Lysine bioconjugation on native albumin with a sulfonyl acrylate reagent

. This protocol details a novel bioconjugation strategy that uses a methanesulfonyl acrylate reagent that is directed to the most reactive lysine on human serum albumin, which enables the construction of chemically defined and stable bioconjugates. The reaction proceeds rapidly and a regioselective modification is achieved using a single molar equivalent of the reagent under biocompatible conditions (37 ºC, pH 8.0). Importantly, the bioconjugate retains both the secondary structural content and function of the unmodified protein. During the reaction of the amino group of lysine and the sulfonyl acrylate reagent, methanesulfinic acid is released after the conjugate addition, which then generates an electrophilic acrylate moiety on the protein. This acrylate can be further used for site-specific protein labelling using a synthetic molecule bearing a reactive amine under biocompatible conditions (21 ºC, pH 8.0).


Introduction
Protein-drug conjugates have been reported and evaluated in clinical trials. 1,2Compared to the parental small molecule drugs, protein-drug conjugates offer several advantages, including halflife extension, localization to a target tissue, avoidance of drug-drug interactions, and toxicity reduction. 3,4,5Site-or residue-specific methods to install drugs on proteins simplify the interpretation of results and yield predictable conjugates. 6However, chemical reactions that can proceed under conditions mild enough to maintain the structure and function of the modified proteins are limited.Even more rare are chemical strategies that can target a single site, leading to products with uniform properties and optimal function without the need for sequence engineering. 7njugation to serum albumin (HSA) has emerged as a powerful approach for adjuvating the immune response triggered by synthetic vaccines and extending the in vivo half-life of many small molecule and peptide/protein drugs. 8,9However, HSA conjugation strategies, can often yield heterogeneous mixtures with inadequate pharmacokinetics, low efficacies, and variable safety profiles.To date, none of these compounds have displayed site specificity for a single lysine (siteselective bioconjugation).Barbas et al. first approached this goal in 2014 by showing that certain lysine residues in albumin reacted more rapidly with α,β-unsaturated sulfonamides than other lysine residues. 10e method reported therein explores a two-step strategy to build protein-drug conjugates: the first step consists of lysine chemo-and regioselective chemical modification utilizing a methanesulfonyl acrylate aza-Michael acceptor; the second step then generates a type-2 alkene that can be further modified through a second aza-Michael ligation with amine-and hydroxylamine-containing molecules (Fig. 1). 11Many relevant conjugation reagents such as fluorescent probes, polyethylene glycol (PEG), carbohydrate, deoxyribonucleic acid (DNA) or drug derivatives are commercially available and feature a free reactive amine handle.This twostep, irreversible and chemoselective approach can be directly used to modify several commercial proteins and antibodies bearing free lysine residues (can be confirmed by protein digestion and analysis via LC-MS/MS analysis -see subheading 3.3), and in some cases even in the presence of potentially reactive cysteines.In this protocol, we describe the method for the modification of recombinant human serum albumin (rHSA) at a single lysine from potential 59 reactive lysines.

Materials
Prepare all reagents and solutions using ultrapure water (prepared by purifying deionized water at 25 ºC to >15 MΩ/cm resistance and filtered through a 0.2-μm disc filter) and analytical grade reagents.Prepare all reagents at room temperature and store them at 4 ºC (unless indicated otherwise).Use all commercially available reagents as received unless otherwise noted.Reagents used in the procedure are potentially dangerous, and appropriate care should be taken during their manipulation.The solid and liquid waste products generated should be disposed of appropriately, as defined locally.95% aqueous acetonitrile with 0.05% formic acid and 10 mM ammonium acetate with 0.1% formic acid; 50% aqueous acetonitrile with 0.25% formic acid.

Solutions for protein modification
1. Formic acid, LC-MS grade.
2. LC-MS system for sulfonyl acrylate reagent characterization: Waters micromass ZQ instrument equipped, electrospray (ESI) ionization, with Waters 2795 HPLC and a Waters 2996 photodiode array detector.The separation technology is based on a 50x4.6 mm C18 column (currently a Phenomenex Kinetix solid core column).

Vortex shaker suitable for short-time operation (touch function), activated by
pressing shaker attachment or continuous operation.

Methods
Carry out all procedures at room temperature unless otherwise specified.
2. Treat the solution with 0.18 g of sodium methanesulfinate (1.5 mmol) portion wise over 10 min at room temperature (note 4).

3.
Stir the solution at room temperature for a period of 1.25 h and concentrate the solution in vacuum by evaporating all the solvent.4. The residue is taken up in water (10 mL) and extracted four times with ethyl acetate (4x 10 mL) using a liquid/liquid extraction flask.Liquid-liquid extraction is a very well-known method to separate compounds (usually the desired compound from impurities) based on their relative solubilities in two different immiscible liquids, usually water (polar) and an organic solvent (non-polar).
5. Wash the combined ethyl acetate solution (40 mL) with saturated sodium chloride solution in an Erlenmeyer flask, dry over anhydrous magnesium sulfate, filter (fluted paper filter) and concentrate in vacuum to give a white solid.
6. Purify the solid residue by flash column chromatography (pre-packed until 16 cm high with silica gel -see subheading 2.1) eluting with hexane/ethyl acetate 8:2, to give desired product (Scheme 1) as a white solid (0.175 g, 65% yield) (notes 5 and 6). 1. Mass spectroscopy is performed using a Waters micromass ZQ (LC-MS) instrument (see subheding 2.4).This system is an automated service utilizing electrospray (ESI) ionization.To prepare the sample, 1 mg of the product is dissolved in methanol and transferred to a mass vial (see subheading 2.5).9.The mobile phases are described in subheading 2.1.The separation technology is based on a 50x4.6 mm C18 column (currently a Phenomenex Kinetix solid core column) (note 7).The system runs using 50% aqueous acetonitrile with 0.25% formic acid as mobile phase and can measure accurate masses from 150 Da to 1500 Da.
6. Analyse a 10 μL aliquot at each reaction time by LC-MS (note 14). 7. Complete conversion to the expected product is observed after 1 h (calculated mass,

7 .
Analyse reaction mixtures by analytical TLC on TLC silica gel plates (see subheading 2.5).Visualization is accomplished with UV light (254 nm) or KMnO4 staining solution.8. 1 H NMR and 13 C NMR spectra are recorded on a Bruker 400 MHz DPX-400 Dual Spectrometer in deuterated CDCl3 as a solvent using tetramethylsilane as an internal standard.To prepare the sample, 5 mg of the product are dissolved in deuterated CDCl3 and transferred to an RMN tube.Chemical shifts are reported in parts per million (ppm) on the δ scale from tetramethylsilane (NMR descriptions: s, singlet).

Figure 2 .
Figure 2. Typical analysis of rHSA conjugation.The total ion chromatogram, combined ion series and deconvoluted spectra are shown.

Figure 3 .
Figure 3. Scheme of reaction for the bioconjugation rHSA with 1.

Figure 4 .
Figure 4. Combined ion series and deconvoluted mass spectrum of the reaction of rHSA (10 μM) with 1 equiv. of 1 after 1 h at 37 ºC.Identical data is obtained at 2 and 24 h.