JPH02101072A - Tetrahydrocannabinol derivative - Google Patents
Tetrahydrocannabinol derivativeInfo
- Publication number
- JPH02101072A JPH02101072A JP25021088A JP25021088A JPH02101072A JP H02101072 A JPH02101072 A JP H02101072A JP 25021088 A JP25021088 A JP 25021088A JP 25021088 A JP25021088 A JP 25021088A JP H02101072 A JPH02101072 A JP H02101072A
- Authority
- JP
- Japan
- Prior art keywords
- thc
- tetrahydrocannabinol
- solution
- expressed
- formula
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- CYQFCXCEBYINGO-IAGOWNOFSA-N delta1-THC Chemical class C1=C(C)CC[C@H]2C(C)(C)OC3=CC(CCCCC)=CC(O)=C3[C@@H]21 CYQFCXCEBYINGO-IAGOWNOFSA-N 0.000 title claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 10
- 239000000126 substance Substances 0.000 claims description 3
- 229960004242 dronabinol Drugs 0.000 abstract description 25
- CYQFCXCEBYINGO-UHFFFAOYSA-N THC Natural products C1=C(C)CCC2C(C)(C)OC3=CC(CCCCC)=CC(O)=C3C21 CYQFCXCEBYINGO-UHFFFAOYSA-N 0.000 abstract description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 16
- 238000002360 preparation method Methods 0.000 abstract description 9
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 abstract description 4
- 230000007062 hydrolysis Effects 0.000 abstract description 3
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 3
- 230000003533 narcotic effect Effects 0.000 abstract description 3
- 150000001875 compounds Chemical class 0.000 abstract 3
- 244000025254 Cannabis sativa Species 0.000 abstract 1
- 235000012766 Cannabis sativa ssp. sativa var. sativa Nutrition 0.000 abstract 1
- 235000012765 Cannabis sativa ssp. sativa var. spontanea Nutrition 0.000 abstract 1
- 235000009120 camo Nutrition 0.000 abstract 1
- 235000005607 chanvre indien Nutrition 0.000 abstract 1
- 239000011487 hemp Substances 0.000 abstract 1
- 238000001727 in vivo Methods 0.000 abstract 1
- 239000004615 ingredient Substances 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- UHBAPGWWRFVTFS-UHFFFAOYSA-N 4,4'-dipyridyl disulfide Chemical compound C=1C=NC=CC=1SSC1=CC=NC=C1 UHBAPGWWRFVTFS-UHFFFAOYSA-N 0.000 description 7
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 125000000524 functional group Chemical group 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000003463 adsorbent Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000012746 preparative thin layer chromatography Methods 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 238000004809 thin layer chromatography Methods 0.000 description 3
- FHTDDANQIMVWKZ-UHFFFAOYSA-N 1h-pyridine-4-thione Chemical compound SC1=CC=NC=C1 FHTDDANQIMVWKZ-UHFFFAOYSA-N 0.000 description 2
- GDRNNGAOPZOKFM-UHFFFAOYSA-N 4-(4-bromobutyl)isoindole-1,3-dione Chemical compound BrCCCCC1=CC=CC2=C1C(=O)NC2=O GDRNNGAOPZOKFM-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000218236 Cannabis Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000011481 absorbance measurement Methods 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 1
- 102100034613 Annexin A2 Human genes 0.000 description 1
- 108090000668 Annexin A2 Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- HAXFWIACAGNFHA-UHFFFAOYSA-N aldrithiol Chemical group C=1C=CC=NC=1SSC1=CC=CC=N1 HAXFWIACAGNFHA-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 125000002228 disulfide group Chemical group 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 229910000104 sodium hydride Inorganic materials 0.000 description 1
- 239000012312 sodium hydride Substances 0.000 description 1
Landscapes
- Pyrane Compounds (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明はテトラヒドロカンナビノール（以下ＴＨＣと記
す）誘導体に関する。ＴＨＣは***の主要麻薬成分であ
る。本発明を用いて作製した抗ＴＨＣ抗体は、生体内や
大気中のＴＨＣの極微量検出に有効である。DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to tetrahydrocannabinol (hereinafter referred to as THC) derivatives. THC is the main narcotic component of cannabis. Anti-THC antibodies produced using the present invention are effective in detecting trace amounts of THC in living bodies and in the atmosphere.
