JP5073238B2 - Method for degrading sugar nucleotides - Google Patents
Method for degrading sugar nucleotides Download PDFInfo
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- JP5073238B2 JP5073238B2 JP2006205426A JP2006205426A JP5073238B2 JP 5073238 B2 JP5073238 B2 JP 5073238B2 JP 2006205426 A JP2006205426 A JP 2006205426A JP 2006205426 A JP2006205426 A JP 2006205426A JP 5073238 B2 JP5073238 B2 JP 5073238B2
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- 238000000034 method Methods 0.000 title claims description 22
- 230000000593 degrading effect Effects 0.000 title claims description 4
- 235000000346 sugar Nutrition 0.000 title description 75
- 239000002773 nucleotide Substances 0.000 title description 52
- 125000003729 nucleotide group Chemical group 0.000 title description 52
- 125000003047 N-acetyl group Chemical group 0.000 claims description 35
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- -1 UDP monosaccharide Chemical class 0.000 claims description 28
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- 238000000354 decomposition reaction Methods 0.000 claims description 21
- 229910001428 transition metal ion Inorganic materials 0.000 claims description 21
- 239000011572 manganese Substances 0.000 claims description 19
- 239000003795 chemical substances by application Substances 0.000 claims description 18
- 229910052723 transition metal Inorganic materials 0.000 claims description 15
- 150000003839 salts Chemical group 0.000 claims description 10
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- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 claims description 3
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- 102100039847 Globoside alpha-1,3-N-acetylgalactosaminyltransferase 1 Human genes 0.000 description 7
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- LFTYTUAZOPRMMI-NESSUJCYSA-N UDP-N-acetyl-alpha-D-galactosamine Chemical compound O1[C@H](CO)[C@H](O)[C@H](O)[C@@H](NC(=O)C)[C@H]1O[P@](O)(=O)O[P@](O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 LFTYTUAZOPRMMI-NESSUJCYSA-N 0.000 description 7
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- 238000004458 analytical method Methods 0.000 description 3
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- 238000010586 diagram Methods 0.000 description 3
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- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
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- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical group [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 2
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Description
本発明は、糖ヌクレオチドの分解方法及び分解剤に関する。 The present invention relates to a sugar nucleotide degradation method and a degradation agent.
まず、本出願書類において用いる略号を説明する。
ＡＤＰ：アデノシン５'−ジリン酸
ＣＤＰ：シチジン５'−ジリン酸
ｄＴＤＰ：デオキシチミジン５'−ジリン酸
ＥＤＴＡ：エチレンジアミン四酢酸
Ｇａｌ：ガラクトース
ＧａｌＮＡｃ：Ｎ−アセチルガラクトサミン
ＧＤＰ：グアノシン５'−ジリン酸
Ｇｌｃ：グルコース
ＧｌｃＮＡｃ：Ｎ−アセチルグルコサミン
ＧｌｃＵＡ：グルクロン酸
ＨＰＬＣ：高速液体クロマトグラフィー
ＵＤＰ：ウリジン５'−ジリン酸
ＵＭＰ：ウリジン５'−モノリン酸
細菌及び哺乳類等の細胞中にはＡＤＰ−ＧｌｃやＵＤＰ−Ｇｌｃ等の様な糖ヌクレオチドを加水分解する事のできる酵素があることが知られている。例えばＡＤＰ−Ｇｌｃホスホジエステラーゼとも呼ばれる酵素は、生体検体中の糖ヌクレオチドの分析による診断やトランスジェニック植物の製造等に応用されている（特許文献１）。ホスホジエステラーゼはリン酸ジエステルを加水分解してリン酸モノエステルとする酵素の総称である。しかしながら、上記ホスホジエステラーゼの様な酵素を含まず、Ｍｎ２＋を単独で糖ヌクレオチドと共存させることにより糖ヌクレオチドを選択的に分解する事は知られていない。
First, abbreviations used in the application documents will be described.
ADP: adenosine 5′-diphosphate CDP: cytidine 5′-diphosphate dTDP: deoxythymidine 5′-diphosphate EDTA: ethylenediaminetetraacetic acid Gal: galactose GalNAc: N-acetylgalactosamine GDP: guanosine 5′-diphosphate Glc: glucose GlcNAc: N-acetylglucosamine GlcUA: Glucuronic acid HPLC: High performance liquid chromatography UDP: Uridine 5′-diphosphate UMP: Uridine 5′-monophosphate In cells such as bacteria and mammals, ADP-Glc, UDP-Glc, etc. It is known that there are enzymes that can hydrolyze such sugar nucleotides. For example, an enzyme called ADP-Glc phosphodiesterase is applied to diagnosis by analysis of sugar nucleotides in biological specimens, production of transgenic plants, and the like (Patent Document 1). Phosphodiesterase is a general term for enzymes that hydrolyze phosphodiester to phosphomonoester. However, it is not known to selectively degrade sugar nucleotides by not containing an enzyme such as the above-mentioned phosphodiesterase and allowing Mn 2+ alone to coexist with sugar nucleotides.
本発明は、Ｎ−アセチル基を保持しない糖ヌクレオチドを特異的に、かつ簡便、迅速、安価に分解する方法及びＮ−アセチル基を保持しない糖ヌクレオチドを特異的に、かつ簡便、迅速に分解することができる安価な分解剤を提供することを課題とする。 The present invention provides a method for specifically, simply, rapidly and inexpensively decomposing a sugar nucleotide which does not have an N-acetyl group, and a method for specifically, simply and rapidly decomposing a sugar nucleotide which does not have an N-acetyl group. It is an object to provide an inexpensive decomposing agent that can be used.
