カチオン性側鎖を持つポルフィリンによるグアニン四重鎖

(1)

カチオン性側鎖を持つポルフィリンによるグアニン四重鎖

DNA

の安定化機構のӕ明

2005 山下健

Stabilization of Guanine Quadruplex DNA by the Binding of Porphyrins with Cationic Side Arms

2005

Takeshi Yamashita

(2)

Stabilization of Guanine Quadruplex DNA by the Binding of Porphyrins with Cationic Side Arms

Takeshi Yamashita

The telomerase is a target for the design of new anti-cancer drugs because telomerase is active in 85-90 % of human tumor cells and this enzyme combines and elongates telomeres by catalyzing the addition of telomere sequence so that cancer cells are immortalized. Telomere is the nucleoprotein structure at the end of chromosomes consisting of guanine-rich tandem repeats of the sequence d(TTAGGG) in human and forms G-quadruplex structure under physiological conditions. One of the approaches to telomerase inhibition is stabilization of G-quadruplex DNA by small molecules and suppression of the interaction between the telomerase and telomere. One of small molecules stabilizing G-quadruplex DNA is tetramethylpyridiniumyl porphyrin (TMPyP4) that binds externally at the end of quadruplex with its porphyrin ring stacked on the G-tetrad, and the four peripheral pyridinium groups are located in the groove of the quadruplex. I synthesized novel porphyrins with cationic pyridinium and trimethylammonium arms at para (pPy, pTm) or meta (mPy, mTm) position of all phenyl groups of tetratolyl porphyrin expecting their stronger interaction with G- quadruplex by both porphyrin ring and cationic side arms than well-characterized TMPyP4. I also synthesized ZnmPy in order to clarify the binding mode of cationic porphyrins with G-quadruplex. The results are summarized below.

Interaction of double-stranded DNAs and cationic porphyrins

Based on the melting temperature (Tm) of double-stranded DNAs, TMPyP4 and novel catonic porphyrins (pPy, pTm, mPy, and mTm) were found to stabilize the DNA almost equally. Absorption spectra and induced CD (iCD) spectra, however, show that TMPyP4 and novel porphyrins bind differently; TMPyP4 binds by self-stacking while pPy, pTm, mPy, and mTm bind mainly groove of double-stranded DNA, although all porphyrins bind to the surface of the DNA.

(3)

Interaction of G-quadruplex and cationic porphyrins

The quadruplex structure was found to be stabilized more by the meta-isomers than by the para-isomers and TMPyP4, and one mole equivalent of all porphyrins was sufficient to the stabilization of the quadruplex, as revealed by the increase of Tm of the quadruplex. Absorption spectra, iCD spectra, and fluorescence resonance energy transfer (FRET) spectra show that all porphyrins bind G-quadruplex at the same position, suggesting that porphyrins bind by external stacking with G-tetrad at the end of G-quadruplex or bind groove of the G-quadruplex.

Interaction of G-quadruplex and Zinc porphyrin

Because zinc porphyrin has five-coordinate structure, it does not favor intercalation and external stacking. Thus I synthesized ZnmPy and studied spectroscopically in order to clarify the interaction between cationic porphyrins and G-quadruplex. ZnmPy stabilized G-quadruplex almost equally to mPy, although 2-3 mole equivalents were required to sufficiently stabilize the quadruplex. ZnmPy binds groove of G-quadruplex as revealed by absorption spectra, iCD spectra and FRET spectra.

Modeling of cationic porphyrin / G-quadruplex complexes

I calculated minimum energy of cationic porphyrin / G-quadruplex complexes in various binding modes. The smallest energy was obtained for the groove binding, indicating that cationic porphyrins energetically favor groove binding to the G- quadruplex in a face-on manner and stabilize the G-quadruplex structure.

Conclusion

I synthesized novel cationic porphyrins and revealed that meta-isomers (mPy and mTm) stabilized G-quadruplex structure more strongly than para-isomers (pPy and pTm), and that cationic porphyrins bind groove of G-quadruplex face-on. All porphyrins bind the same position of G-quadruplex, thus the position of the cationic side arms is very important to stabilize the G-quadruplex structure. Since telomerase inhibition activity of a drug is strongly related to the stabilization of quadruplex DNA structure,

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第一章緒ۗ 1

第二章新֩カチオン性ポルフィリンの合成

第一節序論 10

第二節 5,10,15,20-tetra (a-bromo-p-tolyl)porphyrin (pBr)の合成 11 第三節 5,10,15,20-tetra (a-bromo-m-tolyl)porphyrin (mBr)の合成 12

第四節 pPyの合成 13

第五節 pTmの合成 13

第六節 mPyの合成 14

第七節 mTmの合成 14

第三章二重鎖DNAとカチオン性ポルフィリンとの相互作用

第一節序論 15

第二節ポルフィリン添加に伴う二重鎖DNAのTmの測定 15

第三節 CTDNA添加に伴うポルフィリンの吸収スペクトル変化 18

第四節 CTDNA添加に伴うポルフィリンの誘֬CD 20

第五節小括 24

第四章四重鎖とカチオン性ポルフィリンとの相互作用

第一節序論 26

第二節ポルフィリン添加に伴う四重鎖のTmの測定 28 第三節四重鎖添加に伴うポルフィリンの吸収スペクトル変化 30 第四節四重鎖添加に伴うポルフィリンの誘֬CDスペクトル 32 第五節四重鎖添加に伴うポルフィリンのFRETスペクトル 35

(5)

第五章四重鎖とポルフィリン亜鉛錯体との相互作用

第一節序論 44

第二節 ZnmPyの合成 44

第三節 ZnmPy添加に伴う四重鎖のTmの測定 45

第四節四重鎖添加に伴うZnmPyの吸収スペクトル変化 46 第五節四重鎖添加に伴うZnmPyの誘֬CDスペクトル 46 第六節四重鎖添加に伴うZnmPyのFRETスペクトル 47

第七節小括 49

第六章モデリングڐ算を用いた結合様式の検討

第一節序論 50

第二節ポルフィリンのスタッキングによるG-tetradの安定化 51 第三節四重鎖のݱ格にあるリン酸基の荷஢の分布 53 第四節ポルフィリンの四重鎖に対する結合様式の検討 55

第五節小括 60

第七章総括 61

実験の൉ 64

引用文献 76

ࡤ辞 86

(6)

ફГच ࣜۗ

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භƳƷ¹඄ЂƟƧ DNA ǚ࢑൑ƟƧǒ¹ƥƶ඄ЂƔ࠲ƣƲƌषݜƷ¹ƥƶެභ

࠺ॱƔ࠺ߗơǓƛưƯƔǛҠƟƧެභǚࣩ׫ơǓिҠౖ֞Ɣ੻޲ƟƮƌǓºǆ Ƨ¹ઑఝƶืћެභƳǐƫƮǊƔǛҠƟƧެභƷܫڛƝǔࠑึƟƮƌǓºƟƓ ƟƲƔǑ¹඄ЂƳǐǒƥƶिҠౖ֞Ɣ੾ƲǗǔƮƒǒ(DNA ࢑൑ЋஞࠃǌƔǛ ຫরЋஞࠃƶഩԩ঴Ҡ)¹ǆƧ¹ȒȨȝÓǺÓ(ǡǟȦǴ¹ࠚු¹ѻൟƲƱ)ƶы

؜ƳǐǒืћެභƔƎǆƗ߁ພƟƲƌգ؈ƳƲƫƮƌǓषݜ¹ƔǛެභƔ੢ौ

Ɵ¹ƔǛਭॎƳƲǓºƔǛҠƶӬࠀƷ¹ǟȆǲǣÓǺÓƳǐǓ DNA ƶ඄ЂƯ Ɗǒ¹ƥǔǑƶ඄ЂƳǐǒ¹ƔǛЋஞࠃƶԩ঴Ҡ¹DNA࢑൑ЋஞࠃǌƔǛຫর Ћஞࠃƶഩԩ঴ҠƔࢲƲƫƮƔǛެභưƲǓƛưƓǑ¹ƔǛƷЋஞࠃƶഗ֡ư

ۗƎƛưƔƯƕǓºǆƧ¹ǟȆǲǣÓǺÓưȒȨȝÓǺÓƶંƗƷ¹॒ࠧǌׇ

ѱƲƱശ૫ƶিԩư॥ƗշٱƟƮƌǓƛưƓǑ¹িԩ࢙ՖഗưǊۗƎƛưƔƯ ƕǓº

¸ƔǛƶ࠲໰Ʒ¹ؤࣗ໰඲ưਙॱ໰඲ƳൟƙǑǔǓºؤࣗ໰඲ưƷ¹ӱҭ໰඲

ǌද࡜ਅ໰඲ƲƱƶƛưƯƔǛƔǆƨ஛ЃƟƮषݜƳƷ๺ܑƨƔ¹ਙॱƳ஛Ѓ

(7)

