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1.
J Am Chem Soc ; 141(34): 13619-13624, 2019 08 28.
Article in English | MEDLINE | ID: mdl-31389231

ABSTRACT

Herein, we report the development of a scalable and synthetically robust building block based on norbornadiene (NBD) that can be broadly incorporated into a variety of macromolecular architectures using traditional living polymerization techniques. By taking advantage of a selective and rapid deprotection with tetrazine, highly reactive "masked" cyclopentadiene (Cp) functionalities can be introduced into synthetic polymers as chain-end groups in a quantitative and efficient manner. The orthogonality of this platform further enables a cascade "click" process where the "unmasked" Cp can rapidly react with dienophiles, such as maleimides, through a conventional Diels-Alder reaction. Coupling proceeds with quantitative conversions allowing high molecular weight star and dendritic block copolymers to be prepared in a single step under ambient conditions.


Subject(s)
Cyclopentanes/chemical synthesis , Norbornanes/chemical synthesis , Polymers/chemical synthesis , Click Chemistry , Cycloaddition Reaction , Cyclopentanes/chemistry , Molecular Weight , Norbornanes/chemistry , Polymerization , Polymers/chemistry
2.
Bioconjug Chem ; 30(9): 2340-2348, 2019 09 18.
Article in English | MEDLINE | ID: mdl-31380623

ABSTRACT

The normal electron-demand Diels-Alder (DA) cycloaddition is a classic transformation routinely used in synthesis; however, applications in biological systems are limited. Here, we report a spiro[2.4]hepta-4,6-diene-containing noncanonical amino acid (SCpHK) capable of efficient incorporation into antibodies and subsequent coupling with maleimide via a DA reaction. SCpHK was stable throughout protein expression in mammalian cells and enabled covalent attachment of maleimide drug-linkers yielding DA antibody-drug conjugates (DA-ADCs) with nearly quantitative conversion in a one-step process. The uncatalyzed DA reaction between SCpHK and maleimide in aqueous buffer was rapid (1.8-5.4 M-1 s-1), and the antibody-drug adduct was stable in rat serum for at least 1 week at 37 °C. Anti-EphA2 DA-ADCs containing AZ1508 or SG3249 maleimide drug-linkers were potent inhibitors of tumor growth in PC3 tumor models in vivo. The DA bioconjugation strategy described here represents a simple method to produce site-specific and stable ADCs with maleimide drug-linkers.


Subject(s)
Immunoconjugates/chemistry , Maleimides/chemistry , Animals , CHO Cells , Cell Survival/drug effects , Cricetulus , Cycloaddition Reaction , Humans , Immunoconjugates/pharmacology , Models, Molecular , PC-3 Cells , Protein Conformation , Spiro Compounds/chemistry
3.
Angew Chem Int Ed Engl ; 58(25): 8489-8493, 2019 06 17.
Article in English | MEDLINE | ID: mdl-31018033

ABSTRACT

Here, we describe a diene-containing noncanonical amino acid (ncAA) capable of undergoing fast and selective normal electron-demand Diels-Alder (DA) reactions following its incorporation into antibodies. A cyclopentadiene derivative of lysine (CpHK) served as the reactive handle for DA transformations and the substrate for genetic incorporation. CpHK incorporated into antibodies with high efficiency and was available for maleimide conjugation or self-reaction depending on position in the amino acid sequence. CpHK at position K274 reacted with the maleimide drug-linker AZ1508 at a rate of ≈79 m-1 s-1 to produce functional antibody-drug conjugates (ADCs) in a one-step process. Incorporation of CpHK at position S239 resulted in dimerization, which covalently linked antibody heavy chains together. The diene ncAA described here is capable of producing therapeutic protein conjugates with clinically validated and widely available maleimide compounds, while also enabling proximity-based stapling through a DA dimerization reaction.


