JPS63243101A - Cyclodextrin derivative and coloring - Google Patents
Cyclodextrin derivative and coloringInfo
- Publication number
- JPS63243101A JPS63243101A JP7455087A JP7455087A JPS63243101A JP S63243101 A JPS63243101 A JP S63243101A JP 7455087 A JP7455087 A JP 7455087A JP 7455087 A JP7455087 A JP 7455087A JP S63243101 A JPS63243101 A JP S63243101A
- Authority
- JP
- Japan
- Prior art keywords
- cyclodextrin
- derivative
- coloring
- nitrophenol
- basic functional
- 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
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical class O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 title claims abstract description 18
- 238000004040 coloring Methods 0.000 title claims abstract description 17
- 125000000524 functional group Chemical group 0.000 claims abstract description 14
- 125000000217 alkyl group Chemical group 0.000 claims abstract 2
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical class OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 claims 1
- 239000001257 hydrogen Substances 0.000 claims 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 32
- 239000000758 substrate Substances 0.000 abstract description 21
- 229920000858 Cyclodextrin Polymers 0.000 abstract description 20
- 102000004190 Enzymes Human genes 0.000 abstract description 15
- 108090000790 Enzymes Proteins 0.000 abstract description 15
- 230000007935 neutral effect Effects 0.000 abstract description 11
- 230000002378 acidificating effect Effects 0.000 abstract description 9
- 239000001116 FEMA 4028 Substances 0.000 abstract description 7
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 abstract description 7
- 229960004853 betadex Drugs 0.000 abstract description 7
- RBXVOQPAMPBADW-UHFFFAOYSA-N nitrous acid;phenol Chemical class ON=O.OC1=CC=CC=C1 RBXVOQPAMPBADW-UHFFFAOYSA-N 0.000 abstract description 7
- 229920001450 Alpha-Cyclodextrin Polymers 0.000 abstract description 6
- HFHDHCJBZVLPGP-RWMJIURBSA-N alpha-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO HFHDHCJBZVLPGP-RWMJIURBSA-N 0.000 abstract description 6
- 229940043377 alpha-cyclodextrin Drugs 0.000 abstract description 6
- 235000011175 beta-cyclodextrine Nutrition 0.000 abstract description 6
- 238000012360 testing method Methods 0.000 abstract description 5
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 abstract description 2
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 abstract description 2
- MZVQCMJNVPIDEA-UHFFFAOYSA-N [CH2]CN(CC)CC Chemical group [CH2]CN(CC)CC MZVQCMJNVPIDEA-UHFFFAOYSA-N 0.000 abstract description 2
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 abstract 1
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 abstract 1
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 20
- 238000002835 absorbance Methods 0.000 description 20
- 229940088598 enzyme Drugs 0.000 description 14
- 238000010521 absorption reaction Methods 0.000 description 11
- 238000000862 absorption spectrum Methods 0.000 description 10
- 238000006911 enzymatic reaction Methods 0.000 description 9
- 238000005259 measurement Methods 0.000 description 8
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 8
- 238000011161 development Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- -1 diethylaminoethyl group Chemical group 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 229940097362 cyclodextrins Drugs 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000008351 acetate buffer Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 238000000691 measurement method Methods 0.000 description 3
- 125000006501 nitrophenyl group Chemical group 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- XVMIKRZPDSXBTP-UHFFFAOYSA-N 1,3-dibromobutan-2-one Chemical compound CC(Br)C(=O)CBr XVMIKRZPDSXBTP-UHFFFAOYSA-N 0.000 description 2
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 2
- 108010055851 Acetylglucosaminidase Proteins 0.000 description 2
- 102000013563 Acid Phosphatase Human genes 0.000 description 2
- 108010051457 Acid Phosphatase Proteins 0.000 description 2
- 102100030122 Protein O-GlcNAcase Human genes 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 102000004139 alpha-Amylases Human genes 0.000 description 2
- 108090000637 alpha-Amylases Proteins 0.000 description 2
- 229940024171 alpha-amylase Drugs 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000003028 enzyme activity measurement method Methods 0.000 description 2
