JP5229700B2 - Novel fluorescent compound and method for detecting intracellular cholesterol using the same - Google Patents
Novel fluorescent compound and method for detecting intracellular cholesterol using the same Download PDFInfo
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- FMMNDASGPPDFJA-UHFFFAOYSA-N CC(CCC(NC)=O)C(CC1)C(C)(CC2)C1C1C2C(C)(CCC(C2)O)C2=CC1 Chemical compound CC(CCC(NC)=O)C(CC1)C(C)(CC2)C1C1C2C(C)(CCC(C2)O)C2=CC1 FMMNDASGPPDFJA-UHFFFAOYSA-N 0.000 description 1
- WLRJXZUKHMWWPZ-UHFFFAOYSA-N CC(CCC(NCCN=S(c1c(cccc2N(C)C)c2ccc1)=O)=O)C(CC1)C(C)(CC2)C1C1C2C(C)(CCC(C2)O)C2=CC1 Chemical compound CC(CCC(NCCN=S(c1c(cccc2N(C)C)c2ccc1)=O)=O)C(CC1)C(C)(CC2)C1C1C2C(C)(CCC(C2)O)C2=CC1 WLRJXZUKHMWWPZ-UHFFFAOYSA-N 0.000 description 1
- HIAJCGFYHIANNA-UHFFFAOYSA-N CC(CCC(O)=O)C(CC1)C(C)(CC2)C1C1C2C(C)(CCC(C2)O)C2=CC1 Chemical compound CC(CCC(O)=O)C(CC1)C(C)(CC2)C1C1C2C(C)(CCC(C2)O)C2=CC1 HIAJCGFYHIANNA-UHFFFAOYSA-N 0.000 description 1
- OQDZYCWSQGVRTD-UHFFFAOYSA-N CC(CCC([N+](CCC1)(C1=O)[O-])=O)C(CC1)C(C)(CC2)C1C1C2C(C)(CCC(C2)O)C2=CC1 Chemical compound CC(CCC([N+](CCC1)(C1=O)[O-])=O)C(CC1)C(C)(CC2)C1C1C2C(C)(CCC(C2)O)C2=CC1 OQDZYCWSQGVRTD-UHFFFAOYSA-N 0.000 description 1
- VLLCDGLVBCJGJM-SEMRAHOQSA-N CC1[C@@H](CN(C)c2c(cccc3C(S(NCCN)=O)=O)c3ccc2)C1 Chemical compound CC1[C@@H](CN(C)c2c(cccc3C(S(NCCN)=O)=O)c3ccc2)C1 VLLCDGLVBCJGJM-SEMRAHOQSA-N 0.000 description 1
- VUNUZOAWZLFHGW-UHFFFAOYSA-N CN(C)c1cccc2c1cccc2C(OCCl)S=O Chemical compound CN(C)c1cccc2c1cccc2C(OCCl)S=O VUNUZOAWZLFHGW-UHFFFAOYSA-N 0.000 description 1
- FOPMVXKRQHOLDC-UHFFFAOYSA-N ONC(CCC=O)=O Chemical compound ONC(CCC=O)=O FOPMVXKRQHOLDC-UHFFFAOYSA-N 0.000 description 1
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Description
本発明は新規蛍光化合物に関する。本発明はまた、当該化合物を用いた細胞内コレステロール検出法および検出キットに関する。 The present invention relates to a novel fluorescent compound. The present invention also relates to an intracellular cholesterol detection method and detection kit using the compound.
