US3481952A - N-(1-oxide-2,2,5,5-tetra lower alkyl pyrrolidinyl-3)-maleimides - Google Patents
N-(1-oxide-2,2,5,5-tetra lower alkyl pyrrolidinyl-3)-maleimides Download PDFInfo
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- US3481952A US3481952A US512793A US3481952DA US3481952A US 3481952 A US3481952 A US 3481952A US 512793 A US512793 A US 512793A US 3481952D A US3481952D A US 3481952DA US 3481952 A US3481952 A US 3481952A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
- B23H—WORKING OF METAL BY THE ACTION OF A HIGH CONCENTRATION OF ELECTRIC CURRENT ON A WORKPIECE USING AN ELECTRODE WHICH TAKES THE PLACE OF A TOOL; SUCH WORKING COMBINED WITH OTHER FORMS OF WORKING OF METAL
- B23H3/00—Electrochemical machining, i.e. removing metal by passing current between an electrode and a workpiece in the presence of an electrolyte
- B23H3/08—Working media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/806—Antigenic peptides or proteins
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
- Y10T436/143333—Saccharide [e.g., DNA, etc.]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/24—Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry
Definitions
- the present invention relates in general to electron spin resonance (ESR) labeling of biologically active molecules and more particularly to an improved organic spin labeling compound containing the paramagnetic nitroxide group, and containing the maleimide ring for attachment to the bimolecule.
- ESR electron spin resonance
- ESR labels have been used for labeling biomolecules.
- One example of such a prior label is the positive ion radical of the tranquilizer drug chlorpromazine (CPZ which forms the subject matter of and is claimed in the pending application Ser. No. 496,682 filed Oct. 15, 1965.
- CPZ+ attaches predominantly only to DNA and RNA type biomolecules for labeling same.
- a more general type of spin label for biomolecules employs the nitroxide radical group, which provides a strong electron resonance line spectrum having a simple triplet hyperfine structure.
- Preferred molecular structures involving this radical group are remarkably stable and inert, and show ESR spectral features that are sensitive to molecular motion, and to a lesser extent, sensitive to the polarity of the molecular environment. This sensitivity in turn permits certain chemical, structural and kinetic information concerning biomolecules to be obtained from the paramagnetic resonance of the attached spin labels.
- the use of nitroxide groups for spin labeling is described in the pending application, Ser. No. 496,682 filed Oct. 15, 1965, and Ser. NO. 496,683 filed Oct. 15, 1965.
- the nitroxide radical spin label is attached to the biomolecule via the intermediary of an isocyanate group.
- the principal object of the present invention is to provide still another improved organic spin label for biologically active molecules, especially a label that is not rapidly hydrolyzed by water in competition with the labeling of proteins and synthetic polypeptides.
- One feature of the present invention is the provision of a maleimide ring system as the intermediary for attaching the spin label to proteins and synthetic polypeptides, and the methods for synthesizing such spin labels.
- improved nitroxide compounds are synthesized to contain at least one maleimide ring which serves to form a covalent bond with atoms of the biologically active molecule to be labeled.
- a class of improved nitroXide compounds exhibiting ESR and useful for spin labeling biologically active molecules are those organic free radicals of the general formula,
- C and C are tertiary carbon atoms; C and C are bonded directly to carbon or fluorine atoms; A represents at least one independent organic group and has a total valency of 6 for bonding to said C and C tertiary carbon atoms. (The broken lines between A and C and C representing 6 saturated bonds); and where A containing at least one maleimide ring, which is operative to form a bond with a biologically active molecule.
- A may represent one or more independent organic groups up to a total of 6 and the maleimide functional group serving to form the bond with the biologically active molecule may be present on any one or more of these groups.
- a in the above formula includes a plurality of carbon atoms arranged to form a closed ring with C and C and where C, and C are further substituted with lower alkyl groups so as to provide the requisite tertiary character for C and C
- These materials may be defined as having the general formula
- X is R1 R3 594 2 0 4 II wherein R R R and R are lower alklyl groups i.e., each having 1-5 carbon atoms; B represents a plurality of carbon atoms in a partial cycloalkyl chain, and X is the maleimide ring:
- a in the general formula previously discussed comprises four independent organic groups, namely R R R and R and B, the five groups having a total valency of 6 since B is divalent, whereas the Rs are monovalent.