本発明の発明者らは上記課題を解決すべく鋭意検討した結果、驚くべきことに２価の遷移金属塩がＮ−アセチル基を保持しない糖ヌクレオチドを特異的に分解することを見出し、さらにこれにより当該糖ヌクレオチドを簡便、迅速かつ安価に分解することができることを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the inventors of the present invention have surprisingly found that a divalent transition metal salt specifically degrades a sugar nucleotide that does not have an N-acetyl group. Thus, the present inventors have found that the sugar nucleotide can be easily, rapidly and inexpensively decomposed, and have completed the present invention.
すなわち本発明は、Ｎ−アセチル基を保持しない糖ヌクレオチドと、２価の遷移金属イオンとを共存させるステップを少なくとも含む、当該糖ヌクレオチドの分解方法（以下、「本発明方法」という。）を提供する。 That is, the present invention provides a method for degrading a sugar nucleotide (hereinafter referred to as “the method of the present invention”) comprising at least a step of allowing a sugar nucleotide not retaining an N-acetyl group and a divalent transition metal ion to coexist. To do.
ここにいう糖ヌクレオチドは、ＵＤＰ糖であることが好ましい。またここにいうＮ−アセチル基を保持しないＵＤＰ糖は、ＵＤＰ−グルクロン酸、ＵＤＰ−ガラクトース及びＵＤＰ−グルコースからなる群から選ばれる１又は２以上のＵＤＰ糖であることが好ましい。またここにいう２価の遷移金属イオンは、Ｍｎ２＋、Ｃｄ２＋、Ｃｏ２＋及びＣｕ２＋からなる群から選ばれる１又は２以上のイオンであることが好ましい。また前記の「共存」は、ｐＨ６以上の条件下で行われることが好ましい。
The sugar nucleotide referred to here is preferably a UDP sugar. Moreover, it is preferable that the UDP saccharide | sugar which does not hold | maintain an N-acetyl group here is 1 or 2 or more UDP saccharides chosen from the group which consists of UDP-glucuronic acid, UDP-galactose, and UDP-glucose. Further, the divalent transition metal ion mentioned here is preferably one or more ions selected from the group consisting of Mn 2+ , Cd 2+ , Co 2+ and Cu 2+ . The “coexistence” is preferably performed under a condition of
また本発明は、２価の遷移金属塩を成分とする、Ｎ−アセチル基を保持しない糖ヌクレオチドの分解剤（以下、「本発明の剤」という。）を提供する。 The present invention also provides a sugar nucleotide degrading agent that does not retain an N-acetyl group (hereinafter, referred to as “agent of the present invention”), which comprises a divalent transition metal salt as a component.
ここにいう糖ヌクレオチドは、ＵＤＰ糖であることが好ましい。またここにいうＮ−アセチル基を保持しないＵＤＰ糖は、ＵＤＰ−グルクロン酸、ＵＤＰ−ガラクトース及びＵＤＰ−グルコースからなる群から選ばれる１又は２以上のＵＤＰ糖であることが好ましい。またここにいう２価の遷移金属塩は、マンガン、カドミウム、コバルト及び銅からなる群から選ばれる１又は２以上の金属の塩であることが好ましい。 The sugar nucleotide referred to here is preferably a UDP sugar. Moreover, it is preferable that the UDP saccharide | sugar which does not hold | maintain an N-acetyl group here is 1 or 2 or more UDP saccharides chosen from the group which consists of UDP-glucuronic acid, UDP-galactose, and UDP-glucose. The divalent transition metal salt mentioned here is preferably a salt of one or more metals selected from the group consisting of manganese, cadmium, cobalt and copper.
本発明方法は、Ｎ−アセチル基を保持しない糖ヌクレオチドを特異的に、かつ簡便、迅速、安価に分解することができ、極めて有用である。また本発明の剤は、Ｎ−アセチル基を保持しない糖ヌクレオチドを特異的に、かつ簡便、迅速に分解することができ、しかも非常に安価であることから極めて有用である。 The method of the present invention is extremely useful because it can specifically, simply, rapidly and inexpensively degrade sugar nucleotides that do not have an N-acetyl group. The agent of the present invention is extremely useful because it can specifically, conveniently and rapidly degrade sugar nucleotides that do not have an N-acetyl group, and is very inexpensive.
以下、発明を実施するための最良の形態により本発明を詳説する。
＜１＞本発明方法
本発明方法は、Ｎ−アセチル基を保持しない糖ヌクレオチドと、２価の遷移金属イオンとを共存させるステップを少なくとも含む、当該糖ヌクレオチドの分解方法である。
Hereinafter, the present invention will be described in detail by the best mode for carrying out the invention.
<1> Method of the Present Invention The method of the present invention is a method for decomposing a sugar nucleotide, comprising at least a step of allowing a sugar nucleotide not retaining an N-acetyl group and a divalent transition metal ion to coexist.
