Fluorophore‐Conjugated Holliday Junctions for Generating Super‐Bright Antibodies and Antibody Fragments

The site‐specific modification of proteins with fluorophores can render a protein fluorescent without compromising its function. To avoid self‐quenching from multiple fluorophores installed in close proximity, we used Holliday junctions to label proteins site‐specifically. Holliday junctions enable modification with multiple fluorophores at reasonably precise spacing. We designed a Holliday junction with three of its four arms modified with a fluorophore of choice and the remaining arm equipped with a dibenzocyclooctyne substituent to render it reactive with an azide‐modified fluorescent single‐domain antibody fragment or an intact immunoglobulin produced in a sortase‐catalyzed reaction. These fluorescent Holliday junctions improve fluorescence yields for both single‐domain and full‐sized antibodies without deleterious effects on antigen binding.     

Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

A strategy for the preparation of homogeneous antibody–drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site‐specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug‐linkers that have differing physiochemical properties and exert complementary anti‐cancer activities. Dual‐auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual‐drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site‐specific protein modification.     

Reversible Control over Molecular Recognition in Surface‐Bound Photoswitchable Hydrogen‐Bonding Receptors: Towards Read–Write–Erase Molecular Printboards

The synthesis of an anthracene‐bearing photoactive barbituric acid receptor and its subsequent grafting onto azide‐terminated alkanethiol/Au self‐assembled monolayers by using an Cu^I‐catalyzed azide–alkyne reaction is reported. Monolayer characterization using contact‐angle measurements, electrochemistry, and spectroscopic ellipsometry indicate that the monolayer conversion is fast and complete. Irradiation of the receptor leads to photodimerization of the anthracenes, which induces the open‐to‐closed gating of the receptor by blocking access to the binding site. The process is thermally reversible, and polarization‐modulated IR reflection–absorption spectroscopy indicates that photochemical closure and thermal opening of the surface‐bound receptors occur in 70 and 100 % conversion, respectively. Affinity of the open and closed surface‐bound receptor was characterized by using force spectroscopy with a barbituric‐acid‐modified atomic force microscope tip.     

A Versatile Approach for the Site‐Specific Modification of Recombinant Antibodies Using a Combination of Enzyme‐Mediated Bioconjugation and Click Chemistry

A unique two‐step modular system for site‐specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide–alkyne cycloaddition click reaction. The versatility of the two‐step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron‐emitting copper‐64 radiotracer for fluorescence and positron‐emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor‐made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.     

Star polymers by photoinduced copper‐catalyzed azide–alkyne cycloaddition click chemistry

Well‐defined star polymers consisting of tri‐, tetra‐, or octa‐arms have been prepared via coupling‐onto strategy using photoinduced copper(I)‐catalyzed 1,3‐dipolar cycloaddition click reaction. An azide end‐functionalized polystyrene and poly(methyl methacrylate), and an alkyne end‐functionalized poly(ε‐caprolactone) as the integrating arms of the star polymers are prepared by the combination of controlled polymerization and nucleophilic substitution reactions; whereas, multifunctional cores containing either azide or alkyne functionalities were synthesized in quantitatively via etherification and ring‐opening reactions. By using photoinduced copper‐catalyzed azide–alkyne cycloaddition (CuAAC) click reaction, reactive linear polymers are simply attached onto multifunctional cores to form corresponding star polymers via coupling‐onto methodology. The chromatographic, spectroscopic, and thermal analyses have clearly demonstrated that successful star formations can be obtained via photoinduced CuAAC click reaction. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015 , 53, 1687–1695     

Selective Uptake of Cylindrical Poly(2‐Oxazoline) Brush‐AntiDEC205 Antibody‐OVA Antigen Conjugates into DEC‐Positive Dendritic Cells and Subsequent T‐Cell Activation

