JP2982621B2 - Novel amino acid with photosensitizing function - Google Patents
Novel amino acid with photosensitizing functionInfo
- Publication number
- JP2982621B2 JP2982621B2 JP6171050A JP17105094A JP2982621B2 JP 2982621 B2 JP2982621 B2 JP 2982621B2 JP 6171050 A JP6171050 A JP 6171050A JP 17105094 A JP17105094 A JP 17105094A JP 2982621 B2 JP2982621 B2 JP 2982621B2
- Authority
- JP
- Japan
- Prior art keywords
- compound
- dna
- amino acid
- amino
- light
- 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.)
- Expired - Fee Related
Links
- 150000001413 amino acids Chemical class 0.000 title claims description 15
- 239000003504 photosensitizing agent Substances 0.000 title claims description 8
- 230000002165 photosensitisation Effects 0.000 title description 5
- 238000000034 method Methods 0.000 claims description 8
- 230000001678 irradiating effect Effects 0.000 claims description 2
- 230000003647 oxidation Effects 0.000 claims description 2
- 238000007254 oxidation reaction Methods 0.000 claims description 2
- 230000001590 oxidative effect Effects 0.000 claims 2
- 239000004480 active ingredient Substances 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 description 43
- 108020004414 DNA Proteins 0.000 description 13
- 235000001014 amino acid Nutrition 0.000 description 10
- -1 methoxycarbonylpentyl Chemical group 0.000 description 10
- 238000003776 cleavage reaction Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 230000007017 scission Effects 0.000 description 8
- 239000002904 solvent Substances 0.000 description 7
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 238000001704 evaporation Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 230000007018 DNA scission Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 235000019766 L-Lysine Nutrition 0.000 description 3
- 150000008545 L-lysines Chemical class 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- XJHABGPPCLHLLV-UHFFFAOYSA-N benzo[de]isoquinoline-1,3-dione Chemical compound C1=CC(C(=O)NC2=O)=C3C2=CC=CC3=C1 XJHABGPPCLHLLV-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 3
- 238000010992 reflux Methods 0.000 description 3
- 238000010898 silica gel chromatography Methods 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 239000007978 cacodylate buffer Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000027756 respiratory electron transport chain Effects 0.000 description 2
- IHQKEDIOMGYHEB-UHFFFAOYSA-M sodium dimethylarsinate Chemical compound [Na+].C[As](C)([O-])=O IHQKEDIOMGYHEB-UHFFFAOYSA-M 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- DYWWDZQRZYAPHQ-UHFFFAOYSA-N 3-benzoyl-4-ethyl-4-methyl-2-phenyl-1,3-oxazolidin-5-one Chemical compound O1C(=O)C(CC)(C)N(C(=O)C=2C=CC=CC=2)C1C1=CC=CC=C1 DYWWDZQRZYAPHQ-UHFFFAOYSA-N 0.000 description 1
- LKOZHLXUWUBRDK-UHFFFAOYSA-N 4-nitro-1,8-naphthalic anhydride Chemical compound O=C1OC(=O)C2=CC=CC3=C2C1=CC=C3[N+](=O)[O-] LKOZHLXUWUBRDK-UHFFFAOYSA-N 0.000 description 1
- 230000001482 DNA photocleavage Effects 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- GRSMWKLPSNHDHA-UHFFFAOYSA-N Naphthalic anhydride Chemical compound C1=CC(C(=O)OC2=O)=C3C2=CC=CC3=C1 GRSMWKLPSNHDHA-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 150000003949 imides Chemical class 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 125000001160 methoxycarbonyl group Chemical group [H]C([H])([H])OC(*)=O 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 208000017983 photosensitivity disease Diseases 0.000 description 1
- 231100000434 photosensitization Toxicity 0.000 description 1
- 238000001126 phototherapy Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000001235 sensitizing effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
Landscapes
- Other In-Based Heterocyclic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、光増感機能をもつ新規
アミノ酸、更に詳しくは、光増感機能をもつＬ−リジン
の新規誘導体に関する。The present invention relates to a novel amino acid having a photosensitizing function, and more particularly, to a novel derivative of L-lysine having a photosensitizing function.
【０００２】[0002]
【従来の技術】本発明者は、分子生物学や光生物化学に
応用でき、しかも光をトリガーとしてＤＮＡを切断する
アミノ酸（Photochemical DNA-cleaving Amino Acid, P
CAA ）の開発研究を行なっている。2. Description of the Related Art The present inventors have found that an amino acid that can be applied to molecular biology and photobiochemistry and that cleaves DNA using light as a trigger (Photochemical DNA-cleaving Amino Acid, P
CAA).