- 150000004702 methyl esters Chemical class 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- KPJLHDPHGOCMDS-FCQTXGLASA-N (2r,3r,4s,5s,6r)-2-[(2r,3s,4r,5r,6r)-6-[(2r,3s,4r,5r,6r)-6-[(2r,3s,4r,5r,6r)-6-[(2r,3s,4r,5r,6s)-6-(2-chloro-4-nitrophenoxy)-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@H](O[C@H](O[C@@H]3[C@H](O[C@H](O[C@@H]4[C@H](O[C@@H](OC=5C(=CC(=CC=5)[N+]([O-])=O)Cl)[C@H](O)[C@H]4O)CO)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O KPJLHDPHGOCMDS-FCQTXGLASA-N 0.000 description 1
- LXJXRIRHZLFYRP-VKHMYHEASA-L (R)-2-Hydroxy-3-(phosphonooxy)-propanal Natural products O=C[C@H](O)COP([O-])([O-])=O LXJXRIRHZLFYRP-VKHMYHEASA-L 0.000 description 1
- UFBJCMHMOXMLKC-UHFFFAOYSA-N 2,4-dinitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O UFBJCMHMOXMLKC-UHFFFAOYSA-N 0.000 description 1
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 description 1
- RAGSWDIQBBZLLL-UHFFFAOYSA-N 2-chloroethyl(diethyl)azanium;chloride Chemical compound Cl.CCN(CC)CCCl RAGSWDIQBBZLLL-UHFFFAOYSA-N 0.000 description 1
- BOFRXDMCQRTGII-UHFFFAOYSA-N 619-08-9 Chemical compound OC1=CC=C([N+]([O-])=O)C=C1Cl BOFRXDMCQRTGII-UHFFFAOYSA-N 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- LXJXRIRHZLFYRP-VKHMYHEASA-N D-glyceraldehyde 3-phosphate Chemical compound O=C[C@H](O)COP(O)(O)=O LXJXRIRHZLFYRP-VKHMYHEASA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 102000014384 Type C Phospholipases Human genes 0.000 description 1
- 108010079194 Type C Phospholipases Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- MHSVUSZEHNVFKW-UHFFFAOYSA-N bis-4-nitrophenyl phosphate Chemical compound C=1C=C([N+]([O-])=O)C=CC=1OP(=O)(O)OC1=CC=C([N+]([O-])=O)C=C1 MHSVUSZEHNVFKW-UHFFFAOYSA-N 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- KPDLTBYCZXVKQN-UHFFFAOYSA-N ethyl 2-hydroxy-5-nitrobenzoate Chemical compound CCOC(=O)C1=CC([N+]([O-])=O)=CC=C1O KPDLTBYCZXVKQN-UHFFFAOYSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- UJYAZVSPFMJCLW-UHFFFAOYSA-N n-(oxomethylidene)benzenesulfonamide Chemical compound O=C=NS(=O)(=O)C1=CC=CC=C1 UJYAZVSPFMJCLW-UHFFFAOYSA-N 0.000 description 1
- UBBCYDPSFMEFFL-DHGKCCLASA-N n-[(2s,3r,4r,5s,6r)-2-(2-chloro-4-nitrophenoxy)-4,5-dihydroxy-6-(hydroxymethyl)oxan-3-yl]acetamide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C([N+]([O-])=O)C=C1Cl UBBCYDPSFMEFFL-DHGKCCLASA-N 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
Description
【発明の詳細な説明】
[産業上の利用分野]
本発明は塩基性官能基で修飾されたシクロデキストリン
に関するもので、合r&基質を用いた測定法等に応用さ
れ、臨床化学検査等の分野において効果的に用いられる
。更に本発明はニトロフェノール類を中性ないし弱酸性
条件下で発色させるための試薬及び方法に関するもので
ある。[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to cyclodextrin modified with a basic functional group, and is applied to measurement methods using a combined r&substrate, and is applicable to fields such as clinical chemistry tests. It is effectively used in Furthermore, the present invention relates to a reagent and method for coloring nitrophenols under neutral to weakly acidic conditions.
[従来の技術]
ニトロフェノール類の1種である4−ニトロフェノール
(パラニトロフェノールともいう)は4−ニトロフェノ
ール基をもつ各種合成基質が特定の酵素作用により分解
されるときに生成する物質で、この物質の増加を経時的
に定量することにより当該酵素の活性を容易に測定する
ことができる0例えば、フォスファターゼの活性を測定
するためには4−ニトロフェノールリン酸を基質として
遊離して生じる4−二トロフェノールを定量する。フォ
スフオシエステラーゼの活性測定にはビス(4−ニトロ
フェニル)リン酸を基質として同様に測定する。β−N
−7セチルヘキソサミニダーゼとβ−N−7セチルグル
コサミニダーゼの活性測定では1合成基質として4−ニ
トロフェニルN−7セチルーβ−D−グルコサミニドが
利用される。その他、各種のグリコシダーゼ(グリコシ
ド結合の加水分解を触媒する酵素)、ホスホリパーゼC
、トリプシンやキモトリプシンなどのセリンプロテアー
ゼ、D−グリセルアルデヒド−3−リン酸デヒドロゲナ
ーゼなどに対し、4−ニトロフェニル基をもつ合成基質
が開発され、臨床検査をはじめとするさまざまな分野で
実用に供されている。[Prior art] 4-nitrophenol (also called para-nitrophenol), a type of nitrophenol, is a substance produced when various synthetic substrates containing a 4-nitrophenol group are decomposed by the action of specific enzymes. For example, in order to measure the activity of phosphatase, the activity of the enzyme can be easily measured by quantifying the increase in this substance over time. Determine 4-ditrophenol. The activity of phosphosiesterase is measured in the same manner using bis(4-nitrophenyl) phosphate as a substrate. β-N
In the activity measurement of -7 cetylhexosaminidase and β-N-7 cetylglucosaminidase, 4-nitrophenyl N-7 cetyl-β-D-glucosaminid is used as one synthetic substrate. In addition, various glycosidases (enzymes that catalyze the hydrolysis of glycosidic bonds), phospholipase C
Synthetic substrates with 4-nitrophenyl groups have been developed for serine proteases such as trypsin and chymotrypsin, and D-glyceraldehyde-3-phosphate dehydrogenase, and have been put to practical use in various fields including clinical testing. has been done.
