8/3/2019 Takahiro Inui, Hisafumi Ikeda and Yushin Nakamura- Design of an artificial restriction enzyme having simultaneous

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2000 Oxford University Press Nucleic Acids Sympo sium Series No . 44 141-142

Design of an artificial restriction enzyme having simultaneousDNA cleavage activityTakahiro Inui, Hisafumi Ikeda and Yushin NakamuraDepartment of Biological Science and Technology, Faculty of Industrial Science and Technology, ScienceUniversity of Tokyo, 2641 Yamazaki, Noda, Chiba 278-8510, Japan

A B S T R A C TWe designed dimeric distamycin linked hydroxamicacid (DDHA)-metal complexes and investigated doublestrand simultaneous cleavage. Compared as distamycinlinked hydroxamic acid (DHA)-metal complex,DDHA-metal complex cleaved DNA at the same level ofDHA-metal complex without double strandsimultaneous cleavage.

cooperative molecular recognition between distamycin andhydroxamic acid-metal complex.

1Since Lown et al.demonstrated that a cross-linked lexitropsin has a binding

enhancement of ca. 1000 times compared with that of themonomer, the dimerization of DHA via methylene linker isexpected to recognize DNA with more powerful bindingaffinity.2 We herein report the synthesis of DD HA 5 and theDNA cleavage properties of DDHA-metal complexes.

INTRODUCTIONSequence-specific DNA cleavage by small functionalizedmolecule has attached much attention in connection withthe design of an artificial restriction enzyme. Recently wehave demonstrated that several types of DHA-metalcomplexes sequence-selectively cleave DN A by

V T V ,DHA

DDHA 8

RESULTS and DISCUSSIONThe synthesis of DDHA 5 was shown in scheme 1.Tetrapyrrole derivative 2 was prepared froml,r-(l,4-alkanediyl)bis(p yrrole) 1 according to thepreviously reported method.2 2 was hydrogenolysis andcondensed with carboxylic acid using DCC/HOBt in THFto give pyrrole triamide 3. Hydrogenation of 3 followed bythe alkylation using pentafluorophenyl ester gave themethyl ester 4 in 84 % yield. Finally, 4 was co nverted intoDDHA 5 using NH 2OH . HCl/f-BuOK in MeOH . Thisreaction was quantitatively proceeded, but the yield was18 % because 5 was hardly purified. Introduction ofhydroxamic acid into 5 was confirmed by FeCl 3 test andHRMS.We examined the DNA cleavage activities of DHA andDDHA 5 toward Col El plasmid DNA by monitoring thescission states between supercoiled (form I), nickedcircular (form II) and linear (form III). DDHA 5 has two

8/3/2019 Takahiro Inui, Hisafumi Ikeda and Yushin Nakamura- Design of an artificial restriction enzyme having simultaneous

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142 Nucleic Acids Symposium Series No. 44Scheme 1

R e a g e n t s a n d con d i t i on s : i ) P t O 2 , H 2 , TH F; i i) Af-methyl-4-nitropyrrole- I, HI2-carboxyl ic acid , D C C , H O B t , D M F ; i ii ) methyl pentaf luorophenylad ip a t e , DM F; i v ) NH 2 O H - H C 1 , f- B uO K , M e O H JV

metal complexes by which the double strand simultaneouscleavage was expected. H owever, 1 % agarose gelelectrophoresis showed that DD HA have the same intensityof DHA cleavage activity (Figure 1, Lane 4-7 vs Lane9-12). And also, no linear (form IE) in gel indicated thatDDHA do not cleave double strand. These resultssuggested that two DHA moieties of DDHA were noteffectively placed in the DNA minor groove because thelength of linker (tetramethylene chain) was not enoughlong to fold tw o m oieties.Figure 1 . DN A Cleavage Activities of DHA /DDHA -Fe2+ComplexFe(10/ iM) -DHA /M ) -D D H A ( M ) 5 5 2 0.5 0.1 5 5 2 0.5 0.1

Lane 5 6 7 8 9 10 11 12

A solution of supercoiled (form I) Col E l plasmid DNA(0.3 ^ig), FeSO 4 (10 nM) and DHA/DDHA in 40 mMTris-HCl (pH 8.0) was incubated at 37 C for 1 hr: (Lane 1)intact; (lane 2) incubated DNA without DHA/DDHA; (lane3 and 8) incubated DN A without Fe ? '2+In conclusion, we demonstrated that DDHA-metal complexcan cleave DNA as well as DHA-metal complex. Thedouble strand simultaneous cleavage w ill be realized by theoptimization of the length of DDHA linker. More preciseanalyses for sequence-specific DNA cleavage includingphosphorous hydrolysis are in progress.

R E F E R E N C E S1. (a) Hashimoto, S.; Itai, K.; Takeuchi, Y; Nakamura, Y.

(1997) Heterocycl. Commun. 3, 307-315. (b)Hashimoto, S.; Nakamura, Y. (1998) Chem. Pharm.Bull. 46, 1941-1943. (c) Hashimoto, S.; Inui, T;Nakamura, Y. (2000) Chem. Pharm. Bull. 48, 603-609.

2. Chen, Y-H .; Lown, J.W. (1994) J. Am. Chem. Soc. 116,6995-7005.