JPH0446143A - Synthesis of isotopic multiply labeled amino acid - Google Patents
Synthesis of isotopic multiply labeled amino acidInfo
- Publication number
- JPH0446143A JPH0446143A JP2150033A JP15003390A JPH0446143A JP H0446143 A JPH0446143 A JP H0446143A JP 2150033 A JP2150033 A JP 2150033A JP 15003390 A JP15003390 A JP 15003390A JP H0446143 A JPH0446143 A JP H0446143A
- Authority
- JP
- Japan
- Prior art keywords
- concentrated
- groups
- amino acid
- isotopic
- ammonium chloride
- 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.)
- Pending
Links
- 150000001413 amino acids Chemical class 0.000 title claims description 30
- 230000000155 isotopic effect Effects 0.000 title abstract 3
- 230000015572 biosynthetic process Effects 0.000 title description 4
- 238000003786 synthesis reaction Methods 0.000 title description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims abstract description 8
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 7
- 235000019270 ammonium chloride Nutrition 0.000 claims abstract description 6
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 5
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 5
- 125000005219 aminonitrile group Chemical group 0.000 claims abstract description 4
- KXZJHVJKXJLBKO-UHFFFAOYSA-N chembl1408157 Chemical compound N=1C2=CC=CC=C2C(C(=O)O)=CC=1C1=CC=C(O)C=C1 KXZJHVJKXJLBKO-UHFFFAOYSA-N 0.000 claims abstract description 4
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 claims abstract 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 6
- 125000003277 amino group Chemical group 0.000 claims description 4
- 238000001308 synthesis method Methods 0.000 claims description 4
- QGZKDVFQNNGYKY-OUBTZVSYSA-N Ammonia-15N Chemical compound [15NH3] QGZKDVFQNNGYKY-OUBTZVSYSA-N 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 230000003301 hydrolyzing effect Effects 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 125000000524 functional group Chemical group 0.000 claims 1
- 239000002994 raw material Substances 0.000 abstract description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 abstract description 10
- 150000001875 compounds Chemical class 0.000 abstract description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 abstract description 4
- 238000006243 chemical reaction Methods 0.000 abstract description 4
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 abstract description 3
- 239000003513 alkali Substances 0.000 abstract description 3
- 239000011734 sodium Substances 0.000 abstract description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 abstract description 2
- 238000002955 isolation Methods 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 229910052708 sodium Inorganic materials 0.000 abstract description 2
- 230000007062 hydrolysis Effects 0.000 abstract 1
- 238000006460 hydrolysis reaction Methods 0.000 abstract 1
- 235000001014 amino acid Nutrition 0.000 description 27
- 238000000034 method Methods 0.000 description 12
- 235000018102 proteins Nutrition 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 238000005481 NMR spectroscopy Methods 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 5
- 235000004279 alanine Nutrition 0.000 description 5
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 4
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 4
- 239000000543 intermediate Substances 0.000 description 4
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- LELOWRISYMNNSU-UHFFFAOYSA-N hydrogen cyanide Chemical compound N#C LELOWRISYMNNSU-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000012916 structural analysis Methods 0.000 description 2
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-OUBTZVSYSA-N Carbon-13 Chemical compound [13C] OKTJSMMVPCPJKN-OUBTZVSYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-NJFSPNSNSA-N Carbon-14 Chemical compound [14C] OKTJSMMVPCPJKN-NJFSPNSNSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000003541 multi-stage reaction Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は同位体で多重に標識されたアミノ酸の合成法に
関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for synthesizing multiple isotopically labeled amino acids.
タンパク質は多数のアミノ酸が結合した、生体内で重要
な機能をもつ巨大分子である。その研究法として、溶液
での核磁気共鳴（ＮＭＲ）スペクトルを測定し、各々の
シグナルをタンパク質を構成するアミノ酸と対応づける
ことにより、構造解析を行なう手法が知られている。Ｎ
ＭＲスペクトルは、プロトン、炭素１３（１３Ｃ）、窒
素１５（１５Ｎ）等の原子核を測定対象とする。このう
ち、プロトンはピークの重なりが大きく、タンパク質等
の複雑な分子への適用は困難であることが多い。Proteins are macromolecules made up of many amino acids that have important functions in living organisms. As a research method for this purpose, a method is known in which structural analysis is performed by measuring a nuclear magnetic resonance (NMR) spectrum in a solution and associating each signal with an amino acid that constitutes a protein. N
The MR spectrum measures atomic nuclei such as protons, carbon-13 (13C), and nitrogen-15 (15N). Among these, protons have large overlapping peaks and are often difficult to apply to complex molecules such as proteins.