生体内のコレステロールの分布や動態を解析する上で、蛍光分光法は、高感度な分光法のため有用である。現在では、共焦点レーザー顕微鏡、二光子励起蛍光顕微鏡など測定機器が飛躍的に進歩し、高い時空間分解で分布や動態を解析することができる。しかしながらコレステロールは蛍光を示さないため、これらの技術を用いるためには、コレステロールに蛍光分子を標識させる必要があり、そのような蛍光性コレステロールが開発されている。ここで、重要な要素として、蛍光団を結合させてもコレステロール本来の性質を保持させなければならないことが挙げられる。
以下、これまで開発されている蛍光性コレステロールおよびそれらの問題点を示す。デヒドロエルゴステロール（下記化合物Ａ）はコレステロールに分子構造が類似しているため内因性コレステロールに近い性質を有する。しかしながら，蛍光強度が弱いため精度の高い解析が困難である。Molecular Probes社（米国）において、コレステロールの３位のヒドロキシル基をエステル化して蛍光団を組み込んだ化合物（下記化合物Ｂ）が販売されている。しかしながら、細胞内に存在するコレステロールは３位のヒドロキシル基が遊離して存在しており、細胞内コレステロールの動態解析には不向きである。また、同社はコレステロールの２２位にニトロベンゾフラザンを置換した化合物（下記化合物Ｃ）を市販している。しかしながら、化合物Ｃはミトコンドリアの膜に特異的に集積してしまいコレステロールの性質を保持していないことが報告されている[非特許文献1]。近年、コレステロールの７位にダンシルヒドラゾンを結合させた化合物（下記化合物Ｄ）が開発されている。Ｄは内因性コレステロールの性質に近いことが報告されており、蛍光性コレステロールアナログとして使用されている[非特許文献２]。
Hereinafter, fluorescent cholesterols developed so far and their problems will be shown. Dehydroergosterol (compound A below) has properties similar to endogenous cholesterol because of its molecular structure similar to cholesterol. However, since the fluorescence intensity is weak, high-precision analysis is difficult. Molecular Probes (USA) sells a compound (compound B below) in which a fluorophore is incorporated by esterifying the hydroxyl group at the 3-position of cholesterol. However, cholesterol present in cells is present in a state where the hydroxyl group at position 3 is liberated, and is not suitable for analyzing the dynamics of intracellular cholesterol. In addition, the company sells a compound in which nitrobenzofurazan is substituted at the 22-position of cholesterol (compound C below). However, it has been reported that Compound C specifically accumulates in the mitochondrial membrane and does not retain the properties of cholesterol [Non-patent Document 1]. In recent years, a compound in which dansyl hydrazone is bonded to the 7-position of cholesterol (compound D below) has been developed. D is reported to be close to the nature of endogenous cholesterol, and is used as a fluorescent cholesterol analog [Non-Patent Document 2].
本発明は、細胞内コレステロールの検出などに有用な、新規な蛍光化合物を提供することを課題とする。 An object of the present invention is to provide a novel fluorescent compound useful for detection of intracellular cholesterol and the like.
本発明者は上記課題を解決すべく鋭意検討を行った。その結果、下記一般式（Ｉ）で表される蛍光化合物（以下、本発明の化合物とも呼ぶ）を合成することに成功し、さらに、当該化合物が細胞内でコレステロールと同様の挙動を示すことを見出して本発明を完成するに至った。 The present inventor has intensively studied to solve the above problems. As a result, the inventors succeeded in synthesizing a fluorescent compound represented by the following general formula (I) (hereinafter also referred to as the compound of the present invention), and that the compound exhibits the same behavior as cholesterol in cells. As a result, the present invention has been completed.
すなわち、本発明は以下の通りである。
（１）下記一般式（I）で表される化合物。
（２）ｎが２であり、ｍが０である、（１）の化合物。
（３）（１）または（２）の化合物を細胞に添加し、蛍光を測定することを特徴とする、細胞内コレステロールの検出方法。
（４）前記化合物をmethyl-β-cyclodextrinとともに溶解して細胞に添加することを特徴とする、（３）の方法。
（５）（１）または（２）の化合物を含む、コレステロール検出キット。
（６）さらにmethyl-β-cyclodextrin を含む、（５）のキット。
That is, the present invention is as follows.
(1) A compound represented by the following general formula (I).
(2) The compound of (1), wherein n is 2 and m is 0.
(3) A method for detecting intracellular cholesterol, comprising adding the compound of (1) or (2) to a cell and measuring fluorescence.
(4) The method according to (3), wherein the compound is dissolved with methyl-β-cyclodextrin and added to cells.
(5) A cholesterol detection kit comprising the compound of (1) or (2).
(6) The kit according to (5), further comprising methyl-β-cyclodextrin.
