US4584130A
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Intramolecularly cross-linked hemoglobin and method of preparation
Assignee: University of Maryland, Baltimore
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Filing: 1985-03-29
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Granted: 1986-04-22
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Expires: 2005-03-29
Abstract
Abstract Stroma-free hemoglobin cross-linked with reagents that mimic 2,3 diphosphoglycerate and transform stroma-free hemoglobin into a physiologically competent oxygen carrier which is retained in vivo for adequate periods of time and thus can be used in fluids for transporting oxygen; reagents for cross-linking stroma-free hemoglobin and method for cross-linking stroma-free hemoglobin.
Inventors
- 👤 Enrico Bucci
- 👤 Clara Fronticelli-Bucci
- 👤 Ramachandra Hosmane
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 03:21
CPC Classifications
C; C07; C07D; C07D231/00; C07D231/02; C07D231/10; C07D231/12; C07C; C07C317/00; C07D233/00; C07D233/54; C07D233/56; C07D249/00; C07D249/02; C07D249/08; C07F; C07F9/00; C07F9/02; C07F9/28; C07F9/38; C07F9/40; C07F9/4003; C07F9/4006; C07F9/4012; C07K; C07K14/00; C07K14/795; C07K14/805; A; A61; A61K; A61K38/00
Claims Analysis
Total Claims: 44
Claim 1
Independent Claim
225 words
Cross-linked stroma-free hemoglobin obtained by cross-linking stroma-free hemoglobin with at least one cross-linking reagent selected from the group consisting of compounds of the general formulae (I) and (II): ##STR16## wherein one of R 1 or R 2 represents an electron-withdrawing atom or group, and the other of R 1 or R 2 represents a leaving atom or group or an electron-withdrawing group containing a leaving atom or group; one of R 3 or R 4 represents an electron-withdrawing atom or group, and the other of R 3 or R 4 represents a leaving atom or group or an electron-withdrawing group containing a leaving atom or group; R 5 and R 10 , which may be the same or different, each represents an electron-withdrawing atom or group or an electron-withdrawing group containing a leaving atom or group; one of R 6 or R 7 represents a hydrogen atom, an alkyl group, an aryl group, or a heterocyclic group, and the other of R 6 or R 7 represents a leaving atom or group; one of R 8 or R 9 represents a hydrogen atom, an alkyl group, an aryl group or a heterocylic group, and the other of R 8 or R 9 represents a leaving atom or group; X represents a linking atom or group, n=0 to 4, m=0 to 4 and l=0 to 4.
Claim 2
Dependent Claim
225 words
Cross-linked stroma-free hemoglobin obtained by cross-linking stroma-free hemoglobin with at least one cross-linking reagent selected from the group consisting of compounds of the general formulae (I) and (II): ##STR16## wherein one of R 1 or R 2 represents an electron-withdrawing atom or group, and the other of R 1 or R 2 represents a leaving atom or group or an electron-withdrawing group containing a leaving atom or group; one of R 3 or R 4 represents an electron-withdrawing atom or group, and the other of R 3 or R 4 represents a leaving atom or group or an electron-withdrawing group containing a leaving atom or group; R 5 and R 10 , which may be the same or different, each represents an electron-withdrawing atom or group or an electron-withdrawing group containing a leaving atom or group; one of R 6 or R 7 represents a hydrogen atom, an alkyl group, an aryl group, or a heterocyclic group, and the other of R 6 or R 7 represents a leaving atom or group; one of R 8 or R 9 represents a hydrogen atom, an alkyl group, an aryl group or a heterocylic group, and the other of R 8 or R 9 represents a leaving atom or group; X represents a linking atom or group, n=0 to 4, m=0 to 4 and l=0 to 4.
Claim 3
Dependent Claim
96 words
The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein the electron-withdrawing group represented by R 1 , R 2 , R 3 , R 4 , R 5 and R 10 is a member selected from the group consisting of CN, NO 2 , halogen, S(O)R 11 , S(O 2 )R 11 , C(O)R 11 , wherein R 11 is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C 1 -C 12 alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group, and a substituted or unsubstituted heterocyclic group.
