7-deazaadenine-DNA: Bulky 7-iodo substituents or hydrophobic 7-hexynyl chains are well accommodated in the major groove of oligonucleotide duplexes

Article abstract of DOI:10.1002/(SICI)1521-3765(19980904)4:9<1781::AID-CHEM1781>3.0.CO;2-K

Oligonucleotides containing 7-deaza-7-(hex-l-ynyl)-2'-deoxyadenosine (1) and 7-deaza-7-iodo-2'-deoxyadenosine (2) are described. The corresponding phosphoramidites (3 a,b) were synthesized and employed in solid-phase oligonucleotide synthesis. The modifie

Full text of DOI:10.1002/(SICI)1521-3765(19980904)4:9<1781::AID-CHEM1781>3.0.CO;2-K

FULL PAPER
7-Deazaadenine-DNA: Bulky 7-Iodo Substituents
or Hydrophobic 7-Hexynyl Chains Are Well Accommodated
in the Major Groove of Oligonucleotide Duplexes
Frank Seela* and Matthias Zulauf
Abstract: Oligonucleotides containing 7-deaza-7-(hex-1-ynyl)-2'-deoxyadenosine
(1) and 7-deaza-7-iodo-2'-deoxyadenosine (2) are described. The corresponding
phosphoramidites (3a,b) were synthesized and employed in solid-phase oligonucleo-
tide synthesis. The modified 7-deazaadenine residues show selective base-pairing
with dT. According to the Tm values and the thermodynamic data of duplex
formation, the 7-iodo and the 7-(hex-1-ynyl) residues of a 7-deazaadenine moiety
increase the duplex stability with retention of the particular DNA structure. This is
different from 8-substituted adenine, which destabilizes the nucleic acid duplex
significantly. The nucleoside 1 fluoresces strongly.
Keywords: DNA
duplex stability
oligonucleotides
structures
nucleosides
´
´
´
gonucleotides.[8, 9] Thermal denaturation profiles of oligo-
phosphorothioates containing C-7 propyne derivatives of 7-
deaza-2'-deoxyguanosine and 7-deaza-2'-deoxyadenosine hy-
bridized to RNA have been reported.[10]
Introduction
Modified oligonucleotides are useful tools with which to
probe the influence of specific changes to bases or to sugar
moieties on the structural, spectroscopic, and thermodynamic
properties of nucleic acids. They have also attracted interest as
gene expression inhibitors (antisense oligonucleotides) and
are used as hybridization probes.[1] In particular, the 7-
deazapurine (pyrrolo[2,3-d]pyrimidine) nucleosides, nucleo-
tides, and oligonucleotides (purine numbering is used
throughout the discussion section) have already made a
significant contribution to various areas of this research.[2±4]
Previously, we observed that less bulky substituents intro-
duced at the 7-position of 7-deazaadenine-containing oligo-
deoxynucleotides stabilize the oligonucleotide duplex struc-
ture compared with the parent adenine-containing DNA
fragments.[5] These findings are in line with the effects of 5-
substituted pyrimidine nucleosides with less bulky groups.[6, 7]
Nevertheless, very bulky substituents or lipophilic side chains
in 7-deazapurine nucleosides can affect the major groove in
various ways and can thereby change the DNA structure. Up
to now, such studies have only been performed on 7-
deazaguanine and 2,6-diamino-7-deazapurine containing oli-
This work focuses on the properties of oligonucleotides
with rather bulky iodo or lipophilic (hex-1-ynyl, hxy) sub-
stituents in the 7-position of the 7-deazaadenine moiety (1 and
2, shown in Scheme 1). Evidence for the influence of modified
residues on the stability of duplexes will be presented and
thermodynamic data will be determined. The results of these
studies can have practical applications: a) a 7-iodo substitu-
ent, when used in the form of the isotope 135I, can be valuable
for the isotopic labeling of DNA; b) the lipophilic 7-alkynyl
side-chains can improve the delivery of antisense constructs
through the cell membrane, and c) the rodlike structures of
the 7-(hex-1-ynyl) side-chain have the potential to allow the
identification of particular nucleobases by atomic force
microscopy (AFM).
Here, we report the synthesis of the phosphoramidites 3a,b.