ここにいう「Ｎ−アセチル基を保持しない糖ヌクレオチド」は、糖ヌクレオチドにおける糖残基部分が、Ｎ−アセチル基（ＮＡｃ）を保持しない糖からなっている糖ヌクレオチドである限りにおいて特に限定されない。この「Ｎ−アセチル基（ＮＡｃ）を保持しない糖」は、単糖、オリゴ糖、多糖のいずれであってもよい。なかでも単糖が好ましい。 The “sugar nucleotide that does not retain an N-acetyl group” as used herein is not particularly limited as long as the sugar residue portion in the sugar nucleotide is a sugar nucleotide that is composed of a sugar that does not retain an N-acetyl group (NAc). The “sugar that does not retain the N-acetyl group (NAc)” may be any of monosaccharides, oligosaccharides, and polysaccharides. Of these, monosaccharides are preferred.
またここにいう「糖ヌクレオチド」も、ＡＤＰ糖、ＣＤＰ糖、ＧＤＰ糖、ＵＤＰ糖、ｄＴＤＰ糖のいずれであってもよい。これらのなかでもＵＤＰ糖が好ましい。 The “sugar nucleotide” referred to here may be any of ADP sugar, CDP sugar, GDP sugar, UDP sugar, and dTDP sugar. Of these, UDP sugars are preferred.
また、Ｎ−アセチル基を保持しないＵＤＰ糖は、ＵＤＰ糖における糖残基部分が、Ｎ−アセチル基（ＮＡｃ）を保持しない糖からなっているＵＤＰ糖である限りにおいて特に限定されない。この糖も、単糖、オリゴ糖、多糖のいずれであってもよい。なかでも単糖が好ましい。なかでも、ＵＤＰ−グルクロン酸、ＵＤＰ−ガラクトース及びＵＤＰ−グルコースからなる群から選ばれる１又は２以上のＵＤＰ糖が好ましい。 Further, the UDP sugar that does not retain the N-acetyl group is not particularly limited as long as the sugar residue portion in the UDP sugar is a UDP sugar composed of a sugar that does not retain the N-acetyl group (NAc). This sugar may be any of monosaccharide, oligosaccharide and polysaccharide. Of these, monosaccharides are preferred. Among these, one or more UDP sugars selected from the group consisting of UDP-glucuronic acid, UDP-galactose and UDP-glucose are preferable.
またここにいう「２価の遷移金属イオン」も、元素周期表の３族から１２族までに属する金属のイオンのうち、２価のイオンである限りにおいて特に限定されない。なかでも、Ｍｎ２＋、Ｃｄ２＋、Ｃｏ２＋及びＣｕ２＋からなる群から選ばれる１又は２以上のイオンであることが好ましい。これらのイオンのうち、分解効率が最も高いのはＭｎ２＋である。 Further, the “divalent transition metal ion” here is not particularly limited as long as it is a divalent ion among ions of metals belonging to Groups 3 to 12 of the periodic table. Of these, one or more ions selected from the group consisting of Mn 2+ , Cd 2+ , Co 2+ and Cu 2+ are preferable. Among these ions, Mn 2+ has the highest decomposition efficiency.
Ｎ−アセチル基を保持しない糖ヌクレオチドと２価の遷移金属イオンとを「共存」させる方法も、両者の分子が相互に接触できる状態となる限りにおいて特に限定されない。この「共存」は水性溶液中で行われるが、その際のｐＨは６以上に設定することが分解効率等の面で好ましい。ｐＨが高いほど、Ｎ−アセチル基を保持しない糖ヌクレオチド分解効率も高いことから、これを指標にして、両者を共存させる時間、両者を共存させる際の２価の遷移金属イオンの濃度、両者を共存させる温度、目的とする分解の程度等に応じて当業者が具体的なｐＨを適宜設定することができる。例えば、ｐＨ６以上、ｐＨ６．５以上、ｐＨ７以上、ｐＨ７．５以上、ｐＨ８以上等の条件が例示される。より具体的には、ｐＨ６〜１０の範囲、ｐＨ６．５〜１０の範囲、ｐＨ７〜１０の範囲、ｐＨ７．５〜１０の範囲、ｐＨ８〜１０の範囲等が例示される。
The method of “coexisting” a sugar nucleotide that does not retain an N-acetyl group and a divalent transition metal ion is not particularly limited as long as both molecules can come into contact with each other. This “coexistence” is carried out in an aqueous solution, and the pH at that time is preferably set to 6 or more in terms of decomposition efficiency and the like. The higher the pH, the higher the sugar nucleotide decomposition efficiency that does not retain the N-acetyl group. Therefore, using this as an index, the time for both to coexist, the concentration of the divalent transition metal ion when both coexist, A person skilled in the art can appropriately set a specific pH according to the coexisting temperature, the desired degree of decomposition, and the like. For example, conditions such as
また、両者を共存させる時間も特に限定されない。その時間が長いほど分解の程度を高めることができるので、これを指標にして、両者を共存させる際のｐＨ、両者を共存させる際の２価の遷移金属イオンの濃度、両者を共存させる温度、目的とする分解の程度等に応じて当業者が具体的な時間を適宜設定することができる。例えば、１０分間〜１２時間、１０分間〜６時間、１０分間〜２時間等の範囲を例示することができる。 Also, the time for both to coexist is not particularly limited. The longer the time, the higher the degree of decomposition, so using this as an index, the pH when both coexist, the concentration of divalent transition metal ions when both coexist, the temperature at which both coexist, A person skilled in the art can appropriately set a specific time depending on the target degree of decomposition. For example, the range of 10 minutes to 12 hours, 10 minutes to 6 hours, 10 minutes to 2 hours, etc. can be illustrated.