【０００３】これまで人工制限酵素を目指したＤＮＡ切
断分子の研究が盛んにおこなわれているが、塩基配列特
異的に切断する分子は天然の抗生物質では見られるもの
の、人工的にデザインしたものではほとんど知られてい
ない。[0003] Until now, research on DNA-cleaving molecules aimed at artificial restriction enzymes has been actively conducted. Molecules that specifically cleave nucleotide sequences can be found in natural antibiotics, but not in artificially designed ones. Little is known.
【０００４】[0004]
【発明が解決しようとする課題】前項に記載の従来の技
術の背景下に、本発明は、塩基配列特異的な光ＤＮＡ切
断活性を有する化合物を開発し提供することを目的とす
る。The object of the present invention is to develop and provide a compound having a base sequence-specific photoDNA cleavage activity in the background of the prior art described in the preceding section.
【０００５】[0005]
【課題を解決するための手段】本発明者は、前項に記載
の目的を達成すべく鋭意研究の結果、本発明者の合成し
たＬ−リジンの新規誘導体のなかのいくつかが光照射に
より塩基配列特異的にＤＮＡを切断することを見いだ
し、本発明を完成するに至った。The present inventors have conducted intensive studies to achieve the object described in the preceding section, and as a result, some of the novel derivatives of L-lysine synthesized by the present inventors have been exposed to light to form a base. The inventors have found that the DNA is sequence-specifically cleaved, and have completed the present invention.
【０００６】すなわち、本発明は、Ｌ−リジンから容易
に得られる、塩基配列特異的な光ＤＮＡ切断活性を有す
る非天然型アミノ酸、具体的には、下記一般式（Ｉ）で
表される光増感機能をもつ新規アミノ酸に関する。That is, the present invention provides a non-natural amino acid having a base sequence-specific photoDNA-cleaving activity, which is easily obtained from L-lysine, specifically, a light-emitting amino acid represented by the following general formula (I): It relates to a novel amino acid having a sensitizing function.
【０００７】[0007]
【化２】 Embedded image
【０００８】上記式中、Ｒ1 およびＲ2 はそれぞれ独立
にＨまたはＮＯ2 を表わす。ただし、Ｒ1 およびＲ2 が
同時にＮＯ2 を表わす場合を除く。In the above formula, R 1 and R 2 each independently represent H or NO 2 . However, this excludes the case where R 1 and R 2 simultaneously represent NO 2 .
【０００９】以下、本発明を詳細に説明する。Hereinafter, the present invention will be described in detail.
【００１０】本発明者がデザインし、合成したＬ−リジ
ンの新規誘導体には、次の化合物（１）〜（６）が含ま
れるが、これらの化合物のうち、化合物（１）〜（３）
が特に光増感機能に優れ、延いては塩基配列特異的な光
ＤＮＡ切断活性に優れている。The novel derivatives of L-lysine designed and synthesized by the present inventors include the following compounds (1) to (6). Among these compounds, compounds (1) to (3)
Are particularly excellent in the photosensitizing function and, in addition, are excellent in base sequence-specific photoDNA cleavage activity.
【００１１】[0011]
【化３】 Embedded image
【００１２】これらの新規誘導体の合成法自体には特別
の困難はなく、上に述べたように、Ｌ−リジンから容易
に得ることができる（なお後掲実施例１参照）。There is no particular difficulty in the method of synthesizing these novel derivatives per se, and as described above, they can be easily obtained from L-lysine (see Example 1 below).
【００１３】以上のように、本発明者は、ＤＮＡを光切
断できるアミノ酸（PhotochemicalDNA-Cleaving Amino
Acid 、PCAA）をデザインし、いくつか合成したが、こ
れらのアミノ酸は、（１）Ｌ−リジンより一段階で大量
合成することができる、（２）ＤＮＡに強く結合し、 3
00〜380nm の光照射でＤＮＡやＲＮＡを塩基配列特異的
に切断し、しかもナフタルイミド部位の置換基を変える
ことでＤＮＡ切断の塩基特異性が変わる（化合物（１）
は−ＧＧ−特異的切断、化合物（２）はＴ特異的切断、
そして化合物（３）は−Ｇ−および−Ｔ−の双方で切
断）、（３）この性質を利用して制ガン剤としての用途
が期待される、（４）光治療のための増感剤としての利
用できる、（５）強いケイ光を有するのでケイ光性アミ
ノ酸として利用できる、（６）水溶性の光増感剤、特に
電子移動型光増感剤として広い範囲で光増感反応や生体
分子の酸化反応等に用いられる、そして（７）固相化学
合成で任意のポリペプチドに組み込むことができ、遺伝
子工学的手法を用いれば蛋白質にも組み込むことが可能
なものである。As described above, the present inventors have developed an amino acid capable of photocleaving DNA (Photochemical DNA-Cleaving Amino).
