DNA-Encoded Macrocyclic Peptide Libraries Enable the Discovery of a Neutral MDM2-p53 Inhibitor.

Synthetic macrocyclic peptides are an emerging molecular class for both targeting intracellular protein-protein interactions (PPIs) and providing an oral modality for drug targets typically addressed by biologics. Display technologies, such as mRNA and phage display, often yield peptides that are too large and too polar to achieve passive permeability or oral bioavailability without substantial off-platform medicinal chemistry. Herein, we use DNA-encoded cyclic peptide libraries to discover a neutral nonapeptide, UNP-6457, that inhibits MDM2-p53 interaction with an IC50 of 8.9 nM. X-ray structural analysis of the MDM2-UNP-6457 complex revealed mutual binding interactions and identified key ligand modification points which may be tuned to enhance its pharmacokinetic profile. These studies showcase how tailored DEL libraries can directly yield macrocyclic peptides benefiting from low MW, TPSA, and HBD/HBA counts that are capable of potently inhibiting therapeutically relevant protein-protein interactions.

Stretching Peptides to Generate Small Molecule ß-Strand Mimics.

Advances in the modulation of protein-protein interactions (PPIs) enable both characterization of PPI networks that govern diseases and design of therapeutics and probes. The shallow protein surfaces that dominate PPIs are challenging to target using standard methods, and approaches for accessing extended backbone structures are limited. Here, we incorporate a rigid, linear, diyne brace between side chains at the i to i+2 positions to generate a family of low-molecular-weight, extended-backbone peptide macrocycles. NMR and density functional theory studies show that these stretched peptides adopt stable, rigid conformations in solution and can be tuned to explore extended peptide conformational space. The diyne brace is formed in excellent conversions (>95%) and amenable to high-throughput synthesis. The minimalist structure-inducing tripeptide core (<300 Da) is amenable to further synthetic elaboration. Diyne-braced inhibitors of bacterial type 1 signal peptidase demonstrate the utility of the technique.

Native Chemical Ligation of Peptides and Proteins.

For over 20 years, native chemical ligation (NCL) has played a pivotal role in enabling total synthesis and semisynthesis of increasingly complex peptide and protein targets. Classical NCL proceeds by chemoselective reaction of two unprotected polypeptide chains in near-neutral-pH, aqueous solution and is made possible by the presence of a thioester moiety on the C-terminus of the N-terminal peptide fragment and a natural cysteine residue on the N-terminus of the C-terminal peptide fragment. The reaction yields an amide bond adjacent to cysteine at the ligation site, furnishing a native protein backbone in a traceless manner. This unit highlights a number of recent and powerful advances in the methodology and outlines their particular uses, facilitating application in the synthesis of challenging protein targets. © 2019 by John Wiley & Sons, Inc.

Expedient on-resin synthesis of peptidic benzimidazoles.

The benzimidazole moiety is a ubiquitous pharmacophore present in numerous anthelmintic, antibacterial, antiviral, antineoplastic, and antifungal drugs. While the polypharmacology of this heterocycle has spurred the development of numerous solution-phase syntheses, only a handful of disparate and inefficient methods detailing its synthesis on-resin have been reported. Here we report the concise and expedient syntheses of internal and C-terminal peptidic benzimidazoles - an emerging class of peptide deformylase (PDF)-inhibiting antimicrobials. This method benefits from being performed wholly on solid-phase at room temperature resulting in minimal purification and tolerance of temperature-sensitive functionality.

Rigid Peptide Macrocycles from On-Resin Glaser Stapling.

Peptide macrocycles are widely utilized in the development of high affinity ligands, including stapled α-helices. The linear rigidity of a 1,3-diynyl linkage provides an optimal distance (7 Å) between ß-carbons of the i,i+4 amino acid side chains, thus suggesting its utility in stabilizing α-helical structures. Here, we report the development of an on-resin strategy for an intramolecular Glaser reaction between two alkyne-terminated side chains by using copper chloride, an essential bpy-diol ligand, and diisopropylethylamine at room temperature. The efficiency of this ligation was illustrated by the synthesis of (i,i+4)-, (i,i+5)-, (i,i+6)-, and (i,i+7)-stapled BCL-9 α-helical peptides using the unnatural amino acid propargyl serine. Overall, this procedurally simple method relies on inexpensive and widely available reagents to generate low molecular weight 23-, 26-, 29-, and 32-membered peptide macrocycles.

