Jianmin Gao

Bioorthogonal fluorogenic reactions serve as enabling tools in research and biotechnology. Herein we describe fluorogenic conjugations of semicarbazide with coumarin derivatives that incorporate a 2-acetylphenylboronic acid motif. These designed coumarins rapidly conjugate with semicarbazide to give diazaborine products with significantly enhanced fluorescence. To demonstrate potential applications of this fluorogenic reaction, we synthesized a semicarbazide-presenting amino acid D-Dap-Scz, which readily incorporates into the cell wall of Staphalococcus aureus and serves as a handle for conjugation with the coumarins. The fluorogenic conjugation of the coumarins to cell surface semicarbazide enables facile visualization of D-Dap-Scz treated bacteria.

Iminoboronate-Based Peptide Cyclization That Responds to pH, Oxidation, and Small Molecule Modulators

Co-reporter:Anupam Bandyopadhyay

Journal of the American Chemical Society 2016 Volume 138(Issue 7) pp:2098-2101

Publication Date(Web):February 9, 2016

DOI:10.1021/jacs.5b12301

As a rich source of therapeutic agents, peptide natural products usually adopt a cyclic or multicyclic scaffold that minimizes structural flexibility to favor target binding. Inspired by nature, chemists have been interested in developing synthetic cyclic and multicyclic peptides that serve as biological probes and potential therapeutics. Herein we describe a novel strategy for peptide cyclization in which intramolecular iminoboronate formation allows spontaneous cyclization under physiologic conditions to yield monocyclic and bicyclic peptides. Importantly the iminoboronate-based cyclization can be rapidly reversed in response to multiple stimuli, including pH, oxidation, and small molecules. This highly versatile strategy for peptide cyclization should find applications in many areas of chemical biology.

Fast Diazaborine Formation of Semicarbazide Enables Facile Labeling of Bacterial Pathogens

Co-reporter:Anupam Bandyopadhyay, Samantha Cambray, and Jianmin Gao

Journal of the American Chemical Society 2016 Volume 139(Issue 2) pp:871-878

Publication Date(Web):December 19, 2016

DOI:10.1021/jacs.6b11115

Bioorthogonal conjugation chemistry has enabled the development of tools for the interrogation of complex biological systems. Although a number of bioorthogonal reactions have been documented in literature, they are less ideal for one or several reasons including slow kinetics, low stability of the conjugated product, requirement of toxic catalysts, and side reactions with unintended biomolecules. Herein we report a fast (>103 M–1 s–1) and bioorthogonal conjugation reaction that joins semicarbazide to an aryl ketone or aldehyde with an ortho-boronic acid substituent. The boronic acid moiety greatly accelerates the initial formation of a semicarbazone conjugate, which rearranges into a stable diazaborine. The diazaborine formation can be performed in blood serum or cell lysates with minimal interference from biomolecules. We further demonstrate that application of this conjugation chemistry enables facile labeling of bacteria. A synthetic amino acid D-AB3, which presents a 2-acetylphenylboronic acid moiety as its side chain, was found to incorporate into several bacterial species through cell wall remodeling, with particularly high efficiency for Escherichia coli. Subsequent D-AB3 conjugation to a fluorophore-labeled semicarbazide allows robust detection of this bacterial pathogen in blood serum.

Fast and selective labeling of N-terminal cysteines at neutral pH via thiazolidino boronate formation

Co-reporter:Anupam Bandyopadhyay, Samantha Cambray and Jianmin Gao

Chemical Science 2016 vol. 7(Issue 7) pp:4589-4593

Publication Date(Web):04 Apr 2016

DOI:10.1039/C6SC00172F

Facile labeling of proteins of interest is highly desirable in proteomic research as well as in the development of protein therapeutics. Herein we report a novel method that allows for fast and selective labeling of proteins with an N-terminal cysteine. Although N-terminal cysteines are well known to conjugate with aldehydes to give thiazolidines, the reaction requires acidic conditions and suffers from slow kinetics. We show that benzaldehyde with an ortho-boronic acid substituent readily reacts with N-terminal cysteines at neutral pH, giving rate constants on the order of 103 M−1 s−1. The product features a thiazolidino boronate (TzB) structure and exhibits improved stability due to formation of the B–N dative bond. While stable at neutral pH, the TzB complex dissociates upon mild acidification. These characteristics make the TzB conjugation chemistry potentially useful for the development of drug–protein conjugates that release the small molecule drug in acidic endosomes.