Acid, PCAA) and synthesized some of these amino acids. These amino acids can (1) be synthesized in large quantities from L-lysine in one step, (2) strongly bind to DNA,
The base sequence specificity of DNA cleavage is changed by irradiating light of 00 to 380 nm with DNA and RNA in a base sequence-specific manner and by changing the substituent at the naphthalimide site (compound (1)
Is -GG-specific cleavage, compound (2) is T-specific cleavage,
(Compound (3) is cleaved by both -G- and -T-), (3) Expected to be used as an anticancer agent utilizing this property, (4) As a sensitizer for phototherapy (5) It can be used as a fluorescent amino acid because it has strong fluorescent light. (6) It can be used as a water-soluble photosensitizer, especially as an electron transfer type photosensitizer in a wide range of photosensitization reactions and biomolecules. (7) can be incorporated into any polypeptide by solid phase chemical synthesis, and can also be incorporated into proteins by using genetic engineering techniques.
【００１４】[0014]
【作用】化合物（１）は、ＧＧ配列の５′側のＧを切断
することからＧから化合物（１）への電子移動とそれに
続く酸化により切断を起こしていると考えられる。ま
た、化合物（２）は、チミン塩基のメチル基を酸化して
Ｔ特異的に切断することがわかった。この化合物はＴの
光シークエンシングや遺伝子のＴのみを変異させるポイ
ントミューテーションにも有用であろう。Since compound (1) cleaves G at the 5 'side of the GG sequence, it is considered that the cleavage is caused by electron transfer from G to compound (1) and subsequent oxidation. Compound (2) was found to oxidize the methyl group of the thymine base and cleave T-specifically. This compound would also be useful for light sequencing of T and point mutations where only T in a gene is mutated.
【００１５】[0015]
【実施例】以下、実施例を掲げて本発明を更に説明す
る。The present invention will be further described below with reference to examples.
【００１６】実施例１（合成例） 下記化合物（ａ）〜（ｈ）を合成した。ただし、化合物
（ｇ）を除く。Example 1 (Synthesis Example) The following compounds (a) to (h) were synthesized. However, compound (g) is excluded.
【００１７】[0017]
【化４】 Embedded image
【００１８】（ｉ）Ｎ−（５−アミノ−５−メトキシカ
ルボニルペンチル）−１，８−ナフタルイミド（化合物
（ａ））の合成 Ｂｏｃ−Ｌ−Ｌｙｓ−ＯＭｅ（1.21ｇ，4.69mmol）と
１，８−ナフタル酸無水物（0.977 ｇ，4.69mmol）をト
ルエン20mlに溶解し、３時間加熱還流した。溶媒留去後
得られた粗精製物をシリカゲルクロマトグラフィーによ
り精製してＮ−（５−ブトキシカルボニルアミノ−５−
メトキシカルボニルペンチル）−１，８−ナフタルイミ
ド（化合物（ｂ））を1.81ｇ、収率90.4％で得た。(I) N- (5-amino-5-methoxyca)
Rubonylpentyl) -1,8-naphthalimide (compound
Synthesis of (a)) Boc-L-Lys-OMe (1.21 g, 4.69 mmol) and 1,8-naphthalic anhydride (0.977 g, 4.69 mmol) were dissolved in 20 ml of toluene and heated under reflux for 3 hours. The crude product obtained after evaporation of the solvent was purified by silica gel chromatography to give N- (5-butoxycarbonylamino-5-
1.81 g of (methoxycarbonylpentyl) -1,8-naphthalimide (compound (b)) was obtained in a yield of 90.4%.
【００１９】この化合物（ｂ）1.81ｇ（4.11mmol）をト
リフルオロ酢酸10mlに溶解し、室温で１時間攪拌した。
溶媒留去後Ｎ−（５−アミノ−５−メトキシカルボニル
ペンチル）−１，８−ナフタルイミド（化合物（ａ））
を1.86ｇ、収率 100％で得た。1.81 g (4.11 mmol) of this compound (b) was dissolved in 10 ml of trifluoroacetic acid and stirred at room temperature for 1 hour.
