JPH06504031A - Amino acid transport proteins, amino acid analogs, assay devices, and their use in cancer treatment and diagnosis - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

Amino Acid Transport Proteins, Amino Acid Analogs, Assay Devices and Their Use in the Treatment and Diagnosis of Cancer Background of the Invention The present invention relates to compounds and methods for treating and diagnosing cancer in animals such as humans. Ru. More specifically, the present invention provides a method for treating cancer cells that are common in tumor cells but are Concerns the identification and isolation of human amino acid transporters, including transporters such as the glutamine transporter, which are not commonly found in tumor cells or are less active or present in small amounts. The present invention provides recombinant products and methods useful for the production of such transporters and other biological substances, such as antibodies, and for detecting the presence of such transporters in animals. diagnostic products and assays useful for Can effectively inhibit the passage of amino acids such as glutamine, or glutamine in cells. The present invention relates to relevant compositions such as antibodies, anti-glutamine compounds and glutamine analogs of glutamine transporters that interfere with glutamine metabolism, and various therapeutic compositions comprising such inhibitors. The present invention provides methods for treating cancer by administering such compositions, such transporters and vaccines consisting of its fragments or subunits and the use of such vaccines. It also relates to a method for immunization using the method. Current cancer treatments generally require patients to use three main groups of compounds: (1) (2) anti-metabolites such as methotrexate, 6-mercaptopuri; 6-thioguanine, 5-fluorouranyl, cytosine arabinoside and and (3) antibiotic therapy such as actinomycin D1 and mithramycin; The drug may include administering one or more of the following drugs: cin, mitomycin C1, bleomycin, and daunomycin. Unfortunately, there are problems with the use of such compounds. There may be. The big problem is that such compounds generally kill tumor cells and non-tumor cells. It is cytotoxic to both tumor cells and therefore causes severe side effects in healthy tissues and organs in patients. It is possible that this may lead to adverse effects. Currently, there are few ways to selectively target such cytotoxic drugs to tumor cells. 6-diacetic 5-offso mononorleucine (DON), 0-diazo-acetylene glutamate such as serine (^+sse+ine) and α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (A+i marcil). Min analogs are included in the chemotherapeutic agents mentioned above, and they interfere with intracellular biosynthesis. death However, these compounds have clearly proven to be too toxic and are no longer commercially available for use in cancer treatment. The lack of basic biological knowledge about the behavior of malignant cells has slowed the development of drugs designed to take advantage of the similarities and significant differences between normal and malignant cells. One such difference is the amount of glutamine required for cellular metabolism. Group Lutamine is a major metabolite in cancer cells and is similar to glucose in providing energy. It is also an important source of acid groups in DNA biosynthesis. However, correct Normal cells contain glutamine synthetase and can synthesize the necessary glutamine, whereas in tumor cells, glutamine synthetase is essentially inactive (or in low amounts). Instead, glutamina, a hydrolytic enzyme that destroys glutamine, -ase activity is high. Glutamine in C1 tumor cells is therefore dependent on the surrounding blood supply and tissues. Glutamine is transported into tumor cells by glutamine transport proteins. Therefore, the tumor The ability of these glutamine transporters to move glutamine across the tumor cell plasma membrane is essential for tumor cell proliferation and growth. In this regard, glutamine transporters in tumor cells are much more active than those in normal cells. It has been shown that Researchers have found that by lowering the serum glutamine levels of cancer survivors, they can reduce tumor cell glutamine. Attempts have been made to take advantage of the tamin requirement. This has been done, for example, by the use of glutamine-restricted diets and/or the breakdown of blood glutamine by administration of glutaminase or asparakinase. Guru Deficiency of tamine could possibly weaken cancer cells, thereby making them more sensitive to conventional chemotherapy and radiotherapy. However, this therapy may ultimately destroy both tumor cells and normal cells indiscriminately. There is a potential. In summary, tumor cells take up greater amounts of glutamine from surrounding blood and tissues. Although it is known that increased uptake is required, no significant progress has been made in exploiting this increased uptake to design drugs or methods that selectively act on tumor cells. Similarly, this difference has not been exploited in the field of cancer diagnosis, where reliable assays for diagnosing the presence or absence of cancer are highly desirable. SUMMARY OF THE INVENTION Using a kinetic analysis developed by the inventors, three groups present in tumor cells can be Discovered the lutamine transporter. Additionally, at least one of the transporters is associated with other solid tumors and They also discovered something common to lymphoma. Identifying and isolating tumor glutamine transporters Another human amino acid transporter, ara A nin transporter was also identified. Therefore, the identification, isolation, and characterization of tumor glutamine transporters, as well as other human and mammalian amino acid transporters, will improve glutamine uptake between tumor and non-tumor cells. It can be used to detect tumors by exploiting differences in the A wide range of diagnostic, therapeutic and other applications that can act selectively on tumor cells without A related composition and method was obtained. Therefore, a first embodiment of the present invention provides a method for treating glutamine and aranine found in tumor cells. Substantially purified human amino acid transporters, including amino acid transporters, are provided. Other embodiments of the invention include the ability of a compound to bind to the binding site of a tumor glutamine transporter. compound screening to determine the ability of compounds to inhibit glutamine transport into tumor cells. Provide cleaning methods. Another embodiment of the invention is to perform the binding and transport inhibition assays of the invention. Provide experimental equipment for Other embodiments provide diagnostic products and methods of using such diagnostic products for detecting tumor cells in the first or second order. Also provided are methods of monitoring cancer progression by periodically repeating the assay of the invention. Another embodiment of the invention is to produce the transporter protein of the invention by recombinant DNA methods. The present invention provides biological products useful for the production of monoclonal antibodies that specifically bind to transporters. Other embodiments provide anti-glutamine compounds and glutamine analogs and methods of using such compounds and analogs in cancer treatment. Other embodiments of the invention provide methods of treating cancer, including methods of inhibiting glutamine transport into tumor cells using anti-glutamine compounds, glutamine analogs or antibody compositions. Such methods can be used in conjunction with conventional anti-cancer therapies such as chemotherapy or radiation therapy. The present invention provides at least one antigenic human amino acid transporter or fragment or substrate thereof. We also offer vaccines consisting of buunits. Therefore, it is an object of the present invention to include tumor glutamine transporters useful in the diagnosis and treatment of cancer. An object of the present invention is to provide an amino acid transporter containing the amino acid transporter. Another object of the present invention is to provide a screening method that can identify compounds useful in cancer treatment. A further object is to provide diagnostic products and methods for diagnosing or monitoring cancer progression. Yet another objective is to produce and purify biological products for diagnosing and treating cancer. It is an object of the present invention to provide biological products for producing large amounts of transporters or antibodies that bind to such transporters. Another object of the invention is compounds and compositions for the treatment of cancer, as well as compounds and compositions for the treatment of cancer. Provided are methods of treating cancer comprising administering products and compositions. A further object of the present invention is to selectively transport cytotoxic compounds into tumor cells by inhibiting glutamine transport into tumor cells or using the transport machinery of tumor cells. The aim is to provide a cancer treatment method by delivering cancer. Glutamine uptake into tumor cells This includes methods of weakening tumor cells by inhibiting their uptake and then killing them with chemotherapy, radiation therapy, or any other therapy directed against the tumor cells. Yet another objective is to provide a vaccine that can be used to prevent cancer. Another object is to provide compounds and methods for expanding lymphocytes. Other objects and advantages of the invention will be set forth in part in the description that follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by the compositions and methods particularly pointed out in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a polyacrylamide gel showing the purification of glutamine transport protein, with lane 1 containing the molecular weight standard and lane 2 containing the eluted protein. Areas 3-8 contain gold film samples; Figure 2 is a graph showing glutamine uptake into ReL Hatake cells; Figure 3 is a graph showing glutamine uptake into H*L cells with NEM. FIG. 4 is a graph showing various NEM incubations on glutamine uptake into HeLt cells. FIG. 5 is a graph showing the effect of glutamination time on ReL1 cells. Figure 6 is a graph showing the effect of antiserum on glutamine transport to HcLx cells; Figures 7a-7j are graphs showing the effect of 1mM NEM on glutamine uptake into HcLx cells; Glutamine transport to the line Figure 8 shows the binding of 3H-NEM to the plasma membrane of HeL1 cells. FIG. 9 is a graph showing the binding of 3H-NEM to the substance obtained from beaks A-E of the H+Li cell plasma membrane defined in FIG. 8; FIG. Vis defined in Figure 8 Figure 11 is a graph showing the titration of glutamine transport by 3H-NEM binding to material obtained from beaks A-E; The principle of FIG. 12 is a graph showing protection of 3H-NEM binding by excess substrate; FIG. 13 is a graph showing the effect of various NEM inhibitors on glutamine uptake into ReLt cells FIG. 14 is a graph showing the effect of incubation time; FIG. 14 is a graph showing the differentiation of IIeLx cells into original cells by 3H-NEM; FIG. Autoradiolog of protoga membrane proteins lanes 1 and 2 show separated proteins labeled with 1mM 3H-NEM; lanes 3 and 4 show separated proteins labeled with 1mM 3H-NEM in the presence of 100mM glutamine; The figure is a graph showing the protection of C-NEM binding by excess substrate; Figure 17 is a graph showing the protection of C-NEM binding by excess substrate; Lanes 1 and 2 represent fraction 1, lanes 3 and 4 represent fraction 2, and lanes 5 and 4 represent fraction 2. Lanes 7 and 6 contain fraction 3; lanes 7 and 8 contain fraction 4; Figure 19 shows a polyacrylamide gel of HeLr cell membrane protein under reducing and non-reducing conditions; lane 1 shows a molecular weight standard. lane 2 contains membrane proteins under non-reducing conditions, lanes 3 and 4 contain membrane proteins under reducing conditions; FIG. 21 is a graph showing the effect of sodium on the binding of H-NEM; FIG. 21 is a graph showing the binding of 3H-NEM to the plasma outer membrane site (UPM); FIG. Figure 24 is a graph showing the binding of 3H-NEM to the inner membrane site (LPM); Figure 23 is a graph showing the effect of serum on the binding of 3H-NEM to the plasma membrane of HeLx cells; The figure shows the Line<t matrix H-Bsrk plot for the bonding of representative compounds. FIG. 25 shows a silver-stained 4%-30% 5DS-polyacrylamide gradient gel showing the location of glutamine transport proteins; FIG. Typical immunopros showing response to lane 1 contains molecular weight standards and lane 2 contains ReL* cells. lane 3 contains Del+o order 562 cells and lane 41 contains Moli3 cells. Lane 5 contains Molt-4 cells: FIG. 28 is a graph showing protection of NEM binding; FIG. 28 is a graph showing inhibition of alanine uptake into He Ls cells by NEM that correlates with H-NEM binding to peaks A-E; FIG. 30 is a graph showing the corrected value of the inhibition of alanine uptake into HeL* cells by NEM that correlates with the binding of H-NEM to peak D; FIG. FIG. 31 is a graph showing a comparison of concentration-dependent glutamine uptake curves in spheres; FIG. FIG. 32 is a graph showing the time course of tamine uptake; FIG. Figures 33-38 are graphs showing the initial rate of glutamine uptake: Figure 1139 is a graph showing the relative mitosis measured in MTT absorption plotted against analog concentration; Figure 40a is a plot of the reciprocal label uptake rate using two first order systems; Figure 40b is a plot of the reciprocal label uptake rate using three first order systems. In the plot of Figure 41 is a plot of the reciprocal rate of label uptake using three first-order systems. Figure 42 shows the reciprocal of the label uptake rate in the multiorder system followed by the first order system; Figure 43 is the plot for the -order system then the multiorder system; Figure 44 shows the plot for the two multiorder systems; Figure 15 shows the uptake rates for three systems, two of which are multiorder systems. Figure 46 is a plot of the rate of glutamine uptake in IeLgll follicles; Figure 47 is a plot of l/V+* versus concentration; Figure 48 is a plot of the final equation FIG. 49 is a plot showing glutamine uptake of HeL* cells under Na-deficient conditions; FIG. 50 is a plot of the reciprocal of label uptake rate versus concentration; FIG. Figure 59 consists of reaction schemes 1-9, each representing a synthesis useful in making the glutamine compounds and glutamine analogs of the present invention; Figure 60 shows multiple samples (n =6) Measure the total cellular proteins present in Figure 61 is a graph showing the growth of HeLs cells in 1mM histidine, the growth rate quantified by Figure 62 shows the alanine uptake into Lg cells (incubated in the presence or absence of 1mM NEM). FIG. 63 is a graph showing the titration of alanine uptake into HeL* cells at various NEM concentrations; FIG. 64 is a graph showing the titration of alanine uptake into HeL* cells at various NEM concentrations; FIG. 65 is a graph showing the effect of 1 m MNEM on alanine uptake into HeL* cells incubated in the presence or absence of NEM; FIG. FIG. 66 is a flowchart showing the method for preparing the site (LPM); FIG. Figure 67 is a schematic illustration of a 6-well assay plate placed upside down on a save cover; 68 is a schematic diagram of the assembly; FIG. 68 is an inverted six-well cup of the present invention; Figure 69 shows the 6 well bottom plate inverted and the inverted cover plate containing the solution. Figure 70 shows the 6-well plate and cover plate assembled for solution transfer; Figure 71 shows the 96-well plate of the invention; Figure 72 Figure 73 shows a 96-well cover plate showing tubes containing solutions; Figure 73 shows a 96-well plate inverted and an inverted cover plate containing solutions; Figure 74 shows the 96-well plate and cover plate assembled for solution transfer; Figures 75-86 show Tables 4-15 of the present invention, respectively; show. Description of Preferred Embodiments 1. Definitions and Abbreviations Definitions and abbreviations for some basic terms used herein are shown below. Anti-glutamine compounds are those that act on cells via the glutamine transporter or by any other route. and/or glutamine uptake and/or metabolism, biosynthesis, e.g. It refers to all compounds that act on intracellular degradation (e.g. by enzymes) and/or interfere with the binding of glutamine to transporters of any other substances that bind to glutamine. Anti-receptor refers to a substance that selectively binds to the receptor of a ligand-receptor pair. For example, when the ligand is an antigen and the receptor is an antibody, such as a mouse IgG antibody, the anti-receptor can be an antibody to IgG. An anti-receptor can also be a substance that selectively binds to the portion conjugated to the receptor. For example, this part The anti-receptor can be an antibody against Habten. Also includes fragments and materials derived from them. Glutamine analogs refer to all compounds with structural similarities to glutamine. Taste. Human amino acid transporter refers to a molecule that is present in cell membranes derived from human tissues and transports amino acids into and out of cells. Ligand means the target analyte to be assayed. This term includes antibodies or anti-antibodies. It includes all the members of an immunological binding pair such as the original. Also, the ligand may be e.g. amino acids or their transporters, enzymes or nucleic acid sequences such as RNA or DNA probes. It can be bu. Receptor refers to a molecule that binds directly to a target ligand. For example, if the ligand is an antigen, the receptor may be an antibody, preferably a monoclonal antibody. If the target ligand is an antibody, the receptor can be anti-antibody or anti-antibody. If the ligand is an enzyme, the receptor can be an enzyme substrate. If the ligand is a nucleic acid sequence, the receptor can be a complementary sequence. Also includes fragments and substances derived from them. be caught. Tumor glutamine transporter (TGT) refers to a molecule located within the tumor cell membrane that is capable of transporting the amino acid glutamine from the extracorporeal medium across the membrane to the cell chamber of the cell, and vice versa. Ac-acetyl AC3-aqueous measurement scintillant ATV-antibiotic tribucinbersen Bm-maximum binding constant Bn-benzyl BSA-bovine serum albumin DCC-dicyclohexylcarbodiimide DCHA-dicyclohexylamine DCM-dichloromethane DMF-dimethylformamide DMSO-dimethylsulfoxide dpm - Degree of disintegration EDTA - Ethylenediaminetetraacetic acid EL I SA - Enzyme-linked immunosorbent assay Et - Ethyl FBS - Fetal bovine serum h - 11i11 coefficient HB S S - Hanks' buffered saline solution HE P E S -N -2- -Hido Roxyethylpiperazine-N' -2-ethanesulfonic acid IgG - Immunoglobulin G Km - Michaelis-Menten's constant Ks' - Apparent dissociation constant LPM - Subplasma plasma membrane M2O3 - Medium 199 Me - Methyl Mr - Relative molecular weight N E M -N-Ethylmaleimide PBS A-Phosphate buffered saline without calcium or magnesium PEG-Polyeriene glycol PMSF-Phenylmethylsulfonyl fluoride rpm-Revolutions per minute RPMI 1640-LsewellPr+klleso+1sllnN days++ 1@for cell culture Medium 5DS-sodium dodecyl sulfate 5DS-PAGE-3DS polyacrylamide gel electrophoresis - Tris-buffered saline containing tertiary T BST -Tveen T E N -Th1s -EDTA-N*CITFA-hLifluoroacetic acid THF-tetrahydrofuran Tosyl-para-toluenesulfonyl Tris-Tr rho(hydroxymethylene) ) Aminoethane UPM - Upper plasma membrane v/v - Volume/Volume Vm - Maximum velocity w/v - Weight/Volume 2-Carbobenzyloxy +1. Identification of Amino Acid Transport Proteins A first embodiment of the invention consists of human amino acid transport proteins. Of particular interest are transporters that are present in tumor cells but absent, inactive, or in small amounts in non-tumor cells. Three such imports The receptors TGT1, TGT I+ and TGT Il+ have been discovered, which are present in tumor cells but appear to be absent or inactive in non-tumor cells. Alternatively, TGT 1-111 may be present in normal cells, but only in small amounts. The methods developed to isolate TGT I, TGT I+ and TGT II+ are examples of methods by which other transporters, particularly those specific to tumor cells, may be identified and isolated. This method is based on a series of procedures used to isolate alanine carriers from hepatocytes (Hx7e+ et al., Bioches, 1., 214. (1983) 489-495), but with some important differences. There is a need. The glutamine transporter of the invention was isolated from HeLx cells derived from an established human tumor cell line. This cell line was originally obtained in 1951) from a headache biopsy of Ira+ir++gLsck+. A. HsL1 Cell Protein Isolation Procedure The initial preparation procedure for monolayer culture of HsLs cells and other adherent cell cell lines and the preparation procedure for microparticle carrier cultured cells were as follows. 1. Monolayer culture of HtLs cells and other adherent cell lines on the surface of plastic flasks or glass bottles (Flow Lxbo+tlo+ie+). Cells were allowed to grow and when confluent, they were harvested using the antibiotic trypsin-versene (ATV). The medium of actively growing cultures was replaced every 2 days with fresh medium (M2O3 or RPMT1640) containing 5% (V/V) fetal bovine serum (FBS). 2. Microparticle carrier culture Ph1+ms

By applying the method described by [Me (1981)], glass beads (Si Adherent cells were grown on the gIChtmicxl Comp*al) surface. Bee (0.5 g) was rinsed 3 times with fresh medium containing FBSIO% (v/v), then Harvested by ATV from monolayer culture at The cells were mixed with approximately 106 cells diluted in 1:1. Next, the inoculum was transferred to a microparticle carrier container (5 00ml volume, Thchne, C1mb+idgt, UK) and incubate at 37°C. Place on a magnetic stirrer for 2 minutes at 25 rpm every 30 minutes for the first 24 hours. The culture was then continuously agitated at 35 rpm for the remaining incubation time. with cells After settling the attached beads, decant the old medium and add FB containing 85% (V/V). The medium was changed every 2 days by replacing with fresh medium. After 5 days the beads will be collected. It became dense and was used for plasma membrane preparation. Next, the glutamine transporter of HsLs cells was identified and purified as follows. 3. Preparation of plasma membrane from HsL1 cells until growth on beads becomes sufficiently confluent. Cells were grown as microparticle carrier cultures. Next, examine the plasma membrane as described below. Made: (i) Beads with attached cells were pelleted and the supernatant was discarded. Wash the beads three times with fresh medium, then incubate in hypotonic buffer (10 mM) for 10 min at 4 °C. Cells were swollen and lysed by resuspending them in 20 ml of Lys-HCl, pH 7.4). Ta. (11) Vortex the cell suspension vigorously for 8 seconds, then use MSE ultrasound at the highest setting. Ultrasonication was performed for 8 seconds using a wave decomposer microchip. (ii) Wash the cell suspension three times with hypotonic buffer containing 1mM EDTA. The cells were resuspended in 10 ml of hypotonic buffer and sonicated for 30 seconds. (iv) Collect the released H by differential sedimentation, DImoa/IECB-20A type The pellet was centrifuged at 10,000 g for 15 minutes using a centrifuge to form a pellet. (V) Beret 0. 1 ml of hypotonic Babufa, pH 7.4, containing 25M sucrose and stored at -20'C. Enzyme assay on freshly prepared membrane fractions Sei and binding experiments were performed. Gollib et al., Btochet e(1lio bys, ^ (see 11, 601 (1980) 201-212. 4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis a. Selective extraction of membrane proteins Detergent T+NoII! A method based on the unique properties of -114 (Thoaps++Il et al., Biocbemist B, 26. (19871743-750) , membrane proteins were selectively extracted into four groups. Monolayer cultures were grown in phosphate buffered saline (PBSA). ) Rinse 3 times with 10ml, 1mM EDTASo, 15mM NaCl and 10m M4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEP ES), pH 7, 42ml to complete the first group of membrane proteins. Extracted from the surface of whole Hear cells (Fraction 1). using rubber policeman Scrape the cells from the flask surface and incubate for 5 minutes in a conical polypropylene centrifuge tube. Cells were collected by centrifugation at 500g. Cell pellets were added to 2% (V/V) D+1 in 10mM HEPES 1 pH 7.4. fon Insoluble material was removed. The upper layer, which does not contain detergent, contains hydrophobic proteins (fraction 2). , the lower layer contains hydrophilic proteins (fraction 3). Solubilizing insoluble pellets with SDS Dissolved in buffer (fraction 4). acetate to a final concentration of 90% (V/V). The proteins in fractions 1-3 were precipitated by adding Epp! ndo+I ultracentrifuge (5 414 type) for 10 minutes to form a pellet at 11,600 g. acetone precipitate Dry under vacuum and then dissolve in 100 µI of SDS solubilization buffer. Separated by PAGE. b. Protein separation Vortex vigorously three times for 1 minute each, then store at -20°C and transfer to SDS solubilization buffer. 7 (25mM l-Lis-HCl, 2% (w/v) SDS, 2% (w/v) fission : +-L, 1% (v/v) 2-mercaptoethanol, 2mM EDTA, 5m M PMSF. The membrane preparation (1 mg of protein) was dissolved in 1 ml of pH 6,8). Bio-Rsd Mini-Protein II device (Lxesmli, tiers, 22 12% polyacrylamide using 7° (see 197016110-685) Proteins were separated on a gel. Solubilized membrane (1 mg/ml protein) was added to sample buffer y. (62.5mM Tris-HCl pH 6.8.2% (w/v) S DS, 2% (W/v) Ficoll, 5% (V/V) mercaptoethanol, 0. 00125 % bromophenol blue, 25mM EDTA) at a ratio of 1:2, Heated to 0°C for 10 minutes. A 30μm sample was coated with gel 0. Place in a 75mm well, A constant current of 0 mAmp was applied for 1.5 hours. Stain the gel using one of the methods below. (Osgwi et al., Ansl. Bioehet, H5, (see 19831409-415). (i) Coomassie blue staining Remove the gel from the electrophoresis apparatus and add 70% (w/v) polyethylene glyco One device was placed in PEG 2000 solution. Next, cool the opaque gel. Stain for 15 minutes in Macable Blue-1% (W/V’) solution, then 10% acetic acid. It was bleached by changing the color several times. (11) Silver staining The gel was allowed to shrink overnight in 70% (w/v) polyethylene glycol 2000 and then Silver nitrate solution (20% (w/v) AgNO3, 35% (v/v) ammonia A. Transfer to 4% (W/V) NaOH, used within 5 minutes after making II) and relax for 20 minutes. It was shaken. Rinse the gel 3 times for 1 minute with distilled water and add coloring solution (0,002% (V/ V) Formaldehyde, 0. 005% (W/'V) citric acid), and the band I left it until I could see it. Color development can be stopped with 10% (V/V) acetic acid, and the gel It could be stored in PEG solution. Measurement of C1 radioactivity transparent placed on a transilluminator (visible wavelengths) so that the bands are clearly visible The stained gel was placed on a plastic cut surface. Proteins stained using a scalpel The samples were excised from the gel and placed in 4 ml scintillary silane vials. of gel Sections were incubated overnight at room temperature with 100 μl H2O2 and 100 μm 1. 8mM CuS Dissolved in O4. Next, add 4 ml of aqueous measurement scintillant (AC3) and The radioactivity of Al was measured. (Dong+o, i, Bi’oeh+s, Biopby@, Methods , 15. (See 19881331-336). d. Autoradiography AsplN7 radioactive polyacrylamide gel containing degraded membrane proteins (Ass+5hss) for 30 min at room temperature, then Phwtm It was dried at 60° C. for 2 hours using a sc eye gel dryer (GSD-4 type). Plasti I placed a piece of glue between the gel and the dryer lid to prevent it from sticking. Next, dry The dried gel was contacted with Faii X-ray film at -65°C for 6 days. exposure period When the gel is finished, bring it to room temperature and incubate for 2 minutes with gentle stirring every 15 seconds. Place in Dsveloper solution and wash with tap water for 2 minutes until the film becomes clear. Developed by placing in [od*k] Fixet solution until [od*k]. e, reducing and non-reducing electrophoresis Plasma membrane preparations were transferred to buffers containing or without mercaptoethanol 5% (V/v). It was dissolved in a. (Afloae et al., Mol. Ismwaol,,20. (See +19831383-395). solubilized The membrane was mixed with sample buffer with or without mercaptoethanol and then Proteins were separated by 5DS-PAGE and stained in the same manner as above. f, glycoprotein staining Membrane proteins were separated by 5DS-PAGE and stained for hydrocarbons as above. . (28ebsriws et al., Aail, Biochem, 30°+1969 1118-152). 7. Gel at 4°C for 1 hour. Identification with 5% (V/V) acetic acid , then at room temperature with 0.0% periodic acid. 2% (W/V) solution for 45 minutes. unintentionally Immediately soak the gel in Schiff's reagent (Fnch++n O, 5g% Na in 500ml of water). 2SO39,Og・Next add 110ml of IC, keep the solution protected from light. ) for 45 minutes at 4°C. 10% (V/V) acetic acid was exchanged for 2-3 hours. The color of the gel was removed overnight. 5. Electric elution Electroelution was performed with a BioRld Elecl+o+lot<+ type 422. A membrane protein polyacrylamide gel was prepared at 0. 1% (W/V) Kuman-blue and light The stain was washed several times with 10% (W/V) acetic acid to remove the stain. Guru Cut out the stained band corresponding to the tamine transporter protein with a scalpel and connect it to the dialysis membrane. I put it in a glass hundred. Fill the tank with volatile elution buffer (0.05 MN H, 4 HCO3, ]11% W/'V) SDS) and 6sAvp/'it for 24 hours. A constant current was applied. The eluted protein was collected from the dialysis membrane surface, and O, LM, PM Stored at −20° C. in elution buffer containing 50 μm SF. Eluted protein The quality sample is a Cen1+1eoa micro outlet with a molecular weight cutoff of 10,000. It was concentrated using a rotor. Microconcentrator with 45° fixed angle rotor (1εC/Ds+on B-20A type centrifuge), centrifuge at 5000g for 30 minutes The final volume was 50-100 μl. Purification of the eluted protein band of Mr42000 A silver-stained polyacrylamide gel is shown in FIG. Dialyze the protein solution against PBSA using a microconcentrator and remove the salts. and SDS can be removed. The purity of the eluted protein is 200μ of the concentrated protein. Equilibrate with 1 SDS sample buffer, perform 5DS-PAGE, and silver stain. It was measured by The amount of protein present in each sample was determined by adding 200 μl of protein solution. Using the MieroLovrY method to read the absorbance at 660 nm, measured (Mrrkwell et al., Anzl. Biochet, 87. (19781206). 6. 3H-NEM labeling experiment a1 Labeling with various concentrations of NEM in 10 ml HBss, each containing 0.1 ml of 3H-NE M stock solution. 0. A 25-10 mM NEM solution was prepared. Plasma membrane preparation (protein 0. 2mg/ml) with a 200μm NEM solution at 37°C. Both were incubated for 1 hour. Next, wash and mark in ice-cold 0.9% NaCl. I stopped my consciousness. Pellet the membrane at 11,600 g and then add 200 μm of solubilization buffer as described above. and stored at -20°C. b. Protection of binding by excess substrate Plasma membrane preparation (protein 0. 2mg/m+) of LOOmM glucoin in HBSS. -incubate with D or L isomer and then incubate with 100mM Labeling was carried out for an additional 20 minutes with 1mM 3H-NEM containing 30% chloride. Ice-cold 0.9% Na Labeling was stopped by washing with Cl, and then the membrane was dissolved in solubilization buffer. C, Differential labeling of glutamine binding site Plasma membrane preparation (protein 0. 2mg/ml) to 0. Various concentrations of 5-40mM Incubated with 100 μM glutamine for 5 minutes at 37°C. test gluta Add NEM (1004+) to 2mM in min concentration to make NEM concentration 1mM. , the membrane suspension was incubated for an additional 10 min at 37°C. This incubation After completion of the test, the membrane was centrifuged at 11,600 g rpm for 1 minute, and the pellet was transferred to a Hank's tube. Washed with balanced salt solution (HBSS). Next, the plasma membrane was treated with 1mM 3H-NEM. Both were incubated at 37°C for 1 hour. Wash with ice-cold 0.9% NaCl and label. I stopped understanding. Membranes were dissolved in solubilization buffer and stored at -20°C. d, Labeling of upper and lower plasma membranes. The flowchart in Figure 65 shows the upper plasma membrane (UPM) and lower plasma membrane (LPM). ) The preparation method of fractions and the labeling method with 3H-NEM are shown. This method is ReL Can be used for any adherent cells other than g cells, and can be used on the inner and outer surfaces of the same cell membrane. It can be used in any type of assay that requires 1mM N in HBSS, pH 7.4 100 μI of 3H-N EM stock solution (0.5 mci/ml) in 10 ml of EM was added to prepare a radioactive solution. e, Na+ dependence Aliquot of plasma membrane of HeLr cells containing 100 μg/ml of protein was used in the experiment. All solutions were prepared in sodium-free HBSS; 1 mM NEM solution in HBSS 1 lH-NEM stock solution (0,5 m Radioactive NEM was prepared by adding 100 ul of Ci/ml). 10 The membrane was incubated for 5 minutes at 37°C in the presence of 0mM NaC+ or 100mM choline chloride. Brain-incubated with mM glutamine. (Appropriate concentration of glutamine and to the membrane incubating the NEM solution (containing sodium or choline). In addition, NEM was added to a final concentration of 1 mM and further incubated at 37°C for 10 minutes. . Next, pellet the membrane in an Epp+ndo+f ultracentrifuge at 11,600g for 5 minutes. and then 1,mM H- (containing 100mM sodium or choline) Resuspended in NEM for 1 hour at 37°C. After the labeling period, the membrane was washed with HBSS and It was then dissolved in 100 μl of SDS solubilization buffer. The dissolved membrane sample was added to 12% polyester. A constant current of 50 mAMP was applied for 1 hour on an acrylamide gel. separate The proteins were stained with Coomassie blue, the gel was cut out, and the radioactivity was measured. Ta. [The effect of serum on glutamine transporters] Before isolating both membrane components, glutamine transport IIeLR cells were treated in various ways to translocate carriers into the membrane. Membrane preparation 20’fi (V/’V) FBS-containing or FBS-free Incubate H+Ls cells in fresh llIediomu 199 containing Ta. The membrane was isolated and then labeled with 3H-NEM in a conventional manner. All solutions were in HBSS. 3H-NEM (0.5mC1/ml) in 1mM NEM in HBSS. Radioactive NEM was prepared by adding 100μ+ of stock solution of ). The membrane was diluted with 10mM glucose. Incubate in Tamine for 5 min at 37°C, then incubate in NEM for an additional 10 min. In addition, the final concentration was LmM, and the mixture was further incubated for 10 minutes. The membrane was pelleted in an Eppendorl ultracentrifuge at 11,600 g for 5 min. Resuspended in 3H-NEM for 1 hour at 37°C. Membrane 0. At 99fiNaC1 Washed and dissolved in SDS solubilization buffer for 5DS-PAGE analysis. minutes The released protein was excised from the gel and its radioactivity was measured. B. Identification and properties of isolated glutamine transporter 1. Identification of carrier protein a, 3H-NEM labeling experiment HeLs cells were incubated with 3H-NEM, dissolved in SDS detergent, Proteins were separated on polyacrylamide gel. As shown in Figure 8, many proteins were found to bind to 3H-NEM, and these were labeled A-E. child The binding pattern was found consistently in many membrane preparations. Table 1 below shows peak A- The approximate molecular weight of E and the binding of each peak to 3H-NEM are shown; The specific activity for H-NEM solution is 2. Total as 22x105dpm/nmol It is calculated. Molecular weights were determined by comparison to standard markers and the values were determined at least three times. The average of the measurements is ±S,E. A 95000 0. 416±0. 05B 53000 0. 259±0. 04 C4200G 0. 155±f1. 03D 31000 0. 469±0. 04 E 22000 0. 397±0. 05Specific protein has glutamine NEM-sensitivity To establish that it is involved in passive transport, it is necessary to It was necessary to show that titration of binding is consistent with inhibition of transport by NEM. P The NEM binding peaks for each of A-E are shown in Figure 9. Peak C is about 2 It becomes saturated at m　M　N　E　M, and the binding of 3H-NEM reaches its maximum at 1mM NEM. This indicates that at this level of NEM, glutamine This is consistent with a 50% inhibition of transport (see Figure 5). Combines with peak B At 1mM, the amount of 3H-NEM is slightly larger than that bound to peak C (but on the other hand, , a larger amount of 3H-NEM was bound to peaks A, D, and E. various NE NEM binding to peaks A-E at the same concentration for glutamine transport at M concentration. The plot of is shown in FIG. At peak C, a straight line plot is obtained with a one-to-one correspondence. , indicating that transport inhibition is consistent with binding of the inhibitor to this protein. Binding to peak B-E does not show a one-to-one correspondence with inhibition of glutamine transport by NEM. stomach. A similar plot for only peak C is shown in Figure 11, but for 3H-NEM The rate of glutamine uptake when binding is 100% saturated is the total glutamine uptake. is considered to be equal to the unsaturated component of NEM, and the difference from the uptake rate measured at each NEM concentration I pulled it. Adjustment of saturation rate of carrier protein with 3H-NEM and glutamine uptake Draw the line of the theoretical value that matches the integral ratio. What are measured values and theoretical values? This suggests that peak C is involved in glutamine transport to HeL* cells. is supported. Transport substrates have been shown to protect transport proteins from NEM binding. These results clearly indicate the involvement of peak C in the NEM-sensitive transport of glutamine. It is. Glutamine in significant excess before incubating with unlabeled NEM Membranes were incubated with D-glutamine or D-glutamine. excess glutamine and NEM was removed and proteins were separated by gel electrophoresis. Figure 12 shows the L-glue D-glutamine clearly protects peak C, whereas D-glutamine This indicates that there is no protection from M-bonds. Peak B is also substantially derived from 3H-NEM. This protein may also be involved in glutamine transport. suggested. b. Discrimination between carriers with high and low affinity 1 (for glutamine transport to eLt cells) In order to identify the role of peaks B and C in A novel method was used to identify. Time to brain-incubate with NEM The effect of the differences on glucose transport is shown in Figure 13. Pre-incubation run time At 6 minutes, low-affinity transport is somewhat inhibited, but high-affinity transport is inhibited. It doesn't seem to work at all. However, the pre-incubation time was 12 minutes. Both systems appeared to be inhibited. New experiments are based on these observations. The method involved using unlabeled NEM for 6 min, which It will bond to all sulfhydryl groups. However, protecting the glutamine binding site To do this, a higher concentration of glutamine was also added. Wash away glutamine and excess NEM. The solution was washed away to reveal the glutamine binding sites. Next, apply 3H-NEM for 12 minutes. In addition, the glutamine binding site was specifically labeled. used to protect the binding site As the concentration of glutamine increases, 3 binds to the glutamine binding site. It was expected that the amount of H-NEM would also increase proportionately. At 1mM glutamine High affinity binding sites could be protected due to high affinity transport. Since low-affinity glutamine transport occurs at 1mM or higher, the low-affinity binding site position may require a higher concentration of protected glutamine groups before labeling with 3H-NEM. Dew. The results of this type of identification mark experiment are shown in Figure 14, and Mr42000 The peak C of The figure shows the behavior expected for a tropic transport protein chamber. On the other hand, peak B is gluta It is not labeled with 3H-NEM until the concentration of Mr53000 is 5mM or more. This protein has been implicated in low-affinity transport of glutamine. Polya The positions of peak B and peak C in the acrylamide gel are shown in FIG. C. Autoradiography HeLg cell plasma membranes were transported with 14C-NE in the presence or absence of excess transport substrate. It was incubated with M. Membrane proteins were analyzed by 5DS-gel electrophoresis and then The gel was then exposed to X-ray film. The autoradiography in Figure 15 was performed in the absence of the protective substrate glutamine (lane 1). And in 2), the band of Mr42000 is NEM-? 'Radioactively labeled, but ink The bands are protected from radioactive labeling when glutamine is present in the fermentation medium. (lanes 4 and 5). These results suggest that the mechanism of action of NEM is glutamate on the transport protein. This is due to direct competition with the glutamine binding site, and specific proteins involved in glutamine transport. This shows that it is possible to identify white matter. Marked with 14C-N E M The gel of the identified membrane protein pair was also cut out and the radioactivity was measured. The results obtained are This is shown in Figure 16, which is shown in Figure 12 for tritium-labeled membrane proteins. I support the results. Many peaks were labeled with 14C-NEM, but Mr420 Only peak C at 0 was significantly preserved from C-NEM labeling in the presence of excess glutamine. protected. The energy of 14C is much higher and therefore it has a very high specific activity value. Therefore, the intensity of the radioactive signal is much stronger than that of 3H-NEM. Ta. 2. Fractionation of membrane proteins Membrane proteins were selectively extracted into four fractions depending on their hydrophobicity and location within the cell membrane. . The stained gel in Figure 17 has fraction 1 containing surface or superficial membrane proteins. This shows that there is almost no overlap in proteins between the groups. Fraction 2 is a hydrophilic membrane Fraction 3 contains hydrophobic membrane proteins. The last fraction 4 is an ionic detergent It contained integral membrane proteins that were only lysed in the presence of SDS. stained gel The high-affinity glutamine transport protein of Mr42000 appears mainly in fraction 4. This indicates that the protein fraction overlaps with the small amount of hydrophobic protein fraction in cancer. Mr53000 The low-affinity transport protein mainly appeared in fraction 3, indicating that it is a hydrophobic protein. be done. 3. Characterization of glutamine transporter by 5DS-PAGE Separated by electrophoresis L* cell membrane proteins were stained for hydrocarbons using the Schiff method. Figure 18 Many proteins in the gel, including the glutamine transport protein of M24200, are glycosylated. It shows that Detects the presence of intermolecular and intramolecular disulfide bonds Membrane proteins were separated using SDS under reducing and non-reducing conditions. 1st The gel in Figure 9 shows that the Mr42000 glutamine transport protein is a non-reduced sample (lane 2). ), but it appears in the reduced sample (lane 4), which indicates that This suggests that this protein is a subunit of a protein complex. middle In the lane (lane 3), the side closest to the reduced sample was affected by the reducing agent diffusion. Therefore, the band was partially visualized. 4. Na dependence of NEM binding Establishing whether binding of glutamine transport proteins to NEM is sodium dependent For HeLx cell plasma membrane in the presence or absence of sodium ions, ``We investigated the binding of H-NEM. The graph in Figure 20 shows Mr42, in which the glutamine binding site was protected by excess substrate. The specific binding of NEM to the high-affinity glutamine transport protein of 000 It is shown that there was a 30% decrease in the absence of ON. In the absence of sodium ions The fact that NEM binding was inhibited in the active site of the glutamine transport protein or suggests that it depends on the presence of sodium ions in the vicinity. Mr53 The binding of NEM to the low affinity glutamine transport protein of 000 It was not affected by its presence or absence. These results indicate that the Km of the Na-dependent transporter TI is N 0 if “a” does not exist. 17mM to 1. increased to 5mM, while transporter III Consistent with the kinetic findings of no effect. 