[発明が解決しようとする問題点1
以上の例で示した各種酵素の活性測定が可能となる原理
は、例えば4−ニトロフェニル基をもつ合成基質の場合
は、その合成基質と遊離して生じる4−ニトロフェノー
ルの間で吸光スペクトルに差があるという事実に基礎を
おいている。すなわち、4−ニトロフェニル基をもつ合
成基質は広いpH領域において紫外部(波長300〜3
20 nm)に強い吸光ピークをもち可視部(波長40
On層以上)にはほとんど吸光を示さないが、4−ニト
ロフェノールはアルカリ性pH領域において電離し、4
0Ons付近を中心とする強い吸光ピークをもっ4−ニ
トロフェルレート・アニオンヲ生シル、シたがって、測
定しようとする酵素の活性をアルカリ性pi 領域で
測定できれば、酵素反応の進行にともなう可視部の発色
を400nmの吸光度増加として連続モニターでき、正
確なレートアッセイが可能となる。しかし、測定しよう
とする酵素の最適pHが中性ないし顕著には弱酸性の領
域にあれば、4−ニトロフェノールの吸光スペクトルは
4−ニトロフェニル基をもつ合成基質の吸光スペクトル
とほとんどオーバーラツプしてしまい、発色が微弱で、
反応を経時的に連続モニターすることが著しく不都合と
なる。そこで、現行の測定法では、酵素反応をスタート
させてから一定時間後に、アルカリを添加して反応を停
止させると同時に生成した4−ニトロフェノールを発色
させ、その400n曽付近の吸光度を測定するという方
法が多く採用されている。すなわち、現行測定法では酵
素活性測定法としてより正確で簡便な形式といわれる理
想的なレートアッセイが困難なだけでなく、アルカリ添
加という余分な操作を必要とする。[Problem to be Solved by the Invention 1] The principle that makes it possible to measure the activity of various enzymes as shown in the examples above is that, for example, in the case of a synthetic substrate with a 4-nitrophenyl group, It is based on the fact that there are differences in the absorption spectra among 4-nitrophenols. In other words, a synthetic substrate with a 4-nitrophenyl group can be used in the ultraviolet region (wavelength 300 to 300 nm) in a wide pH range.
It has a strong absorption peak in the visible region (wavelength 40 nm).
4-nitrophenol is ionized in the alkaline pH region, and 4-nitrophenol shows almost no absorption in
The 4-nitropherulate anion has a strong absorption peak centered around 0 Ons. Therefore, if the activity of the enzyme to be measured can be measured in the alkaline pi region, the color development in the visible region as the enzymatic reaction progresses. can be continuously monitored as an increase in absorbance at 400 nm, allowing accurate rate assays. However, if the optimum pH of the enzyme to be measured is in the neutral or markedly slightly acidic region, the absorption spectrum of 4-nitrophenol will almost overlap with the absorption spectrum of the synthetic substrate with 4-nitrophenyl group. The color is weak and the color is weak.
Continuous monitoring of the reaction over time becomes extremely inconvenient. Therefore, in the current measurement method, after a certain period of time after starting the enzyme reaction, alkali is added to stop the reaction, and at the same time, the produced 4-nitrophenol is colored, and its absorbance at around 400 nm is measured. Many methods have been adopted. That is, with the current measurement method, not only is it difficult to perform an ideal rate assay, which is said to be a more accurate and simple format for enzyme activity measurement, but it also requires the extra operation of adding alkali.
中性ないし弱酸性の領域に最適pHをもつ酵素には、例
えば酸性フォスファターゼ、β−N−7セチルグルコサ
ミニダーゼなど臨床化学的に重要な酵素が含まれている
。これら諸酵素の反応を経時的に連続モニターするため
の有用な手段を得ることができれば、測定操作を簡素化
し、測定時間を短縮できるだけでなく、測定精度をも高
め、各種疾患の早期発見や診断に正確な情報を提供でき
る。Enzymes that have an optimum pH in the neutral to slightly acidic range include, for example, enzymes that are important in clinical chemistry, such as acid phosphatase and β-N-7 cetylglucosaminidase. If we could obtain a useful means to continuously monitor the reactions of these enzymes over time, we would not only be able to simplify the measurement operation and shorten the measurement time, but also improve measurement accuracy and enable early detection and diagnosis of various diseases. can provide accurate information.
従って、4−ニトロフェノール及びその他のニトロフェ
ノール類の中性ないし弱酸性のPH領域での効果的な発
色促進作用を有す試薬の開発は、臨床化学検査分野にお
ける実用面に重要な課題となっている。Therefore, the development of reagents that effectively promote color development in the neutral to weakly acidic pH range of 4-nitrophenol and other nitrophenols has become an important practical issue in the field of clinical chemistry testing. ing.
[問題点を解決するための手段]
本発明は、塩基性官能基を共有結合させたシクロデキス
トリン誘導体である。[Means for Solving the Problems] The present invention is a cyclodextrin derivative to which a basic functional group is covalently bonded.
更に本発明は、塩基性官能基を共有結合させたシクロデ
キストリン誘導体を用いて、ニトロフェノール誘導体を
包接するすることを特徴とする発色方法である。Furthermore, the present invention is a coloring method characterized by including a nitrophenol derivative using a cyclodextrin derivative to which a basic functional group is covalently bonded.
以下1本発明の詳細な説明する。The present invention will be explained in detail below.
4−ニトロフェノールをはじめとする芳香族化合物は、
疎水的性質をもつフェニル基がシクロデキストリンに取
り込まれて包接化合物を形成することが知られている。Aromatic compounds including 4-nitrophenol are
It is known that phenyl groups with hydrophobic properties are incorporated into cyclodextrins to form clathrates.