Adapting the Glaser Reaction for Bioconjugation: Robust Access to Structurally Simple, Rigid Linkers.

Copper-mediated coupling between alkynes to generate a structurally rigid, linear 1,3-diyne linkage has been known for over a century. However, the mechanistic requirement to simultaneously maintain CuI and an oxidant has limited its practical utility, especially for complex functional molecules in aqueous solution. We find that addition of a specific bpy-diol ligand protects unprotected peptides from CuII -mediated oxidative damage through the formation of an insoluble CuII gel which solves the critical challenge of applying Glaser coupling to substrates that are degraded by CuII . The generality of this method is illustrated through the conjugation of a series of polar and nonpolar labels onto a fully unprotected GLP-1R agonist through a linear 7 Šdiynyl linker.

Base-catalyzed diastereoselective trimerization of trifluoroacetone.

Amphiphilic fluorocarbons have unique properties that facilitate their self assembly and adhesion to both inorganic and biological substrates. Incorporation of these moieties into valuable constructs typically require complex synthetic routes that have limited their use. Here, the base-catalyzed diastereoselective synthesis of 6-methyl-2,4,6-tris(trifluoromethyl)tetrahydro-2H-pyran-2,4-diol is reported. Trimerization of trifluoroacetone in the presence of 5 mol% KHMDS delivers one of four diastereomers selectively in 81% yield with no column chromatography. Temperature screening revealed the reversibility of this trimerization and the funneling of material into the most thermodynamically stable oxane. Subsequent functionalization with boronic acids is reported.

Rhodium(II)-catalyzed nondirected oxidative alkenylation of arenes: arene loading at one equivalent.

A bimetallic Rh(II) catalyst promoted the C-H alkenylation of simple arenes at 1.0 equivalent without the use of a directing group. A phosphine ligand as well as cooperative reoxidation of Rh(II) with Cu(TFA)2 and V2O5 proved essential in providing monoalkenylated products in good yields and selectivities, especially with di- and trisubstituted arenes.

Mechanistic investigation of the enantioselective intramolecular stetter reaction: proton transfer is the first irreversible step.

A study on the mechanism of the asymmetric intramolecular Stetter reaction is reported. This investigation includes the determination of the rate law, kinetic isotope effects, and competition experiments. The reaction was found to be first order in aldehyde and azolium catalyst or free carbene. A primary kinetic isotope effect was found for the proton of the aldehyde. Taken together with a series of competition experiments, these results suggest that proton transfer from the tetrahedral intermediate formed upon nucleophilic attack of the carbene onto the aldehyde is the first irreversible step.

Stability of MCT/LCT-based total nutrient admixtures for neonatal use over 30 hours at room temperature: applying pharmacopeial standards.

BACKGROUND: United States Pharmacopeial Chapter <729> places a limit on the percentage of large fat globules >5 microm, expressed as a PFAT5 of <0.05% for all native lipid emulsions. Some adult total nutrient admixtures (TNAs) have also remained below this limit for up to 48 hours. In 2003, medium-chain/long-chain triglyceride (MCT/LCT)-based neonatal TNAs with between 2% and 3% amino acid (AA) concentrations were shown to be similarly stable by the PFAT5 parameter. Stability assessment of neonatal TNAs with AA <2% or > or =3% were tested. METHODS: Eight neonatal TNAs with various combinations of AA (1%, 1.5%, 3%, and 4%), glucose (G; 5% and 10%), and MCT/LCT (ML; 2% and 4%) and standard concentrations of additives were tested in triplicate (n = 24) over 30 hours (immediately after mixing, then at 6, 24, and 30 hours) at 25 degrees C +/- 2 degrees C. PFAT5 determinations for all 24 formulations were made in duplicate, immediately after mixing, and then at 6, 24, and 30 hours later. Mean droplet size (MDS) and pH were assessed at the outset and end of the study. RESULTS: The differences in the PFAT5 levels were significant (P < .001) by a 2-way analysis of variance based on formula and time as the independent variables. The TNAs with 1% and 1.5% AA with all Gs and MLs (group 1, n = 12) had PFAT5 levels >0.05% (up to 0.50%) in most samples (68 of 96 samples, or 71% of cases) in the study, whereas in the same TNAs, but made with 3% and 4% AA (group 2, n = 12), 100% of samples (all 96 cases) had PFAT5 levels <0.05% (up to 0.04%), and this difference was significant (P < .001). Pairwise comparisons between groups based on overall values of PFAT5, MDS, and pH showed significant differences between groups for all variables. CONCLUSIONS: For neonatal TNAs, AA level is the most sensitive determinant of stability, and the PFAT5 parameter is the most sensitive indicator of stability.

