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Site-specific isotopically-labeled proteins, amino acids, and biochemical precursors thereforRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Cyclopeptides, 25 Or More Peptide Repeating Units In Known Peptide Chain StructureSite-specific isotopically-labeled proteins, amino acids, and biochemical precursors therefor description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060293229, Site-specific isotopically-labeled proteins, amino acids, and biochemical precursors therefor. Brief Patent Description - Full Patent Description - Patent Application Claims
CONTINUING DATA [0001] This application is an original conversion from the provisional application Ser. No. 60/128,668, filed Apr. 9, 1999. FIELD OF THE INVENTION [0002] The present invention relates to site-specific isotopically-labeled organic compounds and processes for their preparation. More particularly, the present invention concerns site-specific isotopically-labeled biochemical precursors of leucine, isoleucine, and valine, the isotopically-labeled amino acids per se, proteins, protein fragments or polypeptides made therefrom, and related methods of preparation. BACKGROUND OF THE INVENTION [0003] A recently-developed technique for discovering new drug leads involves the use of nuclear magnetic resonance (NMR) spectroscopy to discover compounds that bind to a particular target molecule such as a protein (.see, for example, U.S. Pat. Nos. 5;698,401 and 5,804,390, to Fesik, et al.). The technique involves the determination of a first two-dimensional .sup.15N/.sup.1H NMR correlation spectrum of a protein in which nitrogen atom sites have been isotopically enriched with .sup.15N. This first correlation spectrum is obtained for the protein in the absence of any potential ligand compound(s). Next a suspected ligand compound, or a mixture of such putative ligand compounds, is mixed with the isotopically enriched protein, and a second NMR correlation spectrum is obtained. The two spectra are compared, and differences between the two spectra provide information about 1) the existence of binding between any ligand and the host protein, 2) the site(s) of binding, and 3) the strength(s) of binding. [0004] The technique described in Fesik, et al., supra, employs target molecules which have been isotopically enriched with the NMR-detectable .sup.15N spin nucleus. This method relies upon the genetic modification of a suitable microorganism to express the desired protein, protein fragment, or polypeptide, followed by culturing the modified microorganism in a nutrient medium containing assimilable sources of carbon and nitrogen which include .sup.15N-labeled nutrients. Comparatively inexpensive commercially available .sup.15N ammonium salts provided the .sup.15N source. [0005] However, the application of this NMR drug discovery technique to target molecules isotopically enriched with .sup.13C has been hampered by two drawbacks. First, it is comparatively expensive to produce .sup.13C-enriched target molecules in any useful quantities. For example, the production of proteins by genetically modified microorganisms grown in nutrient media containing commercially available uniformly-labeled glucose (glucose-.sup.13C.sub.6) is expensive. At the time of filing this application, the cost of glucose-.sup.13C.sub.6 was approximately $480/g. Alternatively, the production of .sup.13C-labeled proteins by including uniformly .sup.12C-labeled amino acids in the nutrient medium is similarly expensive. Second, the biomolecules produced using glucose-.sup.13C.sub.6 or commercially available uniformly .sup.13C-enriched amino acids are not ideally suited for the NMR correlation spectra technique. Biomolecules expressed by microorganisms grown in nutrient media containing uniformly .sup.13C-enriched starting materials contain adjacent .sup.13C-labeled carbon atoms. Since the NMR technique depends upon detection of spatial spin coupling (i.e., the nuclear Overhauser effect), the relatively strong spin-spin coupling of adjacent .sup.13C nuclei interferes with the desired observation. There is thus a need for the development of site-specifically .sup.13C-enriched amino acids, proteins and polypeptides. SUMMARY OF THE INVENTION [0006] The instant invention provides biochemical precursors of the site-specific isotopically-enriched amino acids leucine, isoleucine, and valine, as well as the site-specific isotopically-enriched amino acids per se. Additionally, proteins, protein fragments and polypeptides containing site-specific isotopically-enriched aminoacyl residues derived from these amino acids, and methods for their production, are also provided. The amino acids and the amino acid biosynthetic precursors are isotopically enriched with either .sup.13C or .sup.14C at the carbon atoms of methyl groups most remote from their carboxyl group. In the labeled amino acids of the present invention, non-adjacent carbon atoms are labeled. In the case where the label is .sup.13C, the amino acids of this invention are thus ideally suited for use in the NMR drug discovery technique, since there is no interference with the desired signals by adjacent atom .sup.13C-.sup.13C spin-spin interaction. Moreover, since the amino acids are labeled only at methyl groups, the