Hemoglobin-based oxygen carrier compositions

1 . A multimerized hemoglobin (Hb) composition, comprising two or more modified Hb, wherein each modified Hb independently comprises:

at least two subunits of a Hb intramolecularly crosslinked with a compound of formula (I)

or a salt, a stereoisomer, or a deuterated form thereof,

wherein:

X is C 1-24 alkylene, C 2-24 alkenylene, C 2-24 alkynylene, C 3-9 cycloalkylene, C 3-9 cycloalkenylene, 5-12 membered heterocyclylene containing 1 or 2 heteroatoms selected from N, S, or O, heteroarylene, or -L 1 -NR A -L 2 -, wherein X is optionally substituted with 1-6 R 3 as permitted by valency;

R 1 and R 2 are each independently —OH, halogen, or a leaving group;

each R 3 is independently —OH, —CN, —NO 2 , halogen, oxo, —C 1-6 alkyl, —C 1-6 haloalkyl, —O(C 1-6 alkyl), —S(C 1-6 alkyl), —SO(C 1-6 alkyl), —SO 2 (C 1-6 alkyl), —(C 1-12 alkylene)-N 3 , —(C 1-12 alkylene)-(OCH 2 CH 2 ) m —(CH 2 ) n —N 3 , —C(O)NH—(C 1-12 alkylene)-(OCH 2 CH 2 ) m (CH 2 ) n —N 3 , —NHC(O)—(C 1-12 alkylene)-(OCH 2 CH 2 ) m —(CH 2 ) n —N 3 , —O—(C 1-12 alkylene)-(OCH 2 CH 2 ) m —(CH 2 ) n —N 3 , —S—(C 1-12 alkylene)-(OCH 2 CH 2 ) m —(CH 2 ) n —N 3 , or —C(O)—R 1 , wherein each alkyl or alkylene is optionally and independently substituted with —C 1-6 alkyl, —OH, —CN, —NO 2 , or halogen;

R A is H, —C 1-6 alkyl, aryl, —C(O)-aryl, —SO 2 (C 1-6 alkyl), —SO 2 -aryl, or —(C 1-6 alkylene)-(OCH 2 CH 2 ) m —(CH 2 ) n —N 3 , wherein each alkyl, aryl, or alkylene is optionally and independently substituted with —C 1-6 alkyl, —OH, —CN, —NO 2 , or halogen;

L 1 and L 2 are each independently C 1-12 alkylene, C 2-12 alkenylene, C 2-12 alkynylene, C 3-9 cycloalkylene, C 3-9 cycloalkenylene, arylene, or heteroarylene, wherein L 1 and L 2 are optionally and independently substituted with 1-3 R 3 as permitted by valency;

each m is independently an integer of 1-200; and

each n is independently an integer of 0-50.

2 . The multimerized Hb composition of claim 1 , wherein the Hb intramolecularly crosslinked with the compound of formula (I) comprises:

wherein SU1 is a residue on a first subunit of the Hb, and SU2 is a residue on a second subunit of the Hb.

3 . The multimerized Hb composition of claim 2 , comprising a reaction product of:

a first modified Hb comprising a first residue functionalized with a first reactive group for a click reaction; and

a reactant molecule comprising a second reactive group that is complementary to the first reactive group for the click reaction.

4 . The multimerized Hb composition of claim 3 , wherein the reactant molecule is a second modified Hb comprising a second residue functionalized with the second reactive group.

5 . The multimerized Hb composition of claim 3 , wherein the reactant molecule is a scaffold compound comprising the second reactive group.

6 . The multimerized Hb composition of claim 3 , wherein:

the first reactive group is azido, and the second reactive group is cycloalkynyl; or

the first reactive group is cycloalkynyl, and the second reactive group is azido.

7 . The multimerized Hb composition of claim 3 , wherein:

the first reactive group is azido, and the second reactive group is propargyl; or

the first reactive group is propargyl, and the second reactive group is azido.

8 . The multimerized Hb composition of claim 3 , wherein:

the first reactive group is tetrazine, and the second reactive group is TCO; or

the first reactive group is TCO, and the second reactive group is tetrazine.

9 . The multimerized Hb composition of claim 1 , wherein the at least two subunits of each modified Hb are intramolecularly crosslinked with the compound of formula (I) at a residue selected from the group consisting of Lys, Cys, Arg, and an N-termini.