5° labeling of the inner and outer surfaces of the plasma membrane (see 11. above). A.6. ) protoplasm We developed a method to selectively label glutamine transport sites on the inner and outer membrane surfaces. . The binding of 3H-NEM to the outer surface of the plasma membrane is shown in Figure 21; After protecting the binding site with glutamine, the Mr42000 and 53000 peaks were This shows that it is specifically labeled with an isotope. As expected, these peaks The binding to glutamine was proportional to the glutamine concentration used to protect the binding site. The inner surface was also specifically labeled with 3) (-NEM, with its binding pattern shown in Figure 22). The curves are very different from those obtained for the outer surface. Mr42000 and 5300 Although the binding intensity for the 0 peak is still comparable to that of the outer plasma membrane surface, the binding There was no clear correlation between the amount of glutamine and the concentration of glutamine used for protection. The most notable difference is that in the previous experiment it was not shown to bind to 3H-NEM. However, when only the inner surface of the membrane was labeled, the binding and glutamine concentration were good. Another band (designated as peak F) that clearly shows a strong correlation appears. Met. In addition, peak E, which previously showed an inverse correlation with glutamine concentration, is now 3 A positive correlation was shown between H-NEM and glutamine concentration. It is unclear why the binding patterns are so different between the inner and outer surfaces of the plasma membrane. However, the regulation of glutamine transport by sites containing reactive sulfhydryl groups It may be involved in regulation. 6. Translocation of glutamine transporter FBS interacts with growth factors and cells in culture Contains hormones that enhance the active growth of. HeL* cells were prepared before plasma membrane preparation. The cells were incubated in the presence or absence of FBS and transport proteins were incubated with excess substrate. After protecting the glutamine serum site, it was labeled with 3H-NEM. Figure 23 Rough is the binding activity of HtL* cell membrane protein prepared from normal cells and serum-deficient cells. It shows. In the absence of serum, NEM binding to the high-affinity glutamine transporter is 50 %Diminished. Many other proteins normally bind significantly to NEM, i.e. peaks ASB, D and NEM binding was also reduced in the absence of serum. After incubation in serum-free medium, adding serum back to the medium does not The binding of 38-NEM to the rutamine transport protein increased by only 10%. Ta. 7. Dynamic analysis a, derivative of the dynamic equation At an early stage, the metabolic importance of glutamine uptake into tumor cells It was recognized that it was necessary to quantitatively study the transmission characteristics. Therefore, H*L The kinetics of glutamine transport in T cells was studied in detail. 1Ilv as much as possible Optimal transstimulation to obtain parameters related to the state of O. All experiments were performed under normal conditions, and this method Applicable to tamin concentration range in! Data were obtained under il+o conditions. Sara In addition, because the glutamine uptake curve in HeL* cells was complex, the transport mechanism devising or developing new analytical procedures that reveal as much as possible the major components; became necessary. Unmodified Double Reciprocal Plot (Lineves Mar & Buck , 1. As, Chet Socw, 56. (1934165g-666) is frequently discussed as lacking reliability when used with non-metric data from enzyme experiments. Although it has been discussed (Dovd & Rigg, 1. Biol, Chew, 24 0゜(1965) 863-869. Woe, K+ael+cs of En tYme　Mschs+sms. 19751 p. 29. ＾c＊de＊ie　P+es+, LoIldon; Robe+I+, EnsueKineli+3 (197?ip, 287 ．． C15b+idge 1lniye+sself I Press), membrane transport dynamics It is still commonly used in the study of mechanics. Furthermore, the research conditions were If it seems that it does not follow a simple linear hyperbola expressed by the xek rho MenHn equation, Sometimes these plots appear to be even less reliable (BlxB7 et al. 1. Mat, Biol. 31, (19681N-35). There is another problem with intracellular transport. This means that there is rarely just one transporter that is not complex. Therefore, when multiple transporters exist, 1/' S variable (S = substrate concentration) is opposite to the relative distance between the plots, so as the concentration range becomes wider, Lin evesmaer-B+++ plots become increasingly difficult to manipulate. For this reason, the concentration At the highest concentration in the region, S constantly increases, and the 1/S value does not change much. A single plot with good separation between distinct points at the highest concentration of the concentration range. It becomes very difficult to obtain 1/1, which is also the point where the velocity value is the lowest. The plot points at the vertices of the S region are highlighted (RobC+11. 1977), accordingly The overall accuracy of the plot seems to be the lowest. This effect is mainly caused by 1. 5 and The main inflection between 5mM and fleLt is compressed to 1/400 in the 1/'S region. Seen in the double reciprocal plot for glutamine uptake in cells. HeLt For glutamine transport in cells, to include all components of the transport machinery, 0 ．． 005-It is necessary to plot the velocity against the glutamine concentration range of 5mM It turns out. Furthermore, 0. The size of the error range at 01mM is 32 times that at 1mM. Therefore, any meaningful plot becomes speculation. To overcome these difficulties, new A new plotting procedure was developed. From the Michselis-Lnlen equation, constant The following equation for the unidirectional flow of labels under label concentration conditions of is obtained: = Importing the sign from surface 1, 819 - constant label concentration at plane 1 = "labeling rate - substrate concentration coefficient". Reciprocating equation 1: becomes. Therefore, the gradient device /L8 is obtained from the plot of 1/V against S, and the standard Once the sensitization concentration is obtained, Vm can be calculated. (For convenience, the label concentration in these experiments is was the lowest substrate concentration used for a given series of experiments). The intercept on the S axis is -K equal to ugly The intercept at 1/'V' is equal to K/L. For multiple primary transport systems, para The relationship between the meters is as follows: 1/V axis intercept (S=O)= 1/Σ(L/K) (3) +1 (1) [In the formula, the lowest value of K = Kl (1), ■ Number the other transporters in order of increasing value of each K]. Σ for the group sum When used below, all n starts from i=1. Intercept at S- (1/v'-〇) -s=Σ(L/to,)/Σ(L/K12) (4) Final slope at S-■ =1/Σt, *(5) Intercept of the above slope on the S axis = Ne One Σ (K, L) / ΣL (6) ■ 1/v* Intercept of the above slope on axis = $2 Σ(K,L)/ΣL (7) ■ The following equation is obtained from Hill's equation and is applied to multidimensional systems that follow Hill's relationship. do' h = h / (K, ll + Sh) Mamoto L Zero (8) If you make it the reciprocal, llI car [In the formula, L = v, (S”/S)h (10) Therefore, the coefficient of Hll is large. If the field can be calculated, a linear relational expression can be obtained, and then V and K can be calculated. In multiple transport systems, l11 Equations 3 and 9 are particularly useful, and for multiple primary transport systems, Equation 3, multiple multiorder transport For transport systems, 9 may prove useful. Equation 2 is Hsn! + expression and relationship It is immediately obvious that It is multiplied by *. Lil about glutamine uptake in HsL* cells Contrasting the plot at 1eveIte+-fi+ with that obtained from Equation 2, we find that Equation 2 becomes Concentration when used: 0. The error range of 1/v1 at 01-1mM is 1. 6 times less On the other hand, in 1/V using Line 1 mare+-Bark plot, It was double that. Also, the distribution of points in the new plot is directly related to the substrate concentration used. The ability to appropriately separate important points over a wide range of substrate concentrations ( It was easy. In a multi-order relationship, the uptake rate can be expressed by Hill's equation: sign taking The formula for this lever can be modified as follows: If we reciprocate equation (14), we get becomes. Differentiating the above equation with respect to dS, the gradient of 1 is the minimum (d (1/v )/ds->O) l approaches (and d (1/v) /ds=0=(1-h) K h/L, 1/5h-1/I-” and (1-h)KIlh/5h=-1, resulting in K/S=(1/( h-1)) 1/h (17). Therefore, the minimum value (d (1/V)/d s=o) and K. 1/b The relationship between the two simply depends on the H111 coefficient and is expressed as (1/(h-t)). Note that this formula has some interesting and useful properties. First, h becomes 1 As we approach, the expression becomes infinite; secondly, when h is 2, the expression becomes 1; Thirdly, as h increases, the value decreases to about 0. It decreases to 7 and becomes 1 when it reaches infinity. return. When h is from 2 to 10, the formula is from 1 to 0. It is shown that it is between 7. When h is 2 or more, the ratio of concentration and K at the minimum plot point does not change significantly like this. This means that the approximate value of K can be obtained directly from the concentration in the minimum plot. Taste. This value can then be used in Table 2. Table 2 shows the speed versus Km at various concentrations. h is shown in relation to the ratio. Using a pair of velocity ratios at concentrations at the same distance from the Km value, Obtain two calculated values of h from the table. The values of h obtained from paired readings differ by more than 1 Must not be. If the difference between paired readings is significant, the h value will be low at high concentration. In this case, the K value should be adjusted and lowered, and in the opposite case, the should be raised. Continue adjusting Km until the h values are within 1 unit of each other, and Obtain a new calculated value KII(2). Next, the average of the two h values is set as the calculated value h2. Next The calculated value of h is shown in Table 3 (ratio of Kl/'S, S = minimum plot for various h). Roughly specify K by using the concentration at If the calculation of h can be carried out well, , then 1/hV” can be plotted against sh (Equation 9), which gives ■ and K can be further characterized. h is 21 , the above minimum value can be regarded as an inflection point between a pair of positive gradients in a multicomponent system. It turns out that it is possible to do this (see Figure 5, Appendix i). Finally, the calculation of the approximate value of V for the separate transporters in the multivariate system is the slope of the plot. It can be obtained directly from distribution. Therefore, a plot of 1/v against substrate concentration? The number of transporters present, the concentration range in which the transporters act, and the type of uptake can be determined by Which of the multi-order Mieh*eli+−IJente mouth (represented by Milli's formula This provides a first indication as to whether Next, we deal with the essential properties of the existing transporters, A starting point for more accurately measuring all the transporter parameters that appear in the structural hypothesis. This provides a "structural hypothesis" that can be used by Of course, the structural hypothesis also separates K accordingly. and the V value is very high It depends on not being significantly different. These requirements relate to detailed calculation steps. It is included in the set of rules in the Addendum ■. The above calculation is applied to glutamine in l1sL* cells. When applied to protein uptake, the parameters of three major transporters in HeLs cells are determined. Can be determined. K is the best The two transporters (transporters I and II) that are low are Na-dependent, and the Km is the highest. One transporter (transporter 111) is Na-independent. Addendum 1 includes transport vehicles and Analytical procedures used to demonstrate the Na ion dependence of It shows the changes in parameters due to losses. Transporter 2 contains the glutamine blood concentration range. It was shown that it is a transporter with a concentration-velocity curve. Therefore, this transporter It is of scientific importance and is therefore commonly referred to as the upper Na-dependent transporter. Structural Hypothesis: General Description of the Model Used Table 11 shows the parameters of the model selected to test the plotting procedure. ing. In this analysis, each K is one import It is thought to represent a transparent body. model 1 Figure 39 shows the plot for Model 1 (one primary transporter). axis intercept is as follows: , , * Intercept on 1/v axis=to/'L" Intercept on S axis = -K. Figure 39 shows the plot for Model 2 (two primary transporters). this pro is a non-linear combination of two linear relations separated by an inflection of J) The first linear relationship is the slope approaching 1/'L,'' and the second is The relational expression is 1,, ■1. etc. (the final slope is 5). Figure 40 is not a straight line (with respect to the final slope as S becomes infinite) and Ks (starting around 21, point 3006 below the extrapolated final slope (S-, -, =) has a downward inflection across 17·v, and is very clearly seen in most experiments. This shows that there is a difference. On the other hand, the Lin+wetve+-B+uk plot of model 2 deviates from the straight line. There are only a few cases of 1/2) and 0, and 1. 7’3 K, +21, the dispersive solution is only 996 extrapolated lines, less than 1/Km (2) In many experiments, it is sufficient to adjust the slope of this line only slightly to include may be difficult to distinguish from experimental error. Esdie plot of the same model Esdie. 1942+　Hof+lee, 1952), it is farther from the straight line than in Fig. 40. Then, vII(1) and "1(11/ 11(1)+v, (2)/, the value of (2) can be obtained. model 3 Figure 41 shows label uptake for multiple transport mechanisms consisting of three primary transporters. A plot of the reciprocal is shown. This curve consists of three straight lines connected by two inflections. It is the same as that obtained in FIG. 40 except that it is composed of a linear relational expression. first linear relationship When the gradient of (S is close to 0) is cut along the 1/v axis, the relationship shown in Equation 3 is obtained. this is It is usually an accurate intercept, and when calculating Kam/vm(1), Km(21/vm( 2) can be used to determine. First, the slope △( Analyze the curve by obtaining 1/v)ΔS. In multicomponent-order systems, one point The change in slope from one point to the next will reveal the part of the plot that is minimal. These nearly straight parts of the plot and each nearly straight part extend to the next K value. alternating with more widespread areas. The straight line section starts at or just above the continuous K value (see calculations). dark The lowest on the degree scale is (1). The initial gradient underestimates 1/■7 and overestimates (v, (1)). ), the intercept of the S axis when 1/V → 0 is -Km (overestimating II There is. By the last slope before the second minimum change in slope (see data for model 3) 1/(L, + prison Calculated values such as L2) are obtained. The final slope approaches 1/L. Therefore, each V moves along the plot from the lowest concentration. It is obtained by subtracting the calculated value before. model 4 Figure 42 shows that transporter 1 has a multidimensional relationship (all the multidimensional relationships considered here are of the Hill type). ), and the characteristic obtained when there are two transporters in which transporter 2 has a linear relationship is It shows the sex curve. This plot was previously described in the main text by applying equations 2 and 17. , it shows a negative slope that starts from concentration 0 and suddenly reaches the lowest value near I(1). vinegar. Songs following the model of Monod, WBsn and Ch＊nge■x ([65) For lines, the 1/v plot also shows a clear minimum, with an inflection point at 0. 5V(K ). Main points of the two types of plots The difference lies in the nature of the negative slope from zero. Considering the Moaod type relationship, To The dissociation constant of KT% is, and the dissociation constant of RO is, c = K / K SL = T/RO. n=4, C; R1G 0. 01 and L=10,000, and the Mofiod formula is used to calculate ■ When applied, 1. /V can be plotted. This plot The chart will show the following diagnostic indicators:1. Starting from 0 concentration, initially, the area of There is a negative slope that is the largest negative slope in the area. The slope can be seen as a simple Hill-type relationship. is much more gradual than the There are always steep slopes. Formula of HiN where h=4, K=〇, 15, ■ ±20 If the concentration is 0. The slope from 05 is -540, while the equation for 11oI lod In the plot, it was =38. 2. In the plot of Moaod's equation, the negative slope gradually bottoms out and becomes K(0,5 There is an inflection point near V), which is more than 100 ■ It turns out that all L's are correct. 3. As L decreases, the position of the inflection point on the concentration scale decreases, and the inflection widens and becomes a negative curve. The line changes only slightly. As L approaches 1 (h<1. If it is 5 , the type of plot obtained from the I(ill equation and the one obtained from the Monod equation is almost It won't change. model 5 Figure 43 shows the type of curve obtained for a primary transporter followed by a multiorder transporter. in this case , there is a positive slope followed by an abrupt inflection to the final slope. At the initial slope, 1/L,” A calculated value is obtained, and for the second gradient a calculated value of 1/'I-'' is obtained.1. 4mM The plot with the lowest value at (inflection point) gives an estimate of KI(2), and S−>O The intercept on the S axis of the extrapolated plot is -Km (calculated values of 11. These are the 4th A control plot of Figure 4 is shown. Line 1 shows the plot of model 6 L1 This is the curve The first part of the line repeats the essence of Model 4. i.e. negative song There is a line, with an abrupt minimum just above the K11(1) value. Then this curve It took a different course from Del 4, the slope rose rapidly, and the calculated value of 1/L was obtained. After that, it inflects toward the final slope, and the calculated value of 1/'L is obtained in the same way as above. Ru. The curve shown in line 2 is and exhibits a type of curve that is very difficult to quantify. The negative slope is again Shows a multidimensional equation. However, the minimum value is approached more slowly and therefore the inflection point is It's not clearly shown. This means that the value of h is 1. 5 or less or close to it or ■7 close to 1 suggests that there is a 1lonod type relationship, The slow rollover to the final gradient indicates that the next transport system is multiorder, and ■ is Indicates that the value is significantly large, or that the true KIll(2) is greater than or equal to 1. are doing. model 8 Figure 45 shows a 1/V plot for a mixed ternary system. Diagnostic analysis of the previous plot is sufficient to indicate the nature of the transport mechanism represented by this plot. Good to have. Three gradients representing three transporters are shown in the figure. Transporter 1 is a linear relationship , the plot shows the minimum slope between transporters 1 and 2, with transporter 2 in a multiorder relationship. It shows that there is. Finally, the rollover from transporter 2 to transporter 3 is It is shown that the sending body 3 also has a multidimensional relationship. rollover very slowly Therefore, the visible minimum calculation is very approximate. 1. Order of rules specifying parameter limits for multicomponent systems: Transporters are always numbered from 1 to n starting with the lowest Km. All plots was tested as a regression line on H-P15C. Prepare the following data before starting the analysis. Prepare: speed for concentration, 1/v and △(1/v)/ for concentration ΔS0 In the examples discussed below, the maximum number of transporters present cannot be more than 3. stomach. In general, intermediate transporters model all intermediate transport processes, regardless of number. provide. Total concentrations are in mM and total rates are in Bol/min/mg protein. Rules/ 1. (i) In general, the difference in consecutive K value openings is 4 times or more ■ (for multiorder systems this can be close to twice as large). (i) V values should not differ from each other by more than 5 times, −■ as a general rule (V / V) (K / K > 4 (19) mL ah m (t+1) @f the law of nature [In the formula, VIL = lower of the two vI values vJ = higher V value Kl (1411 = higher K value intestine KI (Il = lower K value] (i The cut points must be connected to a concentration that is at least one-fifth of Km (11, The minimum value of at least three points is smaller than K. II (1,) (iv) Plot points are at least 2K (highest K value) l (Il)l The minimum value of at least 3 points is greater than Ks (ml). (V) There should be a minimum of at least 4 plot points between each consecutive pair of K values. It is possible. 2. Rules for structural hypotheses (i) (a) The initial slope is positive: transporter i is linearly related. Final from S to 0 Calculate vI(11) from the slope, and calculate the intercept or slope by S transverse of this slope=”1(1). Calculate Km (11) from the first position of the least change in the arrangement. (b) The initial slope is negative: the transporter ■ is a multiorder system. Km from the concentration at which the initial slope is the lowest (11 initial calculations are obtained, and the -th The slope at the apex of the gradient provides 1/L,′ and v,(1). (11) In the case of a multi-component-order relationship, it is continuous from the position of the minimum change in slope (the straight line) Calculate the remaining transporter by determining the K value, and calculate the slope just below the line of least change in slope. Continuously calculate 1/ΣL from Obtain 1/L,'' from the final slope. (i　i　i) For multicomponent multidimensional relations, continuous Calculate the K value. Negative change layer of slope change rate indicates an inflection point at the position where the slope changes to a positive change. The cumulative 17'L'' slope is obtained from the slope of the vertex of each line of positive gain in the slope. Similar to i), 1/L,' is obtained from the final slope. 3. Rules for detailed parameter analysis in multicomponent-order system relationships (three components) Ne (i) Using Equation 3 (main text) and initial calculated values of Kl fil and Ll 1. TeK , calculate L (2, then net mf2 on i, zv” axis)/2 , zero Calculate the calculation cutoff for the 1/v2 value. Using the initial transporter l parameter, the V value Calculate the vI value by subtracting from bs Obtain the calculated net v[. In the calculation area, 1i (1) to 2Km (1) or? Score points from 0, OLK to 0, 02K, L3+. In this range, 1 ll(3) The effect of transporter T11 velocity would be negligible17, more than four times the continuous K value. This seems to be the difference above. Next, plot 1/v2* against S, and 1/v axis Calculate the intercept at from the regression line. Then calculate this value for the intercept from equation 3 above. Compare with the established one. The plotted intercept is much larger than that calculated by Equation 3. Therefore, it seems that the ratio between the result of plot j7 and the result of equation 3 is (R)>1. R is Repeat the procedure in steps of approximately 10% until constant, then continuously decrease . Then decrease Kmfil by 10% in a single step at this point. R is one again Repeatedly decrease Ll until it becomes constant. Repeat this procedure until R=l. return. This then suggests a way to obtain the parameters of both transporters II and II at the same time. provide The transporter ■1 parameter is further calculated from the final net 1/V' for the S regression line. It can be confirmed. (i) Now repeat the above steps using the net V, value. Insert the previous calculated value of ta into equation 3 to calculate the intercept of net 1/v3*. Here, transportation The parameters of the body If are the above transport bodies! Adjust as for the parameters of the transporter Obtain the final values for II and transporter II+. (ii) To confirm the results of the analysis, obtain the net vI value, from 0 to approximately 3K1 Plot 1/V against S in the range of 1 (1) to obtain the correlation coefficient. This is 0. Should be 98 or higher. 4. Rules for detailed analysis of multicomponent polydimensional relationships (i) If transporter I is a linear relationship, Transport calculated as part of the structural hypothesis by subtracting the calculated v1 from vob+ Calculate the net v2 value using the weight parameter. ”　5Ki (2) to 2KI Plot 1/V2' against S at n(2). The negative slope indicates that transporter II is Indicates that it is the next system. Next, the concentration when 1/v is the minimum is Km (2) (Equation 17). 0. 75K to K and Ks (2) to 1. 25, (2,, ma (2) m (2 ma or 0. 5K to K and Km(2) to 1. m (21m) at “Kif2) (21 Calculate K11(2) and h2 by calculating the V ratio, and use Table 12 to calculate h2. Obtain a fixed value. The values of h obtained from paired readings should not differ by more than one. pair reading When the difference between cuts is significant, if the h value decreases as the concentration increases, then K should be adjusted to lower it, and in the opposite case should be adjusted to raise it. h value is mutual Continue adjusting K until it is within 1 unit, then use the new calculated value of K11(2) get. Let the average of the two h values be the calculated value of h2. Next, Table 13 (using Equation 17) shows the first calculated value using the plot minimum value. indicates the necessary modifications. The two adjusted K values should match within about 20%. (Please note that the corrections in Table 13 are based on calculations of a single plot point. ). Plot 1/hV” against S11. Next, transporter I Use the I value to obtain the net vl value, and calculate the value for S in the range below KII (1). Plot v1'. From this plot, calculate the parameters of transporter I again. , then use this to recalculate the parameters of Transporter II, and for a set of transporters. The above process is repeated until a constant value is obtained. From Km (2) or less” 2Ks The final plot of net 1/hV 2'' against sh for S in the range of (3) is Provides calculated values for Transporter II parameters. (ii) When transporter I is in a multidimensional relationship, the procedure is applied to transporter II in (i) above. It's the same as what I mentioned above. Next hh h zero Plot 1/v against S using the value m(1) and then K Check s (1). Then use these parameters to obtain the net v2 value, If transporter II is a multidimensional relation, then the transporter in (i) above! As for ■ Continue with the steps. Next, use these parameters to obtain a revised estimate of the parameters of the transporter ■ , transporters until the parameters of one or the other transporter are constant! and II positive Repeat the taste value determination process. Calculation of transporter II parameters by plotting final 1/h V against Sh obtain. (i　i　i)Here, using the previously calculated parameters of transporters I and II, Calculate the net V parameter and set it to 0. 5, for S in the range from (3) to 2K Plot the net 1/v3 value. h3 and as above for other multiorder systems. , (3) is calculated and repeated until it becomes constant. Plot 1/hv3* against S Then, calculate K and vm (31. f31 (iv) Subtract the calculated V2 + v3 value from v, and get K. ob+ umbrella 1/V or 1/h for the concentration range where the contribution of high value transporters is minimal Net transporter 1 (Km One round of net calculation is completed by calculating the velocity (lowest transporter). (V) Measure at the middle of the minimum concentration, maximum concentration, K value, and K value open. Check vob+ against the calculated V value and make fine adjustments. Most of the observed velocities Only a small amount of adjustment is required to place the minute on the calculation curve. model 3 Transporter KV IQ, 03 10 2 0. 30 20 3 3. 0 25 Data: Concentration is mM, rate is n1 ole/min/11 mg protein. S(mMl 0. 005 G, OI 0. 02 0. 03 0. 04 G, 05 V1. 8G 3. 23 5. 41 7. 07 8. 4 0. 231/ma" 0 ．． 55 0. 62 0. 74 0. 85 G, 95 1. 05△ (1/ma) /ΔS -H, 911, 910, 9110, 310, 07'1 11. 75N , 53 18. 26 21. 36 23. 7 25. 51/Ma’　1. H1,4 g2. 19 2. 81 3. 38 3. 92△t+/v')/ΔS 9. 0 5 8. 06 7. 1 6, 19 5. 7 5. 41S (mMl 11. 75 1. 0 1. 5 2. 0 2. 5 3. 05V 2g, 9 31. 34 34. 8 37. 24 39. 1 40. H1/v' 5. 19 6J8 g, 62 10. 74 12. 79 14. 78△(1/y” l/△S 5. 08 4, 76 4. 41! 4. 24 4,09 4°O3 (mM) 3. 5 4. O S, 06. OV 41. 8 42, 8 44. 43 45. 6617ma1 16. 75 18. 68 22. 5 26. 27Δ(1/f”)/ΔS 3. 93 3. 87 3, 83 3. 78, A slope between 11 points, a constant velocity decrease occurs between K values. in these parts of the curve is underlined. 2, the curve becomes almost straight (the change in slope is minimal) near the value. these parts The minutes are just above the Km values and are shown in bold. 3. 3 = 0. 005mM. Structural hypothesis/ Kei-shi 2 (i)-a, 0. 005-0. 02Mm’ (7) Regression line 1 / v Intercept=0. 49, slope=12. 57°K, (1)=0. 04mM, Vl, 1) =15. 9゜Rule 2 (ii) K −−−−−−−−−−−−−−−−−−−−1I(I)<0゛04゛Kl(2 )=0°””m(2) Parameters from the structural hypothesis: Rule 3(i) Reciprocal of equation 3: zero zero To Ll 7, (1) + L2 / (2) = 17 intercept = 2. 040, 0810 ．． 04+x=2. 04, x=0. 04. 1/(initial intercept is too high). * Ne Test the (1) ratio to L/ using the net 1/V plot. L /K (111/! Intercept R0,030,04G, 04G, 075 -Ml　m　□ 0. 0710. 04 3. 45 5. 57 1, 61 5. 6 5. 7 5. 7 45,730. 0610. 04 +, 85 2. 69 +, 45 3. 1+3 13 3. 50 3. 820. 0510. 04 1. 27 1. 70 1. 35 2. +5 2. 35 2. 52 2. 870. 04510. 04 1. 09 1. 46 1. 34　1,89　2,052゜212,550. 0510. 03 2 2. G9 2. 39 1. 14 2. 69 2. 812. 923. 161/ The v*transport intercepts match. 1, +=0. 05, v, (1)=2o and Km(ll=0. 03゜1　/　v　 The net 1/v 1 intercept on the 'axis is 2. 66, slope is 7. It is 93. K=0. 33, and V, (2,=25. 2゜Rule 3 (ii) Transporter +71j! ■For use in applying Equation 3 using the above parameters of Net ■Calculate the three values. Equation 3: L2''/, (2, +x=0. 376, therefore 0. 12610. 3 3+x=0. 376, X would be too thick. umbrella I am using a net 1/v3 plot and test (2) in / 1/ma3* 12”/Kl (211/l Intercept RO-30-40,50,755M0. 1 5510. 33 36. 34 45. 2 1. 2444. 5 44, 5 45. 4 45. 07G, IQ510. 33 17j 22. 96 1j3 26. 4 3 27. 65 29. 3 31. 80. 1010J3 13. 7 +8. 07 1. 32 21. 84 23. 24 24. 93 27. 720. 105/Q J15 23. 43 27. 04 1. 15 29. 61 30. 17 31. 48 33. 290. 1010j15 17. 08 20. 65 +, 21 2 3. 87 24. 91　26j8　28. 760. 09510. 315 N, 4 3 16. 53 1. 23 20. 0 2+, 2+ 22. 7 25. 330. 1010. 305 2G, 77 22. 75 1. 10 25, 5 26. 21 27. 5 29. 510. 09510. 305 15. 5 17. 86 1. 15 21. 06 22. 1 23. 4g 25. 90. 1010JO23,4 323,95+,02 26. 43 26. 92 211. 1 29. 91L 2’ = 0. 10-Jute V, +21 = 20 and (FK, (21= 0. 30mM. l/V3' plot: I/V3'' fJ piece = 23. 95. Gradient=7. 97K = 3. 00. V, (3,=25. 1 Rule 3 (iii): Confirmation of transporter parameters. 1/-8 ring intercept = 0. 60. Gradient = 20. 0K = 0. 03. V, (1 )=10. 0. I=1. 0000m (It Final parameter (the numbers in parentheses are the actual values). 1 0. ONo, 031 10 4101 12 0. 03 (0,03120+ 201　13　3,Of3. 0+ 25. H251120, 15202 SO,0050,010,020,030,040,050,+1751. 4 5 2. 59 4. 35 5. 77 7. 04 8. 2511. 141#’ 0 ．． 69 0. 77 0. 92 1. 04 1. 14 1. 21 1. 35△(1 /T　l/△S　-16,515,012,09,67,25,44S　Ll　 0. 15 0. 20 0. 25 0. 30 0. 40 0. 5ON, 85 1g , 33 21. 5 23. 65 25. 13 26. 96 28. 0917ma” ! , 44 1. 64 1. 8G 2. 11 2. 39 2. 97 3. 56Δ (1/l l/Δ3. 77 3. 92 4. 4G 5. 08 5. 55 5. 7 7 5. 9S O, 751, 01, 251, 51, 752, 02, 5 Ma 301 33. 39 37. 61 42. 1 45. 92 48. 73 51. 941 /Ma” 4. 95 5. 99 6. 65 7. 12 7. 62 8. 21 9. 6353. 3g 54, 4 54. 7 54. 81/l' 11. 4 14. 7 0 +11. 2721. 88△(1/ma)/△　3. 22 3, 46 3. 5 73. 61 structure hypothesis: Rule 2 (i) a, positive slope: Linear relationship of transporter I Regression line passing through the three lowest points: L/V ring intercept = 0. 615, slope=15. 29°KI(1)=0. 04,”, (1)=13°Rule 2(iii) The next parameter is 0. It is indicated by a decreasing slope that is lowest at 1mM, and a new increasing slope is 0. 15mM=K11 (starting with 2, 0. 0.0 at 5mM. 59(7) Final gradient It ends with ``tevm(1)+''5(21=33. 9,■,(2)=20. 9 becomes. The next lowest is 1. ．． 5mM, 1, 75mM = K11 (3, rising slope starts and the final slope is 3,61, so vm(0)=55. It becomes 4, vll(3)=21. It becomes 5. Structural hypothesis parameters: Transporter KII V, h Calculate the parameters of the net v2 value using the calculation parameters for transporter 1 above. Set: K = 0. 04,”1ll (ll=13°Sfmll) 0. 05 0 ．． 0?5 0,10 0. 15 [1,20[1,25V21. 03 2. 66 4,56 8. 07 10. 7 12. 44K11 (2,=0. 15. v2 (0,15'?'+7))=to, (,=8. 07°V2 (at 0,11) =0. 75, (1) = 5. 20t. V2 (0, I91'(7)) = 1. 25Kl,,=10. 04 pieces. *Calculated by interpolation. Speed ratio (K, (7) ratio: 0. 75, /KII=6591 and /1. 25 % at each concentration expressed as = 80%). Applying the ratio to Table 12, h2=2. It is shown as 5. Using Table 13, K 10. 15=0. 85, therefore K11+2 1=0. It is 13. Perform a 1/bv* plot of the net v2 value. First net v2 plot S2°5G, 0032 0. GO870, 0180, 031G, 049”h” 392 610 956 1421 2035 (first calculation) 1/v axis intercept=296. Gradient = 35. 785°KII (2,=0. 15. 　 K, (2,=15. 9. h2=2. 5. Correlation coefficient fil = 1. 0 First net v1 plot using the above parameters to recalculate transporter l lc 1 ring intercept = 06. Gradient = 18° K = G, Q33. V,,1)=10. r=mouth. Use the new transporter l parameters to replot v2 Second net 1/v2 chapter plot 1/V warehouse 6. 1 4J7 3,63 3,25 3. 34 3. 61KmB) = 0 from the plot minimum value. 15v (at 0,15) = Km (2) = “”v (>=0 at 0,11) 75K 11F2) = 6. 25 palms. v (at 0,19) = 1. 25K 11 (2, = Il, 29t. Calculation by interpolation Speed ratio (ratio of K: Q, 75K/K = 68% (ratio 1) and m s m Bi/1. 25K = 81% (% at each concentration expressed as ratio 2). ■ Table 12 shows that for ratio 1, h=2. 25, h=2 for ratio 2. I'm 13 It shows. The above values are approximately the same and therefore do not require further adjustment. The average value of h is 2. It is 19. From the minimum value of the plot, (2,=0. 15mM, and the h value in Table 13 The correction is 0. 15xO, 96=0. It becomes 144mM. Second net 1/hV zero plot Adjustment completed. Speed ratio (K ratio・0. 75K/K = 66% (ratio 1) and ■1m BiK /1. 25K = 90% (% at each concentration expressed as ratio 2). Table 12 shows that for ratio 1, h=2. 5. For ratio 2, h=1. that it is 0 It shows. The fact that there is a low value of h above the K value means that the initial calculated value of K is This suggests that it was too high. Fine tune KIll(3) until the h values are equal. Adjust Kl (3) and finally 1. It was set to 6mM. Then, ratio 1=53. 6%, ratio 2=74%. Table 12 shows that for ratio 1, h=3. 5. For ratio 2, h=3. that it is 5 It shows. KII (3); 1, 6 and h snow 3. 5゜Plot 1 / h v 3 *+・ S1. 8 1. 25 1. s1. 7s 10 3. 0 4. 9 5. 01. 0 118 4. 13 7. 1 11. 3 16. 8 H8280 Follow Te, Kl ,,=1. 18. V, (3, -25, 7, and h=3. 5゜(11113 table )K, (3,=t,OxO,775-1,5 5mK At this stage, the calculation parameters are as follows: Transporter KVh 1 0. 034 11. 1 1 2 fi, +5 114 12 Finally calculate the net ■1 1/v+ intercept=0. 60. Gradient=ILS7K=0. 032. and v m(fl　=””嘗11 After completing the detailed analysis, the calculated parameters are as follows 3.1. 41 25. 7 3. 5 Fine adjustment 2K value and observe at a point selected from below the minimum value, above the maximum value, and in between. , check the calculated speed. No further adjustments are necessary. Final parameters (actual values are in parentheses): Transporter K V h 1 0, 032 +6. Hl lfi, 8 +10. 01 1 (1120 ,15(0,15111,4(2012,2+2. 013 1. 48 1. 50 1 25. 7 (2513,5(4,01LL*Glutamine uptake in cells) Line analysis Figures 46 and 47 show the uptake curve and reciprocal label plot, respectively. ing. data 1. 19 2, 15 2. 88 3. 57 4. 29 6. 70 13. 717 Mazero G, 840. 931. 04+, 401. 401. 501. 46Δ (17 mane )/ΔS-2f1. 0 22 36 0 5 -0. 822. 1 24. 1　 29. 1 32. 8 32. 2 33, 0 37. 31/Ma umbrella 1. 81 2. 07 3. 43 4. 57 6. 2 157 g, 0439. 5 47,? 5 0. 8 52. 5 5332 53. 1 52. 91/Mahon 8. 86 8,8 3 9. 84 11. 1 13. 1 15. fl　15. flΔ[+/+1/Δ S 3. 28-1. 92 2, 92 3, 12 3. 1 3. 8 3. 90 buildings Hypothesis Rule 2 (i) a Positive slope Transporter ■-order quantity relationship regression line passing through the three lowest points 1/'v' ring intercept - 0. 74, slope = 20°Km (ll = 0. 0”　”m( Il='''Rule 2 (iii). 0. 25 to 0. There is a zero slope between 1mM and the new rising slope is Ktg +2 0. indicates an approximate value of 1. Starts with 2. This upward slope is 0. Final slope at 5mM5. 4, so there is Vts (1) "Vll (2) = 37, and Vts ( 2) becomes 27. The next smallest rise in the incline is 2. Starting at 0mM, new The rising slope is 2. 5” M=Ki (starts at 31, final slope is 3. Because it is 9 ,” ta (+) 251. 3, and vm(3) is 14. It becomes 3. Structural hypothesis parameters 2 0. 2 27 >+ Calculate the net v2 value parameters using the calculation parameters for transporter 1 above. Set: Niwa (1)″0. 04・Vm (11=10V, 0. 95 6, 1 13. 7 15. 4 19. 8/v* 1G, 5 3,12 2,92 3,25 5 ．． 05, (2,=0. 20. V2 (at 0,15)=Kl(2,=+3. 7 °V (at 0,11) = 0. 75K11 (2,=I0. 51°V (0,1 9) -1,25KII (2,=+5. Calculation by 4-nez interpolation Speed ratio (K ratio 0. 75K/K=73. 3 = ratio 1 and lI m BiK /1. 25K = 89% = % at each concentration expressed as ratio 2). 11m Applying the ratios to Table 12, for ratio 1 h2=2. 0, h2 for ratio 2 =1. 1. This suggests that K's calculated salary was too high. are doing. Finally Km is 0. If you adjust it to 15, it will look like this: V2 (at 0, 15) = to (2, = IO, O5゜1'2 (0, at f175) ) = 0. 5, (2,=3. 675°V2 (at 0,225) = 1. 5 (2,=14. 55 speed ratio (K ratio 0. 5K /K = 37% wealth ratio 1 and above Bi■ K/1. 5K = 69% - % at each concentration expressed as ratio 2). Then h2=2. 25. This is shown in Table 12 for ratio 1. Then h2=2. 25, 0 for ratio 2. 1. The calculated value of Kl(2) at 5mM is confirm. Using the above h2 value, the first Km from the lowest value of the plot as shown in Table 13 (21 Correct the calculated value: K, (2, = 0. 2 xO, 92-0, 18m M0 h zero Plot 1/V of the net v2 value. Initial net v2 plot SO, 00560, 027G, fi14 0. 0211/vKyo 132294 132 15971/vt axis intercept=103. Slope = 7H? K = O, +5. 　 V=21. 1. h2=2. 2. Correlation coefficient +++ = 0. 9999m (21 m (2) This confirms the parameters of the transport vehicle (■). Recalculate transporter l using the above parameters. Initial net v1 plot S(mkll　O,0050,010,0150,0250,030,05■+ 1. 18 2, 10 2, 76 3. 2 3, 74 5. 051/V umbrella 0. 85 0. 95 1. 09 1. 56 1. 6 1. 981/v zero axis intercept = O, ? 1. Gradient=26. 18,,,,=Q,02!1. V,,,=7. SS, r= 0. 9783 Replot v2 using the new transporter I parameters. Second net 1/v2 plot. S [all O, f15 0, 10 0, 20 0, 25 0. 50v21. 86 +, 8 15, 5 17. 3 21. 95K = 0. 15. v (0, 15)””K, +2)=II 65°+1(2)2 v (at 0,11) = 0. 5Km (2, = t 83 zero. v (a, at 19) = 1. 5, (2,=16. Calculation by 0*interpolation Speed ratio (Kffi ratio 0. 5KII/K11l=41% (ratio 1) and K/' 1, 5 K = 71% (9 stones at each concentration expressed as ratio 2)). Table 12 shows that for ratio 1, h2=2. 0, for ratio 2 (h=2. It is 0 It shows. The above values are approximately the same and therefore do not require further adjustment. The average value of h is 2. It is 0. Using the above h2 values, KII ( Correct the initial calculation value of 2)"5(2)="'"xl, o=o, 15m M0 Second net 1 / h v 2 * plotted, K, +21 = 0. 15. VII(2) = 23. 9. and proceed to h2=LD transporter ■. Dark blue 4 (iiil. 1 0. 028 7. 55 1 2 0. 15 23. 9 2. 0 1/v3 meeting 5g, 5518. 23410424. 225. 6 Old calculation value was done * ( 31=”　” ■ (at 20) = Kl (3, = 16. SO ■ (at 15) = Q, at 75 , (3, -6, 22°V (at 15) = 1. 25KII (3,=+9. 5゜ Speed ratio (ratio of K + 0. 75K/K = 38% (ratio 1) and m ugly BiK /1. 25K = 85% (% at each concentration expressed as ratio 2). I11 Table 12 shows that for ratio 1, h3=5. 0, h3=1 for ratio 2. It's 6 It shows. Make fine adjustments until the h values are equal. h to recalculate 1Ki (final adjustment of 31 = 1. 85mM0V (at 1,85 ) to -, (3, -11,60V (at 1,411) -11,75Kl (3 ,=5. 44°v (at 2,31) = 1. 25KII (3,=18. 40 ゜Speed ratio (K ratio: Q, 75K/K = 47% (ratio 1) and 1 ■■ BiK /1. 25K = 63% (% at each concentration expressed as ratio 2). l　■ Table 12 shows h=3''4 for ratio 1 and h=6 for ratio 2. 0 It shows that. Let h3=5. Using the above h3 value, the first K from the lowest value of the plot as shown in Table 13 Correct the calculated value Ks (31,022. Ox■ (3) 0, 71=1. 4mM. b zero 1/ Plot V・3・ S1. 5 1. 75 2. 0 2. 5 3. 0! , 5 4. 0゛7. 6 + 6. 4 310 97. 7 243 525 1024 Jutte, K -1,77 ,V,(3,=20. 9. and h=s, o. Ugly (3) Side shell 4 (1 ma) Recalculate net transport I using the above parameters for transports ■ and ■ . 1. 16 2. 04 2. 64 2, 92 3, 37 4. 311/v umbrella section = f1. 7G, slope = 34. +3K -1,02,V, (1)=5. 9゜so (1) Transporter parameters Transporter KVh ob, 2,153. 576. 713. 7 old 32. 2v (112,873,92 6,612,320,730,2v(211,813,766,7512,92 1,932,1S frill 1. 5 1. 75 2. 0t, 0Vt11 35. 1 39312. +1 50. 2vt2+ 37. 7 ill 15. 1 53. Note 5: 1. The numbers in bold are the numbers that require adjustment. 2. V (1) is obtained from the calculated parameters. 3. V (2) Adjustment is as follows. Second, (1) is O, Q3mM, vIl ( 1) Hello, to 8, vm (21 to 25, V, (31 to 22). 4, v (3) Adjust h3 to 6. Final parameters・ Transporter KVh 1 0. 03 6. 8 1 2 0. 15 25 2 3 tg 22 6 Figure 48 shows the curve calculated in relation to the average of the plot points. Note: Experiments were carried out at different times using two different parameters for speed determination. . Numerically, these two sets of parameters are 1 mom I/min/cell = 1. 12n It turns out that it is equivalent to mol/min/mg protein. Progress chart: a = structural hypothesis, b = detailed analysis, C = fine-tuning. 1 body progress KV h Figures 49 and 50 show normal uptake and Na+ deficient uptake, respectively. Shown are uptake curves and reciprocal label plots. ΔQ/v)/As --100-11-16-6,0-4,0S 3. 5 4. 0 5. 0 Structural hypothesis: Rule 2 (] and i　j　j)　b. The -order transporter ■ was excluded because the initial slope was negative. The minimum slope is 0, 5 m M-K, (minimum near 21, final slope at the end of the next upward slope 0. 75mM point, slope = 12. 0'''1/L' = 1/Vm(2,x O,005, VII(2) = 17 nmol/min/mg protein. tilt The next minimum value after the minimum change in slope is 2. 5mM=, occurs at (3) and after this minimum The maximum slope (at 5 mm) is v, (2) + V, L 31 = 69 n 　m　　　　　　/min/mg protein. Therefore, V, (3) = 52 n m　　　　　/min/　mg protein. The shape of the curve is typical of two multiorder systems (Figure 44). Structural hypothesis parameters Rule 4(1) Kl (calculated value of 21 and h2)'l (final adjusted value of 2+ = 0. 5mM0 velocity ratio (K ratio 0. 75K/K = 72% (ratio 1) and ffi l 11 BiK /1. 25K = 80% (% at each concentration expressed as ratio 2). lpeng Table 12 shows that for ratio 1, h2=2. 0, h=2 for ratio 2. be 3 It shows. h2=2. 15. Using h2 above, first calculate K from the minimum value of the plot as shown in Table 13. Modify the value: K, (2,”’0. 5xl (2) 0, 95=0. 48mM0 Prot 1/bV= 2′ SO, 150, 250100, 350, 400, 500, 6111/'v 〇41　0. 63 0. 62 1. 60 3. 67 7. 65 14. 9? I/bvs intercept -730, slope = 6578° Therefore, KII (2, = 0. 36. V, (2,=+3. 5. and h=2. 15. r=0. 9861 Rule 4 (item 1 ). Calculated value of net ■3. L’k　v3　目5　11. 2 16. 1 28. 8 41, 7 49. 64 51. 6 5! II/　v3''　Ios　22. 38 18, 6 13, 9 1 2. 0 12-1 13. 6 15. The calculated value of 2K and h is the maximum of 2Kmf31. Final value=2. 5. Final adjustment of K11 (3) = 2. 0mM v+2. ) (K, (3,) = 28. 8゜v (at 1,!nM ) (to 0,75, (3,) = 16. 1°V (at 2, Sd) (1,25 K11 (3,) = 41. 7゜speed ratio (K, ratio of 0. 75 to / to = 56% (ratio 1) and Km/1. 25K = 69% (% at each concentration expressed as ratio 2). Table 12 shows that for ratio 1, h3=3. 25. For the ratio 2, 0, l and h=4. At 25 Which indicates that. h3=3. 75. Using h3 above, the first Km (3 ) Correct the calculated value: Kl(3)-2,5xO,79=2. 　0mM0 Initial net 1/’ v reference plot 1 house v zero intercept -6,? 4XIO', slope -11, T89xlo7. r- a,! 999゜Initial net v2 plot Net v 22. 4 4. 2 5-1 5. 9 L 1 net v2 value of 0. 6 to 1 ．． A decrease to 0mM suggests that either h3 or Kl(3) is too low. There is. h3 to 3. 75 to 5. 0 and reproduce net 1/v2* for S. The net v2 value is now 0. 6 to 1. Rising to or near a maximum value of 0mM Find out if it will work. First net 1/v2 umbrella plot S (mill O, 150, 250, 300, 350, 40 net v 2. 4 1. 2 5. 1 5. 9 611/vt 30 11. 9 11, 8 11. 9. 11. 85 (ail) 0. 50 11. 60 G, 65 fl, 75 1. 0 Net V2 19 10, 2 10. 6 io, g 11. 11/v2 umbrella 11. 2 Old +2. 2 Old The minimum value of the old plot is Ks (21 = “5”) It shows. Speed ratio (K ratio: 0. 75K/K = 71% (ratio 1) and l I! 11 % at each concentration expressed as K/1°25K=8696 (ratio 2). Table 12 shows that for ratio 1, h2=2. 0, h=1 for ratio 2. be 5 It shows. h2=1. 75. Using Table 13, for K correction, Kffi(2,=0. 42 is shown. well one I will. Tadashi Hatsuda→　1haV＊””υ, de 5 (all) 0. 15 0, 25 0, 30 0. 35 G, 40 0. 50 31. 75 11. 036　1H80,120゜! G O, 200, 30S (aM) L 60 G, 65 0. 75 1. 0S　O,410,470,601,0 1,75 1/hV2 @ intercept = 135. Gradient = 7SL r = 0. 