JP5597903B2 - Introduction of sugar chains into gold-containing materials - Google Patents
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Description
本発明は、金原子を金含有物に糖鎖を結合させる方法に関する。 The present invention relates to a method for bonding a sugar chain to a gold-containing material with a gold atom.
近年、金ナノ微粒子は光学材料・導電材料への応用など幅広い分野において利用されており、例えば、表面に糖鎖が導入された金ナノ微粒子はバイオセンシングの分野での利用が期待されている。その理由の１つは、金ナノ微粒子表面に局所的に糖ペプチドや糖鎖を高密度で導入すると「クラスター効果」が発現され、より実用的な糖鎖結合金ナノ微粒子の開発が可能となるからである。さらに、金ナノ微粒子は発色性に優れており、凝集などで色調が変化するため、さまざまな用途への応用が検討されている。特に、医療分野においては、妊娠判定キット、糖尿病検査キットなどへの利用の他、インフルエンザウィルスなどと特異的に結合する糖鎖を担持させた金ナノ微粒子を、ウィルス感染の診断薬などに応用する技術なども開発されつつある。とりわけ、金原子は人体に対する毒性が低いと考えられることから、薬剤の担体などとしての使用も検討されている点において他の金属担体とは異なる特徴を有している（例えば、非特許文献１など）。 In recent years, gold nanoparticles have been used in a wide range of fields such as application to optical materials and conductive materials. For example, gold nanoparticles having a sugar chain introduced on the surface are expected to be used in the field of biosensing. One of the reasons is that when a glycopeptide or sugar chain is locally introduced at a high density on the surface of the gold nanoparticle, a “cluster effect” is expressed, and it becomes possible to develop a more practical sugar chain-bonded gold nanoparticle. Because. Furthermore, since gold nanoparticle is excellent in color developability and the color tone changes due to aggregation or the like, application to various uses is being studied. In particular, in the medical field, in addition to use in pregnancy determination kits, diabetes testing kits, etc., gold nanoparticles that carry sugar chains that specifically bind to influenza viruses, etc. are applied to diagnostic agents for viral infections, etc. Technology is also being developed. In particular, since gold atoms are considered to be less toxic to the human body, they have characteristics different from other metal carriers in that they are also considered for use as drug carriers (for example, Non-Patent Document 1). Such).
金粒子に糖鎖を導入する方法は、すでにいくつか報告されている。その代表的な方法として、水素化ホウ素ナトリウム還元法、グリコシル化法を挙げることができる。水素化ホウ素ナトリウム還元法は、２分子の糖鎖がＳ−Ｓ結合した２量体を塩化金酸と共に反応系に加え、そこへ水素化ホウ素ナトリウム水溶液を滴下することで塩化金酸および糖鎖を還元して、所定の微粒子を得る方法である（非特許文献２）。また、グリコシル化法は、金微粒子表面にスペーサーを導入させた後、スペーサー末端の水酸基と糖鎖分子を結合させる方法である（非特許文献３）。これらの方法は、従来法として当該分野で使用されているが、水素化ホウ素ナトリウム還元法は、糖鎖２量体を合成する必要があるため、目的の金粒子を製造するまでに複数の工程を要し、時間と手間が掛かかってしまう。また、グリコシル化法は、水酸基を持つスペーサーを金粒子に予め導入しておく必要があり、煩雑な工程が必要とされる。 Several methods for introducing sugar chains into gold particles have already been reported. Typical methods include sodium borohydride reduction method and glycosylation method. In the sodium borohydride reduction method, a dimer in which two sugar chains are S—S bonded is added to the reaction system together with chloroauric acid, and an aqueous sodium borohydride solution is added dropwise thereto, whereby chloroauric acid and sugar chains are added. Is a method for obtaining predetermined fine particles (Non-patent Document 2). The glycosylation method is a method in which a spacer is introduced on the surface of gold fine particles, and then a hydroxyl group at the end of the spacer is bonded to a sugar chain molecule (Non-patent Document 3). These methods are used in the art as conventional methods, but the sodium borohydride reduction method requires the synthesis of a sugar chain dimer, so that a plurality of steps are required until the desired gold particles are produced. Takes time and effort. In addition, the glycosylation method requires that a spacer having a hydroxyl group be introduced into the gold particle in advance, and a complicated process is required.