A series of oligonucleotides with different sequence patterns
are prepared and their physicochemical properties, as well as
their duplex structure and stability, are studied.
Results and Discussion
[*] Prof. F. Seela, Dipl.-Chem. M. Zulauf
Laboratorium für Organische und Bioorganische Chemie,
Institut für Chemie
1
Monomers: Single-crystal X-ray analysis as well as H NMR
Universität Osnabrück, Barbarastr. 7
D-49069 Osnabrück (Germany)
and NOE measurements have already been used for the
conformational analysis of 7-deaza-7-iodo-2'-deoxyadenosine
(I7c7Ad, 2).[11] It has been demonstrated that the bulky iodo
Fax : (‡ 49)541-969-2370
E-mail: fraseela@rz.uni-osnabrueck.de
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F. Seela and M. Zulauf
FULL PAPER
0.27 and the lifetime 5.8 ns; both were
measured in aqueous solution. A typical
feature of the fluorescence spectrum is
the large Stokes shift, which can be
related to a charged transition state
(1: excitation: 280 nm; emission:
397 nm).[16]
NH2
NH2
(Me)2NHC
N
N
C
C(CH2)3CH3
I
R
N
7
5
1
3
N
N
N
N
N
N
3
9
N
1
7
O
O
O
HO
HO
(MeO)2TrO
HO
HO
O
Regarding oligonucleotide synthesis,
P
it has been reported that the NH2-
protecting groups of 7-deaza-2'-deoxy-
adenosine are more stable than those of
2'-deoxyadenosine.[17] The same was ex-
pected for the 7-substituted derivatives 1
and 2. Several acyl derivatives (4a ± d)
were prepared as well as the (dimethyl-
amino)methylidene derivatives 5a,b (Scheme 2).[18] The in-
creased basicity of the 6-amino group of 2 prompted us to try
the acylation reaction in EtOH solution with acid anhydrides,
NCCH2CH2O
N(iPr)2
purine numbering
systematic numbering
2
1
3a,b
Scheme 1. Nucleosides and phosphoramidite building blocks used for the synthesis of oligonucleo-
tides [a: R ˆ I, b: R ˆ C C(CH2)3CH3].
ꢀ
substituent does not affect the favorable anti conformation of
the nucleoside (2)[11] as has been reported for 8-substituted
purines.[12] The 7-deaza-7-(hex-1-ynyl)-2'-deoxyadenosine
(hxy7c7Ad, 1) was prepared by the palladium-catalyzed
cross-coupling reaction of 7-deaza-7-iodo-2'-deoxyadenosine
(2) with hex-1-yne.[13] It was observed that compound 1
exhibits strong fluorescence.[14] More than 30 7-alkynyl-7-
deazaadenine nucleosides have been synthesized since and
studied with regard to their fluorescence, quantum yields, and
lifetimes.[15] Figure 1 shows the excitation and emission
spectra of compound 1. The quantum yield was found to be
NHR
(Me)2NHC
N
N
I
R
N
N
N
N
N
O
O
HO
HO
HO
HO
5a,b
4a-e
Scheme 2. N6-amino-protected nucleosides [4a: R ˆ Bz, b: R ˆ Ac, c: R ˆ
ꢀ
iBu, d: R ˆ Piv, e: R ˆ Pac; 5a: R ˆ I, b: R ˆ C C(CH2)3CH3].
using conditions developed earlier for the relatively nucleo-
philic amino group of 2'-deoxycytidine.[19] However, these
conditions resulted in low reaction yields ( ꢁ 20%). Higher
yields ( ꢁ 50%) were obtained when compounds 4a ± d were
prepared by the protocol of transient protection.[20] Only the
phenoxyacetyl derivative 4e was prepared by peracylation
followed by selective deprotection of the sugar moiety.[21]
The stability of the various protecting groups (4a ± e and
5a,b) was studied by UV spectrophotometry in 25% aqueous
NH3 (Table 1). From these data it is apparent that the
benzoylated derivative 4a is too stable for oligonucleotide
synthesis, whereas the phenoxyacetyl nucleoside 4e is too
labile. Because the (dimethylamino)methylidene compounds
5a,b were readily available, further investigations were
performed with these derivatives. The intermediates 5a,b
Figure 1. Fluorescence spectra (excitation and emission) of 7-deaza-7-
(hex-1-ynyl)-2'-deoxyadenosine (1) measured in double-distilled H2O with
10 5 m nucleoside concentration.