9658゜net Second calculated value of v3: Net V3 11. 2 16. 0 28. 6 41. 4 19. 26　s+252 ．． 11/V3” 22318-714,0 Old 1221a,y!5jKs(3 Final adjustment value of 1 = 2. 0d V2 (at 2,0-) (K, (3,) = 216° speed ratio (ratio of K: Q, 75K/K = 56% (ratio 1) and l ■ Seagull BiK /1. 25K = 69% (% at each concentration expressed as ratio 2). Table 12 shows that for ratio 1, h3=3. 25, h=4 for ratio 2. It is 25 It is shown that. h3=3. 75 and h3 above, as shown in Table 13. Correct the initial K calculated value from the minimum value of the sea urchin plot K, (3,=2. 5x■ (3) 0, 79-2. 　0mM. Second net 1/’ v zero plot 1/hv3. t71 piece = ]23x1010. Slope-1), 602xlO”, r-0,9999°I/bv, zero intercept-! 43, slope -756, r-0, 9957° Transporter KVh Tweak. S (ml O, 15035G, 75 1. fl　1. 25 2. 0 2. 5 5 ．． 0zb, 2. 4 6. 0 11. s　11,0　23. 8 42. 0 55 U 66. 5vL, 2. 2 6, 4 11, 3 14, 2 19, 4 4 6. OS7i 66. 7 results need 1. to match the observed values. 25. 2. 0 and a value of 50 indicates that an adjustment of approximately 10% is required (λ). This can be achieved by decreasing the value of h. However, this adjustment is 375 to 5 (1), so further fine-tuning was not considered appropriate. . The final parameters were obtained from the above detailed analysis, and Figure 502 shows the final parameters. The calculated curve related to the average of the cut points is shown. The K value is expressed in mm and the velocity is expressed in nm. Expressed in ol/min/mg protein. Progress chart + a = structural hypothesis, b = detailed analysis, c = fine-tuning. b O, 3914, 11, 75 32,552>1 Compare the Na deficiency parameters (in parentheses) with those obtained in the presence of Na ions. Ta. Transporter K V, h l　0. 03(-16,8+-11,031,8(1,8122,0+55. 0 1.6 f5) The results show that the consequences of Na deficiency are related to normal Na-dependent functions. The fruit transporter I is completely removed, and the transporter II is K It is shown that ■ was inhibited to the extent that it was more than doubled, and that ■ was almost halved. Na +-dependent transport is inhibited by leucine, whereas uptake of the remaining transport is inhibited by leucine. It is suggested that this is probably due to a system that has been enhanced to avoid being harmed. observation The uptake transport is caused by the exchange of labels such that the internal and external glutamine concentrations are equal. It is almost certain that this is the case. The parameters for uptake in Na ion deficiencies are very approximate; Emphasize that it can be represented by a series of curves depending on the weight of a particular part of the lot Should. Furthermore, there is no evidence as to the nature of the mechanisms involved in the remaining activity. . The importance of the above data is that the diagram shows that the concentration range of activity normally occupied by the transporter IT is Na This shows that it is severely inhibited under + ion deficient conditions. 8 Exposure of cells to glutamine a0 procedure The procedure consists of a double-labeling experiment in which cells are first labeled with 2 mM H-glutamine (5 0 μCi/ml) for 10 minutes, followed by cell number measurement. We measured the incorporation of the label into proteins (also used in The uptake rate was measured using a 1 minute exposure to 50 μCi/ml). all multiple The same labeling solution was used in the samples, therefore 50 μl of Samples were removed and measured to correct for label dilution. of each labeled incubator. Ink for 3 minutes with labeled glutamine at the same test concentration as the previously labeled glutamine sample. No concentration dilution occurred due to the turbidation. whole incubation The experiments were performed at 37°C and fresh medium containing 10% (v/v) FBS was added before each experiment. Cell cultures were incubated with Di199 for 2 hours. Next, change the medium to medium 199 The medium was replaced with unlabeled 2mM glutamine and incubated for 10 minutes. Next, this Replace the medium with 3H-glutamine in medium 199 and incubate for exactly 30 minutes. I started. Cultures were washed three times with HBSS and then tested with glutamine in HBSS. solution (0.25-4.0 mM) for 3 minutes. Immediately and accurately Exposure to 14C-glutamine for 1 min at 0.5 min and cells in water at 0.5 min. 9% (w/v) NaCI Uptake was stopped by washing three times with water and then three times with water-cold acetone. The culture was then placed in a vacuum desiccator. b Separation of proteins and soluble amino acids Remove cells from the surface of the culture bottle using a spatula and acetone and incubate at 100°C. The protein was precipitated by coagulation (LleEma0!-Bove et al.), Ch+ om+tog,,347. (1985) 199-208). pasteur pi The upper aqueous layer was removed with a pet and stored. This fraction contains cytosolic amino acids 0. Take a 2 ml sample, 8200, 3 ml and A CS (AllIezhsm) 4ml was added and radioactivity was measured. The lower layer was further extracted again with 0.5 ml of acetone, this time the upper layer was discarded. Chlorophor The protein precipitate was washed with 2 ml of 5% (w/V) sulfosalicylate. Then, it was washed twice with 202 ml of H. After each wash, test tubes at 600 g for 10 min. Centrifuged. The protein pellet was dried overnight at 56°C, then the dried protein was injected with 0. 1M NaO Add 81 ml, seal the test tube, and leave it at 100°C overnight to digest the protein. Ta. Measure protein content by reading absorbance at 500 nm using BSA as standard. (LOWIT et al., 1. Biol, Chew, 193. (195++ 265-275). The sample stopped with ACS was programmed to be measured with double label S. Programmed LKBLll++ 1215 Rxekb+1+Liquid scintillation 9-'T: The radioactive sample was measured. C. Calculation of glutamine uptake The number of cells present is determined by the 3H - Measured by glutamine uptake. The amount of glutamine incorporated into cellular proteins per minute is determined by the number of cells using the following formula: Convert to: Number of cells = (2, 2 + 24. 6xnmol　gln/min)xlo5 ．． The amount of glutamine taken into the cytoplasm per minute is the specific radioactivity of glutamine in the medium. , and the results were expressed in the form of cell counts or protein content; They showed almost identical uptake curves. Including a pre-incubation period of 3 minutes or more The uptake rate in inhibition experiments was expressed only in terms of protein content. d. Experimental equipment used for transportation experiments Another embodiment of the invention is a transport assay plate useful for carrying out the assays of the invention. Regarding seat assembly. Another embodiment uses such an assembly. It consists of procedures to be used. Tissue culture or cell culture expansion in clinical chemistry typically requires multiple specimen receiving systems. An assay plate with small wells or wells is used. common usage Multi-well plates are molded with 6-96 wells arranged in a rectangular array. It can be in the form of strip or sew molding. The plate is a flat plate that closes on all wells at the same time. They often have a unique cover. Separately removable covers provide individual protection for each well. You can also make it so that it can be placed in U.S. Patent No. 4,599. In issue 314 , a multi-well plate and lid construction of this type is disclosed. Transport experiments in tissue or cell culture require large numbers of samples to obtain reliable results. and precise timing conditions are often required. Monolayer on culture plate surface Many homogeneous cells are obtained from tissue culture cells grown as a The culture conditions of the cells can be changed instantly and simultaneously to resemble physiological conditions. I can do it. Multi-well plates allow simultaneous transport experiments of large amounts of samples. It is possible to do this by adding the same amount of brain-containing radioactive solution to each well. A method is needed to transfer the data from time to time. The present invention provides downwardly opening wells arranged to match the wells of an assay plate. Provide a transport multi-well assay plate cover consisting of a lid with a frame; When placed on the assay plate, the lid will touch the plate and the opening in the frame will touch the plate. Extends into the well hole. The invention utilizes a plurality of spaced wells and a support surface that is flat when inverted. Transport assay plate consisting of a removable cover that stands freely upright on a surface A cover assembly is also provided, with the cover opening downwards to match the wells of the plate. It has an array of frames, so it should be covered and in contact with the plate. and the opening of the frame extends into the well hole of the assay plate. The present invention involves culturing cells at the bottom of multiple wells of an assay plate and Reverse the cover, which consists of an array of frames that match the wells of the Add incubation medium and invert the assay plate onto the cover. Place the assembly upside down and incubate the assay plate with 6 volumes of incubation run medium. The transport assay procedure, which consists of ensuring transfer to adjacent wells of the provide. Referring now to FIGS. 66 and 67, the present invention will now be described by way of example only. Let us describe an embodiment of this. The transport assay cover described herein can be used in multiple well assay plates of various sizes. Adapted for use with The following two examples (Examples 80 and 8) In 1), the cover is used with 6-well plate and 96-well plate. Compatible with However, the present invention is equally suitable for a variety of multi-well specimen plates. It is understood to include other covers that may exist. Additionally, examples are readily available. describes the manufacture of covers from laboratory equipment that can be sterilized; It is understood that it can be molded from plastic. Figures 66 and 67 have six flat-bottomed wells 22 divided into two rows of three. The assembly of the transport assay plate 20 consisting of a rectangular base structure 21 and the cover 10 It shows the assembly. The cover 10 has the same cross section as the plate and has a downwardly extending peripheral flange 12. It consists of a substantially rectangular lid 11 that grows. A plurality of cylindrical frames 13 are attached to the plate 20. They are formed to extend downward from the lid in an arrangement corresponding to the arrangement of the wells 22. 6 frames 1 3 is 1. 1 has the shape of a closed cylinder facing downwards from the surface of the lid 11. It has an opening 15 at the other end extending at the other end. Cover 10 is a 6-well plate 20, the lid 11 contacts the flat upper surface 25 of the plate 20, and the lid 11 rests on the 6-frame The openings 15 of 13 extend into adjacent well holes. For use, culture cells in a culture plate for 2-3 days until 75%-90% confluency. Grow at the bottom of the plate. At the beginning of the transport experiment, decant the medium from the assay plate. Add fresh incubation medium to the well using the cover above. Ru. Any number of different solutions can be placed in the frame of the upturned cover before starting the experiment. . The solution can be maintained at a given temperature by placing an inverted cover in a water bath at a predetermined temperature. Wear. Cultures containing cells attached to the bottom of flat-bottomed wells 2-3 seconds before the required time Turn the plate upside down and match the corresponding solution in the frame defined by the cover (6th 9 and 73). Then, at the desired time, invert the entire assembly and Transfer the solution from the well to the corresponding well without spilling anything (Figures 70 and 7). (See Figure 4). All wells are transferred immediately, which means less experimental time. This is also desirable in terms of improving the accuracy of experiments. As can be seen from FIGS. 49 and 50, the cross section of the frame 13 of the cover 10 is an assay block. considerably smaller than the cross-section of the flat-bottomed well 22 of the plate 20. The cross section of the frame is quite large in comparison. Although it is not essential that the cover be small, it should be small enough to prevent the contents of the cover from spilling during transfer. This is advantageous because it reduces the amount of sleeping. Also, the cross section of the frame is almost the same as the cross section of the well. It is one. Although not shown in the drawings, a second embodiment is for use with 96-well plates. Includes 96 framed covers for. In this embodiment, the cross section of the frame is The cover may be smaller than or nearly identical to the cross section of the assay plate. Place it so that it fits tightly and reduces the possibility of spillage. like a clip Suitable fasteners can be used to improve the close fit between the cover and the plate. Wear. The 6-well cover is designed to hold 1-3ml of solution within the frame, but the design has been changed. It can also be used to accommodate a larger amount of solution. The opening of the frame is from the bottom of the lid. It is important that it extends at least approximately 5mm and protrudes into the bore of the well. . The 96-well cover allows 100-300 μl or more of solution to fit within the frame. can be designed to Both the 6-well cover and 96-well cover have flat bottoms. Designed with a frame to ensure the cover stands upright when upside down on a flat surface. be able to do so. 9. Results Glutamine transport into ReLt cells was concentration dependent and saturable. Import The mixing rate is a measure of the number of viable cells per milligram of protein in the harvested sample, which is different from the cell number. Expressed as Figure 2 shows that glutamine uptake into HeLx cells is caused by two high-affinity divided into a sexual transport system (transporters ■ and II) and one low-affinity transport system (transporter r [I)]. This indicates that the 0. Uptake of glutamine below 2mM (transporter ■) showed negligible cooperativity, whereas uptake via transporter II showed negligible cooperativity. 2m Michxelii-Menlen kinetics were followed in the M-1mM concentration range. Transporter III is 1. 5-3. For 0mM, it shows cooperative kinetics and is non-saturable. The ingredients are 3. 3 nmol/min/mg protein, which is the reason for all rate measurements. subtracted from. Complete kinetic analysis of glutamine uptake into IIeLs cells According to (see Table 2 below), K of transporter ■ is 0. 03±0. 0015mM Low, ■ also 6. 8B III ±0. This transporter's glutamine content is very low at 0.35 nmol/min/mg protein. The contribution to transport was found to be relatively insignificant. Substrate concentration 0. 2-1゜ At OmM, the K of transporter II is 0. 15 soil 0,075mM, V is 25±2n m011 111! /min/mg protein. Glutamine concentration range 1. 0-3. At 0m M, the transporter K of III is 1. 8±0. 10mM. ■ is 22±L, lnmol/min/mg protein. Each transport to the substrate Body specificity was calculated. The specificity of transporter II is 167 m1/min/mg protein. Although always high, the specificity of transporter II+ was 12. 2ml/min/mg protein and It was very low. As expected, the specificity of transporter ■ is 227 m1/min/mg protein. very high, indicating that this transporter is highly specific for the substrate glutamine. . Table 2 H! Kinetic parameters of glutamine uptake into Ll cells In addition to the kinetic values , the following information about TGT l-111 is also known. TGT 1 One glutamine concentration is 0. Mainly involved in transport when below 2mM - It is monosodium dependent following the kinetics of Mich++li+-Mentsn. Ru Accepts a small amount of lithium ions High stereospecificity -Accepts N-methyl amino acids - Not inhibited by 2-amino-2-norbornanecarboxylic acid - No adaptive adjustment stomach one stimulated trance effect Most likely amino acid transport system A. TGT IT - Glutamine concentration is 2. 0-1. Mainly involved in transport at 0mM is a cooperative carrier monosodium dependent - Does not accept lithium ions -High stereospecificity -Do not accept N-methyl amino acids 0 II) - adaptive regulation is not inhibited by 2-amino-2-norbornanecarboxylic acid I can't do it one stimulated trance effect inhibited by mersaril It is an endogenous (inleg+xll membrane glycoprotein) with one disulfide bond. -The relative molecular weight of one dissociated protein, which is probably the amino acid transport system ASC, is 42,000. TGT II+ which is 0 One glutamine concentration is 1. Primarily involved in transport when above 0mM is a cooperative carrier monosodium dependent - Does not accept lithium ions High stereospecificity -Does not accept N-methyl amino acids -1 adaptive adjustment not inhibited by 2-amino-2-norbornanecarboxylic acid stomach one stimulated trance effect inhibited by mersaril is a monohydrophobic membrane protein -Probably an amino acid transport system The relative molecular weight of one dissociated protein is 530,000. Table 4 (Figure 75) shows Na+-dependent absorption at a glutamine concentration of 0°75mM. rate of inhibition, nature of the major known inhibitors, and derepression in glutamine-deficient media. Glutamine transporters were classified according to their abilities. When glutamine is 1mM, 0. 25-10. 0mM (Glutamine with DNEM Figure 3 shows the titration of the ion transporter. The pre-incubation time used is shown in Figure 4. 20 minutes as shown, which is 1 m for glutamine transport at 1 mM. It is known that M　NE　M is the optimum incubation time that shows the maximum effect. It is being Glutamine concentration range 0-25-4. Glutamine import into HcL1 cells at 0mM The effect of the sulfhydryl reagent N-ethylmaleimide on transport is shown in FIG. N After 20 minutes of incubation with EM, the monolayer was incubated with radioactive glutamine. and uptake was measured for 1 minute. Which of transporter 1, II and r■■ Uptake via 1mM NEM was also inhibited by 1mM NEM. Protect the support from NEM binding By including a large excess of substrate in the pre-incubation substrate, the N The inhibitory effect of EM was found to be in direct competition with glutamine for sites on the carrier. I made it clear. Table 3 shows that 1 m N E M due to excess glutamine It is shown that glutamine transport at 1 mM is partially protected from inhibition. deer However, D-glutamine has no protective effect. Table 3 Substrate-protected incubation conditions for inhibition of glutamine uptake by NFM Glutamine uptake (% of control) Control 110 0NE inhibition (1mM) 53 Protected with L-glutamine (100mM) Protected with 75D-glutamine (100mM) Protected 53 isolated receptors (e.g. M42000) can be subunits. It was recognized that Transporter isolation conditions, especially 2-merase (which breaks disulfide bonds) Conditions using SDS combined with lucaptoethanol (to break non-covalent bonds) can reduce large functional molecules to their lowest molecular weight. From this, the transporter is M bonded via a disulfide bond or a non-covalent bond. , 42,000 in size. Also , it is also possible that it binds to other large and small molecules that have not been detected in this system. Guru The present invention also includes combinations of such subunits that together form the tamine transporter function. include. C1 Identification and isolation of other human amino acid transporters Regarding the tumor glutamine transporter Other human amino acid transporters can be isolated using methods similar to those described. amino acid to tumor cells Transporters involved in the uptake of amino acids are of particular importance, especially if such transporters are Important if absent or not active in tumor cells. For example, HeL The alanine transporter discovered in I cells was also isolated by the following method. 1. 1 (Identification and Isolation of Alanine Transporter in eLi CellsAlanine Concentration1. 0-1 0. When measuring alanine uptake into neLx cells for 0mM, the 61st As shown in the figure, Mich*! in this range! Dynamics of 1 row 11tffiten type seemed to follow. In Figure 61, LLt cells are exposed to 14C-alanine for 1 minute. The amount of label in the cell chamber was quantified as total cell protein. L for alanine uptake The analysis is shown in Figure 62 for in+vexma e+-Bo+, and the K of alanine uptake is is 1. 33mM, V is 19. 6nmol/min/11 It was revealed that it was mg protein. In Figure 62, HcLs cells are treated with radioactive Breincubate with or without 1mM NEM before incubation run. I did it. The diffusion rate of alanine into these cells is 0. 625 nmol/min/mg protein white matter and was subtracted from the saturable component of the kinetic analysis. Alanine transport at 1mM alanine was reduced to 0. Titrate with 25mM-10.0mM NEM The result is shown in No. 63v4. In Figure 63, RtLt cells are 1mM by incubating with various concentrations of NEM before exposure to The uptake rate of ranin was measured. Inhibition of alanine transport is maximum at 2mM NEM. The alanine transport rate at this time was 40% of the control value. l　m　M Alanine transport to HeL* cells incubated with NEM for 15 minutes FIG. 64 shows the effect of 1 m NM N EM on . pre-incubation Preparation of sulfhydryl reagents by including a large excess of substrate in the mixture The mechanism of action was shown to be due to direct competition with alanine for carrier sites. It was done. Table 14 (Figure 85) shows that 100mM alanine is determined by 1mM NEM. partially protects against inhibition of alanine uptake, but the non-specific stereoisomer D-a Ranin was unable to protect the carrier from NEM inhibition. Lin2veswe+ inhibits alanine uptake by NEM (shown in Figure 62) -B*+ Analysis shows that NEM is an irreversible inhibitor, but its mechanism of action is to lower Km. It appears to be a competitive inhibitor. Identification of alanine transport protein in ReLr cells was used to identify glutamine transport protein It is based on the same principle. Alanine transporter is highly sensitive to NEM inhibition It has been reported that NEM is used to label alanine carriers. It seemed possible to do so. IIcL Hatake cell plasma membrane is transported by excess transport substrates. Incubate with 3H-NEM in the presence or absence of ranin and Membrane proteins were analyzed by electrophoresis. As shown in Figure 27, many proteins contain 3H - It was found that it binds to NEM, but only peaks C and D show the presence of L-alanine. It was found that this protection is provided by the existence of In Figure 27, ink with 3H-NEM R5Lr cell plasma membranes were treated with 100 mM alanine before or during pageage treatment. Brain-incubated with or without alanine. D of the untransported stereoisomer -Alanine cannot protect any of these peaks from label binding. won. Breincubate with excess alanine, then incubate with 14C-NEM The plasma membranes were dissolved in SDS and analyzed by 5DS-gel electrophoresis. Next, gel When exposed to X-ray film, the autoradiogram shows the transport substrate L-alanine and The M31000 band (lanes 3 and 4) was pre-incubated with is specifically radiolabeled, but preincubation with D-alanine It was shown that it is not labeled. The binding of higher concentrations of 38-NEM to these peaks was compared with the same concentration of NEM. Titrated with nin transport inhibition. As shown in Figure 28, alanine at various NEM concentrations It was found that there is a one-to-one correspondence between transport and binding of 3H-NEM to the peak. Ta. When the carrier is 100% saturated with 3H-NEM, the uptake rate is the same as that of alanine uptake. It is thought to be equal to the diffusion component of the uptake, and from the uptake rate measured at each NEM density, By subtraction, a titration graph for only peak D in FIG. 29 was obtained. M310 One-to-one correlation between alanine uptake inhibition on NE~1 binding to 00 protein. A theoretical agreement was found, indicating that this protein is involved in the transport of alanine to HeL1 cells. It was shown that it is a suitable carrier. 2. Results Alanine transport into HtLJIM cells is distinct from glutamine transport in these cells. It appears to have unique kinetic properties. The uptake curve follows simple MichIe1-Msalsn kinetics and Km5 , reaching a maximum rate of 15 nmol/min/mg protein at OmM. plain cuvee The presence of 1 m M N E M in the alanine medium increases the uptake was inhibited by 50%. Transport inhibition and binding and binding of NEM to membrane proteins The fact that the protein band of M31000 is correlated with the protection by the substrate of " This indicates that it is involved in alanine transport to the cells. M. The 31,000 protein can be isolated using the same method as described above for TGT If. It seemed like it would. Similarly, antibody production, recombinant DNA cloning methods, diagnostic use The discussion herein regarding therapeutic products, compounds and methods, etc. refers to the alanine transporter. Or it can be applied to any other transporter of the present invention. Furthermore, upon reading this specification, Those skilled in the art will recognize other methods of isolating human amino acid transporters, particularly tumor amino acid transporters. You will be able to understand it. Although we have described the identification and isolation of amino acid transporters using NEM, those skilled in the art will appreciate that other Understand that reagents can be used. For example, those who are susceptible to NEM. Transport systems that have been shown to have a thiol group in the active site include other thiol analytes. Medications such as mersaril, azaserine, DTNB and organomercurial compounds can be used as well. Wear. Transport systems that are not sensitive to NEM are labeled with specific amino acids transported. can be used to identify transporters. D. Identification of transporters in other cell lines Now that we have isolated the human aminotransporter, it is possible that the same or related transporter exists in other cell types. It is desirable to determine whether the For example, in the case of the tumor glutamine transporter isolated from R1Ll cells, the same transporter It would be desirable to know if this is found in other tumor cells. As detailed in Section F, TGTII is associated with other solid tumor cell lines and lymphomas. It was discovered that it exists in cell lines. The obvious advantage of this knowledge is that the same diagnostic and therapeutic compositions can be used to It will become possible to diagnose and selectively treat different types of cancer. 1. Screening of cell lines with antiserum and then using the experimental procedure TGT Antibodies to II may be used in other solid tumor cell lines, lymphoma cell lines, and leukemia cell lines. The reaction with proteins present in cell lines and normal human cell lines was determined. a. Production of antiserum in mice Male BIlb/c mice were used with 500 μm of antigen (purified glutamine transfection). Delivery protein (250μl/m+) 250μm and complete Freund's adjuvant 2 Antiserum was obtained by intramuscular injection of 50 μl). Freund's incomplete a every month After immunizing mice with the antigen in the adjuvant, serum was collected by bleeding from the tail. . The blood was allowed to clot overnight at room temperature, and the serum was transferred into a microfuge tube at 1200 ml. g (Red blood cells were removed by centrifugation for 10 seconds in an Eppendo τ (ultracentrifuge). Serum was stored at -20°C. Control serum from non-injected mice was also obtained. b. Preparation of antiserum in rabbits Using male rabbits, antigen (mixed with complete Freund's adjuvant in a ratio of 1:1) After intramuscular injection of 250μI/m) 22m, 3 months for 2 weeks. Each time, Freund's incomplete adjuvant was subcutaneously injected for booster immunization and antiserum was obtained. . Bleed rabbits from peripheral ear veins, allow blood to clot overnight at 4°C, and collect blood. Ta. Serum was collected and centrifuged at 400g for 10 minutes to remove red blood cells. 500 serum Stored as μl aliquots at −20°C. Control from rabbit before first injection Serum was collected. Standardized ELISA Screening of C8 Antiserum 96-well Microtiter First 5 of each 12 wells of rate (Flow L1bo+xlo+ies) The column was coated with coaching buffer (35mM N+HC015mM Nt2CO3, Anti-3' diluted twice at pH 9.6) Coated with 50 μl of stock solution (50 μl/ml). Add PBSA (50 μl) to the final Added to 3 columns. Plates were incubated for 2 hours at room temperature (20°C). antigen/ Brush off the PBS solution from the wells and replace the plate with wash buffer (0.0% in PBSA). 0 Washed three times with 5% (v/v) Tveen 20). punch the plate I placed it on top of a tasuru ball to dry. T E N /< - SO Fa (θ51J Tt low MCI, 0. [l1M EDT^, 1. 5M NiC Incubate for 2 hours at room temperature with 02% (w/v) casein in 1, pH 9,0). After washing, the cells were washed three times with washing buffer in the same manner as IT. The one that started from 1.10 Serial two-fold dilutions of antiserum were diluted to 0. Made in @TEN buffer containing 2% (W/V) casein. A 100 μm dilution of the antiserum was prepared and added to 11 columns of 8 wells each. contrasting Serum was added to the wells of column 12 and the plate was incubated at room temperature. As before, the antiserum solution is brushed off from the wells and the plate is washed 3 times with wash buffer. Washed twice. 0. Anti-mouse/'mouse in TEN buffer containing 2% (w/v) casein. Heron horseradish peroxidase IgG conjugate (Bio-Rgd) Make a 1/1000 dilution and add 100 μl to each well of the plate and incubate at room temperature. Incubated for hours. Wash the plate three times with wash buffer as before, washing each 100 μl of substrate was added to the wells. The substrate is 0. 05M Sodium Citrate, 2% 1mM BTS dissolved in 0.15M sodium phosphate containing H2O2. It was. Absorb each well at 414 nm and 492 nm using a Flow plate reader. Colored reactions were quantified by reading the difference in light intensity. d. Screening of antiserum After obtaining the optimal conditions for the EL ISA assay from standardization, the antiserum was further screened. A screening assay was performed to test the results. Generally, plate the antigen (50 g/ml) and probed with a 1/100 dilution of antiserum. I was disappointed. The conjugate dilution used was 1/1000 and the plates were read as above. I did. e, Western blotting The antibody titer in the antiserum was low, making it more sensitive to confirm the immune response. Western blotting assay was used. Bio-Rld electrophoresis truck Proteins were transferred onto nitrocellulose using a transfer cell. (Tovb in et al., P+oc, N-sho

[1, ACxd, Sci, U, S, A,, 76 , (1979) 4350). After electrophoresis, transfer the gel to transfer buffer (0,025M Ir-HCl, 0. 192M glycine, 20% (v/v) methanol) for 30 minutes at room temperature. Boil the nitrocellulose cut to size. Moisten the gel with transfer buffer and remove the gel on the cathode and the nitrocellulose on the anode side. I made a sandwich with roulose. Transfer with constant voltage of 60 volts was carried out for 3 hours. Remove nitrocellulose and remove 0. Twe of 05% (V/V) ea 20 (Tris-buffered containing TBSTI and 4% (V/V) FBS) Moisten with saline solution (10mM Tris-HCl, 150mM NaCl, pH 8.0) The mixture was placed on a filter paper and incubated at 4°C overnight. Next, nitrocellulose Transfer to antiserum solution (1/100 dilution in TBST) and incubate for 1 hour at room temperature. After that, it was washed three times with TBST. Anti-mouse/rabbit alkaline phosphatide in TBST. Prepare a 1/10000 dilution of fatase IgG conjugate (Promef*), Incubated with nitrocellulose for 30 minutes at room temperature. nitrocellulose Wash the plots three times with TBST and then add alkaline phosphatase by 77 (10G+Ml+1s-HCl, IHsM NtCl, 5mM MgCl2, Substrate (QJ7mM BCIP, 0.5mM) dissolved in pH 19.5). 5mM NET) added. When the band becomes sufficiently thick, add a stop solution (20 μM l+1i-HCI, 5 Color development was stopped by washing the plots with mMEDTA, pH 9,5). f. Cell line screening (i) Preparation of plasma membrane Prepare plasma membranes from adherent cells as described above for tleL* cell plasma membranes. did. The suspension culture was grown to a confluency of approximately 103 cells/ml and then grown in a sucrose gradient. Both plasma membranes were prepared by centrifugation (SBsl et al.), Ce11. Pt+4 +iol, 100. (1979) 109-118). All operations are 4 The cells were first incubated at 200 g for 5 min (CI! mtnls 2000 Ben Pellet (titop centrifuge) and Wash twice with 9% (w/v) sodium chloride. Ta. Dissolve the pellet in solution (1mM N*HC0, 0.5vM ClC12, pH 7,4) Resuspend in 10 ml and tightly combine Dooaaee homogena. Homogenization was carried out with 30 strokes of the iser (highest setting). cell homogenate Bsck ■■ Swing arm rotor of centrifuge (SV40. 1) for 20 minutes, Centrifugation was performed at 500 g, and the supernatant was collected and stored. Pellet in 10ml of another lysis solution Rehomogenized and pelletized as above. Collect the supernatant and incubate at 12,800 g for 20 minutes. It was allowed to settle for a while. Resuspend the membrane pellet in lysis buffer (prepared in lysis solution). ) Mixed with 15 ml of 40% (w/v) sucrose to make a 30% solution. with a long tube Using the 20ml syringe provided, add 40ml to the bottom of the 30% sucrose solution in the centrifuge tube. A layer of 15 ml of % (W/V) sucrose was carefully made. Next, insert the centrifuge tube into the 5445 After centrifugation at 0g for 4 hours, a white film was visible at the gradient interface. Collect the membrane layers with a pipette. The mixture was precipitated at 45,000 g for 1 hour. Bellet 10m M lri+-f lcl, pH 7.4 and stored at -20°C. (ii) Screening procedure Purified plasma membranes from various cell lines (Table 9 (Figure 80)) were Separate 12% (W/V) polyacrylamide gels dissolved in solubilization buffer. I put it in the well. Propagate the separated proteins onto nitrocellulose as described above. and probed for reaction with glutamine transporter antiserum. 2. Measurement of glutamine uptake in other cell lines The uptake rate of the mineral was also measured using the following procedure. a, Glutamine uptake procedure in other tissues and analogue inhibition experiments Effect of glutamine analogs on the rate of glutamine uptake into HeL1 cells and to measure the rate of glutamine uptake into other types of adherent cells. We developed a new measurement procedure to measure uptake into adherent cells. floating culture described another procedure for measuring glutamine uptake into nutrients. (i) Adherent cells Adherent cells were grown in 6-well culture plates until confluent. incubation All scintillations were performed at 37°C, with 6 x 4 ml scintillator tubes placed directly above each well on the lid. Add new solution using the modified culture plate lid (above) with the vial inserted. Each was moved. The normal procedure is to pour out the old solution from the culture wells and invert the plate. and tighten the lid with fresh solution in the quenching vial. next, Reverse the clamped assembly and simultaneously supply the culture wells with fresh solution. Two hours before each experiment, cell cultures were incubated in fresh medium containing 10% (v/v) FBS. 199. The medium was then diluted with 2mM unlabeled glucose in medium 199. Replaced with luteamine and incubated for 20 minutes. Remove this medium and add HBS Glutamine at the test concentration (0.25-5.0 mM) in S was replaced for 3 min. and then add the test concentration of 140-glutamine (62,5μCi/ ml) for exactly 1 minute. Wash wells three times with water-cooled 0.9% NaCl and remove. The loading was stopped. 5% (w/v) trichloroacetic acid 0. Incubate with 5 ml to remove soluble amino acids. No acids were removed, and radioactivity was measured using a scintillation counter. remaining protein was dissolved in IMNaOH for 2 hours, and the amount of protein present was measured. The result is protein Expressed as nmole of glutamine taken up per minute per mg. (11) Floating culture Suspension culture uptake experiments were B+adfo+d and MeGi Ma I11. Biochimict el Biopb7ii+t^c1. , 69g, (1 9g+155-56. 2mM glutamine to cells pre-added for 20 minutes and then incubated for 3 minutes with the test concentration of glutamine. I did it. Radioactive glutamine (0.5 μCi/ml) was then added to the cell suspension. Add for 1 minute, then agitate the cells through silicone oil to stop uptake. I let it happen. A better procedure for measuring glutamine uptake in suspension cultures is to use a tritium standard. water (final concentration 5 nCi/ml) and 2mM glutamine. Resuspended in containing HBS for approximately 20 minutes. At time 0, aliquot 0 of the cell suspension ．． 002 μm in 1,5 μm containing a glutamine solution of appropriate concentration (in HBSS). ml microfuge tube to give a test concentration of 2mM. After 3 minutes, add 200 μl of 14C-glutamine to the microfuge tube and incubate for the next 1 minute. During this time, 100t11 of this aliquot had been warmed to 37°C and a 12% (V/V) Add to a 300 μl microfuge tube containing 20 μl of perchloric acid; A layer of 50 μl of silicone oil was made. After 4 minutes, remove the 30μm microfuge tube. was rotated for 1 minute and then placed in the freezer overnight. Cut the bottom of the microfuge tube and add a scintillator containing 200 μl of PBSA. The mixture was placed in a vial and shaken vigorously. Add the scintillating liquid, shake the vial again, then add the scintillating liquid. Radioactivity was measured using a radioactivity counter. Ink for 20 min for transport inhibition experiments. Inhibitor/analog with HBSS/2mM glutamine during cubagedine added. 3. Results 0-25-4. Glutamine to various cell lines at 0mM glutamine concentration range The uptake speed was investigated. Uptake curves for various cell lines are shown in Section 7a- It is shown in figure g. 0. Uptake rate at 75mM glutamine (physiological concentration) A summary of the results is given in Table 6 (Table 77) along with results from the literature on various tissues. Figure). All of the solid tumor cell lines studied showed high uptake rates. Except for the normal cell line MRC5 and the lymphoblastic leukemia cell line MOLT3. Most normal and lymphocyte-derived cell lines (both normal and cancerous) have low uptake. The speed was shown. Table 9 (Figure 8) shows the effects of mice and mice on the glutamine transport protein of He1g cells. cites immunoplot analysis of cell lines using rabbit antiserum. positive resistance The reaction is indicated by a colored band appearing on the plot at the same position as molecular weight 42,000. Ta. A representative immunoplot showing both positive and negative results is shown in Figure 26. many others The band reacted nonspecifically with the antiserum, indicating that some cross-reactivity occurred. It suggests. Table 10 (Figure 81) lists cell lines, mouse or rabbit anti- Their response to immunoplot analysis with serum and 0. Glue at 75mM The rate of tamin uptake is mentioned. List only cell lines with complete data Ta. Table 9 (Table 80) on the specificity of different cell types for glutamine transport proteins Figure 6 shows the results shown in Fig. 6 and the glutamine uptake rate in various cell lines. Comparing the results in the table (Figure 77), it is found that the antibody It becomes clear that specificity correlates with glutamine uptake of cell lines. anti-blood Cell lines that responded negatively to the serum also had low glutamine uptake rates. 10th A two-way contingency test for the results in the table (Figure 81) shows that the independent The hypothesis of lack of antibody specificity or antibody specificity is 0. It was rejected with a probability of 005. One goal of these studies was to determine whether the rate of glutamine uptake is associated with a relationship between rapidity and malignancy. The objective was to determine whether a significant correlation existed. Table 6 (Figure 77) shows the six All solid tumor tissues have a glutamine uptake rate of 20 nmo+/min/mg protein. This shows that it was more than white matter. Burkitt's lymph all grown in suspension culture. The tumor appears to be intermediate between a solid tumor and a leukemic/normal cell population, with two of the three showed a positive antigen-antibody reaction against HeLx cell glutamine transporter antibodies. In Table 9 (Figure 80) and Table 10 (Figure 81), the glutamine uptake rate is It was low. One of the normal cultures had a fast glutamine uptake rate (MRC 5, Table 5 (Figure 80)). Normal cells are better than predicted in yield+o. 1n and 1m0 have a much slower growth solubility, and for obvious reasons , the transformation of cultures from slow growth and limited survival to rapid growth and long survival. glutamine transporter activity. In this respect, this is due to one of the natural harms of this type of research. As a result of the above findings, blood supply is usually insufficient and therefore unavailable in solid tumors. Glutamine uptake may be slowed down because all of the glutamine must be taken in. It suggests that. On the other hand, it is readily available to cells normally present in blood or lymph. There is sufficient supply of available glutamine, so the inhibitory mechanisms are not initiated. complete information Among the 3 lymphoblastic leukemias and 3 lymphomas obtained, 3 leukemias All diseases show negative responses to transporter antibodies, one of which is the rate of glutamine uptake. (this is the only example to date showing this relationship); two of the lymphomas are One showed a positive response to the carrier antibody, but not one.・In lymphoma, all three groups Tamin uptake rate was low. Bo+dinEpNein-Bg++ transformation The lymphocytes were placed in the same location as the lymphomas because they are the closest culture equivalents. Thus, one of the lymphomas presented the opposite situation to the one of leukemias. These results suggest that transfection of two or more cells until a fully solid tumor state is achieved. There is an Omeshi rose step, which is one of the above two intermediate states, i.e. is an intermediate state due to a partial increase in the glutamine uptake mechanism, and the other is a partial decrease in the glutamine uptake mechanism. It is suggested that this can be explained. 6 solid tumor cells (cells grown in suspension culture) and 15 other tissues to obtain the mean significant difference between When a 1tadent t-test was performed on the data in Table 6 (Figure 77), it was found that the tumor The average is 39. 4nmol/min/mg protein, S±14. 8 and other organizations 8. Onmol/min/mg protein, S±8. It was 0. From now on, t=6. 0, and with 19 degrees of freedom it becomes 0. There is a probability that the difference between the two averages is far greater than 005. There wasn't. Differences in the antigenicity of glutamine transport proteins between normal and tumor cells result in polyclonal Numerical antibodies and monoclonal antibodies can be used in cancer therapy and diagnosis. Table 9 (8th (Fig. 0), all six solid tumor types were positive, and three Burkittrin cells were positive. 2 of the leukemias are positive, all 5 of the leukemias are negative, and 4 of the normal cells are negative. All were found to be negative. Antiserum against purified glutamine transporter protein The uniformly positive response of solid tumors upon probing with glutamine transporter antibodies Clinical use suggests it is effective against solid tumors and at least many lymphomas are doing. Table 10 (Figure 88) shows that the antibody reaction results indicate the glutamine uptake rate. glutamine transporter derepression is likely to be a key factor in solid tumors. The above suggestion that it is a specific marker was confirmed. 111, antibodies against human amino acid transporters.Another embodiment of the invention is the human amino acid transporter of the invention. Consists of monoclonal and polyclonal antibodies that recognize amino acid transporters. ( The method for producing polyclonal antibodies against TGTII (above) is similar to other human amino acid transporters. It can also be used to produce polyclonal antibodies against the body. In addition to polyclonal antibodies, antibodies specific to epitopes on human amino acid transporters It is also desirable to have clonal antibodies. For example, since TGT I+ is common to other tumor cells, TGTII Monoclonal antibodies directed against the epitope may be useful in diagnosing the presence of cancer. mono Clonal antibodies are produced using recombinant clones to isolate the gene encoding TGTII. It can also be used for cleaning procedures. Any transporter monoclonal antibody can be used to target TGT. It can be produced using a procedure similar to the following method for producing a monoclonal antibody that recognizes it. 1, e.g. purified dissociated TGT, purified raw TGT, raw crude membrane protein or Peptides derived from transporter amino acid sequences can be used to test mice, rats, or other animals. immunizes the substance against the glutamine transporter protein. 