ƟƮƌǓषݜƳƷܕƓƲƌºГධ¹ਙॱ໰඲Ʒ¹ܨƔǛޱǌȖȦȝȮޱƲƱƶ

๣ޱǚພƌƮ࠲໰ơǓධ඲ƯƊǒ¹ਙॱƳ஛ЃƟƧषݜǌƔǛǚՏਙƳ࠲ơƛ ưƔƯƕƲƌषݜƯǊ¹ƔǛƶપƕƝǚࣹƝƗơǓƛưƯѩฮܑүǌ୊ǇƲƱ ƶऔीǚཋǑƚǓƛưƔ֛ગƯƕǓºܨƔǛޱƷ¹ެභमӳ঴ܨƔǛޱưൟࠃ

഍இ࠲໰๣ƶ2ࡾ་Ƴൟ་ơǓƛưƔƯƕǓºެභमӳ঴ܨƔǛޱƷ¹ƔǛެ

භƶದА½੢ौƳഁঅƯƊǓ൝ࡐƶݜ঵ǚਰӳơǓનࡤዓܨޱ¹ƔǛެභƳઓ ƟƮਏવஇƳ௪ƗܨƔǛ঴ܨি൝ࡐ¹ެභƶൟ༡Ƴࢲ຤Ʋ೰ࣹըƶ௪ƕǚࠐǉ ǓƛưƳǐǒƔǛެභǚࠑึƝƣǓ೰ࣹը߁ພ๣¹ƥǔƳҦƐƮ¹DNAǚǺÓ ǭǾȃưƟƧǝȦǨȦҠޱǌǲǴȒȤǼȮƳનഒƝǔǓಋؽߋઑƲƱƔƊǓ²⁾º ƟƓƟƲƔǑ¹ƌƢǔƶ๣ޱǊƔǛެභƳઓơǓਏવ঴ƔහƟƗࡶƳޙি½੢

ौƶଙƟƌެභƳ߁ພƟ¹ڥעۑࣺ¹ેโ¹ऎҠ֎औीƲƱƶ؊ƌ൐߁ພƔ֬

ǓưƌƎڠ஝ƔƊǓºГධ¹ൟࠃ഍இ࠲໰๣Ʒ¹ƔǛެභ۞๺ƶ௾ଦǚൟࠃȧ ȔȦƯ഍இưƟƧ๣ޱƶƛưƯ¹൐߁ພƶړۑƔ֛ગƝǔǓƔ¹ࡑພҠƝǔƮ ƌǓ๣ƔǆƨࣺƲƗ¹ǆƧ¹ࡑްƳࠑළࠧۥƔ֬ǓƲƱࠈǗƴ൐߁ພƔඦݥƝ ǔƮƌǓºǆƧ¹ެභƷ֕ƬǊƶ඄ЂƔ৘ǇࢲƲƫƮƔǛҠơǓƛưƓǑ¹Ɣ ǛެභƷ়ƳંࡾƳǗƧƫƮƌǓºƥƶڤү¹ƥƶিҠԙஇഗ໊ԙஇ঴ࡐƷં

ࡾંບƯƊǒ¹ƔǛެභƶࡾ་ƳǐƫƮܨƔǛޱƳઓơǓՕࢄ঴ƔЂƲƫƮƌ ǓƛưƓǑ¹ƌƢǔƶ๣൝ƳƒƌƮǊ¹உ҃ƯƕǓƔǛƶࡾ་ƳઓƟƮۙӧƔ ƊǓºƥƶƧǉƌƗƬƓƶ๣ޱǚ௬࠯Ƴ¹ǆƧƷݜ໊இƲ್ࣔƳ߹ພơǓഁ຤

ƔƊǒ¹ંޱ൮ພ໰඲Ɣ݉ǗǔƮƌǓºƥƛƯ¹૒ఃƯંƗƶࡾ་ƶƔǛƳ߹

ພƯƕ¹ƔǛެභਏવ঴Ɣݗƌ¹Ƭǆǒ൐߁ພƶࣺƲƌ๣൝ǚӬದơǓƛưƔ כยưƲƫƮƌǓº

¸ȁȨȜǝ(telomere)Ʒ¹२Ԍি൝ƶ৾ॐઑƶง૝൉ൟƶƛưƯ¹ǩȥǲȞ܃Ư íง૝îǚϼฒơǓtelosưí൉ൟîǚϼฒơǓmerosƓǑ߁ǑǔƧ܃ƯƊǓºȁ ȨȜǝƶѻ֏౴༞Ʒ¹1978ైƳȁȃȤȍȜȅǚພƌƧڽףƓǑฯǑƓƳƝǔ³⁾¹

۔޲ǆƯƳંƗƶࡾƶ౴༞ƔൟƓƫƮƌǓ^4-7)ºƥƶ౴༞Ʒ¹ǫǝȆȮƳമǛƨ

౴༞ǚ๺Ɵ¹ȍȃƳƒƌƮ ƶ ѻ֏ƶ٤ǒඉƟ౴༞ƔঝǨȨѻ֏Ƴ

(8)

஬ƫƮ੶ƌƮƒǒ¹ƥƶ3’ง૝൉ൟƷ¹100-200ѻ֏¹Г෭ޒ൉ൟƔఈࢽƟƮƌ

Ǔ^8-10)ºǆƧ¹ƛƶǫǝȆȮƳമǛƨѻ֏౴༞ƷਙƮƶႄస་௫൝⁴⁾ƨƙƯƲƗ

ȁȃȤȍȜȅ³⁾ǌݏඟ⁵⁾¹ǦǟǮ⁶⁾¹ो൝ƶǲȨǟȇȅǵȅ⁷⁾ƲƱǆƯඐ੻ƝǔƮ

ƒǒ(Table 1)¹ƥƶܲ੧ǊࠨƮƌǓƛưƓǑનǗǒƶܑƓƲƌౖ֞ǚ๺ƟƮƌ

ǓƛưƔੀ੡ƝǔǓº

Table 1. Sequence of telomere.

ࡑްƳȁȨȜǝƷ¹৾ॐઑƶक़॒ǌ¹ਭ՘Ɛ¹ง૝ຈݜƲƱƓǑ৾ॐઑǚࡸǓ ưۗƎࢲ຤Ʋ๡ԥǚ૕ƫƮƌǓƛưƔ಴ƫƮƌǓ^{10, 11)}ºƝǑƳ¹৾ॐઑง૝൉

ൟƷRNAȒȤǟșÓ൉ൟǚൖৄƯƕƲƌƧǉ¹ൖৄƶƧƻƳ50-200ѻ֏૜ࢹƟ (ง૝ൖৄ๓ભ¹ Figure 1)^{12, 13)}¹ƊǓ୷ஶ૜ƗƲǓưǝȘȃÓǲǴǚСƕ֬ƛơ ƛưƓǑ¹ȁȨȜǝƷ¹ެභࢆฮƶࠉ഍ưǊ݂ƐǑǔƮƌǓºƥǔƳҦƐƮЋ ஞஇ੆ཀऔƶƌƗƬƓƳƷ¹ઑެභƶൟ༡ࢆฮƔ૜ƌƶư௬࠯Ƴ¹ȁȨȜǝન ࡤƳЂऺƔƊǓषݜƔඦݥƝǔƮƌǓƛưƓǑ^14-17)¹ཀҠ۔नưǊธৣƲշٱ ƔƊǓƛưƔ࠷މƝǔƮƌǓºГධ¹ȁȨȜȤÓǷƷ¹1985ైƳȁȃȤȍȜȅ ǚພƌƧڽףƓǑದۂƝǔƧ¹⁸⁾º1989ై¹ȁȃȤȍȜȅƶȁȨȜȤÓǷଓٸRNA ǚǪȨÓȆȮǫƟƧưƕƓǑג஛࡛ݏਬƯƊǓưຊੀƝǔƮƌƧ¹⁹⁾ºƥƶۼ¹

1997-1998ైƳȍȃǚսǉƧݏਬȁȨȜȤÓǷƶǰȑȡȆǾȃƔ੏࠰ƌƯǪȨÓ

ɒɈ

ɆɭɩɒɡɊ ȫȤȳ

ȷɭȤɌɊȺɊ

ॆ੐C. elegans ڐೠ

(TTAGGG)n (TTGGGG)n (TTAGG)n (TTTAGGG)n

(TG1-3)n (TTAGGC)n

(9)

lack

lack start replication

end replication

: template DNA : RNA primer : product DNA : DNA polymerase

Figure 1. Model of end replication problem. RNA primer binds template DNA and starts replication by polymerase. After replication, product DNA is shorter than template DNA because RNA primer portion is not replication.