three magnetically equivalent hydrogen atoms of the methyl group(s) provide strong NMR signals for observation of any effects of coupling with the .sup.13C atom(s) to which they are attached. [0007] Specifically, the present invention provides compounds of formula I or a salt thereof, wherein R.sup.1 is oxygen or NH.sub.2, and R.sup.2 is selected from the group consisting of In the formulae presented above, R.sup.3 is hydrogen or .sup.nCH.sub.3, the dotted line bonds represent valence bonds, m is zero or one, and n, at each occurrence, is 13 or 14, with the provisos that: a) when R.sup.1 is NH.sub.2, the second valence bond represented by the dotted line bond to R.sup.1 is absent and the hydrogen attached to the dotted line bond is present; b) when R.sup.1 is oxygen, the second valence bond represented by the dotted line bond to R.sup.1 is present and the hydrogen atom attached to the dotted line bond is absent; c) when R.sup.1 is oxygen, R.sup.2 is B and m is zero; and d) when R.sup.1 is NH.sub.2, R.sup.3 is hydrogen or .sup.nCH.sub.3. [0008] The present invention provides the site-specific .sup.13C- and .sup.14C-enriched amino acids isoleucine (formula I above where R.sup.1 is amino, R.sup.2 is A); leucine (formula I above where R.sup.1 is amino, R.sup.2 is B, R.sup.3 is .sup.nCH.sub.3, and m is one), and valine (formula I above where R.sup.1 is amino, R.sup.2 is B, R.sup.3 is .sup.nCH.sub.3, and m is zero), and the site-specific .sup.13C- and .sup.14C-enriched biochemical precursors of these amino acids, 2-keto-4-(.sup.nC)-butyric acid (formula I above where R.sup.1 is oxygen, R.sup.2 is B, m is zero, and R.sup.3 is hydrogen) and 2-keto-3-(.sup.nC-methyl)-4-(C)-butyric acid (formula I above where R.sup.1 is oxygen, R.sup.2 is B, m is zero, and R.sup.3 is .sup.nCH.sub.3). In the foregoing, n represents either 13 (i.e., .sup.13C-enriched compounds) or 14 (i.e., .sup.14C-enriched compounds). [0009] The present invention further provides proteins, protein fragments, and polypeptides containing aminoacyl residues derived from one or more of the amino acids selected from the group consisting of L-2-amino-3-methyl-5-(.sup.13C)-pentanoic acid; L-2-amino-3-methyl-5-(.sup.14C)-pentanoic acid; L-2-amino-4- (.sup.13C-methyl-5-(.sup.13C)-pentanoic; L-2-amino-4-(.sup.14C-methyl-5-(.sup.14C)-pentanoic acid; L-2-amino-3-(.sup.13C-methyl)-5-(.sup.13C)-butanoic acid; and L-2-amino-3-(.sup.14C-methyl)-5-(.sup.14C)-butanoic acid. [0010] Also provided by the present invention are chemical methods of preparing the site-specific .sup.13C- and 14C-labeled biochemical precursors acids, 2-keto-4-(.sup.nC)-butyric acid and 3-(.sup.nC-methyl)-4-(.sup.nC)-butyric acid, or salts thereof, which involves reacting a compound of formula IV with isotopically-labeled methyl iodide (H.sub.3.sup.nCI) to produce a compound of formula V removing the protecting tert-butyl ester and dimethylhydrazino groups of a compound of formula V to produce 2-keto-4-(.sup.nC)-butyric acid; or further reacting a compound of formula V with isotopically-labeled methyl iodide (H.sub.3.sup.nCI) where n is 13 or 14, to produce a compound of formula VI removing the protecting tert-butyl ester and dimethylhydrazino groups to produce 2-keto-3-(.sup.nC-methyl)-4-(.sup.nC)-butyric acid; and optionally salifying the products. [0011] The present invention additionally provides methods for preparing the site-specific .sup.13C- and .sup.14C-labeled amino acids, leucine, isoleucine, and valine. The process involves genetically modifying a microorganism to express a polypeptide containing an amino acid selected from leucine, isoleucine, valine and mixtures thereof; culturing the modified microorganism in a nutrient medium containing assimilable sources of carbon and nitrogen which includes 2-keto-4-(.sup.nC)-butyric acid, 2-keto-3-(.sup.nC-methyl)-4-(.sup.nC)-butyric acid, and salts and mixtures thereof; isolating the resulting expressed polypeptide; and fragmenting the polypeptide and isolating the individual amino acids. The expressed polypeptide is fragmented by conventional methods known in the art including hydrolysis or enzymatic cleavage. [0012] The yield of a particular amino acid may be maximized and the cost minimized by modifying the host microorganism to express a homopolymer of the amino acid, and utilizing the appropriate isotopically enriched biosynthetic precursor in the nutrient medium. [0013] The present invention still further provides a method of preparing a protein, protein fragment, or polypeptide containing amino acyl residues derived from amino acids selected from the group consisting of L-2-amino-3-methyl-5-(.sup.13C)-pentanoic acid; L-2-amino-3-methyl-5-(.sup.14C)-pentanoic acid ; L-2-amino-4-(.sup.13C-methyl-5-(.sup.13C)-pentanoic; L-2-amino-4-(.sup.14C-methyl-5- (.sup.14C)-pentanoic acid; L-2-amino-3-(.sup.13C-methyl)-5-(.sup.13C) -butanoic acid; and L-2-amino-3-(.sup.14C-methyl)-5-(.sup.14C)-butanoic acid which involves genetically modifying a microorganism to express a pre-determined protein, protein fragment or polypeptide; culturing the modified microorganism in a nutrient medium containing assimilable sources of carbon and nitrogen which includes 2-keto-4-(.sup.nC)-butyric acid, 2-keto-3-(.sup.nC-methyl)-4-(.sup.nC)-butyric acid, and salts and mixtures thereof; and