US3453288A - Electron spin resonance labels for biomolecules - Google Patents
Electron spin resonance labels for biomolecules Download PDFInfo
- Publication number
- US3453288A US3453288A US496683A US3453288DA US3453288A US 3453288 A US3453288 A US 3453288A US 496683 A US496683 A US 496683A US 3453288D A US3453288D A US 3453288DA US 3453288 A US3453288 A US 3453288A
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- US
- United States
- Prior art keywords
- group
- biologically active
- nitroxide
- label
- molecule
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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- 238000004435 EPR spectroscopy Methods 0.000 title description 13
- -1 free radical nitroxides Chemical class 0.000 description 22
- 229910052799 carbon Inorganic materials 0.000 description 21
- 125000000524 functional group Chemical group 0.000 description 18
- 238000002372 labelling Methods 0.000 description 16
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical class ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 14
- IQPQWNKOIGAROB-UHFFFAOYSA-N isocyanate group Chemical group [N-]=C=O IQPQWNKOIGAROB-UHFFFAOYSA-N 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 11
- 125000004432 carbon atom Chemical group C* 0.000 description 11
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 8
- 239000000975 dye Substances 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 238000001228 spectrum Methods 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- 239000012948 isocyanate Substances 0.000 description 6
- 150000002513 isocyanates Chemical class 0.000 description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 125000003396 thiol group Chemical group [H]S* 0.000 description 4
- 125000001917 2,4-dinitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C(=C1*)[N+]([O-])=O)[N+]([O-])=O 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- YGYAWVDWMABLBF-UHFFFAOYSA-N Phosgene Chemical compound ClC(Cl)=O YGYAWVDWMABLBF-UHFFFAOYSA-N 0.000 description 3
- 125000000217 alkyl group Chemical group 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 239000007795 chemical reaction product Substances 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 125000000623 heterocyclic group Chemical group 0.000 description 3
- 125000000962 organic group Chemical group 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 229940124530 sulfonamide Drugs 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 125000002947 alkylene group Chemical group 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- ZPEIMTDSQAKGNT-UHFFFAOYSA-N chlorpromazine Chemical compound C1=C(Cl)C=C2N(CCCN(C)C)C3=CC=CC=C3SC2=C1 ZPEIMTDSQAKGNT-UHFFFAOYSA-N 0.000 description 2
- 229960001076 chlorpromazine Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 125000000816 ethylene group Chemical group [H]C([H])([*:1])C([H])([H])[*:2] 0.000 description 2
- 125000001153 fluoro group Chemical group F* 0.000 description 2
- ZBKFYXZXZJPWNQ-UHFFFAOYSA-N isothiocyanate group Chemical group [N-]=C=S ZBKFYXZXZJPWNQ-UHFFFAOYSA-N 0.000 description 2
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 2
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229940125725 tranquilizer Drugs 0.000 description 2
- 239000003204 tranquilizing agent Substances 0.000 description 2
- 230000002936 tranquilizing effect Effects 0.000 description 2
- CGKOJRBANWWHCG-UHFFFAOYSA-N 1-$l^{1}-oxidanyl-2,2,5,5-tetramethylpyrrolidin-3-amine Chemical group CC1(C)CC(N)C(C)(C)N1[O] CGKOJRBANWWHCG-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- YCKRFDGAMUMZLT-UHFFFAOYSA-N Fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- XDXHAEQXIBQUEZ-UHFFFAOYSA-N Ropinirole hydrochloride Chemical compound Cl.CCCN(CCC)CCC1=CC=CC2=C1CC(=O)N2 XDXHAEQXIBQUEZ-UHFFFAOYSA-N 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- XLJMAIOERFSOGZ-UHFFFAOYSA-M cyanate group Chemical group [O-]C#N XLJMAIOERFSOGZ-UHFFFAOYSA-M 0.000 description 1
- 125000000753 cycloalkyl group Chemical group 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012259 ether extract Substances 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 102000034238 globular proteins Human genes 0.000 description 1
- 108091005896 globular proteins Proteins 0.000 description 1
- 150000007857 hydrazones Chemical class 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 150000003949 imides Chemical class 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000000199 molecular distillation Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229940070353 protamines Drugs 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
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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
-
- 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
-
- 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
-
- 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
-
- 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 class of such organic spin labels employing a nitroxide radical as the active labeling group bound to the biomolecule via the intermediary of an isocyanate group.