After evaporating the solvent, N- (5-amino-5-methoxycarbonylpentyl) -1,8-naphthalimide (compound (a))
Was obtained in a yield of 100%.
【００２０】1Ｈ ＮＭＲ（ＣＤ3 ＯＤ）δ：1.40-2.20
(m,6H) ，3.71(s,3H)，3.96(t,1H,J=6.2Hz)，4.17(t,2
H,J=6.3Hz)，7.80(t,2H,J=7.4Hz)，8.84(d,2H,J=7.4H
z)，8.54(d,2H,J=7.3Hz)． ＵＶ（ＣＨ3 ＣＮ）λmax 332nm ：ｌｏｇε＝4.08． ＭＳ ｍ／ｅ（相対強度）：340 （Ｍ＋0.5 ），323
（100 ）． ｍｐ：68〜73℃． ケイ光Ｅm max ：３８０ｎｍ（３３５ｎｍ励起）。 1 H NMR (CD 3 OD) δ: 1.40-2.20
(m, 6H), 3.71 (s, 3H), 3.96 (t, 1H, J = 6.2Hz), 4.17 (t, 2
H, J = 6.3Hz), 7.80 (t, 2H, J = 7.4Hz), 8.84 (d, 2H, J = 7.4H
z), 8.54 (d, 2H, J = 7.3Hz). UV (CH 3 CN) λ max 332 nm: log ε = 4.08. MS m / e (relative intensity): 340 (M + 0.5), 323
(100). mp: 68-73 ° C. Kei light E m max: 380nm (335nm excitation).
【００２１】（ii）Ｎ−（５−アミノ−５−メトキシカ
ルボニルペンチル）−３−ニトロ−１，８−ナフタルイ
ミド（化合物（ｃ））の合成 Ｂｏｃ−Ｌ−Ｌｙｓ−ＯＭｅ（856mg ，2.47mmol）と３
−ニトロ−１，８−ナフタル酸無水物（500mg ，2.05mm
ol）をＤＭＦ20mlに溶解し、３時間加熱還流した。溶媒
留去後得られた粗精製物をシリカゲルクロマトグラフィ
ーにより精製してＮ−（５−ｔ−ブトキシカルボニルア
ミノ−５−メトキシカルボニルペンチル）−３−ニトロ
−１，８−ナフタルイミド（化合物（ｄ））を250mg 、
収率25.1％で得た。(Ii) N- (5-amino-5-methoxyca)
Rubonylpentyl) -3-nitro-1,8-naphthalate
Synthesis of amide (compound (c)) Boc-L-Lys-OMe (856 mg, 2.47 mmol) and 3
-Nitro-1,8-naphthalic anhydride (500 mg, 2.05 mm
ol) was dissolved in 20 ml of DMF and heated under reflux for 3 hours. The crude product obtained after evaporation of the solvent is purified by silica gel chromatography to give N- (5-t-butoxycarbonylamino-5-methoxycarbonylpentyl) -3-nitro-1,8-naphthalimide (compound (d )) 250mg,
Obtained in 25.1% yield.
【００２２】この化合物（ｄ）250mg （0.515mmol ）を
トリフルオロ酢酸10mlに溶解し、室温で１時間攪拌し
た。溶媒留去後Ｎ−（５−アミノ−５−メトキシカルボ
ニルペンチル）−３−ニトロ−１，８−ナフタルイミド
（化合物（ｃ））を242mg 、収率93.9％で得た。250 mg (0.515 mmol) of this compound (d) was dissolved in 10 ml of trifluoroacetic acid and stirred at room temperature for 1 hour. After evaporating the solvent, 242 mg of N- (5-amino-5-methoxycarbonylpentyl) -3-nitro-1,8-naphthalimide (compound (c)) was obtained at a yield of 93.9%.
