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DNAЦCarbohydrate Recognition Design and Synthesis of an Eight-Base Sequence-Selective DNA-Binding Oligosaccharide.

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DNA- Carbohydrate Recognition: Design and
Synthesis of an Eight-Base Sequence-Selective
DNA-Binding Oligosaccharide""
K. C. Nicolaou,* Keiichi Ajito, Hironori Komatsu,
Brian M. Smith, T i a n h u Li, Miles G. Egan, and Luigi
Gomez-Paloma
DNA-binding molecules, particularly those with sequence
specificity, have considerable potential in chemistry, biology,
and medicine." - 'I Such molecules may provide the basis for
further insights in the chemistry and structure of DNA, for the
design and development of tools for molecular and cell biology,
and for the development of antisense and gene
Following calicheamicin Is] as a lead, we have recently synthesized and studied the binding of a series of oligosaccharides to
duplex DNA.[' - ' In these investigations''~ lo] oligosaccharide
1 (Scheme 1) emerged as the most potent and selective molecule,
binding duplex D N A at TCCT sites with an affinity constant of
Ka lo6 M - First synthesized in 1990 in Our 1aboratory,"21
oligosaccharide 1 was recently shown by Crabtree et a1."3i to
specifically inhibit transcription complexes. Appropriately designed oligomers of 1 should exhibit enhanced binding to duplex
D N A in terms of potency and specificity. We now wish to report
the design and chemical synthesis of the head-to-head dimer 2
(Scheme 1) of oligosaccharide 1, which was designed to bind
duplex D N A selectively at TCCTXXAGGA
>J:
Fig. 1 . Computer-generatedmodel ofdimer 2bound to theminor groove ofduplex
DNA [GCATCCTATAGGATGC] along a TCCTATAGGA site.
Based on the detailed interactions of oligosaccharide 1 with a
TCCT site of a duplex 10-mer D N A as determined by NMR
spectroscopy,"'. 15] and footprinting experiments,[', lo. l6I and
by using computer modeling, the head-to-head dimer 2 was
designed to dock properly into the minor groove of duplex D N A along a TCCTXXAGGA tract. The five-atom
tether -CH,CH,OCH,CH,- was found,
by computer modeling, to allow optimum fit of the two oligosaccharide domains into their respective binding sites.
Me0
-, e
Figure 1 depicts a computer-generated
HNM
OMe
model
of dimer 2 bound to duplex DNA
HO
6M'
oMeHO#*
along its target TCCTXXAGGA tract.
MeONH
The synthesis of the designed deca*'
cyclic
oligosaccharide 2 was carried out
Cn
from the previously reportedr"I key intermediate 3 as summarized in
Me
'&'*O
0
Me
Scheme 2.[ls1 Coupling trichloroacetimidate 3 with excess diethylene glycol
in CH,CI, at -78 -+ -40°C in the
Me
presence of BF, Et,O provided compound 4 in 69 % yield together with its
OMe
OH
Me0
O HHN-Me
L
= 3
anomer (4', 11 % yield). The chromato1 - 2
graphically separated 4[191 was then
HO
i.
OMe
coupled with a second molecule of 3 under Similar conditions to afford the
Scheme 1. Structures of calicheamicin ;.:ohgosaccharides 1 (monomer, binds a TCCT site) and 2 (head-to-head
dimer designed to bind a TCCTXXAGGA site). The tether is marked with an arrow
dimeric compound 5[l9] in 59% yield
(plus 18 Yo recovered starting material
4). Desilylation of 5 with I-IF. pyridine
[*I Prof. K. C. Nicolaou, K. Ajito, H Komatsu, 8 . M. Smith, Dr. T. Li.
followed by removal of the 9-fluorenylmethoxycarbonyl
M . G. Egan, Dr. L. Gomez-Paloma
(FMOC) group led to the polyhydroxyl intermediate 7, via comDepartment of Chemistry, The Scripps Research Institute
pound 6 , in 76% overall yield. Finally, NaCNBH, reduction of
10666 North Torrey Pines Road, La Jolla, CA 92037 (USA)
7 catalyzed by BF, . Et,O resulted in the formation of the
and
targeted compound 2[191in 46% yield. The structure of 2 was
Department of Chemistry and Biochemistry, University of California. San
secured by spectroscopic means, particularly ' H N M R specDiego, 9500 Gilman Drive, La Jolla, CA 92093 (USA)
troscopy which defined the stereochemistry at the newly generTelefax: Int. code +(619) 554-6738
ated anomeric and N-bearing centers.