To achieve specific cell targeting by various receptors for oligosaccharides or antibodies, a carrier must not be taken up by any of the very many different cells and needs functional groups prone to clean conjugation chemistry to derive well‐defined structures with a high biological specificity. A polymeric nanocarrier is presented that consists of a cylindrical brush polymer with poly‐2‐oxazoline side chains carrying an azide functional group on each of the many side chain ends. After click conjugation of dye and an anti‐DEC205 antibody to the periphery of the cylindrical brush polymer, antibody‐mediated specific binding and uptake into DEC205⁺‐positive mouse bone marrow‐derived dendritic cells (BMDC) was observed, whereas binding and uptake by DEC205^? negative BMDC and non‐DC was essentially absent. Additional conjugation of an antigen peptide yielded a multifunctional polymer structure with a much stronger antigen‐specific T‐cell stimulatory capacity of pretreated BMDC than application of antigen or polymer–antigen conjugate.     

Alkene–Azide 1,3‐Dipolar Cycloaddition as a Trigger for Ultrashort Peptide Hydrogel Dissolution

An alkene–azide 1,3‐dipolar cycloaddition between trans‐cyclooctene (TCO) and an azide‐capped hydrogel that promotes rapid gel dissolution is reported. Using an ultrashort aryl azide‐capped peptide hydrogel (PhePhe), we have demonstrated proof‐of‐concept where upon reaction with TCO, the hydrogel undergoes a gel–sol transition via 1,2,3‐triazoline degradation and 1,6‐self‐immolation of the generated aniline. The potential application of this as a general trigger in sustained drug delivery is demonstrated through release of encapsulated cargo (doxorubicin). Administration of TCO resulted in 87 % of the cargo being released in 10 h, compared to 13–14 % in the control gels. This is the first example of a potential bioorthogonal‐triggered hydrogel dissolution using a traditional click‐type reaction. This type of stimulus could be extended to other aryl azide‐capped hydrogels.     

Three‐Dimensional Ordered Antibody Arrays Through Self‐Assembly of Antibody–Polymer Conjugates

Three‐dimensional (3D) ordered arrays of human immunoglobulin G (IgG) were fabricated using well‐defined full‐length antibody–polymer conjugates (APCs). The conjugates were prepared through a two‐step sequential click approach with a combination of oxime ligation and strain promoted alkyne–azide cycloaddition. They were able to self‐assemble into lamellar nanostructures with alternating IgG and poly(N ‐isopropylacrylamide) (PNIPAM) nanodomains. As a proof‐of‐concept, these materials were fabricated into thin films and their specific binding ability was tested. The nanostructure not only improves the packing density and the proper orientation of the IgG, but also provides nanochannels to facilitate substrate transport.     

Synthesis and Reversible Hydration of a Pseudoprotein,a Fully Organic Polymeric Desiccant by Multiple Single‐Crystal‐to‐Single‐Crystal Transformations

A diphenylalanine derivative, N3‐Phe‐Phe‐NHCH2CCH, was designed for topochemical azide–alkyne cycloaddition (TAAC) polymerization. This dipeptide adopted β‐sheet arrangement as designed, in its crystals, but the azide and alkyne were not fitly aligned for their topochemical reaction. However, the voids present around these groups allowed them to attain a reactive geometry upon heating and their consequent TAAC polymerization to a pseudoprotein in a single‐crystal‐to‐single‐crystal (SCSC) fashion. This motion led to the creation of channels in the product crystal and it absorbed water from the surroundings to fill these channels as H‐bonded water wire. The pseudoprotein undergo reversible hydration/dehydration in SCSC fashion many times under mild conditions: hydration at low relative humidity and dehydration at low temperature. Vapor sorption analyses suggest that this fully organic polymer might be useful as an energy‐efficient desiccant material for controlling indoor humidity.     

Rapid and Efficient Generation of Stable Antibody–Drug Conjugates via an Encoded Cyclopropene and an Inverse‐Electron‐Demand Diels–Alder Reaction

Homogeneous antibody–drug conjugates (ADCs), generated by site‐specific toxin linkage, show improved therapeutic indices with respect to traditional ADCs. However, current methods to produce site‐specific conjugates suffer from low protein expression, slow reaction kinetics, and low yields, or are limited to particular conjugation sites. Here we describe high yielding expression systems that efficiently incorporate a cyclopropene derivative of lysine (CypK) into antibodies through genetic‐code expansion. We express trastuzumab bearing CypK and conjugate tetrazine derivatives to the antibody. We show that the dihydropyridazine linkage resulting from the conjugation reaction is stable in serum, and generate an ADC bearing monomethyl auristatin E that selectively kills cells expressing a high level of HER2. Our results demonstrate that CypK is a minimal bioorthogonal handle for the rapid production of stable therapeutic protein conjugates.     