10 . The multimerized Hb composition of claim 1 , wherein the two beta subunits of the Hb are intramolecularly crosslinked by the compound of formula (I).

11 . The multimerized Hb composition of claim 10 , wherein amino groups of lysine residues of the beta subunits are intramolecularly crosslinked by the compound of formula (I).

12 . The multimerized Hb composition of claim 11 , wherein the lysine residues of the beta subunits are Lys-81 and Lys-81 of SEQ ID NO: 2, Lys-81 and Lys-94 of SEQ ID NO: 2, or Lys-75 and Lys-81 of SEQ ID NO: 2.

13 . The multimerized Hb composition of claim 1 , wherein the Hb is bovine, human, equine, porcine, ovine, simian, or fish hemoglobin.

14 . The multimerized Hb composition of claim 3 , wherein the first reactive group is azido, and the second reactive groups are cycloalkynyl, and wherein the first modified Hb comprising a first residue functionalized with the first reactive group for a click reaction is obtained by:

reacting the compound of formula (I) and a Hb to form a crosslinked Hb, and reacting the crosslinked Hb and an azido compound; or

reacting an azido compound and a Hb to form an azido-functionalized Hb, and reacting the azido-functionalized Hb and the compound of formula (I),

wherein the azido compound is 4-azidophenylglyoxal (4-APG), 6-azidomethyl pyridine carboxaldehyde (6-AMPC), or a compound of formula (II) or (III)

wherein m 2 and m 3 are each independently an integer of 1-5000.

15 . The multimerized Hb composition of claim 14 , wherein the reactant molecule is a scaffold compound comprising the second reactive group, and the scaffold compound is a compound of formula (IV) or (V)

wherein m 4 and m 5 are each independently an integer of 1-5000.

16 . The multimerized Hb composition of claim 3 , wherein the first reactive group is cycloalkynyl, and the second reactive groups are azido, and wherein the first modified Hb comprising a first residue functionalized with the first reactive group for a click reaction is obtained by:

reacting the compound of formula (I) and a Hb to form a crosslinked Hb, and reacting the crosslinked Hb and a cycloalkynyl compound; or

reacting a cycloalkynyl compound and a Hb to form an alkynyl-functionalized Hb, and reacting the alkynyl-functionalized Hb and the compound of formula (I),

wherein the cycloalkynyl compound is a compound of formula (VI), (VII), (VIII), or (IX)

wherein m 6 , m 7 , m 8 , and m 9 are each independently an integer of 1-5000.

17 . The multimerized Hb composition of claim 16 , wherein the reactant molecule is a scaffold compound comprising the second reactive group, and the scaffold compound is a compound of formula (X) or (XI)

wherein m 10 and m 11 are each independently an integer of 1-5000.

18 . The multimerized Hb composition of claim 1 , wherein the two or more modified Hb are connected by a bifunctional linker selected from 1,4-bis-phenylglyoxal (1,4-PBG) and (PEG) m12 -bis-phenylglyoxal:

wherein m 12 is an integer of 1-5000.

19 . The multimerized Hb composition of claim 1 , wherein:

X is C 1-24 alkylene, C 2-24 alkenylene, C 2-24 alkynylene, C 3-9 cycloalkylene, C 3-9 cycloalkenylene, 5-12 membered heterocyclylene containing 1 or 2 heteroatoms selected from N, S, or O, heteroarylene, or -L 1 -NR A -L 2 -, wherein X is optionally substituted with 1-6 R 3 as permitted by valency;

R 1 and R 2 are each independently —OH, halogen, or a leaving group;

each R 3 is independently —OH, —CN, —NO 2 , halogen, oxo, —C 1-6 alkyl, —C 1-6 haloalkyl, —O(C 1-6 alkyl), —S(C 1-6 alkyl), —SO(C 1-6 alkyl), —SO 2 (C 1-6 alkyl), or —C(O)—R 1 , wherein each alkyl or alkylene is optionally and independently substituted with —C 1-6 alkyl, —OH, —CN, —NO 2 , or halogen;

R A is H, —C 1-6 alkyl, aryl, —C(O)-aryl, —SO 2 (C 1-6 alkyl), or —SO 2 -aryl, wherein each alkyl, aryl, or alkylene is optionally and independently substituted with —C 1-6 alkyl, —OH, —CN, —NO 2 , or halogen;

L 1 and L 2 are each independently C 1-12 alkylene, C 2-12 alkenylene, C 2-12 alkynylene, C 3-9 cycloalkylene, C 3-9 cycloalkenylene, arylene, or heteroarylene, wherein L 1 and L 2 are optionally and independently substituted with 1-3 R 3 as permitted by valency;

each m is independently an integer of 1-200; and

each n is independently an integer of 0-50.