また１３Ｃ，１５Ｎは、同位体の天然依存比が、それぞ
れ約１．１％、約０．４％と小さいため、天然依存比の
ままでは充分なシグナル強度が得られないことが多い。Furthermore, since the natural isotope dependence ratios of 13C and 15N are small, about 1.1% and about 0.4%, respectively, sufficient signal intensity cannot often be obtained with the natural dependence ratios.
このため、人工的に１３Ｃ，１５Ｎを濃縮したアミノ酸
を合成し、これを標識化合物として用いてシグナル強度
を大きくする方法が行なわれている。このようなＩ！識
化合物の合成法は種々の例が知られているが１例えばジ
ャーナル オブバイオロジカルケミストリー １６２巻
（１９４６）年第２９７頁から第３０７頁（Ｊ、Ｂｉｏ
ｌ、Ｃｈｅｍ、、 １６２（３９４６）、ｐｐ２９７−
３０７）には、１３Ｃ，”’Ｎで二重標識されたグリシ
ン（” ５Ｎ Ｈ２１” ＣＯＯＨ〕の合成法が記載さ
れている。これは第２式に示したように、フタルイミド
［”Ｎ〕をホルムアルデヒド、塩化チオニル、シアン化
ナトリウム（１３Ｃ］と順に反応させる方法である。For this reason, methods have been used to synthesize amino acids artificially enriched with 13C and 15N and use this as a labeling compound to increase signal intensity. I like this! Various examples of synthetic methods for biochemical compounds are known. For example, there is a method described in Journal of Biological Chemistry, Vol.
l, Chem, 162(3946), pp297-
307) describes a method for synthesizing glycine double-labeled with 13C and ``N'' (``5N H21'' COOH). This is a method in which formaldehyde, thionyl chloride, and sodium cyanide (13C) are reacted in this order.
上記従来技術では、目的の二重標識されたアミノ酸を得
るまでに多段階の反応を必要とし、さらに各段階ごとに
中間体を精製単離する必要があるこのため１反応途中で
の損失が大きく、目的とするアミノ酸の収率が低下し、
高価な同位体濃縮原料が有効に使われないという問題が
あった。The above conventional technology requires a multi-step reaction to obtain the desired double-labeled amino acid, and it is also necessary to purify and isolate the intermediate at each step, resulting in large losses during one reaction. , the yield of the desired amino acid decreases,
There was a problem that expensive isotope enrichment raw materials were not used effectively.
本発明は、１ｆＩＮ及び１３Ｃで二重標識したアミノ酸
を一段階で簡便に合成する方法を提供することを目的と
し、これにより、同位体濃縮原料の損失を小さくするこ
とを目的とする。An object of the present invention is to provide a method for easily synthesizing an amino acid double-labeled with 1fIN and 13C in one step, thereby reducing the loss of isotope-enriched raw materials.
上記目的を達成するために、以下に記した手段を用いた
。In order to achieve the above objective, the means described below were used.
全体の合成系路は第１式に示した。これはシュドレッカ
ー合成として知られるアミノ酸合成法を応用したもので
ある。アミノ酸の前駆体として、ホルムアルデヒド、ア
セトアルデヒド及びその他の各種アルデヒドＲ−ＣＨＯ
（ＲはＨ，ＣＨａ 。The entire synthesis route is shown in Equation 1. This is an application of the amino acid synthesis method known as Schdrecker synthesis. Formaldehyde, acetaldehyde and other various aldehydes R-CHO as precursors of amino acids
(R is H, CHa.
Ｃ２Ｈ５等のアルキル基、フェニル基、及びアミノ基、
カルボキシル基、水酸基等で置換されたアルキル基誘導
体又はフェニル基誘導体）を用いる。Alkyl groups such as C2H5, phenyl groups, and amino groups,
An alkyl group derivative or a phenyl group derivative substituted with a carboxyl group, a hydroxyl group, etc.) is used.