これにより芳香環のイオン状態に微妙な変化が起きれば
電離の促進が期待される。そこで、4−ニトロフェノー
ル等にα−シクロデキストリンまたはβ−シクロデキス
トリンを添加すると電離が進行し、400n−付近の吸
光度の増大がみられるが、その効果は中性から特に弱酸
性領域ではなお満足できるものではなくそれ自体の溶解
性の低さも合わせ、実用化には多くの問題を含んでいる
。If this causes a subtle change in the ionic state of the aromatic ring, it is expected that ionization will be accelerated. Therefore, when α-cyclodextrin or β-cyclodextrin is added to 4-nitrophenol etc., ionization progresses and an increase in absorbance around 400n- is observed, but the effect is still satisfactory in the neutral to weakly acidic region. There are many problems in putting it into practical use, including the fact that it is not possible to make it, and its solubility is low.
本発明者等は、鋭意検討の結果、シクロデキストリノに
塩基性官能基を共有結合させることにより、フェノール
性水酸基の電離の促進が起こらないかどうか、またそれ
により4−ニトロフェノール類の発色促進が起こらない
かどうかを試みてそこで塩基性基を融合させたシクロデ
キストリン誘導体を合成し、中性ないし微酸性pH領域
で、4−ニトロフェノールおよびその他のニトロフェ/
−ル類の吸光スペクトルに対する効果を調べたところ
、顕著な発色効果をもつことが判明し本発明を達成した
。As a result of intensive studies, the present inventors have determined whether or not covalently bonding a basic functional group to cyclodextrino would promote the ionization of phenolic hydroxyl groups, and whether this would promote color development of 4-nitrophenols. We synthesized a cyclodextrin derivative with a basic group fused to it to determine whether 4-nitrophenol and other nitrophenols would occur in the neutral to slightly acidic pH range.
When the effect of the compounds on the absorption spectrum was investigated, it was found that they had a remarkable coloring effect, and the present invention was achieved.
本発明を更に詳述すると本発明のために、塩基性官能基
としてまずジエチルアミノエチル基を選び、下記の原理
により、そのシクロデキストリン結合体を合成した。To describe the present invention in more detail, for the purpose of the present invention, a diethylaminoethyl group was first selected as a basic functional group, and a cyclodextrin conjugate thereof was synthesized according to the following principle.
同様にしてα−シクロデキストリンのジエチルアミノエ
チル結合体が得られる。 またジエチルアミノエチル基
以外にジメチルアミノエチル、ジメチルアミノプロピル
、アミノエチル基等の結合体も得ることができる。 こ
のように塩基性官能基で体筒されたシクロデキストリン
、例えばジエチルアミノエチル化α−シクロデキストリ
ン(以下DEαと略す、)およびジエチルアミノエチル
化β−シクロデキストリン(以下DEβと略す、)は意
外にも1通常のシクロデキストリンに比らべはるかに水
によく溶は実用に供し易いものであった。現在、特に安
価で一般に用いられているβ−シクロデキストリンは比
較的に水に難溶性でこの性質が臨床化学検査等における
実用上の範囲を決めているものである。A diethylaminoethyl conjugate of α-cyclodextrin is obtained in the same manner. In addition to diethylaminoethyl groups, conjugates of dimethylaminoethyl, dimethylaminopropyl, aminoethyl groups, etc. can also be obtained. Cyclodextrins encapsulated with basic functional groups, such as diethylaminoethylated α-cyclodextrin (hereinafter abbreviated as DEα) and diethylaminoethylated β-cyclodextrin (hereinafter abbreviated as DEβ), surprisingly have 1 Compared to ordinary cyclodextrin, it was much more soluble in water and easier to put into practical use. β-cyclodextrin, which is currently particularly inexpensive and commonly used, is relatively sparingly soluble in water, and this property determines its practical range in clinical chemistry tests and the like.
すなわち、難溶性の目的物質をβ−シクロデキストリン
の包接により溶解せしめる時シクロデキストリン自身の
溶解性によりその溶解度が限定されてしまうのである。That is, when a poorly soluble target substance is dissolved by inclusion of β-cyclodextrin, its solubility is limited by the solubility of the cyclodextrin itself.
具体的には特定酵素の活性を測定するためによく使用さ
れる合成基質の溶解の場合などである。加水分解酵素の
場合は多く難溶性のニトロフェノール誘導体が発色剤と
して酵素反応の経過観察のために結合されている0例え
ば前記のN−アセチル−β−D−グルコサミニダーゼ測
定のための合成基質である、4−ニトロフェニル(アル
いは2−クロロ−4−ニトロフェニル)−N−アセチル
−β−D−グルコサミニドの場合、β−シクロデキスト
リンの助けで必要量の溶解が試みられるが、シクロデキ
ストリン自身の溶解性の悪さのためその目的を充分に果
たし得ない、それに比し、本発明によるDEα、DEβ
等はたやすく水に溶解させることができ、その包括能に
より上記基質等を充分に必要量溶かし得るのでこの面に
おいても本発明に実用的価値を付加するものである。Specifically, this is the case when dissolving synthetic substrates that are often used to measure the activity of specific enzymes. In the case of hydrolytic enzymes, a poorly soluble nitrophenol derivative is often bound as a coloring agent to monitor the progress of enzyme reactions. , 4-nitrophenyl (al or 2-chloro-4-nitrophenyl)-N-acetyl-β-D-glucosaminide, attempts are made to dissolve the necessary amount with the help of β-cyclodextrin, but the cyclodextrin itself In comparison, DEα and DEβ according to the present invention cannot fully achieve their purpose due to poor solubility.
etc. can be easily dissolved in water, and their entrapping ability allows them to dissolve the necessary amount of the above-mentioned substrates, etc., so this aspect also adds practical value to the present invention.