[**I This work was financially supported by the National Institutes of Health
(USA) and the University of California (Biotechnology Grant). Fellowship
Preliminary NMR studies (variable-temperature, NOESY,
from Meiji Seika Kaisha, Ltd. (visiting scientist, K. A.), Mitsui Toatsu ChemTOCSY)
on a 1:l complex of dimer 2 with ds[GCATCCicals, Inc. (visiting scientist. H. K.), and the Universitl Degh Studi Di Napoli
TATAGGATGC1 DNA revealed binding in the minor groove
"Frederico 11" (visiting assistant professor, L. G. P.) are gratefully acknowledged.
along the TCCTATAGGA tract. The data are consistent with a
ox;f l 0
'
"
'
O
.
T
o
Me'&lIH
,*;,w'z*
'#No
576
<C) VCH l+l-iagsgeseli.dufi mhH, 0-69421 Weinfrrirn. / W S
M70-~j833:9Sl0505-0576$10.00
+ ..?.5111
Arigew. Chem. In!. Ed. EngI. 1995. 34, N o . 5
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T E S O ~ O T E S
6Me
3
4
+
OMe
Scheme 2. Synthesis of head-to-head
dimer 2. a ) 4.0equiv diethylene glycol. 0.5 equiv EF, . Et,O, CH,CI,.
- 78 + -40
-C, 4 h. 69% yield plus
11 % c( anomer: b) 2.0 equiv 3.0.3 equiv
BF, . Et,O, CH,CI,. -78'C. 5 h, 59%
yield plus 18% recovered 4: c) HF . py,
CH,CI,. py. -40 + 0 C, 2 0 h ; d)
Et,NH:THF (1:l). 25 C. 3 h. 76%
overall yield from 5 ; e) 60 equiv NaCNBH,. 26 equiv BF, Et,O. CH,CI,.
-60
-40 -C, 2.5 h. 4 6 % yield plus
a number of other. not characterired
isomers. TES = triethylsilyl; FMOC =
9-fluorenylmethoxycarbonyl.
-
model in which two complexes are present at low temperature
corresponding to one tightly bound pentasaccharide unit and
one loosely bound unit; at higher temperatures the two states
recognition and gene regulation. The design, synthesis, and
evaluation of higher oligomers of 1 and their corresponding
enediyne-containing relatives are in progress.
Footprinting experiments with 2 were complicated, presumably owing to the interference of the molecule with the en-
Received: November 2. 1994 [Z 7449 IE]
German version: Angew Chmi. 1995, 107. 614
oligosaccharides
concentration-dependent inhibition of D N A cleavage by t i e
dimer 2 as expected (Fig. 2). In comparison with monomer 1
(lanes 8-10. Fig. 2), dimer 2 (lanes 5-7, Fig. 2) appears to exhibit considerably higher binding affinity to DNA.