Synthesis and Reversible Hydration of a Pseudoprotein,a Fully Organic Polymeric Desiccant by Multiple Single‐Crystal‐to‐Single‐Crystal Transformations

A diphenylalanine derivative, N3‐Phe‐Phe‐NHCH2CCH, was designed for topochemical azide–alkyne cycloaddition (TAAC) polymerization. This dipeptide adopted β‐sheet arrangement as designed, in its crystals, but the azide and alkyne were not fitly aligned for their topochemical reaction. However, the voids present around these groups allowed them to attain a reactive geometry upon heating and their consequent TAAC polymerization to a pseudoprotein in a single‐crystal‐to‐single‐crystal (SCSC) fashion. This motion led to the creation of channels in the product crystal and it absorbed water from the surroundings to fill these channels as H‐bonded water wire. The pseudoprotein undergo reversible hydration/dehydration in SCSC fashion many times under mild conditions: hydration at low relative humidity and dehydration at low temperature. Vapor sorption analyses suggest that this fully organic polymer might be useful as an energy‐efficient desiccant material for controlling indoor humidity.     

Photo‐induced cross‐linking mechanism in azide–novolac negative photoresists: molecular level investigation using NMR spectroscopy

Negative photoresists are composed of a photoactive component (aromatic azides/bisazides) and cyclized rubber or novolac resin dissolved in an organic solvent. Hydrogen abstraction and/or insertion reaction of the reactive nitrene intermediate formed during photoirradiation of the azide result in a cross‐linked network of the novolac resin. The molecular weight of novolac resin in the exposed part of the photoresist film thus increases compared with that of the unexposed part. This makes the exposed part insoluble in the alkaline developer. Exploiting this change in physical property, a pattern can be transferred to a substrate from a mask. A better understanding of the exact mechanism of cross‐linking reactions is very important to the design of a high‐performing negative photoresist. A quinone–imine‐type complex has been proposed earlier involving the aromatic moiety of novolac resin as the reaction site. A more recent study focuses the attack of nitrene on the methylenic bridge and hydroxyl group of novolac resins, which were found to be responsible for the cross‐linking reaction along with the aromatic moiety of novolac resin. However, in our study no evidence was found for the involvement of a methylenic hydrogen or aromatic moiety of novolac resin in the cross‐linking reaction. The ¹H NMR, ¹³C NMR and DEPT‐135 spectra before and after photolysis indicate that the cross‐linking site is predominantly the hydroxyl group of novolac resin. Multiple reaction sites of attack for the nitrene intermediate have been demonstrated in cashew nut shell liquid (CNSL)‐based novolac resin by ¹H NMR spectroscopy, which in turn further increases the cross‐linked network in the exposed part of a negative photoresist. Copyright © 2003 John Wiley & Sons, Ltd.     

Azide‐functionalized nanoparticles as quantized building block for the design of soft–soft fluorescent polystyrene core—PAMAM shell nanostructures

This work deals with the covalent coupling of azide‐functionalized polymeric nanoparticles as a reactive core and amino‐terminated PAMAM dendrons as a reactive shell. The nanoassemblies thereby obtained could be modified after the dendronization step by grafting an alkynyl Bodipy dye on the unreacted azide moieties. Only a few steps are required to attain nanoassemblies that could mimic dendrimers of high generation with sizes of nano‐objects beyond those of dendrimers. The structure of the nanoassemblies are composed of a polystyrene core, an inner shell including the Bodipy dyes along with the internal branches of the PAMAM, and the terminal amino groups from the outer shell. The dendritic shell acts as a protective layer that prevents NP from aggregation in a surfactant free aqueous solution. The nano‐objects display absorption and emission maxima above 500 nm with brightness that are the same order of magnitude than Qdots. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 115–126     