2. Myeloma cells removed from the pancreas of an immunized mouse and cultured in a fusion experiment to be fused with. 3. Plant the fused cells in culture dishes under the conditions selected for hybridoma cell growth. . 4. Once the hybridoma colony starts to grow, screen the hybridoma. Start the process. Many types of monoclonal antibodies that bind to tumor glutamine transporters have been developed. Screening procedures can be used. One screening method is ELr SA The process consists of the following steps: 1. Coat a (eg 96-well) plate with purified transporter protein. 2. Add supernatant from hybridoma colonies to coated wells (positive antibodies will bind to the corresponding epitope of the transporter protein). 34. Wash away excess antibody. 4. 1gG conjugate (anti-mouse antibody conjugated to an enzyme or other reporter molecule) Add to well. 5. Wash away excess conjugate. 6. Add enzyme substrate and incubate wells with supernatant from positive clones. A color reaction can be seen in the sample. 7. Multiply positive clones by culturing. 8. Positive clones can be propagated in large numbers by injection into the ascites of mice. However, due to the unique properties of TGT, glutamine transporter inhibition assays have been used to Can screen for bleedoma. Monoclonal antibodies that bind to transporters are inhibits luteamine transport and can be identified using this method. The screening procedure consists of the following steps: It consists of 1. HeLx grown to confluence in 7596 (e.g. 96-well) plates Inoculate cells. 2. Add high concentration of glutamine (2-3mM) to HtLs cells in advance. 3. Add the upper groove of the proliferating clone to the well (positive monoclonal antibodies should be added to the outside of the membrane). bind to the active site of the transporter protein exposed on the side surface). 4. Wash away excess antibody and add a test concentration of glutamine containing 14C-glutamine. Incubate the cells with min for the correct amount of time. 5. Remove radioactive glutamine by washing with ice-cold 0.9% (w/v) NaCl. stop the crowding. 6. Quantify the amount of label present in the cell chamber by radioactivity measurement (trichloroacetic acid using acid to extract soluble components). 7. Evaluate the total amount of protein present by LowB analysis and analyze the results per mg protein for 1 minute. Expressed in nmol of glutamine taken up per unit. 8. Results for each clone tested (not including glutamine transporter protein) b) Compared to the control using control serum, the glutamine uptake was 2% compared to the control. Samples with 0% or more inhibition are considered positive. 9. Cultivate or inject into mouse ascites to multiply positive clones in large numbers. Producing antibodies against glutamine transporter regions that are distant from the glutamine binding site You can also do that. Such antibodies may also be useful in diagnosing the presence of tumor cells. However, it cannot be screened using the inhibition assay described above. rather the other way It can be identified by First, if the receptor is isolated, the antibody will bind but not to glutamate. It does not inhibit the binding of min. Second, we used cells with many glutamine receptors to and compare the results with other cells with no or few receptors. reactivity This difference suggests that the antibody may be a glutamine receptor. Such monochromes directed against the receptor molecule itself or the binding site of such a molecule. The study of null antibodies is common, usually using a variety of diagnostic tests to determine the extent to which antibodies are present. and to which parts of these molecules it binds. In most cases, These tests include direct serological assays such as cytotoxicity or cell binding. Includes flow cytometry, which uses differential reactions to show which molecules are more susceptible. this Such studies can then be carried out using, for example, immunoprecipitation using radiolabeled cell membranes, or fractionation. When the offspring can be identified, a sea urchin plot (immunoplot) type analysis is performed. You can try it out again. These tests identify the molecules that antibodies bind to. However, it does not specify which parts of the molecule are reactive. Radioactivity to receptor molecules A follow-up test can be performed in which antibodies inhibit the binding of labeled glutamine. Up These tests are often direct, as described above, and measure the binding of the antibody to a functional molecule. It is the very standard of analysis. Apparently, the antibodies obtained depend on the proteins used to immunize mice and other animals. will. For example, when obtaining TGT II from 5DS-PAGE, protein The white matter may be of the "dissociated" or "degenerative" type. Depends on the tertiary structure of the transporter protein No antibodies are formed. Therefore, for example, monochromatic molecules that depend on the tertiary structure of TGTII To obtain national (or polyclonal) antibodies, immunize animals with the raw protein. Must be sensitized. This can be done in at least two ways. First, animals are immunized against a portion of the tumor cell membrane containing TGT II. Can be sensitized. Next, use the same monoclonal antibody against the live form as above. It can be screened by this method. In addition, the above-mentioned recombinant suspension obtained Animals can be immunized with purified raw protein. In summary, the type of monoclonal antibody obtained was determined only by the amino acid transporter used. It will depend on whether the transporter is in native or dissociated form. similarly , whether the resulting antibody uses the whole protein or only fragments or subunits. It may depend on what you use. Monoclonal antibodies against glutamine transporters have been tested in mice, rats, other species and Eventually, it can be produced in humans. These various monoclonal antibodies all target a part of the glutamine transporter or a subspecies thereof. As long as the unit can be recognized, it is within the scope of the present invention. Additionally, antibodies are or as a moiety (e.g. r1b'2 or F+b), here a single chain It even talks about antibodies, where specificity is recognized to be due to retention of hypervariable regions. It is. In addition, such monoclonal antibodies can be produced by genetic engineering, and one Hybrid chimeras are created by copying parts of a sequence from a species onto the genes of the same or another species. can have a child. Has the ability to bind to glutamine transporters (in part or in whole) All such antibodies are within the scope of the present invention. IV. Manufacture of transporters using recombinant methods cDNAs and genes from various sources and species transporter (or its sub-species) using standard methods including blobbing of both The N-terminal and partial amino acid sequence of the oligonuclease unit) can be obtained. Reotide probes can be synthesized and CDNA and genomic clones can be obtained. The isolation of the glutamine transporter described herein naturally results in cD in one species (human). Although cloning of NA and genomic clones can be performed, species cross-hybridization It may also be possible to use rod formation to isolate transporters in other species. gene black There are many methods used for do. These include polyclonal or monoclonal antisera and expression systems. use of; isolation in cells to isolate cDNA by antigen expressed on the cell surface; Transfection method; Metsenger RNA selection procedure: Gene cloning or the identification of restriction fragment length polymorphisms that lead to translocation silanes. can. These methods are now standard and are used in many laboratories around the world. It is used in , and there is no need to describe it in detail. Important features are cDNA and genomic muclones and genetic polymorphisms, histological polymorphisms, gene rearrangements or alternative splats. those by ising or any other procedure that alters the glutamine transporter structure. The nucleotide sequence obtained from a variant of. E. coli cells, other bacteria, yeast, and higher eukaryotic cells can be used to copy cDNA into expression vectors. Synthesize foreign proteins such as glutamine transporter protein by You can make it so that This is first known to express the desired protein. Isolate (total) mRNA from the tissue containing the tissue, generate cDNA from the mRNA, Reverse transcriptase is used to generate the first strand and RNase converts the RNA template. It is carried out by degrading the DNA and synthesizing the second strand with DNA polymerase. . The double-stranded cDNA is then transferred into a suitable expression vector, either a plasmid or a phage. Clone to The cloning sites of these vectors are (1) fusion protein or (2) the 5° coding region of the regulatory gene that synthesizes the non-fusion protein. The insertion is immediately downstream of the appropriate promoter/liposome binding site. next, Transfer colonies (plasmid vectors) or plaques (phage vectors) to appropriate Screen for desired protein production with a probe and produce the desired protein Clones are isolated and expanded in large quantities. Thus, for example, a protein that reacts with a polyclonal antibody against a purified transporter protein. Many λgtll clones can be isolated that synthesize protein. A. Cloning experiment A cDNA clone capable of expressing the glutamine transporter protein (or a portion thereof) in a prokaryotic system. The availability of this protein makes it possible to produce large quantities of this protein. pure substances can be used for diagnostic testing or therapeutic purposes. In addition, (monoclona for the production of antibodies (both polyclonal and polyclonal), and much more New chemical compounds (or novel monochromes) with high glutamine uptake activity can be easily identified. local) can be used to screen. cloned As a first step toward the production of transport proteins, for example, typical HeL* The cell cDNA library was screened with polyclonal antisera against purified transport proteins. One can lean, isolate, and purify several positive clones. The next section describes the properties of cDNA libraries and the screening methods that can be used. As mentioned above, this means that there are many different cloning methods that can be used in accordance with the present invention. Merely one method, the invention is not expressly limited to any one method. do not have. Other such methods will be readily apparent to those skilled in the art and are equally within the scope of the present invention. is within the range of 1. Characteristic value of HeLt cell c DNA library 1 --9x109 pf u/ml capacity +0. 2m1 mRNA1lI: D98-AH2 cells derived from HcL* cells (phenotypically clonal) ・EcoRr Scope of screening (recombinant vs. non-recombinant) Clear plaque vs. blue plaque Proportion of clones that are recombinants in this range: 81% Number of (transparent) plaques: 1. 3 Average insertion size 0. 86kb (range/ 0. 48-3. 1kb) 2, HeLi cell c DNA library with antibody preparation lee screening The basics of one screening method that can be used are as follows. When cDNA is cloned into the EcoR1 site of λgtll, (the phage The encoded β-galactosidase not only deactivates the insertion but also corrects the insertion. When the cDNA insertion is in the correct orientation and reading frame, it is expressed as a fusion protein. Yes, this can be detected with antibodies specific to the protein encoded by the cDNA. Aesop Lopil b-D-thiogalactopyranoside (IPTG) and nitrocellulose filtrate The phage was induced using the fusion protein linked to the target. Next, dilute primary antibody (in this case a polyclonal antiserum against a purified transport protein in the mouse host) to wash away unbound primary antibody, then remove the enzyme-conjugated primary host antibody. Detect the bound primary antibody and screen the filter using a secondary antibody against -ning. In this case, the secondary antibody conjugate is goat anti-mouse IgG (H+L) Affinity purification using alkaline phosphatase conjugate. Next, the colored ho Sphatase substrate cocktail phosphate 5-bromo-4-chloro-3-indole (BC conjugated phosphatide using IP) and nitroblue tetrazolium (NBT). Detect the tase-conjugated secondary antibody. a. Screening protocol You can use the following steps. 1. Proliferation of flat cells. pH 7.5 containing 100 μg 7’m+ ampicillin A single colony of E. coli Y1090r is placed in a striped pattern on an LB agar plate. Incubate overnight at 37°C. cuvate. Starting with a single colony from the plate, at 37°C with air passage Grow Y1090r to saturation in LB (pH 7.5). 2. Infection of cells with cDNA library. per plate for screening Cell culture grown overnight (Y1090r) 0. 2ml and 3x1 library Phage dilution containing 04 pfu (10mMT containing 10mM MgC12) ris-HCl, pH 7.5) O, 1 ml can be used. currently available Using an antibody preparation that requires filling four plates will provide some positives. It's a minute. 3. Flattening of cells. LB soft agar (pH 7.5) (preliminary) was added to the phage and bacteria mixture. per card) 3. Add 5 ml to the 2-day old LB agar plate (pH 7.5). I poured it. Incubate at 42°C for 3 hours. 4. Add a nitrocellulose filter. Place the plate in a 37°C incubator Put it in. Nitrocellulose filter disks with 10mM (aqueous) isopropylene Pre-soaked in β-D-galactopyranoside (TPTG). Dry r　Treat with PTG Place the treated filter on the plate and incubate at 37°C for 3.5 hours. . 5. Processing of filters for screening. Remove the plate and let it come to room temperature. Mark the filters and arrange the filters with plates in succession and place them on the agar plate. Carefully remove the filter and add 150mM NaCl and 0. 05% (v/ v) 100mMT+low HCI (pH 8, ol) containing Tve+n 2G ( Wash the filter with TBST solution to remove any agar attached. non-specific TBS containing 1% bovine serum albumin to saturate protein binding sites. Incubate the filter in T solution for 30 minutes. 7 for each filter. Use 5 ml of solution. 6.-Pretreatment of next antibody. - Antibody solution (i.e. mouse anti-transport protein) to the large intestine Immediately after treatment with the bacterial extract, the serum is used to screen the nitrocellulose filter. training. Antiserum 1/100 μg of E. coli extract for 10,000 dilution /ml and incubation may be carried out for 30 minutes. 7. - Incubation in the swab. TBS containing 1/100 dilution Incubate filters in T for 30 minutes. 7. solution per filter. 5m Use l. 8. Clean the filter. Aliquot 20 ml of TBST for at least 3 min for 10 min. Wash the filter twice. 9. Incubation run in secondary antibody. Next, the secondary antibody diluted to 1/7500 body conjugate (affinity purified goat anti-mouse T gG (H+L) alkaline mouse Transfer the filter to fresh TBST containing (fatase conjugate) and filter Hit 7. Use 5 ml and incubate for 30 minutes at room temperature. 10.Cleaning the filter. Repeat step 8 above. 11. Incubation with phosphatase substrate. Soak the filter onto filter paper. Remove, dry, and then transfer to substrate coloring solution. 100 for 5 ml of substrate solution 100mM Tris-HCI containing mMNaC+ and 5mM MgCI2 ( Add nitro blue tetrazolium (NBT) stock solution (pH 9.5) to 5 ml (pH 9.5). Add 33 μm (50 mg in 1 ml of 0% dimethylformamide). mix and then 5-bromo-4-chloro-3-indolyl phosphate (BCIP) stock solution (dimethyl Formamide 1ml 150mg) 16. Add 5 μl and mix again. strong from a distance Incubate in bright light for 1-4 hours. Positive clones are on the filter It becomes a purple plaque. 12. Stopping color development. Before the plaque becomes visible and the background darkens, the substrate solution Discard the solution and add 20mM blind 1s-HCI containing 5mM E D T A ( Transfer the filter to pH 8.01 to stop the phosphatase reaction. Next, Keep the filter dry or store it in this solution. 51st screening Primary screening generally yields an average of two positives per plate, with - The overall frequency of positive screening is approximately i, per 5 x 10' plaques. It is 1. − Take the putative positive clones from the next screening plate. and transfer to a secondary screening plate of host bacterium E. coli Y1090r. positive Plaques were arranged in a grid mold, and negative (control) plaques were placed on each plate. Add. - Process the plate as for the next screening (steps 4-12) . - Approximately 75% of presumptive positives from the secondary screen The test results are positive. Repeating this purification procedure twice will result in a third screening process. The frequency of positive results is approximately 80-100%, and at the fourth screening All plaques may be positive. Positives from 4 screenings was grown in large quantities, and the standard method (Dttit et al., 8thic Method+ i nMo1ccalt+Biolog7. El+evie+? bli+hc ++, New York). The cloned live glutamine transporter molecule or its fragments are then used to monopolize Both clonal and polyclonal antibodies can be produced. In addition, we determined the sequence of the dissociated protein obtained from 5DS-PAGE and analyzed the amino acids of the transporter. I was able to get the array. Prepare a DNA probe from the amino acid sequence and probe the DNA. A cDNA library of complementary strands can be probed. cDNA clone or using oligonucleotide probes or other techniques known to those skilled in the art. , genomic clones can be isolated from genomic libraries. Also, this reagents can be used to isolate homologous cDNAs or genes from humans or other species. . In this way, a gene encoding the production of raw protein is obtained. As described above Make it manifest. It should be emphasized that there are many different cloning methods that can be tried. above If one method as detailed fails, one skilled in the art will be able to use other methods that work well. You will recognize it. Guidance can be found in the following literature: DNA CIoning; A P+5clicsl^pprastb, Vol, l-1119M, D, G ed., 1. R, L, P+e+s. WstbiB+on D, C, (1985): Cot+eol Proloco l+ In Molccu11rBiolog7. 　Vol, I and II, F, 11. Edited by Aasabcl et al., Wilt7 & So++ (+9!171; M olecwl*t C1oniB, A LIbor [or7 Mt++ul. T, M*a lis et al., Co1d Sp+iB Harbor Lsbozlo ry　f19821゜B, change in arrangement The nucleotide sequence encoding the transporter may vary for the following reasons. 1. Degeneracy in genetic code nucleotide changes does not necessarily result in changes in the amino acid code For example, the codon GUU is similar to the codon GUC%GUA, GUG. identify valine residues, each differing by one nucleotide. 2. Even if two or three nucleotides change, the same amino acid can result, e.g. For example, the codons UUA, UUG, CUU, CUCSCUA and CUG are all leuci code. The codons AGUXUCCSUCU, UCA and UCG represent serine. Code. 3. Changes in one or two nucleotides have no significant effect on protein function. No mild amino acid changes occur. For example, the codon UUC specifies leucine , AUU specifies isoleucine. In addition, UCG has identified tryptophan. However, UUU specifies phenylalanine. All changes are gradual. 4. Allelic variation. Nucleotide and amino acid sequences of encoded proteins Changes in and their consequences can occur between members of the same species. These changes occur in proteins. due to changes in the nucleotide sequence encoding the quality. Therefore, (called an allele) Different types of the same gene have slightly different amino acid sequences but have the same function. The proteins that are present are produced. 5. One gene, mature protein, consisting of many different segments (exons) of the coding sequence The DNA encoding white matter has certain exon-derived RNA parts removed. changes as a result of differential mRNA splicing resulting in RNA spliced in a manner similar to may occur. Exon selection may vary depending on cell type. 6. Proteins with stiffening functions, such as major histocompatibility proteins, are linked to related genes. obtained from. Many protein gene groups have been described, including nucleotide and immunoglobulins that have altered amino acid sequences but retain their primary antigen-binding function. is listed. Such cognate proteins are encoded by cognate genes. child These genes result from duplication or gene conversion of one original gene. Changes can be introduced intentionally as follows: 1. Point mutations, rearrangements, or cDNA or genomic clones encoding functional proteins cDNA or cloned by inserting related or unrelated DNA into the clone. or mutate genomic DNA. Such mutant clones (variants ) is used herein to generate a mutant protein or protein that may have a glutamine transport function. can produce peptides. 2. Proteins (synthesized in mil+o or from proteins synthesized by normal cells) The cleavage product may or may not undergo repair/rearrangement. Ru. 3. Chemical modification 4. Radioactive irradiation The present invention provides fragments (peptides) of glutamine transporter and synthetic or genetically engineered fragments (peptides) of glutamine transporter. It also includes modified variant peptides. Equivalent at the nucleic acid, protein and functional level Other transporters enter the species. Due to substantial sequence homology, c as described herein DNA cloning will allow the isolation of related sequences. ■ Anti-glutamine compounds and glutamine analogs Another embodiment of the present invention is Anti-glutamine compounds and glutamine analogs suitable for the treatment of cancer are provided. this is a compound having the following general formula (1) or a physiologically acceptable salt thereof: Select from CR5R6, NHlNOH and O. W is II II II Choose from. ■Has Select from; ! +3 R is selected from OCH, , Ph, OR5NH2 and NHNH2 13 is RH and Cl-5” substituted or unsubstituted, cyclic or acyclic, saturated or or unsaturated hydrocarbon groups. R is selected from the group defined by RSNH, (C=O)R13, and (C=O)0R13 death: R3 is selected from the group defined by R13; R is R, halogen and (C=O)R'; selected from the group specified; R and R are each independently selected from the group defined by halogen and R13. R is selected from the group defined by OR and NR15R16, and this is 14 + 5 1. R, R and R are independently H-Ct-s substituted or unsubstituted, saturated or unsaturated. , cyclic or acyclic hydrocarbon groups (and substituted or unsubstituted 05-6 aromatic or (heterocycle). R is selected from the group defined by R, OR, amihamide, No and No2; R9 is selected from the group defined by R14: or R8 and R9 together with the nitrogen atom Represents a 3- to 8-membered substituted or unsubstituted, saturated or unsaturated heterocycle; R is R, OR 14, selected from the group defined by amino and amide. 11!2 R and R are independently selected from the group defined by R14 and amide. Mata, V-W-X-CH2 is a group. [wherein R is selected from the group defined by R, SoC1 and (C=O)-R14; and R and R are independently R14, halogen, and the α carbon to the 4-membered ring is a leaving group (e.g. (including but not limited to OMs, OTs and halogens) selected from R14 groups]. In other examples, V-W-X is a group: [wherein R is from the group defined by RSOR and NHR14 and <c=o>R14 choose. or zR21 is hydrogen; Z is selected from 0, S and NH14: or unsubstituted, cyclic or acyclic, saturated or unsaturated hydrocarbon group choose; Or, R20 and R21 are groups: and a 5- or 6-membered heterocycle, which may or may not be further substituted. form]. The important group of compounds defined by formula (1) is general formula (II): [wherein X is CH, N selected from HlNOH or O, R1, R, R and R9 are as defined above] It includes, but is not limited to, the compound or a physiologically acceptable salt thereof. The important compound group defined by formula (+1) is general formula (II+) [In the formula, X is CH2, NHSN is OH or O. R is defined by OH, NHR, (C=O)NHR14 and (C=O)N(R”)OH R23 is H when selected from the group. From the group where R11 is defined as (C=O)NHR” and (C=0)NCR”)OH When selected, R23 is OH. or R23 and R8 are independently selected from the group defined above for R14. Or R23 and R8 together with the nitrogen atom are saturated or unsaturated, substituted or unsubstituted. Forming a 5- or 6-membered heterocycle that may be Not allowed. An important compound defined by formula (II) is formula c, where R and R9 are as defined above. [synonymous] and physiologically acceptable salts thereof; [In the formula, R26 is H, OH or NO, 24 25 13 N + 3 R and Rfi independently HlOR, SRSNR5No2, CHOSCO2H and halo a compound selected from Gen, where R is as defined above] and its physiologically acceptable Salt; C・Formula: Compounds of [wherein R and R28 are independently selected from H and OH] and their products Select from physiologically acceptable salts, where X is OH, OM, OT. or halogen, and R and R are independently selected from the group defined above for R13. and physiologically acceptable salts thereof, but are not limited to these compounds. Not done. rcl-5 substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic hydrocarbon group The term j includes alkyl, alkenyl and alkynyl, containing 1-carbon atoms. 5 linear or branched chain or cyclic groups, which are responsible for the glutaminosis of the tumor plasma membrane. assist in the binding of such compounds to transporter proteins, or Even if the compound is substituted with a substituent that modifies its action, it is still called a "3-8 membered ring hydrocarbon". The term includes 3- to 8-membered saturated or unsaturated hydrocarbon rings; It may contain three heteroatoms (N10 or S), which is a group of tumor plasma membranes. assist in the binding of such compounds to rutamine transporter proteins or It may be substituted with substituents that modify its action after delivery. Preferably, a "3-8 membered ring hydrocarbon" includes one or more polar groups such as OH, SH, NHR, No2, CHOSCOOR or halogen (where R13 is the same as above) selected from C5-6 aromatic or heteroaromatic hydrocarbons substituted with do. The term "amino" refers to primary, secondary and tertiary amino groups NR8R (where R8 and and R9 have the same meanings as above). The term "amide" refers to C(0)NR8R9, where R8 and R9 are as defined above. righteousness). Possible salts of the compounds of general formulas (1), (+r) and (TII) include, for example, acids and Includes all base addition salts. Physiologically acceptable salts are generally inorganic or organic acids. or from bases. Physiologically acceptable salts may, for example, first be added to the process product e.g. Obtained as a process product in the production of the compounds according to the invention on an industrial scale and known as It can be converted into a physiologically acceptable salt by the following method. Examples of physiologically acceptable salts are water-soluble and water-insoluble acid or base addition salts; For example, hydrochloride, bromate, iorate, phosphate, nitrate, sulfate, acetate, citric acid. acid salts, gluconates, benzoates, butyrates, sulfosalicylates, maleates , laurate, malate, fumarate, succinate, oxalate, tartrate, sulphate Thearate, tosylate, mesylate, salicylate, sodium, potassium and ammonium salts. A. Synthesis of anti-glutamine compounds and glutamine analogues Compounds of general formula T-111 The compounds can be synthesized by a number of different methods as outlined below. The synthesis method is generally , X1W and the properties of V. Generally, a protecting group is required and the precursor The choice of protective group for the product will depend on the functionality required in the final product. protecting group The use and methods of protection or deprotection are well known to those skilled in the art (e.g. T, W, Greene, 'P+olctingG+oap+ in O+gxn ic 57nlhe+口’, John WilB & Song, 1981) . Similarly, various groups that can constitute R1-R4 are well known, and the introduction of groups has not been reported. using the synthetic methods described or techniques from the reported methods and disclosures herein. This can be implemented by simply combining the following. Order of substitution and substitution in synthesis The timing of may vary depending on R1-R4 and other substituents. Simply put, the Those skilled in the art will know a variety of ways to prepare the compounds of this invention, including X, W, V and R1- Depending on the particular group chosen for R4, it may be necessary to adapt the synthetic method. Compounds of formula 1-111 in which X is CH2 and W is c=o can be prepared using a suitably protected group. From derivatives of rutamic acid, as shown in Schemes 1 and 2 (Figures 51 and 52, respectively), It can be synthesized by sea urchin. These protected derivatives include pyroglutamic acid derivative 2 and 3 and the semi-monoesters of glutamic acid 6 and 7. Many of these compounds Although some are commercially available, it may be cheaper to synthesize them as needed. As shown in Scheme 2, there are two main methods: mixed anhydride e.g. Activation of compound 6 or 7 by formation of a naturalized ester, e.g. may be suitable for nuclear substitution by minR8R9NH. Both methods It has been reported in the literature (eg, GoodIn et al. (1962) 1. A11 Chew, Sec., 84. 1279; ^ade+soa et al. (+9671 1. ^■, Che■C1, 2896; Kin et al. (1975) 1. 　 Chew, Soe, Chew, Cos++++++++,. 473) and is also described in the Examples below. Compounds of general formula I in which X is CH2 and W is other than C=0 are suitably protected dihydro Derivatives of alanine, for example 10, can be substituted with one phosphoryl carbanion 11 or one sulfonate. It can be produced by reacting with nylcarbanion 12. Also, in diagram 3 (Figure 53) As illustrated, diethyl acetylaminomalonate 13 and appropriately assimilated phosphorus Synthesis of these compounds by reaction with vinyl phonate 14 or vinyl sulfonate 15 (e.g., Mtict et al., (19831 Pbosph) oru+ 5ultn+, 17. (See 1). W is SO and X is CH2 It can also be synthesized by assimilation of homocystine or homocystic acid. Compounds of formula 1-111 where X is O and W is C;0 are generally suitably protected. Acylation of serine derivatives such as 16 (Scheme 4. (Fig. 54). When V is represented by NR8R9 (where R8 and R9 have the same meanings as above) would form a chloroformate by the reaction of phosgene with the protected serine. This product is then added to the nuclear amine R8R9 as illustrated in Scheme 4 (Figure 54). Reaction with NH would give carbamate 17. If W is other than C=0, the formula ■The compound is a serine derivative and a suitable sulfonic acid or phosphonic acid or activated It could be synthesized by reacting with derivatives (eg acid halides). Compounds of formulas I-III, where X is N or N-OH and W is C=O, have the formula 5 (Figure 55), appropriately protected 3-aminoalanine 18 or 3 - Hydroxylaminoalanine 19 with a suitable isocyanate 20 or acyl halide It can be synthesized by reacting with Ride 21. When W is other than C=O, compounds of formula I are It can be synthesized using a procedure similar to that shown in Equation 5. In these cases, appropriately protected 3-aminoalanine derivatives 18 or 3-hydro Appropriately substituted phosphoryl or sulfonyl xylaminoalanine derivative 19 It could be reacted with a halide (Scheme 6. Figure 56). [In the formula, R17, R18 and R19 have the same meanings as above] The compound of the formula , an appropriately protected alkenylglycine 2 as illustrated in Scheme 7 (Figure 57). From 2, r[2+2] cycloaddition with substituted isocyanate 23 under pyrolysis conditions ” can be synthesized by V-W-X is the group [In the formula, Z is 0, and R20 and R21 have the same meanings as the above definitions] Compounds such as 24 and 24 with suitably protected glutamine under dehydrating conditions include tosyl chloride and and pyridine to form nitrile 25. next , as illustrated in Scheme 8 (Figure 58), nitrile is mixed with alcohol R210H. The iminoether 26 can be obtained by partial solvolysis. For example, H i+ot+u et al. (1970) Biochill. Bioph1+, ACll, 222. See 540. iminoether is imine Further assimilation can be achieved by alkylation or acylation of the nitrogen. Also, a Substituted amide 27 can be converted to imino ether by acylation or alkylation of mido oxygen. Alternatively, iminoester 28 can be obtained. Nitriles 25 or simple iminoethers 26 (R21=Me or Et) are It also acts as a precursor for compounds where Z is S or N. Therefore, 25 or 26 (R2'=Me or Et) with thiol (R"SH) or amine (R"' When reacted with R14NH) (where R and R14 have the same meanings as above), each It is expected that derivatives 29 and 30 will be obtained (Scheme 9; Figure 59). In a similar method, R20 and R21 are groups and a 5- or 6-membered heterocycle which may be further substituted or unsaturated. Applicable to the synthesis of compounds of formula I that form. For example, 25 or 26 (R”=Me or Et) is a β-amino alcohol, 1,000-ol or amine (e.g. NH2CH2CH2ZH; Z=O1S, NH ), dihydroxazole, △2-thiazoline or imidazole, respectively. One would expect to obtain the syrin derivative (31; Z=O1S, NH). (for example ,’Comp+eheisiwe Hefe+oe7clic Cbemi+1 +7' ^, R1Ku+N+ky & C, W, Ree+ @, PeBtwon 　Pess, 1984. V, 5°469, V, 6. 228-229. 3 (see 09). These derivatives can, for example, be desorbed as shown in Scheme 9 (Figure 59). Further assimilation by hydrogen reaction gives the corresponding oxazole, thiazole or imidazo. It can be a metal derivative (32; Z=O1S, NH). This method supports It can also be used in 6-membered heterocyclic compounds and further substituted examples. B. Anti-tumor design strategy Chemical synthesis programs are often highly integrated with biochemistry programs and are It can be processed by molecular modeling using a computer. Biological, as discussed below. Assays: Binding Parameters (BmSKs) and Glutamine Transport Inhibition and Tumor Anti-glutamine compounds and anti-glutamine compounds that are effective in the treatment of at least three cancers as measured by proliferation. Glutamine analogs have been identified. Preliminary molecular modeling studies as a means to identify potential synthetic target molecules Comparisons of overall structures can be made. As more data become available examining newly synthesized compounds, , to clarify quantitative structure-activity relationships for each of the above types. become. Such a relationship may be related to the compound's observed biological activity and within the program. Various physical results obtained from the molecular modeling and/or molecular orbital calculations performed and chemical properties such as minimum energy conformation, atomic juxtaposition, critical Correlate polar moment, static voltage, etc. With this method, the resulting analogs still effectively and selectively bind tumor cells. Successful identification of structural modification of glutamine γ-amide group transported by tamine transporter . Appropriate levels of cytotoxicity can then be introduced into such analogs. . Screening method for Vl, anti-glutamine compounds and glutamine analogs Another embodiment of the invention is to determine the binding parameters of glucose in tumor cells and lymphocytes. transport inhibition of tamine transport and the effects of such compounds on cell proliferation. Includes screening methods for anti-glutamine and glutamine analogs to determine I'm here. From the results obtained (described below), potentially useful anti-cancer compounds can be identified. same type Screening methods to screen other compounds for other human amino acid transporters It will be understood that it can be cleaned. A, bond parameters Binding parameters for anti-glutamine compounds and analogs were determined using the following experimental procedure. The data was measured. 1. Solution All solutions were adjusted to pH 7.2 with 5% (w/v) sodium bicarbonate. Created in BSS. The pre-incubation test solution was 0. 25mM? Containing anti-glutamine compounds and analogs at a concentration range of 20mM The incubation solution contained 1mM glutamine. NEM incubation The solution contained the same concentration of analog or control glutamine along with 1mM NEM. I had it. The radioactive solution was 0.1 m containing 3H-NEMo, 05 mC1/ml. It was MNEM. 2 Procedure 0. 11,600 g of membrane preparation containing 2 mg protein/ml (Eppendo+ Centrifuge for 2 minutes in a t-microscope) and incubate for 10 minutes at room temperature with gentle agitation. Resuspend the cells in 100 μl of preincubation contest solution or control solution. Made it muddy. After 10 minutes, add NEM incubation solution for another 10 minutes at room temperature. 100 μm was added to each sample. The membrane suspension was then centrifuged at 11,600 g for 2 min. The pellet was washed twice with 31 ml of fresh HBS. Relax the membrane pellet for 1 hour at room temperature. The mixture was resuspended in 100 μl of 3H-NEM solution while stirring. After the labeling period, the membrane The suspension was centrifuged at 11,600 g for 2 min and then added to water-cooled 0.9% (w/v) NaC. Washed 3 times with 1ml. Vortex the pellet for 1 minute every 5 minutes for 15 minutes. , dissolved in 200 μm of SDS sample buffer and stored at −20°C. total of each sample Protein content was determined by LowB analysis, and 30 μl of each sample was diluted with 12% polyacrylamide. Membrane proteins were separated by 5DS-PAGE and Coomassie gel. - stained with blue. The corresponding stained proteins, including the glutamine transporter protein, Dissolve in 11 solution of 0,9 mM CuSO with 30% HO overnight at room temperature. and radioactivity was measured. The results are nmo 13H-NEM/mg total protein, Expressed as the amount of label bound to the protein band. 3. Estimation and derivation of coupling parameters The method involved incubating the membrane with 1mM NEM in HBSS for 10 min. Anti-glutamine compounds and glutamine amines have the ability to protect the glutamine binding site when We evaluate the two main coupling parameters of analog. The membrane was first incubated in the presence of NEM. incubated with a range of concentrations of glutamine or analogues (ligands) . The reaction between NEM and thiol moieties is covalent and therefore irreversible; The extent to which the reaction of NEM with the tamin sites is interfered with by the ligand is determined by washing the membrane and then by re-exposure to the same concentration of tritium-labeled NEM (NEM”). was measured. Here, NEM” means that the ligand molecules are Occupies the protected area. for a constant concentration of NEM during the initial incubation. By using a ligand with a concentration range of , a square inverse proportionality plot (Fig. ) to obtain the relative dissociation constant (K゛) and the maximum association constant (B') l　I + It will be done. Reaction diagram 10 below shows that each ligand binding reaction is performed competitively with NEM. Therefore, we show that the former is a relative value and that as long as the binding of the ligand is reversible, This shows that the latter would indicate the absolute value of the maximum coupling. The binding of the ligand If it is irreversible, the rate constant is Jijo Illusionary Liao (C5) km To HE HEH Association 2 to υ−+ C, NEM C, NEM Association to form a tighter bond or covalent complex (CS). first incubation The ratio of C8 to the total carrier (Co) present at the end of the reaction is given by the following formula: : C3/C=S/ (K (1+k, [NEM]’) + SO low 2 (1+3)) [In the formula, K = 1/]. From now on, a new constant: K’ = K (1 + k 2 [NEM ko) I can be obtained, where [NEM] is kept constant during the incubation period. . Basically, B is the ability of a compound to reversibly bind to the binding site of the glutamine transporter. Represents force, B=3. 45 (glutamine) is the highest possible value and is , which shows that it becomes non-bonded. Low values (e.g., about 1. less than 0) is a compound shows that glutamine binds irreversibly to the glutamine transporter. Which compound is K'? Indicates whether it is suitable for the transport vehicle. Low value, for example about 1. Less than 5 indicates relatively good compatibility , with higher values indicating a less good fit. B, Transport inhibition assay A given anti-glutamine compound or geltan analog can inhibit glutamine transport. For details, see Chapter I1. C,2,a, can be measured as described above. Transport obstruction In related experiments, glutamine is used to incorporate glutamine into the cytoplasm. Appropriate concentrations of glutamine analogs must be present in all test solutions. usually The incubation time was 3 to 10 minutes. C1 growth inhibition assay The effects of anti-glutamine compounds on the proliferation of HtLt cells in culture were investigated over a period of time. The study was carried out by following the changes in the total protein content of the culture. HeLi cell culture is 2 The seeds were inoculated to 20% confluency in 5 cm 2 plastic flasks. 24 After an initialization period of time, the medium was changed to fresh medium containing 1 mM test compound. every day Fresh medium was added to the culture, and culture conditions were carefully monitored using microscopy and photography. 3 Cultures (n=3) were harvested every 24 hours during the day, and cells were harvested using the following method: 1, culture at 0. Wash 3 times with 9% (w/v) NaCl, then 5 m for 10 min at room temperature. 0.1 of 9% (w/v) NaCl was added. 2. Scrape the cells from the surface of the flask using a rubber policeman and clean the glass. I put it in a test tube. 3. Centrifuge the test tube at 200g for 10 minutes (Clemeaf+ benchtop centrifuge) ) and the pellet at 0. Washed twice with 9% (w/v) NaCl. 4. Incubate the cell pellet overnight at 100°C. Digest in 1 mI of IMNaOH and The total protein content of the samples was determined by LovB analysis. 5. Results are expressed as the amount of protein present relative to a control sample not exposed to analog. Ta. In an experiment using a combination of two antiglutamine compounds in a growth inhibition study, three sets of Separate controls for the effect of each compound and another control for normal culture are required. It was important. D. Glutamine transport in lymphocytes Lymphocytes require large amounts of glutamine during mitosis. Although the amount of glutamine uptake by individual lymphocytes is actually low, many lymphocytes Because of this, a large amount is taken up during the active period. Therefore, glutamine is released It has an important role in the immune system due to its metabolic role within lymphocytes. Therefore, phosphorus The possible effects of anti-glutamine compounds and glutamine analogs on glutamine cells are also discussed. should be investigated by For example, it is toxic to both cancer cells and lymphocytes. Certain compounds may be undesirable. Furthermore, compounds that enhance mitosis are Proven to be useful in boosting the immune system to fight immune-related diseases like AIDS and cancer It can be revealed. Additionally, compounds that inhibit mitosis but are not toxic to the lymphatic system may be useful, for example, as an immunosuppressant. ) Comparison of glutamine uptake in IeLg cells and lymphocytes. It is shown in FIG. Glutamine transport to HeL1 cells was 0. Concentration range of 25-4mM is biphasic and incubated for 20 minutes with the sulfhydryl reagent NEM (1mM). Incubation results in 50% inhibition. This result indicates that the sulfhydryl group is glutamine. Located in the active site of transporter proteins, modification of these groups inhibits glutamine transport It shows that it will be done. 1. Materials and methods Lymphocytes are routinely obtained from fresh whole blood from bovine jugular vein punctures and are not used in experiments. It has also been obtained from fresh human specimens obtained by venipuncture. Ficoll Plqo Lymphocytes were separated from red blood cells by density gradient centrifugation using standard techniques including e. . Tissue culture materials were obtained from Flow L*bo…o+ie+ (IIs^). , U-14C-glutamine was obtained from Aaer+b Hatake (Australia). . Freshly isolated cells were resuspended in RPM11640 medium at a concentration of 107 cells/ml. Made it muddy. Concanavalin A was added to the final concentration of the suspension v2. 5μg/ml And so. A mixture of 5% CO2:95% air was passed through the mixture and incubated at 37°C for 48 hours. Incubated. Tritiated water-containing lymphocytes to measure glutamine transport Resuspended in PBSA for 30 minutes to a final concentration of 5 nCi/mI. . Prepare four 50 μl samples, pre-warm to 37°C, and add 2 sucrose solution (10% w/v). 