ȆȮǫƝǔ¹ƥƶDNA౴༞Ɣڸ௟ƝǔƧڤү¹ȁȨȜȤÓǷƷ¹DNA౴༞ƶ฻

ƓǑǊ¹ƥƶܲ੧ƓǑǊג஛࡛ݏਬƯƊǓƛưƔ಴ฯƟƧ^20-22)ºȁȨȜȤÓǷ Ʒ¹ƔǛެභƶ85-90%Ưದ۔ƟƮƒǒ^{23, 24)}¹৾ॐઑƶง૝൉ൟƯƊǓȁȨȜǝ ƳڤݜƟȁȨȜǝ౴༞ƶॖସǚ॑౷Ɵ¹ƥƶସƝǚГ୩ƳඐƬƛưƯƔǛެභ ǚഩࠑҠƟƮƌǓ(Figure 2)^25-28)ºǆƧ¹ȁȨȜȤÓǷƷ¹ȍȃƯƷ1132ǝȚȉ

߱ƓǑƲǓג஛࡛ݏਬ൉ൟư5’-CUAACCCUAA-3’ưƌƎ11ѻ֏ƶȁȨȜǝ౴༞

ư੏඗இƲ౴༞ǚսǈ451ȇǪȧǥǼȄƶଓٸRNAƶࡶƳమƬƯܲ঵ƝǔƮƒ ǒ¹ƥƶג஛࡛ݏਬȝǼÓȐƷࡾǚଵƐƮǐƗඐ੻ƝǔƮƌǓºȁȨȜȤÓǷ Ʒ¹ࡿᗡದিưธৣƲշٱƔƊǓƛư²⁹⁾¹חƻ¹ȁȨȜȤÓǷƷ়ऺƲઑެභ

(10)

Figure 2. Model of elongation of telomere by telomerase.

C A A U C C C A A U C

3' 5'

T T A G G G T T A G G G T T A G G G

C C A

3' 5'

T T A G G G T T A G G G T T A G G G

C C A

3' 5'

T T A G G G T T A G G G T T A G G G T T A G G G

C C A

3' 5'

C C A

T T A G G G T T A G G G G G

C C A

C C A A A T C C C A A T C C C A A T C

telomerase fixation

telomere elongation by telomerase

telomerase movement

polymerase extension

telomere elongation by telomerase

(11)

ƯƷǃưǛƱದ۔ƟƮƌƲƌƛưƓǑ¹ƔǛਏવ঴ƔݗƗ¹൐߁ພƶࣺƲƌॠ ƟƌܨƔǛޱƶǺÓǭǾȃưƟƮ֛ગƝǔƮƒǒ³⁰⁾¹ࡑްƳȁȨȜȤÓǷƶଓ ٸRNAǚǺÓǭǾȃưƟƧǝȮǼǶȮǴ඲ǌ^{31, 32)}¹ȁȨȜȤÓǷƶԩ঴ǚຫর ơǓǺȮȌǪࡐƶದ۔຃௮^{33, 34)}¹ǆƧƷࣹൟࠃǚພƌƮȁȨȜȤÓǷƔȁȨȜ ǝƳڤݜơǓƶǚਰӳơǓධ඲ƲƱƔڽףƝǔƮƌǓ^{35, 36)}º

¸DNAƷ¹ǝȂȆȮ¹ǫǝȆȮ¹ǲȃǲȮ¹ǼȚȮƶ߾Ƭƶѻ֏ư¹ȥȮ߱¹ ȂǥǨǲȥȗÓǴƓǑܲ঵ƝǔƮƌǓºǝȂȆȮưǼȚȮ¹ǫǝȆȮưǲȃǲ ȮƔƥǔƦǔWatson-Crickٸƶ঎ਬڤݜǚٺ঵Ɵ¹ୋऺమ෭ƶޒƔג݉ƟƮƌ ǓమࢲǑƣǛܲ੧ǚưƫƮƒǒ³⁷⁾¹ȍȃƯƷެභƳƒƌƮ๢30Ғѻ֏ઓƊǓº ƟƓƟƲƔǑ¹ȁȨȜǝ٤ǒඉƟ౴༞ǚ࠮ƬГ෭ޒ൉ൟƔ¹িઑఝऽڨҟƯ߾

ࢲޒܲ੧ǚưǓƛưƔޚؼඦݥƝǔƧ^{38, 39)}º߾ࢲޒƷ¹ǫǝȆȮƳമǛƨГ෭ ޒƔ৥ǒƧƧǆǔƧܲ੧ǚưƫƮƒǒ¹ǆƨিઑఝƯƷƥƶܲ੧ƔԏీƝǔƮ ƌƲƌƔ¹ȁȨȜǝ^{40, 41)}ǌc-mycƶȒȨȝÓǺÓЋஞࠃ^{42, 43)}¹ืћǫȨȑȥȮƶ ǴǟǾǼ໹Џ⁴⁴⁾ƲƱƔ߾ࢲޒܲ੧ǚࡷǒƎǓ౴༞ǚ๺ƟƮƌǓƛư¹ǆƧ¹߾

ࢲޒƳڤݜơǓǺȮȌǪࡐƔƊǓƛư^45-48)¹߾ࢲޒܲ੧ǚӕƕГ෭ޒƳơǓݏ ਬƔƊǓƛư^49-53)ƲƱƓǑ¹߾ࢲޒƷিઑఝƯࢲ຤Ʋ๡ԥǚ૕ƫƮƌǓƛưƔ

࠷މƝǔƮƌǓº߾ࢲޒƷ¹మƬƶ൉ൟƓǑܲ঵ƝǔƮƒǒ¹ГƬƷ¹TTAȦ ÓȒ൉ൟƯ¹ǊƎГƬƷ߾ƬƶǫǝȆȮƔHoogsteenٸƶ঎ਬڤݜƳǐƫƮٺ঵

ƝǔƧǫǝȆȮ߾ࢲޒ൲฻൉ൟáG-tetradâƯƊǓ^{54, 55)}ºԌ࠶֡؁ำǚພƌƧӕ

৖ǐǒ௻ǑǔƧ঎ຜњଇƯƶܲ੧Ʒ¹TTAȦÓȒƔൟࠃյڤݜƟƧG-tetradƶઓ Ԓਅחƻ੩൉ƳƊǒ¹߾෭ƶDNAޒƔג൲݉Ƴ౴ܕƟƮƌǓƛưƔඦݥƝǔƮ ƌǓ⁵⁴⁾ºǆƧ¹Xਅڤईܲ੧ӕ৖ǐǒ௻ǑǔƧڤईଇƯƶܲ੧Ʒ¹ൟࠃյڤݜ

ƟƧG-tetradƶ੩฻ǚTTAȦÓȒƔઓԒਅरƳਜ਼ƫƮƒǒ¹߾෭ƶDNAޒƷ¹൲

݉Ƴ౴ܕƟƮƌǓƛưƔඦݥƝǔƮƌǓ⁵⁵⁾ºƝǑƳ¹ຜњଇưڤईଇ໩ධƳƒ ƌƮ¹ߡƬƶG-tetradƔπ-π੏۶߁ພƳǐƫƮଽਅஇƳ൵ǛƯƒǒ¹ƥƶ൲฻յ ƶِյƳƷؽੳǟǥȮáNa⁺, K⁺ƲƱâƔ੻޲Ɵƥƶܲ੧ǚϩ୩ҠƟƮƌǓºƛ ƶ߾ࢲޒܲ੧ǚϩ୩ҠơǓƛưƳǐƫƮ¹ȁȨȜȤÓǷƶ ȁȮȒȧÓȃƔ

(12)

ȁȨȜǝDNAƳڤݜơǓƛưǚਰӳƝǔ¹ƥƶڤү¹ȁȨȜȤÓǷƶॖସಶ҃

ǚਰӳƯƕǓƛưƔൟƓƫƮƌǓ⁵⁶⁾ºޚؼ¹߾ࢲޒƶง૝ƳƊǓG-tetradƳઓƟ

Ʈπ-πǴǺǾǨȮǫơǓƛưƯ¹߾ࢲޒܲ੧ǚϩ୩ҠơǓමݖգǚ๺ơǓҠݜ൝

Parallel G-quadruplex DNA

G G G

G

G G

G

G G G G

Antiparallel G-quadruplex DNA

G G G

G

G G

G

G G G G

N H H

N N

N N N

H H R

O H

N N N N

N H H

R O

H

N N N N

H H

R O H N

N N N H

R

O

N

G-tetrad

A B C

M+

Figure 3. (A) Chemical structure of G-tetrad formed by Hoogsteen-type hydrogen bonding network (dashed lines). A metal (M) is accommodated at the center of the G-tetrad. (B) Schematic representation of the antiparallel quadruplex DNA. The arrows represent the direction (5' to 3') of the sugar phosphate backbone. (C) Schematic representation of the parallel quadruplex DNA. The arrows represent the direction (5' to 3') of the sugar phosphate backbone.