- R R R and R are each a methyl group
- B represents an ethylene or propylene group so as to form a five or six membered saturated heterocyclic ring, respectively, with the nitrogen atom
- X is the maleimide ring attached to one of the carbon atoms in the ethylene or propylene group.
- the preferred materials include a heterocyclic ring as provided by Formula II the ring structure is not essential so long as the tertiary character of C and C is retained. Further, and as already mentioned, C and C in addition to being tertiary must have all of their valencies satisfied by saturated bonds to either carbon atoms or fluorine atoms. For example, in Formula IV given above, the replacement of the methyl groups by fluorine atoms would provide typical compounds contemplated within the scope of this invention.
- the maleimide group is especially useful for labeling proteins and some synthetic polypeptides, since the maleimide group forms covalent bonds by reaction with both sulfhydryl groups and e-amino groups of proteins.
- the maleimide ring also is not rapidly hydrolyzed by water in competition with the labeling reaction with a great variety of proteins. 5
- Preparation of labels having the maleimide ring follows conventional organic synthesis procedures. In general, there are a number of compounds of various structures known and available containing the requisite nitroxide group with adjacent tertiary carbon atoms.
- the present materials may be conveniently prepared from corresponding compounds having the structure of the end product sought with the difference being the presence of an amino group at the site where the maleimide group is desired. With such a corresponding amino compound the maleimide is obtained by conventional condensation with maleic anhydride to form the maleamic acid and then water is eliminated by heating with a dehydrating agent such as acetic anhydride.
- 2,2,5,5 tetramethyl 3 amino pyrrolidine l oxyl is used as a starting material and may be synthesized from triacetoneamine by the method of Rozantzev and Krivitzkaya, Tetrahedron 21, 491 (1965).
- An equimolar amount (0.40 gm.) of the starting material in one ml. anhydrous ether was slowly added with stirring to a room-temperature solution of 0.25 gm. of maleic anhydride in 5 ml. anhydrous diethyl ether.
- N-(l-oXyl-2,2,5,5-tetramethylpyrrolidinyl) -maleamic acid (V) immediately precipitated and after three hours of stirring at room temperature the precipitate was filtered, washed ten times with 0.4 ml. of anhydrous diethyl ether, and dried; yield 97%.
- N-(l oxyl 2,2,5,5- tetramethylpyrrolidinyl)-maleimide was obtained from the above acid, as follows. A mixture of 0.63 gm. V, 5 ml. acetic anhydride, and 0.2 gm. sodium acetate were stirred in a tightly closed container for twenty-four hours at 25- 35 C. The acetic anhydride was removed in vacuo at room temperature and the crude product was obtained as a viscous oil which solidified on standing. N-(1-oxyl-2,2, 5,S-tetramethylpyrrolidinyl)-ma1eimide VI was purified by sublimation or by recrystallization from benzene (compound V is insoluble in benzene).
- Attachment of the spin label VI to poly nucleic acids is also possible, as, for example, by first reacting VI with an aromatic dye (having amino groups) or antibiotics that in turn bind to nucleic acids.
- NICHOLAS S. RIZZO Primary Examiner molecule is N-(1-oXyl-2,2,5,5-tetramethylpyrrolidiny1-3)- JOSE TOVAR Assistant Examiner maleimide.
Description
United States Patent 3,481,952 N-(1-0XIDE-2,2,5,5-TETRA LOWER ALKYL PYRROLlDINYL-3)-MALEIMIDES Harden M. McConnell, Stanford, and Osbie H. Griffith,
Laverne, Calif., assignors, by mesne assignments to Synbar Associates, Palo Alto, Calif., a partnership of California No Drawing. Filed Dec. 9, 1965, Ser. No. 512,793 Int. Cl. C07d 27/18, 27/06 US. Cl. 260-326.5 2 Claims ABSTRACT OF THE DISCLOSURE Free radical organic nitroxides in which the nitroxide nitrogen atom forms part of a heterocyclic ring. A maleimide ring is attached to the heterocyclic ring and is useful for forming a bond with a biologically active molecule to spin label the biologically active molecule. Nitroxides containing a maleimide ring are particularly good spin labels in that they are not rapidly hydrolyzed by Water in competition with protein and synthetic polypeptides.
