Formation of hydrazones and stabilized boron–nitrogen heterocycles in aqueous solution from carbohydrazides and ortho-formylphenylboronic acids

Abstract

A recent addition to the suite of fast bioorthogonal reactions combines hydrazines and hydroxylamines with ortho-carbonyl substituted phenylboronic acids. Carbohydrazides are easily incorporated into biomolecules, making them appealing substrates in these reactions. Here we show that simple alkyl carbohydrazides form a single product with ortho-formylphenylboronic acid in an organic solvent and in the solid state. The solution structures of the products formed from the carbohydrazides in buffered aqueous solution, however, are markedly different from those identified in the organic solvent and solid state. The reactants form a mixture of hydrazone and heterocyclic products, the relative composition of which varies with pH. The observed reversibility of bioconjugates using carbohydrazide can thus be explained by the reversibility of the boron-nitrogen bond in the heterocycle. In contrast, the inclusion of an α-amine into the carbohydrazide substrate yields a single product in which both nitrogens are bonded to boron. These tricyclic structures are the same in organic solvent, solid state and aqueous solution from pH 4 to pH 9. Bioconjugates formed with α-amino carbohydrazides are stable to SDS-PAGE, while those formed with simple alkyl carbohydrazides are not. We propose that the inclusion of an intramolecular stabilizing ligand into a carbohydrazide substrate is a generally applicable principle that may be exploited to form boronic acid-based bioconjugates with a defined structure and resistance to hydrolysis.

Boronic acids as building blocks for the construction of therapeutically useful bioconjugates

“…171 A similar experiment was performed with the a-amino carbohydrazide in which BSA was functionalized with a fluorescent coumarin through DAB formation. 182 Another example of DAB chemistry was reported by Gao and co-workers. Taking advantage of the dynamic rearrangement of bacterial peptidoglycan, the authors envisioned the possibility of incorporating a 2-ABBA-bearing amino acid, which could be subsequently labelled with a fluorescent semicarbazide.…”

“…However, a detailed study uncovered that, depending on pH and concentration, the reaction of 2-CBBA with carbohydrazides can yield different products. 182 If the reaction is performed at high millimolar concentrations in aqueous or organic solvents, the major product is the DAB anhydrous dimer. Under dilute conditions, at higher pHs (8-9) DAB is the obtained product, preferentially in the tetravalent boronate form.…”

Innovative Linker Strategies for Tumor‐Targeted Drug Conjugates

“…[56] Similarly,t he reaction of ortho-acetyl phenylboronica cid( 10)w ith carbohydrazide derivatives gives rise to as ix-membered benzodiazaborine ring (DAB, such as 13 and 14). [57] The latter shows remarkable serum stability andh ydrophilicity (due to its zwitterionic nature)a nd for theser easonsi th as been mostly exploiteds o far for the fast assembly of highlys table bioconjugates. [58] It is still unclear whether this functional group may also be considered as am ore stable version of the traditional N-acylhydrazone linker, [59] with potentiala pplications in acid-mediated release of potent hydrazide-bearing anticancer drugs.…”

“…The resulting N,O ‐iminoboronates showed improved stability at neutral pH and in human plasma, while efficiently releasing their amine cargoes at low pH values . Similarly, the reaction of ortho ‐acetyl phenylboronic acid ( 10 ) with carbohydrazide derivatives gives rise to a six‐membered benzodiazaborine ring (DAB, such as 13 and 14 ) . The latter shows remarkable serum stability and hydrophilicity (due to its zwitterionic nature) and for these reasons it has been mostly exploited so far for the fast assembly of highly stable bioconjugates .…”

N,O‐Iminoboronates: Reversible Iminoboronates with Improved Stability for Cancer Cells Targeted Delivery

“…[27] In this context, Bane also described the formation of stabilized hydrazones using a-aminec arbohydrazides and 2-formylbenzene boronic acids. [28] Based on these observations, we envisioned that a strategy to improve these construct'ss tabilityw ould be to designaN,O-bidentate ligand that enables the formation of the iminoboronate with an additionalB ÀOb ondScheme 1.…”