また、両者を共存させる際の２価の遷移金属イオンの濃度も特に限定されない。その濃度が高いほど分解の程度を高めることができるので、これを指標にして、両者を共存させる際の時間、両者を共存させる際のｐＨ、両者を共存させる温度、目的とする分解の程度等に応じて当業者が具体的な時間を適宜設定することができる。例えば、両者を共存させる際の終濃度として、０．２ｍＭ以上、０．５ｍＭ以上、１ｍＭ以上、２ｍＭ以上、５ｍＭ以上、１０ｍＭ以上、２０ｍＭ以上等の濃度が例示される。より具体的には、０．２ｍＭ〜５００ｍＭ、０．２ｍＭ〜４００ｍＭ、０．２ｍＭ〜３００ｍＭ、０．２ｍＭ〜２００ｍＭ、０．２ｍＭ〜１００ｍＭ、０．５ｍＭ〜５００ｍＭ、０．５ｍＭ〜４００ｍＭ、０．５ｍＭ〜３００ｍＭ、０．５ｍＭ〜２００ｍＭ、０．５ｍＭ〜１００ｍＭ、１ｍＭ〜５００ｍＭ、１ｍＭ〜４００ｍＭ、１ｍＭ〜３００ｍＭ、１ｍＭ〜２００ｍＭ、１ｍＭ〜１００ｍＭ、２ｍＭ〜５００ｍＭ、２ｍＭ〜４００ｍＭ、２ｍＭ〜３００ｍＭ、２ｍＭ〜２００ｍＭ、２ｍＭ〜１００ｍＭ、５ｍＭ〜５００ｍＭ、５ｍＭ〜４００ｍＭ、５ｍＭ〜３００ｍＭ、５ｍＭ〜２００ｍＭ、５ｍＭ〜１００ｍＭ、１０ｍＭ〜５００ｍＭ、１０ｍＭ〜４００ｍＭ、１０ｍＭ〜３００ｍＭ、１０ｍＭ〜２００ｍＭ、１０ｍＭ〜１００ｍＭ、２０ｍＭ〜５００ｍＭ、２０ｍＭ〜４００ｍＭ、２０ｍＭ〜３００ｍＭ、２０ｍＭ〜２００ｍＭ、２０ｍＭ〜１００ｍＭ等の範囲を例示することができる。 In addition, the concentration of the divalent transition metal ion when both coexist is not particularly limited. The higher the concentration is, the higher the degree of decomposition can be. Therefore, using this as an index, the time when both coexist, the pH when coexisting, the temperature at which both coexist, the degree of target decomposition, etc. Depending on the situation, a person skilled in the art can set a specific time as appropriate. For example, as the final concentration when both coexist, concentrations of 0.2 mM or more, 0.5 mM or more, 1 mM or more, 2 mM or more, 5 mM or more, 10 mM or more, 20 mM or more are exemplified. More specifically, 0.2 mM to 500 mM, 0.2 mM to 400 mM, 0.2 mM to 300 mM, 0.2 mM to 200 mM, 0.2 mM to 100 mM, 0.5 mM to 500 mM, 0.5 mM to 400 mM,. 5 mM to 300 mM, 0.5 mM to 200 mM, 0.5 mM to 100 mM, 1 mM to 500 mM, 1 mM to 400 mM, 1 mM to 300 mM, 1 mM to 200 mM, 1 mM to 100 mM, 2 mM to 500 mM, 2 mM to 400 mM, 2 mM to 300 mM, 2 mM to 200 mM, 2 mM to 100 mM, 5 mM to 500 mM, 5 mM to 400 mM, 5 mM to 300 mM, 5 mM to 200 mM, 5 mM to 100 mM, 10 mM to 500 mM, 10 mM to 400 mM, 10 mM to 300 mM, 10 mM to 200 mM, 10 mM to 100 mM, 0MM～500mM, it 20mM~400mM, 20mM~300mM, 20mM~200mM, be mentioned range, such 20MM～100mM.
また、両者を共存させる際の温度も特に限定されない。その温度が高いほど分解の程度を高めることができるので、これを指標にして、両者を共存させる際のｐＨ、両者を共存させる際の２価の遷移金属イオンの濃度、両者を共存させる時間、目的とする分解の程度等に応じて当業者が具体的な温度を適宜設定することができる。例えば、４℃以上、１５℃以上、３０℃以上、３７℃以上、５０℃以上等の条件が例示される。より具体的には、４℃〜６０℃、１５℃〜６０℃、３０℃〜６０℃、３７℃〜６０℃、５０℃〜６０℃等の範囲が例示される。 Moreover, the temperature at the time of making both coexist is not specifically limited. Since the degree of decomposition can be increased as the temperature is higher, using this as an index, the pH when coexisting both, the concentration of divalent transition metal ions when coexisting both, the time when both coexist, A specific temperature can be appropriately set by those skilled in the art according to the target degree of decomposition. For example, conditions such as 4 ° C. or higher, 15 ° C. or higher, 30 ° C. or higher, 37 ° C. or higher, 50 ° C. or higher are exemplified. More specifically, examples include ranges of 4 ° C to 60 ° C, 15 ° C to 60 ° C, 30 ° C to 60 ° C, 37 ° C to 60 ° C, 50 ° C to 60 ° C, and the like.