Abstract in German: Oligonucleotide, die 7-Desaza-7-(hex-1-
inyl)-2'-desoxyadenosin (1) oder 7-Desaza-7-iodo-2'-desoxya-
denosin (2) enthalten, werden beschrieben. Entsprechende
Phosphoramidite (3a,b) wurden synthetisiert und in der
Festphasensynthese eingesetzt. Die modifizierten 7-Desazaa-
denine gehen eine selektive Basenpaarung mit dTein. Laut den
Tm-Werten und den thermodynamischen Daten der Duplex-
bildung stabilisieren die 7-Iod- und 7-(Hex-1-inyl)-Reste
Oligonucleotid-Duplexe unter weitgehender Erhaltung der
DNA-Struktur. Dies unterscheidet sie von 8-substituierten
Adeninen, die die Duplexstruktur signifikant destabilisieren.
Das Nucleosid 1 weist eine starke Fluoreszenz auf.
Table 1. Half-life values (t1/2) for the deprotection of 7-deazaadenine
2'-deoxyribonucleosides in 25% aq. ammonia.[a]
l [nm]
t1/2 [min]
bz6I7c7Ad (4a)
ac6I7c7Ad (4b)
ibu6I7c7Ad (4c)
piv6I7c7Ad (4d)
pac6I7c7Ad (4e)
fma6I7c7Ad (5a)
fma6hxy7c7Ad (5b)
312
299
299
303
301
323
321
290
7
12
103
4
82
110
[a] Measured at 408C.
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Duplex Stability
1781±1790
were treated with 4,4'-dimethoxytriphenylmethyl chloride to
give the (MeO)2Tr derivatives 6a,b (Scheme 3).[22] The
phosphoramidites 3a,b were prepared from the DMT deriv-
atives under standard conditions from chloro((2-cyanoethyl)-
diisopropylamino)phosphine.
Oligonucleotides: Oligonu-
cleotides were synthesized
on a solid phase by means of
an automated synthesizer
with the phosphoramidites
3a,b, as well as those of the
regular DNA constituents.[24]
The oligonucleotides were
prepared and purified by oli-
gonucleotide purification car-
tridges,[25] and their homoge-
neity was determined by re-
verse-phase HPLC. Figure 2
shows that the mobility of the
parent oligomer 7 was de-
creased by a 7-iodo substitu-
ent (!11) and even more by
the lipophilic hexynyl side
chain of 12. The nucleoside
composition of the oligomers
(Me)2NHC
N
N
R
N
N
Cl
NC(CH2)2OPN(iPr)2
56-61%
(MeO)2TrCl
67-70%
5a,b
3a,b
O
Figure 2. HPLC profiles of
the oligonucleotides 5'-d(A ±
T)6 (7), 5'-d(I7c7A ± T)6 (11),
and 5'-d(hxy7c7A ± T)6 (12)
after purification by reverse-
phase (RP-18) chromatogra-
phy, gradient I; for details see
Experimental Section.
(MeO)2TrO
HO
6a,b
Scheme 3. Synthesis of the 7-deazaadenine nucleoside building blocks for
ꢀ
DNA synthesis [a: R ˆ I, b: R ˆ C C(CH2)3CH3].
was determined by hydrolysis with snake-venom phospho-
diesterase followed by alkaline phosphatase (see Figure 3, for
example), as well as by MALDI-TOF mass spectroscopy
(Table 3).
The structures of the new compounds were determined by
1H, 13C, and 31P NMR spectra and by elemental analyses
(Table 2 and Experimental Section). The 13C chemical shifts
of the derivatives 4a ± e, 5a,b, and 6a,b were assigned from
the coupling pattern of the corresponding gated-decoupled
13C NMR spectra. According to Table 2, carbon-7 of the 7-
iodo derivatives shows a strong upfield shift upon iodination,
which is due to the strong mesomeric effect of the iodo
substituent.
