Recherche Images Maps Play YouTube Actualités Gmail Drive Plus »
Les utilisateurs de lecteurs d'écran peuvent cliquer sur ce lien pour activer le mode d'accessibilité. Celui-ci propose les mêmes fonctionnalités principales, mais il est optimisé pour votre lecteur d'écran.


  1. Recherche avancée dans les brevets
Numéro de publicationUS3453288 A
Type de publicationOctroi
Date de publication1 juil. 1969
Date de dépôt15 oct. 1965
Date de priorité15 oct. 1965
Autre référence de publicationDE1496814A1, DE1620411A1, US3409522, US3481952, US3489522
Numéro de publicationUS 3453288 A, US 3453288A, US-A-3453288, US3453288 A, US3453288A
InventeursHarden M Mcconnell
Cessionnaire d'origineSynvar Ass
Exporter la citationBiBTeX, EndNote, RefMan
Liens externes: USPTO, Cession USPTO, Espacenet
Electron spin resonance labels for biomolecules
US 3453288 A
Previous page
Next page
Description  (Le texte OCR peut contenir des erreurs.)

United States Patent US. Cl. 260326.8 2 Claims ABSTRACT OF THE DISCLOSURE Stable free radical nitroxides in which the nitroxide nitrogen is bonded to 2 tertiary carbon atoms. The molecule further contains an isocyanate group which bonds with biologically active molecules to label the same. The labeled molecules can be studied from the electron spin resonance of the nitroxide.

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. 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.

Heretofore, synthetic organic 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. In addition, 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.

In the present invention ESR labels are provided 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. In a preferred embodiment, of the present invention, 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.

In the preferred embodiment 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.

Thus, in accordance with this invention, 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.

As will appear more fully hereinafter, 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.

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 requi site tertiary character for C and C These materials may be defined as having the general formula wherein R t R1 B R3 ;.c,3. 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.

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 group or propylene group so as to form a five or six membered heterocyclic ring respectively, with the nitrogen atom, and X is an isocyanate group attached to one of the carbon atoms in the ethylene group 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. Thus, stable molecules of the following type (III) have been reported in the literature and are contemplated within the Scope of this 3 nvention, Y representing a functional group operative For bonding the label to a biologically active molecule.


Further, and as already mentioned, 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. For example, in Formula III given above, 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. However, 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.

Preparation of labels having isocyanate functional groups follows conventional organic synthesis procedures. IWhere the isothiocyanate derivative is desired, parallel )rocedures are used as will be clear to those skilled in the art.) In general, there are a number of compounds of vari- )US structure known and available containing the requiiite nitroxide group with adjacent tertiary carbon atoms. The present materials may be conveniently prepared from corresponding compounds having the structure of he end product sought with the difference being the preierve of an amino group at the site where the isocyanate group is desired. With such a corresponding amino compound, the isocyanate is obtained by a conventional conlensation reaction with phosgene in accordance with the iollowing general reaction.

a. typical preparation of an isocyanate-nitroxide label of :his invention is as follows:

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.

The type of functional group to be included as part of the nitroxide label molecule will depend largely upon the character of the biologically active molecule to be labeled. Where the molecule is a protein containing e-amino group, the isocyanate functional group is a logical choice. However, Where the molecule to be labeled has other prevalent reactive sites for bonding purposes, other functional groups may be preferred. For example, where the biologically active molecule has sulfhydryl groups, an imide type functional group may best serve the purpose. For example, a nitroxide label can be prepared with a maleimide functional group according to the following procedure.

Example B.Preparation of N-(2,2,5,5-tetramethyl- 1-oxylpyrrolidone-3 -maleimide The general reaction is as follows:

To a room temperature solution of 0.25 gm. of maleic anhydride in 5 ml. anhydrous diethyl ether was slowly added with stirring an equimolar amount (0.40 gm.) of 2,2,5,5-tetramethyl-3-aminopyrrolidine-l-oxyl (I) in 1 ml. of anhydrous diethyl ether. The N-(nitroxide)-maleamic acid (II) 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%). (Found: C, 56.0; H, 7.5; N, 11.3. C H N O requires: C, 56.5; H, 7.5; N, 11.0%.

In a typical reaction, a mixture of 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.

The above discussion exemplifies two types of biologically active molecules, i.e. protein containing e-amino groups and biologically active molecules containing sulfhydryl groups which may also be protein molecules. However, 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.

TABLE 1 (I) Nucleic acids:

(a) DNA (b) RNA (II) Proteins:

(a) Enzymes (b) Albumins, as serum albumin (c) Globular proteins, as antibodies (d) Structural proteins, as, for example, the proteins in hair (e) Synthetic polypeptides, that are used as, for examples, models of biological molecules (f) Lipo-Proteins, as, for example, in the brain (III) Nucleo-Protein:

(a) Nucleo-Histores (b) Nucleo-Protamines (IV) Other bialogically active molecules:

(9.) Antibodies (b) Drugs, such as for example, the tranquilizer,

chlorpromazine (c) Antigens, such as, for example, molecules concontaining the 2,4-dinitrophenyl group (d) Toxins.

From the diversity of the above exemplary list of biologically active molecules it will be appreciated that the spin label should be synthesized with the properties of the biologically active molecule in mind. In most cases, it will simply be a matter of adapting a known reaction of the biologically active molecule. The nitroxide group with its adjacent tertiary carbon atoms are conveniently formed as part of a molecule that is known to form a bond with the biologically active molecule.