C A A U C C C A AU C

3'

5' T T A G G G

G G C C A

G G G

T T A G G G G G

C C A

G G G

Inhibition of telomerase fixation Formation of G-quadruplex Structure

(13)

ƔඦݥƝǔƮƒǒ^57-67)¹ƥƶƛưƓǑ߾ࢲޒܲ੧ƶϩ୩ҠƳƷ¹Ҡݜ൝ƶದॐ

૥ƶ฻৘Ɣࢲ຤ƯƊǓư݂ƐǑǔƮƌǓº

¸߾ ࢲޒܲ੧ǚϩ ୩ҠơǓҠ ݜ൝ƶГƬƯ ƊǓ5,10,15,20-tetra-(N-methyl-4- pyridyl)porphyrin (TMPyP4, Figure 5A)Ʒ¹߾ࢲޒƳઓƟƮݗƌ९ཋ঴ƔƊǒ¹ԯ Ƭ؊ƌȁȨȜȤÓǷਰӳԩ঴ǚ࠮ƬƛưƔඦݥƝǔƮƌǓ^68-71)ºǆƧ¹ƥƶڤ ݜƷ¹߾ࢲޒƶง૝ƳƊǓG-tetradưȘȦȐǞȥȮգƔǴǺǾǨȮǫƟƮƒǒ¹ գƶࢍඈƳƊǓ߾ƬƶȏȥǳȆǡț֏Ʒ¹ƥǔƦǔ߾ƬƊǓ߾ࢲޒƶܷƳ੻޲

ƟƮƌǓºȘȦȐǞȥȮգƶ฻৘ưG-tetradƶ฻৘ƷǃǄ௬ƠƯƊǓƛưƓǑ¹

TMPyP4ƶࡶƲϩ୩Ҡƶ֞ܲƷ¹ȘȦȐǞȥȮգưǫǝȆȮѻ֏յƯƶπ-π੏۶

߁ພƳǐǓӱ൉ǴǺǾǨȮǫƯƊǓưƝǔƮƌǓºГධ¹TMPyP4ƶϳ૷Ђ঴

ઑƯƊǓ5,10,15,20-tetra-(N-methyl-2-pyridyl)porphyrin (TMPyP2, Figure 5B)Ʒ¹

TMPyP4ƳೝǁƮȁȨȜȤÓǷਰӳԩ঴Ɣୢƌ^{69, 70)}ºƛƶǐƎƳ¹ȁȨȜȤÓǷ

ਰӳԩ঴Ʒ¹ȘȦȐǞȥȮգƳڤݜƟƮƌǓ૷՘֏ƶϳ૷ƔธৣƳշٱƟƮƌ

ǓºTMPyP4ǚսǉƧǦǼǥȮ঴ȘȦȐǞȥȮƷ¹మࢲޒDNAǌߡࢲޒDNAư

ƶ੏۶߁ພƳƬƌƮƷંƗƶ૯ۂƔඦݥƝǔƮƌǓƛư^72-93)¹ǆƧ¹ȘȦȐǞ ȥȮ࠺ॱࡿᗡࢥ৘঴ƔƊǓƛưƓǑ^94-96)¹ȘȦȐǞȥȮƷ¹߾ࢲޒưҠݜ൝ư ƶ੏۶߁ພǚଳǁǓरƯ๺ພƲڽףਬ޳ưƌƎƛưƔƯƕǓº

Figure 5. Chemical structure of (A)TMPyP4 and (B) TMPyP2.

NH N N

HN

N N N

N

A B

N H N N

HN

N N

N

(14)

¸ƥƛƯଙࡣƷ¹߾ࢲޒǚTMPyP4ǐǒ؊Ɨϩ୩ҠơǓƛưƯ¹ǐǒ؊ƌȁȨ ȜȤÓǷਰӳԩ঴ǚ࠷ơƛưǚ֛ગƟƮ¹߾Ƭƶ੩ޒƳǦǼǥȮ঴ƶ૷՘֏ǚ

࠮ƬȘȦȐǞȥȮǚ০ڐ½ݜ঵ƟƧºpTmưpPyƷ¹ȜǸϳƶȐǢȆȦ֏ƶȌ ȤϳƳǦǼǥȮ঴ƶȃȥȜǼȦǝȮȝȆǡț֏ưȏȥǳȆǡț֏ǚƥǔƦǔ੩ ޒƳ࠮ƫƮƒǒ¹TMPyP4ưೝǁƮ੩ޒƔସƌºǆƧ¹mTmưmPyƷ¹ƥǔƦ ǔȜǸϳƶȐǢȆȦ֏ƶȜǺϳƳ૷՘֏ƔƊǒ¹ƥǔƦǔpTmưpPyƶϳ૷Ђ

঴ઑƯƊǓºǆƢ¹మࢲޒDNAưƶ੏۶߁ພƳƬƌƮંƗƶ૯ۂƔƊǓƛưƓ Ǒ¹మࢲޒDNAưȘȦȐǞȥȮưƶ੏۶߁ພǚຈӕҜஶ(Tm)¹טࢌǴȕǪȃȦ¹

Ѥమॐ঴(circular dichroism, CD)ǚ߹ພƟƮଳǁƧº࠰ƳƛǔǑƶȘȦȐǞȥȮ

ƶ߾ࢲޒƶϩ୩Ҡƶ֒ຌǚଳǁǓƧǉƳTmǚ੯୩ƟƧºƥƟƮƛǔǑƶȘȦȐ ǞȥȮƶϩ୩Ҡƶ֞ܲƶЊƌǚଳǁǓƧǉƳטࢌǴȕǪȃȦ¹CDǴȕǪȃȦ¹ fluorescence resonance energy transfer (FRET) ǴȕǪȃȦƶ੯୩¹חƻȝȂȥȮǫ ڐ߬ǚ݉ƫƧº

(15)

ફమच¸ॠ֩ǦǼǥȮ঴ȘȦȐǞȥȮƶݜ঵

ફГ২¸ࣥཉ

¸TMPyP4 Ʒ¹Wheelhouse ǑƳǐƫƮ߾ࢲޒƳઓƟƮڤݜƟ¹ƥƶܲ੧ǚϩ୩

ҠơǓƛư¹חƻݗƌȁȨȜȤÓǷਰӳԩ঴ǚ๺ơǓƛưƔඦݥƝǔƮƌǓ^{68, 69)}º ƥƶࡶƲϩ୩Ҡƶ֞ܲƷȘȦȐǞȥȮգư G-tetrad յƯƶπ-πǴǺǾǨȮǫƳǐ ǓºǆƧ¹Han ǑƳǐƫƮ TMPyP4 ƶϳ૷Ђ঴ઑƯƊǓ TMPyP2 Ʒ¹TMPyP4 ǐǒǊȁȨȜȤÓǷਰӳԩ঴ǊୢƌƛưǊඦݥƝǔƮƌǓºƛƶƛưƷȁȨȜ ȤÓǷਰӳԩ঴ƳƷ¹ȘȦȐǞȥȮգƳǐǓ੏۶߁ພƶǇƲǑƢ¹ȘȦȐǞȥ ȮգƶȜǸϳƓǑࢽƮƌǓ੩ޒƶы؜ƔપƕƌƛưǚϼฒƟƮƌǓºƥƛƯଙ ࡣƷ¹ȘȦȐǞȥȮգƳҦƐƮ੩ޒƳǐǓ੏۶߁ພƳǐƫƮ TMPyP4 ǐǒǊ९ ཋ঴½ڤݜ঴ƔरअơǓƛưǚ֛ગƟ¹ॠ֩ǦǼǥȮ঴ȘȦȐǞȥȮǚ০ڐ½ ݜ঵ƟƧºǆƢ¹߾ࢲޒƶȥȮ߱֏ưƶ੏۶߁ພƔ؊ƗƲǓƛưǚ֛ગƟƮ¹

TMPyP4 ǐǒǊସƌǦǼǥȮ঴ȏȥǳȆǡț੩ޒǚ࠮Ƭॠ֩ȘȦȐǞȥȮ pPy

ưƥƶϳ૷Ђ঴ઑƯƊǓ mPyǚݜ঵ƟƧºǆƧ¹૷՘֏ƶЊƌƳǐǓы؜ǚଳ ǁǓƧǉƳ¹ȃȥȜǼȦǝȮȝȆǡț֏ǚ࠮ƬpTmưmTmǚݜ঵ƟƧº

Figure 6. Chemical structure of synthetic meso-tetraphenylporphyrins, pPy, pTm, mPy, and mTm.

