Click-Based Libraries of SFTI‑1 Peptides: New Methods Using Reversed-Phase Silica

Performing sequential reactions for the orthogonal derivatization of peptides in solution often requires intermediate handling and purification steps. To solve these problems, we have exploited the distinct adsorption kinetics of peptides toward particulate reversed-phase (RP) C18 silica material, enabling consecutive reactions to be performed without intermediate elution. To illustrate this approach, sequential CuAAC/click reactions were used to modify an analog of the bicyclic peptide sunflower trypsin inhibitor 1 (SFTI-1), a potent scaffold for trypsin and chymotrypsin-like enzyme inhibitors. The SFTI-1 scaffold was synthesized containing both β-azido alanine and propargyl glycine residues. Despite the mutual reactivity of these groups, site isolation on RP silica enabled consecutive click reactions and associated washing steps to be performed while the peptide remained immobilized. Importantly, this approach eliminated side products that could form between two peptides or within a single peptide. These studies suggest a broad utility for RP silica in solving both peptide handling problems and in improving synthetic workflows

Temperature Dependence of CN and SCN IR Absorptions Facilitates Their Interpretation and Use as Probes of Proteins

Cyano and thiocyano groups have received attention as IR probes of local protein electrostatics or solvation, due to their strong absorptions and the ability to site specifically incorporate them within proteins. However, interpreting their spectra requires knowing whether they engage in hydrogen bonds (H-bonds). Existing methods for the detection of such H-bonding interactions are based on structural analysis or correlations between IR and NMR signals and are labor intensive and possibly ambiguous. Here, using model systems we show that the absorption frequency of both probes is linearly correlated with temperature and that the slope of the resulting line (frequency–temperature line slope or FTLS) reflects the nature of the probe’s microenvironment, including whether or not the probe is engaged in H-bonds. We then show that the same linear dependence is observed with <i>p</i>-cyano phenylalanine, cyanylated cysteine, or cyanylated homocysteine incorporated at different positions within the N-terminal Src homology 3 domain of the murine adapter protein Crk-II. The FTLSs indicate that <i>p</i>-cyano phenylalanine incorporated at two positions is engaged in strong H-bonding, while it is involved in weaker H-bonding at a third position. In contrast, the FTLS of the cyanylated cysteine or cyanylated homocysteine absorptions indicates that they do not engage in H-bonding at either a buried or surface exposed position. While the differences likely reflect side chain flexibility and the probe’s ability to avoid solvent, the data suggest that the temperature dependence of the absorption provides a simple method to gauge the probe’s environment, including the presence of H-bonding

Boc-SPPS: Compatible Linker for the Synthesis of Peptide <i>o</i>‑Aminoanilides

A protection strategy is described for the efficient synthesis of peptide <i>o</i>-aminoanilides using <i>in situ</i> neutralization protocols for Boc-SPPS. On-resin protection of Boc-protected aminoacyl <i>o</i>-aminoanilides is achieved with 2-chlorobenzyl chloroformate. Activation through a peptidyl-benzotriazole intermediate allows for facile conversion to peptide-thioesters for use in native chemical ligation. In addition to providing a robust alternative to established thioester resins, as a latent thioester, the peptide <i>o</i>-aminoanilide has broad utility in convergent ligation strategies

Chemical Protein Synthesis Using a Second-Generation <i>N</i>‑Acylurea Linker for the Preparation of Peptide-Thioester Precursors