It has already been illustrated how the reactivity of the isocyanate group and maleimide groups are utilized. To illustrate the other types of bonding required with some of the molecules in the above list other than proteins, the labeling of an antibody can be used. To label an antibody with the present nitroxide materials, a functional group will generally be required of a type that forms a non-covalent bond with the antibody. In order to accomplish this 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:

(I) 17TH-NHI N02 H+ CH3 l/CHs CH3 f on O NO:

CH3 CH3 17103 CH CH NO) The end product hydrazone derivative has been demonstrated to label antibodies formed by rabbits which are known to be specific to the 2,4-dinitrophenyl group and to form a bond therewith. The preparation of this derivative illustrates the broader concept of the invention which contemplates selecting a functional group known in the art to be operable for bonding to a particular biologically active molecule. The selected functional group is then included in a molecular structure with the discussed nitroxide grouping by conventional organic synthesis.

It is interesting to observe that 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. In other words, some biologically active molecules may serve as a functional group for bonding the nitroxide group to other biologically active molecules. In using such materials 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. To illustrate this point 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.

In selecting 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. For example, if a dye such as a fluorescent dye molecule is chemically integrated with a molecular structure including the nitroxide group and 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. A great deal of work has been done in the area of conjugating proteins with fluorescent dyes. Reference to such work will quickly reveal those dyes which are suitable for the biologically active molecule at hand and by chemically combining such a dye with the nitroxide structure in a single molecule in conventional fashion, a suitable spin label is obtained. To illustrate this approach to multi-labeling the following example is given.

Example C.1-dimethylaminoaphthalene-f-(N- nitroxide) -sulphon amide CH3 I V 112M 0 morn on, on; on:

A solution of 0.28 gm. 1-dimethylaminoaphthalene-S- sulfonyl chloride (Aldrich Chemical Co.) (I) in 5 ml. reagent grade acetone was filtered and added to 0.20 gm. (20% excess) 2,2,5,5-tetramethyl-3-aminopyrrolidine-1- oxyl (II) in 1 ml. acetone. The solution Was stirred for two hours at 25-35 C. and the acetone was removed in vacuo. The yellow solid was dissolved in ether and extracted repeatedly with an aqueous buffer (pH 6.8) to remove unreacted amine (I). The ether extract was chromographed on a silica column and was eluted with acetone. The resulting sulphonamide (III) contains -1.0 spin/mole and the optical spectrum in ethanol exhibits a maximum A ==337 my, e =4,000 cm. /millimole in the region characteristic of dimethylaminoaphthalene-sulphonamides. In glycerol (25 C.) 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. In addition to the spin label trace, 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). Here the dye group has been utilized for forming a n0n-covalent bond between the bovine serum albumin and nitroxide containing molecule.

Once the label molecule has been synthesized to contain both the nitroxide group and the functional group needed for bonding purposes, 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.

What is claimed is:

1. A nitroxide of the formula:

j 3 f R2 0 R4 wherein 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 and X is an isocyanate group which is operative to form a bond with a biologically active molecule.

2. A free radical organic molecule for spin labeling biologically active molecules comprising 2,2,5,5-tetramethyl-3-isocyanatopyrrolidine-1-oxyl.

References Cited UNITED STATES PATENTS 3,253,015 5/1966 Hoifman 260 465.5

3,163,677 12/1964 Holfmann et a1 260-583 ALEX MAZEL, Primary Examiner.

J. A. NARCAVAGE, Assistant Examiner.

US. Cl. X.R.

Citations de brevets
Brevet cité Date de dépôt Date de publication Déposant Titre
US3163677 *7 août 196129 déc. 1964American Cyanamid CoProcess for preparing n, n, o-trisubstituted hydroxyl amines and n, n-disubstituted nitroxides and products
US3253015 *20 juil. 196424 mai 1966American Cyanamid CoN, n-disubstituted nitroxides and process for preparing same
Référencé par
Brevet citant Date de dépôt Date de publication Déposant Titre
US3853914 *10 juil. 197210 déc. 1974Syva CoLigand determination of spin labeled compounds by receptor displacement
US3887698 *25 févr. 19743 juin 1975Univ Leland Stanford JuniorSacs with epitopic sites on walls enclosing stable free radicals
US3959287 *3 mai 197425 mai 1976Syva CompanyLigand determination of spin labeled compounds by receptor displacement
US4231750 *13 déc. 19774 nov. 1980Diagnostic Reagents, Inc.Methods for performing chemical assays using fluorescence and photon counting
US4240797 *18 oct. 197723 déc. 1980The Governing Council Of The University Of TorontoAssay for reserve bilirubin binding capacity
US5164297 *3 mai 199017 nov. 1992Advanced Magnetics Inc.Solvent mediated relaxation assay system
US5254460 *3 mai 199119 oct. 1993Advanced Magnetics, Inc.Solvent mediated relaxation assay system
WO1984002643A1 *10 janv. 198419 juil. 1984Gordon Robert ThomasMethod for enhancing nmr imaging; and diagnostic use
Classification aux États-Unis548/542, 546/242, 530/405, 530/408, 436/94, 530/806, 548/518, 530/363, 530/404, 546/223, 530/391.5, 435/188, 530/357, 435/177, 436/173, 546/244, 436/88, 530/358, 436/87, 530/409
Classification internationaleC12Q1/68, B23H3/08, G01N33/58
Classification coopérativeC12Q1/68, Y10S530/806, G01N33/58, B23H3/08
Classification européenneB23H3/08, C12Q1/68, G01N33/58