CH2 N(CH3)3

N CH₂

CH₂ N(CH₃)₃ N CH₂ NH

N N HN R₂

R2

R₂

R₁

R₁ R₁

R₁

pTm

R₁=H

R₂= R₂= R₁= mPy

H

mTm

H

R₂= R₁= R₂=H pPy

R₁=

(16)

ફమ২¸5,10,15,20-tetra (α-bromo-p-tolyl)porphyrin (pBr)ƶݜ঵

¥Scheme 1 ƳǦǼǥȮ঴ȘȦȐǞȥȮ pPy חƻ pTmƶۍ໬ưƲǓ pBr ƶݜ঵

ډ༲ǚ࠷ơºp-tolunitrile ǚࢽದ൝ࡐưƟƮພƌ¹benzene ଇƯ p-tolunitrile ƶȜ ǼȦ֏ǚ N-bromosuccinimide ǚພƌƮȑȨȝҠƟα-bromo-p-tolunitrile ǚ௻Ƨº

࠰Ƴ¹α-bromo-p-tolunitrile ƶȆȃȥȦ֏ǚ benzene ଇƯ diisobutylalminium hydride(DIBAL-H)ǚພƌƮճیƟα-bromo-p-tolualdehyde ǚ௻Ƨº࠰Ƴ¹CHCl₃ ଇ Ưα-bromo-p-tolualdehyde ư pyrrole ǚ ௓ ໷ ށ ݜ Ɵ Lindsay ඲ ǚ ພ ƌ Ʈ 5,10,15,20-tetrakis(α-bromo-p-tolyl)porphyrin (pBr)ǚ௻Ƨ⁹⁷⁾º

CN

CN CHO

CHO H

N

CH₃ CH₂Br CH₂Br

CH₂Br

NH N N

HN Br

Br

Br N-Bromosuccinimide

BPO

benzene

triethylamine DDQ

4 eq. 4 eq.

dry benzene reflux

BF₃-Et₂O

DIBAL-H

reflux

p-tolunitrile α−bromo-p-tolunitrile α−bromo-p-tolualdehyde

pyrrole

pBr

CHCl₃

(17)

ફߡ২¸5,10,15,20-tetra (α-bromo-m-tolyl)porphyrin (mBr)ƶݜ঵

¸Scheme 2 ƳǦǼǥȮ঴ȘȦȐǞȥȮ mPy חƻ mTm ƶۍ໬ưƲǓ mBr ƶݜ

঵ډ༲ǚ࠷ơºm-tolunitrileǚࢽದ൝ࡐưƟƮພƌ¹benzene ଇƯm-tolunitrileƶ ȜǼȦ֏ǚN-bromosuccinimideǚພƌƮȑȨȝҠƟα-bromo-p-tolunitrileǚ௻Ƨº

࠰Ƴ¹α-bromo-m-tolunitrileƶȆȃȥȦ֏ǚbenzeneଇƯdiisobutylalminium hydride (DIBAL-H)ǚພƌƮճیƟα-bromo-m-tolualdehyde ǚ௻Ƨº࠰Ƴ¹CHCl₃ ଇƯα- bromo-m-tolualdehyde ư pyrrole ǚ௓໷ށݜƟ Lindsay ඲ǚພƌƮ 5,10,15,20- tetrakis(α-bromo-m-tolyl)porphyrin (mBr)ǚ௻Ƨ⁹⁸⁾º

Scheme 2. Synthetic pathway of mBr CN

CH₂Br

CN CHO

CH₂Br

CHO

CH₂Br

HN

NH N N

HN Br

Br

Br Br

CH₃

triethylamine DDQ benzene

reflux

benzene

BF₃-Et₂O

N-Bromosuccinimide BPO

4 eq. 4 eq.

DIBAL-H

mBr

m-tolunitrile α−bromo-m-tolunitrile α−bromo- m-tolualdehyde

pyrrole

reflux CHCl₃

(18)

ફ߾২¸pPyƶݜ঵

pBrƳӄव໷ƶpyridineǚҦƐ¹1.5࠯յճ໙ƟƮpPyǚ௻Ƨ(Scheme 3)º

Scheme 3. Synthetic pathway of pPy

ફ۵২¸pTmƶݜ঵

pBrƳӄव໷ƶȃȥȜǼȦǝȚȮǣǺȉÓȦຜњǚҦƐ 80˚CƯ14࠯յҦేƟ pTmǚ௻Ƨ(Scheme 4)º

NH N N

HN N

N

trimethylamine pBr

pTm

80 °C, stir

NH

N N HN N

N

pyridine pBr

pPy

reflux

(19)

ફང২¸mPyƶݜ঵

mBrƳӄव໷ƶpyridineǚҦƐ¹1.5࠯յճ໙ƟƮmPyǚ௻Ƨ(Scheme 5)º

Scheme 5. Synthetic pathway of mPy

ફࡆ২¸mTmƶݜ঵

mBr Ƴӄव໷ƶȃȥȜǼȦǝȚȮǣǺȉÓȦຜњǚҦƐ 80˚C Ư 14 ࠯յҦే

ƟmTmǚ௻Ƨ(Scheme 6)º

NH

N N HN N

N

N N

pyridine mBr

mPy

reflux

NH

N N HN N

N

N N

mBr

trimethylamine

mTm

80 °C, stir

(20)

ફߡच¸మࢲޒDNAưǦǼǥȮ঴ȘȦȐǞȥȮưƶ੏۶߁ພ

ફГ২¸ࣥཉ

¸߾ࢲޒưҠݜ൝ưƶ੏۶߁ພƳƬƌƮƷ¹ؼై¹ীǛƳڽףƝǔƮƒǒ¹ƥ ƶڤݜບ࠿ƳƬƌƮǊ¹Ԍ࠶֡؁ำ(nuclear magnetic resonance, NMR)⁵⁴⁾ǌ Xਅ ڤईܲ੧ӕ৖⁵⁵⁾ǚພƌƮӕฯƝǔƮƕƮƌǓºƟƓƟƲƔǑ¹Xਅڤईܲ੧Ʒ¹ ຜњଇƯƶܲ੧ǚಶьƟƲƌषݜƔƊǓƛưǌԌ߱ưҠݜ൝ưƶൖݜઑƶڤई ҠƷຓϾƯƷƲƌƛư¹ǆƧ¹NMR ư X ਅڤईӕ৖ưǊƳƥƶӕ৖ƳƷ௾ࡻ

ƲָౖƔഁ຤ƲƛưƲƱ໅උ঴ƳڠƙǓºГධ¹טࢌǴȕǪȃȦǌ CD¹ڏ܎

ǴȕǪȃȦƲƱƷૐƯǊຓϾƳ੯୩ơǓƛưƔƯƕ¹ޙ۔঴ǊݗƌƛưƓǑ¹ ƛǔǑƶࡹ඲ǚພƌƮҠݜ൝ƶ߾ࢲޒƳઓơǓ੏۶߁ພƳƬƌƮຓϾƳ૯Ǔƛ ưƔƯƕǓǐƎƳƲǔƸ¹߾ࢲޒƳڤݜơǓҠݜ൝ƶӬದǚॳǉƮƌƗरƯ๺

ќƯƊǓºȘȦȐǞȥȮƷ¹26π஢ࠃ؁๡ڈǚ࠮Ʃ¹400 nm പؼƳπ"πਐЃƳ

֬ПơǓȘȦȐǞȥȮ௾๺ƶ Soret ખưǐƸǔǓ؊ƌטࢌƔƊǓºǆƧ¹ȘȦ ȐǞȥȮƷ¹؊ƌڏ܎ǊದơǓºȘȦȐǞȥȮƶ Soret ખƷ¹ȘȦȐǞȥȮƶ գ؈ीઙƳљധƯƊǒ¹ຓϾƳƥƶ඄Ҡǌ¹ڏ܎؊ஶƶ඄ҠǚୈƎƛưƔƯƕ ǓƛưƓǑ¹DNAưƶ੏۶߁ພǚൟ܎ԙஇࡹ඲ƓǑۂƮƌƗरƯ๺ພƲڽףਬ

