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Formation of Stable DNA Loops by Incorporation of Nonpolar Non-Hydrogen-Bonding Nucleoside Isosteres.

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S. Schul/. M. Andruh. T. Pape. T. Heinze. H. W. Roesky, L. Himing. A.
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1968. 1.5. ?7?. b ) D. A. Lesch, T 8 . Rduchfuss. Inorg. C/ir2m.19881. 20, 3583; c)
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778. e ) G. Gervnaio. 1 Orgonon?rt. Chmi. 1993. 445. 147: f ) L. C. Roof. D. M
Smith. Ci. M: Drake. \hi T. Pennington. J. W. Kolis, /norg. C/t%eni.
1995, 34. 337.
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1993. ( ( J i . 754. Angiwv. Chtvn. 1ri1 Ed. G i g / . 1993. 32. 238; c) S. Pohl, U. Opitz,
h i d 1993. /05.950 and l993,32.863: d) S. Dehnen, D. Fenske. hid. 1994, 106.
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199.5. 34. 307
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3721
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2x4. 13
S. W~illenhaucl-.K . Seppelt. Angeii . C/irni. 1994. 104. 1044; Angels. Chrm. 1171
Ed En,?/. 1994. 33. 976.
a ) C'rystal structuredata for 1. C,Cl,,Fe,O,Tee,: A4 = 946.16. monoclinic. space
group P2, ( ' (no. 14). Z = 4. u = 16.401(3). h = 10.549(2). i. = 14.035(3) A. B
= 1 1 5 . 1 3 ( ? ) . r'=2198.4(7)A'.p,~,~, = 2 . 8 5 9 g c m - ' , M o K , . i =0.71069A.
11 = 0 113 mni ' A total of 2406 independent reflections were collected on a
Rigaku AFC'SS diffractometer at 223(2) K with 0 between 2.37 and 22.49
usins 20-1., scans. and an absorption correction by azimuthal ($) scans was
applied. The structure was solved by direct methods (Siemens SHELXTL-PC)
and refined (SHELXL-93. G . M. Sheldrick) by full-matrix least-squares on F z
t o final residuals of R = 0.0255 (F)and IVR = 0.0647 (F') for observed
data ( / > k i f ) j . and G O F =1.064; b) crystal structure data for 2.
Ci,CI,Fe,0,2~~c,,,: A4 =1977.32. triclinic. space group P i (no. 2). 2 = 2 , u
= 9 045(2). h = 13.684(3). c =17.961(4)
Y =72.74(3),
= 82.19(3), y
=75.15(3). b'=1047.7(8)A3.p~.,,,,=3.207gcm-'.MoK,.L =0.71069&p
= X.673 mm ' A total of 6428 independent reflections were collected o n a
Rigaku A F C j S diffrdctometer at 223(2) K with 0 between 2.24 and 25.00 , a n d
;in absorption correction by azimuthal ($)scans was applied. T h e structure was
solved by direct methods and refined by full-matrix least-squares on Fz to final
residuals of K = 0.041X ( F ) and ISR = 0.1167 (F') for observed data
( / > ? n ( / ) ) and
.
G O F = 0.994. Further detailsofthecrystal structure investigation may be obtained from the Fachinformationszentrum Karlsruhe. D-76344
Eggen~tein-Lcopoldshaf~n(Germany). on quoting the depository number
CSD-59195.
a ) A C' Harell. A 1 . f ~C/wm Scond. 1966. 20. 165; b) P. H Collins. M. Webster,
A m C'r:r.$ru//o~r.
ScJrr. B 1972, 28. 1260; c) B. Krebs, B. Buss. D. Altena. Z.
A n n r x . A//,?. ( ' h c n i . 1971.386. 257; d) B. Buss, B. Krebs, lnorg. Chrm. 1971,10,
2795.
a ) J. Miindenberg. M. Noltemeyer. H. W. Roesky, Chrm. Ber. 1989. t22. 1915;
b) H. W. Roesky. A. Mazzah, D. Hesse. M. Noltemeyer. ihid. 1991, /24, 519.
a ) E. S o h , E ('orazzi, C. Floriani, A. Chiesi-Villa, C. Guastini, J. Chem. Suc.,
/M/(JII
7i.oii.\. 1990, 1345: b) D K Mills. Y. M. Hsdio. P. J. Farmer, E. V.