本発明の蛍光化合物は、細胞内に取り込まれ、コレステロールと同様の挙動を示すため、細胞内コレステロールの検出に好適に使用することができる。例えば、細胞内のコレステロール含有小胞の同定など、細胞内コレステロール分布の測定などに使用することができる。同化合物は細胞に添加してもアポトーシスなどの悪影響を起こしにくいという利点も有している。
Since the fluorescent compound of the present invention is taken into cells and exhibits the same behavior as cholesterol, it can be suitably used for detection of intracellular cholesterol. For example, it can be used for measurement of intracellular cholesterol distribution, such as identification of intracellular cholesterol-containing vesicles. The compound also has the advantage that it does not cause adverse effects such as apoptosis even when added to cells.
以下に本発明を詳しく説明する。
本発明の化合物は、以下の一般式（I）で表される。
この中では、ｎが２であり、ｍが０である、下記の化合物が特に好ましい。
The compound of the present invention is represented by the following general formula (I).
Among these, the following compounds in which n is 2 and m is 0 are particularly preferable.
この化合物は下記の合成方法によって合成することができる。なお、ｎが２であり、ｍが０である化合物以外の化合物も原料を代えることによって同様にして合成することがで
きる。
本発明の化合物は、蛍光を発する。したがって、蛍光標識剤や蛍光プローブとして使用することができる。
特に、本発明の化合物は、細胞内に取り込まれ、コレステロールと同様の挙動を示すため、細胞内コレステロールの検出に好適に使用することができる。
The compounds of the present invention fluoresce. Therefore, it can be used as a fluorescent labeling agent or a fluorescent probe.
In particular, since the compound of the present invention is taken up into cells and exhibits the same behavior as cholesterol, it can be suitably used for detection of intracellular cholesterol.
具体的には、本発明の化合物を細胞に添加し、蛍光顕微鏡などで蛍光を測定することによって、細胞内コレステロールの分布などを検出することができる。
検出対象の細胞の種類は特に制限されないが、コレステロールを蓄積する培養細胞が好ましい。
細胞に化合物を添加する場合、本発明の化合物を単独で添加してもよいし、同化合物の溶解を助ける働きをする他の化合物とともに添加してもよい。
本発明の化合物を単独で添加する場合、例えば、同化合物をDMSOなどに溶解させることができる。また、本発明の化合物を水に溶解しやすくするため、methyl-β-cyclodextrinとともに水溶液にしてもよい。
本発明の化合物は培養細胞の培地中に加えることができるが、その濃度は細胞の種類によっても異なるが、好ましくは、０．５μＭ〜５０μＭである。
蛍光顕微鏡などの蛍光測定装置を使用することによって本発明の化合物による蛍光を検出することができる。
なお、本発明の化合物を認識する抗体を用いて検出することも可能である。そのような抗体としては、本発明の化合物に含まれるダンシル基に対する抗体が挙げられる。
Specifically, the distribution of intracellular cholesterol and the like can be detected by adding the compound of the present invention to cells and measuring the fluorescence with a fluorescence microscope or the like.
The type of cells to be detected is not particularly limited, but cultured cells that accumulate cholesterol are preferred.
When the compound is added to the cells, the compound of the present invention may be added alone, or may be added together with other compounds that function to help dissolve the compound.
When the compound of the present invention is added alone, for example, the compound can be dissolved in DMSO or the like. Moreover, in order to make the compound of this invention easy to melt | dissolve in water, you may make it into aqueous solution with methyl- (beta) -cyclodextrin.
Although the compound of this invention can be added to the culture medium of a cultured cell, The density | concentration changes with cell types, Preferably, it is 0.5 micromol-50 micromol.
Fluorescence due to the compound of the present invention can be detected by using a fluorescence measuring device such as a fluorescence microscope.
It is also possible to detect using an antibody that recognizes the compound of the present invention. Examples of such antibodies include antibodies against dansyl groups contained in the compounds of the present invention.
本発明はまた、本発明の化合物を含むコレステロール検出キットに関する。該キットは、本発明の化合物を溶解するための溶媒や、methyl-β-cyclodextrinなどの、本発明の化合物の溶解を助ける働きをする物質をさらに含むものであってもよい。また、本発明の化合物に対する抗体を含むものであってもよい。
The present invention also relates to a cholesterol detection kit comprising the compound of the present invention. The kit may further contain a solvent that dissolves the compound of the present invention and a substance that functions to assist the dissolution of the compound of the present invention, such as methyl-β-cyclodextrin. Moreover, the antibody with respect to the compound of this invention may be included.