【００２３】1Ｈ ＮＭＲ（ＣＤ3 ＯＤ）δ：1.40-2.10
(m,6H) ，3.71(s,3H)，4.05(t,1H,J=6.3Hz)，4.20(t,2
H,J=7.3Hz)，8.00(dd,1H,J=7.3Hz,J=7.5Hz) ，8.62(d,1
H,J=8.2Hz)，8.74(d,1H,J=6.2Hz)，9.18(d,1H,J=2.3H
z)，9.33(d,1H,J=2.3Hz)． ＵＶ（ＣＨ3 ＣＮ）λmax 331nm ：ｌｏｇε＝3.82． ＭＳ ｍ／ｅ（相対強度）：386 （Ｍ+ ，0.5 ），369
（100 ）． ｍｐ：58〜62℃． ケイ光Ｅm max ：４００ｎｍ（３３５ｎｍ励起）。 1 H NMR (CD 3 OD) δ: 1.40-2.10
(m, 6H), 3.71 (s, 3H), 4.05 (t, 1H, J = 6.3Hz), 4.20 (t, 2
H, J = 7.3Hz), 8.00 (dd, 1H, J = 7.3Hz, J = 7.5Hz), 8.62 (d, 1
H, J = 8.2Hz), 8.74 (d, 1H, J = 6.2Hz), 9.18 (d, 1H, J = 2.3H)
z), 9.33 (d, 1H, J = 2.3Hz). UV (CH 3 CN) λ max 331 nm: log ε = 3.82. MS m / e (relative intensity): 386 (M + , 0.5), 369
(100). mp: 58-62 ° C. Kei light E m max: 400nm (335nm excitation).
【００２４】（iii ）Ｎ−（５−アミノ−５−メトキシ
カルボニルペンチル）−４−ニトロ−１，８−ナフタル
イミド（化合物（ｅ））の合成 Ｂｏｃ−Ｌ−Ｌｙｓ−ＯＭｅ（1.21ｇ，4.69mmol）と４
−ニトロ−１，８−ナフタル酸無水物（1.20ｇ，4.93mm
ol）をＤＭＦ50mlに溶解し、３時間加熱還流した。溶媒
留去後得られた粗精製物をシリカゲルクロマトグラフィ
ーにより精製してＮ−（５−ｔ−ブトキシカルボニルア
ミノ−５−メトキシカルボニルペンチル）−４−ニトロ
−１，８−ナフタルイミド（化合物（ｆ））を706mg 、
収率31.0％で得た。(Iii) N- (5-amino-5-methoxy)
Carbonylpentyl) -4-nitro-1,8-naphthal
Synthesis of imide (compound (e)) Boc-L-Lys-OMe (1.21 g, 4.69 mmol) and 4
-Nitro-1,8-naphthalic anhydride (1.20 g, 4.93 mm
ol) was dissolved in 50 ml of DMF and heated under reflux for 3 hours. The crude product obtained after evaporation of the solvent is purified by silica gel chromatography to give N- (5-t-butoxycarbonylamino-5-methoxycarbonylpentyl) -4-nitro-1,8-naphthalimide (compound (f )) 706mg,
Obtained in a yield of 31.0%.
【００２５】同様にして得られた化合物（ｆ）を合し、
その840mg （1.46mmol）をトリフルオロ酢酸10mlに溶解
し、室温で１時間攪拌した。溶媒留去後Ｎ−（５−アミ
ノ−５−メトキシカルボニルペンチル）−４−ニトロ−
１，８ナフタルイミド（化合物（ｅ））を864mg 、収率
100％で得た。The compound (f) obtained in the same manner is combined,
840 mg (1.46 mmol) thereof was dissolved in 10 ml of trifluoroacetic acid and stirred at room temperature for 1 hour. After evaporating the solvent, N- (5-amino-5-methoxycarbonylpentyl) -4-nitro-
864 mg of 1,8 naphthalimide (compound (e)), yield
Obtained at 100%.
【００２６】1Ｈ ＮＭＲ（ＣＤ3 ＯＤ）δ：1.43-2.06
(m,6H) ，3.82(s,3H)，4.05(t,1H,J=6.3Hz)，4.17(t,2
H,J=7.3Hz)，7.91-8.07(m,2H) ，8.44-8.50(m,1H) ，8.
61-8.78(m,3H) ． ＵＶ（ＣＨ3 ＣＮ）λmax 334nm ：ｌｏｇε＝3.92． ＭＳ ｍ／ｅ（相対強度）：386 （Ｍ+ ，1.5 ），369
（100 ）． ｍｐ：66-69 ℃． ケイ光Ｅm max ：４３０ｎｍ（３３５ｎｍ励起）。 1 H NMR (CD 3 OD) δ: 1.43-2.06
(m, 6H), 3.82 (s, 3H), 4.05 (t, 1H, J = 6.3Hz), 4.17 (t, 2
H, J = 7.3Hz), 7.91-8.07 (m, 2H), 8.44-8.50 (m, 1H), 8.