Atnip. J. H. Reibenspies. M. Y. Darensbourg. J: A m Cheni. Suc. 1991, 113,
1421:c)F.A Cot1on.R. L . Luck.K.-A.Son,/norg Chim. Ai.1a1991.179.11;
d ) F. Bitterer. D. J Brauer, F. Dorrenbach. F. Gol. P. C Kniippel, 0 . Stelzer,
C. Kruger. Y.-H TYay, Z.Nu/ii[/or.sc/i.B 1991.44, 1 1 3 1 ; e) K. R. Dunbar, A.
~.
1993, 105,298; Angrit. Chem. / n t . Ed. Engl. 1993.32,
Quillevere. A n ~ q w Chmi.
293: f ) A. Wintcr. L Zsolnai, G Huttner. 1 Orgunomrr. Chrm. 1982,232.47.
R. Knicp. D. Mootz. A. Rahenau. An,qoii,. Chem. 1973.85. 504. A n g w Chem.
/ n r . Ed Eng/ 1973. 12. 499.
a ) S. C' O'Neal. W. T. Pennington, J. W Kolis. Inurg. Chem. 1990.29, 3134; b)
W. A. Flomer. _I W. Kolis. h i d 1989,28, 2513, c ) M. A. Ansari, C. H. Mahler,
G S. C'horghddc. Y -J. Lu. J. A. lbers. ihirl 1990, 29, 3832: d) T. B. Rauchfuss.
S. DeL. S. R M'ilson. ihid 1992, 31. 153; e) M. G . Kanatzidis, S:P. Huang.
ihid 1989. 2X. 4667.
a ) H. Brunner. W. Mcier. B Nuber. J. Wachter. M. L. Ziegler. Angeiv. Chem.
1986. Y X . 907. A n g m Clrem /nr. E d EiigI. 1986, 25, 907; b) H. Brunner, N
Janict/. W Meisr. I . Wachter, E. Herdtweck, W. A. Herrmann, 0. Serhadli.
M L. Ziegler. .I O r , ~ u n o m ~Chem.
r.
1988. 347. 237.
a)
A,
Formation of Stable DNA Loops by
Incorporation of Nonpolar, Non-HydrogenBonding Nucleoside Isosteres**
Xiao-Feng Ren, Barbara A. Schweitzer, Charles J. Sheils,
and Eric T. KooI*
Hairpin loops are ubiquitous structures found in folded RNA
and D N A sequences in nature. Recent reports have noted special stability for certain loop sequences. For example, RNA
tetranucleotide loops (tetraloops) having the sequence G N R A
and U U C G are more stable than other tetraloops and are highly
conserved in nature.[', D N A tetraloops having the sequence
GAAA, in certain contexts, are unusually stable in duplex
DNA;[31 in triplex structures the loop sequence CTTTG has
been reported to be especially stable as
In most or all of
these cases, structural study has either implicated or identified
intraloop hydrogen bonds between bases and/or phosphates as
important structural features. It has long been recognized. however, that hydrogen bonds in aqueous solution are weak. In this
regard a detailed study of hydrogen bonding in the GAAA
tetraloop in RNA concluded that single hydrogen bonds contribute relatively little to the overall stability.'" While less well
understood, base stacking is also known to be at least as important a contribution to nucleic acid stability as hydrogen bonding.16-'1 Studies of loop structures in R N A and D N A have not
generally addressed the relative importance of base stacking and
hydrogen bonding in stabilizing such loops.
We have undertaken a program to design and synthesize nonhydrogen-bonding nucleoside analogues to be used as probes of
the biological noncovalent interactions of oligonucleotides and
nucleic acids in general.['l We now describe the incorporation of
three such isosteres into D N A loops, and we have found that
such substitutions can lead to significant stabilization of doubleand triple-helical folded structures in DNA.
Nucleosides 1 (F), 2 (B), and 3 (D) were designed to act as
nearly perfect isosteres for the natural nucleosides thymidine (T)
and deoxyadenosine (A) (Fig. 1). However, in contrast to the
natural nucleosides, analogues 1-3 have little or no hydrogen
bonding capability.['] Compounds 1-3 are considerably more
hydrophobic and less polar than the natural structures, and
evidence from dangling-base studies in our laboratory indicate
lo] To test
that they are more efficient at base stacking as
the effects of their properties on folded D N A structures, we
incorporated 1-3 at loop positions into D N A hairpin-forming
sequences and evaluated the thermal and thermodynamic stabilities of the folded structures.