Targeting biomolecules with reversible covalent chemistry

Co-reporter:Anupam Bandyopadhyay, Jianmin Gao

Current Opinion in Chemical Biology 2016 Volume 34() pp:110-116

Publication Date(Web):October 2016

DOI:10.1016/j.cbpa.2016.08.011

•Reversible covalent chemistry expands the tool box for molecular recognition.•Reversible covalent inhibitors exhibit appealing characteristics as therapeutics.•α-Cyanoacrylamides display tunable profiles (stability and kinetics) for cysteine targeting.•Boronate ester formation enables selective targeting of activated Ser/Thr nucleophiles.•Iminoboronate formation enables selective targeting of amine-presenting biomolecules.Interaction of biomolecules typically proceeds in a highly selective and reversible manner, for which covalent bond formation has been largely avoided due to the potential difficulty of dissociation. However, employing reversible covalent warheads in drug design has given rise to covalent enzyme inhibitors that serve as powerful therapeutics, as well as molecular probes with exquisite target selectivity. This review article summarizes the recent advances in the development of reversible covalent chemistry for biological and medicinal applications. Specifically, we document the chemical strategies that allow for reversible modification of the three major classes of nucleophiles in biology: thiols, alcohols and amines. Emphasis is given to the chemical mechanisms that underlie the development of these reversible covalent reactions and their utilization in biology.

GramicidinA Mutants with Antibiotic Activity against Both Gram-Positive and Gram-Negative Bacteria

Co-reporter:Breanna L. Zerfas;Yechaan Joo ; Jianmin Gao

ChemMedChem 2016 Volume 11( Issue 6) pp:629-636

Publication Date(Web):

DOI:10.1002/cmdc.201500602

Abstract

Antimicrobial peptides (AMPs) have shown potential as alternatives to traditional antibiotics for fighting infections caused by antibiotic-resistant bacteria. One promising example of this is gramicidin A (gA). In its wild-type sequence, gA is active by permeating the plasma membrane of Gram-positive bacteria. However, gA is toxic to human red blood cells at similar concentrations to those required for it to exert its antimicrobial effects. Installing cationic side chains into gA has been shown to lower its hemolytic activity while maintaining the antimicrobial potency. In this study, we present the synthesis and the antibiotic activity of a new series of gA mutants that display cationic side chains. Specifically, by synthesizing alkylated lysine derivatives through reductive amination, we were able to create a broad selection of structures with varied activities towards Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). Importantly, some of the new mutants were observed to have an unprecedented activity towards important Gram-negative pathogens, including Escherichia coli, Klebsiella pneumoniae and Psuedomonas aeruginosa.

Iminoboronate Formation Leads to Fast and Reversible Conjugation Chemistry of -Nucleophiles at Neutral pH

Co-reporter:Dr. Anupam Byopadhyay ;Dr. Jianmin Gao

Chemistry - A European Journal 2015 Volume 21( Issue 42) pp:14748-14752

Publication Date(Web):

DOI:10.1002/chem.201502077

Abstract

Bioorthogonal reactions that are fast and reversible under physiological conditions are in high demand for biological applications. Herein, it is shown that an ortho boronic acid substituent makes aryl ketones rapidly conjugate with α-nucleophiles at neutral pH. Specifically, 2-acetylphenylboronic acid and derivatives were found to conjugate with phenylhydrazine with rate constants of 10² to 10³ M⁻¹ s⁻¹, comparable to the fastest bioorthogonal conjugations known to date. ¹¹B NMR analysis revealed the varied extent of iminoboronate formation of the conjugates, in which the imine nitrogen forms a dative bond with boron. The iminoboronate formation activates the imines for hydrolysis and exchange, rendering these oxime/hydrazone conjugations reversible and dynamic under physiological conditions. The fast and dynamic nature of the iminoboronate chemistry should find wide applications in biology.