２価の遷移金属イオンは、それ単独で、糖ヌクレオチドを分解する作用を発揮する。したがって、本発明方法における「Ｎ−アセチル基を保持しない糖ヌクレオチドと、２価の遷移金属イオンとを共存させるステップ」においては、糖ヌクレオチドを分解する酵素（例えば、ホスホジエステラーゼ）が共存している必要はない。すなわち、本発明方法は、「Ｎ−アセチル基を保持しない糖ヌクレオチドと２価の遷移金属イオンとを、糖ヌクレオチドを分解する酵素の非存在下で共存させるステップ」を少なくとも含むものであってもよい。 A divalent transition metal ion alone exerts an action of decomposing sugar nucleotides. Therefore, in the “step of coexisting a sugar nucleotide not retaining an N-acetyl group and a divalent transition metal ion” in the method of the present invention, an enzyme (for example, phosphodiesterase) that decomposes the sugar nucleotide needs to coexist. There is no. That is, the method of the present invention may include at least the step of coexisting a sugar nucleotide that does not retain an N-acetyl group and a divalent transition metal ion in the absence of an enzyme that degrades the sugar nucleotide. Good.
本発明方法は、Ｎ−アセチル基を保持しない糖ヌクレオチドと２価の遷移金属イオンとを共存させるステップを少なくとも含む限りにおいて、他のステップを更に含んでいてもよい。例えば、当該ステップを行うに先立ち、２価の遷移金属イオンを生ぜしめる物質を添加するステップ等を更に含んでいてもよい。 The method of the present invention may further include other steps as long as it includes at least a step of allowing a sugar nucleotide not retaining an N-acetyl group and a divalent transition metal ion to coexist. For example, prior to performing this step, a step of adding a substance that generates a divalent transition metal ion may be further included.
ここにいう「２価の遷移金属イオンを生ぜしめる物質」も、水性溶液中において２価の遷移金属イオンを生ぜしめるような物質である限りにおいて限定されない。このような物質としては、例えば「２価の遷移金属塩」が例示される。また、このような塩としては、遷移金属塩のハロゲン化物を例示することができる。この「ハロゲン化物」の一例として、塩化物を例示することができる。このような塩化物としては、ＭｎＣｌ２、ＣｄＣｌ２、ＣｏＣｌ２、ＣｕＣｌ２等を例示することができる。 The “substance that generates a divalent transition metal ion” is not limited as long as it is a substance that generates a divalent transition metal ion in an aqueous solution. Examples of such a substance include “divalent transition metal salts”. Moreover, as such a salt, the halide of a transition metal salt can be illustrated. As an example of the “halide”, a chloride can be exemplified. Examples of such chlorides include MnCl 2 , CdCl 2 , CoCl 2 , CuCl 2 and the like.
また、Ｎ−アセチル基を保持しない糖ヌクレオチドと２価の遷移金属イオンとを共存させるステップの後に、分解により生じた産物や分解されなかった糖ヌクレオチドを分離したり、精製したりするステップ等をさらに含んでいてもよい。 In addition, after the step of allowing a sugar nucleotide not retaining an N-acetyl group and a divalent transition metal ion to coexist, a step of separating or purifying a product produced by decomposition or a sugar nucleotide that has not been decomposed, etc. Further, it may be included.
本発明方法は、Ｎ−アセチル基を保持しない糖ヌクレオチドは分解するが、Ｎ−アセチル基を保持する糖ヌクレオチドは分解しないという特異性を有している。したがって、Ｎ−アセチル基を保持しない糖ヌクレオチドを選択的に分解することが可能である。 The method of the present invention has the specificity that a sugar nucleotide that does not retain an N-acetyl group is degraded, but a sugar nucleotide that retains an N-acetyl group does not degrade. Therefore, it is possible to selectively degrade sugar nucleotides that do not retain an N-acetyl group.
なお、本出願書類における「糖ヌクレオチドの分解」とは、糖ヌクレオチドを分解（切断）する意味である。なかでも、糖ヌクレオチドにおけるピロリン酸部分の分解（切断）であることが好ましい。
＜２＞本発明の剤
本発明の剤は、２価の遷移金属塩を成分とする、Ｎ−アセチル基を保持しない糖ヌクレオチドの分解剤である。
The “degradation of sugar nucleotides” in the present application document means the decomposition (cleavage) of sugar nucleotides. Of these, decomposition (cleavage) of the pyrophosphate moiety in the sugar nucleotide is preferable.
<2> Agent of the Present Invention The agent of the present invention is a sugar nucleotide decomposing agent that contains a divalent transition metal salt as a component and does not retain an N-acetyl group.
ここにいう「Ｎ−アセチル基を保持しない糖ヌクレオチド」については、前記の＜１＞における説明と同じである。したがって、ここにいう「糖ヌクレオチド」はＵＤＰ糖であることが好ましいこととなる。またここにいう「Ｎ−アセチル基を保持しないＵＤＰ糖」も、ＵＤＰ−グルクロン酸、ＵＤＰ−ガラクトース及びＵＤＰ−グルコースからなる群から選ばれる１又は２以上のＵＤＰ糖であることが好ましいこととなる。 The “sugar nucleotide that does not retain an N-acetyl group” here is the same as described in <1> above. Accordingly, the “sugar nucleotide” referred to here is preferably a UDP sugar. In addition, the “UDP sugar that does not retain an N-acetyl group” herein is also preferably one or more UDP sugars selected from the group consisting of UDP-glucuronic acid, UDP-galactose, and UDP-glucose. .
