Fused-ring and bridged-ring tetrahydrofuran scaffolds are found in a number of natural products and biologically active compounds. A new copper-catalyzed intramolecular carboetherification of alkenes ...for the synthesis of bicyclic tetrahydrofurans is reported herein. The reaction involves Cu-catalyzed intramolecular addition of alcohols to unactivated alkenes and subsequent aryl C–H functionalization provides the C–C bond. Mechanistic studies indicate a primary carbon radical intermediate is involved and radical addition to the aryl ring is the likely C–C bond-forming mechanism. Preliminary catalytic enantioselective reactions are promising (up to 75% ee) and provide evidence that copper is involved in the alkene addition step, likely through a cis-oxycupration mechanism. Catalytic enantioselective alkene carboetherification reactions are rare and future development of this new method into a highly enantioselective process is promising. During the course of the mechanistic studies a protocol for alkene hydroetherification was also developed.
Antibiotic resistance of bacterial pathogens poses an increasing threat to the wellbeing of our society and urgently calls for new strategies for infection diagnosis and antibiotic discovery. The ...antibiotic resistance problem at least partially arises from extensive use of broad-spectrum antibiotics. Ideally, for the treatment of infection, one would like to use a narrow-spectrum antibiotic that specifically targets and kills the disease-causing strain. This is particularly important considering the commensal bacterial species that are beneficial and sometimes even critical to the health of a human being. In this contribution, we describe a phage display platform that enables rapid identification of peptide probes for specific bacterial strains. The phage library described herein incorporates 2-acetylphenylboronic acid moieties to elicit dynamic covalent binding to the bacterial cell surface. Screening of the library against live bacterial cells yields submicromolar and highly specific binders for clinical strains of Staphylococcus aureus and Acinetobacter baumannii that display antibiotic resistance. We further show that the identified peptide probes can be readily converted to bactericidal agents that deliver generic toxins to kill the targeted bacterial strain with high specificity. The phage display platform described here is applicable to a wide array of bacterial strains, paving the way to facile diagnosis and development of strain-specific antibiotics.
Fundamental questions regarding collagen biosynthesis, especially with respect to the molecular origins of homotrimeric versus heterotrimeric assembly, remain unanswered. Here, we demonstrate that ...the presence or absence of a single cysteine in type-I collagen's C-propeptide domain is a key factor governing the ability of a given collagen polypeptide to stably homotrimerize. We also identify a critical role for Ca
in non-covalent collagen C-propeptide trimerization, thereby priming the protein for disulfide-mediated covalent immortalization. The resulting cysteine-based code for stable assembly provides a molecular model that can be used to predict, a priori, the identity of not just collagen homotrimers, but also naturally occurring 2:1 and 1:1:1 heterotrimers. Moreover, the code applies across all of the sequence-diverse fibrillar collagens. These results provide new insight into how evolution leverages disulfide networks to fine-tune protein assembly, and will inform the ongoing development of designer proteins that assemble into specific oligomeric forms.
Intracellular procollagen folding begins at the protein's C-terminal propeptide (C-Pro) domain, which initiates triple-helix assembly and defines the composition and chain register of fibrillar ...collagen trimers. The C-Pro domain is later proteolytically cleaved and excreted from the body, while the mature triple helix is incorporated into the extracellular matrix. The procollagen C-Pro domain possesses a single
-glycosylation site that is widely conserved in all the fibrillar procollagens across humans and diverse other species. Given that the C-Pro domain is removed once procollagen folding is complete, the
-glycan might be presumed to be important for folding. Surprisingly, however, there is no difference in the folding and secretion of
-glycosylated versus non-
-glycosylated collagen type-I, leaving the function of the
-glycan unclear. We hypothesized that the collagen
-glycan might have a context-dependent function, specifically, that it could be required to promote procollagen folding only when proteostasis is challenged. We show that removal of the
-glycan from misfolding-prone C-Pro domain variants does indeed cause serious procollagen and ER proteostasis defects. The
-glycan promotes folding and secretion of destabilized C-Pro variants by providing access to the ER's lectin-based chaperone machinery. Finally, we show that the C-Pro
-glycan is actually critical for the folding and secretion of even wild-type procollagen under ER stress conditions. Such stress is commonly incurred during development, wound healing, and other processes in which collagen production plays a key role. Collectively, these results establish an essential, context-dependent function for procollagen's previously enigmatic
-glycan, wherein the carbohydrate moiety buffers procollagen folding against proteostatic challenge.