従来の技術
***の主要麻薬成分であるＴＨＣを微量検出するには抗
ＴＨＣ抗体を用いた免疫反応を用いる方法がを効である
。抗ＴＨＣ抗体を作製するためには免疫原としてＴｌＩ
Ｃを生体に注射する必要がある。ところがＴＨＣは低分
子（Ｍｗ＝３１４）であるため単独では免疫応答を引き
起こせない。このような物質はハプテンと呼ばれる。ハ
プテンはタンパク質等の高分子と結合させ複合体（ハプ
テン−タンパク質コンジュゲート）にすることで免疫原
となる。ＴＨＣをタンパク質と結合させＴＨＣ−タンパ
ク質コンジュゲートにするためには、タンパク質と結合
が可能な官能基をＴＨＣに導入したＴＨＣ誘導体を作製
する必要がある。Conventional Techniques An effective method for detecting trace amounts of THC, the main narcotic component of cannabis, is an immune reaction using anti-THC antibodies. To produce anti-THC antibodies, TlI is used as an immunogen.
It is necessary to inject C into a living body. However, since THC is a low molecule (Mw=314), it cannot cause an immune response alone. Such substances are called haptens. A hapten becomes an immunogen by binding it to a polymer such as a protein to form a complex (hapten-protein conjugate). In order to bind THC to a protein to form a THC-protein conjugate, it is necessary to prepare a THC derivative in which a functional group capable of binding to a protein is introduced into THC.
抗ＴＨＣ抗体を作成するためのＴＩＴＣ誘導体は、Ｐ、
Ｔ。TITC derivatives for producing anti-THC antibodies include P,
T.
ライ らによって報告されている（例えばＰ、 Ｔ。As reported by Lai et al. (e.g. P, T.
Ｔｓｕｌ、 Ｋ、 Ａ、 Ｋｅｌｌｙ ａｎｄ Ａ、
Ｈ，５ｅｈｏｎ、 Ｃａｎ、 Ｊ、 Ｂｊｏｃｅｍ、、
５２（１９７４））。Tsul, K., A., Kelly and A.
H, 5ehon, Can, J, Bjocem,.
52 (1974)).
発明が解決しようとする課題
従来作製されていたＴＩＴＯ誘導体は、ＴＨＣにエステ
ル結合を用いて官能基を導入していた。しかし工ステル
結合は容易に加水分解をするため、動物体内で十分な期
間免疫反応を誘期する点で信頼性に欠けている。タンパ
ク質１分子に結合したＴＨＣの分子数の定量測定を行う
にはＴＨＣ誘導体を放射性同位体でラベルした物質を用
いて測定していた。しかし、この方法はＴＴＩＣの放射
性同位が入手困難であり、さらに放射性物質を扱うので
簡便でなかった。Problems to be Solved by the Invention In the conventionally produced TITO derivatives, a functional group was introduced into THC using an ester bond. However, the ester bond is easily hydrolyzed, making it unreliable in inducing an immune response for a sufficient period of time in the animal body. In order to quantitatively measure the number of THC molecules bound to one protein molecule, a substance labeled with a radioactive isotope was used to measure the THC derivative. However, this method was not convenient because it was difficult to obtain the radioactive isotope of TTIC, and furthermore, it involved handling radioactive substances.
課題を解決するための手段 下記の構造を有するＴｌＩＣ誘導体。Means to solve problems A TlIC derivative having the following structure.
作用
上記構成をとることにより、Ｔｌ（Ｃはエーテル結合で
官能基を導入したため、加水分解が阻止され安定性が向
上する。さらに官能基としてアミノ基を導入することで
、すでに生化学分野の情報として確立されている我々の
定量法が使用可能となり、放射性同位体を用いることな
（定量することができる。Effect By adopting the above structure, Tl(C has a functional group introduced through an ether bond, so hydrolysis is prevented and stability is improved.Furthermore, by introducing an amino group as a functional group, information already available in the biochemical field) Our quantitative method, which has been established as
メチレン基の数ｎは、２０以上ではコンジュゲートの溶
解性が劣るため本発明の用途には不適である。また、良
好な免疫反応を起こすにためには、ハプテンとタンパク
質がある程度離れている方が望ましいという観点から、
２≦ｎ≦２０が理想的である。If the number n of methylene groups is 20 or more, the solubility of the conjugate will be poor and therefore it is unsuitable for the use of the present invention. Additionally, from the perspective that it is desirable for the hapten and protein to be separated by some distance in order to generate a good immune response,
Ideally, 2≦n≦20.
