Paola Laurino

In Vitro Imaging and in Vivo Liver Targeting with Carbohydrate Capped Quantum Dots

PEGylated quantum dots (QDs) capped with d-mannose, d-galactose, and d-galactosamine have been synthesized. The stable, high quantum yield fluorescence of QDs was exploited to study specific carbohydrate-protein interactions in vitro and in vivo.

Asymmetric reactions in continuous flow

Summary An overview of asymmetric synthesis in continuous flow and microreactors is presented in this review. Applications of homogeneous and heterogeneous asymmetric catalysis as well as biocatalysis in flow are discussed.

Synthesis of Carbohydrate‐Functionalized Quantum Dots in Microreactors

Large quantities of monodisperse semiconductor nanocrystals, quantum dots (QDs), are needed for applications in electronics and the life sciences. For biological applications, the surface of QDs is often functionalized with carboxylic acids for the attachment of proteins or directly with carbohydrates. Traditional batch processes are of limited utility for the production of QDs on a larger scale owing to limited temperature control and lack of homogeneous mixing. Continuous-flow microreactors provide precise control over reaction conditions, including temperature, and the production time is independent of the process scale. The high surface-to-volume ratio of the microreactor channels enables precise temperature control as well as efficient mixing, allowing for the preparation of QDs with narrow size distribution. QDs have been prepared using microfabricated gas–liquid and liquid–liquid flow reactors. The preparation of surface-functionalized QDs under mild reaction conditions in the liquid phase remains challenging. Ideally, a continuous process would serve to both produce the quantum dots and to functionalize them. Herein we present a single-phase microfluidic system for the synthesis of highly luminescent, surface-functionalized CdSe and CdTe nanoparticles. In contrast to batch processes, which require temperatures of 250–300 8C, temperatures of 160 8C are sufficient in the flow process. Both the formation of the zinc sulfide shell and the functionalization of the nanoparticles with carboxy groups and carbohydrates were perfomed in a continuous-flow system (Figure 1). Differentsized quantum dots were obtained by simply varying the reaction time in the flow reactor. High reaction temperatures usually result in fast nucleation, and large nanocrystals are quickly obtained. At low temperatures, the size of the nanocrystals and the concentration of the unreacted precursors in the mixture can be balanced. Thus, continuous nucleation is suppressed, the residence time distribution (RTD) is narrowed, and homogeneous QD fractions are obtained by varying the reaction time. The homogenous reaction mixture and slow nucleation results in a mild process for the production of QDs using microreactors. CdSe and CdTe nanoparticles with different emission maxima were prepared by injection of a 1:1 mixture of Cd precursor and Se or Te precursor. The Cd precursor was prepared by the addition of oleic acid and oleylamine to a solution of cadmium oxide dissolved in lauric acid at 150 8C. The Se and Te precursors were prepared by dissolving elemental selenium or tellerium powder in tri-n-octylphosphine (TOP) in a Syrris microreactor. Reaction times ranged from 3 to 30 minutes. The CdSe and CdTe cores were purified by precipitation from methanol/chloroform/n-hexane and dried under vacuum. The average size distribution of each sample was calculated from the absorbance spectra (see Figure 1 in the Supporting Information). The optical properties of the QDs show a time-dependent bathochromic shift in the band-edge emission and enhanced intensity. The photoluminescence peaks of CdSe QDs are sharp, with fwhm (full width at half maximum) values of the band-edge luminescence between 40 and 50 nm (Figure 2), which indicates the narrow size distribution of the QDs. However, after 30 minutes of reaction time the fwhm increased from 40 to 90 nm, and a decrease in quantum yield indicated that saturated nucleation occurred after 20– Figure 1. Microreactor setup for the continuous-flow synthesis of functionalized QDs (OA: oleic acid; TOP: tri-n-octylphosphine).

