3709
J. Am. Chem. SOC.1994,116, 3709-3715
Carbohydrate-Minor Groove Interactions in the Binding of Calicheamicin y l1 to Duplex DNA Tianhu Li,t Zijian Zeng,? Virginia A. Estevez,? Kai U. Baldenius,+K. C. Nicolaou,'*#and Gerald F. Joyce'vf Contribution from the Departments of Chemistry and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, and Department of Chemistry, University of California, San Diego, La Jolla, California 92093 Received January 3, 1994"
Abstract: The sequence-specific D N A cleavage activity of calicheamicin 7,'was studied using a synthetic 20mer D N A substrate that contains a single TCCT-AGGA target site. The cleavage reaction was initiated by addition of 8-mercaptoethanol, which, under conditions of calicheamicin excess, results in burst kinetics. The burst amplitude was used to obtain a KD value of 135 nM for the calicheamicin-DNA interaction. Calicheamicin and calicheamicin oligosaccharide were allowed to compete for binding, which provided a K,value of 4.1 rM for the oligosaccharideDNA interaction. Replacement of the iodo-substituted oligosaccharide by the corresponding bromo-, chloro-, fluoro-, methyl-, and hydrogen-substituted compounds resulted in progressively weaker binding. Replacement of guanine by inosine a t the 5'-most but not the 3'-most position within the AGGA target sequence resulted in greatly diminished D N A cleavage. These results suggest that for this target sequence there is a critical interaction between the iodine substituent on the calicheamicin oligosaccharide and the 5'-most guanine C2-NH2 group within the minor groove of the target DNA.
Scheme 1
Introduction The enediyne antitumor antibiotics, including calicheamicin ylI (1) and esperamicin A I , bind to the minor groove of duplex D N A in a sequence-specific In the case of cali-
0 1. nuclwphlllc altack 2. conjugstr addillon
w O'sugar
Me&
Nu
1: calicheamicin
i,'
Bergman
cycllutlon
1: callcheamlcln yll
cheamicin, binding occurs along short runs of oligopyrimidineoligopurine base pairs, such as TCCT.AGGA.l*2-4J Once bound, calicheamicin promotes cleavage of both strands of the D N A through a remarkable free-radical mechanism that involves a Bergman-typecycloaromatizationtogenerate a reactive diradical ~pecies4.6.~ (Scheme 1). The two strands of the D N A + Department of Chemistry, The Scripps Research Institute.
t Department of Chemistry,The ScrippsResearch Institute, and Department of Chemistry, University of California, San Diego. Departments of Chemistry and Molecular Biology,The Scripps Research Institute. Abstract published in Aduance ACS Abstracts, March 15, 1994. (1) Zein, N.; Sinha, A. M.; Megahren, W. J.; Ellestad, G. A. Science 1988,
*
240, 1198-1201. (2) Zein, N.; Poncin, M.; Nilakantan, R.; Ellestad, G. A. Science 1989, 244, 697-699. (3) (a) Long, B. H.; Golik, J.; Forenza, S.; Ward, B.; Rehfuss, R.;
Dabrowiak, J. C.; Catino, J. J.; Musial, S.T.; Brookshire, K. W.; Doyle, T. W. Proc. Narl. Acad. Sci. U.S.A. 1989, 86, 2-6. (b) Sugiura, Y.;Uesawa, Y.;Takahashi, Y.;Kuwahara, J.; Golik, J.; Doyle, T. W. Proc. Natl. Acad. Sci. U.S.A. 1989,86,7672-7676. (c) Nicolaou, K. C.; Dai, W. M. Angew. Chem. 1991, 30, 1387-1416. (4) Devoss, J. J.; Townsend, C. A.; Ding, W. D.; Morton, G. 0.; Ellestad, G. A.; Zein, N.; Tabor, A. B.; Schreiber, S. L. J . Am. Chem. SOC.1990,112, 9669-9670. .... .. ( 5 ) Drak, J.; Iwasawa, N.; Danishefsky, S.; Crothers, D. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 7464-7468.
(6) Ellestad, G. A.; Hamann, P. R.; Zein, N.; Morton, G. 0.; Siegel, M. M.;Pastel, M.; Borders, D. B.; McGahren, W. J. Tetrahedron Lett. 1989,30, 3033-3036.