also indicated strong inPreliminary in vitro
hibiton of binding for certain transcription factors to D N A and
interference with in vivo gene expression, suggesting potential
application for molecules such as 2 to gene regulation and therapy,[131
Further studies are needed to determine more precisely the
interaction of dimer 2 with D N A and its potential in D N A
Fig. 2. Autoradiogram of denaturing polyacrylamide gel showing products of calicheamicin-induced b
DNA cleavage of' [5'-32P]-labeled. TCCTATACGA-containing DNA duplex in the presence of oligosaccharides 1 and 2. Lane 1 : marker lane containing the products of DNA digestion with Mbo I restriction
enzyme: lane 2 28-mer ladder resulting from partial DNA digestion with nuclease P l ; lane4:
[S'-"P]-labeled 2X-mer; lanes 3. 5--10: products of DNA cleavage with calicheamicin (250 nM) in the
presence of varying concentrations of monomer I and dimer 2 (lane 3: no oligosaccharide, lane 5 : SO nM
2. lane h 5 LIM 2. lane 7 : SO p%t2, lane 8: 50 n M 1. lane 9: 5 p~ 1, lane 10: 50 p~ 1). Methods: DNA
cleavage by calicheainicin was performed as follows: The [5'-32P]-laheled. TCC~ATAGGA-containing
DNA oligoiner was incubated at 95°C for 3 min and then cooled slowly to 25 C over 1 h. An aliquot of
the oligoiner was added to a reaction mixture containing 20 mM NaCI, 30 mM Tris-HCI (pH 7.5). 10%
DMSO ( y v ) , and varying concentrations of oligosaccharide 1 or 2 in a final volume of 40 pL, and the
mixture was incubated at 37 'C for 20 min. Calicheamicin-dependent DNA cleavage was initiated by
addition of 2 pL of 2-mercaptoethanol to attain a final concentration of 50 mM. Ahquots of 3 pL of
reaction inixture were transferred to a dry, ice-cooled Eppendorf tube. An equal volume of gel loading
buffer. containing 96% forrnamide, 7.5 mM Na,EDTA 0.05% (w/v), bromophenol blue, and 0.05 YO( w p )
xylene cqanol. was added and the sample was stored at -70-C until loading. Tris = tris(hydroxyniethyl)aiiiinoethane DMSO = dimethyl sulfoxide, EDTA = ethylenediaminetetraacetic acid.
COMMUNICATIONS
[l] K. C. Nicolaou, W.-M. Dai, Arigew. Cheni. 1991. 103. 1453: Angew. Chem. h i .
Ed. Engl. 1991. 30. 1387.
[2] J. W. Lown. Chem/racrs 1993. 6, 205.
[ 3 ] P. E. Nielsen, Bioconjugute Chem. 1991. 2. 1.
[4] E. Uhlmdnn. A. Peyman, Chem. Rev. 1990, 90, 543.
[5] P. B. Dervan in Nucleic Acids und Molecular Biology, Vol. 2 (Ed.: F. Eckstein.
D. M. J. Lilley), Springer. Heidelberg. 1988, pp. 49-64.
[6] P. B. Dervan in Oligodeo~ynucleoridestAnti.wnsr o/Gene E.rpression (Ed.: J. S.
Cohen), CRC Press. Boca Raton. FL, 1989. pp. 197-210.
[71 C. Helene. J:J. Toulme in Oligodeo.~ynirc/rotid',~:
Antisense of Gene Erpres.rion
(Ed.: J. S. Cohen), CRC Press, Bocd Raton, FL. 1989. pp. 137-172.
[8l M. D. Lee. K. M. Manning, D. R. Williams, N. A. Kuck, R. T. Testa, D. B.
Borders. J Anrrbior. 1989. 42. 1070.
[91 T. Li, Z. Zeng. V. A. Estevez, K.-U. Baldenius. K. C. Nicolaou. G. F. Joyce, J.
Am. Chem. Soc. 1994, 116, 3709.
[lo] K. C. Nicolaou, S:C. Tsay, T. Suzuki, G. F. Joyce, J. Am. Chem. Soc. 1992,
114. 7555.
[Ill L. Gomez-Paloma. J. A. Smith. W J. Chazin, K. C. Nicolaou, J. Am. Chc>m.
Suc. 1994, 116, 3697.
[I21 K . C. Nicolaou, R. D. Groneberg, T. Miyazaki, N. A. Stylianides. T. J. Schulze,
W. Stahl, J. Am. Chem. Soc. 1990, 112.8193. A second synthesis of 1 appeared
later: R. L. Halcomb, S. H. Boyer, S. J. Danishefsky. Angru. Chem. 1992, 104,
314; Angrw. Chern. Inl. Ed. Engl. 1992, 31, 338.