Preparation of Information‐Containing Macromolecules by Ligation of Dyad‐Encoded Oligomers

A simplified strategy for preparing non‐natural information‐containing polymers is reported. The concept relies on the successive ligation of oligomers that contain minimal sequence motifs. It was applied here to the synthesis of digitally‐encoded poly(triazole amide)s, in which propyl and 2‐methyl propyl motifs are used to code 0 and 1, respectively. A library of four oligo(triazole amide)s containing the information dyads 00, 01, 10, and 11 was prepared. These oligomers contain two reactive functions, that is, an alkyne and a carboxylic acid. Thus, they can be linked to another with the help of a reactive spacer containing azide and amine functions. Using two successive chemoselective steps, that is, azide‐alkyne Huisgen cycloaddition and carboxylic acid‐amine coupling, monodisperse polymers can be obtained. In particular, the library of dyads permits the implementation of any desired sequence using a small number of steps. As a proof‐of‐concept, the synthesis of molecular bytes 00000000 and 00000110 is described.     

“Clickable” and Antifouling Block Copolymer Brushes as a Versatile Platform for Peptide‐Specific Cell Attachment

To tailor cell–surface interactions, precise and controlled attachment of cell‐adhesive motifs is required, while any background non‐specific cell and protein adhesion has to be blocked effectively. Herein, a versatile and highly reproducible antifouling surface modification based on “clickable” groups and hierarchically structured diblock copolymer brushes for the controlled attachment of cells is reported. The polymer brush architecture combines an antifouling bottom block of poly(2‐hydroxyethyl methacrylate) poly(HEMA) and an ultrathin azide‐bearing top block, which can participate in well‐established “click” reactions including the highly selective copper‐catalyzed alkyne‐azide cycloaddition (CuAAC) reaction under mild conditions. This straightforward approach allows the rapid conjugation of a cell‐adhesive, alkyne‐bearing cyclic RGD peptide motif, enabling subsequent specific attachment of NIH 3T3 fibroblasts, their extensive proliferation and confluent cell sheet formation after 48 h of incubation. The generally applicable strategy presented in this report can be employed for surface functionalization with diverse alkyne‐bearing biological moieties via CuAAC or copper‐free alkyne‐azide cycloaddition protocols, making it a versatile functionalization approach and a promising tool for tissue engineering, biomaterial implant design, and other applications that require surfaces supporting highly specific cell attachment.     

Copper‐Catalyzed Azide–Ynamide Cyclization to Generate α‐Imino Copper Carbenes: Divergent and Enantioselective Access to Polycyclic N‐Heterocycles

Here an efficient copper‐catalyzed cascade cyclization of azide‐ynamides via α‐imino copper carbene intermediates is reported, representing the first generation of α‐imino copper carbenes from alkynes. This protocol enables the practical and divergent synthesis of an array of polycyclic N‐heterocycles in generally good to excellent yields with broad substrate scope and excellent diastereoselectivities. Moreover, an asymmetric azide–ynamide cyclization has been achieved with high enantioselectivities (up to 98:2 e.r.) by employing BOX‐Cu complexes as chiral catalysts. Thus, this protocol constitutes the first example of an asymmetric azide–alkyne cyclization. The proposed mechanistic rationale for this cascade cyclization is further supported by theoretical calculations.     

Site‐Selective Cysteine–Cyclooctyne Conjugation

We report a site‐selective cysteine–cyclooctyne conjugation reaction between a seven‐residue peptide tag (DBCO‐tag, Leu‐Cys‐Tyr‐Pro‐Trp‐Val‐Tyr) at the N or C terminus of a peptide or protein and various aza‐dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO‐tag increases the rate of the thiol–yne reaction 220‐fold, thereby enabling selective conjugation of DBCO‐tag to DBCO‐linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO‐tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and the antibody trastuzumab. This study demonstrates that short peptide tags can aid in accelerating bond‐forming reactions that are often slow to non‐existent in water.     