61-8.78 (m, 3H). UV (CH 3 CN) λ max 334 nm: log ε = 3.92. MS m / e (relative intensity): 386 (M + , 1.5), 369
(100). mp: 66-69 ° C. Kei light E m max: 430nm (335nm excitation).
【００２７】（iv）Ｎ−（５−（Ｎ−９−フルオレニル
メトキシカルボニル）アミノ−５−メトキシカルボニル
ペンチル）−１，８−ナフタルイミド（化合物（ｈ））
の合成 Ｎ2 雰囲気下Ｎ−（５−アミノ−５−カルボニルペンチ
ル）−１，８−ナフタルイミド（化合物（ｇ））（893.
3mg ，2.15mmol）を10％Ｎａ2 ＣＯ3 水溶液１mlに溶解
し、氷冷下にFmocOSu （869mg ，2.58mmol）を加えた。
反応溶液を室温で１時間攪拌後、塩酸で酸性にして酢酸
エチルで抽出した。有機層を飽和食塩水で洗浄し、無水
硫酸マグネシウムで乾燥後、溶媒を留去して得た粗精製
物をシリカゲルクロマトグラフィーにより精製してＮ−
（５−（Ｎ−９−フルオレニルメトキシカルボニル）ア
ミノ−５−メトキシカルボニルペンチル）−１，８−ナ
フタルイミド（化合物（ｈ））を707mg 、収率56.8％で
得た。(Iv) N- (5- (N-9-fluorenyl)
Methoxycarbonyl) amino-5-methoxycarbonyl
(Pentyl) -1,8-naphthalimide (compound (h))
Synthesis N 2 atmosphere N-(5-amino-5-carbonyl pentyl) -1,8-naphthalimide (Compound (g)) (893.
3 mg, 2.15 mmol) was dissolved in 1 ml of a 10% aqueous solution of Na 2 CO 3 , and FmocOSu (869 mg, 2.58 mmol) was added under ice cooling.
The reaction solution was stirred at room temperature for 1 hour, acidified with hydrochloric acid, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off.
707 mg of (5- (N-9-fluorenylmethoxycarbonyl) amino-5-methoxycarbonylpentyl) -1,8-naphthalimide (compound (h)) was obtained in a yield of 56.8%.
【００２８】1Ｈ ＮＭＲ（ＣＤＣｌ3 ）δ：1.35-2.10
(m,6H) ，4.20(d,2H,J=6.6Hz)，4.33(t,1H,J=7.0Hz)，
7.20-7.60(m,8H) ，7.60-7.75(m,2H) ，8.16(d,2H,J=8.
3Hz)，8.58(d,2H,J=6.3Hz)。 1 H NMR (CDCl 3 ) δ: 1.35-2.10
(m, 6H), 4.20 (d, 2H, J = 6.6Hz), 4.33 (t, 1H, J = 7.0Hz),
7.20-7.60 (m, 8H), 7.60-7.75 (m, 2H), 8.16 (d, 2H, J = 8.
3 Hz), 8.58 (d, 2H, J = 6.3 Hz).
【００２９】実施例２（光化学的なＤＮＡ切断活性の検
査） 化合物（１）、（２）および（４）〜（６）の、それぞ
れの存在下におけるＤＮＡの光化学的な切断活性をｐＢ
Ｒ322 ＤＮＡのform変換により検査した。Example 2 (Test for photochemical DNA-cleaving activity) The photochemical DNA-cleaving activity of compounds (1), (2) and (4) to (6) in the presence of each was determined by pB.
Inspection was performed by form conversion of R322 DNA.
【００３０】切断反応の条件は、次の通りとした。すな
わち、ｐＢＲ３２２ＤＮＡ（５０μM 燐酸塩濃度）のカ
コジル酸ナトリウム緩衝液（ｐＨ 7.0、10ｍＭ）溶液に
化合物（１）、（２）および（４）〜（６）をそれぞれ
を10μＭになるように加えて、これらの溶液を同一条件
下でフナコシ製「フナコシトランスイルミネーターＴＬ
33」（ 366ｎｍ）を用い、10cmの距離から０℃で１時間
光照射し、光照射溶液をアガローズゲル電気泳動し、 f
orm Ｉ（supercoiled circular）から form II（nicked
circular ）への変換を調べた。ＤＮＡ切断活性は
（２）＞（１）＞（４）＞＞（６）＞（５）の順に低下
し、（２）が最も活性高く、（６）および（５）は殆ん
どＤＮＡ切断活性を示さなかった。（２）は10μＭの濃
度でform III（linear ＤＮＡ）まで切断した。The conditions for the cleavage reaction were as follows. That is, the compounds (1), (2) and (4) to (6) were added to a solution of pBR322 DNA (50 μM phosphate concentration) in a sodium cacodylate buffer (pH 7.0, 10 mM) so as to have a concentration of 10 μM, respectively. Under the same conditions, these solutions were prepared using Funakoshi's "Funakoshi Transilluminator TL".