更に本発明の本質である中性ないしは弱酸性条件下での
ニトロフェノール誘導体に対する発色効果を種々の誘導
体を用いて調べた。4−二トロフェノール、2−クロロ
−4−二トロフェノール、5−ニトロサリチル酸、5−
ニトロサルチル酸メチル、2.4−ジニトロフェノール
などについてである0M〈べきことにこれらの中で最も
中性ないしは特に弱酸性下で電離が微弱な、すなわちp
H5,0付近においては、400nm付近での電離性の
可視部発色ピークが認められない4−ニトロフェノール
でさえ、DEαあるいはDEβの1%以下の通常濃度の
存在下でその400nm付近に大きな新しい電離性の発
色ピークを出現させ、DEα、DEβの作用とその効果
が顕著であることが示された0通常のシクロデキストリ
ンではこれ程の効果は認められない。またDEα、DE
βでは水への溶解性がよいため使用量を増やして効果的
な増強できる。また他のニトロフェノールについても1
%以下の通常濃度で、数倍に及ぶ可視部発色効果が示さ
れた。Furthermore, the coloring effect of nitrophenol derivatives under neutral or weakly acidic conditions, which is the essence of the present invention, was investigated using various derivatives. 4-ditrophenol, 2-chloro-4-ditrophenol, 5-nitrosalicylic acid, 5-
0M for methyl nitrosalcylate, 2,4-dinitrophenol, etc. (Of these, 0M should be the most neutral or especially weakly ionized under weak acidity, i.e. p
In the vicinity of H5,0, even 4-nitrophenol, which does not show an ionizing visible color peak near 400 nm, has a large new ionization peak near 400 nm in the presence of a normal concentration of DEα or DEβ of 1% or less. It was shown that the action and effect of DEα and DEβ are remarkable.No ordinary cyclodextrin has such an effect. Also DEα, DE
Since β has good solubility in water, it can be effectively enhanced by increasing the amount used. Regarding other nitrophenols, 1
% or less, the visible region coloring effect was several times greater.
特に2−クロロ−4−二トロフェノールは中性ないし弱
酸性下で4−二トロフェノールに比べ、比較的発色がな
され易いものとして、最近それを結合させた特定酵素の
ための合成基質が供されているが、例えばpH5,0以
下で酵素反応を行う前記のN−アセチル−β−D−グル
コサミニダーゼ等については、酵素反応の結果そのpH
で生成する2−クロロ−4−二トロフェノールの400
n層付近における電離発色は、なお、全く不充分で、そ
の生成量を連続モニターできるほどの感度と精度をもた
ない、しかるに、ここへのDEα、あるいはDEβの添
加は生成する2−クロロ−4−二トロフェノールの吸収
スペクトルをほぼ完全電離に近いかたちに出現せしめ発
色させるので、はぼ理想的な状態での当酵素の連続モニ
ター測定が行える。In particular, 2-chloro-4-ditrophenol is relatively more likely to develop color than 4-ditrophenol under neutral to weakly acidic conditions, and synthetic substrates for specific enzymes to which it is bound have recently been provided. However, for example, for the above-mentioned N-acetyl-β-D-glucosaminidase, etc., which performs an enzymatic reaction at a pH of 5.0 or less, as a result of the enzymatic reaction, the pH
400 of 2-chloro-4-ditrophenol produced in
The ionization color development in the vicinity of the n-layer is still completely insufficient, and does not have the sensitivity and precision to continuously monitor the amount of produced 2-chloro- Since the absorption spectrum of 4-ditrophenol appears in a nearly completely ionized form and is colored, continuous monitoring and measurement of the enzyme can be performed under ideal conditions.
前記のとおり、DEα、DEβないしは充分水に溶は易
く、当合酸基質の必要量を反応液に溶解できることと合
わせ実用面に著しい効果を供するものである。As mentioned above, DEα, DEβ or DEβ is easily soluble in water, and together with the fact that the required amount of the synthetic acid substrate can be dissolved in the reaction solution, it provides a significant practical effect.
以上の効果は、同様に、中性付近で反応を行うα−アミ
ラーゼのための合成基質である、4−二トロフェニルー
マルトへブタオシド、あるいは2−クロロ−4−二トロ
フェニルーマルトペンタオシド等を用いて生成するニト
ロフェニル誘導体を400n■付近で連続モニターする
α−アミラーゼの測定の場合、あるいは、よりpHの低
い領域で酵素反応を行わせる酸性フォスファターゼ測定
のためのニトロフェノール誘導体を結合させた合成基質
を使用する場合などにおいても、通常のシクロデキスト
リンに比し、はるかに効果的に使用される。The above effects are similar to that of 4-ditrophenylmaltopentaoside or 2-chloro-4-nitrophenylmaltopentaoside, which is a synthetic substrate for α-amylase that reacts near neutrality. For α-amylase measurement, which continuously monitors nitrophenyl derivatives produced by using nitrophenyl derivatives at around 400 nm, or for acid phosphatase measurement, where the enzymatic reaction is carried out in a lower pH region. Even when synthetic substrates are used, they are used much more effectively than ordinary cyclodextrins.