前述のごとく、従来法により金粒子に糖鎖を導入する場合、煩雑な工程が必要とされていたため、より簡便に金粒子への糖鎖導入を可能にする技術が望まれていた。さらに、従来法の水素化ホウ素ナトリウム還元法は、金粒子の合成と糖鎖の導入が同時に起こるため、導入する糖鎖の分子量によって合成される糖鎖結合金粒子の大きさが影響を受けてしまい、所望のサイズの糖鎖結合金粒子の作製が困難であった。また、金粒子の合成と糖鎖の導入が同時に起こることから、金粒子の内部に糖鎖が入り込んでしまい、金粒子表面上へ効率的に糖鎖を導入する事が困難であった。一方、グリコシル化法においては、金粒子上のスペーサーに糖鎖を結合させる際に、嵩高い糖鎖の導入が立体障害により難しい場合があった。
本発明はこのような従来法の問題点を解決するために行われたものである。
そこで、本発明は、金原子（Ａｕ）に糖鎖を導入し、金（Ａｕ）含有物を製造する方法、並びに、糖鎖が導入された金含有物の提供を目的とする。
As described above, when a sugar chain is introduced into gold particles by a conventional method, a complicated process is required. Therefore, a technique that enables sugar chains to be introduced into gold particles more easily has been desired. Furthermore, in the conventional sodium borohydride reduction method, since the synthesis of gold particles and the introduction of sugar chains occur simultaneously, the size of the sugar-bonded gold particles synthesized is affected by the molecular weight of the sugar chains to be introduced. Therefore, it is difficult to produce sugar chain-bonded gold particles having a desired size. In addition, since the synthesis of the gold particles and the introduction of the sugar chains occur simultaneously, the sugar chains enter the inside of the gold particles, and it is difficult to efficiently introduce the sugar chains onto the gold particle surface. On the other hand, in the glycosylation method, when a sugar chain is bound to a spacer on a gold particle, it may be difficult to introduce a bulky sugar chain due to steric hindrance.
The present invention has been made to solve such problems of the conventional method.
Accordingly, an object of the present invention is to provide a method for producing a gold (Au) -containing material by introducing a sugar chain into a gold atom (Au) and a gold-containing material into which a sugar chain has been introduced.
本発明者らは、金粒子への糖鎖導入法に関し、鋭意研究を行ったところ、チオアセチル基を有する糖鎖をアルコールとアルコキシドの存在下で金粒子と反応させると、一段階で金粒子へ糖鎖を結合させることに成功した。
すなわち、本発明は、式（Ｉ）で示されるチオアセチル基を有する糖鎖を、エステル交換反応により金原子に結合させることを特徴とする、金含有物への糖鎖結合方法である。
That is, the present invention is a method for binding a sugar chain to a gold-containing material, wherein the sugar chain having a thioacetyl group represented by formula (I) is bonded to a gold atom by a transesterification reaction.
本発明によれば、所望の糖鎖を所望の金含有物に、一段階で、簡易かつ迅速に結合させることが可能である。 According to the present invention, it is possible to easily and quickly bind a desired sugar chain to a desired gold-containing material in one step.
本発明によれば、金含有物の表面に所望の糖鎖を結合させることができる。すなわち、従来法である水素化ホウ素ナトリウム法は、金粒子を合成しながら、糖鎖を結合させる方法であるため、金粒子の内部にも糖鎖が入り込んでしまい、金粒子表面への所望の糖鎖結合率を達成することができなかった。これに対し、本発明による方法は、予め作製した金含有物（粒子状、平板状など）に糖鎖を結合させる方法であるため、その表面に効率よく糖鎖を結合させることができる。 According to the present invention, a desired sugar chain can be bound to the surface of a gold-containing material. That is, the sodium borohydride method, which is a conventional method, is a method of binding sugar chains while synthesizing gold particles, so that sugar chains also enter the inside of the gold particles, and the desired surface of the gold particles is desired. The sugar chain binding rate could not be achieved. On the other hand, since the method according to the present invention is a method of binding sugar chains to a gold-containing material (particle shape, flat plate shape, etc.) prepared in advance, the sugar chains can be efficiently bonded to the surface.