Claim 4
Dependent Claim
57 words
The cross-linked stroma-free hemoglobin as claimed in claim 2, wherein said substituents on said alkyl group, aryl group, and heterocyclic group are selected from the group consisting of a halogen atom, CN, a C 1 -C 12 alkyl group, a substituted or unsubstituted phenyl group, NO 2 , OH, and a C 1 -C 12 alkoxy group.
Claim 5
Dependent Claim
29 words
The cross-linked stroma-free hemoglobin as claimed in claim 2, wherein said heterocyclic group is selected from the group consisting of substituted or unsubstituted imidazolyl and substituted or unsubstituted pyrazinyl.
Claim 6
Dependent Claim
42 words
The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein R 1 , R 2 , R 3 , R 4 , R 5 , and R 10 are selected from the group consisting of CN and SO 2 R 11 .
Claim 7
Dependent Claim
131 words
The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein the electron-withdrawing group containing a leaving group represented by R 1 , R 2 , R 3 , R 4 , R 5 and R 10 is a member selected from the group consisting of R 12 P(O)OR 12 , P(O)(OR 12 ) 2 , OP(O)(OR 12 ) 2 and CO 2 R 12 , wherein R 12 is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C 1 -C 12 alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group, a substituted or unsubstituted heterocyclic group and a halogen atom and P(O)(R 13 ) 2 , S(O)R 13 , S(O 2 )R 13 and C(O)R 13 , wherein R 13 is a halogen atom.
Claim 8
Dependent Claim
54 words
The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein the group other than a leaving group represented by R 6 , R 7 , R 8 and R 9 is selected from the group consisting of a hydrogen atom, a C 1 -C 12 alkyl group, an aryl group, and a heterocyclic group.
Claim 9
Dependent Claim
40 words
The cross-linked stroma-free hemoglobin as claimed in claim 7, wherein R 6 , R 7 , R 8 and R 9 are selected from the group consisting of C 2 H 5 , a phenyl group, or an imidazolyl group.
Claim 10
Dependent Claim
91 words
The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein the leaving group represented by R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 8 and R 9 is selected from the group consisting of a halogen atom and OR 11 , wherein R 11 is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C 1 -C 12 alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group and a substituted or unsubstituted heterocyclic group.
Claim 11
Dependent Claim
37 words
The cross-linked stroma-free hemoglobin as claimed in claim 9, wherein R 7 and R 9 are selected from the group consisting of CH 3 , C 2 H 5 and CH 2 C 6 H 5 .
Claim 12
Dependent Claim
69 words
The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein X is selected from the group consisting of SO 2 , SO, P(O)(OR 11 ), CO, Se, Te and Sr, wherein R 11 is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C 1 -C 12 alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group and a substituted or unsubstituted heterocyclic group.
Claim 13
Dependent Claim
36 words
The cross-linked stroma-free hemoglobin as claimed in claim 11, wherein X is selected from the group consisting of SO 2 , SO, P(O)(OR 11 ) and CO, wherein R 11 is as defined in claim 11.
Claim 14
Dependent Claim
20 words
The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein n=0 to 2, n=0 to 2, and l=0 to 2.
Claim 15
Dependent Claim
30 words
The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein said reagent is selected from the group consisting of ethyl-2,2'-sulfonyl-bis-cyanoacetate, diethyl-2,2'-sulfonyl-bis-malonate, ethyl-2,2'-sulfonyl-2,2'-benzene sulfonyl-bis-acetate, 2,2'-sulfonyl-2,2'-imidazole-N-sulfonyl-bis-acetonitrile, methyl 2,2'-sulfonyl-bis-cyanoacetate, 2,2'-methoxymethylene-2,2'-sulfonyl-bis-acetonitrile and dimethyl 2,2'-methoxyphosphinylidene-2-2'-methoxymethylene-bis-methanephosphonate.
Claim 16
Dependent Claim
29 words
The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein said stroma-free hemoglobin is selected from the group consisting of human, equine, porcine, ovine, bovine, simian and fish hemoglobin.
Claim 17
Dependent Claim
16 words
The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein said stroma-free hemoglobin is bovine hemoglobin.
Claim 18
Dependent Claim
16 words
The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein said stroma-free hemoglobin is human hemoglobin.
Claim 19
Dependent Claim
20 words
The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein said cross-linking is carried out in the presence of oxygen.