- ESR electron spin resonance
- Such improved ESR labels provide strong labeling resonance line and are useful, for example for obtaining a wealth of information about biomolecules and biological systems.
- ESR labels have been used for labeling biomolecules.
- a typical 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 my copending application Ser. No. 496,682, filed Oct. 15, 1965.
- CPZ+ attaches predominately only to DNA and RNA type biomolecules for labeling same. While specificity of the label is desirable in certain instances, there are many additional biomolecules to be ESR-labeled and thus a need for a more universal label.
- the electron resonance line spectrum of CPZ+ includes a substantial amount of fine structure which tends to weaken the resonance lines of the spectrum making measurement of subtle changes in the spectrum of the label difficult.
- ESR labels which employ a nitroxide radical group as the spin labeling group.
- the nitroxide group provides a strong electron resonance line spectrum having little fine structure. This radical has been found to be remarkably stable and inert, to show sharp, well resolved and simple electron resonance spectra that are sensitive to molecular motion, and to a lesser extent, sensitive to polarity of the molecular environment.
- the nitroxide radical group is attached to the biomolecule via the intermediary of an isocyanate group whereby the nitroxide label may be readily bound to such entities as proteins and synthetic polypeptides.
- the principal object of the present invention is to provide an improved class of organic ESR labels for biologically active molecules.
- the labelled molecules can be studied in vivo as Well as in vitro by ESR techniques and this flexibility provides a powerful research tool.
- One feature of the present invention is the provision of a class of ESR biomolecule labeling chemicals and methods of synthesizing same wherein the nitroxide radical is used to provide electron resonance labeling line spectrum.
- Another feature of the present invention is the same as the preceding wherein the nitroxide labeling group is attached to the biomolecule via the intermediary of an iso- ICC cyanate group whereby the label is made specific to certain proteins and synthetic polypeptides.
- novel nitroxide compounds are synthesized to contain at least one isocyanate group which serves to form a covalent bond with atoms of the biologically active molecule to be labeled.
- Isocyanate-containing nixtroxide compounds are especially useful for labeling most proteins through a conventional reaction between the isocyanate group and the e-amino group of the protein molecule.
- a class of 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 a 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 contains a functional group other than a 2,4-dinitrophenyl group, 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 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 requi site tertiary character for C and C
- R2 (5 B4 R R and R are lower alkyl groups, i.e., each having about 1-5 carbon atoms;
- B represents a plurality of carbon atoms in a partial cycloalkyl chain, i.e., an alkylene group, and X is an isocyanate or isothiocyanate group on B.
- 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 group or propylene group so as to form a five or six membered heterocyclic ring respectively, with the nitrogen atom
- X is an isocyanate group attached to one of the carbon atoms in the ethylene group or propylene group.
- C and C in addition being tertiary must have all of their valences satisfied )y saturated bonds to either carbon atoms or fluorine l'tOIIlS.
- the 'eplacement of the methyl groups by fluorine atoms would )rovide typical compounds contemplated within the scope )f this invention.
- Isocyanate and isothiocyanate functional groups are )articularly useful for bonding labels to e-amino groups )f proteins.
- the reaction does not appear to be l00 percent specific for e-amino groups.
- Evidence has :een obtained which indicates that the isocyanate group 1150 attaches to some extent to sulfhydryl groups of proeins although this point of attachment is minor compared 0 the extent of reaction with and attachment to e-amino groups.
- Example A 2,2,5,5-tetramethyl-3-aminopyrrolidone-l-oxyl is used 18 a starting material. It may be synthesized from triicetonamine by the method of Rosantzev and Krivitzkaya, Tetrahedron, 21, 491 (1965).