本発明の剤の有効成分である「２価の遷移金属塩」は、元素周期表の３族から１２族までに属する金属のうち、２価の金属の塩である限りにおいて特に限定されない。なかでも、マンガン、カドミウム、コバルト及び銅からなる群から選ばれる１又は２以上の金属の塩であることが好ましい。なかでも分解効率の面からすると、マンガンの塩であることが好ましい。また、このような塩としては、２価の遷移金属塩のハロゲン化物を例示することができる。この「ハロゲン化物」の一例として、塩化物を例示することができる。このような塩化物としては、ＭｎＣｌ２、ＣｄＣｌ２、ＣｏＣｌ２、ＣｕＣｌ２等を例示することができる。 The “divalent transition metal salt” which is an active ingredient of the agent of the present invention is not particularly limited as long as it is a divalent metal salt among metals belonging to Groups 3 to 12 of the periodic table. Especially, it is preferable that it is a salt of 1 or 2 or more metals chosen from the group which consists of manganese, cadmium, cobalt, and copper. Of these, manganese salts are preferred from the standpoint of decomposition efficiency. Moreover, as such a salt, the halide of a bivalent transition metal salt can be illustrated. As an example of the “halide”, a chloride can be exemplified. Examples of such chlorides include MnCl 2 , CdCl 2 , CoCl 2 , CuCl 2 and the like.
本発明の剤は、このような２価の遷移金属塩を少なくとも含有している限りにおいて、さらに他の成分を含有していてもよい。このような他の成分は、本発明の剤に含有される２価の遷移金属塩や、これから生ずる２価の遷移金属イオンに対して悪影響を与えない物質である限りにおいて特に限定されず、当業者がその目的等に応じて適宜選択することができる。もちろん、２価の遷移金属塩を、他の成分を含有させることなくそのまま本発明の剤としてもよい。したがって本発明の剤は、２価の遷移金属塩をそのまま用い、又は更に他の成分を添加することで、当業者が適宜製造することができる。 The agent of the present invention may further contain other components as long as it contains at least such a divalent transition metal salt. Such other components are not particularly limited as long as they are substances that do not adversely affect the divalent transition metal salt contained in the agent of the present invention or the divalent transition metal ions generated therefrom. The trader can make an appropriate selection according to the purpose. Of course, a divalent transition metal salt may be used as the agent of the present invention as it is without containing other components. Accordingly, the agent of the present invention can be appropriately produced by those skilled in the art by using the divalent transition metal salt as it is or by adding other components.
本発明の剤は、前記＜１＞に記載の方法で使用することができる。すなわち、本発明方法における「Ｎ−アセチル基を保持しない糖ヌクレオチドと２価の遷移金属イオンとを共存させるステップ」を行うに先立ち、本発明の剤を添加すればよい。 The agent of this invention can be used by the method as described in said <1>. That is, the agent of the present invention may be added prior to performing the “step of coexisting a sugar nucleotide not retaining an N-acetyl group and a divalent transition metal ion” in the method of the present invention.
以下、本発明を実施例により具体的に詳説する。 Hereinafter, the present invention will be described in detail by way of examples.
本実施例で用いた糖ヌクレオチド（UDP-GlcUA及びUDP-GalNAc）はいずれもシグマ社より購入した。また、Hydrospher C18カラムはYMC社より購入した。 All of the sugar nucleotides (UDP-GlcUA and UDP-GalNAc) used in this example were purchased from Sigma. Hydrospher C18 column was purchased from YMC.
糖ヌクレオチド（UDP-GalNAC及びUDP-GlcUA）の定量及び安定性は、逆相ＨＰＬＣを用いることによって解析した。具体的には、次の方法によって解析した。 The quantification and stability of sugar nucleotides (UDP-GalNAC and UDP-GlcUA) were analyzed by using reverse phase HPLC. Specifically, analysis was performed by the following method.
50 mM Tris-HCl（pH 7.2）、0.15 M NaCl及び種々の濃度のMnCl2を含有する溶液１０μｌ中で、UDP-GlcUA、UDP-GalNAcその他の糖ヌクレオチド（終濃度３ｎＭ）を、種々の温度（４〜５０℃）や種々の時間（０〜１８時間）でインキュベートした。また、ｐＨ（５〜９）や、金属イオン（塩）の種類（Ca、Mg、Ba、Cd、Co、Cu、EDTA）等の反応条件も種々変化させて解析した。 In 10 μl of a solution containing 50 mM Tris-HCl (pH 7.2), 0.15 M NaCl and various concentrations of MnCl 2 , UDP-GlcUA, UDP-GalNAc and other sugar nucleotides (final concentration of 3 nM) are added at various temperatures ( 4 to 50 ° C.) and various times (0 to 18 hours). In addition, the reaction conditions such as pH (5 to 9) and the types of metal ions (salts) (Ca, Mg, Ba, Cd, Co, Cu, EDTA) were variously analyzed.