Claim 20
Dependent Claim
20 words
The cross-linked stroma-free hemoglobin as claimed in claim 1, wherein said cross-linking is carried out in the absence of oxygen.
Claim 21
Independent Claim
236 words
A method of producing cross-linked stroma-free hemoglobin, comprising: (a) cross-linking stroma-free hemoglobin with at least one cross-linking reagent selected from the group consisting of compounds of the general formulae (I) and (II): ##STR17## wherein one of R 1 or R 2 represents an electron-withdrawing atom or group, and the other of R 1 or R 2 represents a leaving atom or group or an electron-withdrawing group containing a leaving atom or group; one of R 3 or R 4 represents an electron-withdrawing atom or group, and the other of R 3 or R 4 represents a leaving atom or group or an electron-withdrawing group containing a leaving atom or group; R 5 and R 10 , which may be the same or different, each represents an electron-withdrawing atom or group or an electron-withdrawing group containing a leaving atom or group; one of R 6 or R 7 represents a hydrogen atom, an alkyl group, an aryl group, or a heterocyclic group, and the other of R 6 or R 7 represents a leaving atom or group; one of R 8 or R 9 represents a hydrogen atom, an alkyl group, an aryl group or a heterocyclic group, and the other of R 8 or R 9 represents a leaving atom or group; X represents a linking atom or group, n=0 to 4, m=0 to 4 and l=0 to 4; and (b) purifying the resulting cross-linked hemoglobin.
Claim 22
Independent Claim
236 words
A method of producing cross-linked stroma-free hemoglobin, comprising: (a) cross-linking stroma-free hemoglobin with at least one cross-linking reagent selected from the group consisting of compounds of the general formulae (I) and (II): ##STR17## wherein one of R 1 or R 2 represents an electron-withdrawing atom or group, and the other of R 1 or R 2 represents a leaving atom or group or an electron-withdrawing group containing a leaving atom or group; one of R 3 or R 4 represents an electron-withdrawing atom or group, and the other of R 3 or R 4 represents a leaving atom or group or an electron-withdrawing group containing a leaving atom or group; R 5 and R 10 , which may be the same or different, each represents an electron-withdrawing atom or group or an electron-withdrawing group containing a leaving atom or group; one of R 6 or R 7 represents a hydrogen atom, an alkyl group, an aryl group, or a heterocyclic group, and the other of R 6 or R 7 represents a leaving atom or group; one of R 8 or R 9 represents a hydrogen atom, an alkyl group, an aryl group or a heterocyclic group, and the other of R 8 or R 9 represents a leaving atom or group; X represents a linking atom or group, n=0 to 4, m=0 to 4 and l=0 to 4; and (b) purifying the resulting cross-linked hemoglobin.
Claim 23
Dependent Claim
99 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein the electron-withdrawing group represented by R 1 , R 2 , R 3 , R 4 , R 5 and R 10 is a member selected from the group consisting of CN, NO 2 , halogen, S(O)R 11 , S(O 2 )R 11 , C(O)R 11 , wherein R 11 is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C 1 -C 12 alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group, and a substituted or unsubstituted heterocyclic group.
Claim 24
Dependent Claim
60 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 21, wherein said substituents on said alkyl group, aryl group, and heterocyclic group are selected from the group consisting of a halogen atom, CN, a C 1 -C 12 alkyl group, a substituted or unsubstituted phenyl group, NO 2 , OH, and a C 1 -C 12 alkoxy group.
Claim 25
Dependent Claim
32 words
The mthod of producing cross-linked stroma-free hemoglobin as claimed in claim 21, wherein said heterocyclic group is selected from the group consisting of substituted or unsubstituted imidazolyl and substituted or unsubstituted pyrazinyl.
Claim 26
Dependent Claim
45 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 21, wherein R 1 , R 2 , R 3 , R 4 , R 5 , and R 10 are selected from the group consisting of CN and SO 2 R 11 .