The present invention relates in general to electron spin resonance (ESR) labeling of biologically active molecules and more particularly to an improved organic spin labeling compound containing the paramagnetic nitroxide group, and containing the maleimide ring for attachment to the bimolecule.
Heretofore, a limited number of synthetic organic ESR labels have been used for labeling biomolecules. One example of such a prior label is the positive ion radical of the tranquilizer drug chlorpromazine (CPZ which forms the subject matter of and is claimed in the pending application Ser. No. 496,682 filed Oct. 15, 1965. CPZ+ attaches predominantly only to DNA and RNA type biomolecules for labeling same. A more general type of spin label for biomolecules employs the nitroxide radical group, which provides a strong electron resonance line spectrum having a simple triplet hyperfine structure. Preferred molecular structures involving this radical group are remarkably stable and inert, and show ESR spectral features that are sensitive to molecular motion, and to a lesser extent, sensitive to the polarity of the molecular environment. This sensitivity in turn permits certain chemical, structural and kinetic information concerning biomolecules to be obtained from the paramagnetic resonance of the attached spin labels. The use of nitroxide groups for spin labeling is described in the pending application, Ser. No. 496,682 filed Oct. 15, 1965, and Ser. NO. 496,683 filed Oct. 15, 1965. In one preferred embodiment of the above invention, the nitroxide radical spin label is attached to the biomolecule via the intermediary of an isocyanate group.
The principal object of the present invention is to provide still another improved organic spin label for biologically active molecules, especially a label that is not rapidly hydrolyzed by water in competition with the labeling of proteins and synthetic polypeptides.
One feature of the present invention is the provision of a maleimide ring system as the intermediary for attaching the spin label to proteins and synthetic polypeptides, and the methods for synthesizing such spin labels.
In the preferred embodiment improved nitroxide compounds are synthesized to contain at least one maleimide ring which serves to form a covalent bond with atoms of the biologically active molecule to be labeled. Maleimide ring-containing nitroXide compounds are especially useful for labeling most proteins through reaction between the moleirnide ring and sulfhydryl (SH) and e=amino (e=NH groups of the protein molecules.
Thus, in accordance with this invention, a class of improved nitroXide compounds exhibiting ESR and useful for spin labeling biologically active molecules are those organic free radicals of the general formula,
Where C and C are tertiary carbon atoms; C and C are bonded directly to carbon or fluorine atoms; A represents at least one independent organic group and has a total valency of 6 for bonding to said C and C tertiary carbon atoms. (The broken lines between A and C and C representing 6 saturated bonds); and where A containing at least one maleimide ring, which is operative to form a bond with a biologically active molecule.
As will appear more fully hereinafter, A may represent one or more independent organic groups up to a total of 6 and the maleimide functional group serving to form the bond with the biologically active molecule may be present on any one or more of these groups.
Present work has shown that much useful information can be gained where A in the above formula includes a plurality of carbon atoms arranged to form a closed ring with C and C and where C, and C are further substituted with lower alkyl groups so as to provide the requisite tertiary character for C and C These materials may be defined as having the general formula,
X is R1 R3 594 2 0 4 II wherein R R R and R are lower alklyl groups i.e., each having 1-5 carbon atoms; B represents a plurality of carbon atoms in a partial cycloalkyl chain, and X is the maleimide ring:
C III In this latter situation it will be appreciated that A in the general formula previously discussed comprises four independent organic groups, namely R R R and R and B, the five groups having a total valency of 6 since B is divalent, whereas the Rs are monovalent.
Within the group of materials covered by Formula II, an especially useful material is that obtained where R R R and R are each a methyl group, B represents an ethylene or propylene group so as to form a five or six membered saturated heterocyclic ring, respectively, with the nitrogen atom, and X is the maleimide ring attached to one of the carbon atoms in the ethylene or propylene group.
It is to be noted that whereas the preferred materials include a heterocyclic ring as provided by Formula II the ring structure is not essential so long as the tertiary character of C and C is retained. Further, and as already mentioned, C and C in addition to being tertiary must have all of their valencies satisfied by saturated bonds to either carbon atoms or fluorine atoms. For example, in Formula IV given above, the replacement of the methyl groups by fluorine atoms would provide typical compounds contemplated within the scope of this invention.
