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5.  Correction 
doi:10.1021/ja501219s
PMCID: PMC4061606
6.  Pd-Catalyzed Nucleophilic Fluorination of Aryl Bromides 
On the basis of mechanism-driven reaction design, a Pd-catalyzed nucleophilic fluorination of aryl bromides and iodides has been developed. The method exhibits a broad substrate scope, especially with respect to nitrogen-containing heteroaryl bromides, and proceeds with minimal formation of the corresponding reduction products. A facilitated ligand modification process was shown to be critical to the success of the reaction.
doi:10.1021/ja5009739
PMCID: PMC3954505  PMID: 24559304
7.  The Transcriptional Complex Between the BCL2 i-Motif and hnRNP LL Is a Molecular Switch for Control of Gene Expression That Can Be Modulated by Small Molecules 
In a companion paper (DOI: 10.021/ja410934b) we demonstrate that the C-rich strand of the cis-regulatory element in the BCL2 promoter element is highly dynamic in nature and can form either an i-motif or a flexible hairpin. Under physiological conditions these two secondary DNA structures are found in an equilibrium mixture, which can be shifted by the addition of small molecules that trap out either the i-motif (IMC-48) or the flexible hairpin (IMC-76). In cellular experiments we demonstrate that the addition of these molecules has opposite effects on BCL2 gene expression and furthermore that these effects are antagonistic. In this contribution we have identified a transcriptional factor that recognizes and binds to the BCL2 i-motif to activate transcription. The molecular basis for the recognition of the i-motif by hnRNP LL is determined, and we demonstrate that the protein unfolds the i-motif structure to form a stable single-stranded complex. In subsequent experiments we show that IMC-48 and IMC-76 have opposite, antagonistic effects on the formation of the hnRNP LL–i-motif complex as well as on the transcription factor occupancy at the BCL2 promoter. For the first time we propose that the i-motif acts as a molecular switch that controls gene expression and that small molecules that target the dynamic equilibrium of the i-motif and the flexible hairpin can differentially modulate gene expression.
doi:10.1021/ja4109352
PMCID: PMC3985447  PMID: 24559432
8.  The Dynamic Character of the BCL2 Promoter i-Motif Provides a Mechanism for Modulation of Gene Expression by Compounds That Bind Selectively to the Alternative DNA Hairpin Structure 
It is generally accepted that DNA predominantly exists in duplex form in cells. However, under torsional stress imposed by active transcription, DNA can assume nonduplex structures. The BCL2 promoter region forms two different secondary DNA structures on opposite strands called the G-quadruplex and the i-motif. The i-motif is a highly dynamic structure that exists in equilibrium with a flexible hairpin species. Here we identify a pregnanol derivative and a class of piperidine derivatives that differentially modulate gene expression by stabilizing either the i-motif or the flexible hairpin species. Stabilization of the i-motif structure results in significant upregulation of the BCL2 gene and associated protein expression; in contrast, stabilization of the flexible hairpin species lowers BCL2 levels. The BCL2 levels reduced by the hairpin-binding compound led to chemosensitization to etoposide in both in vitro and in vivo models. Furthermore, we show antagonism between the two classes of compounds in solution and in cells. For the first time, our results demonstrate the principle of small molecule targeting of i-motif structures in vitro and in vivo to modulate gene expression.
doi:10.1021/ja410934b
PMCID: PMC3985915  PMID: 24559410
9.  OGlcNAcylation and Phosphorylation Have Opposing Structural Effects in tau: Phosphothreonine Induces Particular Conformational Order 
Phosphorylation and OGlcNAcylation are dynamic intracellular protein post-translational modifications that frequently are alternatively observed on the same serine and threonine residues. Phosphorylation and OGlcNAcylation commonly occur in natively disordered regions of proteins, and often have opposing functional effects. In the microtubule-associated protein tau, hyperphosphorylation is associated with protein misfolding and aggregation as the neurofibrillary tangles of Alzheimer’s disease, whereas OGlcNAcylation stabilizes the soluble form of tau. A series of peptides derived from the proline-rich domain (residues 174–251) of tau was synthesized, with free Ser/Thr hydroxyls, phosphorylated Ser/Thr (pSer/pThr), OGlcNAcylated Ser/Thr, and diethylphosphorylated Ser/Thr. Phosphorylation and OGlcNAcylation were found by CD and NMR to have opposing structural effects on polyproline helix (PPII) formation, with phosphorylation favoring PPII, OGlcNAcylation opposing PPII, and the free hydroxyls intermediate in structure, and with phosphorylation structural effects greater than OGlcNAcylation. For tau196–209, phosphorylation and OGlcNAcylation had similar structural effects, opposing a nascent α-helix. Phosphomimic Glu exhibited PPII-favoring structural effects. Structural changes due to Thr phosphorylation were greater than those of Ser phosphorylation or Glu, with particular conformational restriction as the dianion, with mean 3JαN = 3.5 Hz (pThr) versus 5.4 Hz (pSer), compared to 7.2, 6.8, and 6.2 Hz for Thr, Ser, and Glu, respectively, values that correlate with the backbone torsion angle ϕ. Dianionic phosphothreonine induced strong phosphothreonine amide protection and downfield amide chemical shifts (δmean = 9.63 ppm), consistent with formation of a stable phosphate-amide hydrogen bond. These data suggest potentially greater structural importance of threonine phosphorylation than serine phosphorylation due to larger induced structural effects.