- a saturated solution of the amino compound of Rosantzev and Krivitzkaya in dry benzene is added dropwise at 0 C. to a stirred solution of 12.5 percent phosgene in benzene (1 mole amino compound to 2 moles phosgene).
- the end product in accordance with the above general reaction is 2,2,5,5-tetramethyl-3-isocyanatopyrrolidine-1- oxyl, having the structural formula N00 a i CH3 1? o
- the benzene solvent can be removed in vacuo at room temperature and the isocyanate product is ready for use as a label.
- the isocyanate group while advantageous for its relative specificity for point of attachment to protein as well as for other reasons, is only exemplary of the functional groups which can be synthesized to form part of the label molecule. Any type of functional group capable of bonding in one way or another with a biologically active molecule is contemplated as an alternative to the isocyanate group.
- the functional group of the label and the biologically active molecule can be bonded by a covalent bond such as results from the interaction between isocyanate and an amino group on the biologically active molecule or it may be non-covalent and fall within a diverse category of recognized bonds, such as an ionic bond, a hydrogen bond, a hydrophobic bond, a dispersion or Van der Waals bond, a charge-transfer or dipole-dipole bond, or a combination of a covalent bond and/or any of the other noted non-covalent bonds.
- a covalent bond such as results from the interaction between isocyanate and an amino group on the biologically active molecule or it may be non-covalent and fall within a diverse category of recognized bonds, such as an ionic bond, a hydrogen bond, a hydrophobic bond, a dispersion or Van der Waals bond, a charge-transfer or dipole-dipole bond, or a combination of a covalent bond and/or any of the other noted non-covalent bonds.
- a nitroxide label can be prepared with a maleimide functional group according to the following procedure.
- N-(nitroxide)-maleamic acid (II) 0.63 gm. N-(nitroxide)-maleamic acid (II), 1 ml. acetic anhydride, and 0.12 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.
- the N-(nitroxide)-maleimide may be purified by molecular distillation.
- Both the crude and the purified product III has been shown to combine preferentially with sulfhydryl groups (over amino groups) in bovine serum albumin.
- biologically active molecules i.e. protein containing e-amino groups and biologically active molecules containing sulfhydryl groups which may also be protein molecules.
- the invention is applicable to all biologically active molecules, the term being used in the broadest sense to include all molecules atfecting the life processes from a chemical standpoint.
- Biologically active mole cules within the present context include, for example, the following types of materials.
- Antibodies (b) Drugs, such as for example, the tranquilizer,
- the labeling of an antibody can be used.
- a functional group will generally be required of a type that forms a non-covalent bond with the antibody.
- the nitroxide label can be obtained as part of a molecule structure which includes a ketone group. A dinitrophenyl hydrazone derivative of the ketone will provide the requisite functional group for bonding to many antibodies.
- the preparation of this type of label is illustrated by the following equation:
- the functional group to be integrated with the nitroxide group may be part of a molecule otherwise considered a biologically active molecule within the broad meaning of the term as here used.
- some biologically active molecules may serve as a functional group for bonding the nitroxide group to other biologically active molecules.
- the first step in effect would be the labeling of a. biologically active molecule with the nitroxide group.
- the so labeled biologically active molecule in turn serves as an integral unit as a label for another biologically active molecule.
- an antibiotic (considered as a biologically active molecule herein) can be labeled with a nitroxide grouping by suitable synthesis procedures of the type discussed.
- the so labeled antibiotic can in turn be used to label DNA which is also a biologically active molecule if the antibiotic selected in the first instance is one that reacts specifically with DNA.
- a functional group for bonding purposes it is possible to choose materials which in and of themselves may serve as a label of a diiferent type.
- a dye such as a fluorescent dye molecule
- the dye is of the type which will bond with biologically active molecules
- the result is a biologically active molecule labeled both with the dye and with the resonating nitroxide group.
- the ether extract was chromographed on a silica column and was eluted with acetone.
- the emission maximum occurs at 530 my. and the fluorescent lifetime is approximately 14 nanoseconds.
