Chemically cleavable group
Disclosed is the use of the reactive components of the inverse electron-demand Diels Alder reaction for chemical masking and unmasking in vitro. This can be applied in complex chemical reactions and, particularly in the synthesis of biomolecules, e.g. on solid supports. The reactice components are a dienophile, particularly a trans-cyclooctene, and a diene, particularly a tetrazine.
1 . A dienophile of Formula (1a):
wherein A and P each independently are CH 2 , or CHX D , provided that at least one is CHX D ; X D is (O—C(O)) p -(L D ) n -(C A ), or O—C(S)-(L D ) n -(C A ); wherein n=0 or 1, wherein p=0 or 1; wherein when p=0, L D or C A is bound to the dienophile via O, S, aromatic N or aromatic NH; L D is a self-immolative linker consisting of one or more self-immolative units;
wherein each self-immolative unit is selected from the group consisting of
wherein the wiggly line indicates a bond to (O—C(O)) p or O—C(S) of X D , or to a preceding self-immolative unit; wherein the asterisk indicates a bond to C A or to a further self-immolative unit;
wherein L 2 is O, S, NH, or NR with R being alkyl or aryl; wherein each L 1 is independently H or methyl;
Y, Z, X, Q each independently are selected from the group consisting of CR a 2 , C═O, O, and NR b , with at most three of Y, Z, X, and Q being C═O, wherein two R moieties together may form a ring, and with the proviso that no adjacent pairs of atoms are present selected from the group consisting of O—O, and O—NR b ;
wherein each R a can independently be H, alkyl, aryl, OR′, SR′, S(═O) 2 R′″, S(═O) 2 NR′R″, SO 3 H, PO 3 H, NO 2 , CN, CF 3 , CF 2 —R′, NR′R″, C(═O)R′, C(═O)OH, C(═O)NR′R″, C(═S)NR′R″, NR′C(═O)—R′″, NR′C(═S)—R′″, NR′C(═O)O—R′″, NR′C(═S)O—R′″, NR′C(═O)S—R′″, NR′C(═S)S—R′″, OC(═O)NR′—R′″, SC(═O)NR′—R′″, OC(═S)NR′—R′″, SC(═S)NR′—R′″, NR′C(═O)NR″—R″, or NR′C(═S)NR″—R″, with each R′ and each R″ independently being H, aryl or alkyl and R′″ independently being aryl or alkyl;
wherein each R b is independently selected from the group consisting of H, alkyl, aryl, O-aryl, O-alkyl, OH, C(═O)NR′R″ with R′ and R″ each independently being H, aryl or alkyl, and R′CO-alkyl with R′ being H, alkyl, and aryl;
wherein one R a or R b is optionally bound to a construct C B ; and wherein the self-immolative linker L D , in addition to being bound to C A , is also optionally bound to a construct C B ; wherein T and G denote H;
wherein each C A and C B are individually selected from the group consisting of biomolecules, biomolecule-binding moieties, polymers, particles, gels, fluorophores, and radiolabels.
2 . The dienophile according to claim 1 , wherein p is 1.
3 . The dienophile according to claim 1 , wherein X D is (O—C(O)) p -(L D ) n -(C A ).
4 . The dienophile according to claim 1 , wherein the construct comprises a biomolecule-binding moiety selected from the group consisting of non-covalent binding moieties, and covalent binding moieties.
5 . The dienophile according to claim 4 , wherein the non-covalent binding moiety is biotin or an antibody.
6 . The dienophile according to claim 4 , wherein the covalent binding moiety is an N-hydroxysuccinimide ester, a maleimide, an azide, an alkyne, or a photoreactive group.
7 . The dienophile according to claim 1 , wherein the biomolecule is selected from the group consisting of carbohydrates, peptides, peptoids, lipids, proteins, oligonucleotides, DNA, RNA, PNA, LNA, aptamers, hormones, steroids, and toxins.
8 . The dienophile according to claim 7 , wherein the protein is an enzyme, cytokine, antibody, antibody fragment, antibody fusion, or an antibody fragment fusion.