Nucleosides 1-3 were synthesized by using previously described methods.['] The primary products of coupling of the
bases of 1 and 2 with the a-chlorodeoxyribose toluoyl ester
synthon are a-anomers," I ] which were converted to /I-anomers
by acid-catalyzed epimerization." I b l Compounds I -3 were unknown previous to our reports, although two other substituted
indole nucleoside derivatives have recently been described."
The coupling of 1-3 in oligodeoxynucleotides using standard
phosphoramidite chemistry was efficient, with stepwise yields of
>95 o/o (trityl cation monitoring). For comparison we also syn[*] Prof. E. T. Kool, X.-F. Ren. B. A. Schweitzer. C. J. Sheils
Department of Chemistry, University of Rochester
Rochester, NY 14627 (USA)
Fax: lnt. code +(716)473-6889
e-mail' etk:ii etk.chern.rochester.edu
[**I
This work was supported by the National Institutes of Health (GM 52956).
E. T. K also acknowledges a Camille and Henry Dreyfus Teacher-Scholar
Award and an Alfred P. Sloan Foundation Fellowship.
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Table 1. Effects of natural and nonnatural nucleosides in
tetranucleotide loops on the stability of double-stranded
D N A hairpin structures at pH 7.0 [a].
"CI
- AG&
[kcalmol- '1
5-GC A A T TGC T
3'- C G T T A A C G T T
67.7(0.6)
1.3(0 1)
5'- G c A A T T G c F F
3'~C G T T A A C G
F
7X.S(O.S)
3.6(0.3)
T TGC
Y-C G T T A A C G
78.4(0.5)
3.1 (0.3)
5'- G c A A T T G c A A
3'- C G T T A A C G A A
62.9(0.4)
0.4(0.2)
5'-G c A A T T G c D D
Y-C G T T A A C G D
67.2(1.2)
1 l(O.1)
Sequence
Tm
thymidine and analogues
5'- G C A A
B
B
deoxyadenonsine and
analogues
[a] Conditions. 100 mM NaCl, 10 mM phosphate (pH 7.0).
D N A concentration: 5.0PM The sequences with the natural
nucleotides are the same as those described in ref. [13]. Data
shown are averages; error limits are given in parentheses.
cleosides (A,, T,, C,, G,) have T, values lying
in a narrow range between 62 and 68"C.r131
Free energy values obtained by curve fitting
show that the modified loop containing 1 is
thermodynamically more stable than the most
stable natural loop (the T, loop) (Table 1).
Similarly, the B, hairpin sequence, with
trimethylphenyl nucleosides, is almost as stable
as the sequence with the F, loop.
1,2,3:
a, R, R' = H;
Interestingly, just as the pyrimidine isosteres
give more stable loops than a natural pyrimb, R = DMT, R' = OH;
idine, we find that the purine analogue 3 is
c, R = DMT, R'= P(NiPr,)OCH2CH2CN
more stabilizing
- than its deoxvadenosine counFig. 1. Structural formulas and space-filling models of nonpolar nucleoside isosteres 1 (F), 2 (B), and 3
terpart (ATrn = 4.5 K). As was reported by
(D). Also shown are the structures of the natural nucleosides thymidme and deoxyadenosine.
Breslauer et a1.,[131we find that the T, loop is
more stabilizing than the A, loop. Moreover,
we find that the nonnatural pyrimidine isosteres 1 and 2 are
thesized analogous sequences containing the corresponding natmore stabilizing than the purine isostere 3. This difference in the
ural nucleosides at the same positions (see Tables 1 and 2).
natural hairpin structures has been attributed to cross-loop hyWe first studied the effects of I (F), 2 (B), and 3 (D) in a
drogen bonding. Since in the nonnatural cases this difference
duplex DNA hairpin loop, by using two self-complementary
cannot be due to hydrogen bonding, it seems likely that steric
sequences of eight base pairs bridged by a tetranucleotide
differences in the pyrimidine and purine analogues may play an
loop consisting of four identical residues. Previous studies of
important role both in the natural and nonnatural cases.