また、本発明によれば、所望のサイズの金含有物に糖鎖を結合させることができる。従来法の水素化ホウ素ナトリウム法は、結合させる糖鎖のサイズによって作製される金粒子のサイズがある程度制限されていたが、本発明の方法によれば、所望のサイズの金含有物に糖鎖を結合させることができる。 According to the present invention, a sugar chain can be bound to a gold-containing material having a desired size. In the conventional sodium borohydride method, the size of the gold particles produced is limited to some extent by the size of the sugar chain to be bound. However, according to the method of the present invention, a sugar chain is added to a gold-containing material having a desired size. Can be combined.
本発明によれば、金含有物側にスペーサーを結合させる必要がないため、結合させる糖鎖のサイズによる制限を回避し、所望のサイズの糖鎖を結合させることが可能となる。 According to the present invention, since it is not necessary to bind a spacer to the gold-containing material side, it is possible to avoid a restriction due to the size of a sugar chain to be bound and to bind a sugar chain of a desired size.
本発明の実施形態の１つは、式（Ｉ）で示されるチオアセチル基を有する糖鎖を、エステル交換反応により金原子にスルフィド結合させることを特徴とする、金含有物への糖鎖結合方法である。
本発明は、金含有物の表面上に糖鎖を効率よく結合させる技術である。本発明の方法が適用可能な糖鎖は少なくとも１つのチオアセチル基を含むものであれば、特に限定されるものではなく、単糖及び多糖のいずれも含まれる。また、本発明で使用する糖鎖に含まれる水酸基は、エステル系保護基（アセチル基など）によって保護されていてもよい。例えば、本発明によって製造される金含有物、特に、金微粒子を感染症などの診断に使用する場合は、感染微生物又はその毒素と特異的に結合する糖鎖、例えば、腸管出血性大腸菌Ｏ−１５７が産生するベロ毒素と特異的に結合するグロボ３糖（Ｇｂ３、Ｇａｌα１−４Ｇａｌβ１−４Ｇｌｃβ１−）、インフルエンザウィルス等のウィルス表面に存在するヘマグルチニンと特異的に結合するシアリルラクトース、デング熱ウィルスのスパイク糖タンパク質と特異的に結合するラクトネオテトラオース、他には癌細胞の転移に関与しているガングリオシドＧＭ１やアレルギーエピトープの１種であるフコシルキトビオースの等の導入を挙げることができる。
また、式（Ｉ）のＲの部分は、炭化水素鎖あるいはエチレングリコール鎖を表し、その長さは特に限定しないが、例えば、炭素数０〜２０、好ましくは、０〜１５、より好ましくは、０〜１２程度である。また、式（Ｉ）のＲの部分の炭化水素鎖及びエチレングリコール鎖は、直鎖でも枝分かれしていてもよく、また、飽和結合又は不飽和結合のいずれを含んでいてもよい。
金原子に結合させる糖鎖は、限定はしないが、例えば、金原子１に対し、０．１等量〜２０等量、好ましくは、１等量〜５等量である。
One of the embodiments of the present invention is a method for binding a sugar chain to a gold-containing material, characterized in that a sugar chain having a thioacetyl group represented by formula (I) is sulfide-bonded to a gold atom by a transesterification reaction It is.
The present invention is a technique for efficiently binding sugar chains on the surface of a gold-containing material. The sugar chain to which the method of the present invention can be applied is not particularly limited as long as it contains at least one thioacetyl group, and includes both monosaccharides and polysaccharides. Moreover, the hydroxyl group contained in the sugar chain used in the present invention may be protected by an ester-based protecting group (such as an acetyl group). For example, when the gold-containing material produced by the present invention, particularly, gold fine particles are used for diagnosis of infectious diseases, sugar chains that specifically bind to the infecting microorganism or its toxin, such as enterohemorrhagic E. coli O- Globo trisaccharide (Gb3, Galα1-4Galβ1-4Glcβ1-) that specifically binds to verotoxin produced by 157, sialyllactose that specifically binds to hemagglutinin present on the surface of viruses such as influenza virus, spike sugar of dengue virus Other examples include lactoneotetraose that specifically binds to proteins, ganglioside GM1, which is involved in cancer cell metastasis, and fucosyl chitobiose, which is one of allergic epitopes.