9 . The dienophile according to claim 8 , wherein the antibody fragment is Fab2, Fab, scFV, a diabody, a triabody, or VHH.
10 . The dienophile according to claim 1 , wherein the particle is a bead, a gold particle, a silica-based particle, a glass particle, a polymer particle, an iron oxide particle, a microparticle, or a nanoparticle.
11 . The dienophile according to claim 10 , wherein the bead is a magnetic bead.
12 . The dienophile according to claim 10 , wherein the microparticle is a liposome or polymersome.
13 . The dienophile according to claim 10 , wherein the nanoparticle is a liposome or a polymersome.
14 . The dienophile according to claim 1 , wherein the polymer is a resin.
15 . The dienophile according to claim 1 , wherein one of the bonds PQ, QX, XZ, ZY, YA is part of a fused ring, such that two exocyclic bonds are fixed in the same plane, and provided that PQ and YA are not part of an aromatic 5- or 6-membered ring, or of a conjugated 7-membered ring; when not part of a fused ring P and A are independently CH 2 or CHX D , provided that at least one is CHX D ; when part of a fused ring P and A are independently CH or CX D , provided that at least one is CX D ; the remaining moieties Y, Z, X, and Q of the group (—Y—Z—X-Q-) being independently from each other CR a 2 , S, SO, SO 2 , O, or NR b , such that no adjacent pairs of atoms are present selected from the group consisting of O—O, O—NR b , S—NR b , O—S, O—S(O), O—S(O) 2 , and S—S.
16 . The dienophile according to claim 1 , wherein the dienophile comprises a trans-cyclooctene moiety of formula (1b):
wherein X D is as defined in claim 1 ;
wherein, in addition to the optional presence of at most two exocyclic bonds fixed in the same plane, wherein each R c is independently selected from the group consisting of H, alkyl, aryl, OR′, SR′, S(═O) 2 R′″, F, SO 3 H, PO 3 H, NO 2 , CN, CF 3 , CF 2 —R′, C(═O)R′, C(═S)R′, C(═O)NR′R″, C(═S)NR′R″, NR′C(═O)—R′″, NR′C(═S)—R′″, NR′C(═O)O—R′″, NR′C(═S)O—R′″, NR′C(═O)S—R′″, NR′C(═S)S—R′″, NR′C(═O)NR″—R″, NR′C(═S)NR″—R″, and CR′NR″, with each R′ and each R″ independently being H, aryl or alkyl and R′″ independently being aryl or alkyl;
wherein two R a,e moieties together may form a ring;
wherein optionally one of R a and R c , or the self-immolative linker L D , is bound to C B .
17 . A dienophile of any one of the following formulae:
wherein the dotted line denotes a bond to the rest of C B and wherein the wavy line indicates a bond to the rest of C A or L D -C A , wherein L D may optionally comprise C B ;
wherein the dotted line denotes a bond to the rest of C B and wherein the wavy line indicates a bond to the rest of C A or L D -C A , wherein L D may optionally comprise C B ;
wherein the dotted line denotes a bond to the rest of C B and wherein the wavy line indicates a bond to the rest of C A or L D -C A , wherein L D may optionally comprise C B ;
wherein L D is a self-immolative linker consisting of one or more self-immolative units;
wherein each self-immolative unit is selected from the group consisting of
wherein the wiggly line indicates a bond to (O—C(O)) p or O—C(S) of X D , or to a preceding self-immolative unit; wherein the asterisk indicates a bond to C A or to a further self-immolative unit;
wherein L 2 is O, S, NH, or NR with R being alkyl or aryl; wherein each L 1 is independently H or methyl;
wherein each C A and C B are individually selected from the group consisting of biomolecules, biomolecule-binding moieties, polymers, particles, gels, fluorophores, and radiolabels.
18 . The dienophile according to claim 1 , which is of any one of the following formulae
19 . The dienophile according to claim 1 , which is of any one of the following formulae
20 . The dienophile according to claim 1 wherein n=0 and wherein each instance of R a and R b is bound directly to a construct C B .