Calcium and phosphate compatibility in low-osmolarity parenteral nutrition admixtures intended for peripheral vein administration.

BACKGROUND: Precipitation of calcium (Ca) and phosphate (P) salts can lead to potentially lethal outcomes, especially in low-osmolarity parenteral nutrition (LO-PN) formulations. Three concentrations of amino acids (AA) and 2 concentrations of calcium gluconate and sodium phosphate injections on the compatibility of Ca and P in LO-PN admixtures were studied. METHODS: Final AA concentrations of 1%, 2%, or 3% (n = 3) and 5% glucose (G) were prepared with either 2.5 or 5 mmol/L (5 or 10 mEq) of Ca (n = 2) and 15 or 30 mmol/L of P (n = 2) for a total of 12 base (3 x 2 x 2) formulations. Triplicate bags of each were analyzed for subvisible micro-precipitates using the light obscuration (or extinction) method for particle counts per milliliter (PC) in the size range of 1.8-50 mum at 7 time intervals over 48 hours stored at 30 degrees C +/- 0.2 degrees C. Visual evaluation was performed using a high-intensity lamp against a black background for detection of macro-precipitates. The pH of all 36 admixtures was measured at 0 and 48 hours. Any precipitated material was isolated and characterized by polarized light microscopy and infrared spectroscopy. RESULTS: Of the 12 base LO-PN formulations tested, those containing 1% and 2% AA with 5 mmol/L of Ca and 30 mmol/L of P showed significant increases in PC, and some resulted in visible dibasic calcium phosphate precipitation. Analyses of variance based on concentrations of AA, Ca, P, and time were highly significant independent variables for increases in the PC of potentially embolic particles, that is, sizes >5 mum (P < .0001). The lowest concentrations of Ca and P, 2.5 and 15 mmol/L, respectively, had significantly lower PC (P < .05) for all sizes compared with the other Ca and P combinations. CONCLUSIONS: LO-PN admixtures (AA

Physical compatibility of neonatal total parenteral nutrition admixtures containing organic calcium and inorganic phosphate salts in a simulated infusion at 37 degrees C.

OBJECTIVE: Within an incubator environment at 37 degrees C, we evaluated the physical compatibility of calcium (Ca) and phosphate (P) in simulated neonatal total parenteral nutrition (TPN) infusions. DESIGN: Representative neonatal TPN formulas containing four concentrations of amino acids [AA] (1-4 g/dL) and two levels of dextrose [D] (5 and 10 g/dL) were monitored for calcium-phosphate precipitate formation over 24 hours using a laser-based particle counting technique. SETTING: Research Laboratory. SUBJECTS: None. MEASUREMENTS AND MAIN RESULTS: Of the eight AA-D combinations studied, two formulas containing 1 g/dL AA with either 5 g/dL or 10 g/dL of D and high amounts of Ca and P resulted in significant increases in particle counts and visibly evident precipitation during the 24-hour infusion. Precipitation was noted only after the infusion passed through the incubator, but not in the TPN bags, demonstrating the heightened risk of calcium-phosphate incompatibility with increased temperatures. CONCLUSIONS: Low AA concentrations and high temperatures are significant risk factors for the precipitation of the insoluble dibasic calcium phosphate that may be fatal upon intravenous infusion. When thermoregulation is required, the AA concentration of the TPN formula should not be less than 1.5 g/dL (and ideally > or =3 g/dL) when high intakes of calcium (60 mg/dL) and phosphate (46.5 mg/dL) are prescribed.

Stability of total nutrient admixtures with lipid injectable emulsions in glass versus plastic packaging.