実施例
以下にメチレン基の数ｎを４をとした場合の本発明の実
施例について説明する。Examples Examples of the present invention in which the number n of methylene groups is set to 4 will be described below.
田りＩ遵Ｕ
（Ａ） ＴＨＣ−ＢＰＩの作製
ＴＨＣ８１６ｍｇ （２ＢＯＯμｍｏｌ）を乾燥ベンゼ
ン２ｍｌに溶解し、水素化ナトリウム ｌ０４ｍｇ （
５２００μｍｏｌ）を加えた。この溶液に、ブロモブチ
ルフタルイミド（ＢＢＰＩ） ７３３ｍｇ （２１１ｉ
００μｍｏｌ）を乾燥ベンゼン ２ｍｌに溶解したもの
を徐々に加えた。溶液の温度を８０”Ｃに保ち１０時間
還流し、目的物であるテトラヒドロカンナビノール−〇
−ブチルフタルイミド（ＴＨＣＢＰＴ ）を得た。反応
の確認はＴＬＣでおこなった。(A) Preparation of THC-BPI Dissolve 816 mg of THC (2 BOO μmol) in 2 ml of dry benzene, and dissolve 104 mg of sodium hydride (
5200 μmol) was added. To this solution was added 733 mg of bromobutylphthalimide (BBPI) (211i
A solution of 00 μmol) dissolved in 2 ml of dry benzene was gradually added. The temperature of the solution was maintained at 80"C and refluxed for 10 hours to obtain the target product, tetrahydrocannabinol-0-butylphthalimide (THCBPT). The reaction was confirmed by TLC.
ＴＨＣ−ＢＰＩは分取ＴＬＣ（吸着剤シリカゲル、展開
液ｎ−ヘキサン／ジエチルエーテル＝４／１）で採し、
１４０ｍｇを得た。THC-BPI was collected by preparative TLC (adsorbent silica gel, developer n-hexane/diethyl ether = 4/1),
140 mg was obtained.
モニア飽和）で目的物であるＴＨＣ−Ｎｌ１２を採取し
た。The target product, THC-Nl12, was collected at 100 ml (monium saturation).
反応の確認はＴＬＣでおこなった。The reaction was confirmed by TLC.
Ｈａ ＣＨ。Ha CH.
（Ｂ ） ＴＨＣ−Ｎｌ２の作成
ＴＨＣ−ＢＰ１１４０ｍｇ （２７０μｍｏ１）を９５
％エタノール２ｍｌに溶解したものに、ヒドラジン２７
０ｍｇ （５４０Ｏμｍｏ１）を９５％エタノール２ｍ
ｌに溶解した溶液を徐々に加えた。(B) Preparation of THC-Nl2 1140mg (270μmol1) of THC-BP was added to 95%
% hydrazine 27ml dissolved in 2ml of ethanol.
0mg (540Oμmol1) in 2m of 95% ethanol
1 of the solution was gradually added.
２時間還流した後、分取ＴＬＣ（吸着剤シリカゲル、展
開液 クロロホルム／メタノール＝９５１５、アン（
Ａ ） ＴＨＣ−５ＰＩＩＰの作成
ＴＨｃ−ＮＨ２２０ｍｇ （５２μｍｏｌ）をエタノー
ル２．０ｍｌに溶解したものに、 Ｎ−サクシンイミジ
ル３− （２−ピリジルジチオ）プロピオネート（以下
Ｓ Ｐ ＤＰ、 ファルマシア製、ＦＷ：３１２．４
） １１ｍｇ （３４μｍｏｌ）をエタノール２．０１
に溶解した溶液を徐々に加え、室温で３０分間撹拌した
。ＴＬＣで反応前後の溶液のチェッりを行い、５ＰＤＰ
の消失を確認した。分取ＴＬＣ（吸着剤シリカゲル、展
開液 クロロホルム／メタノール＝＝９５１５、アンモ
ニア飽和）で目的物であるＴＨＣ６ＰＤＰを採取した。After refluxing for 2 hours, preparative TLC (adsorbent silica gel, developing solution chloroform/methanol = 9515, an
A) Preparation of THC-5PIIP 220 mg (52 μmol) of THc-NH was dissolved in 2.0 ml of ethanol, and N-succinimidyl 3-(2-pyridyldithio) propionate (hereinafter referred to as SP DP, manufactured by Pharmacia, FW: 312.4)
) 11 mg (34 μmol) in ethanol 2.01
was gradually added to the mixture, and the mixture was stirred at room temperature for 30 minutes. Check the solution before and after the reaction with TLC, and 5PDP
Confirmed the disappearance of. The target product, THC6PDP, was collected by preparative TLC (adsorbent silica gel, developing solution chloroform/methanol = 9515, saturated with ammonia).