This paper describes our investigation of the structural determinants of a designed cyclic peptide (cLac, cyclic peptide mimicking lactadherin) (Zheng, H.; Wang, F.; Wang, Q.; Gao, J. J. Am. Chem. ...Soc.2011, 133, 15280–15283) for phosphatidylserine (PS) recognition. A highly efficient strategy that takes advantage of the native chemical ligation (NCL) chemistry has been developed for the synthesis and labeling of cyclic peptides in general. Ala scanning of the cLac peptide revealed a sophisticated model for PS binding, in which the peptide scaffold assembles multiple polar residues to balance the desolvation and electrostatic interactions (salt bridge and hydrogen bonding) to achieve lipid selectivity. The results suggest that cLac effectively mimics the membrane binding mechanism of the parent protein lactadherin.
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It has been a long‐standing goal to understand the structure‐stability relationship of proteins, as optimal stability is essential for protein function and highly desirable for protein therapeutics. ...Halogenation has emerged as a minimally invasive strategy to probe the physical characteristics of proteins in solution, as well as enhance the structural stabilities of proteins for therapeutic applications. Although advances in synthetic chemistry and genetic code expansion have allowed for the rapid synthesis of proteins with diverse chemical sequences, much remains to be learned regarding the impact of these mutations on their structural integrity. In this contribution, we present a systematic study of three well‐folded model protein systems, in which their structural stabilities are assessed in response to various hydrogen‐to‐halogen atom mutations. Halogenation allows for the perturbation of proteins on a sub‐angstrom scale, offering unprecedented precision of protein engineering. The thermodynamic results from these model systems reveal that in certain cases, proteins can display modest steric tolerance to halogenation, yielding non‐additive consequences to protein stability. The observed sub‐angstrom sensitivity of protein stability highlights the delicate arrangement of a folded protein core structure. The stability data of various halogenated proteins presented herein should also provide guidelines for using halogenation as a strategy to improve the stability of protein therapeutics.
Cyclic peptides have been proposed as privileged scaffolds that might mimic the folding and function of natural proteins. However, simple cyclic peptides typically cannot fold into well‐defined ...structures. Herein, we describe a foldable cyclic peptide scaffold on which functional side chains can be displayed for targeted recognition of biomolecules. The foldable scaffold is based on prolinomycin, a proline‐rich analogue of valinomycin. We report synthetic mutants of prolinomycin that retain the metal‐assisted folding behavior under physiological conditions. The predictable structure formation of prolinomycin makes it a powerful platform to enable the development of synthetic receptors for biomolecules of interest. We demonstrate the potential of this scaffold by creating prolinomycin mutants that selectively bind anionic vesicles and bacterial cells.
Like a pro: Prolinomycin, a proline‐rich analogue of valinomycin, was found to tolerate various mutations, which enables predictable display of side chains for target binding. Prolinomycin presents a versatile scaffold for developing functional peptides.
Bridged bicyclic rings containing nitrogen heterocycles are important motifs in bioactive small organic molecules. An enantioselective copper‐catalyzed alkene carboamination reaction that creates ...bridged heterocycles is reported herein. Two new rings are formed in this alkene carboamination reaction where N‐sulfonyl‐2‐aryl‐4‐pentenamines are converted to 6‐azabicyclo3.2.1octanes using the complex Ph‐Box‐Cu(OTf)2 or related catalysts in the presence of manganeses dioxide (MnO2) as stoichiometric oxidant in moderate to good yields and generally excellent enantioselectivities. Two new stereocenters are formed in the reaction, and the CC bond‐forming arene addition is a net CH functionalization.