以下に実施例を示し、本発明をさらに具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。なお、以下の実施例では、上記一般式（Ｉ）においてｍ＝０、ｎ＝２の化合物をDCho24と呼ぶ。 The following examples illustrate the present invention more specifically. However, the present invention is not limited to the following examples. In the following examples, a compound having m = 0 and n = 2 in the general formula (I) is referred to as DCho24.
実施例１：DCho24の合成
1-ダンシルスルホンアミド-2-アミノエタン（1）
ダンシルクロリド（312 mg, 1.16 mol）を無水ジクロロメタン（8 ml）に溶解させ，エチレンジアミン（0.4 ml, 6 mmol）を加え，室温で1時間攪拌した。反応液にジクロロメタン（100 ml）加え，水で2回洗浄した。有機層をNa2SO4で乾燥させエバポレーターで減圧乾固させた（収量：320 mg, 収率：94 %）。
Example 1: Synthesis of DCho24
1-dansylsulfonamido-2-aminoethane ( 1 )
Dansyl chloride (312 mg, 1.16 mol) was dissolved in anhydrous dichloromethane (8 ml), ethylenediamine (0.4 ml, 6 mmol) was added, and the mixture was stirred at room temperature for 1 hour. Dichloromethane (100 ml) was added to the reaction solution and washed twice with water. The organic layer was dried over Na 2 SO 4 and evaporated to dryness using an evaporator (yield: 320 mg, yield: 94%).
3β-ヒドロキシ-Δ5-コレン酸（0.201 mg, 0.54 mmol），N-ヒドロキシコハク酸イミド（130 mg, 0.65 mmol）を無水テトラヒドロフラン（15 ml）に溶解させ，氷浴で0℃に冷やす。この溶液にジシクロヘキシルカルボジイミド（130 mg, 0.63 mmol）加え，0℃で1時間，室温で24時間攪拌した。この溶液に1-ダンシルスルホンアミド-2-アミノエタン（1）（157 mg, 0.66 mmol）を加え，室温で24時間攪拌した。反応液をろ過し，N,N'-ジシクロヘキシル尿素を取り除き，ろ液にジクロロメタン（100 ml）を加え，水で2回洗浄した。有機層をNa2SO4で乾燥させエバポレーターで減圧乾固させ，カラムクロマトグラフィー（充填剤：シリカゲル，展開溶媒：クロロホルム：酢酸エチル（7：3））を用いて単離した（収量：180 mg，収率：51 %）。
1H HNR (300 MHz, CDCl3, TMS, RT)：δ8.56-7.18 (6H, m, Ar-H of naphthalene), 5.78
(1H, s, -C(=O)-NH-), 5.30 (1H, m, -C=H-CH2-), 3.53 (1H, m, -CH2-C(OH)H-CH2-), 3.30-3.01 (4H, m, -NH-CH2-CH2-NH-), 2.89 (6H, s, -N(CH3)2), 2.23-0.68 (34H, m, cholesterol moiety)
Dissolve 3β-hydroxy-Δ 5 -cholenoic acid (0.201 mg, 0.54 mmol) and N-hydroxysuccinimide (130 mg, 0.65 mmol) in anhydrous tetrahydrofuran (15 ml) and cool to 0 ° C in an ice bath. To this solution was added dicyclohexylcarbodiimide (130 mg, 0.63 mmol), and the mixture was stirred at 0 ° C. for 1 hour and at room temperature for 24 hours. To this solution was added 1-dansylsulfonamido-2-aminoethane ( 1 ) (157 mg, 0.66 mmol), and the mixture was stirred at room temperature for 24 hours. The reaction solution was filtered to remove N, N′-dicyclohexylurea, dichloromethane (100 ml) was added to the filtrate, and the mixture was washed twice with water. The organic layer was dried over Na 2 SO 4 , evaporated to dryness with an evaporator, and isolated using column chromatography (filler: silica gel, developing solvent: chloroform: ethyl acetate (7: 3)) (yield: 180 mg Yield: 51%).