0 μl and 501 silicone oil (Ve+5ilabe 5-50. G ! ner*l　El+cl+ie. Each sample was added to a microfuge tube containing N, Y, ). When displayed At time (0), an additional 140 to 50 μl of glutamine was added to the tube. Radiation of each solution The activity was kept constant and the glutamine concentration was kept at 0. The concentration was changed from 1mM to 20mM. After the exposure period ends, M, S, E, Mic+ocenl■+centrifuge for 60 seconds was used to centrifuge the cells through a silicone oil layer to terminate transport. lymphocytes Most of it appeared in the sucrose layer within the first 15 seconds. 3H was used as an indicator of the number of cells appearing in the sucrose layer. The tube was frozen by immersing it in liquid nitrogen, and the sucrose chamber at the bottom was cut to remove the plastic material before measurement. 1 mini wrench was placed in a nodition vial. To ensure the dispersion of the lymphocytes, firmly apply the vials containing zinc chloride. The tube was tightly closed and the bottom of the cut tube was shaken overnight on an orbital shaker. Result bc/ Calculate the ratio of 3H, cells, 14 It was used as a measurement of glutamine uptake per (lymphocyte). 2. Using glutamine analogs (and inhibition studies in these experiments, as described) Before measuring uptake, lymphocyte suspensions containing analogs at a series of concentrations Incubated in PBSA. Figure 31 shows 14C-group activation by normal lymphocytes after stimulation with concanavalin A. It shows the time course of rutamine uptake. East map 32 is for Hunkanabarin. Glue to bovine lymphocytes in nmol/mg protein/min in the presence and absence of Initial rate of luteamine uptake is shown. Figure 32 shows uptake by passive diffusion. is also shown. 3. Effects of anti-glutamine compounds or glutamine analogs on lymphocyte metabolism a, Chemosusceptibility testing of normal cell lines using MTT assay Chemical sensitivity using reduction of tetrazolium salts (MTT) as an evaluable endpoint A test was conducted. This particular assay is automated and in the reported literature is further modified to improve the dissolution ability of the formalin product for absorbance measurements. The MTT assay measures the absorbance of a microtiter plate and each well. for a large number of cell lines as it is performed using a scanning reader of Duplicate tests of multiple drugs at multiple concentrations can be performed. Previous researcher correlates highly with clonogenic dye efflux assays by MTT test It was discovered that a response curve could be obtained. Therefore, for any given cell line However, the solubilized formalin obtained after incubating cells with MTT The optical density of the dye product is directly proportional to the number of viable cells per well. b. Method The cell suspension was grown using RPM11640 containing 10% (v/v) FBS. Diluted to xlO' cells/ml. Add 100 μl of cell suspension per well, then Add 50 μl of conA (final concentration 2.5 μg/ml) and 50 μl of analog solution. I got it. Incubate the plates for 48 hours at 37°C in 5% CO2/95% air. 20 μl of MTT was added per well. Incubate for an additional 4 hours at 37°C. continued. The supernatant was removed and 050 μl of DMS was added to dissolve the crystals. solution Read at 560nm. The ability of cells to excrete the pigment TBp*a Blae is a commonly used indicator of cell viability. It is a sign. Approximately 1001 in the field of view of Newb*me + hemacytometer! The cells of I The lymphocyte suspension was diluted as shown. The ratio of dye exporting to dye uptake cells was evaluated several times by direct measurements. Stimulation of lymphocyte mitosis by C8 inhibitory glutamine analogs in 10th column “0” Inhibitory concentrations of glutamine analogs induced by exposure to concanavalin A It was observed that mitosis of reticular lymphocytes was stimulated regularly. generally threaded At the 1/10 level of concentration, where division is significantly inhibited, the rate of cell division generally decreases to normal. A 50% increase was observed. Figures 33-38 were measured using the dye excretion method. M plotted against lymphocyte viability and glutamine analog over a range of concentrations Relative mitosis measured by TT assay is shown. This is a common phenomenon known as ``hormesis,'' i.e. near-inhibitory concentrations of poison. This is considered to be a specific example of stimulation of proliferation by sexual substances. This phenomenon weakens the body's natural immunity. The drug may actually be delivered to the tumor at concentrations that stimulate rather than inhibit. (After this, leakage to other cells of the body is essential) It can be used in different ways. Accordingly, the compounds, products and formulations described herein and This dose enhances hormesis by the immune system rather than inhibiting it, leading to increased hormesis within the tumor. Lumesis can be designed to enhance growth inhibition rather than lumesis. d, inhibition of lymphocyte mitosis by inhibitory glutamine analogs. The results of the MMT assay were compared with those obtained under the same conditions by the dye efflux assay. Ta. The MMT assay follows mitochondrial activity and dye efflux measures viability. Ru. It was observed that all of the analogues tested showed an inhibitory concentration range due to MMT after the stimulatory range. It was. In turn, the zone of inhibition is always before the cell dies, and therefore the zone of inhibition is always present before the cell begins to divide. Some analogues have concentration ranges that are stimulated toward the target. At higher concentrations, cells It forces the cells into a "resting" phase, but at higher concentrations the cells die. The treatment of cancer and other diseases should utilize all of these concentration ranges in which drug action is observed. It is intended that E. Screening test results 1. Bonding parameters Tables 5-8 (Figures 76-79) show the bonding parameters of the various glue gantts tested. data on and how these relate to glutamine transport and cell growth. It shows that. Therefore, under conditions where S is saturated, C, NE at the end of the experiment The quantity M is inversely proportional to 3, and as k3 approaches 0, the formula becomes S/(S+K. (2NEM from 1 to −) l, and all the carriers are converted to cs and C0N E M The formation of is the best. Listed in Table 8 (Figure 89), Gantt has 4 groups (A, - D). Group A consists of compounds 1-3, and the B value of glutamine (2,5n represents a ligand with a B value close to mol/mg protein). (Arai glutamine sites under saturated conditions (as shown by the highest NEM binding during purification). This suggests that the A group ligands bind reversibly to transporter molecules. child Compounds such as glutamine are transported on glutamine carriers in exactly the same way as glutamine itself. , this was also shown in other experiments using 14C-histidine. formation of this group Both compounds have an effect on the proliferation of HξL1 cells. The B value of the B group ligand is 1. 0-2. 5 nmol/mg protein. these In this case, the reaction of the ligand at the binding site is insufficient, and therefore the binding occurs due to competition with NEM. binds to the transporter protein in such a way that the binding site cannot be saturated; or is only slightly reversible and moderately competitive with NEM and thus Compounds that moderately block sites that can be used in the second incubation of didine It is. One of these compounds, glutamic acid γ-hydrazide, has been investigated so far. It is the most potent glutamine transport inhibitor among all, and has an effect on the proliferation of HeLs cells. It can be seen that the use is relatively small. Group 0 ligands have a B value of 1. Less than Onmol/mg protein■ and consists of compounds that significantly (20% or more) inhibit glutamine transport. diagram The mechanism shown in 10 suggests that this group of compounds should consist only of non-disjunctive binders. It is shown that. Three known covalent binders (β-chloroalanine, azaserine Andri. The fact that N) is in this group provides evidence that confirms the above mechanism and current classification. providing. All three compounds strongly inhibit HeLs cell proliferation. other Tight binders strongly inhibit transport but have little effect on proliferation, especially Compound C,2 has low toxicity and is superior when used in the presence of more toxic drugs. May be a glutamine transport blocker. The last group of ligands (D) have weak binding at the glutamine site or It consists of a compound that actively binds at a site on the transporter other than the transporter position. Compounds 9 and 1 2 may no longer be part of this group when the transporter and proliferation experiments are completed. Compound D, 14 enters cells in very small amounts when transported via the glutamine transporter. Further research has shown that this compound is highly toxic. . It should be noted that the properties of compounds A, 3, and 14 are very contrasting. . In other words, adding a methyl group to glutamic acid hydroxamate increases its ability to inhibit transport. disappears, but only 50% of the effect of glutamate hydroxamate increases proliferation. Inhibitory ability is retained. Compound 10, N-acetylglutamine, is a glutamine importer. therefore, this compound does not bind to the glutamine site. However, it was thought that it could act as a positive effector in very close proximity. 2. Growth inhibition assay Results regarding the effect of anti-glutamine compounds on the proliferation of H+L* cells in culture It is shown in Table 4 (Figure 75). Results compared to control cultures not exposed to compound Expressed as the amount of inhibition by the compound after 48 hours of breakthrough. Azase, an established anticancer drug Phosphorus, methotrexate, DON and β-chloroalanine all significantly increase cell proliferation. inhibited. Among the new compounds tested, two compounds viz. -γ-monohydroxamate and glutamic acid-γ-methyl monohydroxame Only 100% of the samples showed a significant effect on proliferation. Antiglucose in combination with methotrexate None of the tamin compounds showed significant synergistic effects. 3. Transport inhibition assay a, Effect of antiserum on glutamine transport in ReL* cells rabbit or mouse Figure 6 shows the action of the glutamine transport protein antiserum obtained in . With 1mM glutamine The rate of glutamine uptake into HeLg cells was inhibited by 25% in the presence of 1% antiserum. harmed. b. Anti-glutamine compounds and glutamine transport in HeLt cells Action of min analogs Anti-glutamine compounds and glutamine on glutamine uptake rate into Re1M cells The effects of the min analogs are shown in Table 5 (Figure 76). Glutamic acid-(2-hydro xyethyl)amide, glutamic acid derivative (see $2 table), glutamic acid-γ- Diethylamide, cyclized glutamate-γ-tris(hydroxymethyl)methyla mido, glutamic acid-γ-bis(hydroxyethyl)amide and glutamic acid- A large number of compounds containing γ-p-nitroanilides are was found to inhibit the rate of min uptake. The compounds in Table 8 (Figure 79) represent a wide range of chemical functionalities. In this experiment, all measurements used were based on a preliminary screening, which It will point the way to a final chemical strategy. Therefore, in most cases, the optimal concentration in human blood is predicted to be 1mM. Inhibition of transport can occur, for example, with both glutamine and its analogs: 1. Performed in mM. subordinate Thus, the results provide an early indication of physiological response. Research based on chemical functionality More detailed dose-response experiments should be carried out once sufficient information has been obtained to develop a research strategy. It can be implemented. The classification of compounds into four groups (A-D) is shown in Scheme 10 by NEM and 3H-NE The proposed mechanism for the reversible and irreversible binding of ligands to M related to the experimental hypothesis. This classification consists of two main measurable variables, 3H-NEM Based on maximum binding test and transport inhibition. Group A compounds are similar to glutamine3 It is a compound having an H-N E M bond. These are reversible and similar to glutamine. It is thought that the reaction occurs at the glutamine site. Group B compounds have moderate 3H-NE It is a compound that has M binding properties and is therefore thought to have moderate reversibility. Ru. Group 0 compounds have very low 3H-N　E　M binding, but their transport is also remarkable (〉 2096) is a compound that inhibits. Such compounds act at the glutamine transporter site. It is thought that the binding can be irreversible. The compounds of group D have 3H-N E M bonding properties. It is a compound that inhibits transport at very low levels (20%). Such a compound It is believed that the substance has minimal binding capacity for the transporter site. The compounds that have the greatest effect on transport and proliferation are three known anticancer compounds: α-chloro alanine, azaserine and DON (compounds C1, 3 and 5). these All compounds have B values of non-■ This means that they all react covalently at the active site and therefore This is explained by the fact that it is an irreversible competitor. From this, these changes It is confirmed that the compound is classified as a C-type compound. These compounds are transporters Covalently binds to the active site, so its ability to kill cells within 48 hours is somewhat uncertain It's true. However, under these conditions covalent binding occurs relatively slowly and therefore from the initial stage of binding until all sites are covalently bound to the ligand, i.e. It should be understood that ligand transport continues until transport other than saturation transport stops. be. Research by Haber et al. (IIl, ), C5ace+, 41. 752-75 5) supports this study and shows that 14C-D in mouse P388 leukemia cells ON uptake was shown to peak within about 3 minutes. D. Since N does not appear to be metabolized in these cells, this final state is a combination of influx and efflux. It should represent the steady state obtained between or the point at which no further inflows or outflows occur. The final concentration of DON is the result of saturation of the transport site by the covalently bound ligand. Represents molecules trapped within fruit cells. If the former is correct, the maximum It should be shown that the retention of 14C-DON becomes O near the final cell concentration. actual The DON decreases by approximately 50% during the first 4 minutes, and after 10 minutes 40% of the DON is still in the cell. The presence of DON within the range indicates that the outflow of DON has virtually stopped. Ru. Therefore, the remaining DON was either internally bound or transported with bound ligand. Body parts are saturated and trapped inside cells. We must conclude that it represents N. In the same literature, when released from cells It has been shown that internal DON can be freely separated by thin layer chromatography. , the latter seems to be the more likely explanation. Furthermore, β-chloroalanine, azaserine, and DON all contain glutamine (K, ’ = 1. 33mM), the value (dissociation constant in competition with NEM) These compounds have poor compatibility with the glutamine binding site and have a low affinity for tumor glucose. This suggests that specificity to the tamine binding site could be improved considerably. The K′ value was the lowest for β-chloroalanine (4,34mM), and the lowest for DON (7,1 (mM), indicating that the latter has the lowest inhibition of glutamine transport. matches. These results for DON were also confirmed by the study by Hlbe et al. Supported. 14C-DO in mouse P388 leukemia cells N uptake is at least partially mediated by the rLJ transport system and therefore We confirmed the lack of specificity for the luteamine transport site. DON's main import The possibility that the transporter is a leucine selective system is based on the structure of DON (norleucine derivative). Not surprising from that point of view. Na-dependent glutamine transport in H+L+ cells is was found to be inhibited by syn and therefore not mediated by rL]-type transporters. Ta. The lowest K' value indicates very tight coupling (low B). The compound shown is compound 0. 2 (glutamic acid-γ-p-nitroanilide) Ta. The structure of this compound automatically participates in tight binding, and from this point of view is also placed in group 0. This compound significantly inhibits glutamine transport but has little effect on proliferation; This suggests that only a small amount of fuel may have been transported. Therefore, such compounds has relatively low toxicity and at the same time significantly reduces the intracellular concentration of glutamine (glutamine glutamine concentration in tl+Li1B is always drastically reduced due to amine deficiency), Increasing cellular tolerance to highly toxic compounds such as Meanbin and other Group 0 compounds It is expected that this will decrease. Compounds A and 2, which are hydrazine derivatives, are glutamate Min transport inhibitory activity was the highest, but the effect on proliferation was only slight. This compound shows a fairly high B value, so a large amount of the compound is imported into cells. and toxic effects should be considered minimal. This latter finding is experimental This is consistent with previous reports showing the effects of hydrazine on cancer in animals. On the other hand, compound Substance A, 3, hydroxamate has a large effect on cell proliferation, but has a negative effect on transport. The effect on Compounds B, 2 and C, 6 inhibit K゛ and transport. shows an excellent reciprocal relationship as well as the relationship between on the γ-amide of the group glutamine If you double the number of substituents, the K value will double $ , and transport inhibition is halved. Apparently minimal transport occurs, but it is important for proliferation. % inhibiting compounds, 14 require further study. These results If confirmed, this compound would be the strongest growth inhibitor of all the compounds tested. From the above considerations, the following conclusions were drawn regarding the activities of the various compounds investigated. 1. Covalently binds to glutamine at the active site and is a potent inhibitor of both transport and proliferation. Known anticancer drugs (compounds C1, 3 and 5) have relatively low ° values and therefore It was shown that this was not the best possible fit for the position. This conclusion suggests that DON transport is partially The study by Haber et al. showed that the rLJ transport system It is highly supported. Therefore, at least one of these compounds, especially DON, may be further After modification, the affinity for glutamine carriers in solid tumor cells was significantly increased (K ’ value is low). 2. Compound A, 3 (glutamic acid-7-hydroxamate) is attached to the glutamine site. It binds reversibly and has been shown to be an excellent inhibitor of both glutamine transport and proliferation. It was revealed. Therefore, this compound has no binding activity to the glutamine site. However, there are related compounds that have shown significant effects on proliferation. 4. The properties of this compound along with methylhydroxomate require further study. Ru. 3. Compound B and 1. Hydrazine derivatives were investigated for their effects on glutamine transport. It was the highest among all compounds, but had the least effect on proliferation. this compound When present, tumor cells become glutamine-deficient and the requirements for tumor growth are compromised. This compound may potentiate other, more toxic compounds. Research should be conducted on the possibility of 4. Compound C,1, nitroanidine, which is a tight binder and is active at the μM level. Phosphorus is a strong inhibitor of glutamine transport. This is because it is a tight bonding agent. The compound has a K value or less. active in the US and therefore any toxic effects would be negligible. to this compound However, the effect on tumor growth in the presence of more toxic drugs should be studied. be. 5. Compound 0. With the exception of 14, D and 7, the rest of the compounds 18 had almost no inhibitory activity. have little or no Although further investigation is required, albidin (Al Bi! riin) and acivicin are in this group. There is one exception to this group. It is the compound 10, N-acetylglutamine. This compound is transported It activates body IiI activity and is completely non-toxic as it is a normal metabolite. . Use this compound to activate a transport system to deliver the preferred analog into tumor cells It has been proposed that the speed at which the The screening tests and results described herein demonstrate that the glutamine transport system Glutamine analogs must have the following properties in order to be transported into cancer cells with maximum efficiency It will be understood that the optimal characteristics that should be achieved can be evaluated. Based on the above results, anti-glutamine compounds or glutamine analytes suitable for cancer treatment Develop a structural model for logs. Therefore, from the above results, it is clear that the tumor cells migrate into tumor cells. Glutamine transport across the plasma membrane of cancer cells at the expense of needed extracellular glutamine The system allows prediction of compounds that will move into cancer cells. When anti-glutamine compounds or glutamine analogs are inside cancer cells, glutamine In particular, it inhibits most of the enzymes involved in DNA synthesis and kills cells. It turns out. Glutamine analogs are transported into cancer cells in large quantities (this transport system (active energy-requiring system), so if the analog is not metabolized, it is non-active to the cell. These compounds are particularly toxic because they are constantly broken down into toxic products. It doesn't have to be gender. ] - Evidence also shows that specifically targeting tumor cells, thereby reducing DNA and RNA A new group of molecules that has the essential property of strongly inhibiting A synthesis either directly or indirectly. Provides the basis for future development of Tamin analogues. Such analogs can be used to treat tumor cells. Due to its specificity for cells, it is less sensitive to normal cells commonly associated with current cancer chemotherapy. It is expected that this product will significantly reduce the cytotoxicity of the cells. of the present invention The application is not limited to the use of a single analogue, but also the use of two or more analogues at the same time. It will be understood that the log can be used. 4. Inhibition and metabolic research using mitochondria Inhibiting glutamine transport within cells In the search for anti-glutamine compounds that harm mitochondria, It is also possible to investigate whether glutamine transport is inhibited. mitochondria Metabolic studies can also be carried out using Previously, materials suitable for experimental purposes were relatively Due to their ready availability, research on tumor mitochondria has largely focused on It was limited to Lurich's ascites cells and hepatoma. On the other hand, mitochondria from cultured tissue It is quite difficult to obtain sufficient material for a preparation. Mitochondrial respiration and A major limiting factor in cell and metabolism experiments is the availability of large numbers of cells. 7. A sufficient amount of mitochondria can be obtained from culture using the large-scale microparticle carrier method described below. was able to be isolated, thus making it possible to carry out experiments. 1 obtained from Flow Libo+m+o+ic+ (zLi cells are at confluence Reached. These cells were grown in MEM containing FBS (10% v/v). I set it. Pass the cells through a 1:2 split ratio and add FBS (10% v/v). Containing medium 199. These cells will adapt to the new medium in 2-3 days. and thereafter exhibited behavior similar to cells cultured in medium 199 for a long period of time. this was confirmed by measuring the glutamine uptake rate. C7todsxl or 3 microparticle carrier beads have adhesive properties, so all Glassware should be treated with silicone before use. 10% (V/V) in toluene The Sa+fmtil solution was applied liberally to the glass surface. Glass after removing liquid The instruments were allowed to dry overnight. Once dry, wash the glassware 10 times with distilled water and remove the orb. I put it in a tank and let it dry. The glassware is now ready for use. When there were too many beads on the container surface, the container was siliconized again. C71odex l or 3 fine particle carrier beads (2g peas/100ml 1PB sΔ) was hydrated overnight in a siliconized bottle. The next day, the old PBSA was discarded and fresh PBSA room was added to the beads. use The beads were previously sterilized (121°C, 1. 20 minutes at 2 ATM). The sterile beads were pelleted and the supernatant was discarded. Resuspended in medium +99 (50 ml/g beads). After 2-3 minutes, the beads were allowed to settle and the supernatant was discarded. After repeating this twice, the beads were finally resuspended in medium 199 (25 　m　　　　　　/　g beads). Harvest cells from 4-5 200 ml confluent culture flasks containing beads. added to the flask. 10% (V/V) FBS was added to neutralize the effect of ATV; promoted the attachment of cells to beads (G+1nell, 1976), The cells were transferred to a 250 ml microcarrier container (Teehne). massive For culture 1. Using 51 microcarrier vessels, 16 200ml culture flasks -20 cells were added to 6 g of beads. Cultures were placed on microcarrier stirrers (Teehnc) and incubated intermittently for 24 hours. (40rpm, stir for 2 minutes, pause for 30 minutes) to maximize cell attachment. The beads were then continuously stirred at 4° rpm. On the second day, the culture was supplemented with FBS (1 An additional 199,100 ml of medium containing 05 v/v) was added. Half the culture medium every day was discarded and replaced with fresh medium to ensure maximum growth rate. Because of the rapid cell growth rate, it was necessary to constantly monitor the pH of the medium. small culture N a HCO3 was added to the culture flask to maintain neutral pH. Na HC It was found that O3 could not maintain the pH of a large-scale culture flask for a long period of time. . This problem can be solved by using [1epei (2QmMl) as a buffer. Resolved. To measure the rate of cell growth on culture beads, remove a small sample from the culture each day. A quantity aliquot was taken. Two broken covers to facilitate cell observation. A glass strip between the slips (acts as a support for the beaded coverslip) Placed a bead on the ride. Cells grown in microcarrier culture can be used for whole cell respiration studies and mitochondria. was used in the preparation of Details of the harvesting method used are described below. Cell culture and media Periodically inspected for biological contamination. Infected cells should be removed and opened. The source of contamination was destroyed by toclave. Breathing and glutamine HeLi cell mitochondria for use in oxidation studies were obtained as follows. . Massive H! L *Raw material used by cell microparticle carrier culture for mitochondria preparation Met. Confluent cultures were harvested and mitochondria were isolated as described below. The beads were pelleted, the medium was decanted, and the beads were then washed two more times with PBSA (pH 7). , 6; 100 ml/g beads). After this, the beads were treated with EDTA (ph 7. 6) 50ml/g beads in PBSA containing (002%w 7v) and stirred for 5-10 minutes (30 rpm). After this period ends, The beads were pelleted and the medium was decanted. Add beads to 50 ml of PBSA containing EDTA/ATV mixture (50:50) /g beads in a siliconized 250 ml conical flask. Moved. Shake the flask occasionally and keep it in a 37°C incubator to incubate the cells. Ensured maximum separation. Take a sample and observe it with a microscope to determine the cell separation rate. was monitored. Pellet the beads, remove the supernatant containing suspended cells, and D*t Cells were spun down at 180 g for 5 min in an IEc centrifuge. Finally, ED the cells The TA-containing H-containing end was resuspended. The above process was repeated three times to maximize yield. Collect cells and digitonin treatment step. Mitochondria were isolated according to the method described in the literature, except that the . Resuspend the resulting mitochondria in 500 μl of H medium and keep on ice before use. It existed. Mitochondria were kept on ice for up to 12 hours before being discarded. Vll, diagnostic use Based on the above, those skilled in the art will be able to understand monoclonal antibodies, polyclonal antibodies, and transporter proteins. It has become clear that many biological products, such as white matter, can be used in cancer assays. It would have been. In the blood (blood, plasma), on cells, urine, tears, milk, stool, spinal fluid, etc. Diagnosing the presence of glutamine and glutamine transporters in secretions from any body containing It will also be recognized that there are many ways to do this. The basis of these steps is to first Use of monoclonal or polyclonal antibodies against tamine transporters, The second is the use of the glutamine transporter or a portion thereof as an antigen in the system. In this way, many different diagnostic systems have been established based on existing immunological techniques. It can be done. For example, an antigen (glutamine transporter) is attached to a plate to which an antibody is bound. Attach and vine. transporter (or antibodies against it) present in serum or other secretions This inhibits the binding of the transporter, which can be used in ELISA assays and radioimmunoassays. b. It can be measured by immunoradioactivity assay or by chemiluminescence or other means. This simple modification of an antibody response is common, and two antibodies - one a 'catcher' - are ”, one to capture circulating or secreted glutamine transporters. including the use as a readout system for "capture tag" assays. I'm reading. In addition, in tissue using fluorescence, immunoperoxidase or radioactivity methods. Transporters can be measured directly. Many of these methods involve plasmatic using rate, adhesion to beads, chemiluminescence, etc. Antigens that are glutamine transporters can be used in whole or in part, synthetic peptide fragments of the receptor or genetic Contains parts obtained from child engineering. Furthermore, as mentioned above, for this transporter A large number of antibodies can be created. The diagnostic product combinations included in the invention can be used in sandwich assays or competitive assays. You can use it for sure. The diagnostic products of the invention are applicable to a wide range of assays and are particularly It is understood that use is not limited to any specific assay or format. Let's go. Examples of assays include: binding assays, sandwich assays, enzymes. immunoassay, fluorescent immunoassay, radioimmunoassay, competitive assay, Immunoradioactivity assay, immunoenzyme assay, immunofluorescence assay or luminescent immunoassay Assay. Diagnostic products can be labeled with conventional labels such as enzymes, radioisotopes, particles, fluorescent molecules, free It can be labeled with groups, chemiluminescent molecules, bioluminescent molecules, phages or metals. other signs will also be clear to those skilled in the art. When using amino acid transporters such as tumor glutamine transporters as diagnostic products For example, the transporter may be present in native or dissociated form, or the entire transporter protein or its It will be appreciated that it may also be a fragment or subunit of. Release diagnostic products such as monoclonal antibodies, glutamine or its analogs. It is also easily understood that they can be used in vivo by conjugating them with radioactive isotopes. Good morning. The diagnostic product can then be injected into the patient and selectively binds to the tumor. , or transported to the tumor. After a suitable period of time, the patient may be subjected to radioimaging, e.g. The location of the tumor can be determined by scanning the tumor. The diagnostic products of the invention are suitable for performing assays on biological tissues such as biopsies. It will also be useful. For example, contacting a tissue sample with a labeled monoclonal antibody can be combined. If the labeled antibody remains bound to the tissue sample, Tumor cells are present in the sample. By repeating the assay process over a period of time and quantifying the detected label, the insect A method of monitoring cancer progression in a patient is provided. The diagnostic product of the invention can be used as a biosensor, especially for the presence of a member of an immune binding pair. It may also be useful as an immunological biosensor useful for detecting. by Osensors are biological molecules or tissues in their raw form, modified forms, or functional fragments thereof. It is a device used as a member of a sensing device in the form of a. These molecules are of various types. The method may be coupled with signal transduction devices, e.g., metal particles, light, incorporated into lipid membranes. can be coated with scientific fibers, piezoelectric crystals, or connected by other methods or means. Therefore, For example, potentiometric, fiber optic, surface plasma resonance and piezoelectric measurements The presence of substances can be measured with high sensitivity. to H+L+ cells, FIeLs cell membranes or fragments thereof or glutamine transporters. antibodies (or fragments thereof) with signal transduction devices in biosensors. can do. Using HeLI cells or HeL* cell membranes (e.g. small II) Then, the presence of ions, amino acids such as glutamine, growth factors, etc. is detected and monitored. can be tarred. Glutamine transporter, fragments or modifications thereof When used as part of an osensor, it can be used to detect or monitor the presence of glutamine. can be done. cells using antibodies or fragments that recognize the glutamine transporter. to detect the presence of the transporter or its fragments in cells or various biological fluid samples. I can do it. These biological materials can be used individually or in combination. can. Vllll, therapeutic use The present invention provides a number of different therapeutic compositions and methods for treating cancer patients. composition Products and methods generally fall under the category of transport-inhibiting and/or cytotoxic agents. Can be done. A, transport inhibitor The main function of transport inhibitors is to deprive tumor cells of glutamine, which is necessary for normal biosynthesis. That is. By effectively depriving a cell of glutamine, the cell weakens and perhaps eventually death and/or susceptibility to cytotoxic drugs or the body's own immune defenses It is believed that this will increase. For example, glutamine is glutamine synthetase glutamine by inhibiting the scales and intracellular glutamine. amine synthetase and other intracellular enzymes such as glutaminase and γ-glutaminase. It is believed to have a positive effect on transferases. This allows for many A variety of problems can occur, some of which are related to cancer growth. Maybe not. Additionally, compounds other than those listed below that have been shown to inhibit glutamine transport The products can also be used according to the invention. That is, another embodiment of the invention merely substantially selectively treats tumors. consists of the use of compounds that inhibit glutamine transport. Therefore, such transformation Although intensive research on all compounds is usually required, e.g. Compounds such as bimersaryl can also be used. 1. Anti-glutamine compounds and glutamine analogs can inhibit glutamine uptake. One group of compounds are the anti-glutamine compounds and glutamine analogs described above. . Compounds of particular interest have high transport inhibition, i.e., at least about 20%, preferably of these exhibiting an inhibition of at least about 40%, more preferably at least about 60%. Compounds and analogs. Such compounds have low K ° values, i.e. about 1 ．． less than 5mM, preferably about 1. Desirably it is less than 0mM. A value like this showed that the analog is a relatively specific binder for the glutamine transporter. are doing. The analog B value is approximately 1. When larger than Onmol/mg protein The analog will be transported into the cell. Otherwise, the analog It probably just binds to the transporter binding site and blocks the transport of other molecules into the cell. cormorant. By depriving cells of glutamine, they weaken the cells and thus cause other cytotoxic effects. Sensitivity to drugs and/or the body's own immune defenses can be increased. 2. Antibodies The same is true for monoclonal or polyclonal antibodies that bind to the active transport site of transporters. would similarly inhibit glutamine transport into cells. Monoclonal antibodies and polycrystalline antibodies Combinations of products with or derived from local antibodies are also transport inhibitors. Similar to the combination of certain glutamine analogs (or other amino acid analogs) with antibodies. Can be used by anyone. Generating the modified antibodies of the invention using relatively bacterially developed recombinant DNA methods can do. For example, antibodies can have one or more binding sites removed from one antibody. It can be modified by taking it out and putting it into another antibody. or Modifying antibodies to add new sites for conjugation with other compounds and can introduce protein toxins as part of the entire antibody molecule. these Many modifications will be apparent to those skilled in the art and are included in the invention. 3. Histidine and other amino acids Histidine is a highly competitive inhibitor of glutamine uptake into Itel* cells. It has been shown to inhibit glutamine uptake by 50% at 1 mM. Thin The presence of high concentrations of histidine in the medium in which cells are growing causes cells to be removed from the medium. It was possible to strongly inhibit glutamine transport to the chamber. Under normal physiological conditions, Histi glutamine concentration is low (so glutamine uptake into HeLi cells is not affected) . However, when there is a high concentration of histidine in the medium, the histidine glutamine entry is prevented, and the cell can subsequently become starved of glutamine. Inhibiting glutamine uptake into tumor cells results in glutamine starvation, which This will result in weakening of the cells. Histidine is a potent competitive inhibitor of glutamine uptake in HeL@ cells; Therefore, when present in sufficiently high concentrations, entry of glutamine into these cells is significantly inhibited. It is expected that In this study, we added histidine to the culture medium of HeLi cells. Well, cell proliferation was monitored over a period of time. The results shown in Figure 60 indicate that high concentration hiss When 1 mM histidine was present, compared to controls that were not exposed to tidine, suggests that HsLt cell proliferation stops within the first 48 hours. deer However, after 48 hours the cells appeared to recover and regained a proliferation rate comparable to controls. Ta. The reason why the cells recovered after 48 hours is not clear, but it appears that glutamine starvation This may be because cellular metabolism changes to compensate. However, this study suggests that blocking glutamine entry into tumor cells may cause some damage to tumors. provide evidence that it is sufficient to cause harm and leaves the cell in a relatively vulnerable situation. ing. In addition, this weakening can be achieved by administering known or new anticancer drugs. This situation can be exploited to induce additive or synergistic effects on cytotoxicity against tumor cells. I hope I can wake you up. Need for known or novel anticancer agents in the presence of blocking agents significantly lower the amount and reduce undesirable cytotoxicity to normal cells during treatment. You can also As can be seen from Table 15 (Figure 86), other amino acids also contribute to glutamate to HeLs cells. It can inhibit the uptake of min and therefore has therapeutic value as an inhibitor. Inhibition of uptake by any mechanism, i.e., TGTllll, diffusion, etc., mediates the inhibition. There is a possibility that B. Cytotoxic drugs The present invention also provides several new types of cytotoxic agents. 1. Anti-glutamine compounds and glutamine analogues showing good proliferation inhibition Analogues can be excellent cytotoxic agents. Such analogs are relatively low Ks' and therefore selectively binds to tumor cells instead of normal healthy cells. It is a particularly good cytotoxic agent when it shows that it will be effective. cytotoxic glutami analogs have high B, which tends to suggest that substantial amounts of analogs enter cells Indicates the value. Low B. The value is that the analogs are simply bound to transport proteins outside the cell membrane, but not within the cell membrane. would indicate that it is not transported. Makes glutamine or its analogs toxic Such modifications are also embodiments of the invention. This may include, for example, known or new toxic molecules, This can be done by incorporating molecules or groups into glutamine or its analogs. 2. Antibodies conjugated to cytotoxic drugs Another embodiment of the invention provides monoclonal or or polyclonal antibodies, which are novel or known antibodies. Conjugated with cytotoxic drugs. Known cytotoxic drugs, e.g. For example, it may be a chemotherapeutic agent or a radioisotope with insufficient selectivity. The antibody is the invention The novel compounds could also be conjugated with anti-glutamine compounds or analogs. chemical treatment When the agent binds to the antibody of the invention, there is a link between the monoclonal antibody and the chemotherapeutic agent. When the bond is broken, the chemotherapeutic agent is chemically removed so that it does not enter any non-target cells. Preferably, the therapeutic agent is modified. Therefore, when the chemotherapeutic agent remains conjugated will be effective and selectivity will be preserved. In addition to using “prodrugs” Similar methods will be apparent to those skilled in the art and are also included in the present invention. 3. Drugs linked to glutamine or analogues of known or new cytotoxic drugs by binding to glutamine, anti-glutamine compounds or glutamine analogs. glutamine or other compounds selectively transport the drug into the tumor cell chamber and This may be able to kill or weaken cells. Such drugs have no release can include radioactive isotopes, drugs or toxins. To be successful, you need glutamine or Binding and drug administration should be done in a way that does not destroy the ability of other compounds to reversibly bind and be transported. It is necessary to design a drug. C0 other compounds An important function of abscess cells is to bind glutamine to glutamine transporters and transport it across the cell. transporting glutamine and subsequently metabolizing glutamine within the cell. Are known. Use of glutamine analogs, antibodies, histidine and cytotoxic drugs A number of compounds, compositions and methods have been described that inhibit this process, such as. However, if you read this specification, you will understand that either method can lead to the glutamine transporter being grouped into cells. Those skilled in the art will know of other substances, including enzymes, that inhibit the transport function of luteamine or analogs. You'll find out right away. Accordingly, such materials are within the scope of this invention. present invention Also includes inhibitors of subsequent metabolism within the cell. Many of the analogs listed are This function, i.e., not just blocking the binding of glutamine to the transporter, It may also act to inhibit the pathway of glutamine metabolism within the cells. Other amino acids are transported by the glutamine transporter, and its analog is the glutamine analog It is a much more powerful inhibitor than other drugs, and is also very likely to be a toxic drug. for example, It was shown above that histidine inhibits glutamine uptake. Therefore, a via amino acids, their analogs or glutamine transporters such as TGT l-111 Therapeutic products such as other compounds that enter into the body are within the scope of this invention. D. Combination therapy Therapeutic uses of the invention include treating combinations of glutamate drugs, particularly using one type of compound. Inhibiting min transport and weakening cells, and the other containing the above-mentioned drugs, weakened tumors. A combination consisting of administering a known or novel cytotoxic agent to the cells. useful for. E. Pharmaceutical compositions and administration methods Another embodiment of the invention provides a physiologically acceptable combination of one or more of the anti-glutamine compounds described above. and a pharmaceutical adjuvant or carrier. Such a composition is It can be manufactured by methods known to those skilled in the art. Therefore, the physiologically active anti-glutamate of the present invention The Min compounds, alone or preferably together with suitable pharmaceutical auxiliaries, can be formulated into tablets, dragees, capsules. It can be used in the form of capsules, suppositories, emulsions, suspensions or solutions. Those skilled in the art will also be familiar with suitable auxiliaries for the desired drug formulation. solvent, gelation In addition to agents, suppository bases, tablet auxiliaries and other excipients for the active ingredient, e.g. , antioxidants, dispersants, emulsifiers, antifoaming agents, flavor modifiers, preservatives, solubilizers and colorants can also be used. Active anti-glutamine compounds, analogs or each specific The optimum dosage and method of administering the composition required for a particular case can be determined by one skilled in the art through routine experimentation. Can be determined. The anti-glutamine compounds, analogs or compositions in the form of pharmaceutical formulations of the invention are of other groups. Physiological active substances of pharmaceuticals, e.g. steroidal and/or non-steroidal anti-inflammatory agents, immunosuppressants, sulfated glycosamines/glycans and other sulfated carbohydrates. It may also contain one or more of analgesics, analgesics, and antipyretics. The anti-glutamine compounds, analogs or compositions of the invention may be solid, semi-solid or Drugs that may be liquid diluents or capsules or aerosol applicators Pharmaceutical preparations consisting of at least one active compound in combination with a pharmaceutically acceptable carrier In the form of a drug, it can be administered orally, rectally or by injection, e.g. by transdermal administration for the treatment of skin cancer. I can give. The active ingredient is in solid/semi-solid/liquid formulations. 