[I31 S. N. Ho, S. H. Boyer, S. L. Schreiber. S. J. Danishefsky. G. R. Crabtree, Prot..
Nail. Acad. Sci. USA, 1994, 91, 9203.
[14] The TCCT site may be interchanged with a TCTC or a TTTT site, see refs.
[9- 121, and references therein.
[15] S. L Walker, A. H. Andreotti. D. E. Kahne, Tetrahedron 1994. 50,
1351.
[16] J. Aiydr. S. J. Danishefsky, D. M. Crothers, J. A m . Chem. Soc. 1992, 114.
7552.
[I71 K. C. Nicolaou, C. W. Hummel. M. Nakada. K . Shibayama, E. N. Pitsinos, H.
Saimoto, Y Mizuno, K.-U. Baldenius. A. L. Smith, J. Am. C h m . Soc. 1993.
115. 7625.
[la] All new compounds provided satisfactory spectroscopic and analytical
data.
[I91 Selected spectroscopic and physical properties of 2.4, and 5.2: Colorless solid:
R, = 0.52 (silica gel. MeOH/CH,CI, l;4); [ZIP= 41 (c = 0.50, MeOH); IR
= 3600-3200,2973.2933,1673,1455,1416,2392.131Y,
1239.1100,
(neat):
1066. 959 c m - ' ; 'H N M R (500 MHz. CD,OD): 6 = 5.62 (br. s, 2H. D-I),
CHHCH,OCH,CHH), 3.81 (s, 6H, CH,O), 3.77 (s, 6H, CH,O). 3.70 (dd,
J=9.0,9.0Hz,2H.D-4),3.67(dd,J=10.5,2.5Hz.2H,B-4),3.63-3.52(m,
8H. D-3, CHHCH,OCH,CHH), 3.39 ( s , 6 H . C H 3 0 ) . 3.32(m,2H. E-4), 3.16
(s. 6 H , CH,O). 2.31 (s, 6H. ArCH,), 2.23 (m, 2 H , E-2,,), 1.95 (m, 4 H ,
B-2,,,..),1.43(d,J=7.0H~,6H,A-6),1.28(d,J=6.5Hz,6H,B-6),1
16(d,
J = 6.5 Hz, 6H. D-6), 0.98-0.90 (m, 72H. 8 x Si(CH,CH,),); MS (positive
2 Na+], calcd for C,,,H,,,I,N,O,,S,Si,:
ion ESI): rnji 3394: [M,,,-2H+
3394.
The details of these NMR studies will be reported elsewhere in due
course.
We thank Drs. Peter Vogt and Chen LIUfor their results. These and related
studies will he reported in due course.
+
Spherical Cyclophosphazene Dendrimers to the
Fifth Generation**
Franqois Sournies, Franqois Crasnier,
Marcel Grdffeuil, Jean-Paul Faucher, Roger Lahana,
Marie-Chris tine Labarre, and Jean-Frangois Labarre*
Dendrimers, that is, highly branched functionalized molecules
formed by iterative reaction sequences, constitute a definite
breakthrough into generations of new materials and are attracting considerable attention in organic, supramolecular, and polymer chemistry.['] Only few of these macromolecules incorporate
main group elements such as silicon,[21germanium,[31or phosp h o r ~ ~ ,and
[ ~ ] only phosphorus dendrimers having charges
within the cascade structure had been de~cribed'~]
till very recently when two kinds of neutral phosphorus dendrimers were
reported concomitantly by Majoral et al.['] and by our
'I Majoral's dendrimers have a structure resembling a
group.[6,
5.43(br.s,2H,E-l),5.09(br.d,J=lO.OHz,2H,B-l),4.49(br.s,2H.D-2).
cauliflower, with SPCI, as the core and possess 46 pentavalent
4.38 (d, J = 8.0Hz, 2 H , A-l).4.25(br. s, 2H. B-3). 4.18 (dq, J = 9.5.6.0Hz.