this sequence by Breslauer et al.['31 showed that, of sequences
Because the nonpolar thymidine isostere 1 was found to be
containing A,, C,, G,, and T, loops, the most stable was
the better stabilizing of the two analogues and because it is the
the T, case, and the least stable, the A, case. We studied
best isosteric substitution, we tested it in a triple helix bridging
the properties of sequences containing F,, B,, and D, loops,
loop. Such folded triplex-forming oligonucleotides are finding
and we synthesized the natural T, and A, analogues for comincreasing use as ligands for single-stranded nucleic acids.['41
parison.
The stability of the loops in such a complex depends largely on
Table 1 shows data obtained for the duplex hairpin sequences.
the nature of the first and last bases in the
The nuBuffer conditions were pH 7.0 (10 mM phosphate) with 100 mM
cleoside 1 was therefore placed at these positions in a pentanuNaC1. All five sequences gave melting transitions that were indecleotide loop bridging the pyrimidine strands in an eight-base
pendent of concentration, confirming the expected hairpin
triplex. For comparison we synthesized the natural T,-bridged
structure as opposed to intermolecular complexes. The data
triplex (see Table2). We examined both 5'- and 3'-type loop
show that the modified hairpin sequences containing 1 (F) and
orientations[41at pH 7.0 with 100 mM NaCl and 10 mM MgC1,.
2 (B) are significantly more stable than the T, analogue. The
The nonpolar nucleoside 1 gives complexes that are both thermelting temperature (T,) for the modified F, sequence, which
mally and thermodynamically more stable than structures concontains difluorotoluene nucleosides, is 78.5 " C , or 10.8 "C
taining the natural nucleosides (Table 2). For example, the hyhigher than that of the T, loop structure, which is the most
drophobic nucleoside F gives complexes that are 4.8-5.4 K more
stable tetraloop containing four identical natural nuthermostable than the T,T-substituted oligonucleotides, and
cleosides.[' 31 The four hairpin structures with natural nu744
G
YCH V e r ~ a ~ . ~ g e . s ~ l lmhH,
, ~ c l ~0-69451
a~l
Weinlieim, 1996
o57o-0833~96~35o7-0744
$ 1 5 . 0 0 i .25/0
Angew Cliem. I n t .
Ed. Engl. 1996. 35, No. 7
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Table 2 Effects of varying the first and last nucleotides in the loop on the stability
of triplexes formed between 21-nucleotide pyrimidine DNAs and complementary
8-nucleotide purine DNA strands at pH 7.0 la].
Complcx with
5’ loop
7,,
[ C]
T T C T T T T c T T -3’
T
GAAAAGAA-3
T T C T T T T C T T -5
27.2
T FCTTTTCTT.3
T
G A A A A G A A T
T C T T T T C T T -5’
32.6
- AG;5
[kcalmol-
Complex with
3‘ loop
Tm - AG;,
[ C] [kcalmol-’1
8.8
5’ T T C T T T T C T T
5’-AAGAAAAG
T
3’ T T C T T T T C T T
256
10.6
5’ T T C T T T T C F T
5’-AAGAAAAG
T
3’- T T c T T T T C T
3 2 4 10.6
‘1
8.4
[a] Conditions. l 0 O m ~NaCI, 1 0 m MgCI,.
~
l O m ~Na-PIPES (pH 7.0); DNA
10% in free energy.
concentration: 7.0 p~ per strand. Errors: i 5 ” C in T, and
free energies are 1 3 - 2 . 2 kcal mol- more favorable. Previous
studies carried out with natural bases in this
have shown
that the T,T case is intermediate in stability; the C,C loop is
the least stable and the G,G loop the most stable. The G,G loop
was found to be more stable than the T,T analogue by
1.5 kcal mol-‘.[41 Thus our F,F loop is more stable than all four
cases doubly substituted with the natural bases.