ＣＨ。CH.
５ＰＤＰ ２０ｍｇ （０，［ｉ４ｍｍｏｌ）を、エタ
ノール１．５ｍＬに緩やかに加熱をしつつ溶解した。Ｃ
ＧＧ溶液５０ｍ１を撹拌しながらこのＳＰ叶温溶液１０
分間にわたって滴下後、さらに−晩４℃で撹拌した。余
分の５ＰＤＰを除くため５ｅｐｈａｄｅｘ Ｇ−２５（
ファルマシア製）のゲルクロマトグラフィー（ＩＧｍｍ
径Ｘ ７０ｃｍ）にこの溶液を流し、ＣＧＧと５ＰＤＰ
の結合物（ＣＧＧ−３ＰＤＰ）を得た。なお、以降の本
実験におけるゲルクロマトグラフィーは全て同一条件で
行った。20 mg (0, [i4 mmol) of 5PDP] was dissolved in 1.5 mL of ethanol with gentle heating. C
While stirring 50ml of GG solution, add 10ml of this SP Kano On solution.
After the dropwise addition over a period of minutes, the mixture was further stirred at 4°C overnight. 5ephadex G-25 (
Gel chromatography (IGmm manufactured by Pharmacia)
Pour this solution through a tube (diameter x 70 cm) and connect CGG and 5PDP.
A conjugate (CGG-3PDP) was obtained. Note that all subsequent gel chromatography in this experiment was performed under the same conditions.
（Ｂ）ＣＧＧ溶液の作製
チキンγグロブリン（ＣＧＧ）
ＮａＣｌを含むｐｈ７．０のリン酸バッ５０ｍ１に分散
し攪拌した。(B) Preparation of CGG solution Chicken gamma globulin (CGG) was dispersed in 50 ml of a pH 7.0 phosphoric acid bag containing NaCl and stirred.
（Ｃ） ＣＧＧ−ＳＰＤＰの作製
１００ｍｇを、 ０．１ｍｏｌの
ファー（以下ＰＢＳ）
（Ｄ ） ＣＧＧ−５ＰＤＰの還元
ｓ、ｅμＭのＣＧＧ−ＳＰＤＰのＰＢＳ溶液５０１に、
１００ｍＭジチオスレイトール（ＤＴＴ）を含むＰＢＳ
溶液溶液１誉ｌっくり滴下し、最終濃度２ｍＭにし、２
−ピリジルジスルフィド基を還元した。３０分後この溶
液をゲルクロマトグラフィーにかけ低分子を除去し、７
．７μＭのＣＧＧ−５Ｒ溶液５０ｍ１を得た。(C) Preparation of CGG-SPDP 100 mg was added to 0.1 mol of fur (hereinafter referred to as PBS) (D) Reduction of CGG-5PDP, eμM of CGG-SPDP in PBS solution 501,
PBS containing 100mM dithiothreitol (DTT)
Add one drop of the solution solution to make a final concentration of 2mM,
-Reduced the pyridyl disulfide group. After 30 minutes, this solution was subjected to gel chromatography to remove low molecules.
．． 50 ml of 7 μM CGG-5R solution was obtained.
めに、ＣＧＧｌＣＧＧ−３ＰＤＰ、 ＣＧＧ−ＴＩＴ
ＣそれぞれのＳｌｌ残基を４−ピリジルヂサルファイド
（４ＰＤＳ）を用いた３２４ｎｍの吸光度測定で定量し
た。その結果ＣＧＣＩ分子当りＴＨＣが９分子結合して
いることがわかった。また酵素免疫測定法によりＣＧＣ
にＴ）ＩＣが導入されていることを確認した。CGGlCGG-3PDP, CGG-TIT
The Sll residue in each C was quantified by absorbance measurement at 324 nm using 4-pyridyldisulfide (4PDS). As a result, it was found that 9 molecules of THC were bound per CGCI molecule. In addition, CGC was determined by enzyme immunoassay.