Subject(s)
Alkadienes/chemistry , Amino Acids/chemistry , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/chemistry , Maleimides/chemistry , Cycloaddition Reaction , Dimerization , Humans , Models, Molecular , Molecular Structure
4.
Bioconjug Chem ; 29(7): 2406-2414, 2018 07 18.
Article in English | MEDLINE | ID: mdl-29932647

ABSTRACT

The thiol-maleimide linkage is widely used for antibody-drug conjugate (ADC) production; however, conjugation of maleimide-drugs could be improved by simplified procedures and reliable conjugate stability. Here, we report the evaluation of electron-rich and cyclic dienes that can be appended to antibodies and reacted with maleimide-containing drugs through the Diels-Alder (DA) reaction. Drug conjugation is fast and quantitative due to reaction acceleration in water, and the linkage is more stable in serum than in the corresponding thiol-maleimide adduct with the same drug. ADCs produced using the DA reaction (DAADCs) are effective in vitro and in vivo, demonstrating the utility of this reaction in producing effective biotherapeutics. Given the large number of commercially available maleimide compounds, this conjugation approach could be readily applied to the production of a wide range of antibody (or protein) conjugates.


Subject(s)
Cycloaddition Reaction/methods , Immunoconjugates/chemistry , Maleimides/chemistry , Alkenes , Antibodies/chemistry , Cross-Linking Reagents/chemistry , Drug Stability , Maleimides/therapeutic use , Pharmaceutical Preparations/chemistry
5.
J Am Chem Soc ; 140(15): 5009-5013, 2018 04 18.
Article in English | MEDLINE | ID: mdl-29613783

ABSTRACT

The development and application of a novel endo furan-protected maleimide building block is reported. The endo isomer undergoes deprotection at temperatures ∼50 °C below the exo derivative. This enables a simple and powerful approach to quantitatively and selectively introduce functional maleimide groups via temperature modulation.

6.
Sci Rep ; 6: 19328, 2016 Jan 14.
Article in English | MEDLINE | ID: mdl-26771074

ABSTRACT

Accurate determination of urinary stone composition has significant bearing on understanding pathophysiology, choosing treatment modalities and preventing recurrence. A need exists for improved methods to determine stone composition. Urine of 31 patients with known renal calculi was examined with nanoscale flow cytometry and the calculi collected during surgery subsequently underwent petrographic thin sectioning with polarized and fluorescent microscopy. Fluorescently labeled bisphosphonate probes (Alendronate-fluorescein/Alendronate-Cy5) were developed for nanoscale flow cytometry to enumerate nanocrystals that bound the fluorescent probes. Petrographic sections of stones were also imaged by fluorescent and polarized light microscopy with composition analysis correlated to alendronate +ve nanocrystal counts in corresponding urine samples. Urine samples from patients with Ca(2+) and Mg(2+) based calculi exhibited the highest alendronate +ve nanocrystal counts, ranging from 100-1000 nm in diameter. This novel urine based assay was in agreement with composition determined by petrographic thin sections with Alendronate probes. In some cases, high alendronate +ve nanocrystal counts indicated a Ca(2+) or Mg(2+) composition, as confirmed by petrographic analysis, overturning initial spectrophotometric diagnosis of stone composition. The combination of nanoscale flow cytometry and petrographic thin sections offer an alternative means for determining stone composition. Nanoscale flow cytometry of alendronate +ve nanocrystals alone may provide a high-throughput means of evaluating stone burden.


Subject(s)
Urinary Calculi/chemistry , Flow Cytometry , Fluorescent Dyes , Humans , Kidney Calculi/diagnosis , Microscopy, Fluorescence , Microtomy , Urinalysis/methods
7.
Org Biomol Chem ; 13(31): 8465-9, 2015 Aug 21.
Article in English | MEDLINE | ID: mdl-26154503

ABSTRACT

This article describes the aza-Piancatelli rearrangement with hydroxylamines to 4-aminocyclopentenones and subsequent transformations that highlight the versatility of the cyclopentene scaffold and the value of the hydroxylamine nucleophile in this transformation.


Subject(s)
Cyclopentanes/chemistry , Hydroxylamines/chemistry , Methanol/chemistry , Catalysis , Stereoisomerism
8.
Mol Imaging ; 142015.
Article in English | MEDLINE | ID: mdl-25762192

ABSTRACT

A fluorescein-GLP-1 (7-37) analog was generated to determine GLP-1R distribution in various cell types of the pancreas in both strains of mice and receptor-specific uptake was confirmed by blocking with exendin-4. Biodistribution studies were carried out using 68Ga-labeled GLP-1(7-37) peptides in CD1 and C57BL/6 mice. In addition, immunocompromised mice bearing GLP-1R-expressing insulinomas were evaluated by positron emission tomography (PET) imaging and ex vivo biodistribution studies. The optical GLP-1 probe strongly colocalized with immunofluorescence for insulin and glucagon, and more weakly with amylase (exocrine pancreas) and cytokeratin 19 (ductal cells), confirming its application for in situ GLP-1R imaging in various pancreatic cell types. Insulinomas were clearly visualized by in vivo PET. Reducing the peptide positive charge decreased renal retention as well as tumor uptake. Results demonstrate the application of the developed GLP-1 peptide analogues for in situ (optical) and in vivo (PET) imaging of GLP-1R expression.