33 ”(366 nm) at a temperature of 0 ° C. for 1 hour from a distance of 10 cm, and the light-irradiated solution was subjected to agarose gel electrophoresis.
orm I (supercoiled circular) to form II (nicked
circular). The DNA cleavage activity decreases in the order of (2)>(1)> (4) >>(6)> (5), with (2) having the highest activity, and (6) and (5) being almost DNA cleavage. No activity was shown. (2) was cleaved to form III (linear DNA) at a concentration of 10 μM.
【００３１】実施例３（ＤＮＡの塩基配列特異的光切
断） Ｎ−（５−アミノ−５−メトキシカルボニルペンチル）
−１，８−ナフタルイミド（化合物（ａ）＝化合物
（１））50μM 、約 0.1μM リン酸濃度の32Ｐ５′末端
ラベルＤＮＡフラグメント（32Ｐ−５′−end-labeled
261-bp fragment ofhuman c-Ha-ras-1 protooncogene
、約0.1 Ｃｉ／mmol）及び50μM リン酸濃度仔牛胸線
ＤＮＡを10ｍＭカコジル酸ナトリウム緩衝液（ｐＨ7.0
） 100μＬに溶解した。得られた溶液を冷却下ＵＶラ
ンプ「フナコシトランスイルミネーターＴＬ33」（フナ
コシ社製）によりＵＶ光を10cmの距離から20分間照射し
た。このようにして光反応させた後、ＤＮＡをエタノー
ル沈殿により回収し、これを再度１Ｍピペリジン水溶液
100μL に溶解し、90℃で20分間加熱した。エタノール
沈殿により回収したＤＮＡをＤＮＡシーケンシングシス
テム「ＬＫＢ2010 Macrophor」（ファルマシア社製）を
用いてゲル電気泳動法によりＤＮＡ切断の塩基配列特異
性をMaxam-Gilbert 法で解析した。Example 3 (Base sequence-specific photocleavage of DNA) N- (5-amino-5-methoxycarbonylpentyl)
1,8-naphthalimide (Compound (a) = Compound (1)) 50μM, 32 P5 ' end-labeled DNA fragment of approximately 0.1μM phosphoric acid concentration (32 P-5'-end- labeled
261-bp fragment ofhuman c-Ha-ras-1 protooncogene
Calf chestline DNA in 10 mM sodium cacodylate buffer (pH 7.0).
) Dissolved in 100 μL. The obtained solution was irradiated with UV light from a distance of 10 cm for 20 minutes by a UV lamp “Funakoshi Transilluminator TL33” (manufactured by Funakoshi) under cooling. After the photoreaction in this manner, the DNA was recovered by ethanol precipitation, and this was again reconstituted with a 1 M aqueous piperidine solution.
It was dissolved in 100 μL and heated at 90 ° C. for 20 minutes. The DNA recovered by ethanol precipitation was analyzed for the nucleotide sequence specificity of DNA cleavage by the Maxam-Gilbert method by gel electrophoresis using a DNA sequencing system "LKB2010 Macrophor" (Pharmacia).
【００３２】Ｎ−（５−アミノ−５−メトキシカルボニ
ルペンチル）−３−ニトロ−１，８−ナフタルイミド
（化合物（ｃ）＝化合物（２））10μM 、およびＮ−
（５−アミノ−５−メトキシカルボニルペンチル）−４
−ニトロ−１，８−ナフタルイミド（化合物（ｅ）＝化
合物（３））25μM の場合もそれぞれ同様に行なった。N- (5-amino-5-methoxycarbonylpentyl) -3-nitro-1,8-naphthalimide (compound (c) = compound (2)) 10 μM;
(5-amino-5-methoxycarbonylpentyl) -4
-Nitro-1,8-naphthalimide (compound (e) = compound (3)) was also used in the case of 25 μM.