It was confirmed that T) IC was installed in the factory.
（Ｅ ） ＣＧＧ−ＴＨＣの作製
７．７μＭのＣＧＧ−５Ｈ溶液５０ｍＬに、７．７μＭ
のＴ）ＩＣ−ＳＰＤＰのエタノール溶液１．５ｍＬをゆ
っくり滴下し、室温で２０分間攪拌ののち冷蔵室で一晩
攪拌した。カラムクロマトグラフィーで精製し１１．３
μＭのＣＧＧ−ＴＨＣのＰＢＳ溶液３０ｍ１を得た。(E) Preparation of CGG-THC Add 7.7 μM to 50 mL of 7.7 μM CGG-5H solution.
T) 1.5 mL of an ethanol solution of IC-SPDP was slowly added dropwise, and after stirring at room temperature for 20 minutes, the mixture was stirred overnight in a refrigerator. Purified by column chromatography 11.3
30 ml of μM CGG-THC in PBS solution was obtained.
（Ｆ）ＣＧＣＩ分子当りのＴｌ１Ｃ量の測定ＣＧ０１分
子当りに結合したＴＨＣの分子数を知るた（Ａ）ＢＳＡ
溶液の作製
牛血清アルブミン（ＵＳＡ） １００ｍｇを０．１ｍｏ
ｌノＮａＣｌ含むｐ■７．θのリン酸バッファー（以下
ＰＢＳ） ３０ｍ１に分散し撹拌した。(F) Measurement of the amount of Tl1C per CGCI molecule. Find out the number of THC molecules bound per CG0 molecule. (A) BSA
Preparation of solution Bovine serum albumin (USA) 100mg to 0.1mo
p■7. Contains l-NaCl. The mixture was dispersed in 30 ml of θ phosphate buffer (hereinafter referred to as PBS) and stirred.
（Ｂ ） ＢＳＡ−ＴＨＣの作製
ＢＳＡ溶液３０ｍＬに、２７ｍＭのＴＨＣ−ＳＰＤＰの
エタノール溶液０．１５ｍＬをゆっくり滴下し、室温で
２０分間攪拌ののち４℃で一晩攪拌後、カラムクロマト
グラフィーテ精製し、３５ μＭ＋７） ＢＳＡ−ＴＨ
ＣノＰＢＳ溶液３８ｍ１を得た。(B) Preparation of BSA-THC 0.15 mL of 27 mM THC-SPDP in ethanol was slowly added dropwise to 30 mL of BSA solution, stirred at room temperature for 20 minutes, stirred overnight at 4°C, and purified by column chromatography. , 35 μM+7) BSA-TH
38 ml of C-PBS solution was obtained.
（Ｃ）ＢＳＡ−分子当りのＴｕＩｃ量の測定ＢＳＡ １
分子当りに結合したＴＨＣの分子数を知るたメニ、ＢＳ
ＡｌＢＳＡ−５ＰＤＰ１ＢＳＡ−Ｔ）ＩＣソれぞレノｓ
Ｈ基量を４−ピリジルジサルファイド（４ＰＤＳ）を用
いた３２４ｎｍの吸光度測定で調べた。その結果ＢＳＡ
Ｓモー当りＴＨＣが０．４モル結合できていることがわ
かった。(C) BSA-Measurement of TuIc amount per molecule BSA 1
Meni, BS, who knows the number of THC molecules bound per molecule.
AlBSA-5PDP1BSA-T) IC sorenos
The amount of H groups was determined by absorbance measurement at 324 nm using 4-pyridyl disulfide (4PDS). As a result, BSA
It was found that 0.4 mol of THC was bound per S mo.
また酵素免疫測定法によりＢＳＡにＴＨＣが導入されて
いることを確認した。Furthermore, it was confirmed by enzyme immunoassay that THC was introduced into BSA.