޳ƯƊǓº۔޲ƶưƛǕ¹ƛǔǑƶࡹ඲ǚພƌƧ߾ࢲޒưȘȦȐǞȥȮưƶ੏

۶߁ພƳƬƌƮƶඦݥƷࣺƲƌºƥƛƯ¹ଙࡣƷ¹మࢲޒ DNA ưȘȦȐǞȥ Ȯưƶ੏۶߁ພƳƬƌƮƷơƯƳંƗƶ૯ۂƔඦݥƝǔƮƌǓƛưƓǑ ^72-96)¹ ǆƢॠ֩Ƴݜ঵ƟƧȘȦȐǞȥȮưమࢲޒ DNA ưƶ੏۶߁ພƳƬƌƮ¹మࢲ ޒ DNAƶ Tm¹טࢌǴȕǪȃȦ¹຃֬ CDǴȕǪȃȦǚພƌƮڸ௟Ɵ¹ƥƛƓ Ǒ௻ǑǔƧऻඦǚ֏Ƴ߾ࢲޒưƶ੏۶߁ພƳƬƌƮڸ௟ơǓƛưƳƟƧº

ફమ২¸ȘȦȐǞȥȮ஗ҦƳಳƎమࢲޒDNAƶTmƶ੯୩

¸ஔਘƶమࢲޒ DNA ƯƊǓ߷ǡǲؖਈ DNA(Calf thymus DNA, CTDNA)ư חƻݶӖݜ঵ƟƧ ¹ ¹ ¹ ưƶൖݜઑƶϩ୩঴ǚଳǁ

(21)

ǓƧǉƳTmǚ20 mM sodium phosphate buffer, 20 mM NaCl pH 7.0ଇƯ੯୩ƟƧº మࢲޒ DNA ƷǑƣǛܲ੧ǚٺ঵ơǓưѻ֏ઓյƶǴǺǾǨȮǫƳǐǒט܎ஶ ƔۑࣺơǓºҜஶरअƳưǊƲƌГ෭ޒ DNA Ɣ඄঴ƟƮГ෭ޒƳƲƫƮƌƗ ƳƟƧƔƫƮט܎ஶƶरअƔհ੯ƝǔǓºTmƷ¹మࢲޒ DNAƔՏਙƳГ෭ޒ ƳǃƱƙƧưƕƶଇ஝ƶҜஶǚ࠷Ɵ^{89, 99)}¹TMPyP4ǌݶӖݜ঵ƟƧpPy¹pTm¹ mPy¹mTmƶమࢲޒDNAƳઓơǓϩ୩Ҡǀƶ֒ຌǚ૯ǓƛưƔƯƕǓº

¸ǆƢ¹CTDNAƨƙƶưƕƷ¹Tm = 75.0 ˚CưƲƫƧ(Figure 7)º࠰ƳƥǔƦǔ ƶȘȦȐǞȥȮưCTDNAƔR = 0.1 (R = [porphyrin] / [base pair])ƶưƕƶൖݜઑ ƶ Tm ǚ੯୩ƟƧºǆƧ¹ȘȦȐǞȥȮ੻޲ҟƯ CTDNA ƨƙƶ࠯ƳೝǁƮƱ ǔƨƙTmƔरअƟƧƓǚ∆TmƯഒƟƧºTMPyP4ƷTm = 81.0 ˚CưƲǒ∆Tm Ɣ6.0˚CƯƊƫƧ(Table 2)ºГධ¹ݶӖݜ঵ƟƧ4ࡾ་ƶҠݜ൝pPy¹pTm¹mPy¹ mTmƷ¹ƥǔƦǔ∆TmƔ6.0¹5.5¹4.5¹3.5˚CưƲƫƧ(Table 2)ºpPyưpTm

ƯƷ∆Tmƶ૮ƔTMPyP4ưǃǄ௬Ơ૮ưƲǒ¹௬୷ஶϩ୩ҠƟƮƌǓư݂ƐǑ

ǔ¹mPy ư mTm Ʒ∆Tm ƶ૮ƔࡲՒୢƗƲƫƮƒǒϩ୩Ҡǀƶ֒ຌƷ੏ઓஇƳ

ୢƗƲƫƮƌǓºǆƧ¹૷՘֏ƳǐǓϩ୩Ҡǀƶ֒ຌƷ¹ȏȥǳȆǡț֏੩ޒ ǚ๺ơǓpPy ưmPyƷ¹ȃȥȜǼȦǝȮȝȆǡț֏੩ޒǚ๺ơǓpTmưmTm ǐǒƥǔƦǔ 0.5¹1.0˚C ưǃǛƶǗƢƓƨƙݗƗƲƫƧºƛƶƛưǐǒ૷՘

֏ƳǐǓы؜ƷǃưǛƱƲƌƛưƔǗƓƫƧº

¸ϱरƶƛưƓǑమࢲޒ DNA ƶϩ୩ҠƳƷ੩ޒƶସƝǌƥƶϳ૷ƳշٱƲƗ ǃǄ௬ƠƯƊǓƛưƔൟƓƫƧºƛƶƛưƓǑ¹ƌƢǔƶȘȦȐǞȥȮǊమࢲ ޒDNAƳઓƟƮ௬ƠǐƎƲڤݜǚƟƮƌǓƛưƔ࠷މƝǔƧº

(22)

Figure 7. Normalized absorbance changes of 38.6 µM base pair CTDNA at 260 nm in the absence (closed squares) or presence of 3.9 µM TMPyP4 (R = 0.1, closed diamonds) and mPy (closed circles) against temperature in 20 mM sodium phosphate buffer, 20 mM NaCl (pH 7.0).

¸¸¸Table 2#T_{m and ∆}Tm of CTDNA in the absence or presence of cationic porphyrins 0

0.5 1

30 40 50 60 70 80 90 100

Temperature (°C)

Normalized Absorbance Change at 260 nm

TMPyP4 81.0 6.0

pPy 81.0 6.0

mPy 79.5 4.5

CTDNA only 75.0

pTm 80.5 5.5

mTm 78.5 3.5

Tm (°C) ∆Tm (°C) porphyrin

(23)

ફߡ২¸CTDNA஗ҦƳಳƎȘȦȐǞȥȮƶטࢌǴȕǪȃȦ඄Ҡ

¸DNA ஗ҦƳಳƎȘȦȐǞȥȮƶ Soret ખƶ඄ҠƓǑȘȦȐǞȥȮƶڤݜບ࠿

ǚ঍୩ơǓƛưƔƯƕǓºDNA ѻ֏ઓյ੃హƝǔǓषݜ(intercalation)Ʒ¹Soret ખƶئપטࢌౡସƔપƕƗȧǾȄǲȐȃƟ(≥ 15 nm)¹ǆƧ؊ƌ૙ॐܑү(≥ 35%) ǚ࠷Ɵ¹DNA ƶǫȦÓȑƳڤݜƟƮƌǓषݜ(groove binding)Ʒ¹ࣹƝƗȧǾȄ

ǲȐȃƟ(≤ 8 nm)¹ǆƧࡴƌ૙ॐܑү(≥ 10%)ǊƟƗƷ౔ॐܑүǚ࠷ơƛưƔ૯

ǑǔƮƌǓ 72-74, 85, 100-103)ºǆƧ¹Soret ખƶ඄Ҡƶӄ୷ƓǑ DNAưƶڤݜӄ୷ǚ

঍੯ơǓƛưƔƯƕǓº

¸ǆƢޚࣖƳTMPyP4ưమࢲޒDNAưƶڤݜ঴ƳƬƌƮଳǁƧº20 mM sodium phosphate buffer, 20 mM NaCl pH 7.0ଇƯTMPyP4ƳCTDNAǚ஗ҦƟƮƌƗư