PURPOSE: The physical stability of two emulsions compounded as part of a total nutrient admixture (TNA) was studied in lipids packaged in either glass or plastic containers. METHODS: Five weight-based adult TNA formulations that were designed to meet the full nutritional needs of adults with body weights between 40 and 80 kg were studied. Triplicate preparations of each TNA were assessed over 30 hours at room temperature by applying currently proposed United States Pharmacopeia (USP) criteria for mean droplet diameter, large-diameter tail, and globule-size distribution (GSD) for lipid injectable emulsions. In accordance with conditions set forth in USP chapter 729, the higher levels of volume-weighted percent of fat exceeding 5 microm (PFAT(5)) should not exceed 0.05% of the total lipid concentration. RESULTS: Significant differences were noted among TNA admixtures based on whether the lipid emulsion product was manufactured in glass or plastic. The plastic-contained TNAs failed the proposed USP methods for large-diameter fat globules in all formulations from the outset, and 60% had significant growth in large-diameter fat globules over time. In contrast, glass-contained TNAs were stable throughout and in all cases would have passed proposed USP limits. CONCLUSION: Certain lipid injectable emulsions packaged in plastic containers have baseline abnormal GSD profiles compared with those packaged in glass containers. When used to compound TNAs, the abnormal profile worsens and produces less stable TNAs than those compounded with lipid injectable emulsions packaged in glass containers.

Establishing a stability window for medium- and long-chain-triglyceride lipid-based total nutrient admixtures using USP standards.

PURPOSE: The stability window of medium-chain triglyceride (MCT) and long-chain-triglyceride (LCT) lipid-based total nutrient admixtures (TNAs) was studied. METHODS: Sixteen different admixtures were selected for study. Of these, eight base macronutrient concentrations representing low and high concentrations were selected, along with low and high concentrations of electrolytes. All TNAs studied contained 2 mg of elemental iron as part of the trace-element formulation, an amount previously shown to produce unstable TNAs with pure LCT-based lipid injectable emulsions. All admixtures were prepared in triplicate and analyzed over five time intervals: time 1 (immediately after preparation), time 2 (after four days of storage at 6 +/- 2 degrees C), and times 3, 4, and 5, corresponding to 6, 24, and 30 hours of storage at 25 +/- 2 degrees C, respectively, after time 2. Stability was measured by comparing results with USP standards for fat globule size in lipid injectable emulsions. RESULTS: A total of 48 admixtures were studied. Samples at each time interval showed an inconsistent but general increase in the number of globules with a diameter of >1.8 microm over time. All admixtures met both the proposed pharmacopoeial criteria for stability with respect to mean droplet size and volume-weighted proportion of fat globules with a diameter of >5 microm. CONCLUSION: A wide range of macronutrients and micronutrients were tested in a series of MCT-LCT-based TNAs and found to be stable. The use of MCTs and LCTs in lipid injectable emulsions confers greater stability to TNAs than has been achieved with pure LCT-based formulations.

Physicochemical stability of highly concentrated total nutrient admixtures for fluid-restricted patients.

PURPOSE: The physicochemical stability of highly concentrated total nutrient admixtures (TNAs) for fluid-restricted patients was studied. METHODS: Five TNAs made from lipid injectable emulsions (50:50 mixture of medium-chain and long-chain triglycerides) designed to meet the full nutritional needs of adults with body weights of 40-80 kg were chosen. Protein was included in the TNAs at 1.5 g/kg for each body weight and was supplied from a concentrated 16% mixture containing the essential and non-essential amino acids. All admixtures were contained in ethylene vinyl acetate bags and were aseptically prepared. Triplicate preparations of each TNA were investigated over 30 hours at room temperature by dynamic light scattering (DLS) and light extinction with single-particle optical sensing (LE-SPOS). RESULTS: No significant changes in the physicochemical stability of the TNAs were observed by DLS (mean droplet size) or LE-SPOS (large-diameter tail) from time 0 (immediately after compounding) to 30 hours. All TNAs met the mean-droplet-size criteria outlined by USP for 20% lipid injectable emulsions. CONCLUSION: Concentrated TNA formulations made from lipid injectable emulsions were stable for 30 hours at room temperature.