１５Ｎの原料として、ＩＩ）Ｎを濃縮した塩化アンモニ
ウム［１５Ｎ］（”ＮＨ４ＣＰ）を用イル。１３ｃの原
料として１３Ｃを濃縮したシアン化アルカリ（１３（：
）（Ｍ！”ＣＮ、Ｍはカリウム、ナトリウム等のアルカ
リ金属）を用いる。上記のアルデヒドＲ−Ｃ）１０と”
５Ｎ Ｈ４Ｃβ の混合物にＭ１８ＣＮを加えること
により、各種のアミノニトリル［１５Ｎ Ｈ２，１３Ｃ
Ｎ ］（Ｒ−ＣＨ（１３Ｎ ８２）”ＣＮ ）が生成す
る。これを塩酸又は硫酸で加水分解することにより、目
的とする各種の二重ｓＷｔされたアミノ酸（１５ＮＨ２
゜１３ＣＯＯＨ）を得ることができる。得られたアミノ
酸は種々の既知の方法で精製する。As a raw material for 15N, ammonium chloride [15N] (NH4CP), which is concentrated with II)N, is used.As a raw material for 13c, alkali cyanide (13 (:
) (M!"CN, M is an alkali metal such as potassium or sodium) is used.The above aldehyde R-C)10 and"
By adding M18CN to a mixture of 5N H4Cβ, various aminonitriles [15N H2,13C
N](R-CH(13N82)"CN) is produced. By hydrolyzing this with hydrochloric acid or sulfuric acid, various desired double-sWt amino acids (15NH2
゜13COOH) can be obtained. The resulting amino acids are purified by various known methods.
同位体濃縮原料として、塩化アンモニウム、シアン化ア
ルカリは、それぞれ濃縮率が１５Ｎ≧９９％ｌ １３Ｃ
≧９９％であることが理想的であるが、より低い濃縮率
の原料でもかまわない。濃縮率１０％程度でもＮＭＲ測
定に役立てることは充分可能である。As raw materials for isotope enrichment, ammonium chloride and alkali cyanide each have a concentration rate of 15N≧99%l 13C
Ideally, it is ≧99%, but raw materials with lower concentration rates may be used. Even a concentration ratio of about 10% is sufficient to be useful for NMR measurements.
ここで採用したシュドレッカー合成法はアルデヒドから
アミノ酸を合成する簡便な方法で、中間体の精製・単離
は必要ではない。従って操作手順は少なくなり、中間段
階での試料の損失は少なくすることができる。アミノ酸
の前駆体に用いたアルデヒドは比較的安価な原料で入手
しやすい。同位体の原料であル１５Ｎ Ｈ４ＣＤ や
Ｎ ａ ’ ３ＣＣＮ ｔＫ”ＣＮなとは、同位体濃縮
率の高いものが市販されており、安価で入手しやすい。The Schdrecker synthesis method employed here is a simple method for synthesizing amino acids from aldehydes, and does not require purification or isolation of intermediates. There are therefore fewer operating steps and less loss of sample in intermediate steps. The aldehyde used as the amino acid precursor is a relatively inexpensive raw material and easily available. Raw materials for isotopes such as 15N H4CD and N a '3CCN tK''CN are commercially available with high isotope enrichment rates, and are inexpensive and easily available.
（実施例１）
アラニン［” Ｎ Ｈ２、’ ” Ｃ○○Ｈ〕塩酸塩の
合成法を第３図により以下に説明する。(Example 1) A method for synthesizing alanine ["N H2,'" C○○H] hydrochloride will be explained below with reference to FIG.
アセトアルデヒド１３ｇをエーテル１０ｃｃに加入でフ
ラスコ１に入れて氷で冷却する。これに１５ＮＨ４ＣＤ
（”Ｎ２９９％）１８ｇを水５５ｃｃに溶カシテ加える
。次ニＮ ａ ”３ＣＮ （１３Ｃ≧９９％）１５ｇを
水４０ｃｃに溶かし、滴下ロート２がら徐徐に加える。13 g of acetaldehyde was added to 10 cc of ether in flask 1 and cooled with ice. This includes 15NH4CD
Dissolve 18 g of N299% in 55 cc of water. Next, dissolve 15 g of N a 3CN (13C≧99%) in 40 cc of water and slowly add it through dropping funnel 2.
このとき、混合物の温度は１０’Ｃ以下に保つようにす
る。次に混合物を室温にもどし、４時間以上充分かくは
んを続ける。次にフラスコｌをトラフト内に入れ、シア
ン化水素の発生に注意しながら、濃塩酸６０ｃｃを加え
る。蒸留して固型物が残るまで充分に濃縮する。２％の
塩酸を含むエタノール１０．０ｃｃを加えて固型物を溶
がす。At this time, the temperature of the mixture is maintained at 10'C or less. The mixture is then allowed to return to room temperature and stirred thoroughly for at least 4 hours. Next, flask 1 is placed in the trough, and 60 cc of concentrated hydrochloric acid is added while being careful not to generate hydrogen cyanide. Distill and concentrate thoroughly until a solid remains. Add 10.0 cc of ethanol containing 2% hydrochloric acid to dissolve the solids.