Azido‐Functionalized 5′ Cap Analogues for the Preparation of Translationally Active mRNAs Suitable for Fluorescent Labeling in Living Cells

The 7‐methylguanosine (m⁷G) cap structure is a unique feature present at the 5′ ends of messenger RNAs (mRNAs), and it can be subjected to extensive modifications, resulting in alterations to mRNA properties (e.g. translatability, susceptibility to degradation). It also can provide molecular tools to study mRNA metabolism. We developed new mRNA 5′ cap analogues that enable the site‐specific labeling of RNA at the 5′ end using strain‐promoted azide–alkyne cycloaddition (SPAAC) without disrupting the basic function of mRNA in protein biosynthesis. Some of these azide‐functionalized compounds are equipped with additional modifications to augment mRNA properties. The application of these tools was demonstrated by labeling translationally active mRNAs in living cells.     

Polar Red‐Emitting Rhodamine Dyes with Reactive Groups: Synthesis,Photophysical Properties,and Two‐Color STED Nanoscopy Applications

The synthesis, reactivity, and photophysical properties of new rhodamines with intense red fluorescence, two polar residues (hydroxyls, primary phosphates, or sulfonic acid groups), and improved hydrolytic stability of the amino‐reactive sites (NHS esters or mixed N‐succinimidyl carbonates) are reported. All fluorophores contain an N‐alkyl‐1,2‐dihydro‐2,2,4‐trimethylquinoline fragment, and most of them bear a fully substituted tetrafluoro phenyl ring with a secondary carboxamide group. The absorption and emission maxima in water are in the range of 635–639 and 655–659 nm, respectively. A vastly simplified approach to red‐emitting rhodamines with two phosphate groups that are compatible with diverse functional linkers was developed. As an example, a phosphorylated dye with an azide residue was prepared and was used in a click reaction with a strained alkyne bearing an N‐hydroxysuccinimid (NHS) ester group. This method bypasses the undesired activation of phosphate groups, and gives an amphiphilic amino‐reactive dye, the solubility and distribution of which between aqueous and organic phases can be controlled by varying the pH. The presence of two hydroxyl groups and a phenyl ring with two carboxyl residues in the dyes with another substitution pattern is sufficient for providing the hydrophilic properties. Selective formation of a mono‐N‐hydroxysuccinimidyl ester from 5‐carboxy isomer of this rhodamine is reported. The fluorescence quantum yields varied from 58 to 92 % for free fluorophores, and amounted to 18–64 % for antibody conjugates in aqueous buffers. The brightness and photostability of these fluorophores facilitated two‐color stimulated emission depletion (STED) fluorescence nanoscopy of biological samples with high contrast and minimal background. Selecting a pair of fluorophores with absorption/emission bands at 579/609 and 635/655 nm enabled two‐color channels with low cross‐talk and negligible background at approximately 40 nm resolution.     

A CXCR4‐Targeted Site‐Specific Antibody–Drug Conjugate

A chemically defined anti‐CXCR4–auristatin antibody–drug conjugate (ADC) was synthesized that selectively eliminates tumor cells overexpressing the CXCR4 receptor. The unnatural amino acid p‐acetylphenylalanine (pAcF) was site‐specifically incorporated into an anti‐CXCR4 immunoglobulin G (IgG) and conjugated to an auristatin through a stable, non‐cleavable oxime linkage to afford a chemically homogeneous ADC. The full‐length anti‐CXCR4 ADC was selectively cytotoxic to CXCR4⁺ cancer cells in vitro (half maximal effective concentration (EC₅₀)≈80–100 pM ). Moreover, the anti‐CXCR4 ADC eliminated pulmonary lesions from human osteosarcoma cells in a lung‐seeding tumor model in mice. No significant overt toxicity was observed but there was a modest decrease in the bone‐marrow‐derived CXCR4⁺ cell population. Because CXCR4 is highly expressed in a majority of metastatic cancers, a CXCR4–auristatin ADC may be useful for the treatment of a variety of metastatic malignancies.