Artificial metalloenzymes containing noble metal-porphyrins

Patent metadata

Field	Value
Publication number	US-11518768-B2
Application number	US-201815953331-A
Country	US
Kind code	B2
Filing date	Apr 13, 2018
Priority date	Oct 14, 2015
Publication date	Dec 6, 2022
Grant date	Dec 6, 2022

Abstract

The present invention is drawn to artificial metalloenzymes for use in cyclopropanation reactions, amination and C—H insertion.

First claim

What is claimed is:

1. A catalyst composition comprising:
a porphyrin;
M(L), wherein the porphyrin and M(L) form a complex;
M is a metal, wherein the metal is Ir;
L is a ligand selected from the group consisting of alkyl, haloalkyl, halogen, CO, and CN; and
a heme apoprotein, wherein the porphyrin-M(L) complex is bound to the heme apoprotein, wherein the heme apoprotein comprises at least one mutation, wherein the at least one mutation is close to an active site of the heme apoprotein,
wherein the heme apoprotein is a cytochrome P450 or a myoglobin, and
wherein the cytochrome P450 has at least 70% sequence identity to the P450 region of SEQ ID NO:3 and comprises a substitution of C317G/A and a second substitution selected from the group of substitutions consisting of T213G/V/A, L69V/Y/W/F, V254L/A/V/G, A209G, A152F/W/Y/L/V, L155T/W/F/V/L, F310G/A/L, and L318G/A/F, as determined with reference to SEQ ID NO:1, wherein when the cytochrome P450 comprises the substitution T213A, the cytochrome P450 additionally comprises a third substitution selected from said group of substitutions.

2. The catalyst composition of claim 1 , wherein the ligand L is selected from the group consisting of methyl, ethyl, F, Cl and Br.

3. The catalyst composition of claim 1 , wherein M(L) is Ir(Me).

4. The catalyst composition of claim 1 , wherein the porphyrin-M(L) complex is a porphyrin-Ir(L) complex comprising the formula:

wherein R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a and R 4b are each independently selected from the group consisting of hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl and C 6-10 aryl, wherein the alkyl can be optionally substituted with —C(O)OR 5 wherein R 5 is hydrogen or C 1-6 alkyl.

5. The catalyst composition of claim 4 , wherein
R 1a , R 1b , R 2a , R 2b , R 3a , and R 4a are each independently selected from the group consisting of hydrogen, C 1-6 alkyl and C 2-6 alkenyl; and
R 3b and R 4b are each independently selected from the group consisting of C 1-6 alkyl, wherein the alkyl is substituted with —C(O)OR 5 wherein R 5 is hydrogen or
R 1a , R 1b , R 2a , R 2b , R 3a ,and R 4a are each independently selected from the group consisting of hydrogen, methyl, ethyl and ethenyl; and
R 3b and R 4b are each —CH 2 CH 2 —C(O)OH.

6. The catalyst composition of claim 4 , wherein the porphyrin-Ir(L) complex is selected from the group consisting of:

7. The catalyst composition of claim 4 , wherein the porphyrin-Ir(L) complex is selected from the group consisting of:

8. The catalyst composition of claim 1 , wherein the at least one mutation close to the active site is at a position corresponding to an axial ligand position of a naturally occurring heme apoprotein.

9. The catalyst composition of claim 1 , wherein the heme apoprotein is the cytochrome P450 and the cytochrome P450 has at least 70% sequence identity to the P450 region of SEQ ID NO:3; and comprises substitutions C317G, L69V, T213G, and V254L;
substitutions C317G, T213G, and F310G; substitutions C317G, L69Y, T213G, and A152W;
substitutions C317G, T213A, and V254L; substitutions C317G, L69F, T213G, and V254L;
substitutions C317G, L69Y, T213G, and A152W; substitutions C317G, L69W, T213G, and L318G; substitutions C317G, T213G, and V254L; substitutions C317G, L69V, T213G, and V254L; substitutions C317G, T213G, and V254L; substitutions C317G, L69W, and T213G; or substitutions C317G, L69V, T213A, V254L, and A152W, wherein the positions are numbered with reference to SEQ ID NO:1; or
the cytochrome P450 has at least 70% sequence identity to the P450 region of SEQ ID NO:3;

and comprises substitutions C317G and V254A; C317G, L69F, and T213V; C317G, V254A, and L155W; C317G, L69F, T213V, and V254L; T213G and V254L; C317G, L69F, T213V, and L155T;
C317G, V254A, and A152L; C317G, L69F, T213V, and V254L; C317G, V254A, and A152V;
C317G, T213G, V254L, and A152Y; C317G, V254A, and L155W; C317G, L69F, T213V, V254L, and L155T; C317G, L69F, T213V, and L155W; C317G and A209G; C317G, L69F, T213V, and V254L; C317G, V254A, F69L, L318F, and L155W; or C317G, L69F, T213V, and L155W; wherein the residues are numbered with reference to SEQ ID NO:1.