本発明によるＴＨＣ誘導体（Ｉ）と、ライらの作製した
ＴＨＣ誘導体（ＩＩ）の安定性を比べる評価実験をした
。ＰＨ７，０に調整したＰＢ３５０ｍｌに１１Ｉｆを溶
解し、この溶液それぞれを室温（２０’Ｃ）でゆるく攪
拌し５日間放置した。その後溶液中のＴＨＣ誘導体量を
定量した結果、■に比べＩがはるかに安定であることが
確認できた。An evaluation experiment was conducted to compare the stability of the THC derivative (I) according to the present invention and the THC derivative (II) prepared by Lai et al. 11If was dissolved in 350 ml of PB adjusted to pH 7.0, and each of these solutions was gently stirred at room temperature (20'C) and left for 5 days. After that, the amount of THC derivative in the solution was quantified, and it was confirmed that I was much more stable than ①.
タンパク Ｉ の’ｒｕｅの 量ＴＨＣ誘導体が
結合するときのタンパク質１分子当りのＳＨ基量の変化
を測定することにより、ＴｌＩＣ量を測定した。ＳＲ基
の変化量は４〜ピリジルヂサルフアイド（４ＰＤＳ）試
薬を用いた吸光度より求めた。Amount of 'rue of protein I The amount of TlIC was measured by measuring the change in the amount of SH groups per protein molecule when the THC derivative was bound. The amount of change in the SR group was determined from absorbance using a 4-pyridyl disulfide (4PDS) reagent.
４ＰＤＳはピリジンニ分子がジスルフィド結合しており
、ＳＨ基と一方のピリジンは結合し、もう一方は４−チ
オピリジン（４−ＴＰ）となる。この４ＴＰの３２４ｎ
ｍの吸光度変化を測定してＴＨＣの結合数を測定した。In 4PDS, pyridine molecules are disulfide bonded, and one pyridine is bonded to the SH group, and the other becomes 4-thiopyridine (4-TP). 324n of this 4TP
The number of THC bonds was determined by measuring the change in absorbance of m.
その結果ＣＧＣＩ分子当りＴＨＣが９分子結合できてい
ることがわかった。As a result, it was found that 9 molecules of THC could be bound per CGCI molecule.
発明の効果
本発明によれば、加水分解の影響なく安定で、タンパク
質と結合して、容易に定量が行えるＴＨＣ誘導体が得ら
れ、さらにこれをタンパク質と結合することにより、抗
Ｔ）ＩＣ抗体の作製が可能なＴｌＩＣ−タンパク質フン
シュゲートが提供される。Effects of the Invention According to the present invention, a THC derivative is obtained that is stable without the influence of hydrolysis, binds to proteins, and can be easily quantified. Provided are TlIC-protein funshugates that can be produced.
Claims (2)
誘導体。 ▲数式、化学式、表等があります▼(1) A tetrahydrocannabinol derivative having the following structure. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼
導体とタンパク質とを結合したテトラヒドロカンナビノ
ール−タンパク質コンジュゲート。(2) A tetrahydrocannabinol-protein conjugate comprising the tetrahydrocannabinol derivative according to claim 1 and a protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25021088A JP2600850B2 (en) | 1988-10-04 | 1988-10-04 | Tetrahydrocannabinol derivative |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25021088A JP2600850B2 (en) | 1988-10-04 | 1988-10-04 | Tetrahydrocannabinol derivative |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02101072A true JPH02101072A (en) | 1990-04-12 |
| JP2600850B2 JP2600850B2 (en) | 1997-04-16 |
Family
ID=17204459
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP25021088A Expired - Fee Related JP2600850B2 (en) | 1988-10-04 | 1988-10-04 | Tetrahydrocannabinol derivative |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2600850B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5817766A (en) * | 1995-04-05 | 1998-10-06 | Roche Diagnostic Systems, Inc. | Reagents for a cannabinoid immunoassay |
| WO2006029089A3 (en) * | 2004-09-03 | 2007-05-24 | Oakville Hong Kong Company Ltd | Tetrahydrocannabinoid- protein conjugates for the production of antibodies for the detection of tεtrahydrocannabinoid components in saliva |
-
1988
- 1988-10-04 JP JP25021088A patent/JP2600850B2/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5817766A (en) * | 1995-04-05 | 1998-10-06 | Roche Diagnostic Systems, Inc. | Reagents for a cannabinoid immunoassay |
| WO2006029089A3 (en) * | 2004-09-03 | 2007-05-24 | Oakville Hong Kong Company Ltd | Tetrahydrocannabinoid- protein conjugates for the production of antibodies for the detection of tεtrahydrocannabinoid components in saliva |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2600850B2 (en) | 1997-04-16 |
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