The broad utility of native chemical ligation (NCL) in protein synthesis has fostered a search for methods that enable the efficient synthesis of C<i>-</i>terminal peptide-thioesters, key intermediates in NCL. We have developed an <i>N-</i>acylurea (Nbz) approach for the synthesis of thioester peptide precursors that efficiently undergo thiol exchange yielding thioester peptides and subsequently NCL reaction. However, the synthesis of some glycine-rich sequences revealed limitations, such as diacylated products that can not be converted into <i>N</i>-acylurea peptides. Here, we introduce a new <i>N-</i>acylurea linker bearing an <i>o-</i>amino­(methyl)­aniline (MeDbz) moiety that enables in a more robust peptide chain assembly. The generality of the approach is illustrated by the synthesis of a pentaglycine sequence under different coupling conditions including microwave heating at coupling temperatures up to 90 C, affording the unique and desired <i>N-</i>acyl-<i>N</i>′-methylacylurea (MeNbz) product. Further extension of the method allowed the synthesis of all 20 natural amino acids and their NCL reactions. The kinetic analysis of the ligations using model peptides shows the MeNbz peptide rapidly converts to arylthioesters that are efficient at NCL. Finally, we show that the new MeDbz linker can be applied to the synthesis of cysteine-rich proteins such the cyclotides Kalata B1 and MCoTI-II through a one cyclization/folding step in the ligation/folding buffer

Chemical Protein Synthesis Using a Second-Generation <i>N</i>‑Acylurea Linker for the Preparation of Peptide-Thioester Precursors

The broad utility of native chemical ligation (NCL) in protein synthesis has fostered a search for methods that enable the efficient synthesis of C<i>-</i>terminal peptide-thioesters, key intermediates in NCL. We have developed an <i>N-</i>acylurea (Nbz) approach for the synthesis of thioester peptide precursors that efficiently undergo thiol exchange yielding thioester peptides and subsequently NCL reaction. However, the synthesis of some glycine-rich sequences revealed limitations, such as diacylated products that can not be converted into <i>N</i>-acylurea peptides. Here, we introduce a new <i>N-</i>acylurea linker bearing an <i>o-</i>amino­(methyl)­aniline (MeDbz) moiety that enables in a more robust peptide chain assembly. The generality of the approach is illustrated by the synthesis of a pentaglycine sequence under different coupling conditions including microwave heating at coupling temperatures up to 90 C, affording the unique and desired <i>N-</i>acyl-<i>N</i>′-methylacylurea (MeNbz) product. Further extension of the method allowed the synthesis of all 20 natural amino acids and their NCL reactions. The kinetic analysis of the ligations using model peptides shows the MeNbz peptide rapidly converts to arylthioesters that are efficient at NCL. Finally, we show that the new MeDbz linker can be applied to the synthesis of cysteine-rich proteins such the cyclotides Kalata B1 and MCoTI-II through a one cyclization/folding step in the ligation/folding buffer

Carbon−Deuterium Bonds as Site-Specific and Nonperturbative Probes for Time-Resolved Studies of Protein Dynamics and Folding

Carbon−deuterium (C−D) bonds are nonperturbative spectroscopic probes that absorb in a region of the IR spectrum that is free of other protein absorptions. We explore the use of these probes under time-resolved conditions to follow the unfolding of cytochrome <i>c</i> from a photostationary state that accumulates after CO is photodissociated from the protein’s heme prosthetic group. Our results clearly show that C−D bonds are well-suited to characterize protein folding and dynamics

Evidence of an Unusual N–H···N Hydrogen Bond in Proteins

Many residues within proteins adopt conformations that appear to be stabilized by interactions between an amide N–H and the amide N of the previous residue. To explore whether these interactions constitute hydrogen bonds, we characterized the IR stretching frequencies of deuterated variants of proline and the corresponding carbamate, as well as the four proline residues of an Src homology 3 domain protein. The C_δD₂ stretching frequencies are shifted to lower energies due to hyperconjugation with N_<i>i</i> electron density, and engaging this density via protonation or the formation of the N_<i>i</i>+1–H···N_<i>i</i> interaction ablates this hyperconjugation and thus induces an otherwise difficult to explain blue shift in the C–D absorptions. Along with density functional theory calculations, the data reveal that the N_<i>i</i>+1–H···N_<i>i</i> interactions constitute H-bonds and suggest that they may play an important and previously underappreciated role in protein folding, structure, and function