1-99% by weight, more specific Generally speaking, injectable preparations are 0. 5-20% by weight, 2-5% in formulations suitable for oral administration. It constitutes 0% by weight. Unit dosage containing at least one compound or composition of the invention for oral administration Pharmaceutical preparations in the form of solid powder carriers such as lactose, sucrose, sorbitol, etc. Thor, mannitol, derivatives such as potato derivatives, corn derivatives or amylo pectin, cellulose derivatives, or gelatin, and lubricants such as stearic acid Magnesium, calcium stearate, polyethylene glycol, wax and Can be manufactured by mixing selected compounds or compositions and then compressing them to form tablets . Coated tablets contain ingredients like gum arabic, talcum, and titanium dioxide By coating the tablets prepared as above with a thick sugar solution that or dissolved in an easily volatile organic solvent or mixture of organic solvents. It can also be coated with lacquer. Soft gelatin capsules contain selected compounds or compositions mixed with vegetable oil. It can be produced by wrapping it in a thick gelatin shell. Gelatin hard capsules are solid powder Terminal carriers such as lactose, saccharose, sorbitol, mannitol, differential examples For example, potato differential or corn differential, or amylopectin, cellulose derivative. Containing selected compounds or compositions mixed with conductors or gelatin Can be done. Preparations in unit dosage form for rectal administration contain the active compound mixed with vegetable or paraffin oil. Can be manufactured in the form of suppositories consisting of: Liquid preparations for oral administration may be in the form of syrups or suspensions; such solutions may The selected compound or composition is about 0. Contains from 2% to about 2% by weight, with the remainder being sugar. and ethanol, water, glycerol and propylene glycol. Solutions for parenteral administration by injection preferably have a concentration of about 0. at a concentration of 5 to about 10% by weight. Can be prepared as an aqueous solution of the selected compound or composition. These solutions are stable may also contain buffering agents and/or buffering agents and are provided in ampoules of various dosages. It is convenient. A preferred daily dose for transdermal administration of selected compounds or compositions of the invention is 100-5 00 mg, preferably 200-300 mg - should administration in weekly doses be It can be 25-2000 mg every 1-3 weeks. The anti-glutamine compounds and analogs of the present invention are expected to be useful in cancer treatment. Ru. Accordingly, the present invention also relates to a method of treating animals suffering from cancer. This method can be used to treat animals. a therapeutically active, physiologically acceptable amount of one or more of the compounds or compositions described above; or by administering combinations and compositions of the therapeutic compounds described above. F. Immunosuppressants and immunostimulants Glutamine has been shown to be taken up by stimulated lymphocytes. Therefore, The anti-glutamine compounds and analogs of the present invention can also be used as immunosuppressants and immunostimulants. Useful. Such immunosuppressants and immunostimulants may cause transplant rejection and transplant recipients. It will be useful for diseases caused by transplantation, autoimmune diseases, immune diseases such as AIDS. Current Numerous applications will be readily apparent to those skilled in the art. Thus, the lymphocyte-transporting glutamine, anti-glutamine compounds and glutamine of the present invention Analogs are transported to lymphocytes and removed by proliferating lymphocytes depending on the desired effect. or stimulate lymphocytes. Anti-glutamine compounds and gluta Min analogues were tested using a simple screen as described above to determine their effect on lymphocytes. It is necessary to do some leaning. For example, for TG Ding I+, as mentioned above, The glutamine transporter can be isolated and identified. Pharmacy that treats humans The methods and methods may be the same or similar as above. IX, vaccine Another embodiment of the invention provides a vaccine that immunizes against cancer. The vaccine composition contains the entire transporter protein in its live form, or at least the active form of glutamine. Preferably, it consists of subunits involved in transport. several different transporters, Its fragments or subunits can be administered simultaneously to transport several different transporters in the host. can generate an immune response against. For example, TGT I, TGT I I and TGT I or fragments or subunits thereof involved in active transport. A three-component vaccine can be administered containing the following: Next, the clay is transported to There will be an immune response against it. When such transporters first appeared, the host provides antibodies that bind to the transporter and transport glutamine into tumor cells at an early stage. will inhibit the Tumor cells may then become susceptible to host immune defenses. X, Example Examples 1-79 below demonstrate the use of anti-glutamine compounds and glutamine analogs of the present invention. Although it describes manufacturing, it is not intended to be limiting in any way. Example 1-79 The general experimental procedures, equipment, solvents and reagents used were as follows. Diethyl ether and tetrahydrofuran are distilled from sodium, then a small amount of A second distillation was carried out from sodium in the presence of benzophenone. pyridine is water Dehydrated over potassium oxide pellets and distilled before use. Triethylamine is hydroxyl The solution was dried over potassium chloride and distilled before use. The distilled product is stored in a brown bottle. Stored on Lil1de131 type molecular sieves. All iodine disappears in methanol It was dehydrated by heating it with a small amount of magnesium flakes and iodine until it was dry. More methano was added and the product was distilled under nitrogen. Depending on the experiment, HPLC grade methanol Anhydrous methanol was used instead of alcohol. Absolute ethanol, 95% ethanol, Ethyl acetate (AR grade) and gasoline with a boiling point of 30-40℃ (AR grade) are left as is. used. Dimethylformamide is Lind! Dehydrated on a one-person molecular sieve, It was vacuum distilled (boiling point 76°C/'40mm). Distilled product until use Stored on molecular sieves in an amber bottle. Is aniline a potassium hydroxide pellet? vacuum distilled and stored over Linds JA molecular sieves in an amber bottle before use. did. Benzyl bromide is vacuum distilled (boiling point 85-87℃/'10mm) and used. It was stored at 4°C in a bottle covered with aluminum foil. Distill acetic anhydride before use. (boiling point 138°C). Distilled acetic anhydride was stored under nitrogen. chromatography Thin layer chromatography (t, 1. C,) above using one of the systems outlined below. An elevated solvent system was used. Spots were performed with standard samples for which possible Rf values are indicated. (i) Commercially available silica gel plate (M@+, DC-P18sfikfolk ieaKie+e1gel 6OF254); The solvent system is shown in parentheses. pre The sheet was viewed under ultraviolet light and exposed by immersion in a 5% phosphomolybdic acid ethanol solution. It was then heated to 100° C. in an oven. (ii) Commercially available cellulose plates (Merck. DC-Pl++1iklolieo Ce1lulose); Solvent system n-bu Tanol/Pyridine 10. 4% aqueous acetic acid solution (22:10:10). 1% pi Develop by immersing the plate in 1% ninhydrin in acetone with lysine and Next, it was heated to 100°C. Vacuum-assisted chromatography (flash chromatography sintered glass disk) TLC silica gel (M!+ek, kie+e1gel) was added to a column equipped with 60G) dry-filled with an abstinent. Vacuum assisted elution was used in the usual manner. ion exchange chromatography Both anion and cation exchange media were used for the separation. these media is packed into an exchange column as a slurry of water, and a 1 molar solution of a soluble salt of the appropriate ion is added to the exchange column. It was converted to the desired ionic form by treatment with Next, wash the column with distilled water and remove the mixture. I placed it. Elution systems are shown in parentheses. The ion exchange medium used is shown below. T, Aml+erlile IR-120 (analytical edge), BDIIT I, Amb e+1ile IRc-50 (analytical edge), IIDIIT I1. Asbt+ 1ile CG-120 (analytical edge), B [1III V, Asb! rI mouth e C G-120 (analysis edge), BDI+V, Bio-Get B10-rex 5 ( Analysis edge), Bio-Red gel filtration (ion ejection) chromatography Se phgdet LH-20 was stored in methanol until use and subjected to gravity chromatography. It was placed on a feed column as a methanol slurry. The gel was washed with the eluate until the bed settled, then the mixture was added as a thin layer. Eluate is a mixture of dichloromethane and gasoline, the ratios of which are shown in parentheses. High performance liquid chromatography (HPLC) U6 with injector and R4G +-(4021 differential refractometer and 481 variable wavelength 1v/vis, detection Uses a WATERS IIPLC system consisting of a 590 type pump with did. The HPLC column is as shown below, and the solvent system is shown in parentheses. v1 Gradient elution was performed using the same injector/detector arrangement as the two solvent pumps. 10 Analytical silica Il, manufacturing silica IIT, analytical reversed phase The purity of the final amino acid derivative was determined by HPL using a WATERS amino acid analyzer. Measured at C. Moderate Pressure Liquid Chromatography (MPLC)MPLC is Rheody++ e FMI-11P pump and WATE with 5G type Teflon injector It was carried out using a differential refractometer for RS R402 preparation. The columns used are below. The solvent system is shown in parentheses. 1, Fe++ig+5ule Ki+5ellel 6G, size C (VE RCK) l I, Lobsr FerliHsile Kie+elle1 6G, size B, 4G-63m (VERCKI [11, Lobsr LiChromop+ep RP-8, size ^, 43- 6f1m43-6f1 [l Infrared absorption (IR) All IR spectra are from PetkiIl-E1m@+297 type grating. Recorded using a photometer. The sample is liquid film (], f,) or sodium chloride. As nuiol between the lium discs or 1 mm of salt Carbon tetrachloride (CCI 4) or Jude in a sodium chloride cavity cell Recorded as a solution in dichloroform (CDCl2) (7.0 cm). Report on. Nuclear magnetic resonance (NMR) spectrum 'HNMR spectrum BTuktt AM-300 spectrophotometer (300,133MHz) A secondary NMR spectrum was recorded. Chemical shifts are deuterochloroform (CDC l2) Values are expressed relative to an internal standard of tetramethylsilane (TMS) in solvent. use Other solvents used were deuteromethanol (CD30D) and deutylium oxide ( D20). Solvents and standards are shown in parentheses. The coupling constant (J) is in Hertz The signal multiplicity is 1 (s), 2 (d), 3 (t), 4 (q), or 5. (qi), six (6), many (m), and wide (b). The coupling shown is Shown diagrammatically. No+lei+Ows+hgase+Execute effect experiment (NOE) Indicate when done. ”’CNMR spectrum Carbon-13 NMR was recorded using a B+wke+ AM-3N spectrophotometer. Perform both complete IH deflipping and DEPT or off-resonance spectra did. All parameters are shown as well as proton spectra. CoB and XY-correlation experiments Indicate when this has been carried out. Melting point measurement (m, p,) Melting point using Reicbert hot stage apparatus and O17mpa+ microscope was measured. All melting points are uncorrected and are given in °C. optical rotation Perkin-El■! Optical rotation of chiral compounds using r　141 polarimeter was measured. The sodium D line was used in all measurements unless otherwise specified. Solvents are shown in parentheses and concentrations are given in mg/m+. All measurements are made with a transmission length of 10 cm. Performed in a covered quartz cell of ml. Each sample was measured 5 times and the average rotation was calculated. others Unless otherwise specified, all reactions were performed at room temperature (average 25°C) and under a dry nitrogen (N2) atmosphere. Stir under ambient atmosphere. Compound 1-78 produced in Example 1-79 is as follows: Compound number U I N-carbobenzyloxy-glutamic anhydride 2 Z-L ~ pyrogluta Minic acid 3 Z-L-pyroglutamic acid t-butyl ester 4 Z-L-glutamic acid γ -Methyl ester 5 Z-L-glutamic acid α-t-butyl-γ-methyl ester le 6 Z-L-glutamic acid α-t-butyl ester 7 Z-L-glutamic acid α -Benzyl ester 8 Z-L-glutamate α-benzyl, γ-2°-pyri Dyl ester 9 Z-L-glutamic acid γ-hydroxamate 10 L-glutamic acid γ-hydroxamate Droxamate 11 Z-L-glutamic acid γ-2゛-hydroxyethylamide de 12 L-glutamic acid γ-2゛-hydroxyethylamide 13 Z-L-g Rutamic acid α-t-butyl ester γ-methylamide 14 Z-L-glutamic acid γ-methylamide 15 L-glutamic acid γ-methyamide Ruamide 16 Z-L-glutamic acid α-t-butyl ester γ-hydrazide 17 Z-L-glutamic acid α-t-butyl ester γ-N′-methylhydrazide de 18 Z-L-glutamic acid γ-N'-methylhydrazide 19 L-glutami α-t-butyl glutamate γ-N-methyl hydrazide 20 Z-L-glutamate Ester γ~2°-hydroxyethyl hydrazide 21 Z-L-glutamic acid γ-2′-hydroxyethyl hydrazide 22 L-Glutamic acid γ-2゛-Hydroxyethyl hydrazi 23 Z-L- Glutamic acid α-t-butyl ester γ-hydroxamate 24 Z-L-glutamic acid α-t-butyl ester γ-anilide 25 Z-L-glutamic acid γ-anilide 26 L-glutamic acid γ-anilide 27 Z-L-glutamic acid α-benzyl ester γ-2′-hydroxydiethyl Ruamide 28 Z-L-glutamic acid α-benzyl γ-2゛-aminoethyl ester 29 L-glutamic acid γ-2'-aminoethyl ester 30 Z-L-glue Tamic acid α-benzyl ester γ-tris(hydroxymethyl)methylamide 3LL-Glutamic acid γ-tris(hydroxymethyl)methylamide 32 L-glutamic acid γ-tris(hydroxymethyl)methylamide cyclization product 33 Z-L ~ Glutamic acid α-benzyl ester Arbis (hydroxyethyl ) amide 34 L-glutamic acid γ-bis(hydroxyethyl)amide 35 Z-L-g Rutamic acid α-benzyl ester γ-1′. 1°-dimethyl-2°-hydroxyethylamide 36L-glutamic acid γ- 1°, 1゛　-dimethyl-2゛　-hydroxyethylamide 37 Z-L-glutamic acid α-benzyl ester γ-ethylamide 38 L-glutamic acid γ-ethylamide 39 Z-L-glutamic acid α-ben Dyl ester γ-diethylamide 40 L-glutamic acid γ-diethylamide 41 Z-L-glutamic acid α-be ester γ-N-(2°-hydroxyethyl)piperazide 42 L-glue γ-N-(2'-hydroxyethyl)biverazide tamate 43 Z-L-glutamic acid α-benzyl ester γ-morpho 44 L-gluta Mic acid γ-morpholide 45 Z-L-glutamic acid α-benzyl ester γ-0 -Methylhydroxamate 46 L-glutamic acid γ-O-methylhydroxamate 47 Z-L-gluta Minic acid α-benzyl ester γ-2゛thioethylamide 48 L-glutamic acid γ-2° thioethylamide 49 Z-L-glutamine Acid α-benzyl ester γ-2° methoxyethylamide 50 L-glutamic acid γ-2° methoxyethylamide 51 Z-L-gluta Minic acid α-benzyl ester γ-2° chlorethylamide 52 L=Glutamic acid γ-2 Chlorethylamide 53 Z-L-Glutami α-benzyl ester γ-2′ hydroxyanilide 54 L-glutamic acid γ-2° hydroxyanilide 55 Z-L-glutami α-benzyl ester γ-3-chloroanilide 56 L-glutamic acid γ-3゛-chloranilide 57Z-L-glutamic acid α-benzyl ester γ-4′-chloranilide 58 L-glutamic acid γ-4°-chloranilide 59 Z-L-glutamine Acid α-benzyl ester γ-piperidide 60 L-glutamine γ-piperidide 61 Z-L-glutamic acid α-benzi ester γ-3゛　-hydroxypiperidide 62 L-glutamic acid γ-3°-hydroxypiperidide 63 Z-L-glu Tamic acid α-benzyl ester γ-[1(4-methylpiperazino)]amide 64 L-glutamic acid γ-[1(4-methylpiperazino)]amide 65 Z-L-glutamic acid α-benzyl ester γ-(4-morpholino)ami de 66 L-glutamic acid γ-(4-morpholino)amide 67 Z-L-glutami alpha-benzyl ester gamma-2'fluoroethylamide 68 Z-L-glutamic acid α-benzyl ester γ-2′. 2", 2'-1-lifluoroethylamide 69 L-glutamic acid γ-2", 2 ',2'-trifluoroethylamide 70 Z-L-glutamic acid α-t-butyl ester γ-〇-methylhydroxy mate 71 Z-L-glutamic acid γ-〇-methylhydroxymate 72 L-gluta Minic acid γ-0-methyl hydroxymate 73 Z-L-glutamic acid α-bendi ester γ-(2-pyridyl)methylamide 74 L-glutamic acid γ-(2-pyridyl)methylamide 75 Z-L-glu Tamic acid α-benzyl ester γ-prob-2-enamide (allylamide) 76 L-glutamic acid γ-propylamide 77 Z-L-glutamic acid α-be ester γ-(2-menloxyethyl)amide 78 Dihydroxazole analog Example I N-carbobenzyloxy-glutamic anhydride (1) (Z-L-glutamic anhydride tamic acid) (e.g. Le Qwe+ne, jJ, & YoOng, G, T, '1954) I, Cbtm, Soc, 195G: K11Be+, E, &G +bi+n　H,! 1961) JoI+as Liebig'+ Ann, C hew,,640. (See 145-157) Z-L-glutamic acid (25.0 g 10.0 g in acetic anhydride (150 mL)) 089m The mixture of ol) was stirred for 6 hours until all the Z-L-glutamic acid was dissolved. The reaction was stopped by removing excess acetic anhydride under vacuum (30°C, 2 mm) and the oil was gotten. The oil was poured into anhydrous ether (2 x 50 mL), then anhydrous gasoline (2 x 5 0 mL) and vacuum drying of the pure product yielded a white crystalline solid (23, 1g, 99%), melting point 91-94°C (literature value, KIiege+, E, &G ibiln, H, (196+1 Jwl1msLiebig’+ Aen, Chel, Shaku 145-157, melting point 92-93°C) was obtained. This preparation Repeating gave the desired product in 95-99% yield. At this stage, anhydrous No further purification attempts were made. Implementation M2 Z-L-pyroglutamic acid (2) (e.g. K11der, E & GibiI n, H, (186211 uslus Liebig's 8me, C to ew,, 6 55. (See 195-211) Anhydride (1) (1) in anhydrous ether (60 mL) and anhydrous THF (40 mL) 9.1g, 0. 072 mol) and dicyclohexylamine (14.5 mL, 0 ．． 088 mol) was stirred under nitrogen overnight. The solid formed in this reaction After filtering and washing with ether, recrystallization from boiling methanol yields a pure white DCHA salt of Z-L-pyroglutamic acid (21.0 g 180%) was obtained. DCHA salt (21 ,0g) The solution was stirred overnight to form the free acid. Filter and fix DCHA hydrochloride The material was extracted with ethyl acetate (3x 100 mL). The combined organic phases were diluted with sodium sulfate. The solvent was removed under vacuum to give a white crystalline product (17.50g). , 68% in total), melting point 136-138°C (literature value, Hisffi', E, A Gibth a, H, (1962) + Iwslo + Liebig’s ＾oa , Chew,, 655. 195-21. 1 3 7 - 1 3 9 °C) was obtained. Successive repetitions of this reaction yield the desired product with an overall yield of 65-75%. A product was obtained. Proton and 13C NMR spectra were in agreement with literature values (K IiBe+, E, & Gibi*Il, H, (196211e+lv+L icbig'+^an, Chew, 655. 195-211). 'HNMR (CDC13)j 62. 10 JIHm), 2. 50 (38m l, 4. 72 (IH, dd J-9, 3, 2,, 7), 5. 30 (28s ), 7. 35 (5M + n). "CN Aj ((CDC1sl:621. 72 (CH2), 30. 99(C)! 2 ). 175. 62 (pass)・ Example 3 Z-L-pyroglutamic acid t-butyl ester (3) (e.g. K11B2+, E, & Gibth n, H, (19G2) Iw+la+ Liebig’+ Ann. Ch+m,,655. 195-211: A11d1 mouth on, GJ, & Cs111h*n, F, 11. (1962) J, ^m, Chet Soc,... ! 12. 3359-3363) Method A: Z-L- in dichloromethane (DCM) (120 mL) saturated with isobutylene. Pyroglutamic acid (Log. 0. 038 mol) was dissolved. Add sulfuric acid (98%, 3 mL) slowly; Isobutylene was bubbled into the solution for 1 hour. After this, t, 1. c, (silica, 2 (0% gasoline/ether) no starting material was observed. The DCM solution was diluted with water (2 x 50 mL), then washed with sodium bicarbonate solution (10%, 2 x 50 mL), Dehydrated over sodium sulfate. Removal of the solvent under vacuum results in a mixture of the two products A product was obtained, which was subjected to flash chromatography (20% gasoline/ether). ) to give ester (3) (RfO, 3.6 g, 50%), mp 55-57 °C, [a] 0-29. 0 (c=2. 04, DMF) and α. γ-di-t-butyl-Z-L-glutamate (Rfo, 3. 6g118%) is obtained. It was done. This reaction is subsequently repeated with varying amounts of diester byproduct and 40- The desired ester was obtained with a yield of 75%. Add sulfuric acid catalyst to perchloric acid or palate Yield even if replaced with luenesulfonic acid or boron trifluoride diethyl ether was not improved. Method B Z-L-pyroglutamic acid (20g, 0. 076mo1) to t-butyl acetate ( 300 mL). Add perchloric acid (70%, 1 mL) to this solution and stock The mixture was stirred overnight at 0° C. in a stoppered flask. Ethyl acetate (150mL) Dilute with water (2 x 50 mL), sodium bicarbonate solution (10%, 2 x 50 mL) ) and saturated sodium chloride solution (1×50 mL) to stop the reaction. Drying the reaction solution over sodium sulfate and removing the solvent under vacuum yields pure (3 ) (RfO, 32006 Gasoline/Ether, 9. 31g, 39%), melting point 57 -59°C was obtained. NMR ('H and 13C) indicates that this compound is isobutylene. It was identified as a product of 1non reaction. 1H release (CDCLs): 51. 38 (98s), 2. 03 (IHdd ddJdo2. 7. 3. 2. 9. 5. 13. 1), 2. 31 (IHdddd J Tomi 9. 2. 9. 3゜10. 5. 13. 1), 2. 49 (IHddd J Tan 3. 2. 9. 2. 17. 51. 2. 63 (18ddd J’-9,5,10,5 , 17, 51, 4, 54 (IHd, d J-2, 7°9. 3), 5. 23. 5 ．． 29 (28AB Jtomi 12. 3), 6. 50 (IHbl 7. 36 (5 8ml. “CNMR (CDCl2): 621. 58 (CH2), 27. 52 (C H31°30. 70 (CI(2), 59. 14 (CI(), 67. 85 (C1(21,82,18(C1゜127. 86 Practice 4-Note C1(), 12 8. 09 Injury C) (), 128. 25 Example 4 Z-L-glutamic acid γ-methyl ester (4) (e.g. 11tab7. W , E, , W*Iey, S, G, , W*1soi 1. & Asbtos s, E, I. (1950) 1. C1us, Soc, 3239-3249) method A: Protection of monoglutamic acid γ-methyl ester by benzyl chloroformate (Hsn b7. W, E,, Wsle7. S, G2. LlsoIII, &1mb +ose, E,], (1950)], Chew, Soc,, 3239-32 49) L-glutamic acid γ-methyl ester (5.0 g. 0. 031mo+) in sodium bicarbonate solution (20%, 20 mL) and the pH 9. Adjusted to 0. To this solution was added benzyl chloroformate (4.84 mL, 0.5 mL, 03 4 mol) was added and the mixture was cooled to 5°C. Stir the reaction mixture overnight; t, that no starting ester remains; 1. c, (cellulose, Rfo, 2 4). pH 2. Adjust to 0 and extract with ethyl acetate (3x 60 mL) did. The combined organic layers were washed with water (10 mL) and dried over sodium sulfate. Removal of the solvent under vacuum gave a white crystalline solid (8.0 g, 87. 5%), melting point 7 1-73℃ (literature value, HsnJ, W, E,, W*lB, S, G,, Wt l+onj　^　^mb+o+e, E, 1. f1950) J, Cksm , Sot,, 3239-3249. 72-73℃), [α] -14,3 (c=10. 60. methanol) (Reference 5, [α] -15,3 (c=7. 46 , 1. 4M KHCO3)) was obtained, which was obtained at t, 1. c, (cellulose, Rf o, 80) was pure. 8. 0), 5. 11 (2Hsl, 5. 61 (LM d J-8,0), 1. 30 (5) 1 m). 8. 53(l)Ib). Method B: Attempt to selectively esterify Z-L-glutamic acid using methanol (2 0 mL). Add acetyl chloride (1,60 mL, Q, 024 m) to this solution. o+) was added and the mixture was stirred overnight (Hsnb7. W, E,, W*l e2. S, G,, Wtl+on 1. &^mbtose. El, (195011, Chew, Soc, 3239-3249). this The resulting solution turned pale yellow in color. Pyridine (2 mL) was added and the mixture was stirred for another 24 hours. Stir for a while. t, l, C, (silica, 5% acetic acid (0,1 )) shows that the main product is dimethyl ester (Rfo, 58). This was also confirmed by 'HNMR analysis. Example 5 Z-L-glutamic acid α-t-butyl-methyl ester (5) (e.g. Ts+ bnt+, E,, L+i+1evski, C,, 5okolovs1. T , &Bi! +Il,], F, (1961) Jotla+Li Yamapaku Aa n, Chsm, 646°127-133) Methyl ester (4) (7,000 g) as above. 0. When 024mol was treated with t-butyl acetate, t, 1°c, (silica, 60 % ether/gasoline, RfO, 84,022). A compound was obtained. These components were separated by flash chromatography (silica, 20% % ether/gasoline), a viscous oil (RfO, 22, t, 1. c, silica, 60% ether/gasoline) as α-t-butyl ester (5) (4 ,90g,58. 7%) was obtained. The components with high Rf values are α, γ-di-t-butylene. It appeared to be Luester. 'HNMR (CDCl: 6 1. 45 (98S), 1. 97 (18d ddd J-7, 9, 8, 1, 14, 2, 14, 3), 2. 23 (lh) bddd J -6,5,8,3゜14. 31. 2. 39 (2Hml, 16 6 (3Hs), 4. 2El　(IHddd　J　-6,5,7,5,7,9 1,5,10428sl, 5. 37 (18d J = 7. 5). 7. 34 (58ml. Example 6 Z-L-glutamic acid α-t-butyl ester (6) (Ta+hne+, E, , Ws+1elev+ki, C,, 1 to 5okolov+. T, & B ierosl. ], F, (196111u+Ia+Liebig’+AlIn, Chel l,, 646. 127-N3) Method A (3) ring-opening attempts (e.g., Kl+ige+, ε & Gibixn, H. (1962Ha+ta+Li+big'+Ann, Che+a,, 655 ．． 195-211) t-butyl ester (3) (500 mg, 1. 56m mol) in acetone (7 mL), and add sodium hydroxide solution (LM, 1. 9 mL) was added. The solution was refluxed for 1 h, t, 1°C (silica 80% ether (gasoline/gasoline). After this, almost no starting material was observed, and hydrochloric acid (0, 2M) to pH 1.5 and extracted with DCM (3x 25 mL). If The combined organic layer was dried over sodium sulfate and the solvent was removed under vacuum to yield two products. A mixture of products was obtained (RfO, 05,078, silica 80% ether/gasoline). Rin). The mixture was subjected to flash chromatography (Shitsuka, DCM, then acetic acid). The starting ester (3) (220 mg, 44%) and t -butyl ester (6) (328 mg, 65%) was obtained. methano as a solvent Repeating this experiment using the 5-methyl ester in 35-50% yield was gotten. Example 7 Method B: Hydrolysis of (5) (e.g. Ts+bae+, E,. L+1elev+ki, C,, Sokolovsk, T, & Bie+ ■l, I, F, (1961) Iu+I++ Liebig’+ Ann, C hell,, 646. 127-133: 5hin, C, G,. Yone+xwg, Y, & Wit*n*be, E, (19851Tzl+zh +d+on Lell. 26, 85-88) Diester (5) (4.90g, 1. 39 mmol) in methanol (20 mL ) and water (5 ml). To this solution was added lithium hydroxide hydrate (0.64 g, 1. 42 mmol) was added and the mixture was stirred at 0° C. for 1 hour. After this, remove the solvent It was removed under vacuum (approximately 20° C.) and the residue was taken up in water (50 mL). Add this solution to acetic acid. Extracted with ethyl acetate (2 x 20 mL), then acidified to pH 1,5 and diluted with ethyl acetate ( Extracted again with 3×20 mL). The latter extract was dehydrated over sodium sulfate and Removal of the solvent under vacuum yielded t-butyl ester (6)-, (3,27 g, 70% , RfO, 50t, 1. c, silica, 60% ether/gasoline) was obtained. Subsequent repetition of this preparation gave (6) in 70-80% yield. (LHd J-8, 11, 7, 31 (58ml, 7. 75 (18bs). Example 8 Z-L-glutamic acid α-benzyl ester (7) (Mo+Ie7°1, S, (1967) 1. Chess, Soc, fc), 2410-2421 ) Add Z-L-gluta to dimethylformamide (DMF) (50 mL) at 15°C. Minic acid (50.0g, 0. 178 mol) was dissolved. Add triethyl to this solution. Amine (24.64 mL. 0. 178 mo+) and benzyl bromide (23.24 mL. 0. 195 mo+) was added. The reaction mixture was maintained at 15°C for 6 hours and then brought to room temperature. Warmed overnight. After this time, ice water (300 mL) was added and the mixture was dissolved in ethyl acetate (3 g 1 60 mL). The combined extracts were washed with ice water (2x 50 mL) and diluted with sulfuric acid. Dehydrated over sodium. The solvent was removed under vacuum and the residue was dissolved in ethyl acetate (180 m I took it to L). Dicyclohexylamine (38.67 g, 42. 36 mL, 0. 214 mol) was added and the mixture was stirred for 6 hours. After this, obtained The solid was collected by filtration, washed with cold ethyl acetate (60 mL), and dried in a vacuum oven. Upon drying, the dicyclohexylamine salt (97.8 g, 99%) was obtained. Recrystallize the crude product from boiling ethanol (250 mL) The pure salt (66.5 g, 68%) was obtained, which was dissolved in ethyl acetate (300 g, 68%). mL) and hydrochloric acid (3.5 M, 70 mL, 2 eq.) and stirred for 2 hours. blend was filtered and the layers were separated. The aqueous layer was extracted with ethyl acetate (2 g 50 mL) and combined. The combined organic layers were washed with cold water (2 g, 50 mL) and dried over sodium sulfate. true Removal of the ethyl acetate under vacuum gave a colorless oil, which was dissolved in diethyl ether/gasoline. (1: 1. 25 mL) for crystallization. Pump this product for 4 hours. After drying for a while, α-benzyl ester (7) (44.5 g, 99.9 g) was obtained. 5%, total So 67. 5%), melting point 96-98°C (literature value, Mo+lsr 1. s, (19 671J, ChetSot, (C1, 2410-2421, melting point 97-98 ℃), [α] 20-〇 18. 9 (c=10. 60, DMF) was obtained. 5. 57 iIHJ J = 8. 1), 7. 33 (IOHml, 8. 8 (IHbl. Z-L-glutamic acid α-benzyl γ-2°-pyridyl ester (8) (S , K111. 1. 1. Lee and Y., Ko, (1984). Tef+zhed+on Left,, 25 (431, 4943-4946) or ^S, Dwll* and "S, Mo+ler, (19811, 1, Chew , Soc, (see C1, 2896-2902) Z-L-glutamic acid α-ben Dyl ester (7) (881mgz 2. 37 mmol) to anhydrous pyridine ( 7 mL) and 2-hydroxypyridine (405 mg, 4. 26 mmol) and dichlorohexyllupodiimide (DCC) (645 mg). 3. 08 mmol) (!Reacted at 15°C for 24 hours. After this, the pyridine was removed under vacuum. Gin was removed. The crude product was redissolved in ethyl acetate (4 mL) and filtered. Soluble dicyclohexyl urea was removed. Sodium bicarbonate solution in ethyl acetate (2 g 3 ml) and then extracted with saturated sodium chloride solution (1 g 3 ml). Yes Removal of the solvent from the organic layer under vacuum gave the crude product (8). Clean the crude product. When subjected to chromatography (silica; 50% gasoline/ethyl acetate), 2゛ -pyridyl ester (8) (1,Olg, 91%) was obtained, which still contained a small amount of Contains dicyclohexyl urea. 'HNMR (CDC13) + 62. 12 (2Hml, 2. 34 (2M) ml, 4. 55 (1M in), 5. 115. 18 (4H2x AB’s ), 5. 60 (IHd J-6,81,7,04 (IHml, 7. 2( j-7,74 (121 (m), 8. :17 (LMml. 1. 3 Intermediates and nuclear reaction reactions The following example describes the preparation of the compound of the invention defined by formula Acid γ-hydroxamate (9) Z-L-glutamic acid (500 mg, 1. 78m mol) was placed in THF (2 mL). Add hydroxylamine hydrochloric acid to this solution. The salt was dissolved in water at 5°C and adjusted to pH 8.0 with sodium hydroxide solution. Created by adjusting to 0 Hydroxylamine aqueous solution (1.8 mL, 4M, 7. 12 mmo I) was added to this solution. added to the liquid. t, l. c, (cellulose, RfO, 49, starting material Rfo, 63) for 4 hours. Afterwards the reaction was completed. pH 2. 0 and remove the solvent under vacuum at approximately 15°C. , then the residue was extracted with THF (2x10mL) and ethyl acetate (2xlOmL). did. Removal of the solvent from the combined extracts gave a white solid. this substance Recrystallization from chloroform gave (9) as a white crystalline solid ( 535 mg, 96%). Repeating this experiment resulted in 80-95% yield of hydroxa. mate (9) is obtained ■, -glutamic acid γ hydroxamate (10) Z-L-g Rutamic acid γ-hydroxamate (0,50g)) (P L C class methanol 10% palladium on charcoal (0.05 g) was added. Siri The sample spotted on the chromatography plate no longer shows u, v, activity. The mixture was stirred under an atmosphere of hydrogen until the mixture was dissolved. After this (30 minutes to 1 hour), The reaction mixture was filtered through a Kaiser gel pad and the pad was washed with water (5 ml). filter When the liquid and washing liquid were combined and the solvent was removed under vacuum, γ-hydroxamate (10) was obtained. (270 mg, 98%) was obtained. Melting point 145-146°C. The proton NMR spectrum and melting point of (10) are the authentic compound purchased from 51gm5. It was the same as that of Example 12 Z-L-glutamic acid γ-2"-hydroxyethylamide Z-L-pyroglutami acid (2) (2.0 g, 7. 6 mmol) in THF (20 mL) and water (1 m I put it in L). Ethanolamine (1.4 mL) was added to this solution and the reaction was stirred overnight. Up Treatment of the reaction as outlined above gave the crude product. This substance is Sξp It was purified by applying h*dtxSP-C25 chromatograph 1E and eluting with water. Removal of water gave (11) as an oil (2. 0g, 81%). 'HMMR (020/DCI) + J 1. 7 (18ml 1. 9 (IHm l　2. 1 (2Hml 3. 05 (28q) 3. 17 (LM tl 3. 40 (2Htl 3. 95 (l) Im) 4. 90 (2Hs) 7. 17 (5Hml. Example 13 L-glutamic acid γ-2'-hydroxyethylamide (12) derivative (11) (600 mg) was placed in HPLC grade methanol (5 mL), and the charcoal was supported on this. 10% palladium (60 mg) was added. This solution was hydrogenated as described above. Upon processing, (12) was obtained as a white powder (340 mg, 97%). melting point 210-210. 5℃, [α +7. 3 (e=10. 96, Wednesday). HPLC Analysis (amino acid analyzer) showed that the purity of the product was over 99%, with glutamine Acid impurity was less than 1%. 'HN)[R(D20/DCI)+J2. 17 (2Hdddd J-2, 2,4,7゜3. 3. 14. El) 2. 46 (2Hddd J Dew 3. 3 ．． 1. 8. 14. 8) 3. 32 (2Ht J-5, 3) 3. 64 ( 28t J-5, 4) 3. 92 (LM dd J-2, 2゜4. 8). “CNXR (D, 0) I J 27. 40 (CH2) 32. 80 (C1( 2) 42. 91 (CH2) 54. 72 (CHI 61. 33 (CH21 174,11 (Ami H’) 176. 91i1Z-L-glutamic acid α-t- Butyl ester γ-methylamide (13) t-Butyl ester (3) (89 mgq 0.00 mg in methanol (1 mL)). 28m To a stirred solution of mol) was added methylamine (24% aqueous solution, 50 mLq 0. 37m mol) was added. t, I, c. (silica, 75% ether/gasoline), starting material was observed after 30 minutes. It wasn't done. Removal of solvent and methylamine under vacuum yields t, 1. Indicated by C. A mixture of two products was obtained as shown. Flash chromatography of crude product graphy (45% ether/gasoline) and removal of the solvent under vacuum. γ-Methylamide (59 mg, 60%) was obtained as a white crystalline solid. Melt Points 109-109. 5℃, [α] D 2G+ 3. 9 (c = 1-1, chloroform). The main byproduct of the reaction was formed by competitive ring opening with methoxide γ-methyl ester (34 mg, 35%). Repeat reaction in THF Removal of leverage side reactions improved the yield to 88%. KS H+ 350. m/@249 (55%), 174 (27%), 9B (27%), 92 (15%), 91 (loo%l, 84 (12%) ), 13 (15%), 57 (28%l, 42 (16%), 19 (1 3%). 'HMKR (CDCIs) + J 1. 45 (98ml 1. 19 (LM) ml 2. 22 (28ml 2. 78 (38bd J = 4. 614. 20 (LM ml 5. 10 (28sl5. 60 (18bd J accent 7. 5 )6. 03 (IHbs)7. 34 (5H town. Eye CNXR (CDC13) i526. 28 (CH3127, 90 (CH312 9,11 (CH2) 32. 44 (CH2154, 01 (CH) 66. 93 (CH2) E12. 43 (C1128,0212B, 13128. 47 (%4-Taku C8's) 136. 21 (#411 town C1156,57 (voice) ri, bame)) 171. 02 (Pumi γ゛) 172゜02 (Amido) 1 F2.5F (j-ste Jshi) Z-L glutamic acid γ-methylamide (14) (Aads mouth 0 mouth. G, W, & Cs1ltbsa, F, M, (1862) 1. At Chew ,Sac,,82. 3359-3363) γ-Methylamide (13) (33 mg, 0. 10 mmol) in cold trifluoro in acetic acid (TFA) (e.g. BrHn. D, B, , Hrll, R, F, , HoldeI1. ni, G,, Hul la*nm, W, F, &Gle*+on. 1, G, (19771, 1, AL Chl, Soc,, 99. 　2353- 2355) and the mixture was stirred in a water bath for 30 minutes. After this, t, 1. C . (Nrika, 5% methanol/chlorohelm), the starting ester is It didn't exist. Remove TFA under vacuum. and the crude product as a pale yellow oil. Obtained. This product was purified by flash chromatography (5% methanol/chromatography). After removal of the solvent in vacuo, a white crystalline solid (24 mg 18 5%) was obtained. When acid (14) is recrystallized (chloroform/gasoline), min. A pure sample was obtained by optical analysis. Melting point 117-118°C. 6. 12 (IHbd J = 7. 2) 6. 67 (18bs) 7. :1 0 (5Hrn) 9. 36 (lHbil. Example 16 L-glutamic acid γ-methylamide (15) (Licblen+1ein. N, (+9421 1. ＾m, CbemSoc,,64. 1021-111 22) Z-L-ri/lz-tami:/acid y-1 tyramide (14) (2,40g 58. 15 mmo I) was placed in absolute ethanol (50 mL), and charcoal-supported 1 0% palladium catalyst (260 mg) was added. This mixture is hydrogenated using standard methods and the disappearance of UV activity is determined by t, l. It was monitored with C. After 4 hours no starting material remained and the mixture was filtered. . The catalyst was washed with water (2xlOmL) and the solvent was removed from the combined washings and filtrate under vacuum. Removed below. Recrystallization of the residue (80% ethanol/water) yields pure amino acids. (15) (1,Olg, 79%) was obtained as a white crystalline solid. Melting point 193-194°C (literature value, Licbl+n+1ein, N, (1942 )　I. ^-Ch! m, Soe,,64. 1021-1022, melting point 192℃), [α ] +4. 1 (c=1. 23, water) (literature value, [α] +6. 5). this substance The purity of Ho Confirmed by PLC. 'EI NIKR (p, o) + 62. 12 (28ml 2. 39 (2Hm l　2. 71 (3) 1sl 3. 75 (18bt J-6,7). “CNIMR (D, O): 626. 49 (CH3) 27. 24 (CH2 132, 12 (CH2) 5B, 8B (CM) 174. 81 Gemito) 175. 7B (front plate). (3) was opened with a series of other nuclear materials as outlined below. Experimental hands on each side The order was the same as above. Example 17 Z-L-glutamic acid α-t-butyl ester γ-hydrazide (16) (e.g. , K11eH+, E, & Gibitn, H, (1962)] a+1usL i+big’i Ann, Chew,, 655. 195-211 and T*+b ne+, E,. W*+i+lev+ki, C,, Sokolow++, T, Bie+mouth 81 1 1. F, (1961)]n5lo+Liebig'+A++n,C1 u1. , 646. 127-133) Ester (3) (118 mg, 0. 3 7 mmol) was added to hydrazine hydrate (16 μl, 0.7 mmol). 32 mmol) and γ-hydrazide (16) (124 mg-95%) as a white crystalline solid. Obtained. Melting point: 95-100°C (literature value, 5 h. C, G, Yonc+svg, Y, &’isfxlIgbe, E, (1985 ) Tsl+th+d+onLu11. , 26. 85-8N, melting point 112-1 13°C)'HNMR (CDCl2) + J 1. 45 (98sl 1. 90 (18ml 2. 22 (3Hml 3. 87 (18bs) 4. 22 (IH bt J -7,4) 5. 1i (21(s) 5. 54 (IHbd J =7. 417. 37 (58ml. Example 18 Z-L-glutamic acid α-t-butyl ester γ-No-methylhydrazide ( 17) Ester (3) (288 mg, 0. Add 9 mmol of methylhydrazine (60 μl, 1. 13 mmol), it reacted with glass in low yield (103 mg, 31%). γ-N°-methylhydrazide (17) was obtained. MS　M+　365. m/e 320 (2%) 309 j71%l 29 2 (16%) 136 (32%) 130 (59%l 113 (76J) 9 1 (59%) 84 (71%) 57 (100%146 (95%l) 42 (63% 129 (51%) 19 (63%) 1 day (57%). 'l'l　) DIR (CDC13): 61. 43 (9Hsl 1. 92 ( IHm) 2. L8 (3Hml 2. 57 (3Hm) 3. 57 (IHbs ) 4. 20 (IHk)t: -8,0) 5. 0B (2Hsl 5. 8 0 (IHd J = 8. 0) 1. 32 (5Hm17. 87 (IHbs ). ■C trace (CDCl2) i J 27. 90 (CH312B, 91 (CH2) 30. 66 (CH2) 39. 16 (CH3153, 93 (CM) 67. 01 (CH2182・49 (C) 12B, 06 (Fragrance C) I) 1 2B, 15 (ji-Yoshibee CH1128, 47 (3J# attack CM) 136. 17 (C1156j7 (/jlu/'me-) 1 170. 30 +7? )”+171. 93 (@). Example 19 Z-L-glutamic acid γ~N'-methyl hydrazide (18) 7-N'-methyl Hydrazide (17) (77mgS0. When 21 mmol) is reacted with TFA, Free acid (18) (28 mg, 90%) was obtained as a clear glass. [α]2 0-10. 0 (c=1. 0, water). HNMR spectrum shows t-butyl group Confirmed the removal of. Example 20 L-glutamic acid γ-N'-methylhydrazide (19) 10% palladium supported on charcoal acid (18) (23 mg, 0.5 mg) in the presence of 07 mmol) was hydrogenated. When added, amino acid (19) (10 mg, 83%) was obtained as a transparent glass. Ta. 'HM (D, O) + J 2. 09 (28ml 2. 33 (2Hm) 2. 53 (3Hbsl 3. 65 (IHrnl 7. 43 (LM 81゜implementation) Example 21 Z-L-glutamic acid α-t-butyl ester γ-2′-hydroxyethyl Drazid (20) Protected pyroglutamate (3) (4.0 g, 12. ．． 5mmo+) Ethylhydrazine (2.09g. 27. hydrazide (20 mmol) as a hygroscopic white solid. ) (4.90 g, 99%) was obtained, which was obtained at t, 1. C, (silica, 10% Methanol/chloroform, Rf 0. 53) was more pure. 'HNMR (CDC1sll 61. 45 (9) 1 sl 1. 90 (IH ml 2. 23 (3Hm) 2. 90 (28 towns 3. 55 (2Hml 4. 2 0 (18ml 5. 0942Hs) 5. 70 (18bd J = 7. 9 ) 7. 34 (5Hml 8. 02 (IHbsl. “CNMR (CDC1x) = 627. 93 (CH3) 29. 31 (CH 2130,6,6 (CH2) 53. 51 (CHI 5B, 75 (CH21 60,05 (CH2)67. 16 (CH2182, 76 (C112B, 12 (#l-Nod CHI 12E1. 27frr4i1: CHI 12B, 53 (1 person Ji CHI 136. 07 (Ff area C) 154. 54 (:n le Bame-11170,84 (Allop') 17153Z-L-glutamic acid γ- 2゛　-Hydroxyethylhydrazide Hydrazide (20) (4 , 7g) to give the acid (20), which was flash chromatinized. Purification by matography (10% methanol/chloroform) yields pure (21 ) was obtained (3. 8g, 94%). Melting point 88-95°C (decomposed). L-glutamic acid γ-2°-hydroxyethyl hydrazide acid (21) by standard method Hydrogenation of (2.1 g) gives the amino acid (22) as a hygroscopic white solid ( 1.24 g, 99%) force (obtained). 'HXKR (DtOl: 62. 00 (2Hm) 2. 36 (28ml 3 ．． 18 (2Ht J-4,9) 3. 583. 60 (28d AJI J = 12. 6613. 87 (LM t (ridge)・ Example 24 Z-L-glutamic acid α-t-butyl ester γ-hydroxamate (23) Protected pyroglutamic acid (3) (5.0 g, 15. 6mmo+) by HPLC The solution was dissolved in grade methanol (20 mL). Add hydroxylamine hydrochloric acid adjusted to pH 8.0 with sodium hydroxide to this solution. Salt (27 mL, 2. 4M, 62. 5 mmol) was added. The reaction mixture was heated for 24 hours. After stirring for t, no starting material remains.1. c, (silica, 80% ether/gasoline). pH 2. 0 and the mixture Extracted with ether (3xlOOmL). Evaporating the dehydrated ether extract A viscous oil (4.80 g, 87%) was obtained, t, 1. C, (silica, 80% a Tel/Gasoline, RfO, 21) indicates that this is a compound. Ta. bd) 7. 30 (5Hml. Example 25 Z-L-glutamic acid α-t-butyl ester-γ-anilide acid (6) (3,6 1g, 10. 7 mmol) was dissolved in anhydrous THF (25 mL) and cooled to 5°C. . Triethylamine (1.63 mL, 11. 7 mmol) and reduce the mixture to approx. Stir for a minute. After this, ethyl chloroformate (1.13 mL). 11. 7 mmo+) was added and the mixture was stirred for a further 3 minutes. Next, add aniline (2.92 mL, 32 mmo+) in THF (5 mL). , the mixture was allowed to warm to room temperature over 30 minutes. After 2 hours, t, 1. c, (silica, (50% ether/gasoline) with almost no starting acid observed. under vacuum Removal of the solvent gave the crude product, which was dissolved in DCM (50 mL) and Water (20 mL), 0. 1M hydrochloric acid (10 mL) and saturated sodium chloride (20 mL) ) was washed. Evaporation of the solvent from the dehydrated solution yielded the crude anilide (24). and purified by flash chromatography (70% ether/gasoline). A pale yellow solid (2.58 g, 59%) was obtained. Reconstituted from ether/gasoline A white solid was obtained upon crystallization. Melting point 136-137°C. 'HMKR (Cχis) + J 1. 44 (9HJ 1. 97 (l) l m l　2. 35 (LM　rnl　2. 43 (28rn) 2. 80 (IHbs l 4. 30 (18ml 5. 11 (2Hs) 5. 66 (18d J - 7,617,10 (IHml 7. 34 (7Mm17. 60 (2Hm). Example 26 Z-L-glutamic acid γ-anilide (25) at 0°C, ester (24) (206 mg, 0. 5 mmol) was treated with excess TFA (1 mL). t, 1. c. , (70% ether/gasoline). Noticeable amount even after 2 hours Since the starting ester remained, the mixture was allowed to warm to room temperature. Another 2 o'clock After that, t, 1. Since the starting ester was not detected by C, the true TFA was removed under vacuum to obtain crude product (25). When ground with carbon tetrachloride, Pure acid (25) (158 mg, 89%) was obtained as a white powder. Melting point 155-158°C. (2Hml 4. 23 (AHml 5. 06 (28s) 7. 06 (IH m) 7. 29 (8Mml 7. 51 (1M ml. Example 27 L-glutamic acid γ-anilide (26) acid (2 5) (1.02g. 2. 5% palladium on charcoal (203 mg) was added to a solution of 87 mmol). child The mixture was hydrogenated for 2 hours, after which time t, 1. C, (25% methanol/c (loloform), no starting acid was detected. When the mixture is treated with standard methods , pure (26) (636 mg, 99%) was obtained as a white crystalline solid. Melting point 210-210. 5℃. ). ]. '60B, 9 (c-8, 90, 7 (). 'HKKR (D, O) + 82. 202. 24 (2HdAB Jtomi 7. 712 ．． 59 (2Hdt J = 7. 7. 5. 013. B2 (IHt J-5, 0) 7. 25 (1Mm)7. 42 (4Hml. Example 28 Z-L-glutamic acid α-benzyl ester γ-2゛-hydroxyethyl ester Mido (27) α-benzyl ester (7) (1,27g, 3. 42 mmol) in THF (6 mL) and cooled to 5°C. Triethylamine (0.48 mL) was added to this solution. , 3. 5 mmol) was added and the mixture was stirred for about 2 minutes. After this, chloroformic acid Ethyl (0.38 mL, 3. 5 mmol) was added and the mixture was stirred for an additional 3 minutes. Ta. React ethanolamine (0.41 mL) in THF (1.5 mL). was added slowly to the reaction mixture and the mixture was allowed to warm to room temperature. After 10 minutes, t, 1. No starting material ester was detected in c, (silica, 80% ether/gasoline). It was. The solvent was removed under vacuum and the resulting oil was dissolved in ethyl acetate (50 mL). acetic acid The ethyl was washed with 5% sodium bicarbonate (10 mL) and water (10 mL), then with sulfuric acid. Dehydrated over sodium chloride. Removal of the solvent under vacuum gave (27) (1.10 g 175%) as a colorless oil. t, 1. C, (silica, 80% ether/gasoline, Rfo, 10 and silica Rica, 10% methanol/chloroform, Rfo, 60), it is pure. there were. 6. 38 (l) I bsl 7. 