The maleimide group is especially useful for labeling proteins and some synthetic polypeptides, since the maleimide group forms covalent bonds by reaction with both sulfhydryl groups and e-amino groups of proteins. The maleimide ring also is not rapidly hydrolyzed by water in competition with the labeling reaction with a great variety of proteins. 5 Preparation of labels having the maleimide ring follows conventional organic synthesis procedures. In general, there are a number of compounds of various structures known and available containing the requisite nitroxide group with adjacent tertiary carbon atoms. The present materials may be conveniently prepared from corresponding compounds having the structure of the end product sought with the difference being the presence of an amino group at the site where the maleimide group is desired. With such a corresponding amino compound the maleimide is obtained by conventional condensation with maleic anhydride to form the maleamic acid and then water is eliminated by heating with a dehydrating agent such as acetic anhydride.
2,2,5,5 tetramethyl 3 amino pyrrolidine l oxyl is used as a starting material and may be synthesized from triacetoneamine by the method of Rozantzev and Krivitzkaya, Tetrahedron 21, 491 (1965). An equimolar amount (0.40 gm.) of the starting material in one ml. anhydrous ether was slowly added with stirring to a room-temperature solution of 0.25 gm. of maleic anhydride in 5 ml. anhydrous diethyl ether. The N-(l-oXyl-2,2,5,5-tetramethylpyrrolidinyl) -maleamic acid (V) immediately precipitated and after three hours of stirring at room temperature the precipitate was filtered, washed ten times with 0.4 ml. of anhydrous diethyl ether, and dried; yield 97%.
Analysis.-Calc. for C H N O C, 56.5; H, 7.5; N, 11.0%. Found: C, 56.0; H, 7.5; N, 11.3%.
The reaction is The desired improved spin-label, N-(l oxyl 2,2,5,5- tetramethylpyrrolidinyl)-maleimide was obtained from the above acid, as follows. A mixture of 0.63 gm. V, 5 ml. acetic anhydride, and 0.2 gm. sodium acetate were stirred in a tightly closed container for twenty-four hours at 25- 35 C. The acetic anhydride was removed in vacuo at room temperature and the crude product was obtained as a viscous oil which solidified on standing. N-(1-oxyl-2,2, 5,S-tetramethylpyrrolidinyl)-ma1eimide VI was purified by sublimation or by recrystallization from benzene (compound V is insoluble in benzene).
Analysis.Calc. for C H N O C, 60.7; H, 7.2; N, 11.8%. Found: C, 60.6; H, 7.3; N, 11.8%.
The reaction is In typical labeling experiments involving proteins, the solid labeling compound V1 is added to a stirred solution of the protein, and non-attached paramagnetic radicals removed from the solution by dialysis, or by use of a sephadex column. In this way the following proteins and synthetic polypeptides have been spin labeled using VI:
alcohol dehydrogenase aldolase atale carbogrypeptidase ceruloplasmin e-gh m yp creatin kinase cytochrome C haemoglobin lactate dehydrogenase lipase lysozyme paaa pgly-L-lysine ry s soluble rat brain proteins tobacco mosaic virus.
Attachment of the spin label VI to poly nucleic acids is also possible, as, for example, by first reacting VI with an aromatic dye (having amino groups) or antibiotics that in turn bind to nucleic acids.
What is claimed is:
1. A nitroxide of the formula X 1% R1 RI QQK R2 0 RA wherein R R R and R are lower alkyl groups, B is an alkylene group having 2-3 carbon atoms forming a heterga y c ring with C and 3; and X is a ma eim dc ng.
8,481,952 5 6 2. A nitroxide in accordance with claim 1 wherein said NICHOLAS S. RIZZO, Primary Examiner molecule is N-(1-oXyl-2,2,5,5-tetramethylpyrrolidiny1-3)- JOSE TOVAR Assistant Examiner maleimide.