冷却後、エーテル３０ｃｃを加え、沈殿する固体を濾別
する。濾液を蒸留し、減圧下でエタノール。After cooling, 30 cc of ether is added and the precipitated solid is filtered off. Distill the filtrate with ethanol under reduced pressure.
エーテルを充分留去させると、目的とするアラニンＣ”
Ｎ Ｈｚ、”Ｃ００Ｈ）塩酸塩の＠製物を得る。When the ether is sufficiently distilled off, the target alanine C”
N Hz, "C00H)" hydrochloride product is obtained.
（実施例２）
実施例１のアセトアルデヒドを他のアルデヒドに変更す
ることにより、各種のアミノ酸（”ＮＨｚ。(Example 2) Various amino acids ("NHz.
”Ｃ００Ｈ）を合成することができる。例を下表にまと
める。"C00H)" can be synthesized. Examples are summarized in the table below.
また、上表に記載されていないアルデヒドについても、
同様の方法で対応するアミノ酸［１５ＮＨ２゜１３ＣＯ
ＯＨ］を合成することができる。Also, regarding aldehydes not listed in the above table,
In a similar manner, the corresponding amino acid [15NH2゜13CO
OH] can be synthesized.
（実施例３） ＮＭＲ測定用試料への応用。(Example 3) Application to samples for NMR measurement.
実施例１又は２で合成したアミノ酸［”ＮＨ２゜１３Ｃ
○○Ｈ〕を原料として、タンパク質を合成する。この合
成は、通常の化学的方法や生物学的方法によって行なう
ことができる。このとき、通常は一種類のアミノ酸（例
えばアラニン）のみを１５Ｎ Ｈ２，１３ＣＯＯＨで標
識したものを使い、他のアミノ酸には通常の未標識のも
のを用いる。合成したタンパク質を水溶液とし、ＮＭＲ
を測定すると、第４図のように、標識されたアラニンの
１５Ｎ Ｈ２，１３Ｃ００Ｈのみが強く観測される。こ
のシグナルの位置、数を解析することにより、試料とし
たタンパク質中のアラニン残基についての構造情報が得
られる。また、標識したアミノ酸を二種類以上（例えば
グアニンとアラニン）用いてタンパク質を合成し、同様
にＮＭＲを測定することもできる。この場合は、タンパ
ク質中のグアニン残基、アラニン残基についての構造情
報が得られ、更にグアニン残基とアラニン残基の間の相
互的位置情報を得ることもできる。例えば第５図のよう
に、細かく分裂した１５Ｎ、１３Ｃシグナルが得られた
時は、標識したアミノ酸どうしが第６図（ｂ）のように
隣りあっていることを示す。The amino acid synthesized in Example 1 or 2 [”NH2゜13C
Synthesize protein using ○○H] as a raw material. This synthesis can be carried out by conventional chemical or biological methods. At this time, usually only one type of amino acid (for example, alanine) is labeled with 15N H2,13COOH, and the other amino acids are normally unlabeled. The synthesized protein is made into an aqueous solution and NMR
As shown in FIG. 4, only the labeled alanine 15N H2, 13C00H is strongly observed. By analyzing the position and number of these signals, structural information about the alanine residues in the sample protein can be obtained. It is also possible to synthesize a protein using two or more types of labeled amino acids (for example, guanine and alanine) and measure NMR in the same manner. In this case, structural information about guanine residues and alanine residues in the protein can be obtained, and furthermore, mutual positional information between guanine residues and alanine residues can also be obtained. For example, as shown in Figure 5, when finely divided 15N and 13C signals are obtained, it indicates that the labeled amino acids are adjacent to each other as shown in Figure 6(b).
また、タンパク質以外でも、アミノ酸を構成要素とする
化学物質であるならば、アミノ酸［：”ＮＨ２，１３Ｃ
ＯＯＨ）はＮＭＲの測定の対象として使用することがで
き、構造解析に役立てることができる。In addition to proteins, if it is a chemical substance whose constituent elements are amino acids, amino acids [:”NH2, 13C
OOH) can be used as a target for NMR measurement and can be useful for structural analysis.