33 (IOHml Example 29 L-glutamic acid γ-2°-hydroxyethylamide (12) 10% charcoal-supported paste Amide (27) (60 mg) in methanol (5 mL) using radium (60 mg) Hydrogenation of amino acid (11) (210 mg, 76%) According to 'HNMR, it was prepared by ring opening of Z-L-pyroglutamic acid. It matched the thing. Melting point 210-211°C, (# consideration, Example 13, melting point 210-2 10. 5℃), [α] +6. 88 (c=9. 39, Water), (Reference, Example 1 3. [α] +7. 3 (c=10. 96, Wednesday). Example 30 Z-L-glutamic acid α-benzyl ester γ-2°-aminoethyl ester (28) α-benzyl ester (7) (2.0 g, 5.0 g in THF (9 mL) at 5° C. Add 39 mmol of the solution to triethylamine (0.75 mL, 5. 40 mmol)’ ? It was treated for 1'2 minutes. Next, ethyl chloroformate (0.52 mL, 5. 4mmo ) was added and the mixture was stirred for about 3 minutes. 2-ethano in THF (5 mL) triethylamine (1.16 g, 6 mmol) and triethylamine (1-5 mL, 1 0. 8 mmol) solution was slowly added to the above mixture and the reaction mixture was brought to room temperature. The mixture was stirred for 30 minutes while warming. The reaction mixture was stirred for a further 24 hours, after which t, 1. C, (silica, 0. The starting ester (1% acetic acid/ether) 7) could not be detected. When the reaction mixture is treated as described above, 'HNMR shows As confirmed, Z-ethanolamine and diester (28) (1:4) (2, 8 g, 91%) of various solvents (80% ether/gasoline; 6 Flash chromatography using 0% ether/1% acetone in gasoline) Fee (t, 1. C, silica, 0. 1 acetic acid/ether, Rfo, 517. 0. Attempts to separate the two components at 510) failed, so the mixture was hydrogenated. s) 5. 03 (18bd) 7. 24 (10Hml. Example 31 Synthesis of L-glutamic acid γ-2°-aminoethyl ester (29) methanol (10%) using 10% palladium on charcoal (200mg) as a catalyst Hydrogenation of diester (28) (2,0 g) in m L ) produces a single product L-glutamic acid (460 mg, corrected yield 88%) was obtained as a product. crude production t of things. 1, c, (silica, 10% methanol/chloroform) or by 'HNMR , γ-2°-aminoethyl ester was detected as Z-L-glutamic acid α-benzi ester γ-tris(hydroxymethyl)methylamide (30) α-Henodyl ester (7) (1.4g, 3. 77 mmol) in THF (5 m Triethylamine dissolved in THF (5 mL) and water (4 mL) (600mL, 4. 5 mmol), ethyl chloroformate (410 mL, 4. 5mm ol) and tris(hydroxymethyl)methylamine (2 g, 18. 9mmo 1). The crude product was subjected to flagge chromatography (100% chloro pure (30) (1,46g) as a hygroscopic white glass , 79%) was obtained. 3. 63 (6HAB) 4. 41 (LM ml・4. 70 (3Hbsl) 5. 10 (2Hs15. 105. 14 [28ABI 5. 9j (IHd) J-8, 3) 6. 66 (Li(bs17. 31 (108ml. I3CNKR (CDC1sl+! 2B, 03 (CH2132, 12 (CH 2153, 21 (CM) 61. 71 (C) 62. 82 (CH2) 67 ．． 15 (CH2167, 32 (CH2) 127. 9 (flirt, CM ) 12B, 20 (i sound line CH1128, 47 (Sae 4Q CHI 128. 5 6 (: Kri f, CHI 135. 03 (Sae) 135 ．． 89 (,1i-4-kana,,,C) 156. 35 (Katana Runo N'Me) )171. 85　(A?)″l　173. 62 (Niskutu V). Synthesis of L-glutamic acid γ-tris(hydroxymethyl)methylamide (31) The amide (30% ) (1,10 g), filter the mixture through a bed of celite and vacuum A single compound was obtained in low yield after removing the solvent below. Celite floor 0. 1M salt Most of the product was recycled by washing with acid. When the solvent is removed under vacuum, the hygroscopic Cyclized amino acid (32) (520 mg) as a white solid. 92%) was obtained. If the above experiment is repeated without hydrochloric acid, cyclized amino acids (32 ) (70%) was obtained as a hygroscopic white powder. 4. 32 peaks If large C, J, JK, 1 knee 2 or - ^ and upper device 1 δ . Z-L-Glutamic acid α-benzyl ester γ-bis(hydroxyethyl)amide Do (33) Acid (7) (3.76 g, 10.0 g in THF (10 mL)) 1mmo+) solution , at 15° C. for 1 hour, triethylamine (1.55 mL, 11. 1 mmo +) , ethyl chloroformate (1.06ml, 11. 1mmol) and bis(hydro) xethylamine) (3.4 mL, 30. 3 mmol). When this reaction solution is subjected to standard finishing treatment, t, 1. C, (10%, methanol/ Chloroform, Rfo, 63, Rf (starting material acid) 0. 48) shows that pure Bis(hydroxyethyl)amide (33) (2,81g, 59%) was obtained. (2H Town 3. 00 (2Hrn) 3. 07 (2Hml 3. 55 (4Hr nl　3. 96 (LMml 4. 62 (2Hs) 4. 624. 75 (2 8ABI 7. 20 (108ml. Example 35 L-glutamic acid γ-bis(hydroxyethyl)amide (34) supported on charcoal as a catalyst in methanol (20 mL) in the presence of 10% palladium (250 mg) Bis(hydroxyethyl)amide (33) (2.15 g) was hydrogenated. child Standard processing of a mixture of amino acids (34) (1, 08g, 92%) was obtained. 'II NMR (DtO/DCI)t 62. 02 (2Hml 2. 17 ( 2Hml 3. 06 (CH2152, 14 (CH2153, 35 (CHI 60 ．． 54 (CH2) 60. F4 (CM2+ 172. 31 Gemi list) 17 3. 53 (@) Example 36 Z-L-glutamic acid α-benzyl ester γ-1′, 1°-dimethyl-2′ -Hydroxyethylamide (35) α-benzyl ester (7) (3,57g, 9. 61 mmol) was dissolved in THF (10 mL). Add triethyl to this solution. Amine (1.47 mL, 10. 57 mmol), ethyl chloroformate (1 mL, 10. 6 mmol) and 1. 1-dimethyl-2-hydroxyethylamine (2 , 75 mL, 29 mmol) was added and the mixture was stirred at 15° C. for 1 hour. Standard laboratory After treatment, the crude product was chromatographed (70% ether/gasoline). (35) (4.08 g, 96%) was obtained as a colorless oil. (3Hm) 3. 483. 50 (2HAB J-11,5) 4. 36 ( IHm) 5. 05(2H815,055,12(2HABI　6. 01 [I Hd J-8,016,20(11(817,30(IOHml. ”CMMR(CDCI,)+624. 03 (C) 131 24. 18 (C H3128,02 (CH2132,51 (CH2) 53. 33 (C) 55 ．． 74 (CHI 66. 87+CM2167. 09 (CH2) 69. 61 (CH21127, 89 (7) #Saka CHI (amide l 172. 50 (9% l. Example 37 L-glutamic acid γ-1',1'-dimethyl-2°-hydroxyethylamide Do (36) Using 10% palladium supported on charcoal (220 mg) as a catalyst, HP L C grade metal was used. Hydrogenation of amide (35) (2.20 g) in ethanol (20 mL) resulted in the absorption of Amino acid (36) (1, Log, 99%) was obtained as a wet white solid. ml 3. 43 (2) I sl 3. 89 (18t J-6,45). 130 order (D) O): J 22. 93 (CH3125, 53 (CH2131 , 66 (CH2) 52. 11 (CHI 54. 91 (C) 66. 87 (CH21L71. 40 (Pumito) 173. 59 (oppression). Example 38 Z-L-Glutamic acid α-benzyl ester γ-ethylamide THF at 5°C Acid (7) (5,27 g) in (40 mL). 14. A solution of 2 mmo]) was added to triethylamine (2.23 mL). 16. 0 mmo]), ethyl chloroformate (1.53 mL. 16. 0 mmo I) and anhydrous ethylamine (1.9 mL. 28. 0 mmol) for 1 hour. Standard processing of this reactant yields the crude ester. Thylamide (37) was obtained, which was purified from chloroform/ether (1:4). When recrystallized, t, 1. C. (8096 ether/gasoline, Rfo, 19) homogeneous ethylamide (37) (5.01 g, 88%) was obtained. Melting point 121-121. 5℃. [. lD2°-6,02 (c Proverb 13. 13. 70 mouths f, +1/to). 7. 31. 4. 39 (IHml, 5. 10'(2Hsl, 5. 12. 5 ．． 17 (2HAB J-12, 41, 5, 70 (28bd J r 7. 01 ．． 7. 33 (10) + +TI). ”CMMR(CDC1sl+J14. 7 (CHsl, 2B, 4 (CH 2). 32. 4 (CHt), 34-4 (CHi), 516 (CH), 67 ．． 0 (CH2). 67. 3 (CH)l, 12B, 0 (Poor Gorge CM), 128. 2 (, 11-Shakan C1 (l, 128. 4 (342 exercises C1 (), 128. 5 ( That line CM). 128. 6 (Sotone board CH), 135. 2 (Current number C1, 136, 1+ 1! -, Narami C), 156. 3 (-p), 8-me) C mushroom 0), 1 71. 5 (C-01゜171. 8 (C wealth 0). Example 39 I, -Glutamic acid γ~ethylamide (38) 10% palladium supported on charcoal as a catalyst γ-ethyl acetate in methanol (20 mL) in the presence of Mido (37) (1,1,3 g) was hydrogenated. Standard processing of this mixture results in Amino acid (3B) (460 mg, 9296) was obtained as a crystalline white solid. . Melting point 211-214. 5℃. ml, 2. 25 (28ml, 3. 00 (2Hq J = 7. 31. 3 ．． 80 (18t J-in-law 6. 3). 13CNMR (D, O1+611. 1 (CH Col, 23. 7(CH,l,29. 1 (CD), 32. 2 (CHt), 50. 6 (CM), 170. 0 (C=O), 171. 6(C-O). Example 40 Z-L-glutamic acid α-benzyl ester γ-diethylamide (39) α-benzyl ester (7) (3.04g, 8. 19 mmol) in THF (15 mL). To this solution was added tritylamine (1.25 mL, 9. Ommo I), ethyl chloroformate (0.86 mL, 9. Ommo +) and diethyl Amine (1.86 mL, 18 mmol) was added and the mixture was stirred at 15 °C for 1 h. did. Standard treatment gave the crude product (38) as a colorless oil, which , 1. homogeneous by C, (silica, 80% ether/gasoline, RfO, 24) there were. 171. 9 (C-01, 172, 6 (C-0). HPLC-grade methane using 1096 palladium (120 mg) supported on charcoal as a catalyst. Nord (20m 1. ,) in water. When added, amino acid (40) (600 mg, 95% )was gotten. Melting point 201-203°C. 2. 34 (2Hm), 3. 01 (28q J = 7. 41. 3. 11 (2Hq J - (C)!, 1. 29. 3 (C)l, ), 30. 6 (CM , l, 51. 1 (CM), 16B・9 (C depth 0), L71. l (C trout 0). Example 42 Z-L-glutamic acid α-benzyl ester γ-N-(2’ hydroxyethyl ester ) Biperazide (41) Method A Mixed anhydride α-benzyl ester (7) (4.07 g, 10.0 g in THF (25 mL)) 9 A solution of 7 mmol) was added to triethylamine (1.68 mL, 12. Ommol) , ethyl chloroformate (1, 1, 5 mL, 12. 0 mmol) and N-2゛ -Hydroxyethylpiperazine (1.6 mL, 14. Ommol) and 1 at 5℃ Allowed time to react. Standard treatment of this reaction product yields crude N-2'-hydroxyethyl Lupiperazine (41) was obtained. Flash chromatography (10% meta alcohol/chloroform), then column chromatography (Sepl+ed) sx Lll-20, 60% DCM/gasoline), t, 1. c, (1 Homogeneous (41) (4,54 g, 86%) was obtained. [α] -5,03 (C=9. 54, DCM). Method B: Use of ester (8) activated with 2-hydroxypyridine Activated ester (8) (5.34 g, 11. The 91 mmol solution was diluted with 1-(2'-hydrochloride) in dichloromethane (20 mL). xyethyl)piperazine (2.5 mL, 14. Add to the stirred solution of 3 mmo I) Ta. The reaction mixture was stirred for 3.5 hours, after which time t, 1. C, (silica, 3% methanol No starting ester was detected with chloroform/chloroform). Pour the reaction solution into water. (3xlOml) and remove the solvent under vacuum to produce the desired product as a pale yellow oil. Product (41) (3.28 g, 57%) was obtained. Optical rotation and 'HNMR spectrum The values were consistent with those measured above. This is the correlation of IH-130 in a short range. Measured with compound. “CNMR (CDCIs) 1 527. 2 (CH21, 28, 8 (C) + 21. 41. 5 (C)+2)+ 45. 1 (Delivery home - C), 156・O (, 1 nreno (me) C"O), 170. 0 (Puno Y゛C-0), 171. 8 (zs5wC-0). Example 43 Charcoal-supported L-glutamic acid γ-N-(2' hydroxyethyl)piperazide catalyst HPLC grade methanol (10ml) using 5% palladium (71mg) ) Hydrogenation of amide (41) (0.32 g) in was gotten. Ion exchange chromatography (system II, NH4+ type, water, The crude mixture was then partially separated with 1M ammonia). This experiment on a large scale Repeated steps also made it difficult to purify the final product. γ-Hydroxyethylpipe Radides are relatively unstable at high temperatures and can produce a mixture of decomposition products that are difficult to separate. It's good. C son o). Example 44 Z-L-Glutamic acid α-benzyl ester γ-morpholide α-benzyl ester Le (7) (1,80g, 4. 84 mmol) was dissolved in THF (15 mL), and T Triethylamine (0.74 mL, 5. 3 mmol), Ethyl chloroformate (0.51 mL, 5. 3 mmol) and morpholine (0,5 1 mL, 6. 0mmo+). standard treatment and recrystallize the product (A pure (43) (1,81) as a white solid. g, 85%) was obtained. Melting point 62-65°C. (28m), 3. 58 (6Hml, 4. 40 (IHml, 5. 15 (4Hml, 5. 73 (IHbd J-7,2), 7. 34 (IOHm l”CM (CDC1sl+J27. 4 (CHZ), 28. 7 (CH2 ). 42. 0 (CH,), 45. 6 (CM, l, 53. 7 (CHI, 6 6. 4 (CH,). 66 = 7 (CHt) + 66-9 (CHil + 67-2 (CHz), 1 28. 1 (fGD, CM), 128. 2 Iff swimming CHI, 128. 4 (Trashso CHI, 128. 5 Ifsha X Trowel CM), 128. 6 ( 3 fragrances (CM). 135. 3 (Fang 4 paper C1 + 135' (14-translation C1, 156, 0L force w , p-) C-01,170,3 (C-01,171,8 (C=O). Example 45 ■, -Glutamic acid γ-morpholide (44) Method A Catalytic hydrogenation HP L C grade using 10% palladium supported on charcoal (242 mg) as a catalyst. Morpholide (43) (2.42 g, 5. 49mm Hydrogenation of l) yields the crude amino acid (44) as a white isomer; This was recrystallized from water/acetone (12) (1. 175g, 92%). Melting point 1 66-169℃. t, ], C, (cellulose; 70% ethanol/water), Rf o, 61, with a slight amount of L-glutamic acid (Rf O, 69) mixed in. . (4Ht), 3. 14 (5Hml. I3CNMR (DtO) + 62 [1, 24 (CHi), 32. 06 (CH 2). 44. 62 (2X CHi), 48. 32 (CHil, 56. 52 (C HI, 68. 613 (2x C) fil, 175. 16 (fpJqc dew o), 176. 35　(jp　G’　Cool. Method B: Transfer in ethanol (I order n5ter) hydrogenated morpholide (43) (223mg, 0. 5 mmol) was placed in absolute ethanol (10 mL). child 10% palladium on charcoal (24 mg) and cyclohex-1,4-di Enene (480 μI, 10 eq.) was added. The reaction mixture was heated at room temperature under dry nitrogen. Stirred for 1 hour. After this time, the mixture was filtered and the charcoal was washed with water. Remove solvent under vacuum Then, t, 1. c, (cellulose, 70% ethanol/30% water) One compound, amino acid (44), was obtained. Example 46 Z-L-glutamic acid α-benzyl ester γ-O-methylhydroxamate (45) Pyridine (19, L) distilled from 0-methylhydroxylamine hydrochloride (5 g) and distilled at 70°C. The distillate (4 mL) collected at -78 °C contained free O- It contained large amounts of pyridine along with methylhydroxylamine. α-benzyl ester (7) (5.0 g, 13. 4 mmol) in THF (30 mL) and triethylamine (1,42 g51. 94mL, 14. 8mm o I), ethyl chloroformate (1.52 g, 1. 34mL, 14. 8 mmol ) and the distillation product of O-methylhydroxylamine in pyridine. The mixture was stirred for 1 h, then the solvent was removed under vacuum and t. A mixture of three products is obtained as shown in 1, c, (80% ether/gasoline). It was done. Chromatography (silica, 50% ether/gasoline then 80% ether) The desired product (45) ( 4.15g (177%) was obtained. Melting point 143-444. 5℃. [α co-8,01 (c=11. 67, chloroform). Others The compound is the starting material ester (7) and the ethylamine of O-methylhydroxylamine. It was identified as carbamate. 'Ii NMR (CDCl5) (45) +52. 19 (48mβand 7 (CHZ), 53. 4 (CM), 64. 2 (CH31167, 2(C) hl, 67. 4 (CHt) + 12LOof branch male CH) + 1211121 'ifsha tribe CHI. 12” (’154’ff1cH) + 128. 5 (i*nee, CM), 12 8. 6IJF-m-RCH), 135. 0 (%4r-xlp=Cl, 1 35. 9 (7 incense C1 + 15L5 (sword t<')-) C-01,169 , 8(C-0), 171. 6(C-O). Example 47 L-glutamine γ-0-methylhydroxamate (46) as catalyst supported on charcoal 10 % palladium (400 mg) in methanol (50 mL). Hydroxamate (45) (3.4 g) was hydrogenated for 2 hours. the resulting mixture The material was filtered and the charcoal was washed with hot water (3 x 5 mL) to give the product (46). solvent Amino acid (46) (1.35g) is removed as a hygroscopic white solid when removed under vacuum. , 90%) was obtained. Z-L-glutamic acid α-benzyl ester γ-2° thioethyl amide (47) α-Benzyl ester (7) (4.67g, 12. 6 mmol) in anhydrous THF ( 40 mL) and cooled to 5°C. This solution was mixed with triethylamine (3,82 g, 5. 30 mL, 36 mmol), then ethyl chloroformate (1.63 g). 1. 44 mL, 14 mmol) and stirred for 5 minutes. Next, 2-thioethylamine hydrochloride (7 g, 14 mmol) was added as a solid. , the mixture was stirred for 4 hours. Removal of the solvent under vacuum yielded the crude mixture, which This was redissolved in dichloromethane (50 mL) and washed with water (2x 40 mL). . The aqueous extract was re-extracted with dichloromethane (70 mL) and the organic extracts were combined. After drying over sodium sulfate, the solvent was removed under vacuum. Chromatograph the crude product Graphite (silica, 95% ether/'gasoline 100% ether and 5% ether) (tanol/chloroform) to give the desired product (47 )was gotten. Melting point 95-97°C. 7. 31 fIOHm Gshazume). Example 49 L-glutamic acid γ-2'1000 nitylamide (48) was used as a catalyst on 10% carbon supported. Radium (98 mg) was used to prepare a 2°- A solution of thioethylamide (199 mg) was hydrogenated for 4 hours. Next, add ammonia Evaporated and the resulting mixture was suspended in methanol. Methanolic (5 mL) suspension The turbid liquid was filtered through a plug of Celite, then the plug was washed with water (5 mL). true solvent Removal in vacuo gave a mixture of starting amide and desired product (48). The crude mixture was dissolved in chloroform (5 mL), extracted with water (2x5 mL) and diluted with water. Lyophilization of the sex extract yielded amino acid (48) (28 mg). organic layer The solvent was removed in vacuo to give unreacted starting material amide (47) (159 mg ). Example 50 Z-L-glutamic acid α-benzyl ester γ-21 methoxyethylamide (4 9) α-benzyl ester (7) (4,11g, 11. 07 mmol) in THF (3 0 mI-). To this solution was added triethylamine (1.80 mL, 12. 17 mmol), ethyl chloroformate (1.30 mL, 12. ’　17mm　1 ) and 2-methoxyethylamine (1.0 g, 12. Add 4 mmol) and mix. The mixture was stirred at 15°C for 3 hours. Next, t, 1. C, (silica; 5% gasoline/ (ether) to complete the reaction. Recrystallize the crude product after standard processing (50% ether/gasoline) as a crystalline solid (49) (4,91 g. 99%) was obtained. Melting point 80-84°C. [ol　”-5・5　(Cw　8. +16. Schiff aaH71・’HN) IR ( CDC131+ 61. 99 (28ml 2. 21 (2Hm) 3. 33( 38sl, 3j6 (48bt J-5,4), 4. 40 (IHml, 5. 105. 16 (4H Town, 5. 75 (IHd J = 7. 6), 7. 34 (108ml. “CMKR (CDC1sl+628. 3 (CH2), 32. 2 (CH2 ). 39. 2 (CFII), 53. 6 (CHI, 58. 7 (CHsl, 67. 0(C H,). 67. 3 (CHtb) 0. 9 (CHZ), 128. 1 (146CM). 128. 2 (￥’14f niece CHI, 12f1. 3 (aromatic C HI, 128. 5 (Aromatic Pregnancy CM), 128. 6 (ゎ1KO CHI, 135 ．． 0 (Fragrance #, C1,135,5 (tongue*$-C1,156,2(:)'J)k /:)-to C-01゜169. 5 (C-0), l fo, e (c-o). Example 51 L-glutamic acid γ-2゛methoxyethylamide (50) supported on charcoal as a catalyst γ-Methoxyethylamine (49 mg) in the presence of 10% palladium (264 mg) ) (2.51 g) was hydrogenated in methanol (20 mL). This mixture is labeled After semi-treatment and recrystallization from water/methanol, the amino acid is produced as a white crystalline solid. Acid (50) (1.19 g, 96%) was obtained. Melting point 183-185°C. [α] +1. 6 (c-6,96, H2O), [α] Hg(436)+17. 7 (C=6. 96, H2O). RfO, 30 (Se Lulose; n-butanol/pyridi/10. 4% acetic acid aqueous solution 22:10:10) RfO, 56 (silica; 70% ethanol/water). '! ! MMR (020) = 12. 12 (21m), 2. 42 (28m l, 3. 35 (38ml, 3. 38 (2Ht J 6. 2), 3. 55 (28t J■6. 21°3. 75 (IHt J 6. 9). “CIGot (DtO)=627. 8 (CHs), 32. 9 (CHZ) ), 40. 3 (CM, ), 55. 6 (CHI, 59. 4 (CHsl+7 1-7 (CHt), 175,4 (C-01,176,2 (C-0). Example 52 Z-L-glutamic acid α-benzyl ester γ-2° chloroethylamide (5 1) 2゛　-Hydroxyethylamide (27) (3.89g, 9゜39mmol) Dissolved in dichloromethane (30 mL). To this solution was added triethylamine (1.85 mL, 12. 5 mmol), Jime Thylaminopyridine (DMAP) (132mg% 11mo 1%) and salt methanesulfonyl (1.08 mL, 12. 5 mmo I) was added and the mixture was Stirred at ℃ for 3 hours. The reaction mixture was then warmed to room temperature and stirred for 2 days. Standard laboratory After treatment, chromatography (silica; 10% gasoline/ether, 10% gas) Solin/ethyl acetate, 100% ethyl acetate) gives a white solid; When this is recrystallized (50% chloride/carbon tetrachloride) and further purified, crystals are obtained. (51) (2.05 g, 5696) was obtained as a solid. 'Fi NMR (CDC1+]l 61. 9[1 (18ml, 2. 19 ( 38m), 3. 55 (4Hrnl, 4. 42 (18ml, 5. 065. 16 (4H2x AB’sl, 5. 65+IHd J-7,81,7,3 4 (108ml. ”CNXR(CDCI>)+62111. 5 (CH2), 32. 2 (C H21,41,3L, -glutamic acid γ-2' chloroethylamide (52) catalyst 2′-chloroethyl in the presence of 10% palladium on charcoal (226 mg) as a medium. Ruamide (51) (2.04 g) was hydrogenated in methanol (30 mL). This mixture was subjected to standard treatment to obtain amino acid (52) (863 g, 100%). It was. 'H) Dot (010): J 2. 16 (2H Town, 2. 46 (2Hm l, 3. 54 (2Ht J-5,6), 3. 66 (28t J-5, 6), 3. 84 (LM t J (C-0). Example 54 Z-L-glutamic acid α-benzyl ester γ-2°-hydroxyanilide ( 53) α-benzyl ester (7) (3.95g, 10. 64 mmol) in THF ( 30 mL). To this solution was added triethylamine (1.63 mL, 12 m mol), ethyl chloroformate (1,11 mL, 12 mmol) and 2-hydroxy Cyaniline (1,74g, 12mmo+) was added and the mixture was stirred at 15°C for 3 hours. did. Next, t, 1. c. The reaction was monitored with (silicami 10% methanol). Completed. After standard treatment, the crude product was chromatographed (silica; ioo% (chloroform) to give (53) (2.29 g, 51%) as a white solid. It was done. 'HNMR (CDCIs) + J 1. 9f (18ml, 2. 34 (IHm l, 2. 46 (2Hrn), 4. 48 (LM ml, 5. 105. 16 (482x AB'sl, 5. 73 (IHd J = 6. 2), 6. 8- 7. 3 (148m). Example 55 L-glutamic acid γ-2'-hydroxyanilide (54) supported on carbon as a catalyst 1 2′-hydroxyanilide (53) in the presence of 0% palladium (300 mg) (1.54 g) was hydrogenated in methanol (30 mL). This mixture is standard After treatment, the aqueous solution is extracted with chloroform and freeze-dried to obtain the amino acid (54) (0.73g, 93%) was obtained. ilHml. Cold example 56 Z　-L-glutamic acid α-benzyl ester γ-3゛゛　-chloroamine Do (55) α-benzyl ester (7) (3.81g, 10. 25mmof) in THF ( 45 mL). To this solution was added triethylamine (1.57 mL, 12 m mo +), ethyl chloroformate (1,11 mL, 12 mmol) and 3-k Loroanilide (1.29 mL 112 mmol) was added and the mixture was heated at 15°C for 1 hour. Stirred. Next, the reaction was monitored with t, I, c, (silica; 10% methanol). completed the response. After standard processing, as pale yellow oil (55) (5.20 g, 9 6%) was obtained. 4. 43 (IH Town, 5. 105. 16 (4H2x AJI’s), 5. 69 (IHd J=8. 01. 7. 04-7. 66 (14Hml. L-glutamic acid γ-3゛-chloroanilide (56) 10% supported on charcoal as a catalyst Protected γ-3°-chloroanilide (55) using palladium (101 mg) ( 1.0 g) was hydrogenated in methanol (20 mL) for 1.5 hours. the resulting mixture The material was filtered and the charcoal was washed with hot water (3 x 5 mL) to give the product (56). solvent Removal under vacuum yields amino acid (56) (280 mg) as a hygroscopic white solid. , 52%) was obtained. G [α] +7. 10 (c=10. 2.Wednesday). t, 1. C. (Cellulose; n-butanol/pyridine 10.4% acetic acid aqueous solution 22:10:1 0, Rf product 0. 62, Rf glutamic acid 0. 20), a small amount of glue Tamic acid (approximately 5%) is also present, which can be removed by washing with a small amount (approximately 500 μm) of warm water. It was done. '11 NMR (D, O/DCI) + J 2. 19 (28ml, 2. 61 (28ml. 199 (IHt J-6, 71, 7, 02-7, 17 (3Hml, 7. 66 (IHsl. Example 58 Z-L~Glutamic acid α-benzyl ester γ-4°-chloroanilide (57 ) α-benzyl ester (7) (4.16g, 11. 20 mmol) to T! (F( 30 mL). To this solution was added triethylamine (1.72 mL, 12. 3 mmol), ethyl chloroformate (1.18 mL, 12. 3 mmol) and 4 -Chloroaniline (1.79 g, 14 mmol) was added and the mixture was heated at 15° C. for 1 hour. Stir for a while. Next, t, 1. c, measured in (silica: 80% ether/gasoline) The reaction was completed as expected. After standard treatment, removal of the solvent under vacuum yields (57) as a crystalline solid (5,21 g, 93%) was obtained. Melting point, [α] -5,21 (c=12. 0, chloro Holm). 'HMMR(CDCl2)! J 1. 99 (IHml, 2. 30 (3H rnl, 4. 43418 ml, 5. 115. 16 (4H2x AB’s ), 5. 63 (l) l d J -8,01,7,22-7,48' (1 4Hm). CM), 128. 1 (Group 34 CHI, 128. 3 (74 books CH], 1 28. 41flkr answer, CM)l 12B, 514tRCHl. 128. 7 (4F group CH), 128. 9 (fragrant dislike C) 11. 129 ．． 0 (Eshaq CHI, 134. 9 (J41 Katsura C), 135. 8 (2x ■, -glutamic acid γ-4゛-chloroanilide (58) supported on charcoal 1 as a catalyst Protected γ-4″-chloroanilide in the presence of 0% palladium (101 mg) (57) (2,00 g) in methanol (10 mL) and ethyl acetate (5 mL) Hydrogenated with. Standard processing of this mixture results in amino acids (58 ) (1 O1 g, 94%) was obtained, which is t. 1, c, (silica, 70% ethanol/water) was pure. "]o"-" (e-9, 10, 7m). Z-L-glutamic acid α-benzyl ester γ-4゛-piperidide (59) α-benzyl ester (7) (5.30g, 14. 27 mmol) in THF ( 30 mL). To this solution was added triethylamine (2.30 mL, 15 m mo I), ethyl chloroformate (1.60 mL, 15 mmol) and piperidi (1.60 mL, 15 mmol) was added and the mixture was stirred at 15° C. for 3 hours. Next, t, 1. The reaction is complete as determined by C, (silica; 100% ether). Completed. After standard work-up, the crude product was chromatographed (silica; 100% chloro holm), t, 1. c, (silica; 100% chloroform, Rfo, A homogeneous yellow oil according to 16.25% ethyl acetate/gasoline, RfO, 50). (59) (5.37 g, 86%) was obtained. 10% Palladium on Charcoal as L-Glutamic Acid γ-Piperidide (60) Catalyst (430 mg), γ-piperidine (59) (4,03 g) was added to methanol. Hydrogenation was carried out in a 20 ml. Standard treatment of the reaction mixture after 4 hours results in t , 1. C, (silica, 70% ethanol/water, Rfo, 65: cellulose, n- Butanol/pyridine 10° 4% acetic acid aqueous solution 22:10:10, Rfo, 50) A homogeneous amino acid (60) (1.68 g, 85%) was obtained. melting point 16 2-162. 5℃. Z-L-glutamic acid α-benzyl ester γ-3′-hydroxypiperizide (61) α-Benzyl ester (7) (8.53g, 22. 96 mmol) in THF ( 30 mL). To this solution was added triethylamine (3.60 mL, 25. 5 mmol, ethyl chloroformate (2.40 mL, 25. 5 mmol) and 3 -Hydroxypiperidine (2.65g, 26mmol) was added and the mixture was Stirred at ℃ for 5 hours. Then, t, ], C, (silica. The reaction was complete as measured by 1 (+00 fy ether). After standard processing , the crude product was chromatographed (silica: 100% ether, 100% acetic acid) ethyl) gives t, 1. C, (Silica: 100% ethyl acetate, Rf O, According to 11), (61) (8.70 g, 83%) was obtained as a homogeneous colorless oil. It was done. [αl　20-3. 24 (c=10. 5. Chloroform). 'HNMR spectrum shows that the product (61) is a mixture of two diastereoisomers. It was shown that HPLC (Silica. Attempts to separate the two isomers using 1096 gasoline/ethyl acetate were unsuccessful. won. L-glutamic acid γ-3゛-hydroxypiperidide (62) carbon supported as catalyst In the presence of 10% palladium (800 mg), γ-3'-hydroxypiperid (61) (7.30 g) was hydrogenated in methanol (20 mL). 5 o'clock After standard treatment of the reaction mixture, t, i, c, (silica; 70% ethanol /water, Rfo, 62 cellulose; n-butanol/visino 210. 4% acetic acid Homogeneous amino acid (62) (3 , 49g, 95%) was obtained. Melting point 151-152°C. [α1 4. 00 (c=10. 96, Wednesday). Assignment of chemical shifts in proton spectra Confirmed by Pulling. 'TI NXR (D20)! J 1. 4-2. 0 (4H Beisuke ml, 2. 1 1 (2Hdt J -6, 7, 6, 61, 2, 61 (2Ht J -6, 71, 3,2-3,95 (6H 哩け　m). “CMMR (D, O)! J22. 5 bar - 23,5 (CHtl, 27. 4 (CH2), 30. 2 (CHII, 32. 2 Da L-32,5 (CHt), 44. 0 Z tile n”47. 44CM21. 49. 4 and α”53. 3 (CHtl , 55. 4 (CHI, 66. 91fi-' 67. 0 (CM-QH), 174. 1 (-y, =) = C-0), 175. 2 (瑣1jC寥0). Example 64 Z-L--Glutamic acid α-benzyl ester γ-[1-(4-methylpiperazi) )] Coamide 63) α-benzyl ester (7) (5.80g, 15. 60 mmol) in THF ( 40 mL). To this solution was added triethylamine (2.45 mL, 17. 1 mmol), ethyl chloroformate (1,,70 mL, 17. 1 mmol) and 1 -amino-4-methylpiperazine (2.14 mL, 17. Add 1 mmol) , the mixture was stirred at 15° C. for 16 hours. During this time, a precipitate formed in the reaction vessel. . Standard treatment of the reaction mixture yields the crude product (63), which is recrystallized to yield (50% chloroform/carbon tetrachloride), t, 1. c, (silica; 5% methanol /Chloroform, Rf 0. According to 30), as a homogeneous crystalline solid (63) (6.96g, 95%) was obtained. Melting point 174℃, [α] -6,00( c=11. 0, chloroform). 'H and 13 in chloroform and pyridine Both CNMR experiments show that two major configurations of the piperazine ring are present in solution. C)+2 92:1), 28. 86jlhi' 31. 30 (7CH21 j. 2+l), 45. 65i’iK” 45. 77 (N-Me41 CH3K 2 +ll. 54. 99 (CHx), 55. 00 (CHt), 55. 07 (2X) Chzl, 56. 32 (αCHI, 66.55 (Hendzuru CH2), 66 ．． 83 (Example 65 Charcoal support as L-glutamic acid γ-[1-(4-methylpiperazino)]amide catalyst γ-[1-(4-methylpiperazide) in the presence of 10% palladium (322 mg) h) Coamide (63) (3.04 g) was hydrogenated in methanol (20 mL). Ta. Standard treatment of the reaction mixture after 3 hours yields t, ], c, (silica; 70% ethanol alcohol/water, Rfo, 10 cellulose; n-butanol/pyridine 10. 4% vinegar Acid aqueous solution 22: 10: 10, Rf 0. According to 05) homogeneous amino Acid (64) (1,56 g. 98%) was obtained. Melting point 190-192℃ mq 74'j)l 2. 38 +38s N-Me41L/)+2. 75 (48ml. 2. 87448 ml, 3. 70 (IHm ap, ),). 13(岨(D,O)+　827. 9 (CHi), 31. 2 (CHtl, 45. 2 (CH3N-Melyl, 54. 3 (2X CH21,54,6( 2X C) It) 155. 5 (CM), 173. 2 (A3) Pa C-0), 175. 111 (Ya 1-C-0). Example 66 Z-L-glutamic acid α-benzyl ester γ-(4-morpholy)) a Mid (65) α-Benzyl ester (7) (4.04g, 10. 87 mmol) in THF ( 30 mL). To this solution was added triethylamine (1.70 mL, 12. 0 mmo I), ethyl chloroformate (1.13 mL, 12. 0 mmol) and 4 -Aminomorpholine (1.19 mL, 12. 0 mmo]) and the mixture was reduced to 1 The mixture was stirred at 5°C for 2 hours. During this time, a precipitate formed in the reaction vessel. Standard processing, The crude product was chromatographed (silica; 15% ethyl acetate/petrol). (65) was obtained, which was recrystallized (50% chloroform/carbon tetrachloride). When purified, t, 1. c, (silica; 10% gasoline/ethyl acetate + 1 drop of acetic acid , Rf 0. According to 22), a homogeneous crystalline solid (3.16 g, 64%) was obtained. It was. Melting point 150-151℃, [αE　-3,80 (c=10. 4. Chlorophor ). Both H and 13c NMR experiments at 300 K and 325 It shows that two major configurations of are present in solution. 'HMIOL (CDC1sp J2. 02 (we), 2. 13 (38 ml, 2. 52C-O1 (13+21゜ Example 67 L-glutamic acid γ-(4-morpholino)amide (66) as catalyst supported on carbon 1 In the presence of 0% palladium (222 mg), γ-(4-morpholino)amide (65 ) (1.97 g) was hydrogenated in methanol (20 mL). 2 hours after birth Standard treatment of the reaction mixture results in t, 1. C, (silica; 7o% ethanol/water, R fO, 63: cellulose; n-butanol/pyridine 10. 4% acetic acid aqueous solution 22 : 10 : 10, Rfo, 15), homogeneous amino acids (66) (99 1 mg, 99%) was obtained. Melting point 200℃ %3 1. 2. 33 (2Hm q%) l, 2. 83 (48t J = 3. 81, 3. 75 (lHtJ-6,901%”51. 1. 80 (4H tJ-3,81° I3C (DfuO): 627. 6 (CHt), 31. 3 (CHi), 55. 5 (CMI, 56. 2 (2x CH, l, 67. 4 (2x CH zl, 1713(j'6)'C-0), 175. 3 ((r) Soybean C-0 1゜Example 68 Z-L-glutamic acid α-benzyl ester γ-2′ fluoroethylamide ( 67) α-benzyl ester (7) (2.50g, 6. 70 mmol) in THF (30 mL). To this solution was added triethylamine (1.03ml7. 7. 40m mol), ethyl chloroformate (0.65 mL, 7. 40 mmol) and chicken 2-Fluoroethylamine hydrochloride (0,72g) in ethylamine (1ml) ,7. 4 mmol I) was added and the mixture was stirred at 15° C. overnight. Next, t, 1. c, the reaction was complete as determined by (silicania 0% ether/gasoline) . The crude product was subjected to standard treatment and chromatography (silicania 0% ether/gasoline phosphorus), t, 1. C, (silica; 7o% ether/gasoline) and homogeneous crude product (67) (2.71 g. 91%) was obtained. (2Hml, 3. 46 (18ml, 3. 55 (18ml, 4. 37 (2Hm CHt-F), 4. 53 418 ml, 5. 09 5. 17 (4 H2x AB’s/’;>n)714. 5), 5. 69 (lHbs N H% 3), 6. 11 (lHbs NHA'S l, 7. 34 (1 0Hm X, 4tn-4-# 1” CMMR (CDC13) + 4 28. 4. (Cut), 32. 2 (CHtl. 39. 8 (CM,), 40. 1 (CHtl, 53. 5 (CH), 67. 1R run, 171. 7 (Ami Y'C stomach 01,172. 0 (Aesthetics + I/C -01゜Example 69 Z-L-Glutamic acid α-benzyl ester γ-2゛, 2°. 2°-trifluoroethylamide (68) α-benzyl ester (7) (6, Log, 16. 43 mmol) was dissolved in THF (50 mL). in this solution Triethylamine (2,6OL, 7. 40 mmol), ethyl chloroformate ( 1.8mL, 17. 8 mmol) and 2,2. 2-trifluoroethylamine (1,45mL, 17. 8 mmol) was added and the mixture was stirred at 15° C. overnight. After this, t, 1. C. The reaction was complete as determined by (silica: 50% ethyl acetate/gasoline). The crude product was subjected to standard treatment and chromatography (silica: 50% ethyl acetate/gas When applied to phosphorus), t, l. C, (silica; 50% ethyl acetate/gasoline) as a homogeneous crystalline solid. (68) (6.81 g, %) was obtained. 'HMKR (CDC13) + J 1. 96 (IHml, 2. 25 (3H ml, 3. 81 (2Hml, 5. 10 (sl, 5. 17 (4HrIl l, 5. 65 (l) (d, J-8,0), 6. 43 (18b), 7. 27 (IOHml. 13CMKR (CDC1x) + 628. 6 (CHZ), 32. 1 (CH *l. 40. 3 (C) I, q, J 35. 01. 53. 4 (CM), 67. 2 (CH, l. 67. 4 (CH, l, 124. 0 (Cq, J-2!D), 128. 1 (Meka Castle CH,, 1211, 3 (Dianfu CHI, 128. 4 (Delicious frog Ml 128. 6+ zshabee CHI, 128. 7t fm group CH), 1 35. 117r4-Cl, 136. 0 (7f$ Cl, 156. 5 (-l Le/17-) C-0). 171. 67; pc=O), 172. 1 (Nispl C-01゜Example 70 L-glutamic acid γ-2°, 2°, 2゛-trifluoroethylamide ( 69) In the presence of 10% palladium on charcoal (478 mg) as a catalyst, trifluorocarbon Loethylamide (68) (3.26 g) was dissolved in water in methanol (50 mL). Added element. Standard treatment of the reaction mixture after 1.5 hours yields t, 1. c, (silica ;70% ethanol/water, Rfo, 70: cellulose; n-butanol/pyridine According to 10.4% acetic acid aqueous solution 22:10:10, RfO, 57) High quality amino acids (69) (1.65 g, 99%) were obtained. Melting point 215℃ 'H field (DtO) i 6' 2. 13 (214m), 2. 48 (28dt , J-7, 7, 3, 3), 3. 75 (IHt, L-6, 2), 3 ．． 93 (2Hq, J -Z-L-glutamic acid α-t-butyl ester γ- 0-Methyl hydroxymate (70) In a water bath, water (5 mL); ethylenol alcohol monomethyl ether (15 mL) and a stirred solution of potassium hydroxide (2.8 g) in ether (19 mL). Diazomethane was added dropwise to a stirred solution of Gizard (9 g) in ether (60 mL). (Hedlickt, M. (1980)), OB, Chew, 4 5. 5377-5378). The mixture was warmed to room temperature, then heated to 50°C in a water bath and the diazomethane was evaporated in the conventional manner. I stayed. A solution of distilled diazomethane in ether (20 mL) was added to A solution of doloxamate (23) (4.0 g) was added and the mixture was cooled in a water bath. The mixture was allowed to stand for 3 hours, after which the excess diazometha precipitated out. The solution was decomposed with acetic acid and the resulting crystalline solid was filtered and reused. t of this substance, 1. C, (silica, 80% ether/gasoline) analysis shows that this is the starting material hydro It showed that it was a xamate, so the whole mixture was put into fresh diazomethane and One device was at room temperature. After this, no diazomethane remains and t, 1. C, (C Rica, 45% ether/gasoline, Rfo 36. 023. 010) by 3 A mixture of products was observed. Removal of the solvent gave the crude mixture as a pale green oil ( 3.72 g, 93%) was obtained. The mixture was chromatographed (silica, 45 % ether/gasoline) to give pure samples of the three compounds (Rf O, 36, 2, 35g. 59%, Rfo, 23. 600mg, 15%; RfO, 10,350mg, 8% ). The NMR spectrum shows that the component with the highest Rf is O-methylhydroquine. (70), while the component with the lowest Rf value was the dimethylation product. It turns out that Synthesis of 0-methylhydroxamate by various methods (Examples) 46) have shown that methylation does not occur with hydroxamate oxygen. Ta. Spectral analysis shows that the isolated compound is O-methyl hydroxymate. 1 degree suggested that. Product (70) was isolated as a viscous oil. [α] D20 -7. 0 (c=10. 19, methanol). If this experiment is repeated continuously, the yield will be low. (23), the dimethylated product was obtained in high yield. 'HMKR (CDCl2)! g1. 38 (91 (S) 1. 88 (18 ddd J = 8. 1. 14. 2. 14. 212. 10 (IHddd J - 6, 6, 13, 2, 13, 312, 29 (ZHd AB J mouth 6. 1. 8. 1 ) 3. 57 (38sl 4. 20 (11 (ddJ = 7. 7,12. 9 )5. 02 (2Hs)5. 42 (IHbd J-6)7. 2B (5H town. 13CM (CDC13)! 627. 64 (CH2) 27. 73 (CH 3129,78 (CH2151,51 (CH3) 53. 63 (CH) 66 ．． 68 (CH2) Now r1 [write lj-(Rf O, 23) + 0. 0->- Mejzification’B MMR (CDCIs) + J 1. 46 (9Hs) 1. 9 7 (18ml 2. 06 (1H Town 2. 38 (28m) 3. 62 (3FI s) 3. 71 (3Hsl 4. 29 (LMdd J = 7. 4. 12 ．． 8) 5. 01 (28sl 5. 69 (18bd J-617,33( 5M ml. 13CMMR(CDCIs)t622. 93 (CH2) 27. 61 (C )! 3127. 72 (CH2) 53. 76 (CH3) 5185 (CH ) 61. 20 (CM,)66. 46 (CH2181, 74 (C) 127 ．． 75 (fr ji-d, CMl 12B, 17 (fr** CM) 136. 2 1 (i-chi 4pl, C) 155. 64 /y) Reno, /-1-)163 ．． 65 (Imi’y Pa) 170. 80 (z7. Te J Shil. 71p,.../f), (Rf O, 10) + O, N-3z-Me1lndi'F i MMR (CDC131=61. 43 (981S) 1. 96 (18n l)　2. 15 (18ml 2. 47 (2Hml 3. 12 (38g) 3 ．． 59 (3Hsl 4. 26 (l) 1m) 5. 065. 09 (28AB I 5. 65 (LM bd J-6,517,31(5) 1 ml. 13C)OGj　(CDC131!　!　22. 10 (CH2) 27. 33 (CH3131, 63 (C1 (2) 53. 41 (CHI 53. 62 ( CH3) 60. 52 (CH3) 66. 16 (CH2) 131. 49 ( C) 127. 43 (-1f7-Jj CM) 327. 84 Example 72 Z-L-glutamic acid γ-0-methylhydroxymate (71) fully protected Hydroxymate (70) (2.10g. 5. 73 mmol) was dissolved in TFA (9 mL) at 0°C. reaction mixture for 1 hour Stir for t, 1. c, (silica; 8o% ether/gasoline) monitored . The TFA is then removed under vacuum at approximately 20°C and the resulting solid is dissolved in ether/gasoline. Recrystallization from (60.40) gives pure (71) (1.73 g, 9896) was gotten. L-glutamic acid γ-0-methyl hydroxymate (72) free acid (71) (1 , 45g, 4. 69 mmol) was added to methanol (18 mL), and 10% charcoal-supported Palladium (145 mg) was added. This mixture was mixed with water in a conventional manner for 1 hour at room temperature. After addition, no starting material remained. Filter the hydrogenation mixture , the solid was washed with water (2xlOmL). Remove the solvent from the combined washing liquid and washing liquid. Removal gave crude product. When recrystallized from boiling methanol, a white Pure amino acid (72) (670 mg, 82%) was obtained as a crystalline solid of . Melting point 186-187°C. 7. 91 3. 56 (3H513,62(l)l t J -6,3) +t 7177, n7'9>'7-1ff (CH3)52. 81　(C1()172j l (Rumiy) 174. 18 (11,). Example 74 Z-L-glutamic acid α-benzyl ester γ-(2′-pyridyl)methyla Mido (73) α-Benzyl ester (7) (7.87g, 21. 19m　m　o　+　　　T Dissolved in HF (100 mL). Triethylamine (5.91 mL) was added to this solution. , 42. 4mmo+), ethyl chloroformate (2.22mL, 23. 4mmo l) and 2-aminomethylpyridine (2-picolylamine) (2.40 mL. 23. 4 mmol) was added and the mixture was stirred at 15° C. overnight. After this, t, 1. The reaction is complete as determined by c, (silica; ethyl acetate). Ta. After standard treatment, the crude product was chromatographed (silica:ether 400 mL , 500 mL of ethyl acetate. 10% methanol/chloroform (500 mL), t. 1, c, (silica; 100% ethyl acetate) as a homogeneous crystalline solid (7 3) (9,5798%) was obtained. Melting point 132-134°C. in CDC13 Attempts to record an NMR spectrum resulted in the compound decomposing. 'HNMR(CDsOD)t62. 0-2. 35 (2Hml, 2. 39 (2Hml, 4. 35 (LM dd J = 4. 2. 4. 3), 4. 4 3 (2Hsl, 5. 02-5. 09 (4HAn thread 1. 7. 36 (1 2Hml, 7. 74 (IHml. 111. 42 (l) i d J Maro 7. 41゜ljCNMR(CDsOD)+ J27. 9 (CH2), 32. 0 (CHt)+43. 4 (CHHz), 53. 6 (CM), 66. 9 (GHz), 67. 2 (CH2). 123. 0 (＾7t7tJJt CHI, 123-3 (H?a’Jf 4tJfi. C++ “6-” (Hete af*l layer CHI, 156. 5 (77/i-Hasort C -01,,171,6-7p1. -C-0), lf2. 1 (Esthetics J Shicw . ). Example 75 L-glutamic acid γ-(2'-pyridyl)notylamide (74) as catalyst 2-aminomethyl pyridine in the presence of 10% palladium on charcoal (650 mg) Amide (73) (4.76 g) was hydrogenated in methanol (50 mL). . Standard treatment of the reaction mixture after 24 hours yields t, 1. C, (Silica, 70% E Tanol/water, Rfo, 60 Cellulose: n-butanol/pyridine/'0. According to 4% acetic acid aqueous solution 22 + 10 + 10, Rfo, 38), a homogeneous amino acid Noic acid (74) (2,43g. 99%) was obtained. Melting point 185-189°C. 'FI NIKR (DtO): J 2. 17 (2Hml, 2. 53 (2 Hm), 3. 80 (18t, J = 6. 21. 4. 42 (2Hd J -7,41,7,31 (28ml. 7. 77 (LM ml, 8. 39　(18d　J　-7,31゜13CNM R(DtO)+J27. 8 (CHtl, 32. 9 (CH21,45, 7 (CH21, 55, 6 (CJ, 123. 2 (to 9ry aroma tp, CH I, 124. 5 (f: fa 7f4-If, CHI, 139. 8 + hair lol 4 luxury,, CM). 149. 8 (-,901klfF-CH), 157. 8 (Haete c7 Aromatic Hayao C 1,175,4 (7s'h-C-0l, 176. 3 (Free time C-01゜Example 7) 6 Z-L-glutamic acid α-benzyl ester γ-prob-2-enamide (Allylamide) (75) α-benzyl ester (7) (3.33g, 8. 90 mml) was dissolved in THF (50 mL). This solution is Ruamine (1.24 mL, 9. 0mmo I), ethyl chloroformate (0,86 mL, 9. 'Ommol) and allylamine (0.68 mL, 9. 0mmo+ ) was added and the mixture was stirred at 15°C overnight. After this, t, 1. c, (silica; vinegar The reaction was complete as determined by ethyl acetate). Standard treatment and chromatography of crude product torography (silica: 80% ether/gasoline) gives t, 1. c, (Silica; 100% ether) as a homogeneous crystalline solid (75) (3, 31gm, 85%) (1OHml. “CMMR (CDC13p 628. 2 (℃Hs), 32. 1 (CH)) . 41. 9 (CHtl, 53. 6' (CM), 66. 9 (CHil, 67. 2 (C!hl. 116. 3 (7Hz-71' CH21, 12E1. , o (ifQ CH I, 128. 1 (141-stop CM), 128. 4 ($4. Taku CH) 12 8:S (Wan Yu J 毫CHI, 133. 9 (;f fin CM), 135. 1 (fr4W, C1,136,1(%dJi”i, C1,156,3(,7 full 1,)-)C-o)1 17"f;)"C=O1,172,1(bu, scale Bar C-01/Example 77 L-glutamic acid γ-propylamide (76) supported on charcoal as a catalyst] 0% paradi Oriuramide (75) (3,00 g) was dissolved in methanol in the presence of Hydrogenation was carried out in a 50 ml. Standard treatment of the reaction mixture after 24 hours results in t, l, e, (silica, 70% ethanol/water cellulose, n-butanol/pi According to a lysine 10.4% acetic acid aqueous solution 22:10:10), homogeneous aE/ 1 l (76) (1.06 g) was obtained. Melting point 205-207°C. Example 78 Z-L-glutamic acid α-benzyl ester γ-(2°-mesyloxyethyl )amide (77) 2'-Hydroxyethylamide (27) (4.45g. 10. 8 mmol) was dissolved in DCM (150 mL). To this solution, add Ethylamine (3.03 mL, 21. 6mmol) and methanesulfonyl chloride (Mesyl chloride) (1,26-ml, 15. 8 mmol) and the mixture was heated to 25°C. It was stirred with Immediately after the addition of mesyl chloride, the solution turned completely yellow. After 15 minutes, t. The reaction is complete as determined by 1.c. (silica, 85% ethyl acetate/gasoline). Completed. DCM was removed under vacuum and the resulting oil was taken up in ethyl acetate. This solution was washed with saturated sodium chloride solution and the organic layer was dried over sodium sulfate. organic layer Removal of the solvent gave the crude product (77) as a yellow oil. The crude product was chromatographed (silica; 85% ethyl acetate/petrol). Then, t, 1. C, (silica, 85% ethyl acetate/gasoline) homogeneous (77) (2.67 g, 62%) was obtained as a white solid. . 1. 33 (IOHm). Example 79 Dihydroxazole analogue (78) 2′-Mesyloxyethylamide (7 7) (872 mg. 1. 77 mmol) was dissolved in anhydrous DCM (5.0 mL). Add triethylamine (0.5 mL, 4 mmo+) to this solution and bring the solution to room temperature. Stir at room temperature for 16 hours. The solvent was then removed under vacuum and the resulting oil was dissolved in ethyl acetate. (10 mL). The ethyl acetate solution was washed with saturated sodium chloride solution and dried over sodium sulfate. . Removal of the solvent from this solution results in t, l. C, (silica: 85% ethyl acetate/gasoline) as a homogeneous white solid (689 m g, 98%) was obtained. 'l'! NIKR (CDC1n) + 62. 05-2. 30 (48ml, 3. 16 (28t J = 9jl, 4. 17 (28t J-9,3), 4. 47 (18ml, 5. 05-5. 18 (48AB Ran), 7. 30 (IOHml. Examples 80-81 describe the construction of multi-well plate lids of the present invention, It is not limited at all. Example 80 This example describes the construction of a 6-well plate lid of the present invention. Transport assay Tissue culture multiple well plate with 6 flat bottom round wells as the base of the plate Liabro was used. inside each well when assembling the plate. A mark was made on the top of the lid corresponding to the center. Use a hole drilling tool with a 14mm attachment. Use 6 holes (1 cm diameter) corresponding to each mark on the plate assembly cover. I opened it. Be careful not to damage the plastic plate cover while drilling holes. need to pay attention to. The opening of each vial should be facing down and the body of the vial should be Place the scintillator vial ( (K volume + 1 tll, 4 ml volume) was passed through each hole. Each vial is placed on the underside of the cover. Approximately 0. It has been extended by 5 cm, and cyanoacrylate adhesive (Nelveo+, 8 00E100s (for rubber and plastics) was used to adhere it in place. about The adhesive was allowed to air dry for 24 hours. Figure 68 shows the lid of the 6-well plate. The figure and FIG. 70 show the relationship between the 6-well plate and the lid. Example 81 This example describes the construction of a lid for a 96-well plate of the present invention. transportation assay The lower half of the plate contains multiple tissue culture wells containing 96 flat-bottomed wells. Luplate (Linbro) was used. A 96-well plate was used for the transport experiment. Made from internal rack of pipette tip rack holder (NOV) instead of bar A modified cover was used. The internal rack (approximately 3cm long and open at one end) The tubes contain 96 tubes, which are completely filled into the 96 wells of the tissue culture plate. handle. The winter bottom of the pipette rack also has legs, which at the same time are directly below the containers. It was firmly tightened to a 96-well plate. transparent perspex as follows The upper end of the pipette rack was sealed by gluing it with water. 