20 . The multimerized Hb composition of claim 19 , wherein R 1 and R 2 of formula (I) are a leaving group, and wherein:

the leaving group is

M is O or S;

each R 4 is independently halogen or —C 1-6 alkyl; and

a is an integer of 0-4.

21 . The multimerized Hb composition of claim 19 , wherein X of formula (I) is C 1-6 alkylene, C 2-6 alkenylene, or C 2-6 alkynylene, each of which is optionally substituted with 1 or 2 R 3 as permitted by valency.

22 . The multimerized Hb composition of claim 19 , wherein the compound of formula (I) is:

or a salt, a stereoisomer, or a deuterated form thereof, wherein DBS represents

23 . A multimerized Hb composition, comprising a reaction product of:

a modified Hb comprising a residue functionalized with a first reactive group for a click reaction; and

a reactant molecule comprising a second reactive group that is complementary to the first reactive group for the click reaction, wherein the modified Hb comprises:

at least two subunits of a Hb intramolecularly crosslinked with a compound of formula (I)

or a salt, a stereoisomer, or a deuterated form thereof,

wherein:

X is C 1-24 alkylene, C 2-24 alkenylene, C 2-24 alkynylene, C 3-9 cycloalkylene, C 3-9 cycloalkenylene, 5-12 membered heterocyclylene containing 1 or 2 heteroatoms selected from N, S, or O, heteroarylene, or -L 1 -NR A -L 2 -, wherein X is optionally substituted with 1-6 R 3 as permitted by valency;

R 1 and R 2 are each independently —OH, halogen, or a leaving group;

each R 3 is independently —(C 1-12 alkylene)-N 3 , —(C 1-12 alkylene)-(OCH 2 CH 2 ) m —(CH 2 ) n —N 3 , —C(O)NH—(C 1-12 alkylene)-(OCH 2 CH 2 ) m —(CH 2 ) n —N 3 , —NHC(O)—(C 1-12 alkylene)-(OCH 2 CH 2 ) m —(CH 2 )~-N 3 , —O—(C 1-12 alkylene)-(OCH 2 CH 2 ) m —(CH 2 ) n —N 3 , or —S—(C 1-12 alkylene)-(OCH 2 CH 2 ) m —(CH 2 ) n —N 3 , wherein each alkylene is optionally and independently substituted with —C 1-6 alkyl, —OH, —CN, —NO 2 , or halogen;

R A is —(C 1-6 alkylene)-(OCH 2 CH 2 ) m —(CH 2 ) n —N 3 , wherein each alkylene is optionally and independently substituted with —C 1-6 alkyl, —OH, —CN, —NO 2 , or halogen;

L 1 and L 2 are each independently C 1-12 alkylene, C 2-12 alkenylene, C 2-12 alkynylene, C 3-9 cycloalkylene, C 3-9 cycloalkenylene, arylene, or heteroarylene, wherein L 1 and L 2 are optionally and independently substituted with 1-3 R 3 as permitted by valency;

each m is independently an integer of 1-200; and

each n is independently an integer of 0-50.

24 . The multimerized Hb composition of claim 23 , wherein the compound of formula (I) is:

or a salt, a stereoisomer, or a deuterated form thereof, wherein DBS represents

25 . The multimerized Hb composition of claim 15 , wherein:

the azido compound is a compound of formula (II)

 and

the scaffold compound is a compound of formula (V)

wherein m 2 is an integer of 1-10, and m 5 is an integer of 1-12.

26 . The multimerized Hb composition of claim 25 , wherein m 2 is 4, and m 5 is 5.

27 . The multimerized Hb composition of claim 25 , wherein X of the compound of formula (I) is

28 . The multimerized Hb composition of claim 15 , wherein the multimerized Hb composition has a molecular weight of about 192 kDa to about 800 kDa.