In addition, the R portion of the formula (I) represents a hydrocarbon chain or an ethylene glycol chain, and the length thereof is not particularly limited, but for example, 0 to 20 carbon atoms, preferably 0 to 15 carbon atoms, more preferably It is about 0-12. Further, the hydrocarbon chain and the ethylene glycol chain in the R portion of the formula (I) may be linear or branched, and may contain either a saturated bond or an unsaturated bond.
The sugar chain to be bonded to the gold atom is not limited, but is, for example, 0.1 equivalent to 20 equivalents, preferably 1 equivalent to 5 equivalents, relative to the gold atom 1.
本発明において、使用される金含有物とは、金原子がその表面上に存在している物のことであり、金原子のみからなるものでもよく、その他の原子（例えば、Ａｇ、Ｃｕ、Ｆｅなど）、分子を含む化合物であってもよい。金含有物の形状は、特に限定されず、平板状、粒子状などいかなる形状でもよい。また、金含有物の大きさについても、特に限定されず、その用途によって自由に選択することが可能である。例えば、金含有物が金微粒子の場合、細胞に内包させる用途では、１〜１０ｎｍ、好ましくは３ｎｍ以下である。
糖鎖を結合させる反応混合物中から糖鎖が結合した金含有物を分離する方法は、使用する金含有物の大きさ、形状に依存して異なるが、当業者であれば、最適な方法を容易に選択することができる。例えば、金微粒子の場合、反応系の混合物を適当な×ｇで遠心分離して金微粒子のみを取得してもよく、あるいは、ゲル濾過カラムなどで精製してもよい。また、金含有物が平板状であれば、その表面を洗浄することで反応夾雑物と金微粒子を分離することが可能である。さらに、金含有物が金と鉄などとの合金である場合には、磁力により金含有物のみを分離することも可能である。
In the present invention, the gold-containing material used is a material in which gold atoms are present on the surface, and may be composed only of gold atoms, or other atoms (for example, Ag, Cu, Fe Etc.), or a compound containing a molecule. The shape of the gold-containing material is not particularly limited, and may be any shape such as a flat plate shape or a particle shape. Moreover, it does not specifically limit about the magnitude | size of a gold containing material, It is possible to select freely by the use. For example, when the gold-containing material is gold fine particles, it is 1 to 10 nm, preferably 3 nm or less for use in cells.
The method for separating the gold-containing material having the sugar chain bound thereto from the reaction mixture to which the sugar chain is bound differs depending on the size and shape of the gold-containing material to be used. Easy to choose. For example, in the case of gold fine particles, the reaction mixture may be centrifuged at an appropriate xg to obtain only gold fine particles, or may be purified by a gel filtration column or the like. If the gold-containing material is a flat plate, it is possible to separate the reaction contaminants and the gold fine particles by washing the surface. Furthermore, when the gold-containing material is an alloy of gold and iron, it is possible to separate only the gold-containing material by magnetic force.
本発明では、エステル交換反応を利用して、チオアセチル基を有する糖鎖を金原子に結合させることができる。エステル交換反応とは、エステルとアルコールを反応させた場合、各々のアシル基部分が入れ替わる反応である。例えば、ツェンプレン (Zemplen) 法を使用する場合、メタノール中、ナトリウムメトキシドの存在下で反応を行い、アシル基がメタノールへ転移するため、エステル化されていた化合物はアルコールとなる。
ここで使用する金属アルコキシドのカチオンは、特に限定はしないが、例えば、Ｌｉ、Ｋ、Ｎａなどのアルカリ金属、Ｍｇ、Ｃａなどのアルカリ土類金属などが好ましい。また、アルコールとしては、反応条件下で液体であるものであればいかなるものも使用することができ、例えば、メタノール、エタノール、プロパノール、ブタノールなどが好適に使用可能である。
エステル交換反応を行う温度条件としては、例えば、１０℃〜４０℃、好ましくは、１５℃〜３５℃、より好ましくは、２０℃〜２５℃である。なお、使用する糖鎖の溶解性及び溶媒である各アルコールの沸点・粘度との関係を考慮し、液体条件下で反応が進行する温度条件を予備的な実験などにより適宜選択することは、当業者であれば容易である。
In the present invention, a sugar chain having a thioacetyl group can be bonded to a gold atom using a transesterification reaction. The transesterification reaction is a reaction in which each acyl group moiety is replaced when an ester and an alcohol are reacted. For example, when the Zemplen method is used, the reaction is performed in the presence of sodium methoxide in methanol, and the acyl group is transferred to methanol, so that the esterified compound becomes an alcohol.