Previous studies have described the use of simple nonnucleotide linkers as loop replacements in nucleic acids.”51 Such
loops are often less stabilizing than natural nucleotide loops;
this may be due both to the greater rigidity of nucleotide loops
relative to the highly flexible nonnucleotide linkers and to favorable base stacking interactions in natural loops, which are missing in simple linkers. The nucleoside analogues in this study are
designed to stabilize nucleic acid helices as a result of both
rigidity and base stacking. In contrast to natural DNAs, loops
containing nucleosides 1-3 have the added advantage of being
resistant to degradation by nucleases (data not shown). In addition, such nonpolar nucleosides have been shown to pair with
low affinity and low specificity with natural sequences[9b1and
thus would not interfere with the expected pairing in biological
systems.
The present results on the incorporation of hydrophobic nucleoside analogues in oligonucleotide loops show that in general
these compounds are significantly stabilizing. Because they d o
not undergo measurable hydrogen bonding interaction^,"^^ it
would seem that the best explanation for this behavior is superior base stacking propensity. This suggests a general strategy for
stabilizing nucleic acid complexes containing loops: the use of
arenes instead of natural bases, especially at positions adjacent
to the helix, where stacking interactions are expected to make
the greatest contributions. Although the present compounds
cannot form stabilizing cross-loop hydrogen bonds, they may
have favorable hydrophobic or van der Waals interactions. Detailed structural and physical studies will be necessary to investigate the specific origins of this stability.
(3H. 5); I3C NMR (CDCI,).6 =13.8 (d). 22.0 (d), 40 1, 64.9.74.9, 83 0, 103.0 (t).
120.1 (d), 124.5(d), 127.3(d),128.6, 128.8(d), 128.9(d), 144.0(d), 156.5(d), 158.0
(d), 155.9 (d). 162.3 (d), 166.1 (d); HRMS (FAB. 3-nitrobenzyl alcohol matrix, M
+H+):calcd for C,,H,,F20, 481.1827, found 481.1853
2a (8-epimer, 54% yield). ’ H NMR (CDCI,): 6 = 8.02 (2H. d, J = 8.0 Hz). 7.97
(2H,d,J=8.0Hz).7.30-7.37(1H,m,obscured)7.32(2H,hrd,J=10.7Hz),7.31
(2H,brd,J=11.2Hz),7.24(1H,s),6.95(1H,s),5.65(1H.hrd,J=5.6Hz),5.42
( l H , dd, J = 5.0, 10.8 Hz), 4.76 (lH,
dd, J = 3.9. 11.8 Hz). 4.70 ( l H , dd, J = 3.6,
11.8 Hz), 4.55 (1H. m). 2.56 ( t H , m), 2.46 (3H, s). 2.43 (3H. s). 2.31 (3H, s), 2.23
(3H,s).2.18 (lH,m),2.13(3K.s); ‘3CNMR(CDC13):6 =lX 2.19.5.22.1 (d),41.0,
65.0.82.5. 126.2.127.0(dj. 128.6(d). 128.81d). 132.1 (d), 135 5.136.2.141.4,144.5,
165.5,166.0; HRMS (FAB. 3-nitrobenzyl alcohol matrix, M + l’calcd for C,,,H,,O,
472.2250. found 472.2234.
Conversion of nucleoside his-p-toluoyl esters of la, 2a t o phosphoramidite derivatives lc, 2c: The hydrolysis of the his-p-toluoyl esters, subsequent conversion to
5’-O-dimethoxytrityl derivatives, and 3’-O-phosphitylation in preparation for automated DNA synthesis were carried out by using the same methods previously
described for the I-isomers [9b]. Analytical data for these compounds were in
complete accord with their assigned structures 11 lb].
Oligonucleotide synthesis: DNA oligonucleotides were synthesized on an Applied
Biosystems 392 instrument by using standard p-cyanoethylphosphoramidite chemistry [16]. Methods for deprotection, tritylation, and phosphitylation of nucleosides
3a-c were as described previously [9b]. Oligomers were purified by preparative gel
electrophoresis with 20% denaturing polyacrylamide (detection at 260 nm) Molar
extinction coefficients for sequences containing the natural nucleosides were calculated by the nearest neighbor method [17]. The molar extinction coefficients measured for nucleosides l, 2, and 3 were 1200, 851. and 6361. respectively; molar
absorptivities for oligonucleotides containing these residues were derived as previously described [9b]. Oligodeoxynucleotides were obtained after purification as the
sodium salts. Intact incorporation of residues 1-3 was confirmed by synthesis of
short oligomers of sequence 5’-T-X-T (where X = 1,2. o r 3). The ‘ H N M R spectra
(500 MHz) indicated the presence of the intact aromatic ring structures with the
expected integration relative to thymine C-5 protons and methyl groups. Enzymatic
degradation analysis could not be performed because the nonniitural residues were
found to inhibit enzymatic cleavage.