- the sulphonamide prepared in the above example contains both the nitroxide label group and a fluorescent dye label grouping. This molecule has been shown to bond with bovine serum albumin and the double label characteristics observed.
- Dyes illustrate one possibility of a second label.
- Other possibilities include the synthesis of a nitroxide molecule with the requisite functional group for bonding purposes wherein the molecular structure also includes a radioactive element.
- a radioactive trace can be obtained in the usual fashion.
- the above example also illustrated the selection of a functional grouping for purposes of bonding the nitroxide to another type of biologically active molecule (bovine serum albumin).
- the dye group has been utilized for forming a n0n-covalent bond between the bovine serum albumin and nitroxide containing molecule.
- the mechanics of combining the label with the selected biologically active molecule is straightforward.
- the mere combination of the label molecule and the biologically active molecule to permit contact between the two in a suitable solvent such as an aqueous medium is suflicient to achieve attachment of the label to the biologically active molecule.
- the amount of label utilized will be dependent upon the sensitivity of the equipment used for detecting the electron spin resonance of the label. Using conventional ESR spectrometers it has been found that one labelling molecule per biologically active molecule provides an adequate signal to noise ratio.
- R R R and R are lower alkyl groups
- B is an alkylene group having 2-3 carbon atoms and forming a heterocyclic ring with C and C
- X is an isocyanate group which is operative to form a bond with a biologically active molecule.
- a free radical organic molecule for spin labeling biologically active molecules comprising 2,2,5,5-tetramethyl-3-isocyanatopyrrolidine-1-oxyl.
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mechanical Engineering (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Electrochemistry (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- General Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Pyrrole Compounds (AREA)
- Plural Heterocyclic Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- ing And Chemical Polishing (AREA)
- Electrical Discharge Machining, Electrochemical Machining, And Combined Machining (AREA)
- Electroplating And Plating Baths Therefor (AREA)
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 |
---|---|
US3453288A true US3453288A (en) | 1969-07-01 |
Family
ID=27541776
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 After (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
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 |
Country Status (4)
Country | Link |
---|---|
US (4) | US3453288A (enrdf_load_html_response) |
BE (1) | BE686616A (enrdf_load_html_response) |
DE (2) | DE1496814A1 (enrdf_load_html_response) |
GB (2) | GB1154559A (enrdf_load_html_response) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3853914A (en) * | 1971-01-11 | 1974-12-10 | Syva Co | Ligand determination of spin labeled compounds by receptor displacement |
US3887698A (en) * | 1973-03-12 | 1975-06-03 | Univ Leland Stanford Junior | Sacs with epitopic sites on walls enclosing stable free radicals |
US3959287A (en) * | 1972-07-10 | 1976-05-25 | Syva Company | Ligand determination of spin labeled compounds by receptor displacement |
US4231750A (en) * | 1977-12-13 | 1980-11-04 | Diagnostic Reagents, Inc. | Methods for performing chemical assays using fluorescence and photon counting |
US4240797A (en) * | 1977-10-18 | 1980-12-23 | The Governing Council Of The University Of Toronto | Assay for reserve bilirubin binding capacity |
WO1984002643A1 (en) * | 1983-01-10 | 1984-07-19 | Robert Thomas Gordon | Method for enhancing nmr imaging; and diagnostic use |
US5164297A (en) * | 1990-05-03 | 1992-11-17 | Advanced Magnetics Inc. | Solvent mediated relaxation assay system |