以上DEα、DEβについて本発明の効果を述べたが他
の塩基性官能基を修飾したシクロデキストリン及び他の
酵素反応のためニトロフェノール誘導体を結合した他の
合成基質を使用する場合においても本発明の本質は、同
様である。The effects of the present invention have been described above for DEα and DEβ, but the present invention also applies when using cyclodextrins modified with other basic functional groups and other synthetic substrates bound with nitrophenol derivatives for other enzymatic reactions. The essence is the same.
[実施例] 以下、実施例により本発明を更に具体的に説明する。[Example] Hereinafter, the present invention will be explained in more detail with reference to Examples.
実施例1
(発色試薬の合成法)
1.2−(N、N−ジアルキルアミノエチル)−α−シ
クロデキストリン(以下、DEαと略記):α−シクロ
デキストリン2.4gを4 tanの蒸留水に懸濁させ
、攪拌しながら水酸化ナトリウム1.5 gを含む4
tanの水溶液を滴下していくと透明な液、が得られる
。さらに攪拌を続けながら、ジエチルアミノエチルクロ
リド塩酸塩1.4gを含む2 mlの水溶液を滴下し、
30〜40℃で数時間攪拌を続けると反応は完全に終結
する。この反応混合液より、セファデクスG−25カラ
ムクロマトグラフィーにより低分子物質を除去すること
によりi、e gのDEαが得られる。得られたDBα
はジエチルアミノエチル基をl(Iないし2個共有結合
していることが中和滴定およびプロトン核磁気共鳴より
判明した。Example 1 (Synthesis method of coloring reagent) 1.2-(N,N-dialkylaminoethyl)-α-cyclodextrin (hereinafter abbreviated as DEα): 2.4 g of α-cyclodextrin was added to 4 tan of distilled water. 4 containing 1.5 g of sodium hydroxide while suspending and stirring.
When the tan aqueous solution is added dropwise, a clear liquid is obtained. While continuing to stir, 2 ml of an aqueous solution containing 1.4 g of diethylaminoethyl chloride hydrochloride was added dropwise.
The reaction is completely terminated by continuing stirring at 30-40°C for several hours. From this reaction mixture, low molecular weight substances are removed by Sephadex G-25 column chromatography to obtain DEα of i and e g. Obtained DBα
It was found by neutralization titration and proton nuclear magnetic resonance that 1 to 2 diethylaminoethyl groups were covalently bonded.
2 、 ’2− (N、N−ジアルキルアミノエチル)
−β−シクロデキストリン(以下、DEβと略記)二上
記合tlにおいてα−シクロデキストリンのかわりにβ
−シクロデキストリンを用いることにより、はぼ回収率
でDEβを合成することができる。2, '2-(N,N-dialkylaminoethyl)
-β-cyclodextrin (hereinafter abbreviated as DEβ) β-cyclodextrin instead of α-cyclodextrin in the above combination tl
- By using cyclodextrin, DEβ can be synthesized with a low recovery rate.
実施例2
DEαの4−二トロフェノールに対する可視部発色効果
と酵素反応への応用。Example 2 Visible coloring effect of DEα on 4-nitrophenol and its application to enzymatic reactions.
第1図はp)15.0における4−二トロフェノールの
吸光スペクトルと、発色試薬としてDEαを濃度1%に
添加した場合の吸光スペクトルを比較したものである。FIG. 1 is a comparison of the absorption spectrum of 4-nitrophenol at p) 15.0 and the absorption spectrum when DEα was added to a concentration of 1% as a coloring reagent.
この図より明らかなように4−二トロフェノールは40
0n腸付近においてほとんど吸光度をもたないが、DE
αを含む溶液中で顕著な発色促進効果が示され400n
mにおける吸光度は、4−ニトロフェノールの紫外部吸
光度の30%以上に達し、十分に測定可能となる。一方
、4−ニトロフェニール合成基質の1つである4−ニト
ロフェノールN−7セチルーβ−D−グルコサミニドの
吸光スペクトルは、pH5,0における4−二トロフェ
ニールのスペクトルよりわずかに低波長にシフトしてい
る以外はほとんど同じで、これにDEαを濃度1%に添
加しても、吸光ピークの微少な長波長シフトが観察され
るだけで400nm付近における吸光度は全く影響を受
けない、したがって、DEαを含むこの反応液中で4−
ニトロフェニールN−アセチル−β−D−グルコサミニ
ドを基質として、N−7セチルーβ−D−グルコサミニ
ダーゼを作用させ生成する4−ニトロフェノールを41
0nmの吸光度変化量として連続モニターすることによ
り当酵素活性を測定することができる。なお、DEα共
存下で可視部吸光度の極大を与える波長は410nmで
ある。また、使用する分光光度計が36onI11にお
ける吸光度を測定する性能をも有していれば、同波長に
おける吸光度変化をモニターすることによりpH5,0
における測定感度を更に、約25%増大させることも可
能である。DEaの4−二トロフェノールに対する可視
部吸光度増大効果はp)14 、0においてもいくらか
観察され、中性ないし微アルカリ性までの広いpH領域
においてその効果を保持している。As is clear from this figure, 4-nitrophenol is 40
0n It has almost no absorbance near the intestine, but DE
A remarkable color development promotion effect was shown in a solution containing α, and 400n
The absorbance at m reaches 30% or more of the ultraviolet absorbance of 4-nitrophenol, making it fully measurable. On the other hand, the absorption spectrum of 4-nitrophenol N-7 cetyl-β-D-glucosaminide, which is one of the 4-nitrophenyl synthetic substrates, is slightly shifted to a lower wavelength than the spectrum of 4-nitrophenyl at pH 5.0. Even if DEα is added to this at a concentration of 1%, only a slight shift in the longer wavelength of the absorption peak is observed, and the absorbance near 400 nm is not affected at all. In this reaction solution containing 4-
4-Nitrophenol is produced by the action of N-7 cetyl-β-D-glucosaminidase using nitrophenyl N-acetyl-β-D-glucosaminid as a substrate.