420 nmƶט܎ஶƔࣦÐƳۑࣺƟ¹ئપౡସƷȧǾȄǲȐȃƟƧºǆƧ¹௖טࢌ

஝ƔۂǑǔƧƧǉƛƶ൲݊ಶ҃ƷГ૫ӭƯƊǓƛưƔ࠷މƝǔƧ(Figure 8A)º

¸࠰ƳݶӖݜ঵ƟƧŏࡾ་ƶҠݜ൝ pPy¹pTm¹mPy¹mTm ưమࢲޒ DNA ư ƶڤݜ঴ƳƬƌƮଳǁƧ(Figure 8B)ºԇȘȦȐǞȥȮƳ CTDNA ǚ஗ҦƟƮƌ Ɨư¹ƥǔƦǔ 410 nm പؼƶט܎ஶƔۑࣺƟئપౡସƔȧǾȄǲȐȃƟƧº ƟƓƟƲƔǑ¹ƥƶ඄ҠƷரଇǆƯƷ௖טࢌ஝ǚ࠷ƟƧƔ¹TMPyP4 ưƷЂƲ

ǒR = 0.20പؼƓǑ௖טࢌ஝ƷӱǔƮƌƫƧƛưƓǑ¹൲݊ಶ҃ƷࣺƲƗưǊ

మ૫ӭƊǓƛưƔǗƓƫƧº

¸ǆƧ¹ƥǔƦǔƶȘȦȐǞȥȮƶȧǾȄǲȐȃ(∆λ)ư૙ॐܑү(H)ǚ Table 3 ƳǆưǉƧºƌƢǔƶȘȦȐǞȥȮǊȧǾȄǲȐȃƷǟȮǺÓǦȧÓǲȢȮǚ

࠷ơ15 nmǐǒǊࣹƝƓƫƧ (8.0-13.0 nm)ºǆƧ¹૙ॐܑүƷ¹TMPyP4ưpPy¹ mTm ƯƷƥǔƦǔ 50.1¹44.0¹43.0%ưƲǒǟȮǺÓǦȧÓǲȢȮǚ࠷ơ 35%

ǐǒǊપƕƗƲƫƧƔ¹pTmưmPyƷ¹ƥǔƦǔ32.5¹32.3%ưࣹƝƗƲƫƧº ƛǔǑƶڤүƓǑ¹TMPyP4 ư pPy¹mTm Ʒ¹ǟȮǺÓǦȧÓǲȢȮưమࢲޒ DNA ƶӱ൉ƳڤݜƟƮƌǓ໩ධƶҧౖ঴Ɣ¹pTm¹mPy Ʒ¹మࢲޒ DNA ƶǫ ȦÓȑƳڤݜƟƮƌǓҧౖ঴Ɣ࠷މƝǔƧº

(24)

Figure 8. Absorption spectral change of TMPyP4 (A) and mPy (B) on the addition of CTDNA.

The porphyrin concentration was 1.5 µM. The spectra were recorded in 20 mM sodium phosphate buffer, 20 mM NaCl (pH 7.0). A, R values were as follows ∞, 1.08, 0.52, 0.39, 0.31, 0.10. B, R values were as follows ∞, 0.90, 0.60 ,0.40, 0.05, 0.01. Insets show enlarged spectra in the Soret region.

0 0.1 0.2 0.3 0.4 0.5

300 400 500 600 700

Wavelength (nm)

0 0.1 0.2 0.3 0.4

350 400 450 500

wavelength (nm)

0 0.1 0.2 0.3 0.4 0.5

350 400 450 500

wavelength (nm)

A

B AbsorbanceAbsorbance

(25)

Table 3. Spectroscopic data for the porphyrins bound to CTDNA. ∆λ, bathochromic shift.

Hypochromicity (H) was determined by the equation H = (εf — εb) / εf × 100, where εf and εb

represent the molar absorptivities of free and bound porphyrins, respectively.

ફ߾২¸CTDNA஗ҦƳಳƎȘȦȐǞȥȮƶ຃֬CD

¸CTDNA ưƶڤݜƳಳƎȘȦȐǞȥȮƶ຃֬ CD ǴȕǪȃȦǚ੯୩ƟƧºȘ

ȦȐǞȥȮ࠺ઑƷഩ্૛ਬǚ࠮ƫƮƌƲƌƧǉƳ CD Ʒհ੯ƝǔƲƌƔ¹ǨȤ ȦƲൟࠃƯƊǓ DNAưڤݜơǓƛưƯ SoretખപؼƳCDƔ຃֬ƝǔǓºƛƶ ƛưǚ໅ພƟƮȘȦȐǞȥȮƔమࢲޒ DNA ƳઓƟƮƱƶǐƎƳڤݜƟƮƌǓ Ɠ಴୩ơǓƛưƔƯƕǓºГೃƳ¹়ƶȏÓǪǚ࠷ƟƧषݜƷǫȦÓȑڤݜ¹

ാƶȏÓǪǚ࠷ƟƧषݜƷѻ֏ઓյǀƶǟȮǺÓǦȧÓǲȢȮ¹়ưാƶȏÓ Ǫǚ࠷ƟƧषݜƳƷ DNA ഒ฻रƯƶ࠺ۡǴǺǾǨȮǫ(self-stacking)ư֟ੳƝ ǔǓ72, 73, 75, 83, 85, 100)º

¸ǆƢ¹TMPyP4ƶǴȕǪȃȦǚۂƮǇǓư¹R = 0.1ưR = 0.05¹R = 0.01ƶư ƕƌƢǔǊ়ưാƶȏÓǪƔ຃֬ƝǔƧƛưƓǑ¹TMPyP4 Ʒ DNA ഒ฻रƯ

࠺ۡǴǺǾǨȮǫƟƮƌǓƛưƔ࠷މƝǔƧ(Figure 9)º࠰ƳݶӖݜ঵ƟƧpPy¹ pTm¹mPy¹mTmƶǴȕǪȃȦǚۂƮǇǓư¹pPyƯƷ¹R = 0.1¹0.05ƶưƕ

়ƶȏÓǪƨƙƲƶƳઓƟƮ¹R = 0.01 ƶưƕ়ƶȏÓǪưƥƶସౡସ੩Ƴࡴ

TMPyP4 pPy pTm mPy mTm

13.0 8.0 8.5 9.0 8.0

50.1 44.0 32.5 32.3 43.0

∆λ (nm) H (%) porphyrn

(26)

ƌാƶȏÓǪƔհ੯ƝǔƧºpTm ƯƷ R = 0.1ư 0.05¹0.01ƶ࠯ƌƢǔǊ؊ƌ

়ƶȏÓǪưƥƶସౡସ੩ƳࡴƌാƶȏÓǪƔհ੯ƝǔƧ(Figure 10)ºǆƧ¹mPy

ưmTmƷ¹R = 0.1ư0.05¹0.01ƶ࠯ƌƢǔǊ؊ƌ়ƶȏÓǪưƥƶସౡସ੩

ư૜ౡସ੩ƳࡴƌാƶȏÓǪƔհ੯ƝǔƧƔ¹R = 0.1ƶưƕƷସౡସ੩Ƴೝǁ Ʈ૜ౡସ੩ƶാƶȏÓǪƔ¹R = 0.01 ƶưƕƷ૜ౡସ੩ƳೝǁƮସౡସ੩ƶാ

ƶȏÓǪƔƥǔƦǔ؊ƗƲƫƧ(Figure 11)ºƌƢǔƶȘȦȐǞȥȮǊࡶƳ়ƶ ȏÓǪǚ࠷ƟƮƌǓƛưƓǑ¹ƥǔƦǔࡶƳǫȦÓȑڤݜǚƟƮƌǓư݂ƐǑ ǔǓºƟƓƟƲƔǑ¹૒࣐Ƴ়ƶȏÓǪƶǇƯƷƲƗƥǔƦǔࡴƌാƶȏÓǪ Ɣସౡସ੩Ɠ૜ౡସ੩ƶƱƩǑƓǆƧƷ໩ධƳۂǑǔƧƛưƓǑ¹ƌƗƬƓƶ ڤݜບ࠿ƔށƞƫƮƌǓƛưƔ࠷މƝǔƧº

Figure 9. Induced CD spectra of 5 µM TMPyP4 in the presence of CTDNA at R = 0.1 (solid line), 0.05 (dashed line), and 0.01 (chained line). The spectra were recorded in 20 mM sodium phosphate buffer, 20 mM NaCl (pH 7.0).

-20 -10 0 10

∆ε (M cm )-1-1

350 400 450 500

Wavelength (nm)

(27)

Figure 10. Induced CD spectra of 5 µM porphyrins in the presence of CTDNA at R = 0.1 (solid lines), 0.05 (dashed lines), and 0.01 (chained lines). A, pPy; B, pTm. The spectra were recorded in 20 mM sodium phosphate buffer, 20 mM NaCl (pH 7.0).