2H. D-5), 4.06 (dq, J=10.5. 6.5Hz. 2H. B-5). 4.01-3.92 (m. 4H. E-5.
phosphorus atoms and 48 terminal functions (aldehydic groups
CHHCH,OCH,CHH), 3.96 (dd, J = 9.5, 9.5 Hz. 2H. A-3), 3.92 (s. 6H.
or phosphorus-chlorine bonds; molecular weight 1 1 268 or
CH,O), 3.87 (s, 6 H , CH,O). 3.84-3.71 (m, 12H, B-4. D-3, E-5'.
15 381). His group is now synthesizing further generations havCHHCH,OCH,CHH). 3.69(dq.J = 9.5. 6.5 Hz,2H,A-5).3.61 ( d d , J = 9.5,
ing up to 384 functional groups (molecular weight 94 146).C8I
9.5 Hz. 2 H . D-4), 3.57 (s. 6H. CH,O), 3.42 ( s , 6 H . CH,O), 3.37 (dd, J = 9.5,
8.0 Hz, 2H, A-2). 2.89 (m. 2H. E-4). 2.80 (m. 4 H , 2 x CH,CH,N), 2.47 (m,
Our dendrimers are spherical architectures which were de2H. E-2,J. 2.38 (s. 6 H , ArCH,), 2.28 (dd. J = 9.5, 9.5 Hz, 2H. A-4). 2.00 (m,
signed from previously skillfully synthesized D,, cyclophos2H. B-2,,). 1.79 (m, 2 H , B-2,,,). 1.50 (m, 2H. E-2,,), 1.41 (d, J = 6.5 Hz. 6 H ,
phazene cores. Indeed, we recently reportedL6.'I pure cyclophosB-6), 1.39 (d, J = 6.5 Hz, 6H. A-6), 1.28 (d, J = 6.0 Hz. 6H, D-6), 1.22 (br. t,
phazenic hexapodanes (termed sexapus because of the six
J = 6 . 8 Hz, 6 H , 2xCH,CH2N); " C N M R (125MHz, CD,OD): 6 =194.1,
153.2, 152.0, 144.6, 134.5, 132.5, 99.8, 94.2. 81.7. 80.6, 77.1, 72.6. 72.3, 72.1.
tentacles, in analogy to an octopus) synthesized through a re71.6, 71.3, 70.5, 69.4, 69.3. 69.3, 68.2, 62.3, 61.5, 59.9, 57.5. 56.2, 42.7, 41.6,
giospecific peraminolysis of hexachlorocyclotriphosphazene
39.0, 35.2, 25.7, 19.4, 18.8. 14.7. 10.0; MS (positive ion EN): mjr 1996:
(N,P,CI,) by long-chain diamines, H,N(CH,),NH, ( n 2 6), on
[ ( M + H)i],calcdforC,,H,2412N40,,S,:
1996.-4: Colorlesssolid; R, = 0.15
ALPOT (50: 1I), that is, on alumina impregnated with a certain
(silica gel, 2 % MeOH in CH,CI,): [m];' = - 21 ( c = 1.7, CHCI,): IR (neat):
C = 2953, 2926, 2876, 1694, 1682, 1454, 1260. 1084. 1017 cm-'; ' H NMR
amount of potassium hydroxide.['] These sexapus units are 3-di(500 MHz, [DJDMSO, 80°C): 6 ~ 7 . 8 (d,
4 J =7.5 Hz. 2H, FMOC), 7.63 (d.
mensional polyfunctional cores suitable for generating spherical
J =7.5 Hz, 2H, FMOC), 7.39 (dd, J =7.5, 7.5 Hz, 2H. FMOC), 7.31 (dd,
cyclophosphazene dendrimers (that is, resembling the structure
J =7.5, 7.5 Hz, 2H. FMOC), 5.42-5.37 (m, 2 H , B-I, D-1). 5.08 (br. s. 1 H.
of a dry dandelion flower and not of a cauliflower like Tomalia's
E-I), 4.75 (9. J = 6.5 Hz, 1 H, A-5), 4.47-4.38 (m, 5H, A-1, 8-3, D-2. CH,FMOC). 4.28 (t. J = 6.0 Hz, 1 H, benzyl. FMOC), 4.22 (br. s. 1 H, A-3). 4.05
dendrimers['I). An attempt at the production of spheroid den(dq,J=9.0,6.5HZ,1H,D-5),4.00(dq,J=10.0,6.5Hz.lH,B-5),3.84-3.72
drimers belonging to carbon chemistry was recently made by
~
(m, SH, A-2. E-3, E-5. E-S, CHHCH,OCH,CH,OH). 3.X2 (s, 3H, CH,O).