（実施例４）
本発明で合成した標識アミノ酸はＮＭＲに限らず、同位
体を利用する他の分析方法の測定対象へ応用することが
可能である。(Example 4) The labeled amino acid synthesized according to the present invention can be applied not only to NMR but also to measurement targets of other analysis methods that utilize isotopes.
例えば、質量スペクトルに用いて、天然同位体存在比か
らのズレを測定して分析に用いることができる。For example, it can be used in a mass spectrum to measure the deviation from the natural isotope abundance ratio and use it for analysis.
また、Ｎａ１３ＣＮやＫ１３ＣＮのかわりに炭素１４を
濃縮したＮａ”ＣＮやＫ”ＣＮを用いて、１４Ｃの放射
能測定による放射分析へ応用することができる。必要に
応じて、窒素、炭素を更に短寿命の核種（例えば１１Ｃ
，１３Ｎなど）で標識し、放射分析を行なう方法へも応
用できる。Furthermore, by using Na''CN or K''CN, which are enriched with carbon-14, instead of Na13CN or K13CN, it can be applied to radioanalysis by measuring the radioactivity of 14C. If necessary, nitrogen and carbon may be replaced with shorter-lived nuclides (e.g. 11C).
, 13N, etc.) and can be applied to a method of radioanalysis.
本発明によれば、以上に説明したように、アミノ基を１
５Ｎで、カルボキシル基を１３Ｃで同時に標識した各種
のアミノ酸を簡便に合成することができる。また、中間
での処理が少ないので、高価な同位体濃縮原料の損失を
少なくし、目的のアミノ酸（”Ｎ Ｈ２，”ＣＯＯＨ）
の収率を上げることができる。According to the present invention, as explained above, the amino group is
5N and various amino acids whose carboxyl groups are simultaneously labeled with 13C can be easily synthesized. In addition, because there is less intermediate processing, the loss of expensive isotope enrichment raw materials is reduced, and the desired amino acids ("NH2," COOH) are
yield can be increased.
第１図は本発明を説明する反応式第１式を示す図、第２
図は従来技術を説明する反応式を示す図、第３図は本発
明による合成方法を示す図、第４図は本発明により合成
したアミノ酸（１５ＮＨ２゜”Ｃ００ＨＩをＮＭＲ測定
に応用したときのスペクトル図、第５図は第４図と同じ
＜ＮＭＲスペクトル図、第６図はタンパク質中のアミノ
酸の結合を示す図である。
１・・フラスコ、２・・滴下ロート、３・・・温度計、
４・・氷水浴、５・・磁気かくはん子、６・・・かくは
ん機、第
図
ＮＨ４ＣＬ Ｍ′３ＣＮ
尺ｃｔ−ｔ。
第
図
ＲＣＨ（ＩＳＮＨ２）”ｃｏｏＨ
第３図
第４図
第５図
（ｂ）
身
一一一一、−ｍ−
と
第
図Figure 1 is a diagram showing the first reaction equation explaining the present invention, and the second
The figure shows a reaction formula explaining the prior art, Figure 3 shows the synthesis method according to the present invention, and Figure 4 shows the spectrum of the amino acid synthesized according to the present invention (15NH2゜"C00HI) when applied to NMR measurement. Figure 5 is the same as Figure 4. Figure 6 is a diagram showing the bonding of amino acids in proteins. 1. Flask, 2. Dropping funnel, 3. Thermometer,
4. Ice water bath, 5. Magnetic stirrer, 6. Stirrer, Figure NH4CL M'3CN length ct-t. Figure RCH (ISNH2) "cooH Figure 3 Figure 4 Figure 5 (b) Body 1111, -m- and Figure
Claims (1)
＿５等のアルキル基、フェニル基、及びアミノ基・水酸
基・カボキシル基等の官能基で置換されたアルキル基誘
導体又はフェニル基誘導体）で表わされる各種アルデヒ
ドに、窒素１５を濃縮した塩化アンモニアムと、炭素１
３を濃縮したシアン化ナトリウム又はシアン化カリウム
を反応させ、生成する各種のアミノニトリル Ｒ−ＣＨ（＾１＾５ＮＨ＿２）＾１＾３ＣＮを加水分解
することにより、アミノ基を窒素１５で、カルボキシル
基を炭素１３で、同時に標識したアミノ酸 Ｒ−ＣＨ（＾１＾５ＮＨ＿２）＾１＾３ＣＯＯＨを合成
することを特徴とする同位体多重標識アミノ酸合成法。[Claims] 1. Chemical formula R-CHO (R is H, CH_3 or C_2H
Ammonium chloride with nitrogen 15 concentrated in various aldehydes represented by alkyl groups such as _5, phenyl groups, and alkyl group derivatives or phenyl group derivatives substituted with functional groups such as amino groups, hydroxyl groups, carboxyl groups, etc. carbon 1