Design, synthesis and biological evaluation of carbohydrate-functionalized cyclodextrins and liposomes for hepatocyte-specific targeting†

Targeting glycan-binding receptors is an attractive strategy for cell-specific drug and gene delivery. The C-type lectin asialoglycoprotein receptor (ASGPR) is particularly suitable for liver-specific delivery due to its exclusive expression by parenchymal hepatocytes. In this study, we designed and developed an efficient synthesis of carbohydrate-functionalized β-cyclodextrins (βCDs) and liposomes for hepatocyte-specific delivery. For targeting of ASGPR, rhodamine B-loaded βCDs were functionalized with glycodendrimers. Liposomes were equipped with synthetic glycolipids containing a terminal D-GalNAc residue to mediate binding to ASGPR. Uptake studies in the human hepatocellular carcinoma cell line HepG2 demonstrated that βCDs and liposomes displaying terminal D-Gal/D-GalNAc residues were preferentially endocytosed. In contrast, uptake of βCDs and liposomes with terminal d-Man or D-GlcNAc residues was markedly reduced. The d-Gal/d-GalNAc-functionalized βCDs and liposomes presented here enable hepatocyte-specific targeting. Gal-functionalized βCDs are efficient molecular carriers to deliver doxorubicin in vitro into hepatocytes and induce apoptosis.

Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability

Summary Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.

Detection of bacteria using glyco-dendronized polylysine prepared by continuous flow photofunctionalization.

Biocompatible glyco-dendronized poly-l-lysine (PLL) polymers carry either three or nine mannose- or galactose-bearing dendrons that selectively bind, and thus can be used to detect, bacteria. Central to the synthesis of glyco-dendronized polymers was the development of a continuous flow [2 + 2] photocycloaddition reaction to connect the dendrons and PLL. Glycodendronized polymers cluster bacteria by binding to cell-surface carbohydrate receptors and thereby result in an easy read-out using microscopic analyses.

Snowballing Radical Generation Leads to Ultrahigh Molecular Weight Polymers

Styrene is the classical monomer obeying zero-one kinetics in radical emulsion polymerization. Accordingly, particles that are less than 100 nm in diameter contain either one or no growing radical(s). We describe a unique photoinitiated polymerization reaction accelerated by snowballing radical generation in a continuous flow reactor. Even in comparison to classical emulsion polymerization, these unprecedented snowballing reactions are rapid and high-yielding, with each particle simultaneously containing more than one growing radical. This is a consequence of photoinitiator incorporation into the nascent polymer backbone and repeated radical generation upon photo-irradiation.

DddD Is a CoA-Transferase/Lyase Producing Dimethyl Sulfide in the Marine Environment

Dimethyl sulfide (DMS) is produced in oceans in vast amounts (>10(7) tons/year) and mediates a wide range of processes from regulating marine life forms to cloud formation. Nonetheless, none of the enzymes that produce DMS from dimethylsulfoniopropionate (DMSP) has been adequately characterized. We describe the expression and purification of DddD from the marine bacterium Marinomonas sp. MWYL1 and its biochemical characterization. We identified DMSP and acetyl-coenzyme A to be DddDs native substrates and Asp602 as the active site residue mediating the CoA-transferase prior to lyase activity. These findings shed light on the biochemical utilization of DMSP in the marine environment.

Continuous-flow reactor-based synthesis of carbohydrate and dihydrolipoic acid-capped quantum dots

A detailed protocol for the large-scale synthesis of carbohydrate and dihydrolipoic acid (DHLA)-coated CdSe/ZnS and CdTe/ZnS nanoparticles using continuous flow reactors is described here. Three continuous flow microreaction systems, operating at three different temperatures, are used for the synthesis of mannose-, galactose- or DHLA-functionalized quantum dots (QDs). In the first step of synthesis, the CdSe and CdTe nanoparticles are prepared. The size and spectral properties of the CdSe core of the nanoparticles are controlled by adjustment of the residence time and the temperature. As a second step, the zinc sulfide capping under homogenous conditions is carried out at a substantially lower temperature than is required for nanoparticle growth in batch processes. Finally, the trioctylphosphine/oleic acid ligand is effectively replaced with either carbohydrate PEG-thiol moieties or DHLA at 60 °C. This new protocol allows the synthesis of biologically active fluorescent QDs in 4 d.

Spontaneous Emergence of S‐Adenosylmethionine and the Evolution of Methylation

S-Adenosylmethionine (SAM) is an essential methylation cofactor. The origins of SAM methylation are complex, seemingly demanding the simultaneous emergence of an enzyme that makes SAM and enzyme(s) that utilize it. We report that both ATP and adenosine spontaneously react with methionine to yield SAM, thus suggesting that SAM could have emerged by chance. SAM methylation thus exemplifies how metabolites and pathways can co-emerge through the gradual recruitment of individual enzymes in reverse order.