Subject(s)
Glucagon-Like Peptide 1/analogs & derivatives , Glucagon-Like Peptide-1 Receptor/metabolism , Molecular Imaging , Peptides/chemistry , Positron-Emission Tomography , Animals , CHO Cells , Cricetinae , Cricetulus , Cyclic AMP/metabolism , Gallium/chemistry , Gallium Radioisotopes/chemistry , Glucose/metabolism , Insulin/metabolism , Insulinoma/metabolism , Male , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Neoplasm Transplantation , Radioimmunoassay
9.
Artif DNA PNA XNA ; 4(1): 4-10, 2013.
Article in English | MEDLINE | ID: mdl-23422048

ABSTRACT

The synthesis of an azide containing PNA monomer is described. The monomer was incorporated into two PNA sequences for the purpose of synthesizing an intercalating fluorophore-labeled PNA and a metal binding hairpin using a solid phase copper catalyzed azide-alkyne Huisgen cycloaddition (CuAAC). Click chemistry was performed using 2-ethynylfluorene or 1-ethynylpyrene to add a fluorophore to the PNA, which were tested for their ability to recognize an abasic site on a DNA target. A PNA hairpin possessing azide monomers at each termini was synthesized and reacted with 2-ethynylpyridine to form a hairpin that is stabilized by Ni²âº.


Subject(s)
Alkynes/chemistry , Azides/chemical synthesis , Peptide Nucleic Acids/chemical synthesis , Azides/chemistry , Click Chemistry , Cycloaddition Reaction , Molecular Structure , Nucleic Acid Conformation
10.
Org Biomol Chem ; 10(32): 6521-5, 2012 Aug 28.
Article in English | MEDLINE | ID: mdl-22752020

ABSTRACT

The synthesis of nucleoside analogues incorporating 4-(5-pyrimidinyl)-1,2,3-triazole aglycons as expanded purine nucleobase mimics were accessed using the copper-catalyzed azide-alkyne Huisgen cycloaddition between a ribosyl azide and 5-alkynylpyrimidines. Depending on the nature of the alkyne employed, other nucleoside analogues that possess fluorescence or potential metal-binding properties were prepared. Computational studies were undertaken on the purine analogues and indicate that the heterocycles of the unfused nucleobase prefer a coplanar arrangement and the anti-glycosidic conformer is favoured in most instances.


Subject(s)
Copper/chemistry , Purine Nucleosides/chemistry , Ribonucleosides/chemistry , Catalysis , Click Chemistry , Computer Simulation , Copper/metabolism , Fluorescent Dyes/chemistry , Heterocyclic Compounds/chemistry , Molecular Conformation , Molecular Structure , Purine Nucleosides/metabolism
11.
Org Biomol Chem ; 10(4): 876-81, 2012 Jan 28.
Article in English | MEDLINE | ID: mdl-22159214

ABSTRACT

A Boc-protecting group strategy for Fmoc-based PNA (peptide nucleic acid) oligomerization has been developed for thymine, 2,6-diaminopurine (DAP) and 2-aminopurine (2AP). The monomers may be used interchangeably with standard Fmoc PNA monomers. The DAP monomer was incorporated into a PNA and was found to selectively bind to T (ΔT(m)≥ +6 °C) in a complementary DNA strand. The 2AP monomer showed excellent discrimination of T (ΔT(m)≥ +12 °C) over the other nucleobases. 2AP also acted as a fluorescent probe of the PNA:DNA duplexes and displayed fluorescence quenching dependent on the opposite base.


Subject(s)
2-Aminopurine/analogs & derivatives , 2-Aminopurine/chemistry , Peptide Nucleic Acids/chemical synthesis , Thymine/chemistry , 2-Aminopurine/chemical synthesis , 2-Aminopurine/metabolism , DNA/metabolism , Peptide Nucleic Acids/chemistry , Peptide Nucleic Acids/metabolism , Spectrometry, Fluorescence , Thymine/chemical synthesis , Thymine/metabolism
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