Intracellular collagen assembly begins with the oxidative folding of ∼30-kDa C-terminal propeptide (C-Pro) domains. Folded C-Pro domains then template the formation of triple helices between ...appropriate partner strands. Numerous C-Pro missense variants that disrupt or delay triple-helix formation are known to cause disease, but our understanding of the specific proteostasis defects introduced by these variants remains immature. Moreover, it is unclear whether or not recognition and quality control of misfolded C-Pro domains is mediated by recognizing stalled assembly of triple-helical domains or by direct engagement of the C-Pro itself. Here, we integrate biochemical and cellular approaches to illuminate the proteostasis defects associated with osteogenesis imperfecta-causing mutations within the collagen-α2(I) C-Pro domain. We first show that “C-Pro-only” constructs recapitulate key aspects of the behavior of full-length Colα2(I) constructs. Of the variants studied, perhaps the most severe assembly defects are associated with C1163R C-Proα2(I), which is incapable of forming stable trimers and is retained within cells. We find that the presence or absence of an unassembled triple-helical domain is not the key feature driving cellular retention versus secretion. Rather, the proteostasis network directly engages the misfolded C-Pro domain itself to prevent secretion and initiate clearance. Using MS-based proteomics, we elucidate how the endoplasmic reticulum (ER) proteostasis network differentially engages misfolded C1163R C-Proα2(I) and targets it for ER-associated degradation. These results provide insights into collagen folding and quality control with the potential to inform the design of proteostasis network-targeted strategies for managing collagenopathies.
My graduate research career has focused on studying the principles that underlie molecular recognition, which include protein folding, protein-membrane interactions, structural preoranization for ...target binding and non-covalent interactions. This thesis will present an overview of this work through three different projects. I) Synthetic receptors for target binding in water. Molecular interactions in water provide the foundation for life. More specifically, the interactions between one or more molecules, through hydrogen bonding, π-effects, hydrophobic interactions and electrostatic interactions, all play a significant role essential to biological processes. This chapter will present an overview of supramolecular chemistry in water, with a focus on small molecule receptor “warheads” that target biomolecules of interest. The discussion will then move towards the ability to preorganize these “warheads” on a scaffold to improve their potency towards a target. The fundamental principles discussed in this section will provide a foundation for the following chapter in this thesis. II) Understanding Phosphatidylserine Recognition Using the Model cLac Peptide. The plasma membrane serves as a defining feature of the cell membrane, acting as a barrier for material exchange between a cell and its local environment. More importantly, membrane lipids are involved in mediating numerous cell-signaling events and acting as receptors to recruit proteins that carry out a specific function. Due to the important role that lipids play, it is highly desirable to develop affinity ligands for the diverse range of lipid headgroup structures on a cell membrane. Although prevalent, proteins have intrinsic limitations due to their size, low stabilities and slow clearance rates. This chapter will focus on the model peptide, cLac, which was previously developed as an affinity ligand for phosphatidylserine recognition. We will focus on understanding the key properties that contribute to PS selectivity and affinity, then attempt to improve this scaffold through structural preorganization. III) A prolinomycin-based scaffold for developing functional peptides. Nature has evolved proteins to bind cell-signaling molecules with exquisite affinity and specificity, making molecular recognition an essential part of biology. It has been a highly sought after goal within the chemistry field to be able to mimic the structure and function of certain proteins with smaller molecules, such as peptides. Specifically, cyclic peptides are showing promise as therapeutic agents due to their high proteolytic stabilities, faster clearance rates and ease of synthesis compared to proteins. One challenge, however, is that peptides generally do not possess the ability to properly fold and display their side chains for target binding, as proteins do. In this chapter, I will present a prolinomycin-based scaffold, which can fold in the presence of K+ ions to preorganize its side chains for target binding. Moreover, the focus will be on the structural aspects of this cyclic peptide, along with proof-of-concept studies demonstrating its ability to recognize a target under physiological conditions. The findings in this study will be useful in developing peptide-based tools that recognize various targets. IV) Dissecting the energetic consequences of fluorinating a protein core. Proteins have emerged as a powerful class of therapeutic agents due to their superior properties over small molecules in the clinic. Some of the key advantages include their large surface areas and highly defined structures, which allow them to perform very specific functions that are generally not reproducible with traditional small molecule scaffolds. In addition, proteins possess the ability to properly fold under physiological conditions through precise, noncovalent interactions between their side chain residues. Perhaps the most relevant interactions arise from aromatic side chains, which can interact in a variety of ways to help proteins fold. In this chapter, we will focus on the model protein, VHP35, which contains a hydrophobic core of three interacting Phe residues, to study the effects of fluorination on an edge-face interaction.
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![6-Azabicyclo[3.2.1]octanes ...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/1615-4150/small "6-Azabicyclo[3.2.1]octanes via Copper-Catalyzed Enantioselective Alkene Carboamination")
