2.6 CuAAC Applications in Macromolecules, Polymers, Nanoparticles, and Supramolecular Chemistry

In this chapter we describe applications of copper-catalyzed azide–alkyne cycloaddition (CuAAC) in macromolecular synthesis and polymer functionalization. This entails the synthesis of polymers with different architectures and the conjugation of polymers to surfaces and particles.

Exfoliated black phosphorous-mediated CuAAC chemistry for organic and macromolecular synthesis under white LED and near-IR irradiation

The development of long-wavelength photoinduced copper-catalyzed azide–alkyne click (CuAAC) reaction routes is attractive for organic and polymer chemistry. In this study, we present a novel synthetic methodology for the photoinduced CuAAC reaction utilizing exfoliated two-dimensional (2D) few-layer black phosphorus nanosheets (BPNs) as photocatalysts under white LED and near-IR (NIR) light irradiation. Upon irradiation, BPNs generated excited electrons and holes on its conduction (CB) and valence band (VB), respectively. The excited electrons thus formed were then transferred to the CuII ions to produce active CuI catalysts. The ability of BPNs to initiate the CuAAC reaction was investigated by studying the reaction between various low molar mass alkyne and azide derivatives under both white LED and NIR light irradiation. Due to its deeper penetration of NIR light, the possibility of synthesizing different macromolecular structures such as functional polymers, cross-linked networks and block copolymer has also been demonstrated. The structural and molecular properties of the intermediates and final products were evaluated by spectral and chromatographic analyses.

The Block Copolymer Shuffle in Size Exclusion Chromatography: The Intrinsic Problem with Using Elugrams to Determine Chain Extension Success

Size exclusion chromatography (SEC) based on direct homopolymer calibration is the preferred method for molecular weight determination in macromolecular synthesis. However, using the same method and calibration in block copolymer...

3D visualization of macromolecule synthesis

Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole-mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.

3D Visualization of Macromolecule Synthesis

Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here, we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.

Macromolecular Synthesis Shutoff Resistance by Myeloid Cells Is Critical to IRF7-Dependent Systemic Interferon Alpha/Beta Induction after Alphavirus Infection

ABSTRACT Alphavirus infection of fibroblastic cell types in vitro inhibits host cell translation and transcription, leading to suppression of interferon alpha/beta (IFN-α/β) production. However, the effect of infection upon myeloid cells, which are often the first cells encountered by alphaviruses in vivo, is unclear. Previous studies demonstrated an association of systemic IFN-α/β production with myeloid cell infection efficiency. Murine infection with wild-type Venezuelan equine encephalitis virus (VEEV), a highly myeloid-cell-tropic alphavirus, results in secretion of very high systemic levels of IFN-α/β, suggesting that stress responses in responding cells are active. Here, we infected myeloid cell cultures with VEEV to identify the cellular source of IFN-α/β, the timing and extent of translation and/or transcription inhibition in infected cells, and the transcription factors responsible for IFN-α/β induction. In contrast to fibroblast infection, myeloid cell cultures infected with VEEV secreted IFN-α/β that increased until cell death was observed. VEEV inhibited translation in most cells early after infection (<6 h postinfection [p.i.]), while transcription inhibition occurred later (>6 h p.i.). Furthermore, the interferon regulatory factor 7 (IRF7), but not IRF3, transcription factor was critical for IFN-α/β induction in vitro and in sera of mice. We identified a subset of infected Raw 264.7 myeloid cells that resisted VEEV-induced translation inhibition and secreted IFN-α/β despite virus infection. However, in the absence of IFN receptor signaling, the size of this cell population was diminished. These results indicate that IFN-α/β induction in vivo is IRF7 dependent and arises in part from a subset of myeloid cells that are resistant, in an IFN-α/β-dependent manner, to VEEV-induced macromolecular synthesis inhibition. IMPORTANCE Most previous research exploring the interaction of alphaviruses with host cell antiviral responses has been conducted using fibroblast lineage cell lines. Previous studies have led to the discovery of virus-mediated activities that antagonize host cell antiviral defense pathways, such as host cell translation and transcription inhibition and suppression of STAT1 signaling. However, their relevance and impact upon myeloid lineage cell types, which are key responders during the initial stages of alphavirus infection in vivo, have not been well studied. Here, we demonstrate the different abilities of myeloid cells to resist VEEV infection compared to nonmyeloid cell types and begin to elucidate the mechanisms by which host antiviral responses are upregulated in myeloid cells despite the actions of virus-encoded antagonists.