Although the cation of the metal alkoxide used here is not particularly limited, for example, alkali metals such as Li, K, and Na, and alkaline earth metals such as Mg and Ca are preferable. Any alcohol can be used as long as it is liquid under the reaction conditions. For example, methanol, ethanol, propanol, butanol and the like can be suitably used.
As temperature conditions which perform transesterification, it is 10 to 40 degreeC, for example, Preferably, it is 15 to 35 degreeC, More preferably, it is 20 to 25 degreeC. In consideration of the relationship between the solubility of the sugar chain used and the boiling point / viscosity of each alcohol as a solvent, it is appropriate to appropriately select the temperature conditions under which the reaction proceeds under liquid conditions by preliminary experiments. It is easy for a contractor.
また、反応時間は、例えば、３時間〜１２時間、好ましくは、1時間〜２時間、より好ましくは、１０分〜３０分である。糖鎖がすでに完全に溶解している場合、反応そのものはナトリウムメトキシドなど金属アルコキシド投入後、ごく短時間で終了することが予想されるが、反応開始時に糖鎖が完全に溶解していない場合などは、より長い反応時間が必要とされ、使用する糖鎖、アルコール及びアルコキシドに応じて、最も適した反応時間を予備的な実験により設定することが望ましく、当業者であれば容易に実施可能である。
使用する金属アルコキシドの糖鎖（より具体的には糖鎖に存在する−ＳＡｃ）に対する比率は、限定はしないが、例えば、糖鎖分子１に対し、０．１等量〜２等量、好ましくは１等量〜１．１等量である。使用する金属アルコキシドのアルコールに対する濃度は、例えば、０．０１Ｍ〜０．１０Ｍ、好ましくは、０．０２Ｍ〜０．０５Ｍである。
The reaction time is, for example, 3 hours to 12 hours, preferably 1 hour to 2 hours, and more preferably 10 minutes to 30 minutes. If the sugar chain is already completely dissolved, the reaction itself is expected to be completed in a very short time after adding metal alkoxide such as sodium methoxide, but the sugar chain is not completely dissolved at the start of the reaction. For example, a longer reaction time is required, and it is desirable to set the most suitable reaction time through preliminary experiments depending on the sugar chain, alcohol, and alkoxide to be used. It is.
The ratio of the metal alkoxide to be used to the sugar chain (more specifically, -SAc present in the sugar chain) is not limited, but for example, 0.1 equivalent to 2 equivalents, preferably 1 equivalent to the sugar chain molecule 1. Is 1 equivalent to 1.1 equivalents. The density | concentration with respect to the alcohol of the metal alkoxide to be used is 0.01M-0.10M, for example, Preferably, it is 0.02M-0.05M.
本発明の他の実施形態には、上記実施形態により製造された金含有物、及び、上記実施形態を実施するためのキットが含まれる。
本発明のキットには、所望の糖鎖及び所望の金含有物の他、反応に必要な試薬類（例えば、金属アルコキシド、アルコールなど）などが含まれていてもよい。また、所望の糖鎖の代わりに、該糖鎖を合成するための出発原料となる化合物などが含まれていてもよい。本発明のキットの作製にあたり、糖鎖及び試薬類の安定性を保持し、キット輸送時における安全性を確保するために必要な保存方法及び保存容器が適宜選択される。また、キットに使用説明書が添付される場合、その使用説明は、紙又は他の材質上に印刷されて供給されてもよく、フロッピー（登録商標）ディスク、ＣＤ−ＲＯＭ、ＤＶＤ−ＲＯＭ、Ｚｉｐディスク、ビデオテープ、オーディオテープなどの電気的又は電磁的に読み取り可能な媒体として供給されてもよい。あるいは、キットの製造者によって指定され又は電子メール等で通知されるウェブサイトに掲載されていてもよい。
Other embodiments of the present invention include the gold-containing material produced by the above embodiment and a kit for carrying out the above embodiment.
