Acyl Fluorides: Fast, Efficient, and Versatile Lysine-Based Protein Conjugation via Plug-and-Play Strategy

Abstract

We report a plug-and-play strategy for the preparation of functionally enhanced antibodies with a defined average degree of conjugation (DoC). The first stage (plug) allows the controllable and efficient installation of azide groups on lysine residues of a native antibody using 4-azidobenzoyl fluoride. The second step (play) allows for versatile antibody functionalization with a single payload or combination of payloads, such as a toxin, a fluorophore, or an oligonucleotide, via copper-free strain-promoted azide-alkyne cycloaddition (SPAAC). It is notable that in comparison to a classical N-hydroxysuccinimide ester (NHS) strategy, benzoyl fluorides show faster and more efficient acylation of lysine residues in a PBS buffer. This translates into better control of the DoC and enables the efficient and fast functionalization of delicate biomolecules at low temperature.

Synthesis of carboxy‐polyethylene glycol‐amine (CA (PEG)n) and [1‐14C]‐CA (PEG)n via oxa‐Michael addition of amino‐polyethylene glycols to propiolates vs to acrylates

“…The O ‐terminus of 5 is then converted to an amino group giving intermediate 6 , a protected CA (PEG) n . Alternatively, addition of amino‐polyethylene glycol 3 to acrylate 4 gives directly the intermediate 6 [7a–e]. Deprotection and hydrolysis of the intermediate 6 complete the synthesis of CA (PEG) n ( 1 ).…”

“…To be efficient and practical, synthesis plan A demands that the acrylate [1‐ 14 C]‐ 4 be the limiting reagent in the oxa‐Michael addition and the yield of the adduct be good. It came to our attention that the reported oxa‐Michael addition of amino‐polyethylene glycol 3 to acrylate 4 has been achieved using an excess (greater than or equal to 1.5 Eq) of acrylate [7a–e]. Furthermore, the yield of the adduct in the limited number of reported cases was generally moderate,[7a–d] yet low yields and failed reactions have also been reported.…”

“…It came to our attention that the reported oxa‐Michael addition of amino‐polyethylene glycol 3 to acrylate 4 has been achieved using an excess (greater than or equal to 1.5 Eq) of acrylate [7a–e]. Furthermore, the yield of the adduct in the limited number of reported cases was generally moderate,[7a–d] yet low yields and failed reactions have also been reported. [9e] In response, the use of phase transfer catalysts has addressed such issues, resulting in excellent yield; however, large excesses (up to 5 Eq) of acrylate were normally used [7e].…”

Acyl Fluorides in Late‐Transition‐Metal Catalysis

“…Acyl fluorides are carboxylic acid derivatives that contain af luorocarbonyl group.B ecause of the electrostatic stability of the CÀFb ond, [1] acyl fluorides are more stable than comparable acyl halides,s uch as acyl chlorides,t owards solvolysis,i ncluding hydrolysis, [2] alcoholysis, [3] and aminolysis. [4] Accordingly,t hey are regarded as easy-to-handle and versatile potential alternatives to carboxylic acids.A cyl fluorides have traditionally been used as acylationr eagents for nucleophilic molecules,w hereby the acyl CÀFb onds are converted into the corresponding acyl CÀXbonds.Especially in peptide chemistry,t he derivatization of amino acids into the corresponding acyl fluorides is aw ell-documented strategy for peptide coupling [5,6] through the formation of acyl C À Nb onds, [7,8] as the use of a-amino acid fluorides generates the corresponding amides in good yields without racemization. Aside from peptide coupling reactions,numerous reactions have been reported that involve various nucleophiles and the formation of acyl CÀXb onds,f or example,the formation of acyl C À C [9][10][11] and C À O [12,13] bonds, and other related reactions.…”

Carbonsäurefluoride in der Katalyse durch späte Übergangsmetalle

“…Carbonsäurefluoride fanden traditionell Verwendung als Acylierungsmittel fürn ucleophile Moleküle,w obei die C-F-Bindungen der Carbonsäurederivate in die jeweiligen C-X-Bindungen umgewandelt werden. Besonders in der Peptidchemie ist die Derivatisierung von Aminosäuren zu den entsprechenden Säurefluoriden eine gut dokumentierte Strategie der Peptidkupplung [5,6] über die Bildung von C(Acyl)-N-Bindungen, [7,8] da der Einsatz von a-Aminosäurefluoriden die jeweiligen Amide in guten Ausbeuten ohne Racemisierung liefert. Außer Peptidkupplungen wurden auch zahlreiche Reaktionen unter Beteiligung diverser Nucleophile und der Bildung von C(Acyl)-X-Bindungen, beispielsweise von C-(Acyl)-C- [9][10][11] und C(Acyl)-O-Bindungen, [12,13] sowie verwandte Reaktionen [14][15][16] beschrieben.…”