0002-7863/94/1516-3709$04.50/0
are cleaved in a concerted fashion following abstraction of a C5' hydrogen from the oligopyrimidine strand and a C4' hydrogen from the oligopurine ~ t r a n d . 4 .The ~ cycloaromatization reaction and subsequent D N A cleavage are triggered by reduction of the trisulfide moiety that is attached to the enediyne ring. In the absence of a reductant, calicheamicin binds to the D N A but does not promote strand cleavage. Previous studies have shown that calicheamicin binds to the target sequence with high affinity. The dissociation constant, KD,for binding of calicheamicin to duplex D N A has been M for TCCT-AGGA sites5 estimated to be on the order of and 10" M for a mixture of sites within pBR322 DNA.* Most but not all of the binding energy is thought to derive from interactions involving the carbohydrate domain of calicheamicin within the minor groove of the DNA. Calicheamicinone, which lacks the carbohydrate domain, binds to D N A with low affinity and in a non-sequence-specific manner.5 In contrast, the (7)Hangeland, J. J.; De Voss,J. J.; Heath, J. A.; Townsend, C. A. J. Am. Chem. SOC.1992, 114,9200-9202. (8) Ding, W.-D.; Ellestad, G. A.J. Am. Chem.Soc. 1991,113,6617-6620.
0 1994 American Chemical Society
3710 J. Am. Chem. SOC..Yo/. 116. No. 9. 1994
5'
14
Li et a/. M PI
0
25 I O 100 200 400 1000
~ G C T G A T G A T C C T A G A C T G C C 3' ~
~~
Figure 1. Synthetic2Omerduplex DNAcontainingasinglcTCCT.AGGA target site (bored region) far rccognitiion by calicheamicin. Arrows denote sitcs of calicheamicin-induced DNA cleavage. The TCCTcontaining strand was [S'-"P]-labeled (see Experimental Scction). carbohydrate domain alone exhibits much of the binding affinity and sequence specificity that is characteristic of the intact moIecule.9JO In the present study, wecarriedouta kineticanalysistomeasure the binding affinity between calicheamicin and a single TCCT.AGGA site within a synthetic duplex DNA. W e also carried out a competitive binding analysis involving both calicheamicin and the calicheamicin oligosaccharide (2). enabling us to measure the DNA binding affinity of the carbohydrate domain alone. Previously, we showed that the iodinesubstituent
2R-l 3:R-EBr 4R-CI
5R-F 6 RICH, 7:R-H
on the aromatic C ring of the carbohydrate domain is critical for itsability to bind tocertain DNAs ina sequence-specificmanner.10 This result prompted us to prepare a variety of analogues of the calicheamicin oligosaccharide (3-7) that differ with respect to the substituent at the position of iodination. We then measured theDNA bindingaffinityofeachoftheseanalogues.It has been suggested that the iodine moiety interacts with the exocyclic C2NHz of both guanine residues that lie within the oligopurine strandofthetarget DNA." Recent NMRdatasuggest that the iodine is in a position to interact with the first (5'-most) but not the second guanine C ~ - N H Z T . ~o ~evaluate this issue, we measured the binding affinity between calicheamicin and duplex DNA that containseither AGGA, AIGA, AGIA, or AlIA within the oligopurine region of the target DNA (I = inosine, lacking the C 2 amine).
Results Determination of the Calicheamicin-DNA Binding Constant. I n order tosimplify the kineticanalysisoftheinteractionbetween
calicheamicin and a DNA target, we designed a 2Omer duplex DNA thatcontainsa singleTCCT.AGGA site flanked b y a total of 16 base pairs (Figure I). The TCCT-containing strand was [5'-1zP]-labeled and hybridized with a 100-fold molar excess of theunlabeled AGGA-containingstrand. Varyingwncentrations of calicheamicin. always in large excess over the duplex DNA. were added to the reaction mixture and allowed to equilibrate a t 31 OC for 30 min in the absence of a reducing agent. @-Mercaptoethanol (30 mM final concentration) was added to trigger the cycloaromatization ofcalicheamicin, and samples were taken (9) Aiyar. J.; Danishcfsky.S. J.:Crothcrr. D. M.J. Am. Chrm.Soe. 1992.
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