isolating the resulting expressed polypeptide. DETAILED DESCRIPTION OF THE INVENTION [0014] The natural isotopic abundance of .sup.13C is 1.11%, and that of .sup.14C is negligibly low. Thus the probability that any given carbon atom within an organic molecule is .sup.13C is normally about 0.0111, and the probability that any given carbon atom is .sup.14C is quite low. When target proteins are prepared for use in the adapted NMR "screening" or drug discovery process as described by Fesik, et al., supra, it is desirable that the .sup.13C NMR signal be enhanced by increasing the natural .sup.13C content of the target molecule being studied. This is accomplished by either uniformly or selectively enriching the target molecule with .sup.13C. As used throughout this specification and the appended claims, the terms "uniform enrichment," "uniformly enriching," "uniformly enriched," uniform labeling" and "uniformly labeled" mean increasing to a value greater than 0.0111, by synthetic means, the probability that a carbon atom randomly selected throughout the target molecule will be .sup.13C. The terms "specific enrichment," "site-specific enrichment," "specifically enriching," "specifically enriched," "specifically labeling" and "specifically labeled" mean increasing to a value greater than 0.0111, by synthetic means, the probability that carbon atoms at one or more specific pre-selected site(s) within the target molecule will be .sup.13C. [0015] For example, biomolecules expressed by genetically modified microorganisms grown in a nutrient medium containing uniformly .sup.13C-enriched glucose will be uniformly .sup.13C enriched. A protein expressed by a genetically modified microorganism grown in a nutrient medium containing an amino acid which is .sup.13C-enriched only on the methyl side chain would be specifically enriched by .sup.13C at the alanyl residues contained within the expressed protein. Similarly, proteins expressed by the method of this invention will be site-specifically enriched by .sup.13C or .sup.14C at the side-chain terminal methyl groups of leucine, isoleucine, and valine. [0016] The method of the present invention also permits the preparation of site-specifically labeled leucine, isoleucine and valine, proteins, protein fragments, or polypeptides made from these labeled amino acids, and the amino acid biosynthetic precursors with labeled with .sup.14C as well as .sup.13C. Such compounds are useful, for example, in studies of protein metabolism where it is desirable to follow the course and fate of protein degradation by radiometric methods. [0017] Further terms used throughout this specification and the appended claims have their usually accepted meanings. The following specific terms have the ascribed meanings: [0018] "DTT" means dithiothreitol. [0019] "HEPES" denotes N-2-hydroxyethylpiperazine-N'-2-ethylsulfonic acid. [0020] "IPTG" means isopropyl-.beta.-D-thiogalactopyranoside. [0021] "PMSF" refers to .alpha.-toluenesulfonyl fluoride. [0022] "SCD" refers to the catalytic domain (residues 81-256) of stromelysin. [0023] The preparation of an exemplary site-specific .sup.nC-enriched protein fragment target molecule is set forth below. The particular example shown demonstrates the preparation of the so-called "catalytic domain" of human stromelysin ("SCD"), labeled with site-specific .sup.13C-enriched leucine, valine, and isoleucine. While shown with .sup.13C-labeled amino acid precursors, the method is equally applicable starting with .sup.14C-labeled amino acid precursors. A preferred means of preparing adequate quantities of specifically .sup.nC-enriched polypeptide-containing target molecules involves the transformation of a host cell with an expression vector containing a polynucleotide encoding the desired polypeptide. The protein or polypeptide protein fragment is expressed by culturing the transformed cell line in a medium containing assimilable sources of carbon and nitrogen well known in the art and including the .sup.nC-enriched biochemical precursors of this invention. For site-specific labeling of the protein or protein fragment in accordance with the present invention, assimilable sources for .sup.nC labeling of a target polypeptide include .sup.nC-labeled biosynthetic precursors of amino acids. [0024] For example, it is known that .alpha.-keto-butyrate is the biosynthetic precursor of isoleucine, and that .alpha.-keto-isovalerate is the biosynthetic precursor of both valine and leucine. Scheme I below shows how the specifically .sup.nC-enriched biosynthetic precursors of leucine, isoleucine, and valine can be synthesized. The Scheme employs the comparatively inexpensive .sup.nC-enriched methyl iodide, H.sub.3.sup.nCI, as the source for isotopic enrichment to produce .sup.nC-terminally-labeled .alpha.-keto-butyric acid and .alpha.-keto-isovaleric acid. Continue reading about Site-specific isotopically-labeled proteins, amino acids, and biochemical precursors therefor... Full patent description for Site-specific isotopically-labeled proteins, amino acids, and biochemical precursors therefor Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Site-specific isotopically-labeled proteins, amino acids, and biochemical precursors therefor patent application. 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