Thermal denaturation studies: Buffers used for optical melting experiments were
100 mM NaCI, 10 mM Na-phosphate (for duplex hairpins) or 100 mM NaCI. 10 mM
MgCI,, 10 mM Na-PIPES (1,4-piperazine(hisethanesulfonate))(for triplexes). The
buffer pH is that of a 1 . 4 stock
~
solution at 25 ‘ C containing the buffer and salts.
After the solutions were prepared they were heated to 90 ‘C and allowed to cool
slowly to room temperature prior to the melting experiments.
The melting studies were carried out in Teflon-stoppered quartz cells (1 cm pathlength) under nitrogen atmosphere on a Varian Cary 1 UV/Vis spectophotometer
equipped with a thermoprogrammer. Absorbance was monitored at 260 nm while
temperature was increased at a rate of 0.5 ”Cmin-’ ; a slower heating rate with this
instrument does not affect the results. In all cases the complexes displayed sharp,
apparently two-state melting transitions. Melting temperatures ( Tm)were determined by computer fit of the first derivative of absorbance with respect t o l/T.
Uncertainty in individual T,, values is estimated at k0.5 K. Free energy values were
derived by computer-fitting the denaturation data with an algorithm employing
linear sloping baselines by using the two-stateapproximation for inelting[7]. Uncertainty i n individual free energy measurements is estimated at & 5 to 10%.
Received: April 4, 1995
Revised version: December 27. 1995 [Z7865 IE]
German version: Angen. Chem. 1996, 108, 834-837
-
Keywords: base stacking DNA - hydrogen bonds . nucleosides
-
triple helices
[I] D. R. Groebe, 0.C Uhlenbeck, Nurlerc Acid.: Res. 1988. / 6 , 11725- 11 735.
[2l H. A. Heus. A. Pardi, Science 1991,253. 191 - 194.
I31 I. Hirao, Y. Nishimura, Y. Tagawa, K. Watanabe, K. Miura. Nucirrc Acid.: Res.
1992,20, 3891-3896.
Exprirnmtcil Procedure
[41 S. Wang, M. A. Booher, E. T. Kool, Biochemistry 1994. 33. 4639-4644
Epimerization of 1-anomers of la and 2a (bis-p-toluoyl esters) to p-anomers.
151 J. SantaLucia. R. Kierzek, D. H. Turner, Science 1992,256. 217-219.
la. To a solution of the 1-anomer of the his-p-toluoyl ester ofcompound la (synthe161 C . R. Cantor, P. R. Schimmel, Biophvsical Chemistrj, Purr 111 The Behuvior
sis described in ref. [9a]) (780 mg. 1.62 mmol) in toluene (50 mL) was added a
of Biological Macromolecules. W. H. Freeman. San Francisco. 1980,pp. 11 17catalytic amount of benzenesulfonic acid (ca. 10%). one drop of concentrated
1133.
[7] M . Petersheim, D. H. Turner, Biochemistrj 1983,22, 256-263.
H,SO,. and 11\10 t o l’our drops of H,O. The reaction mixture was stirred vigorously
a t reflux for 4 6 h. and the mixture was then poured into 5 % aqueous NaHCO,
[8] M. Senior. R. A. Jones, K . J. Breslauer, Biochemistrr 1988.27, 3879-3885.
(50 mL) and extracted uith EtOAc (3 x 50 mL) The combined organic layers were
I
Org. Chen?. 1994,59, 7238 -7242; b) J Am.
191 a) B. A. Schweitzer, E. T. Kool, .
dried over anhydrous MgSO, and concentrated to dryness. Silica column chroChem. SOC.1995,117, 1863-1872.
matography (eluent 8.1 + 2/1 hexanes/EtOAc) of the crude mixture gave 430 mg of
(101 X.-F. Ren, B. A. Schweitzer, C. J Shiels, N. C. Chaudhuri. E T. Kool, unpublished
la (p-epimer. 4 6 % yield). ‘ H NMR (CDCI,): 6 = 8.02 (2H, d , J = 8.0 Hz), 7.97
(2H. d. J = 8.0 Hz). 7.30-7.37 (1H. m, obscured), 7.31 (2H, d, J = 8.0 Hz), 7.25
[I11 a) N. C. Chaudhuri. E. T. Kool, Terrahedrun L e f t . 1995. 36. 1795-1798; ihid.