References Cited UC CL X R UNITED STATES PATENTS 5 23-230; 195-1035; 260294.7, 112, 112.5; 424 2 1,915,334 6/1933 Salzberg et a1. 260243 2,075,359 3/1937 Salzberg ct a1. 16722 3, 48l,952 Dated December 2, 1969 Inve:::or(S) Harden .H
1 s in the above-identified patent te s Patent are hereby corrected as shown below:
It is certif and that said Let ed that error appear Column 1, line 6, the word "Synbar" should be Synvar SIGNED AND SEALED was 1970 am Amt:
Efiward M. newb mm: 2 601mm g m: Atteating Offioer l enn- 02 3 am:
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US49668365A | 1965-10-15 | 1965-10-15 | |
US49662265A | 1965-10-15 | 1965-10-15 | |
US49668265A | 1965-10-15 | 1965-10-15 | |
US51279365A | 1965-12-09 | 1965-12-09 | |
US513083A US3409522A (en) | 1965-10-15 | 1965-12-10 | Electrochemical machining of titanium and alloys thereof |
Publications (1)
Publication Number | Publication Date |
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US3481952A true US3481952A (en) | 1969-12-02 |
Family
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Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US496683A Expired - Lifetime US3453288A (en) | 1965-10-15 | 1965-10-15 | Electron spin resonance labels for biomolecules |
US496622A Expired - Lifetime US3489522A (en) | 1965-10-15 | 1965-10-15 | Electron spin resonance labeling of biomolecules |
US512793A Expired - Lifetime US3481952A (en) | 1965-10-15 | 1965-12-09 | N-(1-oxide-2,2,5,5-tetra lower alkyl pyrrolidinyl-3)-maleimides |
US513083A Expired - Lifetime US3409522A (en) | 1965-10-15 | 1965-12-10 | Electrochemical machining of titanium and alloys thereof |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US496683A Expired - Lifetime US3453288A (en) | 1965-10-15 | 1965-10-15 | Electron spin resonance labels for biomolecules |
US496622A Expired - Lifetime US3489522A (en) | 1965-10-15 | 1965-10-15 | Electron spin resonance labeling of biomolecules |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
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US513083A Expired - Lifetime US3409522A (en) | 1965-10-15 | 1965-12-10 | Electrochemical machining of titanium and alloys thereof |
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US (4) | US3453288A (en) |
BE (1) | BE686616A (en) |
DE (2) | DE1496814A1 (en) |
GB (2) | GB1154559A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4332946A (en) * | 1981-04-03 | 1982-06-01 | Vanderbilt University | Resolution enhancing maleimide spin label for biological EPR studies |
US4680272A (en) * | 1985-10-23 | 1987-07-14 | University Of California | Method for detecting molecules containing amine or thiol groups |
Families Citing this family (24)
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US3853914A (en) * | 1971-01-11 | 1974-12-10 | Syva Co | Ligand determination of spin labeled compounds by receptor displacement |
US3959287A (en) * | 1972-07-10 | 1976-05-25 | Syva Company | Ligand determination of spin labeled compounds by receptor displacement |
US3850578A (en) * | 1973-03-12 | 1974-11-26 | H Mcconnell | Process for assaying for biologically active molecules |
US3887698A (en) * | 1973-03-12 | 1975-06-03 | Univ Leland Stanford Junior | Sacs with epitopic sites on walls enclosing stable free radicals |
CA1001232A (en) * | 1973-11-30 | 1976-12-07 | Ivan Hrvoic | Nuclear dynamic polarization magnetometer |
US4099918A (en) * | 1977-04-18 | 1978-07-11 | State Board Of Higher Education For And On Behalf Of The University Of Oregon | Proxyl nitroxides as spin labels |
US4240797A (en) * | 1977-10-18 | 1980-12-23 | The Governing Council Of The University Of Toronto | Assay for reserve bilirubin binding capacity |
US4231750A (en) * | 1977-12-13 | 1980-11-04 | Diagnostic Reagents, Inc. | Methods for performing chemical assays using fluorescence and photon counting |
US4604365A (en) * | 1981-06-02 | 1986-08-05 | Electro-Nucleonics, Inc. | Immunoprecipitation assay |
WO1984002643A1 (en) * | 1983-01-10 | 1984-07-19 | Robert Thomas Gordon | Method for enhancing nmr imaging; and diagnostic use |