doi:10.1021/ja407156m
PMCID: PMC4004249  PMID: 24559475
10.  NHC–Cu-Catalyzed Addition of Propargylboron Reagents to Phosphinoylimines. Enantioselective Synthesis of Trimethylsilyl-Substituted Homoallenylamides and Application to the Synthesis of S-(−)-Cyclooroidin 
A catalytic method for the efficient and enantioselective addition of a 1-trimethylsilyl-substituted allene moiety to phosphinoylimines is presented. Transformations are promoted by 5.0 mol % of a copper complex of an N-heterocyclic carbene in the presence of a propargylboron reagent that can be readily prepared in multigram quantities. Within 10 min of reaction, products are obtained in up to 91% yield, 98% allene (vs propargyl) selectivity, and 98:2 enantiomeric ratio. An assortment of aldimines serve as suitable substrates. The phosphinoyl and silyl groups can be removed efficiently and orthogonally. The silylallene moiety may be transformed to versatile derivatives that are difficult to access via nonsilylated allenes. The special features and utility of the approach are highlighted through a succinct enantioselective synthesis of marine alkaloid S-(−)-cyclooroidin.
doi:10.1021/ja500373s
PMCID: PMC3954525  PMID: 24555471
11.  Reverse micelles as a platform for dynamic nuclear polarization in solution NMR of proteins 
Despite tremendous advances in recent years, solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this non-equilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule. Unfortunately, these EPR transitions lie in the microwave range of the electromagnetic spectrum where bulk water absorbs strongly often resulting in catastrophic heating. Furthermore, the residence times of water on the surface of the protein in bulk solution are generally too short for efficient transfer of polarization. Here we take advantage of the properties of solutions of encapsulated proteins dissolved in low viscosity solvents to implement DNP in liquids. Such samples are largely transparent to the microwave frequencies required and thereby avoid significant heating. Nitroxide radicals are introduced into the reverse micelle system in three ways: attached to the protein; embedded in the reverse micelle shell; and free in the aqueous core. Significant enhancements of the water resonance ranging up to ~−93 at 0.35 T were observed. We also find that the hydration properties of encapsulated proteins allow for efficient polarization transfer from water to the protein. These and other observations suggest that merging reverse micelle encapsulation technology with DNP offers a route to a significant increase in the sensitivity of solution NMR spectroscopy of proteins and other bio-molecules.
doi:10.1021/ja4107176
PMCID: PMC3955360  PMID: 24456213
12.  Total Synthesis and Stereochemical Assignment of (±)-Sorbiterrin A 
A concise, biomimetic approach to sorbiterrin A has been developed employing consecutive Michael additions of a 4-hydroxypyrone to a sorbicillinol derivative and silver nanoparticle-mediated bridged aldol/dehydration to construct the [3.3.1] ring system. The relative stereochemistry of sorbiterrin A was unambiguously confirmed by X-ray crystallographic analysis.
doi:10.1021/ja500854q
PMCID: PMC3978410  PMID: 24547688
13.  Detection of Late Intermediates in Virus Capsid Assembly by Charge Detection Mass Spectrometry 
The assembly of hundreds of identical proteins into an icosahedral virus capsid is a remarkable feat of molecular engineering. How this occurs is poorly understood. Key intermediates have been anticipated at the end of the assembly reaction, but it has not been possible to detect them. In this work we have used charge detection mass spectrometry to identify trapped intermediates from late in the assembly of the hepatitis B virus T = 4 capsid, a complex of 120 protein dimers. Prominent intermediates are found with 104/105, 110/111, and 117/118 dimers. Cryo-EM observations indicate the intermediates are incomplete capsids and, hence, on the assembly pathway. On the basis of their stability and kinetic accessibility we have proposed plausible structures. The prominent trapped intermediate with 104 dimers is attributed to an icosahedron missing two neighboring facets, the 111-dimer species is assigned to an icosahedron missing a single facet, and the intermediate with 117 dimers is assigned to a capsid missing a ring of three dimers in the center of a facet.