(2H.d,J=80Hz).h75(1H,dd.J=8.0.8.0Hz).5.64(1H,brd.J=5.8Hz).5.47
1995.36.4910: b) X -F. Ken, N. C. Chaudhuri, E T. Kool. unpublished.
~1H.dd.J=5.1.10XH~).4.78(lH.dd.J=3.8.11.8Hz),4.66(1H,dd.J=3.7, 1121 a) R. S. Coleman, Y Dong, J. C. Arthur, Tetrahedron Lett. 1993,34, 6867ll.~Hz).4.54(lH.m).2.64(1H,m),2.46(3H,s),2.43(3H.s),2.23(1H,m),2.176870; b) D. Loakes. D. M. Brown, Nucleic Acids Res. 1994.22. 4039-4043.
Angeii.. C%rm 1111.G I . Engl. 1996. 35, No. 7
Q VCH &4ag.:gesellschqfr mhH. D-6945i Wemheim, 1996
0570-0833!96!3507-0745 S 15.00+ 2 S i O
745
COMMUNICATIONS
[13] M. M. Senior, R. A. Jones. K. J. Breslauer. Proc Nu/!. Acud. Sci. USA 1988,
85,6242 -6246.
1141 a) L. E. Xodo. G. Manzini. F. Quadrifoglio. G. A. van der Marel, J. H. van
Boom, Nucleic Acids Res. 1991. 19, 5625-5631; b) E.T. Kool. J. Am. Chem.
Soc. 1991, 113. 6265-6266; c) G. Prdkdsh. E. T. Kool. J. Chem. Soc. Chem.
Commurn. 1991, 1161-1162; ;hid. 1994. 646; d) C. GiOVdnnangeh, T Montenay-Garestier, M. Rougee. M. Chassignol. N. T. Thuong, C . Helene. J Am.
Chent. Soc. 1991, 113, 7775-7776; e) G. Prakash, E. T. Kool, ihid. 1992, 114,
3823-3528, f) M. Salunkhe, T. Wu. R. L. Letsinger, ihM 1992. 114, 87688772; g) D. J. D'Souza, E. T. Kool, J. Biomol. Strucr. Dyn. 1992, l(J, 141-152;
h) S. Rumney, E T. Kool. Angew. Chem. 1992, 104,1686-1689; A n p w . Chem.
h r . Ed. Engl. 1992,31.1617-1619; 1)T. A. Perkins. J. L.Goodman, E. T. Kool,
J. Chem. Soc. Chetn. Contmun 1993, 215-217; J ) C. Giovannangeli. N. T.
Thuong, C. Helene, Proc. Null. Acud Sci. USA 1993. 90. 10013-10017, k)
S. M. Gryaznov. D. H. Lloyd, Nuc!eic Acids Re.:. 1993. 21. 5909-5915; I)
R. H. E. Hudson, M. J. Damha. Nucleic Acids S.vmp. Ser. 1993, 29.97- 99; m)
D. J. D'Souzd, E. T. Kool. Bioorg. Med. Chem. Lett. 1994, 4, 965-970; n) S.
Wang, E. T. Kool, Nucleic Acids Res. 1994, 22, 2326-2333; 0)S. Wang, E. T
Kool, J. A n . Chem. Soc. 1994, 116, 8857-8858; p) E. R. Kandimal. S.
Agrawal, Gene 1994,149, 115-121; q) D. M. Noll, J. L. O'Rear, C. D. Cushman, P. S. Miller, Nucleosides Nucleatides 1994. 13, 997- 1005; r ) T L. Trapane, M. S. Christopherson, C. D. Roby, P. 0 P. Ts'o, D. Wang, 1 Am. Chmt.
Soc. 1994, 116.8412-8413. s) R. Bdndaru. H. Hashimoto. C. Switzer. J. O g .
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1995. 117, 10434-10442.