Multimodal Characterization of a Linear DNA-Based Nanostructure

Designer DNA structures have garnered much interest as a way of assembling novel nanoscale architectures with exquisite control over the positioning of discrete molecules or nanoparticles. Exploiting this potential for a variety of applications such as light-harvesting, molecular electronics, or biosensing is contingent on the degree to which various nanoarchitectures with desired molecular functionalizations can be realized, and this depends critically on characterization. Many techniques exist for analyzing DNA-organized nanostructures; however, these are almost never used in concert because of overlapping concerns about their differing character, measurement environments, and the disparity in DNA modification chemistries and probe structure or size. To assess these concerns and to see what might be gleaned from a multimodal characterization, we intensively study a single DNA nanostructure using a multiplicity of methods. Our test bed is a linear 100 base-pair double-stranded DNA that has been modified by a variety of chemical handles, dyes, semiconductor quantum dots, gold nanoparticles, and electroactive labels. To this we apply a combination of physical/optical characterization methods including electrophoresis, atomic force microscopy, transmission electron microscopy, dynamic light scattering, Förster resonance energy transfer, voltammetry, and structural modeling. In general, the results indicate that the differences among the techniques are not so large as to prevent their effective use in combination, that the data tend to be corroborative, and that differences observed among them can actually be quite informative

Modifications of a Nanomolar Cyclic Peptide Antagonist for the EphA4 Receptor To Achieve High Plasma Stability

EphA4 is a receptor tyrosine kinase with a critical role in repulsive axon guidance and synaptic function. However, aberrant EphA4 activity can inhibit neural repair after injury and exacerbate neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer’s. We previously identified the cyclic peptide <b>APY-d2</b> (APYCVYRβASWSC-nh₂, containing a disulfide bond) as a potent and selective EphA4 antagonist. However, <b>APY-d2</b> lacks sufficient plasma stability to be useful for EphA4 inhibition <i>in vivo</i> through peripheral administration. Using structure–activity relationship studies, we show that protecting the peptide N-terminus from proteolytic degradation dramatically increases the persistence of the active peptide in plasma and that a positively charged peptide N-terminus is essential for high EphA4 binding affinity. Among several improved <b>APY-d2</b> derivatives, the cyclic peptides <b>APY-d3</b> (<u>βA</u>PYCVYRβASWSC-nh₂) and <b>APY-d4</b> (<u>βA</u>PYCVYRβA<u>E</u>W<u>E</u>C-nh₂) combine high stability in plasma and cerebrospinal fluid with slightly enhanced potency. These properties make them valuable research tools and leads toward development of therapeutics for neurological diseases

Cytotoxicity of Quantum Dots Used for <i>In Vitro</i> Cellular Labeling: Role of QD Surface Ligand, Delivery Modality, Cell Type, and Direct Comparison to Organic Fluorophores

Interest in taking advantage of the unique spectral properties of semiconductor quantum dots (QDs) has driven their widespread use in biological applications such as <i>in vitro</i> cellular labeling/imaging and sensing. Despite their demonstrated utility, concerns over the potential toxic effects of QD core materials on cellular proliferation and homeostasis have persisted, leaving in question the suitability of QDs as alternatives for more traditional fluorescent materials (e.g., organic dyes, fluorescent proteins) for <i>in vitro</i> cellular applications. Surprisingly, direct comparative studies examining the cytotoxic potential of QDs versus these more traditional cellular labeling fluorophores remain limited. Here, using CdSe/ZnS (core/shell) QDs as a prototypical assay material, we present a comprehensive study in which we characterize the influence of QD dose (concentration and incubation time), QD surface capping ligand, and delivery modality (peptide or cationic amphiphile transfection reagent) on cellular viability in three human cell lines representing various morphological lineages (epithelial, endothelial, monocytic). We further compare the effects of QD cellular labeling on cellular proliferation relative to those associated with a panel of traditionally employed organic cell labeling fluorophores that span a broad spectral range. Our results demonstrate the important role played by QD dose, capping ligand structure, and delivery agent in modulating cellular toxicity. Further, the results show that at the concentrations and time regimes required for robust QD-based cellular labeling, the impact of our in-house synthesized QD materials on cellular proliferation is comparable to that of six commercial cell labeling fluorophores. Cumulatively, our results demonstrate that the proper tuning of QD dose, surface ligand, and delivery modality can provide robust <i>in vitro</i> cell labeling reagents that exhibit minimal impact on cellular viability