‐substituent of an internal amide bond is reported. The approach comprises the one‐pot conjugation of two peptide chains (or amino acid derivatives) to aminosteroids by means of the Ugi reaction to give a unique family of N‐steroidal peptides. The conjugation efficiency of a variety of peptide sequences and steroidal amines, as well as their consecutive head‐to‐tail cyclization to produce chimeric cyclopeptide–steroid conjugates, that is, macrocyclic lipopeptides, was assessed. Determination of the three‐dimensional structure of an acyclic N‐steroidal peptide in solution proved that the bulky, rigid steroidal template is capable of both increasing significantly the conformational rigidity, even in a peptide sequence as short as five amino acid residues, and inducing a β‐turn secondary structure even in the all‐s‐trans isomer. This report provides the first evidence of the steroid skeleton as β‐turn inducer in linear peptide sequences.     

Characterization of collagen model peptides containing 4-fluoroproline; (4(S)-fluoroproline-pro-gly)10 forms a triple helix,but (4(R)-fluoroproline-pro-gly)10 does not

Collagen model peptide (Pro-Pro-Gly)10 has a triple helical structure and undergoes a thermal transition to a single random coil structure. The transition temperature of the analogous model peptides depends largely on amino acid substitution. Substitution of Pro by 4-hydroxyproline (Hyp) or 4-fluoroproline (fPro) has especially attracted attention because the position of substitution and chirality of the hydroxyl group or fluorine atom affect the transition temperatures. Here, we demonstrated that (4(S)-fPro-Pro-Gly)10 takes a triple helical structure, but (4(R)-fPro-Pro-Gly)10 exists in a single chain structure. This is not consistent with the case of Hyp substitution in our previous report where both (4(S)-Hyp-Pro-Gly)10 and (4(R)-Hyp-Pro-Gly)10 are in a single random coil state.     

Synthesis and structural analysis of nonstoichiometric ternary fulleride K 1.5 Ba 0.25 CsC 60

The existence of cation-vacancy sites in fullerides might lead to long-range ordering and generate a new vacancy-ordered superstructure. The purpose of this work is to search whether or not long-range ordering of vacant tetrahedral sites, namely superstructure emerges in nonstoichiometric K _1.5 Ba _0.25 CsC ₆₀ fulleride. Therefore, K _1.5 Ba _0.25 CsC ₆₀ with cation-vacancy sites is synthesized using a precursor method to avoid inadequate stoichiometry control and formation of impurity phases within the target composition. For this purpose, first, phase-pure K ₆ C ₆₀ , Ba ₆ C ₆₀ and Cs ₆ C ₆₀ precursors are synthesized. Stoichiometric quantities of these precursors are used for further reaction with C ₆₀ to afford K _1.5 Ba _0.25 CsC ₆₀ . Rietveld analysis of the high-resolution synchrotron X-ray powder diffraction data of the precursors and K _1.5 Ba _0.25 CsC ₆₀ confirms that K ₆ C ₆₀ , Ba ₆ C ₆₀ and Cs ₆ C ₆₀ are single-phase and they crystallize in a body-centered-cubic structure ( Im 3) as reported in the literature. The analysis also shows that K _1.5 Ba _0.25 CsC ₆₀ phase can be perfectly modeled using a face-centered cubic structure. No new peaks appear which could have implied the appearance of a superstructure. This suggests that there is no long-range ordered arrangement of vacant tetrahedral sites in K _1.5 Ba _0.25 CsC ₆₀ .