Understanding lipid recognition by protein-mimicking cyclic peptides

Co-reporter:Azade S. Hosseini, Hong Zheng, Jianmin Gao

Tetrahedron 2014 70(42) pp: 7632-7638

Publication Date(Web):

DOI:10.1016/j.tet.2014.07.104

Exploring and Exploiting Polar−π Interactions with Fluorinated Aromatic Amino Acids

Co-reporter:Christopher J. Pace and Jianmin Gao

Accounts of Chemical Research 2013 Volume 46(Issue 4) pp:907

Publication Date(Web):October 24, 2012

DOI:10.1021/ar300086n

Fluorination has become an increasingly attrac-tive strategy in protein engineering for both basic research and biomedical applications. Thus researchers would like to understand the consequences of fluorination to the structure, stability, and function of target proteins. Although a substantial amount of work has focused on understanding the properties of fluorinated aliphatic amino acids, much less is known about fluorinated aromatic residues. In addition, polar−π interactions, often referred to as aromatic interactions, may play a significant role in protein folding and protein–protein interactions. Fluorination of aromatic residues presents an ideal strategy for probing polar−π interactions in proteins.This Account summarizes the recent studies of the incorporation of fluorinated aromatic amino acids into proteins. Herein we discuss the effects of fluorinating aromatic residues and rationalize them in the context of polar−π interactions. The results strongly support the proposal that polar−π interactions are energetically significant to protein folding and function. For example, an edge–face interaction of a pair of phenylalanines contributes as much as −1 kcal/mol to protein stability, while cation−π interactions can be much stronger. Furthermore, this new knowledge provides guidelines for protein engineering with fluorination. Importantly, incorporating perfluorinated aromatic residues into proteins enables novel mechanisms of molecular recognition that do not exist in native proteins, such as arene-perfluoroarene stacking. Such novel mechanisms can be used for programming protein folding specificity and engineering peptide-based materials.

A split ligand for lanthanide binding: facile evaluation of dimerizing proteins

Co-reporter:Yue Zhao and Jianmin Gao

Chemical Communications 2012 vol. 48(Issue 24) pp:2997-2999

Publication Date(Web):30 Jan 2012

DOI:10.1039/C2CC17891E

Luminescence of lanthanides is attractive for biological applications due to its long lifetime and sharp emission profiles. We describe the split display of a lanthanide binding ligand that allows facile evaluation of dimerizing proteins. The split lanthanide ligand is cysteine reactive, and therefore should be readily applicable to a variety of protein systems.

Synthesis of Tetrafluorinated Aromatic Amino Acids with Distinct Signatures in 19F NMR

Co-reporter:Luoheng Qin, Christopher Sheridan, and Jianmin Gao

Organic Letters 2012 Volume 14(Issue 2) pp:528-531

Publication Date(Web):December 23, 2011

DOI:10.1021/ol203140n

Fluorinated amino acids serve as powerful tools in protein chemistry. We synthesized a series of para-substituted tetrafluorophenylalanines via the regioselective SNAr chemistry of the commercially available pentafluorophenylalanine Boc-Z. These novel unnatural amino acids display distinct 19F NMR signatures, making them powerful tools for analyzing protein–membrane interactions with NMR spectroscopy.

Stacked Fluoroaromatics as Supramolecular Synthons for Programming Protein Dimerization Specificity

Co-reporter:Christopher J. Pace;Hong Zheng;Ruben Mylvaganam;Diane Kim ; Jianmin Gao

Angewandte Chemie 2012 Volume 124( Issue 1) pp:107-111

Publication Date(Web):

DOI:10.1002/ange.201105857

Experimental Evaluation of CH Interactions in a Protein Core

Co-reporter:Christopher J. Pace;Diane Kim ; Jianmin Gao

Chemistry - A European Journal 2012 Volume 18( Issue 19) pp:5832-5836

Publication Date(Web):

DOI:10.1002/chem.201200334

Stacked Fluoroaromatics as Supramolecular Synthons for Programming Protein Dimerization Specificity

Co-reporter:Christopher J. Pace;Hong Zheng;Ruben Mylvaganam;Diane Kim ; Jianmin Gao

Angewandte Chemie International Edition 2012 Volume 51( Issue 1) pp:103-107

Publication Date(Web):