The nucleosides 1 and 2 were analyzed by 1H NOE
spectroscopy to elucidate the base conformation around the
1
N-glycosylic bond. Vicinal H,1H coupling constants of the
sugar protons revealed the most favored puckering (N vs S-
conformer populations) of these nucleosides. It was found that
the 7-(hex-1-ynyl) chain influences the conformation of the
parent 7-deazaadenine in a similar way to the bulky iodo
substituent. The conformation of the 7-(hex-1-ynyl) nucleo-
side 1 around the N-glycosylic bond is anti and the sugar
confomation is 69% S-type compared to 71% determined for
the iodo compound 2.[23] Thus, it can be anticipated that both
nucleosides form B-like DNA structures if incorporated in a
DNA-duplex.
Figure 3. HPLC profiles of the oligonucleotides a) 11 and b) 24 after
enzymatic hydrolysis with snake-venom phosphodiesterase, followed by
alkaline phosphatase in 1m Tris ± HCl buffer (pH 8.3), gradient II; for
details see Experimental Section.
Table 2. 13C NMR chemical shifts of 7-deazaadenine 2'-deoxyribofuranosides.[a,b]
C(2)[c]
C(2)[d]
C(6)[c]
C(4)[d]
C(5)[c]
C(4a)[d]
C(7)[c]
C(5)[d]
C(8)[c]
C(6)[d]
C(4)
C(7a)[d]
C(1')
C(3')
C(4')
C(5')
c7Ad
1
2
151.6
152.5
152.0
151.9
150.9
150.8
150.7
151.1
151.5
151.6
151.2
151.9
157.5
157.5
157.3
167.3
151.1
151.3
151.5
157.6
160.2
160.9
160.2
161.0
102.9
102.3
103.2
114.5
112.7
113.3
114.0
112.0
110.3
110.1
110.3
110.2
99.6
95.5
51.9
54.6
53.8
54.0
54.0
53.2
53.6
97.5
53.9
98.0
121.6
125.4
126.9
131.9
131.3
131.4
131.4
131.6
128.6
127.1
128.3
127.1
149.6
149.0
149.8
151.1
151.5
151.6
152.0
151.7
150.9
150.7
151.0
151.0
83.3
83.1
83.0
83.1
83.0
82.9
82.9
83.1
83.0
82.9
82.8
82.7
71.1
70.9
71.0
71.0
70.8
70.8
70.8
70.9
71.1
71.0
70.9
70.8
87.3
87.4
87.5
87.8
87.5
87.5
87.5
87.7
87.5
87.4
85.6
85.5
62.1
61.9
62.0
61.8
61.7
61.7
61.7
61.8
61.9
61.9
64.3
64.3
4a
4b
4c
4d
4e
5a
5b
6a
6b
[a] Measured in [D6]DMSO at 258C. [b] Superimposed by DMSO. [c] Purine numbering. [d] Systematic numbering.
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Table 3. Molecular weights determined from MALDI-TOF mass spectra
of oligonucleotides.
shows a significant Tm increase, is stabilized by a more
favorable entropy term. Some of these observations on the
duplexes derived from the analogues of 5'-d(A ± T)6 (7) can
also be made for oligomers in which larger segments alternate
(Table 4, Figure 4).
‡
‡
M
(calcd)
M (found)
5'-d(I7c7A ± T)6 (11)
4391.9
4391.9
5070.5
4117.3
4117.3
4567.1
4171.4
3804.7
4144.0
4392.0
4394.8
5071.2
4117.1
5'-d[(I7c7A)6-(T)6] (14)
5'-d[(I7c7A11)-A] (29)
The retention of the helix geometry of the duplexes, shown
in Table 4, can be seen from their CD spectra. The oligonu-
5'-d(hxy7c7A ± T)6 (12)
5'-d[(hxy7c7A)6-(T)6] (15)
5'-d(hxy7c7A11)-A] (30)
4116.5
[a]
5'-d(hxy7c7A-A)6 (31)
4173.0
3801.2
4144.3
5'-d(CGCG(hxy7c7A)2TTCGCG) (20)
5'-d(GTI7c7AG(I7c7A)2TTCTI7c7AC) (23)
[a] Not detected.
