Multi-arm Linker Units

The design and synthesis of multi-arm linker units are among Immunwork’s core technologies.  Generally, a linker unit has one core, multiple linking arms, and one or two coupling arms.  The core may be a peptide or another type of compound.  The peptide cores, which are frequently used in our molecular design, contain multiple lysine (K) residues and an amino acid residue with an azide or alkyne group in its side chain or/and a cysteine (C) residue.  A commonly used amino acid with azide in its side chain is L-azidohomoalanine (A^AH), and the one with alkyne group is L-homopropargylglycine (G^HP).  The A^AH/ G^HP residue and the C residue are often at the N- or C-terminus of the core, although not necessarily at the termini.  The K residues, the A^AH/ G^HP residue, and the C residue are separated by spacers, designated as “Z”.  The Z is generally a peptide of 2-12 hydrophilic amino acid residues or a PEGylated amino acid residue with 2-12 ethylene glycol (EG) units, i.e. NH₂-EG_2-12-COOH.  The Z’s in a peptide core need not be the same.  The NH₂ group at the N-terminus of the peptide core is protected by an acetyl group (Ac).
 

The azide or alkyne group of the peptide core serves as a functional group for coupling between two multi-arm linker units via a click reaction; in such a configuration, the coupling arm is synthesized as part of the peptide core.  The cysteine provides an SH group to conjugate with a coupling arm, maleimide (or vinyl sulfone)-EG_2-12-X, via a maleimide (or vinyl sulfone)-SH reaction. X can be, but not limit to, azide, alkyne, tetrazine, TCO (trans-cyclooctene), or a strained alkyne, i.e. DBCO (dibenzyl-cyclo-octyne) or BCN (bicyclo[6,1,0] non-4-yn-9-yl).  X serves as a functional group for coupling between two multi-arm linker units or between a multi-arm linker unit and a targeting or effector element.

The lysines in a peptide core provide NH₂ groups to conjugate with linking arms, NHS-EG_2-15-Y, via an NHS (N-hydroxysuccinimide ester)-NH₂ reaction. Y is usually maleimide, but can also be vinyl sulfone, azide, alkyne, tetrazine, TCO, or a strained alkyne. Y serves as a functional group for conjugation with a targeting or effector element.  
 

In a multi-arm linker unit, the Y is different from the functional group(s) used for coupling between multi-arm linker units.  When two coupling groups are present in a multi-arm linker unit, one of them is azide or alkyne group and the other is X, which is not azide or alkyne and is different from Y.  Here are some examples of multi-arm linker units.