The kit of the present invention may contain reagents necessary for the reaction (for example, metal alkoxide, alcohol, etc.) in addition to the desired sugar chain and the desired gold-containing material. Further, instead of the desired sugar chain, a compound or the like as a starting material for synthesizing the sugar chain may be included. In producing the kit of the present invention, a storage method and storage container necessary for maintaining the stability of the sugar chain and reagents and ensuring the safety during transportation of the kit are appropriately selected. In addition, when the instruction manual is attached to the kit, the instruction manual may be printed on paper or other material and supplied, such as a floppy (registered trademark) disk, CD-ROM, DVD-ROM, Zip. It may be supplied as an electrically or electromagnetically readable medium such as a disk, video tape, audio tape or the like. Alternatively, it may be posted on a website designated by the manufacturer of the kit or notified by e-mail or the like.
以下に実施例を示してさらに詳細に説明するが、本発明は以下の実施例により何ら限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.
本実施例は、糖鎖の１例としてラクトース誘導体を金ナノ微粒子に結合させる方法を示すものである（図１）。
１．試薬類
（ａ）ＨＡｕＣｌ４・４Ｈ２Ｏ（分子量４１１．８５）（キシダ化学株式会社製 特級）
１０．５ｍｇ （２．５６×１０−５ｍｏｌ）
この内元素としての金が占める重量は、５．０２ ｍｇ（１００ｍｇのＨＡｕＣｌ４・４Ｈ２Ｏ（黄色固体）を１０ｍｌのＨ２Ｏに溶解させた溶液を１．０５ ｍｌ使用した）
（ｂ）ＮａＢＨ４（分子量３７．８３）（東京化成工業株式会社製 純度＞９３．０％）
２１．３ｍｇ （５．６３×１０−４ｍｏｌ）
対Ａｕ比２２等量
（ｃ）ラクトース誘導体（下記化学式を参照のこと）
Ｃ３３Ｈ４８Ｏ１９Ｓ （分子量 ７８０．７９）
１００ｍｇ （１．２８×１０−４ ｍｏｌ）
対Ａｕ比５等量
１．３０×１０−４ｍｏｌ（１ＭのＮａＯＭｅ／ＭｅＯＨ溶液を０．１３ｍｌ使用）
ラクトース誘導体比は等モル。
This example shows a method of binding a lactose derivative to gold nanoparticles as an example of a sugar chain (FIG. 1).
1. Reagents (a) HAuCl 4 · 4H 2 O (molecular weight 411.85) (special grade by Kishida Chemical Co., Ltd.)
10.5 mg (2.56 × 10 −5 mol)
The weight occupied by gold as an inner element was 5.02 mg (1.05 ml of a solution in which 100 mg of HAuCl 4 .4H 2 O (yellow solid) was dissolved in 10 ml of H 2 O was used)
(B) NaBH 4 (molecular weight 37.83) (Tokyo Chemical Industry Co., Ltd., purity> 93.0%)
21.3 mg (5.63 × 10 −4 mol)
22 equivalents to Au ratio (c) Lactose derivative (see chemical formula below)
C 33 H 48 O 19 S (molecular weight 780.79)
100 mg (1.28 × 10 −4 mol)
5 equivalents to Au ratio
1.30 × 10 −4 mol (using 0.13 ml of 1M NaOMe / MeOH solution)
Lactose derivative ratio is equimolar.