-5 0 5 10 15 20 -10

0 10 20 30

350 400 450 500

Wavelength (nm)

∆ε (M cm )-1-1 ∆ε (M cm )-1-1

A

B

(28)

Figure 11. Induced CD spectra of 5 µM porphyrins in the presence of CTDNA at R = 0.1 (solid lines), 0.05 (dashed lines), and 0.01 (chained lines). A, mPy; B, mTm. The spectra were recorded in 20 mM sodium phosphate buffer, 20 mM NaCl (pH 7.0).

350 400 450 500

-10 -5

0 5 10 15 20 25

Wavelength (nm) -10

-5 0 5 10 15 20

A

B

∆ε (M cm )-1-1 ∆ε (M cm )-1-1

(29)

ફ۵২¸ࣹԨ

¥TMPyP4 Ʒమࢲޒ DNA ஗ҦƳಳƎטࢌǴȕǪȃȦ඄ҠƳƒƌƮ¹௖טࢌ஝

ƔۂǑǔƧƛưƓǑ¹ڤݜӄ୷ƔГ૫ӭƯƊǓƛưƔǗƓƫƧºǆƧ¹ݶӖݜ

঵ƟƧ pPy¹pTm¹mPy¹mTm ƶטࢌǴȕǪȃȦƶ඄ҠƷ¹ƌƢǔǊரଇǆƯ Ʒ௖טࢌ஝ǚ࠷ƟƧƔ¹మࢲޒ DNA ǚҦƐƮƌƗư࠰ફƳ௖טࢌ஝ƓǑӱǔ ƮƌƫƧºƛƶƛưƓǑ¹ݶӖݜ঵ƟƧҠݜ൝ƯƷڤݜӄ୷Ɣమ૫ӭϱरƯƊ ǓƛưƔǗƓƫƧºǆƧ¹טࢌǴȕǪȃȦƶڤүưమࢲޒ DNA ஗ҦƳಳƎ຃

֬CDǴȕǪȃȦƶڤүǚ੒ݜஇƳ಴૨ƟƮ¹TMPyP4ƷDNAഒ฻रƯ࠺ۡǴ ǺǾǨȮǫƟƮ DNA ƳڤݜƟƮƌǓưڤཉƭƙƧ(Figure 12A)ºГධ¹ݶӖݜ

঵ƟƧȘȦȐǞȥȮƷƌƢǔǊڤݜບ࠿ƷࡶƳǫȦÓȑڤݜƯƊǓưڤཉƭƙ

Ƨ(Figure 12B)ºƟƓƟƲƔǑ¹ǴȕǪȃȦƔ়ƶȏÓǪƨƙƯƷƲƌƛư¹R

ƶ૮ƳǐƫƮȏÓǪƶౡٺƔ೰พƳ඄ҠơǓƛưƓǑ¹ƌƗƬƓƶڤݜƔށ޲

ƟƮƌǓƛưƔ࠷މƝǔƧºƛƶƛưƷ¹טࢌǴȕǪȃȦƔమ૫ӭϱरƳƲƫ ƧƛưưǊઓ҃ƟƮƌǓº

¸Tm੯୩ƶڤү¹TMPyP4ƯƷ∆Tm = 6.0 ˚CưƲƫƧºǆƧ¹ݶӖݜ঵ƟƧҠ ݜ൝ƯƷ¹∆Tm = 3.5-6.0 ˚C ưƲǒTMPyP4 ưપƕƲތƷƲƓƫƧºƛǔǑƶ ڤүǐǒȘȦȐǞȥȮƶڤݜບ࠿ưȘȦȐǞȥȮƶమࢲޒ DNA ƶϩ୩Ҡǀƶ

֒ຌƷ੏շƔƲƌƛưƔ࠷މƝǔƧº

¸ϱरƶƛưƓǑ TMPyP4 ưݶӖݜ঵ƟƧȘȦȐǞȥȮƷƥǔƦǔ࠺ۡǴǺǾ ǨȮǫ¹ǫȦÓȑڤݜưڤݜບ࠿ƷЂƲǓǊƶƶ¹ƌƢǔǊ DNA ƶഒ฻रƯ ڤݜƟƮƌǓƛưƔǗƓƫƧºDNAƶȥȮ߱֏Ʒຜ౷੩ǚܕƌƮƒǒȘȦȐǞ ȥȮƶ়ƶҼ஢੩ޒưؼƌϳ૷Ƴ੻޲ơǓºƛǔǑƶƛưƓǑ¹TMPyP4 ưݶ Ӗݜ঵ƟƧ 4 ࡾ་ƶȘȦȐǞȥȮƷ¹؁Ƴ੩ޒƶସƝǌϳ૷¹૷՘֏ƶࡾ་ƶ ы؜ƷƲƗ¹ࡶƳమࢲޒ DNA ݱԋƶȥȮ߱֏ƶാƶҼ஢ǚȘȦȐǞȥȮƶ੩ ޒƶ়ƶҼ஢ƯଇཋơǓƛưƯ¹ȥȮ߱֏௬߿ƶಶದǚۑࣺƝƣమࢲޒ DNA ǚϩ୩ҠƟƮƌǓư݂ƐǑǔƧº

(30)

Figure 12. Model of binding mode of TMPyP4 (A) and pPy, pTm, mPy and mTm (B) against CTDNA. Closed boxes denote the porphyrins.

Groove binding Self stacking

: porphyrin

A B

(31)

ફ߾च¸߾ࢲޒưǦǼǥȮ঴ȘȦȐǞȥȮưƶ੏۶߁ພ

ફГ২¸ࣥཉ

¸ફГचƯ֪ࢿƟƧǐƎƳ¹߾ࢲޒǚ؊Ɨϩ୩ҠơǓƛưƔƯƕǓҠݜ൝Ʒ¹

ࡿᗡਏવ঴ƶݗƌܨƔǛޱǀƶ҃ພƔ֛ગƯƕǓºǆƧ¹ƛǔǑƶҠݜ൝ƶ߾

ࢲޒǀƶ੏۶߁ພǚधެƳ૯ǓƛưƔƯƕǔƸ¹ǐǒ๺ພƲܨƔǛޱƶ০ڐ½ ӬದƳ๡໔ƮƮƌƗƛưƔƯƕǓº߾ࢲޒƳઓơǓ๣൝ƶڤݜưƟƮ¹ƥƶܲ

੧ƓǑǟȮǺÓǦȧÓǲȢȮ¹ӱ൉ǴǺǾǨȮǫ(external stacking)¹ǫȦÓȑ ڤݜ¹ȦÓȒǀƶڤݜƶ߾Ƭƶڤݜບ࠿Ɣ݂ƐǑǔǓ ¹⁰⁴⁾ºමݖգǚ๺ơǓҠݜ

൝ƯƷ¹߾ࢲޒƳઓơǓڤݜບ࠿ƷࡶƳӱ൉ǴǺǾǨȮǫƯƊǒ¹ƥƶමݖգ

ư G-tetrad յƯƶπ-π੏۶߁ພƔࢲ຤ƯƊǓưƝǔƮƌǓ ^57-60)ºƟƓƟƲƔǑ¹

ƥƶ੩ޒƳǐǓ߾ࢲޒǀƶ੏۶߁ພƳƬƌƮƶधެƲඦݥƷࣺƲƌºƥƛƯ¹ ݱԋƷ௬ƠƯ੩ޒƶЂƲǓҠݜ൝ǚພƌƮ߾ࢲޒǀƶ੏۶߁ພƶЊƌǚଳǁǓ ƛưƯ¹੩ޒƳǐǓ߾ࢲޒǀƶ੏۶߁ພƳƬƌƮڸ௟ƟƧº

¸෭चƯƷ¹G4:5'-CATGGTGGTTTGGGTTAGGGTTAGGGTTAGGGTTACCAC-3' ưƌƎ߾ӖƶȁȨȜǝ٤ǒඉƟ౴༞ǚ࠮Ƭ39 ໷ઑƶГ෭ޒǥȥǯșÓ(G4)ǚ¹ 4 mM Tris HCl, 1 mM EDTA, 100 mM KCl pH 7.5 ƶऽڨƯǝȆÓȥȮǫƟଳৄƟ ƧǊƶǚ߹ພƟƧº߾ࢲޒƶٺ঵Ʒ¹࠘ӱ൉ CD ǚ੯୩ơǓƛưƯԏీơǓƛ ưƔƯƕǓ^105-109)ºG4ƔГ෭ޒƯ੻޲ơǓժढњऽڨ(4 mM Tris HCl, 1 mM EDTA