US5254460A (en) * | 1990-05-03 | 1993-10-19 | Advanced Magnetics, Inc. | Solvent mediated relaxation assay system |
US10022812B2 (en) | 2014-10-09 | 2018-07-17 | General Electric Company | Methods for the electroerosion machining of high-performance metal alloys |
Families Citing this family (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3850578A (en) * | 1973-03-12 | 1974-11-26 | H Mcconnell | Process for assaying for biologically active molecules |
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 |
US4332946A (en) * | 1981-04-03 | 1982-06-01 | Vanderbilt University | Resolution enhancing maleimide spin label for biological EPR studies |
US4604365A (en) * | 1981-06-02 | 1986-08-05 | Electro-Nucleonics, Inc. | Immunoprecipitation assay |
US4622952A (en) * | 1983-01-13 | 1986-11-18 | Gordon Robert T | Cancer treatment method |
US4622953A (en) * | 1983-01-13 | 1986-11-18 | Gordon Robert T | Process for the treatment of atherosclerotic lesions |
US4689295A (en) * | 1983-01-20 | 1987-08-25 | Integrated Genetics, Inc. | Test for Salmonella |
US4765179A (en) * | 1985-09-09 | 1988-08-23 | Solid State Farms, Inc. | Radio frequency spectroscopy apparatus and method using multiple frequency waveforms |
US4679426A (en) * | 1985-09-09 | 1987-07-14 | Fuller Milton E | Wave shape chemical analysis apparatus and method |
US4680272A (en) * | 1985-10-23 | 1987-07-14 | University Of California | Method for detecting molecules containing amine or thiol groups |
US5035781A (en) * | 1989-07-19 | 1991-07-30 | Jenoptik Jena Gmbh | Electrolyte for the production of black surface layers on light metals |
KR100916479B1 (ko) * | 2007-11-30 | 2009-09-08 | 삼성전기주식회사 | 금속제품 전해가공용 전해액 |
MX2012005909A (es) * | 2009-11-23 | 2012-11-12 | MetCon LLC | Soliucion de electrolitos y metodos de electropulido. |
CN102234834A (zh) * | 2010-04-20 | 2011-11-09 | 深圳富泰宏精密工业有限公司 | 电解退镀液及使用该电解退镀液退除含钛膜层的方法 |
US8580103B2 (en) | 2010-11-22 | 2013-11-12 | Metcon, Llc | Electrolyte solution and electrochemical surface modification methods |
WO2020128304A1 (fr) * | 2018-12-17 | 2020-06-25 | Safran Aircraft Engines | Electrolyte pour l'usinage electrochimique de superalliages bases nickel type gamma-gamma prime |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2075359A (en) * | 1930-10-16 | 1937-03-30 | Du Pont | Insecticide |
US1915334A (en) * | 1930-10-16 | 1933-06-27 | Du Pont | Fluosilicate of organic heterocyclic bases and process of making it |
US2939825A (en) * | 1956-04-09 | 1960-06-07 | Cleveland Twist Drill Co | Sharpening, shaping and finishing of electrically conductive materials |
-
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/de active Pending
- 1966-10-14 DE DE19661620411 patent/DE1620411A1/de active Pending
- 1966-10-14 GB GB46169/66A patent/GB1169872A/en not_active Expired
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
US3887698A (en) * | 1973-03-12 | 1975-06-03 | Univ Leland Stanford Junior | Sacs with epitopic sites on walls enclosing stable free radicals |
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 |
WO1984002643A1 (en) * | 1983-01-10 | 1984-07-19 | Robert Thomas Gordon | Method for enhancing nmr imaging; and diagnostic use |
US5164297A (en) * | 1990-05-03 | 1992-11-17 | Advanced Magnetics Inc. | Solvent mediated relaxation assay system |
US5254460A (en) * | 1990-05-03 | 1993-10-19 | Advanced Magnetics, Inc. | Solvent mediated relaxation assay system |
US10022812B2 (en) | 2014-10-09 | 2018-07-17 | General Electric Company | Methods for the electroerosion machining of high-performance metal alloys |
Also Published As
Publication number | Publication date |
---|---|
GB1169872A (en) | 1969-11-05 |
US3409522A (en) | 1968-11-05 |
US3489522A (en) | 1970-01-13 |
GB1154559A (en) | 1969-06-11 |
DE1620411A1 (de) | 1970-04-30 |
DE1496814A1 (de) | 1969-07-17 |
US3481952A (en) | 1969-12-02 |
BE686616A (enrdf_load_html_response) | 1967-02-15 |
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