Claim 27
Dependent Claim
135 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein the electron-withdrawing group containing a leaving group represented by R 1 , R 2 , R 3 , R 4 , R 5 and R 10 is a member selected from the group consisting of R 12 P(O)OR 12 , P(O)(OR 12 ) 2 , OP(O)(OR 12 ) 2 , and CO 2 R 12 , wherein R 12 is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C 1 -C 12 alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group, a substituted or unsubstituted heterocyclic group and a halogen atom and P(O)(R 13 ) 2 , S(O)R 13 , S(O 2 )R 13 and C(O)R 13 , wherein R 13 is a halogen atom.
Claim 28
Dependent Claim
57 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein the group other than a leaving group represented by R 6 , R 7 , R 8 and R 9 is selected from the group consisting of a hydrogen atom, a C 1 -C 12 alkyl group, an aryl group, and a heterocyclic group.
Claim 29
Dependent Claim
43 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 26, wherein R 6 , R 7 , R 8 and R 9 are selected from the group consisting of C 2 H 5 , a phenyl group, or an imidazolyl group.
Claim 30
Dependent Claim
94 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein the leaving group represented by R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 8 and R 9 is selected from the group consisting of a halogen atom and OR 11 , wherein R 11 is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C 1 -C 12 alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group and a substituted or unsubstituted heterocyclic group.
Claim 31
Dependent Claim
43 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 28, wherein R 11 is selected from the group consisting of CH 3 , C 2 H 5 , CH 2 C 6 H 5 , and C 6 H 5 .
Claim 32
Dependent Claim
72 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein X is selected from the group consisting of SO 2 , SO, P(O)(OR 11 ), CO, Se, Te and Sr, wherein R 11 is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C 1 -C 12 alkyl group, a substituted or unsubstituted mono- or bicyclic aryl group and a substituted or unsubstituted heterocyclic group.
Claim 33
Dependent Claim
37 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 30, wherein X is selected from the group consisting of SO 2 , SO, P(O)(OR 11 ) and CO, wherein R 11 is as defined above.
Claim 34
Dependent Claim
23 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein n=0 to 2, n=0 to 2, and l=0 to 2.
Claim 35
Dependent Claim
33 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein said reagent is selected from the group consisting of ethyl-2,2'-sulfonyl-bis-cyanoacetate, diethyl-2,2'-sulfonyl-bis-malonate, ethyl-2,2'-sulfonyl-2,2'-benzene sulfonyl-bis-acetate, 2,2'-sulfonyl-2,2'-imidazole-N-sulfonyl-bis-acetonitrile, methyl 2,2'-sulfonyl-bis-cyanoacetate, 2,2'-methoxymethylene-2,2'-sulfonyl-bis-acetonitrile and dimethyl 2,2'-methoxyphosphinylidene-2-2'-methoxymethylene-bis-methanephosphonate.
Claim 36
Dependent Claim
32 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein said stroma-free hemoglobin is selected from the group consisting of human, equine, porcine, ovine, bovine, simian and fish hemoglobin.
Claim 37
Dependent Claim
19 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein said stroma-free hemoglobin is bovine hemoglobin.
Claim 38
Dependent Claim
19 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein said stroma-free hemoglobin is human hemoglobin.
Claim 39
Dependent Claim
23 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein said cross-linking is carried out in the presence of oxygen.
Claim 40
Dependent Claim
23 words
The method of producing cross-linked stroma-free hemoglobin as claimed in claim 20, wherein said cross-linking is carried out in the absence of oxygen.
Claim 41
Dependent Claim
24 words
The method of producing cross-linked hemoglobin as claimed in claim 20, wherein said cross-linking is carried out at about 10° C. to 37° C.
Claim 42
Dependent Claim
24 words
The method of producing cross-linked hemoglobin as claimed in claim 39 wherein said cross-linking is carried out at about 25° C. to 30° C.
Claim 43
Dependent Claim
25 words
The method of producing cross-linked hemoglobin as claimed in claim 20, wherein the molar ratio of said reagent to said hemoglobin is 1:1 to 1:10.
Claim 44
Dependent Claim
25 words
The method of producing cross-linked hemoglobin as claimed in claim 41, wherein the molar ratio of said reagent to said hemoglobin is 1:2 to 1:6.
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1989-04-21 | 1989-10-21 | 1990-04-22 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-04-21 | 1993-10-21 | 1994-04-22 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-04-21 | 1997-10-21 | 1998-04-22 | $7700.00 | Window Closed / Lapsed |