doi:10.1021/ja411460w
PMCID: PMC3985884  PMID: 24548133
14.  A Catalytic Enantiotopic-Group-Selective Suzuki Reaction for the Construction of Chiral Organoboronates 
Catalytic enantiotopic-group-selective cross-couplings of achiral geminal bis(pinacolboronates) provide a route for the construction of nonracemic chiral organoboronates. In the presence of a chiral monodentate taddol-derived phosphoramidite ligand, these reactions occur with high levels of asymmetric induction. Mechanistic experiments with chiral 10B-enriched geminal bis(boronates) suggest that the reaction occurs by a stereochemistry-determining transmetalation that occurs with inversion of configuration at carbon.
doi:10.1021/ja500029w
PMCID: PMC4021567  PMID: 24564423
15.  Genetically Encoded Optochemical Probes for Simultaneous Fluorescence Reporting and Light Activation of Protein Function with Two-Photon Excitation 
Journal of the American Chemical Society  2014;136(44):15551-15558.
The site-specific incorporation of three new coumarin lysine analogues into proteins was achieved in bacterial and mammalian cells using an engineered pyrrolysyl-tRNA synthetase system. The genetically encoded coumarin lysines were successfully applied as fluorescent cellular probes for protein localization and for the optical activation of protein function. As a proof-of-principle, photoregulation of firefly luciferase was achieved in live cells by caging a key lysine residue, and excellent OFF to ON light-switching ratios were observed. Furthermore, two-photon and single-photon optochemical control of EGFP maturation was demonstrated, enabling the use of different, potentially orthogonal excitation wavelengths (365, 405, and 760 nm) for the sequential activation of protein function in live cells. These results demonstrate that coumarin lysines are a new and valuable class of optical probes that can be used for the investigation and regulation of protein structure, dynamics, function, and localization in live cells. The small size of coumarin, the site-specific incorporation, the application as both a light-activated caging group and as a fluorescent probe, and the broad range of excitation wavelengths are advantageous over other genetically encoded photocontrol systems and provide a precise and multifunctional tool for cellular biology.
doi:10.1021/ja5055862
PMCID: PMC4333581  PMID: 25341086
16.  Non-canonical 3′-5′ Extension of RNA with Prebiotically Plausible Ribonucleoside 2′,3′-Cyclic Phosphates 
Ribonucleoside 2′,3′-cyclic phosphates (N>p’s) are generated by multiple prebiotically plausible processes and are credible building blocks for the assembly of early RNA oligomers. While N>p’s can be polymerized into short RNAs by non-enzymatic processes with variable efficiency and regioselectivity, no enzymatic route for RNA synthesis had been described. Here we report such a non-canonical 3′-5′ nucleotidyl transferase activity. We engineered a variant of the hairpin ribozyme to catalyze addition of all four N>p’s (2′,3′-cyclic A-, G-, U-, and CMP) to the 5′-hydroxyl termini of RNA strands with 5′ nucleotide addition enhanced in all cases by eutectic ice phase formation at −7 °C. We also observed 5′ addition of 2′,3′-cyclic phosphate-activated β-nicotinamide adenine dinucleotide (NAD>p) and ACA>p RNA trinucleotide, and multiple additions of GUCCA>p RNA pentamers. Our results establish a new mode of RNA 3′-5′ extension with implications for RNA oligomer synthesis from prebiotic nucleotide pools.
doi:10.1021/ja4127714
PMCID: PMC4333585  PMID: 24660752
17.  Concerted, Rapid, Quantitative, and Site-Specific Dual Labeling of Proteins 
Rapid, one-pot, concerted, site-specific labeling of proteins at genetically encoded unnatural amino acids with distinct small molecules at physiological pH, temperature, and pressure is an important challenge. Current approaches require sequential labeling, low pH, and typically days to reach completion, limiting their utility. We report the efficient, genetically encoded incorporation of alkyne- and cyclopropene-containing amino acids at distinct sites in a protein using an optimized orthogonal translation system in E. coli. and quantitative, site-specific, one-pot, concerted protein labeling with fluorophores bearing azide and tetrazine groups, respectively. Protein double labeling in aqueous buffer at physiological pH, temperature, and pressure is quantitative in 30 min.