DOI:10.1002/anie.201105857

Solubilized Gramicidin A as Potential Systemic Antibiotics

Co-reporter:Fang Wang;Luoheng Qin;Christopher J. Pace;Patrick Wong;Ryan Malonis ; Jianmin Gao

ChemBioChem 2012 Volume 13( Issue 1) pp:51-55

Publication Date(Web):

DOI:10.1002/cbic.201100671

Cofactor-Free Detection of Phosphatidylserine with Cyclic Peptides Mimicking Lactadherin

Co-reporter:Hong Zheng ; Fang Wang ; Qin Wang

Journal of the American Chemical Society 2011 Volume 133(Issue 39) pp:15280-15283

Publication Date(Web):September 7, 2011

DOI:10.1021/ja205911n

Cyclic peptides (cLacs) are designed to mimic the natural phosphatidylserine (PS) binding protein lactadherin. Unlike annexin V or its small molecule mimics, the cLac peptides selectively target PS-presenting membranes with no need for metal cofactors. We further show that a fluorophore-labeled cLac effectively stains early apoptotic cells. The small size and facile conjugation with a variety of imaging tracers make the cLac design promising for imaging cell death in vitro as well as in living organisms.

A FlAsHTetracysteine Assay for Quantifying the Association and Orientation of Transmembrane -Helices

Co-reporter:Christopher J. Pace;Qiongying Huang;Fang Wang;Kanwal S. Palla; Amelia A. Fuller; Jianmin Gao

ChemBioChem 2011 Volume 12( Issue 7) pp:1018-1022

Publication Date(Web):

DOI:10.1002/cbic.201000736

Highly sensitive amyloid detection enabled by thioflavin T dimers

Co-reporter:Luoheng Qin, Julian Vastl and Jianmin Gao

Molecular BioSystems 2010 vol. 6(Issue 10) pp:1791-1795

Publication Date(Web):07 Jul 2010

DOI:10.1039/C005255H

Fluorescent molecules that specifically target amyloid structures are highly desirable for amyloid research. Herein, we show a dimeric design of thioflavin T improves its binding affinity to Aβ amyloid by up to 70 fold, while not sacrificing the specificity and the “light-up” feature upon amyloid binding.

Highly Specific Heterodimerization Mediated by Quadrupole Interactions

Co-reporter:Hong Zheng ; Jianmin Gao

Angewandte Chemie 2010 Volume 122( Issue 46) pp:8817-8821

Publication Date(Web):

DOI:10.1002/ange.201002860

Highly Specific Heterodimerization Mediated by Quadrupole Interactions

Co-reporter:Hong Zheng ; Jianmin Gao

Angewandte Chemie International Edition 2010 Volume 49( Issue 46) pp:8635-8639

Publication Date(Web):

DOI:10.1002/anie.201002860

Facile Synthesis of Tetrafluorotyrosine and Its Application in pH Triggered Membrane Lysis

Co-reporter:Fang Wang ; Luoheng Qin ; Patrick Wong

Organic Letters () pp:

Publication Date(Web):December 10, 2010

DOI:10.1021/ol102610q

“Smart” peptides that lyse membranes in response to biological stimuli are of considerable interest. Herein, we report a facile synthesis of l-tetrafluorotyrosine (l-f4Y) and its utilization as a pH sensing element. The synthetic route affords gram quantities of l-f4Y within two days. Incorporation of this unnatural amino acid into magainin 2 gives a peptide that turns on its membrane-lytic activity upon mild acidification.

A split ligand for lanthanide binding: facile evaluation of dimerizing proteins

Co-reporter:Yue Zhao and Jianmin Gao

Chemical Communications 2012 - vol. 48(Issue 24) pp:NaN2999-2999

Publication Date(Web):2012/01/30

DOI:10.1039/C2CC17891E

Fast and selective labeling of N-terminal cysteines at neutral pH via thiazolidino boronate formation

Co-reporter:Anupam Bandyopadhyay, Samantha Cambray and Jianmin Gao

Chemical Science (2010-Present) 2016 - vol. 7(Issue 7) pp:NaN4593-4593

Publication Date(Web):2016/04/04

DOI:10.1039/C6SC00172F