US4622953A (en) * | 1983-01-13 | 1986-11-18 | Gordon Robert T | Process for the treatment of atherosclerotic lesions |
US4622952A (en) * | 1983-01-13 | 1986-11-18 | Gordon Robert T | Cancer treatment method |
US4689295A (en) * | 1983-01-20 | 1987-08-25 | Integrated Genetics, Inc. | Test for Salmonella |
US4679426A (en) * | 1985-09-09 | 1987-07-14 | Fuller Milton E | Wave shape chemical analysis apparatus and method |
US4765179A (en) * | 1985-09-09 | 1988-08-23 | Solid State Farms, Inc. | Radio frequency spectroscopy apparatus and method using multiple frequency waveforms |
US5035781A (en) * | 1989-07-19 | 1991-07-30 | Jenoptik Jena Gmbh | Electrolyte for the production of black surface layers on light metals |
US5254460A (en) * | 1990-05-03 | 1993-10-19 | Advanced Magnetics, Inc. | Solvent mediated relaxation assay system |
US5164297A (en) * | 1990-05-03 | 1992-11-17 | Advanced Magnetics Inc. | Solvent mediated relaxation assay system |
KR100916479B1 (en) * | 2007-11-30 | 2009-09-08 | 삼성전기주식회사 | Electrolyte for electro-chemical machining of metal product |
EA021898B1 (en) * | 2009-11-23 | 2015-09-30 | МЕТКОН, ЭлЭлСи | Use of an aqueous electrolyte solution for electrochemical machining a surface of anode metal workpiece, and corresponding methods |
CN102234834A (en) * | 2010-04-20 | 2011-11-09 | 深圳富泰宏精密工业有限公司 | Electrolytic stripping liquid, and method for removing titanium-containing film by using electrolytic stripping liquid |
US8580103B2 (en) | 2010-11-22 | 2013-11-12 | Metcon, Llc | Electrolyte solution and electrochemical surface modification methods |
US10022812B2 (en) | 2014-10-09 | 2018-07-17 | General Electric Company | Methods for the electroerosion machining of high-performance metal alloys |
US11548085B2 (en) * | 2018-12-17 | 2023-01-10 | Safran Aircraft Engines | Electrolyte for electrochemical machining of gamma-gamma prime nickel-based superalloys |
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US2939825A (en) * | 1956-04-09 | 1960-06-07 | Cleveland Twist Drill Co | Sharpening, shaping and finishing of electrically conductive materials |
US3163677A (en) * | 1961-08-07 | 1964-12-29 | American Cyanamid Co | Process for preparing n, n, o-trisubstituted hydroxyl amines and n, n-disubstituted nitroxides and products |
US3253015A (en) * | 1962-06-13 | 1966-05-24 | American Cyanamid Co | N, n-disubstituted nitroxides and process for preparing same |
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1965
- 1965-10-15 US US496683A patent/US3453288A/en not_active Expired - Lifetime
- 1965-10-15 US US496622A patent/US3489522A/en not_active Expired - Lifetime
- 1965-12-09 US US512793A patent/US3481952A/en not_active Expired - Lifetime
- 1965-12-10 US US513083A patent/US3409522A/en not_active Expired - Lifetime
-
1966
- 1966-08-19 GB GB37279/66A patent/GB1154559A/en not_active Expired
- 1966-09-08 BE BE686616D patent/BE686616A/xx unknown
- 1966-09-10 DE DE19661496814 patent/DE1496814A1/en active Pending
- 1966-10-14 DE DE19661620411 patent/DE1620411A1/en active Pending
- 1966-10-14 GB GB46169/66A patent/GB1169872A/en not_active Expired
Patent Citations (2)
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US1915334A (en) * | 1930-10-16 | 1933-06-27 | Du Pont | Fluosilicate of organic heterocyclic bases and process of making it |
US2075359A (en) * | 1930-10-16 | 1937-03-30 | Du Pont | Insecticide |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4332946A (en) * | 1981-04-03 | 1982-06-01 | Vanderbilt University | Resolution enhancing maleimide spin label for biological EPR studies |
US4680272A (en) * | 1985-10-23 | 1987-07-14 | University Of California | Method for detecting molecules containing amine or thiol groups |
Also Published As
Publication number | Publication date |
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US3489522A (en) | 1970-01-13 |
DE1620411A1 (en) | 1970-04-30 |
US3409522A (en) | 1968-11-05 |
BE686616A (en) | 1967-02-15 |
US3453288A (en) | 1969-07-01 |
GB1169872A (en) | 1969-11-05 |
GB1154559A (en) | 1969-06-11 |
DE1496814A1 (en) | 1969-07-17 |
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