doi:10.1021/ja4129789
PMCID: PMC4333588  PMID: 24857040
18.  Genetic Encoding of Photocaged Cysteine Allows Photoactivation of TEV Protease in Live Mammalian Cells 
We demonstrate the evolution of the PylRS/tRNACUA pair for genetically encoding photocaged cysteine. By characterizing the incorporation in Escherichia coli and mammalian cells, and the photodeprotection process in vitro and in mammalian cells, we establish conditions for rapid efficient photodeprotection to reveal native proteins in live cells. We demonstrate the utility of this approach by rapidly activating TEV protease following illumination of single cells.
doi:10.1021/ja412191m
PMCID: PMC4333589  PMID: 24479649
19.  Efficient Multisite Unnatural Amino Acid Incorporation in Mammalian Cells via Optimized Pyrrolysyl tRNA Synthetase/tRNA Expression and Engineered eRF1 
Journal of the American Chemical Society  2014;136(44):15577-15583.
The efficient, site-specific introduction of unnatural amino acids into proteins in mammalian cells is an outstanding challenge in realizing the potential of genetic code expansion approaches. Addressing this challenge will allow the synthesis of modified recombinant proteins and augment emerging strategies that introduce new chemical functionalities into proteins to control and image their function with high spatial and temporal precision in cells. The efficiency of unnatural amino acid incorporation in response to the amber stop codon (UAG) in mammalian cells is commonly considered to be low. Here we demonstrate that tRNA levels can be limiting for unnatural amino acid incorporation efficiency, and we develop an optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system, with optimized tRNA expression for mammalian cells. In addition, we engineer eRF1, that normally terminates translation on all three stop codons, to provide a substantial increase in unnatural amino acid incorporation in response to the UAG codon without increasing readthrough of other stop codons. By combining the optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system and an engineered eRF1, we increase the yield of protein bearing unnatural amino acids at a single site 17- to 20-fold. Using the optimized system, we produce proteins containing unnatural amino acids with comparable yields to a protein produced from a gene that does not contain a UAG stop codon. Moreover, the optimized system increases the yield of protein, incorporating an unnatural amino acid at three sites, from unmeasurably low levels up to 43% of a no amber stop control. Our approach may enable the efficient production of site-specifically modified therapeutic proteins, and the quantitative replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function.
doi:10.1021/ja5069728
PMCID: PMC4333590  PMID: 25350841
20.  Testing the Vesicular Morphology to Destruction: Birth and Death of Diblock Copolymer Vesicles Prepared via Polymerization-Induced Self-Assembly 
Small angle X-ray scattering (SAXS), electrospray ionization charge detection mass spectrometry (CD-MS), dynamic light scattering (DLS), and transmission electron microscopy (TEM) are used to characterize poly(glycerol monomethacrylate)55-poly(2-hydroxypropyl methacrylate)x (G55-Hx) vesicles prepared by polymerization-induced self-assembly (PISA) using a reversible addition–fragmentation chain transfer (RAFT) aqueous dispersion polymerization formulation. A G55 chain transfer agent is utilized to prepare a series of G55-Hx diblock copolymers, where the mean degree of polymerization (DP) of the membrane-forming block (x) is varied from 200 to 2000. TEM confirms that vesicles with progressively thicker membranes are produced for x = 200–1000, while SAXS indicates a gradual reduction in mean aggregation number for higher x values, which is consistent with CD-MS studies. Both DLS and SAXS studies indicate minimal change in the overall vesicle diameter between x = 400 and 800. Fitting SAXS patterns to a vesicle model enables calculation of the membrane thickness, degree of hydration of the membrane, and the mean vesicle aggregation number. The membrane thickness increases at higher x values, hence the vesicle lumen must become smaller if the external vesicle dimensions remain constant. Geometric considerations indicate that this growth mechanism lowers the total vesicle interfacial area and hence reduces the free energy of the system. However, it also inevitably leads to gradual ingress of the encapsulated water molecules into the vesicle membrane, as confirmed by SAXS analysis. Ultimately, the highly plasticized membranes become insufficiently hydrophobic to stabilize the vesicle morphology when x exceeds 1000, thus this PISA growth mechanism ultimately leads to vesicle “death”.
doi:10.1021/ja511423m
PMCID: PMC4333598  PMID: 25526525
21.  Catalytic, Enantioselective, Intramolecular Carbosulfenylation of Olefins. Mechanistic Aspects: A Remarkable Case of Negative Catalysis 
In the course of developing an enantioselective, Lewis base/Brønsted acid co-catalyzed carbosulfenylation of alkenes, a seemingly impossible conundrum arose: How could a catalyst inhibit a stoichiometric reaction? Despite the observation of very good enantioselectivities, the rate of the uncatalyzed reaction (i.e., no Lewis base) was found to be comparable to or slightly faster than that of the catalyzed process. A combination of detailed kinetic and spectroscopic studies revealed that the answer is not the direct involvement of the Lewis base catalyst, but rather the secondary consequences of its conversion to the catalytically active sulfenylating agent. Generation of the chiral sulfenylating species is accompanied by the formation of equimolar amounts of sulfonate ion and phthalimide which serve to buffer the remaining Brønsted acid and thus inhibit the racemic background reaction. Thus, the actual background reaction operative under catalytic conditions is not well mimicked by simply removing the catalyst.
