Intra Q-body: an antibody-based fluorogenic probe for intracellular proteins that allows live cell imaging and sorting

Although intracellular biomarkers can be imaged with fluorescent dye(s)-labeled antibodies, the use of such probes for precise imaging of intracellular biomarkers in living cells remains challenging due to background noise from unbound probes. Herein, we describe the development of a conditionally active Fab-type Quenchbody (Q-body) probe derived from a monoclonal antibody (DO-1) with the ability to both target and spatiotemporally visualize intracellular p53 in living cells with low background signal. p53 is a key tumor suppressor and validated biomarker for cancer diagnostics and therapeutics. The Q-body displayed up to 27-fold p53 level-dependent fluorescence enhancement in vitro with a limit of detection of 0.72 nM. In fixed and live cells, 8.3- and 8.4-fold enhancement was respectively observed. Furthermore, we demonstrate live-cell sorting based on p53 expression. This study provides the first evidence of the feasibility and applicability of Q-body probes for the live-cell imaging of intrinsically intracellular proteins and opens a novel avenue for research and diagnostic applications on intracellular target-based live-cell sorting.

A fluorescent immunosensor that lights up tumor biomarker p53 in living cells was developed based on the Q-body technology. The technology was further applied to the live cell monitoring of p53 levels, and live cell sorting based on p53 expression.     

Recruitment of enzyme activity in albumin by molecular imprinting

The catalytic activity of bovine serum albumin (BSA) modified physically by molecular imprinting using transition-state analogue (TSA) as a template molecule was studied. The resultant imprinted serum albumin (Imp-BSA) showed the rate acceleration of dehydrofluorination reaction from (4R,4S)-4-fluoro-4-(4-nitrophenyl)butan-2-one ( 1 ) and followed the type of Michaelis-Menten reaction in ethyl acetate solution. The enzymatic activity of Imp-BSA was competitively inhibited by (4R,4S)-4-hydroxy-4-(4-nitrophenyl)butan-2-one ( 4 ).     

Synthesis of β‐monosubstituted α,β‐unsaturated amides with Z‐selectivity using diphenylphosphonoacetamides

The utility of diphenylphosphonoacetamides [(PhO)₂P(O)CH₂CONRR′] as Horner–Wadsworth–Emmons reagents was examined with five different patterns of substitution upon the amide nitrogen atom ( 2a : R, R′ = CH₂Ph; 2b : R = CH₂Ph, R′ = H; 2c : R = Me, R′ = OMe; 2d : R, R′ = Ph; 2e : R, R′ = (CH₂)₄). The reaction of 2a was found to be Z‐selective for aromatic aldehydes with selectivities up to 95:5. Reagent 2b led to reasonable selectivity for both benzaldehyde (85:15) and 3‐phenylpropionaldehyde (87:13), while 2c was somewhat effective for only the latter alkyl aldehyde (83:17). Compounds 2d and 2e exhibited slightly lower selectivities compared with 2a . © 2004 Wiley Periodicals, Inc. Heteroatom Chem 15:515–523, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.20054     

One‐Pot Synthesis of Novel Branched Polylactide Through the Copolymerization of Lactide with Mevalonolactone

Polylactide, which is a biodegradable and bio‐absorbable polymer having low immunogenicity and good biocompatibility, has been mainly studied for biomedical applications. Branched polymers have different rheological and mechanical properties compared with their linear counterparts owing to their molecular architectures. We synthesized novel biodegradable polylactide having a branched structure composed of metabolically degradable and/or absorbable materials only. The branched polylactide was obtained from a one‐pot copolymerization of L ‐lactide using metabolic intermediate dl ‐mevalonolactone as a bifunctional comonomer having both lactone ring and pendant hydroxy group. The glass transition point, melting point and crystallinity of the branched polylactide are lower than those of linear polylactide.