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(Ed.. G. D. Fasman), CRC Press. Cleveland, OH, USA, 1985. p. 589.
1,3-Didehydrobenzene (m-Benzyne)""
Ralph Marquardt, Wolfram Sander,* and Elfi Kraka*
Of the three isomeric didehydrobenzenes, only 1,2-didehydrobenzene (1) has been characterized unequivocally by direct
spectroscopic methods.['] The only derivative of 2 that has been
characterized by matrix IR spectroscopy is
2,4-didehydrophen01.~'~
Early reports on the
t
2
3
UV/Vis spectroscopic
characterization of 2
and 3 are questionable, since the reaction conditions and reported properties suggest that a more stable isomer, such as enediyne
4, was observed.
A barrier of only 19.8 kcalmol- was determined for the ring
opening of 3 by Roth et al.,I3]which compares well with barriers
of 19.4 and 20.5 kcalmol-' for this reaction calculated at the
0
1 0. 0
.
[*j Prof. Dr. W. Sander, Dip1.-Chem. R. Marquardt
Lehrstuhl fur Organische Chemie I1 der Universitft
D-44780 Bochum (Germany)
Fax: Int. code +(234) 709-4353
Prof. Dr. E. Kraka
Department of Theoretical Chemistry
University of Goteborg
Kemigiirden 3, S-41296 Goteborg (Sweden)
Fax. Int. code f(31) 772-2933
[**I This work was supported by the Deutsche Forschungsgemeinschaft and the
Fonds der Chemischen Industrie as well as the Swedish Natural Science Research Council (NFR) and the Swedish Institute (SI). We owe special thanks
to the Nationellt Superdatorcentrum (NSC), Linkoping, Sweden, for a generous allotment of computer time.
746
8 VCH
Veriagsgesellschaft mbH, 0-69451 Weinheim, 1996
CCSD(T) level of theory by Kraka and creme^.[^. Flash vacuum pyrolysis of both 1,3-diiodobenzene (5)[61and isophthaloyl
diiodide (6)"l produced 4 as the principal product, which suggests
that 2 also undergoes thermal ring opening to give 4 (Scheme 1).
!
&' \
5
[a]
4
2
Scheme 1. Thennochemistry of 1.3-diiodobenzene (5) and isophthaloyl diiodide
(6).
Squires et al. determined the heats of formation of the arynes
1-3 from collision-induced dissociation measurements to be
106.6 i3.0, 122.0 t 3.1, and 137.3& 3.3 kcalmol- respectively.['. 91 These thermodynamic data have been confirmed by
Kraka and Cremer r4. and Lindh et aI.[''l using high-level ab
initio methods. Here we describe the matrix isolation and IR
spectroscopic characterization of 1 ,3-didehydrobenzene (2),
generated from two independent precursors.
UV photolysis (2. > 305 nm) of matrix-isolated'"' [2.2]metaparacyclophane-2,9-dione (7)[12]results in the formation of carand a new compound with an
bon monoxide, p-xylylene (8),[131
intense IR absorption at 547 cm-' and several weak absorptions (Scheme 2, Table 1). The new compound was stable to-
'@
___,
LAr,
> 3 010K
5nm
0-0
t2CO
-
8
2
7
0
0
9
2
Scheme 2. Photochemistry of [2.2]metaparacyclophdne-2,9-dione
(7) and thennochemistry of isophthdloyl diacetyl peroxide (9).
ward prolonged irradiation at 248 nm, and based on the comparison of the IR spectrum obtained experimentally with the ab
initio calculated IR spectrum (vide infra) it was assigned the
structure of 2 (Fig. 1).
Flash vacuum pyrolysis (300 " C ) of isophthaloyI diacetyl peroxide (9)[14'with subsequent trapping of the products in argon
at 10 K produced carbon dioxide and methyl radicals
(Scheme 2).[15]In addition, the same intense IR absorption at
547 cm-' assigned to 2 was observed. Annealing of the matrix
at 30-35 K resulted in the loss of the methyl radicals and a
simultaneous decrease in intensity of the absorptions assigned
to 2. In solid argon at temperatures above 30 K the diffusion of
0570-0833/9613507-0746$ l S . O O + .25/0
Angew. Chem. Int. Ed. EngI. 1996, 35, No 7
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