糖ヌクレオチド類の分離・定量は、Hydrosher C18 カラム（4.6 ｘ 150 cm；YMC社製）を用いた逆相ＨＰＬＣを用いて行った。具体的には、試料溶液10μlをカラムにアプライし、１％アセトニトリル（CH3CN）を含有する20 mM トリエチルアミン−酢酸緩衝液（pH 5.7）を溶媒として流速 0.7 ml/分で流し、溶出液について波長262 nmにおける吸光度を測定することにより行った。標準品を同条件で解析した結果、溶出時間はそれぞれ、UDP-GlcUAが15.95分、UDP-GalNAcが11.47分、UDPが9.58分、UMPが7.91分であった。 Separation and quantification of sugar nucleotides were performed using reverse phase HPLC using a Hydrosher C18 column (4.6 × 150 cm; manufactured by YMC). Specifically, 10 μl of sample solution was applied to the column, and 20 mM triethylamine-acetic acid buffer (pH 5.7) containing 1% acetonitrile (CH 3 CN) was used as a solvent at a flow rate of 0.7 ml / min. The measurement was performed by measuring the absorbance at a wavelength of 262 nm. As a result of analyzing the standard product under the same conditions, the elution times were 15.95 minutes for UDP-GlcUA, 11.47 minutes for UDP-GalNAc, 9.58 minutes for UDP, and 7.91 minutes for UMP, respectively.
糖ヌクレオチドを、20 mM MnCl2の存在下又は非存在下、３０℃で種々の時間インキュベートした結果を図１に示す。図１中、黒丸は20 mM MnCl2の存在下でUDP-GlcUAをインキュベートした結果を、黒四角は20 mM MnCl2の存在下でUDP-GalNAcをインキュベートした結果を、白丸はMnCl2の非存在下でUDP-GlcUAをインキュベートした結果をそれぞれ示す。また図１の縦軸は、インキュベート前の溶液中の糖ヌクレオチド濃度を１００％としたときの、インキュベート後の溶液中の糖ヌクレオチド濃度の割合を示す。また横軸は、インキュベート時間を示す。 The results of incubating the sugar nucleotides at 30 ° C. for various times in the presence or absence of 20 mM MnCl 2 are shown in FIG. In Figure 1, a black circle shows the results obtained by incubating the UDP-GlcUA in the presence of 20 mM MnCl 2, the results black squares were incubated UDP-GalNAc in the presence of 20 mM MnCl 2, open circles absence of MnCl 2 The results of incubating UDP-GlcUA are shown below. Moreover, the vertical axis | shaft of FIG. 1 shows the ratio of the sugar nucleotide concentration in the solution after incubation when the sugar nucleotide concentration in the solution before incubation is 100%. The horizontal axis indicates the incubation time.
図１より、Mn2+によってUDP-GlcUAが分解されることが示され、６時間インキュベートするとほとんど消失することが明らかになった。一方、UDP-GalNAcは、Mn2+の存在下で１２時間インキュベートしても分解されなかった。 From FIG. 1, it was shown that UDP-GlcUA was degraded by Mn 2+ , and it was revealed that it almost disappeared after 6 hours of incubation. On the other hand, UDP-GalNAc was not degraded even when incubated for 12 hours in the presence of Mn 2+ .
UDP-GlcUAを、種々の濃度（0.002mM〜20mM）のMnCl2の存在下、３０℃で種々の時間インキュベートした結果を図２に示す。図２中、白丸は２時間、黒四角は６時間それぞれインキュベートした結果を示す。また図２の縦軸は、インキュベート前の溶液中のUDP-GlcUA濃度を１００％としたときの、インキュベート後の溶液中のUDP-GlcUA濃度の割合を示す。また横軸は、MnCl2の濃度を示す。 FIG. 2 shows the results of incubating UDP-GlcUA at 30 ° C. for various times in the presence of various concentrations (0.002 mM to 20 mM) of MnCl 2 . In FIG. 2, white circles indicate the results of incubation for 2 hours, and black squares indicate the results of incubation for 6 hours. Moreover, the vertical axis | shaft of FIG. 2 shows the ratio of the UDP-GlcUA density | concentration in the solution after incubation when the UDP-GlcUA density | concentration in the solution before incubation is made into 100%. The horizontal axis shows the concentration of MnCl 2.
図２より、Mn2+によるUDP-GlcUAの分解は濃度依存的であることが示された。Mn2+濃度が低下するとUDP-GlcUAの分解は抑制され、Mn2+が0.2mM以下の場合には６時間インキュベートしてもほとんど分解されなかった。 FIG. 2 shows that the degradation of UDP-GlcUA by Mn 2+ is concentration-dependent. When the Mn 2+ concentration decreased, UDP-GlcUA degradation was suppressed, and when Mn 2+ was 0.2 mM or less, it was hardly degraded even after 6 hours of incubation.
UDP-GlcUAを、種々の温度で、20mM MnCl2の存在下又は非存在下、種々の時間インキュベートした結果を図３に示す。図３中、白丸は20mM MnCl2の存在下で２時間、黒丸は20mM MnCl2の存在下で６時間、白四角は20mM MnCl2の非存在下で２時間、黒四角は20mM MnCl2の非存在下で６時間それぞれインキュベートした結果を示す。また図３の縦軸は、インキュベート前の溶液中のUDP-GlcUA濃度を１００％としたときの、インキュベート後の溶液中のUDP-GlcUA濃度の割合を示す。また横軸はインキュベート時の温度を示す。
The results of incubating UDP-GlcUA at various temperatures in the presence or absence of 20 mM MnCl 2 for various times are shown in FIG. In Figure 3,
図３より、Mn2+によるUDP-GlcUAの分解は温度にも依存的であり、温度が高くなるほど分解が激しくなることが示された。一方、Mn2+の非存在下では、５０℃でインキュベートしても分解は見られなかった。 FIG. 3 shows that the decomposition of UDP-GlcUA by Mn 2+ is also dependent on the temperature, and that the decomposition becomes more severe as the temperature increases. On the other hand, in the absence of Mn 2+ , no degradation was observed even when incubated at 50 ° C.
