of any other derivatives such as deoxyadenosine or deoxy-
inosine (data not shown). This establishes that the postsynthetic
addition of the amine proceeds with high (!95%) conversion.
The iodopurine 3 is thus an efficient vehicle for incorporation of
amine nucleophiles into DNA.6
mixed sequence (5A-pdGTCAGTCAA*GTCAGTCAp) (data
not shown) showed that this was more pronounced for purine-
containing DNA. This nonselective iodine reaction was some-
what surprising because of the common use of iodine in
DNA synthesis; however, it seems possible that altering
conditions beyond those we examined, or using a more
selective electrophile, might lead to increased specificity for
allyladenine.
The expected products of self-cleavage of the sequence
A-dTTTTTTA*TTTTT are 5A-dTTTTTTp and 5A-pdTTTTT
5
(Scheme 2). We synthesized these fragments as authentic
samples for comparison on high-resolution gels. The second
modified strand had the sequence 5A-dTTTTTTTA*
TTTTTTA*TTTTT, containing two possible cleavage sites.
The result of iodine treatment with ABI oxidizing solution
followed by heating with piperidine was efficient and clean
chain cleavage for both sequences (Scheme 2). Experimentation
with solvents and reaction conditions established that most
A few other reports of methods for generating abasic
(depurinated or depyrimidinated) sites or for site-directed
7
cleavage in DNA exist in the literature. The present method
represents an alternative cleavage strategy which, unlike most
of the previous approaches, allows essentially normal base
pairing prior to the cleavage. Studies aimed at finding strategies
for decreasing associated nonspecific cleavage are currently
underway.
selective cleavage occurred using 20 mm I and 66 mm DNA
2
strand concentration.† High-resolution polyacrylamide gels
show the cleavage (Fig. 1), and separate gels show that the
products co-migrate with the authentically synthesized frag-
ments. Reversed-phase HPLC analysis also confirmed this
finding. Control reactions show (Fig. 1) that the presence of the
allyl function in the DNA is necessary for cleavage, as is iodine
treatment of the DNA. Examination of the reaction products
indicates that cleavage is not completely selective for the allyl-
substituted base, with some background cleavage also occurring
at other bases. Experiments with a third oligonucleotide of
Footnotes
*
†
E-mail: etk@etk.chem.rochester.edu
Conditions for reactions on DNA: (1) Allylamine treatment—1 ml of
allylamine (neat) was added to CPG bound DNA (1 mmol synthesis) and the
mixture was kept at 65 °C for 18 h. It was lyophilised and treated with
4
NH OH at 55 °C for 12 h, lyophilised and gel purified. (2) Iodine
treatment—5A-32P labelled DNA sequence (200 pmol, 66 mm) was dissolved
in 3 ml of 20 mm I solution (20% v/v pyridine, 10% v/v water, 70% v/v
2
THF), and incubated at 4 °C for 10 min. The mixture was then diluted with
1
50 ml H
a 1.0 m aq. piperidine solution in H
0 °C for 30 min, and lyophilised.
2
O and heated at 90 °C for 6 h. (3) Piperidine treatment—100 ml of
2
O was added and the mixture heated at
9
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5
1
It may be possible to directly place the allyl-dA in the strand during
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2
6, 1217.
6
The ribofuranose version of iodopurine has been synthesized previously.
Fig. 1 Cleavage assay of two DNA sequences containing 1 by denaturing
See V. Nair and S. G. Richardson, J. Org. Chem., 1980, 45, 3969.
3
2
gel electrophoresis. All the sequences were 5A- P labelled. Lanes 1–5 show
the reactions with the sequence 5A-T A*T , lanes 6–8 the reactions with
control DNA molecules, lane 9 the product marker and lanes 10–11 the
reactions with DNA sequence 5A-T A*T A*T . Lane 1: no reaction. Lane 2:
reaction of allyl untreated 5A-T A*T with both I and piperidine. Lane 3:
reaction of 5A-T A*T only with I . Lane 4: reaction of 5A-T A*T only with
piperidine. Lane 5: reaction of 5A-T A*T with I as well as piperidine.
Lanes 6–8: reaction of 5A-T AT , 5A-T CT and 5A-T GT respectively with
both I and piperidine. Lane 10: no reaction. Lane 11: reaction of
A-T A*T A*T with both I and piperidine. Lanes 3, 5 and 11 clearly show
7 J. A. Iocono, B. Gildea and L. W. McLaughlin, Tetrahedron Lett., 1990,
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6
5
7
6
5
6
5
2
6
5
2
6
5
6
5
2
6
5
6
5
6
5
2
5
7
6
5
2
the cleavage of modified DNA to the expected products. Some non-specific
cleavage can be seen in lanes 2, 6, 7 and 8.
Received in Corvallis, OR, USA, 27th January 1997; 7/00626H
1426
Chem. Commun., 1997