Diels-alder conjugation methods

A method of producing the compound having a structure according to Formula (I):

• wherein:
• BA is a binding agent;
• Gln is a glutamine residue of said BA;
• SP is absent or a spacer connected to the side chain of a glutamine residue of said BA;
• Z is a Diels-Alder adduct;
• L is a linker;
• D is a therapeutic moiety, wherein the therapeutic moiety is a maytansinoid, a tubulysin, an auristatin, a dolastatin, a camptothesin, a pyrrolobenzodiazepine, an antibiotic, an antiviral agent, an anti-inflammatory agent, an immunomodulator, an antifungal agent, a steroid, or an analogue or derivative thereof, or D is an imaging agent moiety;
• n is an integer from 1 to 3;
• wherein when n is 2 or 3, Z, L and D may be the same or different;
• and
• d is an integer from 1 to 4,
• the method comprising the steps of:
• a.) contacting:
• i) a compound having a structure according to Formula (V-x):
• wherein:
• X is a moiety that comprises a diene;
• and
• 1 is an integer from 1 to 6;
• with ii) a compound according to Formula (VI-y):
• wherein:
• Y is a moiety that comprises a dienophile;
• and
• b.) isolating the produced compound.

A method of producing the compound of Formula (V-y):

• wherein:
• BA is a binding agent;
• Gln is a glutamine residue of said BA;
• SP is absent or a spacer;
• Z is a Diels-Alder adduct;
• Y is a dienophile moiety;
• 1 is a number from 1 to 6;
• and
• n is 1 or 2,
• the method comprising the steps of:
• 1.) contacting i) the binding agent having at least one acceptor glutamine residue and ii) a compound according to formula V-y1:
• NH2-SP-Y (V-y1) in the presence of transglutaminase (TGase),
• and
• 2.) isolating the produced compound.

A method of producing the compound of Formula (V-y):

• wherein:
• BA is a binding agent;
• Gln is a glutamine residue of said BA;
• SP is absent or a spacer;
• Z is a Diels-Alder adduct;
• Y is a dienophile moiety;
• 1 is a number from 1 to 6;
• and
• n is 1 or 2,
• the method comprising the steps of:
• 1.) contacting i) the binding agent having at least one acceptor glutamine residue and ii) a compound according to formula V-y2:
• in the presence of transglutaminase (TGase),
• Y1 is a moiety that comprises a dienophile;
• Y2 is a moiety that comprises a dienophile, wherein Y1 and Y2 can be the same or different;
• and
• 2.) isolating the produced compound.