1 H HNR (300 MHz, CDCl 3 , TMS, RT): δ8.56-7.18 (6H, m, Ar- H of naphthalene), 5.78
(1H, s, -C (= O) -N H- ), 5.30 (1H, m, -C = H -CH 2- ), 3.53 (1H, m, -CH 2 -C (OH) H -CH 2- ), 3.30-3.01 (4H, m, -NH-CH 2 -CH 2 -NH-), 2.89 (6H, s, -N (CH 3 ) 2 ), 2.23-0.68 (34H, m, cholesterol moiety )
実施例２：DCho24の吸収・蛍光スペクトル
図１にDCho6（前出の化合物Ｄ），DCho24のエタノール中における吸収および蛍光スペクトルを示す。これらの化合物は，ダンシル基を有しているため，340nm付近に吸収極大波長を示す。蛍光極大波長は，DCho6では527nm，DCho24では526nmであり，いずれも緑色蛍光が観測される。蛍光量子収率（Φf）は，DCho6では0.52，DCho24では0.59である。DCho24は，DCho6よりも蛍光量子収率が大きいため，蛍光顕微鏡下での観測に有利である。
Example 2: Absorption / fluorescence spectrum of DCho24 FIG. 1 shows absorption and fluorescence spectra of DCho6 (compound D mentioned above) and DCho24 in ethanol. Since these compounds have a dansyl group, they exhibit an absorption maximum wavelength around 340 nm. The fluorescence maximum wavelength is 527 nm for DCho6 and 526 nm for DCho24, and green fluorescence is observed in both cases. The fluorescence quantum yield (Φ f ) is 0.52 for DCho6 and 0.59 for DCho24. DCho24 is advantageous for observation under a fluorescence microscope because it has a higher fluorescence quantum yield than DCho6.
実施例３：培養細胞での蛍光測定
マウス膵β細胞由来MIN6細胞に75μM（溶媒DMSO）でdansyl-cholesterol probe (DCho6, DCho24)を添加して、5分、30分、1時間、2時間培養し、蛍光顕微鏡で各プローブの自家蛍光を観察した。その結果、図２に示されるように、DCho24は細胞内に取り込まれ、公知のコレステロールプローブであるDCho6よりも強い蛍光を示すことがわかった。
Example 3: Measurement of fluorescence in cultured cells Add dansyl-cholesterol probe (DCho6, DCho24) at 75 μM (solvent DMSO) to mouse pancreatic β cell-derived MIN6 cells, and culture for 5 minutes, 30 minutes, 1 hour, 2 hours The autofluorescence of each probe was observed with a fluorescence microscope. As a result, as shown in FIG. 2, it was found that DCho24 was taken up into the cells and showed stronger fluorescence than DCho6 which is a known cholesterol probe.
次に、マウスマクロファージ細胞由来RAW264.7細胞において、75μM（溶媒DMSO）でdansyl-cholesterol probe (DCho6, DCho24)を添加して5分、30分、1時間、2時間培養し、蛍光顕微鏡で各プローブの自家蛍光を観察した。その結果、図３に示されるように、RAW264.7細胞においてもDCho24は細胞内に取り込まれ、公知のコレステロールプローブであるDCho6よりも強い蛍光を示すことがわかった。 Next, in mouse macrophage cell-derived RAW264.7 cells, dansyl-cholesterol probe (DCho6, DCho24) was added at 75 μM (solvent DMSO) and cultured for 5 minutes, 30 minutes, 1 hour, 2 hours, The autofluorescence of the probe was observed. As a result, as shown in FIG. 3, it was found that DCho24 was taken up into cells even in RAW264.7 cells and showed stronger fluorescence than DCho6, which is a known cholesterol probe.
次に、マウスマクロファージ細胞由来RAW264.7細胞に5 μMのmethyl-β-cyclodextrin（MβCD）とdansyl-cholesterol probe (DCho24)の複合体（溶媒PBS）を添加して5分間培養し、同様に10分間添加培養したナイルレッド（細胞内脂質滴マーカー：SIGMA）との局在を顕微鏡で比較した。
結果を図４に示す。細胞内コレステロールはエステル化され脂質滴に貯えられるため、ナイルレッドで染色されるが、DCho24も同様の局在を示した。
Next, 5 μM methyl-β-cyclodextrin (MβCD) and dansyl-cholesterol probe (DCho24) complex (solvent PBS) was added to RAW264.7 cells derived from mouse macrophage cells and incubated for 5 minutes. The localization with Nile Red (intracellular lipid droplet marker: SIGMA) added and cultured for 1 minute was compared with a microscope.
The results are shown in FIG. Intracellular cholesterol is esterified and stored in lipid droplets, so it is stained with Nile Red, but DCho24 showed similar localization.