Self-complementary oligonucleotides with alternating bases or
alternating base tracts: Oligonucleotide duplexes with alter-
nating 5'-d(A ± T)n, for example poly[5'-d(A ± T)] ´ poly[5'-
d(A ± T)], have a more flexible secondary structure than
other oligonucleotide duplexes. They have a small helical
twist on the 5'-d(A ± T) steps and a larger one on the 5'-d(T±
A) steps.[26, 27] For this kind of duplexes [5'-d(A ± T)n],
environmental factors such as dehydration and counterion
binding will influence their secondary as well as their tertiary
structure. Therefore, structural modification of the base
residues has a major impact on the stability of such duplexes.
In order to study this behavior, alternating self-comple-
mentary oligonucleotides 5'-d(A ± T)6 (7), 5'-d[(A)6 ± (T)6]
(13), and 5'-d[(A)3 ± (T)3 ± (A)3 ± (T)3] (16) were prepared and
the influence of 7-substituents on the Tm values as well as on
the thermodynamic data was determined (Table 4). From
Table 4, it is apparent that the alternating oligonucleotides
with 7-halogenated 7-deazaadenine residues have Tm values
generally about 208C higher than those with the nonsubsti-
tuted 7-deazaadenine residue. This amounts to a Tm increase
of about 1.78C per modified base residue, which is almost
independent of whether the halogen substituent is Cl, Br, or I.
The stabilization of duplexes containing 7-halogenated 7-
deazaadenines reflects a favorable enthalpic term compared
with the duplex of 5'-d(A ± T)6 (7). However, when the
halogenated compounds 9, 10, and 11 are compared with 5'-
d(c7A ± T)6 (8), the change of entropy seems to play the major
role. The duplex of 5'-d[(hxy7c7A)6 ± (T)6] (15), which also
Figure 4. Melting profiles of the alternating duplexes a) 5'-d[(I7c7A ± T)6]2
(11 ´ 11), and b) 5'-d[(hxy7c7A ± T)6]2 (12 ´ 12), measured at 270 nm in 1m
NaCl, 100mm MgCl2, and 60mm Na cacodylate (pH ˆ 7.0) with 10mm
oligomer concentration.
Table 4. Tm values and thermodynamic data for self-complementary oligonucleotides with alternating bases or base tracts.
1
1
1
Tm [8C][a,b]
DH [kcalmol
]
DS [calmol 1 K
]
DG8 [kcalmol ]
5'-d[(A ± T)6]2 (7 ´ 7)
33(26)
36(27)
57(51)
57(51)
59(53)
45( 44)
65( 53)
60( 63)
62( 55)
65( 70)
125( 127)
186( 153)
159( 173)
165( 148)
174( 192)
6.3( 5.5)
6.8( 5.5)
10.6 ( 9.8)
10.9( 9.4)
11.4( 10.6)
10.8)[c]
9.3( 9.4)
9.1( 7.4)
11.9( 10.4)
11.3( 9.0)
5'-d[c7A ± T)6]2[5] (8 ´ 8)
[5]
5'-d[(Cl7c7A ± T)6]2 (9 ´ 9)
5'-d[(Br7c7A ± T)6]2[5] (10 ´ 10)
5'-d[(I7c7A ± T)6]2 (11 ´ 11)
(
71)[c]
(
193)[c]
(
5'-d[(hxy7c7A ± T)6]2 (12 ´ 12)
5'-d[(A)6-(T)6]2 (13 ´ 13)
5'-d[(I7c7A)6-(T)6]2 (14 ´ 14)
5'-d[(hxy7c7A)6-(T)6]2 (15 ´ 15)
52(49)
46(40)
58(52)
58(52)
47( 62)
81( 75)
76( 76)
64( 54)
123( 170)
232( 219)
206( 212)
169( 144)
5'-d[(A)3-(T)3-(A)3-(T)3]2 (16 ´ 16)
40(35)
61(57)
71( 69)
66( 57)
203( 201)
175( 149)
7.8( 6.4)
12.1( 10.8)
5'-d[(hxy7c7A)3-(T)3-(hxy7c7A)3-(T)3]2 (17 ´ 17)
[a] Measured at 270 nm in 1m NaCl containing 100mm MgCl2 and 60mm Na cacodylate (pH ˆ 7.0) with 10mm oligonucleotide concentration. [b] Values in
parentheses measured at 270 nm in 0.1m NaCl, 100mm MgCl2, and 10mm Na cacodylate (pH ˆ 7.0) with 10mm oligonucleotide concentration. [c] Determined
from the concentration dependence of the Tm values.