The enzyme activity can be measured by continuously monitoring the amount of change in absorbance at 0 nm. Note that the wavelength that gives the maximum absorbance in the visible region in the coexistence of DEα is 410 nm. In addition, if the spectrophotometer used has the ability to measure the absorbance at 36 on I11, it is possible to measure the absorbance at pH 5,0 by monitoring the absorbance change at the same wavelength.
It is also possible to increase the measurement sensitivity by approximately 25%. The visible absorbance increasing effect of DEa on 4-ditrophenol was also observed to some extent in p)14,0, and the effect was maintained in a wide pH range from neutral to slightly alkaline.
実施例3
DEaの4−二トロフェノールに対する可視部発色効果
実施例1におけるDEaのかわりにDEaを用いること
により、DEaの約60%の発色効果を得ることができ
る。Example 3 Visible coloring effect of DEa on 4-ditrophenol By using DEa in place of DEa in Example 1, a coloring effect of about 60% of that of DEa can be obtained.
実施例4
DEa、DEaの2−クロロ−4−二トロフェノールに
対する可視部発色効果と酵素反応への応用
2−クロロ−4−二トロフェノールは、PH5,0にお
いて10%以上も電離しているので、400nm付近に
も吸収を示す。0.I IL−酢酸緩衝液中それぞれ0
.8%以上の濃度でDEa、DEaを加えた時、2−ク
ロロ−4−二トロフェノールの吸収スペクトルはほぼ安
全電離に近いかたちを示し、400nmで(7)吸光度
はDEaで3,8倍、DEaで3.4倍であった。Example 4 Visible coloring effect of DEa, DEa on 2-chloro-4-ditrophenol and its application to enzyme reaction 2-chloro-4-ditrophenol is ionized by more than 10% at pH 5.0 Therefore, it also exhibits absorption near 400 nm. 0. I IL-0 each in acetate buffer
.. When DEa is added at a concentration of 8% or more, the absorption spectrum of 2-chloro-4-nitrophenol shows a shape almost similar to safe ionization, and the absorbance at 400 nm (7) is 3.8 times that of DEa. It was 3.4 times higher in DEa.
一方、合成基質のひとつである2−クロロ−4−ニトロ
フェニール−N−アセチル−β−D−グルコサミニドは
PH5,0において400r++s付近にほとんど吸収
を示さない、DEaあるいはDEaにおいても微妙な長
波長シフトが観察されるだけで400nm付近の処理に
はほとんど影響がない、従って、この基質溶液にN−ア
セチル−β−D−グルコサミニダーゼを作用させ、生産
する2−クロロ−4−二トロフェノールを410nmで
の吸光度の変化量として連続モニターすることができた
。On the other hand, 2-chloro-4-nitrophenyl-N-acetyl-β-D-glucosaminide, which is one of the synthetic substrates, shows almost no absorption around 400r++s at pH 5.0, and a subtle long wavelength shift in DEa or DEa. is observed, and there is almost no effect on the treatment at around 400 nm.Therefore, by treating this substrate solution with N-acetyl-β-D-glucosaminidase, the produced 2-chloro-4-ditrophenol can be detected at 410 nm. It was possible to continuously monitor the amount of change in absorbance.
実施例5
DEaおよびDEaの他のニトロフェノール類に対する
可視部発色効果
実施例2〜4の方法でPH5,0において、いろいろな
ニトロフェノール類に対し、DEa及びDEaが可視部
吸光度を増加させる効果をもつかどうかについて調べた
結果を示す、なお、用いたDEaおよびDEaの濃度は
0.8%とした。Example 5 Visible coloring effect of DEa and DEa on other nitrophens This shows the results of an investigation to see if it lasted. Note that DEa used and the concentration of DEa were 0.8%.
(i) 2.4−ジニトロフェノールはpH5,0で約
90%’iff殖しているから、そのままでも400n
■において強い吸光を示している。しかし、この吸光は
、紫外部にある強い吸光の肩を形成するにすぎない、こ
こにDEaを添加すると、40On騰に独立した吸光ピ
ークを形成し、その吸光度は35%増大した。(i) 2.4-dinitrophenol grows approximately 90% at pH 5.0, so even if it is as it is, 400n
3 shows strong light absorption. However, this absorption merely forms a shoulder of strong absorption in the ultraviolet region; when DEa is added thereto, an independent absorption peak is formed at 40 On, and its absorbance increases by 35%.
(ii) 5−ニトロサリチル酸はpH5,0テ40
0 nmの吸光度は紫外部吸光極大値の2.5%にすぎ
ない、これにDEaまたはDEaを添加すると、その吸
光度を3ないし4倍に増大させることができる。(ii) 5-nitrosalicylic acid has a pH of 5.040
The absorbance at 0 nm is only 2.5% of the ultraviolet absorption maximum, and when DEa or DEa is added to this, the absorbance can be increased 3 to 4 times.