【００３３】解析結果から得られる結論は、次の通りで
ある。すなわち、化合物（１）の場合は、ＧＧ配列の
５′側のＧで特異的な切断が起り、化合物（２）の場合
は、ＡＴリッチ部位のところのＴで特異的な切断が起
り、そして化合物（３）は上記のＧおよびＴの双方で切
断が起る。このようにニトロ基を導入するだけで塩基配
列特異性がドラマチックに変化する。The conclusions obtained from the analysis results are as follows. That is, in the case of compound (1), specific cleavage occurs at the 5'-side G of the GG sequence, and in the case of compound (2), specific cleavage occurs at T at the AT-rich site, and Compound (3) undergoes cleavage at both G and T described above. Thus, the nucleotide sequence specificity changes dramatically only by introducing a nitro group.
【００３４】因みに、化合物（１）および（２）のMaxa
m-Gilbert 法での解析結果を図１および図２にそれぞれ
示す。図１から化合物（１）は５′−ＧＧ−配列の５′
側のＧの位置でＤＮＡを切断することが判り、また、図
２から化合物（２）は５′−ＸＴ−（Ｘ＝Ａ，Ｔまたは
Ｃ）の位置で切断することが判る。Incidentally, the Maxa of compounds (1) and (2)
The results of analysis by the m-Gilbert method are shown in FIGS. 1 and 2, respectively. From FIG. 1, compound (1) is 5 ′ of the 5′-GG sequence.
It can be seen that the DNA is cleaved at the position G on the side, and that the compound (2) is cleaved at the position 5'-XT- (X = A, T or C) from FIG.
【００３５】[0035]
【発明の効果】本発明により、光増感機能を有し、ＤＮ
Ａを塩基配列特異的に光切断する新規化合物がＬ−リジ
ンから容易に合成され、提供されるところとなった。According to the present invention, DN having photosensitizing function
A novel compound that photocleaves A in a base sequence-specific manner has been easily synthesized from L-lysine and provided.
【図１】Maxam-Gilbert 法による解析結果を示す（実施
例３）。FIG. 1 shows the results of analysis by the Maxam-Gilbert method (Example 3).
【図２】Maxam-Gilbert 法による解析結果を示す（実施
例３）。FIG. 2 shows an analysis result by the Maxam-Gilbert method (Example 3).
───────────────────────────────────────────────────── フロントページの続き (58)調査した分野(Int.Cl.6，ＤＢ名) C07D 221/14 ＣＡ（ＳＴＮ) ＲＥＧＩＳＴＲＹ（ＳＴＮ)──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int. Cl. 6 , DB name) C07D 221/14 CA (STN) REGISTRY (STN)
Claims (4)
2を表わす。ただし、Ｒ1およびＲ2が同時にＮＯ2を表わ
す場合を除く。1. An amino acid represented by the following general formula (I). Embedded image In the above formula, R 1 and R 2 are each independently H or NO
Indicates 2 . However, this excludes the case where R 1 and R 2 simultaneously represent NO 2 .
して含有する光増感剤。2. A photosensitizer comprising the amino acid according to claim 1 as an active ingredient.
含む溶液に光を照射して該生体分子を特異的に酸化する
生体分子の酸化方法。3. A method for oxidizing a biomolecule, wherein the solution containing the amino acid and the biomolecule according to claim 1 is irradiated with light to specifically oxidize the biomolecule.
はＲＮＡを含む溶液に光を照射して該ＤＮＡまたはＲＮ
Ａの特定の塩基を酸化することにより、ＤＮＡまたはＲ
ＮＡを位置特異的に切断しまたは変異せしめる請求項３
に記載の酸化方法。4. A method comprising irradiating a solution containing the amino acid according to claim 1 and DNA or RNA with light to obtain the DNA or RN.
By oxidizing a specific base of A, DNA or R
4. The NA is site-specifically cleaved or mutated.
Oxidation method according to 1.
Priority Applications (1)
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| JP6171050A JP2982621B2 (en) | 1994-07-22 | 1994-07-22 | Novel amino acid with photosensitizing function |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6171050A JP2982621B2 (en) | 1994-07-22 | 1994-07-22 | Novel amino acid with photosensitizing function |
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| Publication Number | Publication Date |
|---|---|
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| JP2982621B2 true JP2982621B2 (en) | 1999-11-29 |
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| Title |
|---|
| Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｓｙｍｐ．Ｓｅｒ．，１９９４（３１）（２１ｓｔ Ｓｙｍｐｏｓｉｕｍ ｏｎ Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｃｈｅｍｉｓｔｒｙ，１９９４），１４７−８（１９９４） |
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