doi:10.1021/ja413270h
PMCID: PMC3985927  PMID: 24548006
22.  An Fc Domain Protein–Small Molecule Conjugate as an Enhanced Immunomodulator 
Proteins as well as small molecules have demonstrated success as therapeutic agents, but their pharmacologic properties sometimes fall short against particular drug targets. Although the adenosine 2a receptor (A2AR) has been identified as a promising target for immunotherapy, small molecule A2AR agonists have suffered from short pharmacokinetic half-lives and the potential for toxicity by modulating nonimmune pathways. To overcome these limitations, we have tethered the A2AR agonist CGS-21680 to the immunoglobulin Fc domain using expressed protein ligation with Sf9 cell secreted protein. The protein small molecule conjugate Fc-CGS retained potent Fc receptor and A2AR interactions and showed superior properties as a therapeutic for the treatment of a mouse model of autoimmune pneumonitis. This approach may provide a general strategy for optimizing small molecule therapeutics.
doi:10.1021/ja5006674
PMCID: PMC3954559  PMID: 24533830
23.  Concise Enantiospecific Total Synthesis of Tubingensin A 
We report the enantiospecific total synthesis of (+)-tubingensin A. Our synthesis features an aryne cyclization to efficiently introduce the vicinal quaternary stereocenters of the natural product and proceeds in only nine steps (longest linear sequence) from known compounds.
doi:10.1021/ja501142e
PMCID: PMC3985696  PMID: 24524351
24.  The Feasibility of Formation and Kinetics of NMR Signal Amplification by Reversible Exchange (SABRE) at High Magnetic Field (9.4 T) 
1H NMR signal amplification by reversible exchange (SABRE) was observed for pyridine and pyridine-d5 at 9.4 T, a field that is orders of magnitude higher than what is typically utilized to achieve the conventional low-field SABRE effect. In addition to emissive peaks for the hydrogen spins at the ortho positions of the pyridine substrate (both free and bound to the metal center), absorptive signals are observed from hyperpolarized orthohydrogen and Ir-complex dihydride. Real-time kinetics studies show that the polarization build-up rates for these three species are in close agreement with their respective 1H T1 relaxation rates at 9.4 T. The results suggest that the mechanism of the substrate polarization involves cross-relaxation with hyperpolarized species in a manner similar to the spin-polarization induced nuclear Overhauser effect. Experiments utilizing pyridine-d5 as the substrate exhibited larger enhancements as well as partial H/D exchange for the hydrogen atom in the ortho position of pyridine and concomitant formation of HD molecules. While the mechanism of polarization enhancement does not explicitly require chemical exchange of hydrogen atoms of parahydrogen and the substrate, the partial chemical modification of the substrate via hydrogen exchange means that SABRE under these conditions cannot rigorously be referred to as a non-hydrogenative parahydrogen induced polarization process.
doi:10.1021/ja501052p
PMCID: PMC3985893  PMID: 24528143
25.  Circularly Polarized Luminescence by Visible-Light Absorption in a Chiral O-BODIPY Dye: Unprecedented Design of CPL Organic Molecules from Achiral Chromophores 
Circularly polarized luminescence (CPL) in simple (small, nonaggregated, nonpolymeric) O-BODIPYs (R)-1 and (S)-1 by irradiation with visible light is first detected as proof of the ability of a new structural design to achieve CPL from inherently achiral monochromophore systems in simple organic molecules. The measured level of CPL (|glum|) in solution falls into the usual range of that obtained from other simple organic molecules (10–5–10–2 range), but the latter having more complex architectures since axially chiral chromophores or multichromophore systems are usually required. The new design is based on chirally perturbing the acting achiral chromophore by orthogonally tethering a single axially chiral 1,1′-binaphtyl moiety to it. The latter does not participate as a chromophore in the light-absorption/emission phenomenon. This simple design opens up new perspectives for the future development of new small-sized CPL organic dyes (e.g., those based on other highly luminescent achiral chromophores and/or chirally perturbing moieties), as well as for the improvement of the CPL properties of the organic molecules spanning their use in photonic applications.
doi:10.1021/ja412294s
PMCID: PMC3984031  PMID: 24524257

Results 1-25 (5141)