1 perspex Cut a piece of pipette rack (0,550m thick) to the same size as the pipette rack, and place it on the top surface of the rack. placed on an open tube. Parts in direct contact with the pipette rack plastic. On Perspec corresponding to the part of the spec (i.e. around the opening of the vessel) A grid is drawn on the . Scrape off the numbers and letters on the top of the pipette rack and use the adhesive surface. Made the surface smooth. Glue (＾Cτ1lix　92. Rohm) perspec on the grid marks, then place the pipette rack and Perspex together. Press onto the glue on the pipette rack and integrate with the plastic around the tube at the top of the pipette rack. did. Let the assembly sit for 2 hours, then expose to UV light for 2 hours to set the glue. did. Figures 71 and 72 show the lid for a 96-well plate; Figure 4 shows the relationship between the 96-well plate and the lid. It will be apparent to those skilled in the art that various modifications and variations may be made to the compositions and methods of the present invention. Will. Therefore, within the scope of the appended claims and their equivalents, , modifications and variations of the present invention are also intended to be included in the present invention. FIG f FIG. 2 ゛　q゛Rhumin (mM) FIG.3 NEM (mM) Incubation Uma Qυ FIG. 5 Zor7min (m-fight) FIG. 6 7) Rewamin (mM) [Tsurirefmin] (mM) [Gunshitamin (mM) Door) Retamiyl (mM) [/'7 turetamine] (mM) [77 Retamine l (mM) FIG.8 Japanese lanterns * oscillation rate, RF FIG.9 NEM (mM) FIG. 10 FIG. 11 FIG. 12 phase￥T杓ψ〃戊’RF t-te, ty In'renetamine (mM) FIG, /4 Relative erection (RF) F/, 15 FIG. 16 Japanese fencing movement term (RF) F/ni, /7 FIG, /θ FIG, /9 Kiguchi Fukka Dou (RF) FIG. 21 ire Palace dynamic drama (RF) FIG. 22 /1: Floating motion (RF) FIG. 23 Combined ladle traces (RF) 1/! Trade (1/mM) FIG. 25 FIG 26 FIG. 29 FIG. 30 〈Nrenrefmin (mM) FIG. 31 time,) FIG. 32 FIG. 39 FIG, 40σ FIG, 4F (1-#・1-泗・]0 old)↓＾ FIG. 52 FIG. 53 FIG. 54 FIG. 56 FIG, 5B FIG. 59 Time (HR) FIG. 61 Alanine (mM) FIG. 63 8EM (mM) FIG. 64 7-7nin (mM) FIG. 65 Culture HeLa cells until confluence in 200Id glass culture bottles. Wash the cells three times with 09% (w/vl N a Cl). Incubate the cells for 30 min in water-cooled 09% NaCl5d on an orbital shaker. Medium, shake at 120 RPM. UPM LPM ↓ Incubate pellets in NEM for 20 minutes at 37°C with 1mM NaHCO2. Rotate. Wash, suspend in 25% (w/v) sucrose, and then suspend in 40% (w/vl sucrose 3 The radioactive solution was removed, and the membrane was cooled in water at 0.05°C. 1 hour distance at 9125g I care. % (w/v) N a Wash 3 times with CI, then 30 minutes with Gallo 00g Turn into a beret. ↓Membrane pellet was treated with 5DS-solubilization buff 1, and 40% (w/ Dissolve in a layer directly above the vl sucrose 3 and store at -20°C. and 1 for 125g Centrifuge for an hour. The membrane was collected at the interface, diluted with 09% (W/vl NaCl) and pelleted at 600 g for 30 min. Solubilize the leg pellet with SO3-solubilization buffer and store at 20°C. F/to 6θ FI6. 69 FIG. 73 F/, 74 FIG. 75 Tissue Na+ dependent V wA harm suppression release 0. 75 He1a cells + 31 8 IS - Fetal liver +11. 5+ H35 hepatoma + 7. 5+ Intestinal cells +11. 5 ALA N, D. Bovine lymphocytes + 5.1 Colon cells +41 Hepatocytes + 4. 06 HIS + Rat lymphocytes + 3. 43 SEP + renal brush border membrane vesicle + 1. 28 P HE, VAL N, D. Renal basal adventitial membrane vesicles +0. 085 LYS, ARG N, D. FIG. 76 Glutamine 1. OO Glutamic acid-γ-hydrazide 1. 0 90 Histidine 1. 0 50 Glutamic acid-(2-hydroxyethyl)amide 1. 0 50 Glutamic acid- γ-p-nide O anilide 1. 0 50 Glutamic acid-γ-diethylamide 1 ．． 0 45 β-ri O low 7 lanin 5. 0 54' Azaserine 5. 0 461 Glutamic acid derivative” 1. 0 33 Glutamic acid-γ-monohuman 0 xamate 0. 5 1510. 0 53 Acivicin 0. 5 172 6-diaso-5-oxo-norleucine 1. 0 306-Methyl Remerca Butopurine riboside 1. 0 30FIG, 76 (continued) Glutamic acid-γ-BIS-(hydroxyethyl)amide 1. 0 25N- GBZ-L-glutamine 1. 0 10 Glutamic acid-γ-2-methoxy-ethyl Ruamide 1. 0 10 alanine 1. 05 Glutamic acid-γ-ethylamide 1. 05 Glutamic acid-γ-methylamide 1. 00 Glutamic acid-γ-morpholide 1. 00 Alpitin 1cetylglutamine 20. 0 - Glutamic acid-γ-3'-hydroxypiperidinamide 1. 0　0Iboten! ! 40μ level γ-Glutamylglycine 1. 00 Glutamic acid-γ-methylhydroxamate 1. 00 dithiothreitol 5 ．． 〇－ [-Methionine sulfoximine 5. 0-γ-hydroxylysine 5. 0 0 FIG. 77 HFp-266,0 MM 253 C147,0 KB 35. 0 DETROI T-56232,0 HeLa　3LO MRC528,0 M0LT-327,0 m11 cell 115 Fetal rabbit liver 115 RBG 9. 0 M0LT-46,5 1 bovine lymphocytes 51 NAMALWA 5. 0 JIYOYE 5. 0 9 human lymphocytes 45 Condensation cyst 41 Cell correction 406 Rattlin lymphocytes 343 CCRF-8B 3. 2 *aysi Mamoru Elementary School 1a128 Adventitial small gl10 at renal base. 085 After palm stimulation FIG. 78 Glutamine 0. 70 0 Azaserine 0. 16μH95 Methotrexate (MTX) 4uH706-Jiaci5-Offso (-Norlo) Ishin 0. 5 65 Glutamic acid-γ-monohydroxamate 0. 25 10 β-chloro-alanine 1029 Glutamine MT-methylhydroxamate 1. 0 22 Histidine 1,0 15 Glutamine fli-r-hydrazide 1. 0 10 Glutamic acid-γ-morpholy Do 1. 0 10 Glutamine ll-7-dinitylamide 1. 0 10 Glutami phosphoric acid-γ-p-nitroanilide 1. 04 Glutamic acid-(2-hydroxyethyl ) Amide 10 0FIG, 78 (continued) Glutamic acid derivative 1. 00 Glutamic acid-γ-medylamide 1. 00 Fulvitin 1. 00 Tofcetylglutamine 1. 00 Glutamic acid-γ-31-hydroxypiperidinamide 1. 00 glutamic acid -γ-2-methoxy-ethylamide 1. 00 ibotenic acid γ-glutamyl glycylate Shin 1. 00 Methyl 7 Mido Quality-CBZ-1-glutamine 1. 00 dithiothreitol 2,00 Glutamic acid-7-bis(hydroxyethyl)amide 1. 00 Acibicin - − Glutamic acid-γ-(3-carboxy-4-hydroxy) 1o O anilide Glutamic acid-γ-ethylamide 1,00[-methionine sulfoximine] −　−Alanine 10 0 Glutamic acid-γ- (1,1-dimethyl-2-hydroxyethyl)amide 100FIG, 79 Compound Bm K transport proliferation NMOL/NGPROT, (-H)χGLN-GLIIA, highly reversible binding agent 1. Glutamine 3,45 1. 33 100 1002 Histidine 2. 8 6’3. 86 50 153, glutamic acid-T- Monohydroxamate 2,63 1. 35 32 40B, moderately reversible binder 1 Glutamic acid-γ- Hydrazide 2. 27 1. 28 90 102, glutamine 5 (2-hydroxy S Ethyl)amide 1. 89 4. 17 50 03 Glutamic acid derivative 21. 28 0. 2 33 0C1 irreversible binder 1 β-kun 0 loalanine 0. 14 4. 34 543292, glutamic acid -T- p-nitroanilide 0. 136 0. 136 50 43 Azaserine 0. 21 5. 0 463954 Glutamic acid-γ- Diethylamide 0. 55 10. 0 45 1056-Gia Sea 5-Offsew [-noruicine (DONI O, 3757, 130656 acivicin 0. 1 4 10. 7 47'-7 Glutamic acid-γ-bis (Hydroxyethyl)amide 0. 14 8. 0 25 011G, 79 (continued) ) 01 Weak binders or irreversible binders at other sites 111 [Glutamic acid-γ- Tris (hydroxymethyl) -Methylamide 0. 33 7. 75 16 02, glutamic acid-γ-2- Methoxy 1delamide 1,01 5. 26 10 03, N-CB1- L-glutamic acid 0. 25 1. 18 10 04 Glutamic acid-γ-(1 ,1- dimethyl-2-hydroxy Ethyl)amide 0. 1 4. 0 10 05, glutamic acid-T- Ethylamide 0. 10 5. 33 5 06 Alanine 0. 017 1. 8 2.5.07 Glutamic acid-γ- Morpholide 125 0. 91 0 108, glutamic acid Methylamide 1. 25 0. 34 0 09 Fulvicin 1. 15 0. 5 8010, N-acedelglutamine 112 1. 06 0 011 Gluta Minic acid-7-3"- Human []xypiperidine 1,07 5. 98 0 012 Iboten! ! 1 ,00 4. 01--13 γ-glutamylglycine 0. 36 3. 45 0 014 Glutamic acid-γ- Methyl human [l xamate 0. 35 2. 51 0 2215 Dithiotri Thor 0. 24 2. 5--16, 7- L-glutamyl-3- carboxy-4-hydroxy Abrid 0. 11 0. 405　0　0111-Methionine sulfoximine 0. 82 1. 82--FIG, 80 Cell line species Antigen/v'L body reaction HM253C1 human melanoma + B Hydrocavity Carcinoma 10 DETROI T-562 Human pharyngeal cancer 10 HeLa Human cervical cancer 10 T24 Human bladder cancer 10 NAHAL-^ Human Burkitt lymphoma 10EB2 Human Burkitt lymphoma 　10 BORDIN Transformed Lymphocytes 10 JIYOYE Human Burkitt's Lymphoma - MOLT-3 and Tricibablastic Leukemia Disease - MOLT-4 human lymphoblastic leukemia - CCRF-3B human lymphoblast Cytic leukemia -H [60 Human lymphoblastic leukemia - 〇EH-C3 human lymphoblastic leukemia -HRC5 normal human lungs- RBC normal human red blood cells - Lymphocytes Normal person - F! 1Iss3T3 Human-Mouse-FIG, 81 KB Hydrocavitary carcinoma + 35. 0 DETROIT-562 Human pharyngeal cancer + 32. 0HeLa Human cervical cancer + 31. 0T24 Human bladder cancer + 25. 5 HAM^[-^ Human Burkitt lymphoma +5. OJ[YOYE Hitobah Kit lymphoma 5. 0HRC5 Normal Person II 28. 0 HOLT-3 Human Lymphoblastic Leukemia 27. 0RBC Normal human red blood cells 9. 0 HOLT-4 Human Lymphoblastic Leukemia 6. 5 Lymphocytes Normal person 4. 5 CCRF-SB Human Lymphoblastic Leukemia 3. 2FIG, 82 + 1 0. 15 20 1 2 0. 75 20 1 3 3. 00 25 1 4 1 0. 15 20 4 2 1. 50 20 1 5 1 0. 15 20 1 21. Father 204 6 1 0. 15 20 4 2 1. 50 20 4 7 1 0. 50 20 1. 5 2 2. 00 50 5 8 1 0. 03 To 1 2 0. 15 20 2 3 1. 50 25 4 FIG.83 1 66. 7 83 85°7 90 1. 2 60. 6 81 82. 9 88t3 57. 8 79. 5 81. 5 8715 52. 2 77. 2 78. 7 86L7 47 75. 1 76 ．． 0 84+, 8 44, 6 74, 1 74. 7 832. 0 40. 0 7 2. ! 72. 0 822. 5 30, 0 66, + 65. 5 793. 0 22. 2 64, 8 59. 3 763. 5 16, 2 62, 1 53. 5　 734. 0+1. 8 60, 0 48. 1 704. 5 8. 5 58. 1　 43. 0 6B5. 0 6. 1 56. 6 38. 36 666. 0 3. 1　 54,4 30. 2 637. 0-53. 0 23. 5 608〇-father 0 1L2 5B 9. 0-51. 3 14. 0 57FIG, 84 10. 0 0. 8 100+-Lealalanine 581 FIG.86 Amino acid uptake rate (101N) (ratio to control) Pear 100 a＾lB58±6 He^IB83±8 Histidine 25±4 Asparagine 20±3 Alanine 11±5 Serine 12±2 Cysteine 16±4 Medionine 18±5 Methionine sulfone 32±5 Methionine sulfoxide 42±4 Glutamine 85±7 Asbaltate 82±11 Arginine 72±10 Lysine 100±6 Proline 98±1 Valin 33±2 Tryptophan 33±6 Isoleucine 27±5 0 Ishin 33±4 BCH38±2 DL-2-amino-4-arsonobutyrate 51±14 International Search Report mW APPLJCAT Engineering αl fcT/AU 891004271C1ass ification tan C1assification IC07D 2 95/18. 295/22. 211/42. 213/40°C07C day 3108 ,83/10,101/20,103150,109108゜1091097, u51065゜ AIJ+ C07D 295100. 211/42. 213/40M'IQ M　IaTICall, 5EARO(m　Gl’rMmmTX　APPLJCA TION, NITE'll0BE01aT expression πD INaNATIG! Input L APPLICATICN No, PCr ALI89 00427 GIMTNLIEDtE 4125626 CE 280176 2 GB 1598016 JP5310L330W’ERNA’r’lαM WFLXCkTXαl No, PCT AU 8900427 σ sum nail 0L Is3903147 us3947590AU16491/88EP29222 Continued from BJP1052fu43 front page (51) Int, C1,5 identification code Office WITW number A61K 31/40 ABY 9360-4C31/675 8314-4C 37102ABA 8314-4C 49102A 7252-4C CO7C229/26 8930-4H237/22 7106-4H 243/34 9160-4H 2751067188-4H 275/46 7188-4H 323/60 7419-4H C07D 203/18 7019-4C2071068314-4C 207/46 8314-4C 207/48 8314-4C 213/40 6701-4C 295/14 A 6701-4C 295/22 A 6701-4C Z 6701-4C C07F 9153 7537-4H C07K 3102 8517-4H 151048517-4H I Continuation of front page 15/14 8517-4H C12P 21108 8214-48GOIN 33153 D 8310- 2J331574 Z 9015-2J (31) Priority claim number PJ3258 (32) Priority date March 17, 1989 (33) Priority claim country: Australia (AU) (31) Priority claim number: PJ 4948 (32) Priority date June 28, 1989 (33) Priority claim country Ors Tralia (AU) (31) Priority claim number PJ5364 (32) Priority date 19 July 20, 1989 (33) Priority claim country: Australia (AU) (31) Priority Claim number: PJ5611 (32) Priority date: August 4, 1989 (33) Priority claim country: Australia (AU) (31) Priority claim number: PJ 5640(32) Priority date: August 7, 1989 (33) Priority claim country: Australia (AU) (31) Priority claim number: PJ 5872 (32) Priority date: August 21, 1989 (33) Priority claim country: Ors Tralia (AU) (31) Priority claim number PJ6315 (32) Priority date 19 September 12, 1989 (33) Priority claiming country: Australia (AU) (81) Designated -EP (AT, BE, CH, DE. FR, GB, IT, LU, NL, SE), 0A (BF, BJ, CF, CG, CM , GA, ML, MR, SN, TD, TG), AT, AU, BB, BG , BR, CH, DE, DK, FI, GB, HU, JP, KP, K R, LK. LU, MC, MG, MW, NL, No, RO, SD, SE, SU , U.S. (72) Inventor: M.C. Epoybou, Nidward Australia, Ku Einsland 4811, Uluguru, Light Street 98 (72) invention Meehan George, Victor Australia, Queensland 48 17, Ruperts Wood, Romsey Court 5 (72) Inventor Piva, Terenos UP Riva, Home Hill, 4806 Queensland, Australia - (no address) (72) Inventor Rigano, Donna Perugian Gardens, 4810 Queensland, Australia den street 2 (72) Inventor Fabot, Paula Planet Bulle, Griver, 4812, Queensland, Australia Isu (72) Inventor West, Michael Australia, Queensland 4818, Salanders Peach, Ara Tolu Street 13 (72) Inventor Matsukaby, Michael Glenpil Peter Rupertswood, Schringowa, 4817, Queensland, Australia Do, Apotuka Court 6 (72) Inventor Miller, David John Australia, Queensland 4817, Kirwan, 56 Parma Street

Claims (1)

[Claims] 1. a substantially purified human amino acid transporter or a fragment or subunit thereof; to. 2. a substantially purified mammalian glutamine or alanine transporter or fragment thereof; piece or subunit. 3. a substantially purified tumor glutamine transporter or a fragment or subunit thereof; t. 4. 4. The substantially purified transporter of claim 3, a fragment thereof, or a fragment thereof, which is present in a solid tumor. is a subunit. 5. 4. The substantially purified transporter of claim 3, which is present in lymphoma or leukemia. fragment or subunit of. 6. 4. The substantially purified transporter, fragment or subunit thereof of claim 3, which is Na+ dependent. 7. 4. The substantially purified transporter, fragment or subunit thereof of claim 3, which is capable of extracting glutamine from the blood and surrounding tissues and is also capable of acting as a nitrogen trap. 8. against glutamine transporters or their fragments or subunits from solid tumors. 4. The substantially purified transporter, fragment or subunit thereof of claim 3, which reacts with a mouse, rabbit or other antiserum that reacts with a mouse, rabbit or other antiserum. 9. against the glutamine transporter or its fragment or subunit in HeLa cells. 9. The substantially purified transporter, fragment or subunit thereof of claim 8, which reacts with a mouse, rabbit or other antiserum that reacts with a mouse, rabbit or other antiserum. 10. Lymphoma-derived glutamine transporter or its fragments or subunits 4. The substantially purified transporter, fragment or subunit thereof of claim 3, which exhibits a positive antibody-antigen reaction with mouse, rabbit or other antisera directed against the transporter. 11. 4. The substantially purified transporter, fragment or subunit thereof of claim 3 exhibiting an Mr of about 42,000 as determined by SDS-PAGE under reducing conditions. 12. The fruit of claim 3 exhibiting an Mr of about 53,000 as determined by SDS-PAGE. A qualitatively purified transporter, fragment or subunit thereof. 13. 4. The substantially purified transporter, fragment or subunit thereof of claim 3, present in a cell line selected from the group consisting of HEp-2, MM253C1, KB, Delroil-562, HeLa and T24. 14. 4. The substantially purified transporter, fragment or sub thereof of claim 3, wherein the transporter is present in a cell line selected from the group consisting of NAMALWA, EB2, HEp-2, MM253Cl, KB, Detrit-562, HeLa and T24. unit. 15. The transporter is active in HeLa cells, Vm about 6.8±0.35 nmol/min/mg protein, Km about 0.03±0.0015mM, as measured when the intracellular glutamine concentration is at least about 10mM. 4. The substantially purified transporter TGTI of claim 3 exhibiting a coefficient of about 1.0±0.05. 16. The transporter is active in HeLa cells, as measured when the intracellular glutamine concentration is at least about 10mM, with a Vm of about 25±2 nmol/min/mg protein, a Km of about 0.15±0.0075mM, and a Hill coefficient of about 2. .0±0.2. 17. The transporter is active in HeLa cells, Vm about 22.0±1.1 nmol/min/mg protein, Km about 1.8±0.10mM, as measured when the intracellular glutamine concentration is at least about 10mM. 4. The substantially purified transporter TGTIII of claim 3 exhibiting a coefficient of about 6.3±0.31. 18. A. M1 is approximately 42,000 as determined by SDS-PAGE; B. It is Na+ dependent; C. It can extract glutamine from the blood and surrounding tissues and can also act as a nitrogen trap; Shows a positive antigen-antibody reaction with mouse or rabbit antiserum; E. Has histidine as the main amino acid inhibitor;F. Adaptive regulation is lost; G. Has a carbohydrate residue; H. Substantially purified tumor glutamine transporter II can be isolated from HeLa cells. 19. The transporter is active in HeLa cells, as measured when the intracellular glutamine concentration is at least about 10mM, with a Vm of about 25±2 nmol/min/mg protein, a Km of about 0.15±0.03mM, and a Hill coefficient of about 2. 19. The substantially purified transporter of claim 18 exhibiting a .0±0.2. 20. A. Labeled compounds that bind to active sites of amino acid transporter-containing cells and transporters contacting the substance to bind the label to the binding site of the transporter; and B. A substantially purified human amino acid transporter, or a fragment or subunit thereof, obtained by a method for isolating a labeled transporter. 21. 16. The substantially purified human amino acid transporter of claim 15, wherein the cell is a tumor. or its fragments or subunits. 22. 21. The human amino acid transporter or fragment or subunit thereof of claim 20, wherein the cell is a lymphocyte. 23. 24. The human amino acid transporter of claim 23, or a fragment or subunit thereof, which is isolated by electrophoresis. 24. The human amino acid transporter or fragment thereof according to claim 20, which is a glutamine transporter. piece or subunit. 25. 25. The human amino acid transporter of claim 24, or a fragment or subunit thereof, found in solid tumor cells or lymphoma cells. 26. 26. The substantially purified transporter or fragment or subunit thereof of any one of claims 1 to 25, wherein the purified transporter, fragment or subunit thereof is in its native form. 27. The purified transporter, its fragments or subunits are in dissociated form. a substantially purified transporter of any one of claims 1 to 25 or a fragment or substantive thereof; unit. 28. The subunits are involved in active transport of amino acids or glutamine. 28. The substantially purified transporter subunit of claim 26 or 27. 29. 28. The substantially purified transporter of claim 26 or 27, wherein the purified transporter consists of whole transporter protein and the fragment consists of a fragment of whole transporter protein. 30. A method for measuring the binding ability of a ligand to a binding site of a tumor glutamine transporter, comprising: A. A tumor containing the glutamine transporter or a fragment or subunit thereof. B. providing a physical sample; B. B. contacting the sample with a ligand; C. A method comprising the step of determining whether the ligand binds to the transporter. 31. 31. The method of claim 30, wherein step A is performed by providing a substantially pure plasma membrane preparation from a cell line containing the tumor glutamine transporter. 32. After carrying out step B, the sample is washed and then contacted with a labeled ligand that irreversibly binds to the binding site of the transporter, and step C is carried out in the presence of the label. 31. The method of claim 30, wherein the method is performed by measuring 33. Step C is performed by separating the proteins of the biological sample into bands by gel electrophoresis and measuring the presence of the label in bands corresponding to the molecular weight of the transporter or its fragments or subunits. 33. The method of claim 32, carried out by determining. 34. Gel electrophoresis is SDS-PAGE and transporters, fragments or subunits are 34. The method of claim 33, wherein the Mr of the band corresponding to the molecular weight of the compound is about 42,000 or 53,000. 35. Method for measuring the binding ability of the primary ligand to the binding site of tumor glutamine transporter A. A tumor containing a glutamine transporter or a fragment or subunit thereof. providing at least one biological sample; B. contacting said sample with an amount of a first ligand; C. capable of irreversibly binding at least one biological sample to a non-specific reactive group; contact with a second ligand; D. washing the biological sample to remove excess second ligand and releasing any first ligand that can be removed by said washing; E. Place the biological sample in the contact with a labeled ligand that irreversibly binds to the position; and F. A method comprising the steps of: determining the presence of said label. 36. Step A: 1. 2. Cultivating glutamine transporter-containing cells on microparticle carrier beads until fully confluent; 2. 3. Lyse the cells; Remove intracellular debris by shaking; 4. The purified membrane is released from the beads. Se;5. 36. The method of claim 35, carried out by: collecting the released membrane. 37. Step B is performed by inking multiple biological samples in the first ligand at a predetermined concentration range. 36. The method of claim 35, carried out by culturing. 38. Perform steps A to F using glutamine as the first ligand to 36. The method of claim 35, wherein a control sample is run. 39. 36. The method of claim 35, wherein the second ligand is N-ethylmaleimide. 40. 36. The method of claim 35, wherein the labeled ligand is labeled N-ethylmaleimide. 41. The labeled ligand is 3H-N-ethylmaleimide or 14C-N-ethylmaleimide. 41. The method of claim 40, wherein the method is Reimide. 42. Step F 1. Removing unbound labeled ligand from the biological sample;2. Dissolve proteins in biological samples; 3. Separate the dissolved proteins into bands by gel electrophoresis; 4. Stain the gel to visualize protein bands; 5. Glutamine transporter or its fragment or subunit 6. Take out the band corresponding to the molecular weight of 36. The method of claim 35, carried out by: measuring the amount of label. 43. The label is a radioactive label; step 2 is performed by dissolving the protein in sodium dodecyl sulfate; step 3 is performed by polyacrylamide gel electrophoresis. and step 5 is to cut out the appropriate band from the gel, dissolve the band, and irradiate it. 43. The method of claim 42, carried out by measuring the presence of the marker using a line measuring instrument. Law. 44. Step B is performed by contacting a plurality of biological samples with a range of predetermined concentrations of the first ligand; Step F is performed by contacting a plurality of biological samples with a range of predetermined concentrations of the first ligand; 36. The method of claim 35, wherein said value is used to calculate a binding parameter for the first ligand. 45. A method for determining whether a first ligand inhibits glutamine transport in a tumor glutamine transporter, the method comprising: A. B. providing a cell sample containing a tumor glutamine transporter; B. contacting the cell with labeled glutamine in the presence of a concentration of the first ligand; C. A method comprising the steps of: determining the presence of a label in a cell. 46. In step B, cells are contacted with labeled glutamine for a predetermined period of time, then glutamine is Stopping or reducing the transport of min and measuring the amount of intracellular label in step C. The method of claim 45. 47. 47. The method of claim 46, wherein the cells are incubated with a concentration of glutamine prior to step B. 48. Prior to said pre-incubation, the cells are preloaded with a sufficient concentration of glutamine to be transported by the glutamine transporter at a near maximum rate, and then the standard of step B. The pre-incubation in a predetermined concentration of glutamine approximately equal to the concentration of glutamine 48. The method of claim 47, wherein: 49. Step C 1. Extracting labeled glutamine from the cell cytoplasm;2. Quantify the amount of extracted labeled glutamine; 3. 46. The method of claim 45, carried out by: quantifying cellular protein weight. 50. The transporter is TGT I. TGT II. TGT III or a combination? 54. The method of any one of claims 50-53. 51. a labeled or unlabeled human amino acid transporter or a fragment or subunit thereof having an antigenic determinant capable of specifically binding to a complementary receptor; or an anti-receptor capable of specifically binding to said complementary receptor; receptor or its origin Diagnostic products consisting of fragments or products of 52. 52. The diagnostic product of claim 51, wherein the amino acid transporter is a glutamine transporter. 53. Glutamine transporter enriched tumor glutamine transporter or lymphoma glutamine 53. The diagnostic product of claim 52, which is a drug transporter. 54. 53. The diagnostic product of claim 52, wherein the product consists of a fragment of a glutamine transporter capable of binding a complementary receptor to the glutamine transporter. 55. 53. The diagnostic product of claim 52, which is surface bound or dissolved. 56. 53. The diagnostic product of claim 52 conjugated to a label for use in vivo or in vitro. 57. 52. The diagnostic product of claim 51, wherein the anti-receptor is an antibody or an antibody-derived fragment or product. 58. 58. A diagnostic product according to any of claims 51 to 57, wherein the complementary receptor is a monoclonal antibody or a fragment or product derived therefrom. 59. In diagnostic test kits, complementary antibodies and specific 59. The diagnostic product of any of claims 51 to 58, comprising a labeled or unlabeled anti-antibody fragment or product capable of differential binding and a labeled or unlabeled complementary antibody. 60. 60. The diagnostic product of claim 56 or 59, wherein the label consists of an enzyme, a radioisotope, a particle, a fluorescent molecule, a free radical, a chemiluminescent molecule, a bioluminescent molecule, a phage or a metal. A product. 61. 61. A diagnostic product according to any of claims 52 to 60, wherein the transporter or fragment or subunit thereof is in its native form. 62. 61. A diagnostic product according to any of claims 52 to 60, wherein the transporter or a fragment or subunit thereof is in dissociated form. 63. 63. A diagnostic product according to any of claims 50 to 62, wherein the fragment or subunit is involved in active transport of amino acids. 64. 63. A diagnostic product according to any of claims 50 to 62, wherein the fragment or subunit is not involved in active transport of amino acids. 65. 64. The diagnostic product of any of claims 50 to 63, wherein the transporter or fragment or subunit thereof is TGTI, TGTII, TGTIII or a combination thereof. A product. 66. Assays for determining the presence of target ligands in fluid or solid samples of biological origin A. contacting said sample with the diagnostic product of any of claims 46 to 58 to cause the diagnostic product to bind target ligands in the sample; and B. Step A: Measuring Binding; A Method Consisting of the Steps. 67. 67. The method of claim 66, wherein the binding of step A is also quantitatively determined. 68. The assay can be a sandwich assay, an enzyme immunoassay, or a fluorescent immunoassay. radioimmunoassay, competitive assay, immunoradioactivity assay, immunofermentation 68. The method of claim 66 or 67, wherein the method is selected from the group consisting of an elementary assay, an immunofluorescent assay, a luminescent immunoassay, or a binding assay. 69. The assay can be performed using enzymes, radioisotopes, particles, fluorescent molecules, free radicals, or chemiluminescent molecules. 69. The method of any of claims 66 to 68, comprising a label consisting of a child, a bioluminescent molecule, a phage or a metal. 70. Repeat steps A and B at intervals for patient-derived samples; Monitor the progression of the patient's cancer by monitoring binding measurements at How to do it. 71. A method for detecting the presence of tumor cells in vivo, comprising: A. B. administering to an animal subject an effective amount of a diagnostic product according to any one of claims 61 to 65, comprising a specific binding complementary receptor coupled to an in vivo indicating means; retaining the administered analyte for a predetermined period of time sufficient for the receptor to immunoreact with the transporter in vivo and form an immune reaction product; and B. A method comprising the step of assaying for the presence of any immune reaction products formed in step B to assay for the presence of tumor cells in said specimen. 72. Monoclonal antibodies whose receptors bind to tumor glutamine transporters or 72. The method of claim 71, wherein the fragment or product is derived from. 73. 73. The method of claim 72, wherein the receptor is a glutamine analog or other amino acid analog that binds to a tumor glutamine transporter. 74. An isolated DNA segment defining a structural gene encoding at least a portion of an amino acid transporter, or a nucleotide corresponding to a strand of said DNA segment. A biological product consisting of an otide sequence. 75. Amino acid transporter is glutamine transporter in solid tumor or glutamate in lymphoma 75. The biological product of claim 74, which is a amine transporter or a leukemic glutamine transporter. 76. 76. A biological product comprising a recombinant DNA vector according to claim 75, wherein the vector is capable of directing the replication of said DNA segment. 77. 76. A biological product comprising a recombinant DNA vector according to claim 75, wherein the vector is capable of expressing said structural gene in a host cell. 78. A transformed host containing the recombinant DNA vector of claim 76 or 77. Biological products consisting mainly of 79. A biological product comprising a transporter protein produced by the transformed host of claim 78. 80. Amino acids corresponding to human amino acid transporters or fragments or subunits thereof A biological product consisting of a sequence of amino acids. 81. 81. The amino acid sequence of claim 80, wherein the amino acid transporter is a solid tumor glutamine transporter, a lymphoma glutamine transporter, or a leukemia glutamine transporter. biological products. 82. Essentially, antibody molecules that are immunoreactive with human amino acid transporters or derived therefrom are Biological products consisting of antibody compositions consisting of fragments or products of 83. 83. The antibody set of claim 82, wherein the antibody molecule is immunoreactive with a tumor cell glutamine transporter. biological product consisting of components. 84. 84. The biological composition of claim 82 or 83, wherein the antibody composition consists of a polyclonal antibody or a fragment or product derived therefrom. 85. 84. The biological product of claim 82 or 83, wherein the antibody composition consists of a monoclonal antibody or a fragment or product derived therefrom. 86. A biological product consisting of a hybridoma that produces antibodies against human amino acid transporters. 87. Glutamine transporters in solid hybridoma tumors or glutamate in lymphomas 87. The biological product of claim 86, which produces antibodies against the min transporter. 88. A monomer comprising an antibody molecule produced by the hybridoma of claim 86 or 87. Clonal antibody composition. 89. 89. A biological product according to any of claims 74 to 88, wherein the transporter is a subunit involved in active transport or binding of said amino acid or glutamine. 90. the transporter is involved in active transport or binding of the amino acid or glutamine; 89. The biological product of any of claims 74 to 88, wherein the biological product is a subunit that is not a subunit. 91. 89. The product of any of claims 74 to 88, wherein the transporter consists of an entire transporter protein. physical product. 92. The transporter is TGT I, TGTII, TGTIII or a combination thereof. 92. The biological product of any of claims 74-91. 93. A method of forming a monoclonal antibody immunoreactive with a human amino acid transporter or a fragment or subunit thereof, comprising: A. immunizing a mammal with said transporter, fragment, peptide or subunit; B. C. removing antibody-producing cells from the immunized mammal to create a cell suspension; B. D. producing transformed antibody-producing cells by treating the cells with a transforming agent; D. Process in Step C by limiting dilution in tissue culture medium that does not support non-transformed cells. cloning the treated cells to create a cloned transformant; E. The presence of secreted antibody molecules that immunoreact with the transporter or fragment F. Evaluating the enriched tissue culture medium; Cloned transformants producing secreted antibody molecules are selected and cultured in tissue culture medium; and G. select claw harvesting secreted antibody molecules from the culture medium of the transformed transformant. 94. A method of forming a monoclonal antibody immunoreactive with a human amino acid transporter or a fragment thereof, comprising: A. Immunize mice with the transporter, fragment or subunit. Infection; B. C. Remove the spleen from the mouse and create a suspension of spleen cells; Antibody-secreting hybrids are produced by fusing spleen cells and mouse myeloma cells in the presence of a fusion promoter. D. Diluting and culturing the fused cells in separate cells in a medium that does not support unfused myeloma cells and spleen cells; E. Assess the supernatant of each well containing hybridomas for the presence of secreted antibody molecules that immunoreact with the transporter or fragment. Worth; F. selecting and cloning hybridomas that secrete antibody molecules; G. A method consisting of harvesting antibody molecules from the supernatant on a clone. 95. 95. The method of claim 93 or 94, wherein the transporter is a tumor glutamine transporter. 96. 96. The method of claim 93, 94 or 95, wherein the transporter is a subunit involved in active transport or binding of amino acids or glutamine. 97. 96. The method of claim 93, 94 or 95, wherein the transporter is a whole transporter protein. 98. 98. The method of any of claims 95 to 97, wherein the transporter is in its native form. 99. 98. The method of any of claims 95 to 97, wherein the transporter is in dissociated form. 100. The diagnosis according to any one of claims 51 to 65, wherein the antibody is a polyclonal antibody. Discontinued product. 101. 84. The biological product of claim 82 or 83, wherein the antibody composition is a genetically engineered antibody, fragment or product derived therefrom. 102. A biosensor comprising a diagnostic product as defined in claims 51 to 58. 103. 103. The biosensor of claim 102, wherein the transporter or fragment or subunit thereof is in its native form. 104. The transporter or its fragment or subunit is in dissociated form. The biosensor of claim 102. 105. 105. The biosensor of any of claims 102 to 104, wherein the fragment or subunit is involved in active transport of amino acids. 106. 105. The biosensor of any of claims 102 to 104, wherein the fragment or subunit is not involved in active transport of amino acids. 107. 107. The biosensor of any of claims 102 to 106, wherein the transporter, fragment or subunit is TGTI, TGTII, TGTII I or a combination thereof. -. 108. 108. The biosensor of any of claims 102 to 107, wherein the antibody is a polyclonal antibody. 109. The antibody composition may be a genetically engineered antibody or a fragment or product derived therefrom. 108. The biosensor of any of claims 102 to 107, consisting of a composition of the invention. 110. A biosensor comprising a biological product as defined in any of claims 74, 75, 80-85 and 88-92. 111. General formula (I): ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) [In the formula, X is selected from CR5R6, NH, NOH or O; W is ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. Select from ▼; V is ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼ R1 is selected from H, OR13, NH2, NHNH2 and benzyl, where R13 is selected from H and C1-5 substituted or unsubstituted cyclic or acyclic, saturated or unsaturated hydrocarbon group; ; R2 is selected from the group defined by R13, NH2, O(C=O)R13, (C=O)OR13 and (C=O)NR13AR13B, where R13A and R13B are independently defined by R13; R3 is selected from the group defined by R13; R4 is selected from the group defined by R13, halogen and (C=O)R1; R5 and R6 are each independently defined by halogen and R13; R7 is selected from the group defined by OR14 and NR15R16, where R14, R15 and R16 are independently H, C1-5 substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic; selected from a hydrocarbon group, or a substituted or unsubstituted C5-6 aromatic or heterocyclic ring; R8 is selected from the group defined by R14, OR14, amino, amido, NO and NO2; R9 is selected from the group defined by R14; or R8 and R9 together with the nitrogen atom represent a 3- to 8-membered substituted or unsubstituted saturated or unsaturated heterocycle; -CH2 is a group: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R17 is selected from the group defined by R14, SO2Cl and -O-(C=O)-R14; and R18 and R19 are independently R14, halogen, and R14 group in which the α carbon to the 4-membered ring is substituted with a leaving group], or V-W-X is a group: ▲ Numerical formula, chemical formula, table, etc. ▼ [wherein R20 is selected from the group defined by R14, OR14 and NHR14 and -O-(C=0)R14; or ZR21 is a halogen; Z is selected from O, S and NH14 R21 is selected from the group defined by mesyl, tosyl, -O-(C=O)-R14 and C1-5 substituted or unsubstituted cyclic or acyclic, saturated or unsaturated hydrocarbon group; or R20 and R21 is a group: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ Together with this, it forms a 5- or 6-membered heterocycle which may or may not be further substituted, and R1-R21 may be the same or different. ] or its Physiologically acceptable salts of. 112. General formula (II): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) [In the formula, X is selected from CH2, NH, NOH or O, and R1, R2, R8 and R9 have the same meanings as above] 112. The compound of claim 111, or a physiologically acceptable salt thereof. 113. General formula (III): ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (III) [In the formula, X is CH2, NH, NO H or O; R8 is OH, NHR14, -(C=O)NHR14 or R23 is H when selected from the group defined by (C=O)N(R14)OH; when R8 is selected from the group defined by -(C=O)NHR14 and -(C=O)N(R14)OH Sometimes R23 is OH; or R23 and R8 are independently the same as above for R14. or R23 and R8 together with the nitrogen atom may be saturated or unsaturated, substituted or unsubstituted; forming a 5- or 6-membered heterocycle. Physiologically acceptable salts of. 114. Formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ The compound or compound of claim 113, which has [In the formula, R8 and R9 have the same meanings as above] or its physiologically acceptable salts. 115. Formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R26 is H, OH or NO, and R24 and R25 are independently H, O R13, SR13, NR132, NO2, CHO, CO2H and halogen. Selection and wherein R13 is as defined above. is its physiologically acceptable salt. 116. 114. The compound of claim 113, or a physiologically acceptable salt thereof, having the formula: ▲A mathematical formula, a chemical formula, a table, etc.▼ [wherein R27 and R28 are independently selected from H and OH]. 117. Formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R29 is ▲There are mathematical formulas, chemical formulas, tables, etc.▼,▲There are mathematical formulas, chemical formulas, tables, etc.▼,▲Number There are formulas, chemical formulas, tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Select from ▼, where X is OH, OMs, OTs or is a halogen, and R30 and R31 are independently from the group defined above for R13. or a physiologically acceptable salt thereof. 118. 118. The compound of any of claims 11-117, which inhibits glutamine transport into tumor cells by at least about 20%. 119. 120. The compound of claim 118, which inhibits transport by at least about 40%. 120. 120. The compound of claim 119, which inhibits transport by at least about 60%. 121. 121. The compound of any of claims 111-120, wherein Bm is greater than about 1. 122. 122, wherein Ks' is less than about 1.5. Compound. 123. 123. A method of treating cancer comprising administering a therapeutic dose of a compound of any of claims 111-122 to a patient in need thereof. 124. A chemotherapeutic composition comprising a compound as defined in any of claims 111 to 122. 125. 123. A method of treating multidrug resistant tumors comprising administering to a patient in need thereof a therapeutic dose of a compound of any of claims 111-122. 126. A method of suppressing or activating a patient's immunity by administering therapeutic doses of an immunosuppressant or stimulant designed to be transported via the lymphoma glutamine transporter. 127. A method of treating cancer by administering to a patient in need thereof a therapeutic dose of a compound that is substantially selectively transported by a tumor-associated transporter. 128. 128. The method of claim 127, wherein the transporter is a tumor glutamine transporter. 129. consisting of a lid having a row of downwardly extending apertures positioned to coincide with the wells of the assay plate; when the cover is on the assay plate, the lid extends into the plate and the well holes of the plate; Cover for multi-well assay plates. 130. 130. The cap of claim 129, wherein the border of the lid is a downwardly curved marginal flange. bar. 131. 130. The cover of claim 129, wherein the cover is adapted to stand freely on a flat support surface when inverted. 132. It consists of a number of spaced apart wells and a removable cover that stands freely on a flat support surface when inverted; It has a row of downwardly extending open frames positioned to coincide with the wells of the assay plate so that when the cover is on and in contact with the assay plate, the openings of the frames are in the well holes of the assay plate. Extended transport assay plate assembly. 133. The assay plate and cover are each made of sterilizable plastic. 133. The assembly of claim 132, wherein the assembly is shaped. 134. Grow cells in the base of multiple wells of the assay plate, invert the cover consisting of a row of multiple frames that match the wells of the assay plate, add incubation medium to each frame of the cover, and invert the assay plate. Place on the cover and invert the assembly to transfer the incubation medium in each frame to the assay plate. Transport assay procedure consisting of ensuring transfer to adjacent wells of the cell. 135. A method of cancer treatment by inhibiting glutamine transport to tumor cells, in which an antibody composition consisting of an antibody that binds to tumor glutamine transporters is administered to patients in need. A method consisting of administering therapeutic doses. 136. 136. The method of claim 135, wherein the transporter is TGTI, TGTII or TGTIII. 137. Claims that the antibody consists of a monoclonal antibody, a fragment or product thereof; The method of claim 135 or 136. 138. 137. The method of claim 135 or 136, wherein the antibody is a polyclonal antibody, fragment or product therefrom. 139. It is conjugated to cytotoxic drugs and selectively binds to glutamine transporters. Composition consisting of children. 140. 140. The composition of claim 139, wherein said molecule consists of a monoclonal antibody, fragment or product therefrom. 141. 140. The composition of claim 139, wherein said molecule consists of a polyclonal antibody, fragment or product therefrom. 142. 140. The composition of claim 139, wherein said molecule consists of glutamine. 143. 140. The composition of claim 139, wherein said molecule consists of a glutamine analog. 144. 144. The composition of claim 143, wherein said analog is as defined in any of claims 111-120. 145. 145. The composition of claim 143 or 144, wherein the Bm of the glutamine analog is greater than about 1.0 nmol/mg protein. 146. 146. The composition of claim 143, 144 or 145, wherein the K of the analog is less than about 1.5 mM. 147. 145. The composition of any of claims 137-144, wherein the glutamine transporter consists of TGTI, TGTII or TGTIII or a combination thereof. 148. 148. The composition of any of claims 139-147, wherein the cytotoxic agent is a chemotherapeutic agent. 149. Any of claims 139 to 147, wherein the cytotoxic agent is a radioisotope. composition. 150. Glutamine analogs and antiseptics with formulas that are relatively harmless when the bond is broken 142. The composition of claim 140 or 141, wherein the composition binds to the body. 151. as defined in any of claims 101 to 122, together with a pharmaceutically acceptable carrier. A pharmaceutical composition comprising at least one compound of the following. 152. as defined in any of claims 139 to 150, together with a pharmaceutically acceptable carrier. A pharmaceutical composition comprising at least one compound of the following. 153. A method of inhibiting lymphocyte proliferation in a patient comprising inhibiting the transport of glutamine uptake within lymphocytes. 154. 54. The method of claim 153, comprising administering a glutamine analog that inhibits proliferation. 155. 153. A method of treating cancer comprising administering a therapeutic amount of at least one composition as defined in claims 139 to 152 to a patient in need thereof. 156. 156. The method of treating cancer of any of claims 135-138 and 155, also comprising another step of administering conventional chemotherapy or radiation therapy to the patient. 157. A method of cancer treatment that consists of administering a substance designed to inhibit glutamine uptake into tumors. 158. 158. The method of claim 157, wherein the substance consists of an amino acid. 159. 158. The method of claim 157, wherein the substance substantially selectively binds TGTII. 160. at least one antigenic human amino acid transporter or a fragment or substantive thereof; A vaccine consisting of buunits. 161. 161. The vaccine of claim 160, wherein the transporter, fragment or subunit consists of a tumor glutamine transporter, fragment or subunit. 162. If the tumor glutamine transporter is TGTI, TGTII, TGTIII or 162. The vaccine of claim 161, consisting of a combination of. 163. 163. The vaccine of any of claims 160 to 162, wherein the transporter, fragment or subunit is in its native form. 164. Claim 160, wherein the transporter, fragment or subunit is in dissociated form. 162 vaccines. 165. 163. The vaccine of any of claims 160 to 162, wherein the subunits are involved in active transport of amino acids or glutamine. 166. administering the vaccine composition of any of claims 160 to 165. Immune sensitization method against different cancers.