２．合成法
２０ｍｌのガラス製サンプル管中のＨＡｕＣｌ４・４Ｈ２Ｏ水溶液１．０５ｍｌ溶液に対し、マグネティックスターラー攪拌下、ＮａＢＨ４水溶液（ＮａＢＨ４ ２１．３ｍｇ／Ｈ２Ｏ ０．５０ｍｌ）を滴下した。金微粒子が生成すると、黄色溶液が瞬時に黒色の溶液へと変化する。そのまま２０分間攪拌を行なった後、ラクトース誘導体１００ｍｇを２ｍｌのＭｅＯＨに溶解させた溶液を滴下した。滴下した直後に、１ＭＮａＯＭｅ溶液０．１３ｍｌを滴下。そのまま室温（約２０℃）で１４時間攪拌した。
生成した金ナノ微粒子を含む溶液をメタノール１０ｍｌに滴下し、エッペンドルフチューブ中・遠心分離で沈殿させた。遠心分離後の上澄みを廃棄した後、さらにメタノールを加えて超音波をかけ微粒子を分散させた。当該エッペンドルフチューブを再度遠心分離にかけた。このメタノール洗浄の操作を５回繰り返して得られた黒色の金ナノ微粒子に、水１ｍｌを加えて凍結乾燥を行い、４．０４ｍｇの糖鎖含有金ナノ微粒子を得た。
金微粒子への糖鎖の導入状態は、重水中の１Ｈ−ＮＭＲで確認し（図２Ａ）、微粒子のサイズ・形状は走査型電子顕微鏡にて観測した（図２Ｂ）。
１Ｈ−ＮＭＲによる観測では、糖鎖（ラクトース）由来の非常にシャープなシグナルが得られ、本発明の方法により金ナノ微粒子上に糖鎖導入が可能であることが確認された。
なお、上記ラクトース誘導体の他、Ｎ−アセチルグルコサミン誘導体も同様の手法で金ナノ微粒子上へ導入可能であることを確認している。
2. Synthesis Method An aqueous NaBH 4 solution (NaBH 4 21.3 mg / H 2 O 0.50 ml) was added dropwise to a 1.05 ml aqueous solution of HAuCl 4 .4H 2 O in a 20 ml glass sample tube while stirring with a magnetic stirrer. When gold fine particles are generated, the yellow solution is instantly changed to a black solution. After stirring as it was for 20 minutes, a solution in which 100 mg of a lactose derivative was dissolved in 2 ml of MeOH was added dropwise. Immediately after the dropwise addition, 0.13 ml of 1M NaOMe solution was dropped. The mixture was stirred at room temperature (about 20 ° C.) for 14 hours.
The solution containing the produced gold nanoparticles was dropped into 10 ml of methanol and precipitated by centrifuging in an Eppendorf tube. After discarding the supernatant after centrifugation, methanol was further added and ultrasonic waves were applied to disperse the fine particles. The Eppendorf tube was centrifuged again. To black gold nanoparticles obtained by repeating this methanol washing operation 5 times, 1 ml of water was added and lyophilized to obtain 4.04 mg of sugar chain-containing gold nanoparticles.
The state of sugar chain introduction into the gold fine particles was confirmed by 1 H-NMR in heavy water (FIG. 2A), and the size and shape of the fine particles were observed with a scanning electron microscope (FIG. 2B).
In observation by 1 H-NMR, a very sharp signal derived from a sugar chain (lactose) was obtained, and it was confirmed that the sugar chain can be introduced onto the gold nanoparticle by the method of the present invention.
In addition, it has been confirmed that in addition to the lactose derivative, an N-acetylglucosamine derivative can be introduced onto the gold nanoparticle by the same method.
本発明によれば、所望の糖鎖を所望の形状及びサイズを有する金含有物上へ結合させることが可能となる。本発明の方法で製造された糖鎖結合金含有物は様々な分野において利用が期待される。特に、本発明により製造された糖鎖結合金ナノ微粒子は、感染症の診断や治療など医療分野における技術開発に対し大きく貢献することが期待できる。 According to the present invention, it is possible to bind a desired sugar chain onto a gold-containing material having a desired shape and size. The sugar chain-bound gold-containing product produced by the method of the present invention is expected to be used in various fields. In particular, the sugar chain-bonded gold nanoparticles produced according to the present invention can be expected to greatly contribute to technological development in the medical field such as diagnosis and treatment of infectious diseases.
Claims (5)
It claims 1 to be produced by the method according to any one of 4, the gold-containing material which has a sugar chain bound.
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