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Duplex Stability
1781±1790
cleotide duplexes 9, 10, and 12 exhibit the characteristics of a
B-like DNA structure, with a positive band at 275 nm and a
negative lobe around 250 nm (Figure 5). Only the CD
spectrum of the iodine-containing duplex 11 differs signifi-
cantly from the others (Figure 5).
Figure 6. 1/Tm vs log c plot of self-complementary duplexes 19 ´ 19, 20 ´ 20,
23 ´ 23, and 24 ´ 24; for sequences see Table 5.
agreement. This underlines the fact that duplex formation
follows a two-state process.
In the case of the oligomers related to the Dickerson ±
Drew dodecamer 5'-d(CGCGAATCGCG) (18) (Table 5),
the replacement of dA residues by either 1 (!20 ´ 20) or 2
(!19 ´ 19) increases the Tm values above the value of the
parent duplex 18 ´ 18. The same is observed for 5'-d(GTA-
GAATTCTAC) (21). The Tm increase is between 1 ± 1.58C per
modified base residue, depending on the sequence and the
type of substituent. A 7-(hex-1-ynyl) residue has less of a
stabilizing effect than the 7-iodo substituent in the case of the
sequence type 21. Again, the stabilization by the alkynyl
residue is caused by more favorable entropy, even when the
enthalpy change is unfavorable.
Figure 5. CD spectra of the alternating duplexes 5'-d[(Cl7c7A ± T)6]2 (9 ´ 9),
5'-d[(Br7c7A ± T)6]2 (10 ´ 10), 5'-d[(I7c7A ± T)6]2 (11 ´ 11), and 5'-d[(hxy7-
c7A ± T)6]2 (12 ´ 12), measured at 108C in 1m NaCl, 100mm MgCl2, and
60mm Na cacodylate (pH ˆ 7.0) with 10mm oligomer concentration.
Self-complementary oligonucleotides with palindromic se-
quences: The next oligonucleotides to be investigated were
also self-complementary but contained a palindromic se-
quence, which represents the recognition site of the endo-
deoxyribonuclease Eco RI 5'-d(GAATTC).[28] Hairpin for-
mation has been previously reported, in particular for the
Dickerson ± Drew oligomer 5'-d(CGCGAATTCGCG)[29]
measured at low salt concentration in the absence of
Mg2‡.[30] Since our measurements were performed at 1m or
0.1m NaCl in the presence of 0.1m MgCl2, only duplex
formation can be anticipated for all cases. The thermody-
namic data of the oligonucleotides were determined by curve
shape analysis of the melting profiles by means of the program
Meltwin 3.0[31] and in a few cases also from the concentration
dependence of the Tm values (Table 5, Figure 6). The thermo-
dynamic data obtained by these two methods are in sufficient
Non-self-complementary Oligonucleotides: homooligonucleo-
tides: For the studies of homooligonucleotide duplexes, the
oligomers 5'-d[(I7c7A)11 ± A] (29) and 5'-d[hxy7c7A)11-A] (30)
were hybridized with d(T)12 (26), and their Tm values were
compared with those of d(A)12 ´ d(T)12 (25 ´ 26) (Table 6).
Stabilization for the hybrid 29 ´ 26 (Tm 548C) containing 7-
iodo-7-deazaadenine residues was similar to alternating 5'-
d(I7c7A ± T)6 (11), compared with the parent duplex d(A)12 ´
d(T)12 (25 ´ 26) (Tm 448C). As can be seen from Table 6, the
Table 5. Tm values and thermodynamic data of self-complementary oligonucleotides containing 7-iodo- and 7-(hex-1-ynyl)-7-deazaadenine.