3.78 (s. 3H, CH,O), 3.70 (dd, J = 9.0, 9.0 Hz. 1 H, D-4). 3.68 (dd, J =10.0,
2.5 Hz, 1 H, 8-41? 3.66-3.42 (m%8 H . D-3, CHHCH,OCH,CH,OH). 3.40 (s.
['I
Dr. JLF. Labarre, Dr. F. Sournies. Dr. F. Crasnier, Dr. M. Graffeuil,
3H.CH,O),3.31(m,lH,E-4),3.17(s,3H,CH,O),2.31(s,3H,ArCH,),2.24 Dr. J.-P. Faucher, Dr. M.-C. Labarre
Institut de Chimie Molkculaire Paul Sabatier, Ldboratoire Structure & Vie
Universite Paul Sabatier. 118 route de Narbonne
F-31062 Toulouse Cedex (France)
Telefax: Int. code + 61 251733
Dr. R. Lahana
Oxford Molecular SA. X-Pole. Ecole Polytechnique
F-91128 Pabaiseau Cedex (France)
[**I On the Scent of Dandelion Dendrimers. Part 3 . The authors are greatly indebt7.38(dd.J=7.5.7.5H~.4H.FMOC).7.30(dd,J=7.5.7.0Hz,4H.FMOC). ed to the Universite Paul Sabatier (ACRU 1993-1994) and the French Minis5.42-5.37 (m, 4 H , B-1, D-I), 5.08 (br. s. 2H. E-I), 4.73 (4, J =7.0 Hz, 2H.
tery of Education (66.71.50) for their generous financial support to this work.
A-5). 4.45-4.37 (m, IOH, A-I, 8-3. D-2. CH,-FMOC). 4.26 (t. J = 6.0 Hz,
Sincere thanks are due to Dr. Suzanne Richelme who performed DCIINH,
2H.benzyl. FMOC).4.22(br.s,2H.A-3),4.05(dq.J=9.0,6.5Hz,2H,D-5).
and FAB measurements and to Dr. Akdin Dall'Ava for 3'PNMR spectra.
3.99(dq,J=10.5.6.5Hz,2H.B-5),3.83-3.73(m,10H,A-2,E-3,E-5,E-5', Part 2: [7].
(m.1 H. E-2,,), 1.98 (m, 2H, B-2eQ.J. 1.44 (d. J = 6.5 Hz. 3H, A-6), 1.29 (d.
J = 6 . 5 H z , 3H, 8-6) 1.17 (d, J = 6 . 5 H z . 3H. D-6). 0.99-0.91 (m. 36H,
4 x Si(CH,CH,),), 0.67-0.58 (m. 27H. 4 x Si(CH,CH,),, CH,CH,N): MS
(positive ion ESI): m/r 1750; [ ( M + Na)']. calcd for C,,H,,,IN,O,,SSi,:
1750. - 5: Colorless solid; R, = 0.46 (silica gel, ether/petroleum ether l / l ) :
[ZIP= 14 (c = 0.99. CHCI,); IR (neat): i. = 2954, 2936. 2876, 1700, 1456.
1417, 1239, 1140, 1085, 1016. 1004cm-', 'H NMR (500MHz, [DJDMSO,
8O'C):S =7.83(d.J =7.5 Hz,4H, FMOC),7,62(d, J =7.0 Hz,4H, FMOC),
~
578
8
VCH Verlugsgrsrllschaft mhH. 0-69451 Weinheim. 1995
0570-0833~9SjOSOS-0578
$10.00+ ,2510
Angew. Chem. Inr. Ed. Engl. 1995, 34, No. 5
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