10. The catalyst composition of claim 1 , wherein the heme apoprotein is the myoglobin and the myoglobin comprises a substitution at position H93 as determined with reference to SEQ ID NO:2;
the myoglobin comprises a substitution at positions H93, F43, and V68 as determined with reference to SEQ ID NO:2;
the myoglobin comprises an amino acid substitution at positions H64, F43, F33, L32, V68, H97, 199, Y103, and 5108 as determined with reference to SEQ ID NO:2, optionally wherein the myoglobin comprises a substitution H93A/G, H64L/V/A, F43L/Y/W/H/I, L32F, F33V/I, V68A/S/G/T, H97W/Y, I99F/V, Y103C, and S108C as determined with reference to SEQ ID NO:2; or
the myoglobin has at least 70% sequence identity to the myoglobin region of SEQ ID NO:6;
and comprises substitutions H93A, H64L, F43L, and F33V; substitutions H93A, H64V, F43Y, V68A, and H97W; substitutions H93A, H64L, F43W, V68A, and H97Y; substitutions H93G, H64L, F43L, and I99F; substitutions H93A, H64V, V68A, Y103C, and S108C; substitutions H93A, H64L, F43W, V68A, and F33I; substitutions H93A, H64V, F43H, and V68S; substitutions H93A, H64A, F43W, and V68G; substitutions H93A, H64A, F43W, and V68T; substitutions H93A, H64A, F431, and V68T; substitutions H93G, H64L, V68A, and I99V substitutions H93A, H64L, V68A, and I99V; substitutions H93A, H64V, V68A, F33V and H97Y; or substitutions H93G and H64L.

11. The catalyst composition of claim 1 , wherein the heme apoprotein is the cytochrome P450 that has at least 80% identity to the P450 region of SEQ ID NO:3, wherein:
(a) the cytochrome P450 comprises substitutions at positions:
(i) C317, L69, T213, and V254; C317, T213, and F310; C317, L69, T213, and A152;
C317, T213, and V254; C317, L69, T213, and V254; C317 and T213; C317, L69, T213, and A152; C317, L69, T213, and L318; C317, T213, and V254; C317, L69, T213, and V254; C317, T213, and V254; C317, L69, and T213; or C317, L69, T213, V254, and A152, wherein the positions are numbered with reference to SEQ ID NO:1; or
(ii) C317 and V254; C317, L69, and T213; C317, V254, and L155; C317, L69, T213, and V254; T213 and V254; C317, L69, T213, and L155; C317, V254, and A152; C317, L69, T213, and V254; C317, V254, and A152; C317, T213, V254, and A152; C317, V254, and L155; C317, L69, T213, V254, and L155; C317, L69, T213, and L155; C317 and A209; C317, L69, T213, and V254; C317, V254, F69, L318, and L155; or C317, L69, T213, and L155, wherein the positions are numbered with reference to SEQ ID NO:1; or
(b) the cytochrome P450 comprises:
(i) substitutions C317G, L69V, T213G, and V254L; substitutions C317G, T213G, and F310G; substitutions C317G, L69Y, T213G, and A152W; substitutions C317G, T213A, and V254L; substitutions C317G, L69F, T213G, and V254L; substitutions C317G, L69Y, T213G, and A152W; substitutions C317G, L69W, T213G, and L318G; substitutions C317G, T213G, and V254L; substitutions C317G, L69V, T213G, and V254L; substitutions C317G, T213G, and V254L; substitutions C317G, L69W, and T213G; or substitutions C317G, L69V, T213A, V254L, and A152W, wherein the positions are numbered with reference to SEQ ID NO:1; or
(ii) substitutions C317G and V254A; C317G, L69F, and T213V; C317G, V254A, and L155W; C317G,

Assignees

• Univ California

Inventors

• Hartwig John F
• Key Hanna M
• Dydio Pawel
• Clark Douglas S

Classifications

• C07K14/805

  Haemoglobins; Myoglobins · CPC title

• B01J2531/827

  Iridium · CPC title

• B01J31/18

  containing nitrogen, phosphorus, arsenic or antimony {as complexing atoms, e.g. in pyridine ligands, or in resonance therewith, e.g. in isocyanide ligands C=N-R or as complexed central atoms (double metal cyanides B01J27/26; N-heterocyclic carbenes B01J31/2265)} · CPC title

• C07K14/80

  Cytochromes · CPC title

• C07K14/795

  Porphyrin- or corrin-ring-containing peptides · CPC title