UDP-GlcUAを、種々のｐＨで、20mM MnCl2の存在下又は非存在下、３０℃で種々の時間インキュベートした結果を図４に示す。ｐＨ5.0及び6.0についてはいずれも50mM 酢酸ナトリウム緩衝液中で、ｐＨ7.0、7.5、8.0及び9.0についてはいずれも50mM Tris-HCl緩衝液中でインキュベートした。図４中、白丸は20mM MnCl2の存在下で２時間、黒丸は20mM MnCl2の存在下で６時間、白四角は20mM MnCl2の非存在下で２時間、黒四角は20mM MnCl2の非存在下で６時間それぞれインキュベートした結果を示す。また図４の縦軸は、インキュベート前の溶液中のUDP-GlcUA濃度を１００％としたときの、インキュベート後の溶液中のUDP-GlcUA濃度の割合を示す。また横軸はインキュベート時のｐＨを示す。
The results of incubating UDP-GlcUA at various pH's in the presence or absence of 20 mM MnCl 2 at 30 ° C. for various times are shown in FIG. Both pH 5.0 and 6.0 were incubated in 50 mM sodium acetate buffer, and pH 7.0, 7.5, 8.0 and 9.0 were all incubated in 50 mM Tris-HCl buffer. In Figure 4, a
図４より、中性から塩基性になるほど（ｐＨ６〜７以上で）UDP-GlcUAの分解は激しくなった。 From FIG. 4, the degradation of UDP-GlcUA became more severe as the pH became neutral to basic (at pH 6-7 or higher).
なお、図１〜図４における値は、それぞれ独立に３回実験した際の「平均値±Ｓ．Ｄ．」で示した。 In addition, the value in FIGS. 1-4 was shown by "average value +/- SD" at the time of experimenting 3 times independently, respectively.
また、種々の糖ヌクレオチド（UDP-GalNAc、UDP-GlcNAc、UDP-GlcUA、UDP-Gal、UDP-Glc）を用いて、同様にMnCl2による影響を調べた。結果を表１に示す。なお、MnCl2の濃度は20mMとし、インキュベート温度は３０℃、インキュベート時間は２時間とした。また実験はそれぞれ独立に３回行い、表１中にその平均値とＳ．Ｄ．を示した。 Further, various sugar nucleotides used (UDP-GalNAc, UDP-GlcNAc , UDP-GlcUA, UDP-Gal, UDP-Glc) to examine the effects of MnCl 2 as well. The results are shown in Table 1. The concentration of MnCl 2 was 20 mM, the incubation temperature was 30 ° C., and the incubation time was 2 hours. In addition, each experiment was performed three times independently. D. showed that.
表１より、UDP-GlcNAcはUDP-GalNAcと同様にMn2+に対して安定であることが示された。一方、UDP-Glc及びUDP-Galは、Mn2+イオン存在下でUDP-GlcUAと同様に分解された。また、逆相ＨＰＬＣの解析から、UDP-GlcUA分解後にはUMPのピークが現れたことから、UDP-GlcUAのピロリン酸部分が切断されていることが示された。 From Table 1, it was shown that UDP-GlcNAc is stable to Mn 2+ like UDP-GalNAc. On the other hand, UDP-Glc and UDP-Gal were decomposed in the same manner as UDP-GlcUA in the presence of Mn 2+ ions. In addition, the analysis of reverse phase HPLC showed that the UMP peak appeared after UDP-GlcUA decomposition, indicating that the pyrophosphate part of UDP-GlcUA was cleaved.
また、Mn2+（MnCl2）に代えて、他の種々の金属イオン（塩）の存在下でUDP-GlcUAをインキュベートした結果を表２に示す。それぞれの金属イオン（塩）の濃度を20mMとし、インキュベート温度は３０℃、インキュベート時間は６時間とした。また実験はそれぞれ独立に３回行い、表１中にその平均値とＳ．Ｄ．を示した。なお、実験に用いた金属塩は、MnCl2、CaCl2、MgCl2、BaCl2、CdCl2、CoCl2、CuCl2である。 Table 2 shows the results of incubating UDP-GlcUA in the presence of other various metal ions (salts) instead of Mn 2+ (MnCl 2 ). The concentration of each metal ion (salt) was 20 mM, the incubation temperature was 30 ° C., and the incubation time was 6 hours. In addition, each experiment was performed three times independently. D. showed that. The metal salts used in the experiment are MnCl 2 , CaCl 2 , MgCl 2 , BaCl 2 , CdCl 2 , CoCl 2 , and CuCl 2 .
表２より、Mn2+と同じ二価イオンでもアルカリ土類金属（Ca2+、Mg2+、Ba2+）は実質的に分解には影響せず、遷移金属であるCd2+、Co2+、Cu2+はUDP-GlcUAを分解した。しかし、Mn2+が最も分解作用が強い金属イオンであった。金属キレートであるEDTAをMn2+を含有する溶液に添加すると、Mn2+による分解は完全に阻害された。 Table 2 shows that alkaline earth metals (Ca 2+ , Mg 2+ , Ba 2+ ) have substantially no effect on decomposition even with the same divalent ions as Mn 2+, and transition metals Cd 2+ , Co 2+ and Cu 2+ decomposed UDP-GlcUA. However, Mn 2+ was the metal ion with the strongest decomposition action. The addition of EDTA which is a metal chelate to a solution containing Mn 2+, degradation by Mn 2+ was completely inhibited.
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