Site-selective installation of BASHY fluorescent dyes to Annexin V for targeted detection of apoptotic cells

Fluorophores are indispensable for imaging biological processes. We report the design and synthesis of azide-tagged boronic acid salicylidenehydrazone (BASHY) dyes and their use for site-selective labelling of Annexin V. The Annexin V-BASHY conjugate maintained function and fluorescence as demonstrated by the targeted detection of apoptotic cells.We thank FCT Portugal (Doctoral Fellowship, SFRH/BD/94779/2013 to F. M. F. S., Postdoctoral Fellowship, SFRH/BPD/103172/2014 to P. M. S. D. C.; projects PTDC/QUI-QUI/118315/2010 and PTDC/BBB BQB/0506/2012; PTDC/QEQ-QOR/1434/2014: PTDC/SAUFAR/119389/2010; FCT Investigator to G. J. L. B. and P. M. P. G.; iMed.ULisboa grant UID/DTP/04138/2013), EU (Marie-Curie CIG to G. J. L. B.; Marie-Sklodowska Curie ITN ProteinConjugates to G. J. L. B. and P. M. P. G.), DFG (SI 2117/1-1 to F. S.), CNPq Brazil (fellowship 200456/2015-6 to J. B. B.); Ministerio de Economía y Competitividad, Madrid, Spain (grant CTQ2014-54729-C2-1-P), Junta de Andalucía (grant P12-FQM-2140) and the EPSRC (G. J. L. B.) for financial support. G. J. L. B. is a Royal Society University Research Fellow and the recipient of a European Research Council Starting Grant (TagIt)

Rh(II) catalysed intramolecular C-H insertion of diazo substrates in water: a simple and efficient approach to catalyst reuse

Water is an efficient solvent for the Rh-2(OAc)(4) catalysed intramolecular C - H insertion of a range of diazo substrates without competitive water insertion. Due to the high solubility and stability of the catalyst in water, the catalyst can be efficiently reused.publishedVersionPeer reviewe

Cysteine-Selective Reactions for Antibody Conjugation

Moving tracks from maleimide: New site-selective protein modification reactions at cysteine have been developed. Unlike conventional maleimide conjugation, which results in a labile thioether succinimide, the new bioconjugation reactions result in stable conjugates and provide opportunities to develop a new generation of homogeneous, stable, and therapeutically useful conjugates

Iminoboronates are efficient intermediates for selective, rapid and reversible N-terminal cysteine functionalisation

We show that formyl benzeno boronic acids (2FBBA) selectively react with N-terminal cysteines to yield a boronated thiazolidine featuring a B-N bond. The reaction exhibits a very rapid constant rate (2.38 ± 0.23 × 102 M-1 s-1) under mild aqueous conditions (pH 7.4, 23 °C) and tolerates different amino acids at the position adjacent to the N-cysteine. DFT calculations highlighted the diastereoselective nature of this ligation reaction and support the involvement of the proximal boronic acid in the activation of the imine functionality and the stabilisation of the boronated thiazolidine through a chelate effect. The 2FBBA reagent allowed the effective functionalisation of model peptides (C-ovalbumin and a laminin fragment) and the boronated thiazolidine construct was shown to be stable over time, though the reaction was reversible in the presence of benzyl hydroxylamine. The reaction proved to be highly chemoselective, and 2FBBA was used to functionalize the N-terminal cysteine of calcitonin in the presence of a potentially competing in-chain thiol group. This exquisite selectivity profile enabled the dual functionalisation of calcitonin and the interactive orthogonal modification of this peptide when 2FBBA was combined with conventional maleimide chemistry. These results highlight the potential of this methodology to construct complex and well-defined bioconjugates

Construction of homogeneous antibody-drug conjugates using site-selective protein chemistry

Systemic chemotherapy, the current standard of care for the treatment of cancer, is rarely curative and is often accompanied by debilitating side effects. Targeted drug delivery stands as an alternative to chemotherapy, with the potential to improve upon its low efficacy and systemic toxicity. Among targeted therapeutic options, antibody-drug conjugates (ADCs) have emerged as the most promising. These conjugates represent a new class of biopharmaceuticals that selectively deliver potent cytotoxic drugs to cancer cells, sparing healthy tissue throughout the body. Despite this promise, early heterogenous ADCs suffered from stability, pharmacokinetic, and efficacy issues that hindered clinical development. Recent advances in antibody engineering, linkers for drug-release, and chemical site-selective antibody conjugation have led to the creation of homogenous ADCs that have proven to be more efficacious than their heterogeneous predecessors both in vitro and in vivo. In this minireview, we focus on and discuss recent advances in chemical site-selective modification strategies for the conjugation of drugs to antibodies and the resulting potential for the development of a new generation of homogenous ADCs

Site-selective installation of BASHY fluorescent dyes to Annexin V for targeted detection of apoptotic cells

Fluorophores are indispensable for imaging biological processes. We report the design and synthesis of azide-tagged boronic acid salicylidenehydrazone (BASHY) dyes and their use for site-selective labelling of Annexin V. The Annexin V-BASHY conjugate maintained function and fluorescence as demonstrated by the targeted detection of apoptotic cells

Analysis of human papillomavirus prevalence and TP53 polymorphism in head and neck squamous cell carcinomas

Head and neck squamous cell carcinoma is a disease associated with tobacco and alcohol abuse. There is evidence that the oncogenic human papillomavirus (HPV) may also be a risk for upper aerodigestive tract cancers. High-risk HPVs encode two early proteins, E6 and E7, that can bind to p53 and pRb, respectively, and induce its degradation or inactivation. The TP53 gene has a single polymorphism at codon 72 of exon 4 that encodes either arginine (Arg) or proline (Pro). The purpose of this study was to evaluate the role of HPV infection and TP53 polymorphism in head and neck cancer. We analyzed 50 tumors, as well swabs of oral mucosa front 142 control individuals, with a polymerase chain reaction technique. The prevalence of HPV in controls was 10.6% and in cancer specimens 16%. The frequency distribution of genotypes in controls was 50% Arg/Arg, 43% Arg/ Pro and 7% Pro/Pro; in tumors, it was 52% Arg/Arg, 32% Arg/Pro, and 16% Pro/Pro. Contrary to the results of some studies on cervical cancer, no association between any TP53 genotype or allele and the development of head and neck cancer was observed, regardless of HPV status, except for the Pro/Pro genotype, which is associated with the absence of HPV. The arginine allele appears to protect against head and neck cancers. Also, the data showed that HPV infection results in no increased risk of developing head and neck tumors. (C) 2004 Elsevier B.V. All rights reserved