(iii) 5−ニトロサリチル酸メチルのpH5,0
における400nmの吸光度は紫外部吸光極大値の3.
6%である。これにDEaを添加すると400nmを中
心に新しい吸光ピークが出現し、吸光度は8.4倍に増
大する、DEaはDEaより弱いが類似の効果をもつ。(iii) pH of methyl 5-nitrosalicylate 5.0
The absorbance at 400 nm is 3.
It is 6%. When DEa is added to this, a new absorption peak appears around 400 nm, and the absorbance increases 8.4 times. DEa is weaker than DEa, but has a similar effect.
(ii)5−ニトロサリチル酸エチルに対しても、DE
aおよびDEaはメチルエステルに対するのと類似の効
果を示す、しかし、その効果はメチルエステルの場合の
方が顕著である。(ii) Also for ethyl 5-nitrosalicylate, DE
a and DEa show similar effects as on methyl esters, but the effect is more pronounced in the case of methyl esters.
[発明の効果]
以上から明らかな如く、本発明によれば塩基性官能基を
結合したシクロデキストリンは、ニトロフェノール誘導
体の可視部における発色を著しく助長する効果を有し、
また、更には、それ自身通常のシクロデキストリンより
水に易溶のためニトロフェノール誘導体を結合した合成
基質を用いる各種の酵素活性測定法に応用する時、従来
にない実用面での有用性を持つ。[Effects of the Invention] As is clear from the above, according to the present invention, the cyclodextrin bonded with a basic functional group has the effect of significantly promoting color development in the visible region of the nitrophenol derivative,
Furthermore, since it is more soluble in water than ordinary cyclodextrin, it has unprecedented practical utility when applied to various enzyme activity measurement methods using synthetic substrates bound with nitrophenol derivatives. .
第1図は4−ニトロフェノールの吸光スペクトルのDE
αによる影響を示すグラフ図である。
イ・・・pH5,020m p−酢酸緩衝液中口・・・
DEα1%を含むpH5,020mg酢酸緩衝液中
坂+(nm)→Figure 1 shows the DE of the absorption spectrum of 4-nitrophenol.
It is a graph diagram showing the influence of α. A...pH 5,020m p-acetate buffer medium...
pH 5,020 mg acetate buffer containing 1% DEα Nakasaka+ (nm) →
Claims (4)
ン誘導体。(1) Cyclodextrin derivative to which a basic functional group is covalently bonded.
請求の範囲第1項記載の化合物。 ▲数式、化学式、表等があります▼ (式中、R_1、R_2はそれぞれ炭素数1〜3個の低
級アルキル基もしくは、水素を示し、nは1〜3の整数
を表わす。)(2) The compound according to claim 1, wherein the basic functional group is represented by the following formula. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R_1 and R_2 each represent a lower alkyl group having 1 to 3 carbon atoms or hydrogen, and n represents an integer of 1 to 3.)
ン誘導体を用いて、ニトロフェノール誘導体を包接する
することを特徴とする発色方法。(3) A coloring method characterized by including a nitrophenol derivative using a cyclodextrin derivative to which a basic functional group is covalently bonded.
記誘導体を共有させ発色を行わせるところの特許請求の
範囲第3項記載の方法。(4) The method according to claim 3, wherein the reaction system for producing the nitrophenol derivative is made to share the derivative to develop color.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP62074550A JPH0819162B2 (en) | 1987-03-30 | 1987-03-30 | Color development method using cyclodextrin derivative |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP62074550A JPH0819162B2 (en) | 1987-03-30 | 1987-03-30 | Color development method using cyclodextrin derivative |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS63243101A true JPS63243101A (en) | 1988-10-11 |
JPH0819162B2 JPH0819162B2 (en) | 1996-02-28 |
Family
ID=13550466
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP62074550A Expired - Fee Related JPH0819162B2 (en) | 1987-03-30 | 1987-03-30 | Color development method using cyclodextrin derivative |
Country Status (1)
Country | Link |
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JP (1) | JPH0819162B2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006104423A (en) * | 2004-10-08 | 2006-04-20 | National Institute Of Advanced Industrial & Technology | Modified cyclodextrin and nano-sized particles comprising modified cyclodextrin |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS60152502A (en) * | 1983-12-17 | 1985-08-10 | コンゾルテイウム・フユール・エレクトロケミツシエ・インヅストリー・ゲゼルシヤフト・ミツト・ベシユレンクテル・ハフツング | Water-soluble ether of beta-cyclodextrin and manufacture |
-
1987
- 1987-03-30 JP JP62074550A patent/JPH0819162B2/en not_active Expired - Fee Related
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS60152502A (en) * | 1983-12-17 | 1985-08-10 | コンゾルテイウム・フユール・エレクトロケミツシエ・インヅストリー・ゲゼルシヤフト・ミツト・ベシユレンクテル・ハフツング | Water-soluble ether of beta-cyclodextrin and manufacture |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006104423A (en) * | 2004-10-08 | 2006-04-20 | National Institute Of Advanced Industrial & Technology | Modified cyclodextrin and nano-sized particles comprising modified cyclodextrin |
JP4534012B2 (en) * | 2004-10-08 | 2010-09-01 | 独立行政法人産業技術総合研究所 | Polyethylene glycol-aminoalkylcarbamoylcyclodextrin copolymer, process for producing the same, and inclusion complex compound using the same |
Also Published As
Publication number | Publication date |
---|---|
JPH0819162B2 (en) | 1996-02-28 |
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