次に、マウスマクロファージ細胞由来RAW264.7細胞に5 μMのmethyl-β-cyclodextrinとdansyl-cholesterol probe (DCho24)の複合体（溶媒PBS）を添加して5分間培養し、同様に10分間添加培養したナイルレッドとの局在を、コレステロールエステル合成阻害剤（ACAT阻害剤：SIGMA58-035）存在下（＋）、非存在下（−）で比較した。結果を図５に示す。ACAT阻害剤はコレステロールの3位のアシルCoAから脂肪酸を転移し、コレステロールエステル合成を触媒する細胞内小胞体酵素阻害剤で、この阻害剤存在下ではコレステロールはエステル化されず、脂質滴に局在できないが、DCho24も同様の挙動を示した。 Next, 5 μM methyl-β-cyclodextrin and dansyl-cholesterol probe (DCho24) complex (solvent PBS) was added to RAW264.7 cells derived from mouse macrophage cells and cultured for 5 minutes. Localization with Nile Red was compared in the presence (+) and absence (-) of a cholesterol ester synthesis inhibitor (ACAT inhibitor: SIGMA58-035). The results are shown in FIG. ACAT inhibitor is an intracellular endoplasmic reticulum enzyme inhibitor that transfers fatty acid from acyl CoA at the 3-position of cholesterol and catalyzes cholesterol ester synthesis. In the presence of this inhibitor, cholesterol is not esterified and is localized in lipid droplets. Although DCho24 did not show the same behavior.
実施例４：細胞毒性試験
チャイニーズハムスター卵巣由来CHO細胞に10 μg/mlで種々の蛍光プローブ（DCho6, DCho24, dehydroergosterol (DHE), 22-NBD-cholesterol (NBD-chol), 7-ketocholesterol）を添加して培養し、24時間後に蛍光プローブ非存在下（control）との細胞毒性（アポトーシス）を比較した。具体的には、添加２４時間後に、Hoechst 33342 (HO342)とPI試薬（プロピジウムアイオダイド）でアポトーシスが起こっている細胞数をカウントとした。HO342 はUV励起可能な核酸染色色素で青色蛍光を示し、アポトーシス細胞の凝縮された核では特に明るい蛍光を発する。一方、PI は原形質膜が損傷している細胞にのみ取り込まれる。このふたつの核酸染色剤を同時に使用した時の染色パターンから、健康な細胞群、アポトーシス細胞群、死細胞群を蛍光顕微鏡法で区別し、カウントした。
なお、DHE, NBD-cholは既存のコレステロール類似蛍光試薬である。7-ketocholesterolはアポトーシスのコントロールとして加えた。
結果を図６に示した。グラフのバーが大きいほど細胞毒性が高いことを示す。
その結果、DCho24は既存の蛍光プローブよりも細胞毒性が少ないことがわかった。
Example 4: Cytotoxicity test Various fluorescent probes (DCho6, DCho24, dehydroergosterol (DHE), 22-NBD-cholesterol (NBD-chol), 7-ketocholesterol) were added to CHO cells derived from Chinese hamster ovary at 10 μg / ml. After 24 hours, the cytotoxicity (apoptosis) was compared with that in the absence of the fluorescent probe (control) after 24 hours. Specifically, 24 hours after addition, the number of cells in which apoptosis occurred with Hoechst 33342 (HO342) and PI reagent (propidium iodide) was counted. HO342 is a UV-excitable nucleic acid staining dye that exhibits blue fluorescence and is particularly bright in the condensed nucleus of apoptotic cells. PI, on the other hand, is taken up only by cells with damaged plasma membranes. From the staining pattern when these two nucleic acid stains were used simultaneously, healthy cell groups, apoptotic cell groups, and dead cell groups were distinguished by fluorescence microscopy and counted.
DHE and NBD-chol are existing cholesterol-like fluorescent reagents. 7-ketocholesterol was added as a control for apoptosis.
The results are shown in FIG. The larger the bar in the graph, the higher the cytotoxicity.
As a result, DCho24 was found to be less cytotoxic than existing fluorescent probes.
Claims (6)
ｎは２、ｍは０〜３の整数を示す。 The compound represented by the following general formula (I).
n represents 2 and m represents an integer of 0 to 3.
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