By reacting 3 with concentrated sodium cyanide or potassium cyanide and hydrolyzing the various aminonitrile R-CH (^1^5NH_2)^1^3CN, the amino group is converted into nitrogen 15 and the carboxyl group is converted into carbon. 13, an isotope multiple labeled amino acid synthesis method characterized by simultaneously synthesizing labeled amino acids R-CH(^1^5NH_2)^1^3COOH.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2150033A JPH0446143A (en) | 1990-06-11 | 1990-06-11 | Synthesis of isotopic multiply labeled amino acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2150033A JPH0446143A (en) | 1990-06-11 | 1990-06-11 | Synthesis of isotopic multiply labeled amino acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0446143A true JPH0446143A (en) | 1992-02-17 |
Family
ID=15488032
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2150033A Pending JPH0446143A (en) | 1990-06-11 | 1990-06-11 | Synthesis of isotopic multiply labeled amino acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0446143A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6111066A (en) * | 1997-09-02 | 2000-08-29 | Martek Biosciences Corporation | Peptidic molecules which have been isotopically substituted with 13 C, 15 N and 2 H in the backbone but not in the sidechains |
| EP1191033A3 (en) * | 1993-02-05 | 2003-07-30 | Martek Biosciences Corporation | Compositions and methods for protein structural determinations |
| JP2005314398A (en) * | 2004-03-30 | 2005-11-10 | Wako Pure Chem Ind Ltd | Heavy-nitrogenized nitrobenzenesulfonamide, its derivative and production method thereof |
| JP2011502920A (en) * | 2007-11-06 | 2011-01-27 | コミサリア ア レネルジィ アトミーク エ オ エネルジィ アルタナティブ | Method for radiolabeling carbon nanotubes, radiolabeled carbon nanotubes, and applications thereof |
| JP2018054622A (en) * | 2017-11-20 | 2018-04-05 | 国立大学法人京都大学 | Probe for multiple resonance |
| JP2019048774A (en) * | 2017-09-08 | 2019-03-28 | 住友化学株式会社 | Manufacturing method of methionine and/or 2-hydroxy-4-(methylthio)butanoic acid |
-
1990
- 1990-06-11 JP JP2150033A patent/JPH0446143A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1191033A3 (en) * | 1993-02-05 | 2003-07-30 | Martek Biosciences Corporation | Compositions and methods for protein structural determinations |
| EP1193271A3 (en) * | 1993-02-05 | 2004-03-24 | Martek Biosciences Corporation | Compositions and methods for protein structural determinations |
| US6111066A (en) * | 1997-09-02 | 2000-08-29 | Martek Biosciences Corporation | Peptidic molecules which have been isotopically substituted with 13 C, 15 N and 2 H in the backbone but not in the sidechains |
| US6335196B1 (en) | 1997-09-02 | 2002-01-01 | Martek Biosciences Corp. | 13C,15N ,2H labeled proteins for NMR structure determinations and their preparation |
| US6340578B1 (en) | 1997-09-02 | 2002-01-22 | Martek Biosciences Corporation | 13C, 15N, 2H labeled proteins for NMR structure determinations and their preparation |
| US6376253B1 (en) | 1997-09-02 | 2002-04-23 | Martek Biosciences Corporation | 13C, 15N, 2H labeled proteins for NMR structure determinations and their preparation |
| JP2005314398A (en) * | 2004-03-30 | 2005-11-10 | Wako Pure Chem Ind Ltd | Heavy-nitrogenized nitrobenzenesulfonamide, its derivative and production method thereof |
| JP2011502920A (en) * | 2007-11-06 | 2011-01-27 | コミサリア ア レネルジィ アトミーク エ オ エネルジィ アルタナティブ | Method for radiolabeling carbon nanotubes, radiolabeled carbon nanotubes, and applications thereof |
| JP2019048774A (en) * | 2017-09-08 | 2019-03-28 | 住友化学株式会社 | Manufacturing method of methionine and/or 2-hydroxy-4-(methylthio)butanoic acid |
| JP2018054622A (en) * | 2017-11-20 | 2018-04-05 | 国立大学法人京都大学 | Probe for multiple resonance |
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