1
1
1
Tm [8C][a,b]
DH [kcalmol
]
DS [calmol 1 K
]
DG8 [kcalmol ]
5'-d[(CGCGAATTCGCG)]2 (18 ´ 18)
64(63)
65(63)
83( 73)
94( 90)
95[c]
89( 84)
94[c]
224( 195)
255( 247)
258[c]
238( 225)
253[c]
13.7( 12.6)
14.5( 13.9)
14.5[c]
15.5( 14.8)
16.0[c]
5'-d[(CGCG(I7c7A)2TTCGCG)]2 (19 ´ 19)
5'-d[(CGCG(hxy7c7A)2TTCGCG)]2 (20 ´ 20)
5'-d[(GTAGAATTCTAC)]2 (21 ´ 21)
70(69)
46(43)
79( 84)
75)[c]
82
80( 81)
85( 85)[c]
56( 63)
62[c]
226( 225)
215)[c]
231
221( 229)
236( 242)[c]
149( 173)
168[c]
9.2( 8.6)
8.5)[c]
9.9
(
(
(
5'-d[(GTAGI7c7AATTCTAC)]2 (22 ´ 22)
47
55(50)
5'-d[(GTI7c7AG(I7c7A)2TTCTI7c7AC)]2 (23 ´ 23)
11.1( 10.0)
12.0( 10.3)[c]
9.4( 9.1)
10.3[c]
5'-d[(GThxy7c7AG(hxy7c7A)2TTCThxy7c7AC)]2 (24 ´ 24)
52(48)
[a] Measured at 270 nm in 1m NaCl, 100mm MgCl2, and 60mm Na cacodylate (pH ˆ 7.0) with 10mm single-strand concentration. [b] Measured at 270 nm in
0.1m NaCl, 100mm MgCl2, and 10mm Na cacodylate(pH ˆ 7.0) with 10mm single-strand concentration. [c] Determined from the concentration dependence of
the Tm values.
Chem. Eur. J. 1998, 4, No. 9
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Table 6. Tm values and thermodynamic data for non-self-complementary oligonucleotides containing consecutive base residues.
1
1
1
Tm [8C][a,b]
DH [kcalmol
]
DS [calmol 1 K
]
DG8 [kcalmol ]
d(A)12 ´ d(T)12 (25 ´ 26)
44(37)
30(24)
54(48)
84( 91)
73( 67)
85( 79)
98( 80)
238( 267)
215( 199)
234( 219)
275( 222)
9.8( 7.9)
6.3( 5.0)
12.7( 10.7)
13.7( 10.9)
[5]
5'-d[(c7A)11-A] ´ d(T)12 (27 ´ 26)
5'-d[(Br7c7A)11-A] ´ d(T)12[5] (28 ´ 26)
5'-d[(I7c7A)11-A] ´ d(T)12 (29 ´ 26)
54(49)
5'-d[(hxy7c7A)11-A] ´ d(T)12 (30 ´ 26)
5'-d[(hxy7c7A-A)6] ´ d(T)12 (31 ´ 26)
5'-d[(hxy7c7A)3-(A)3-(hxy7c7A)3-(A3)] ´ d(T)12 (32 ´ 26)
[c]
47
50
59
59
160
157
9.5
10.3
[a] Measured at 270 nm in 1m NaCl, 100mm MgCl2, and 60mm Na cacodylate (pH ˆ 7.0) with 5mm single-strand concentration. [b] Values in parentheses
measured at 270 nm in 0.1m NaCl, 100mm MgCl2, and 10mm Na cacodylate (pH ˆ 7.0) with 5mm single-strand concentration. [c] No melting was observed.
stabilization by iodinated bases reflects a favorable enthalpy
term. In contrast to 29, no cooperative melting profile was
observed for 5'-d[(hxy7c7A)11 ± A] ´ d(T)12 (30 ´ 26). This phe-
nomenon has already been observed in the case of 5-
substituted pyrimidines, for example by the introduction of
a hexyl tether into synthetic DNA, which led to an unfavor-
able effect on the duplex stability.[32] These results also agree
with our finding that a consecutive arrangement of bulky and
hydrophobic 7-substituents, such as hex-1-ynyl of hxy7c7Gd
along the major groove of a double helix hybridized with
oligo(dC), leads to duplex destabilization. The destabilization
is not found for c7Gd.[33] This is probably due to desiccation of
the molecule, causing a shrinking of the major groove.
However, when alternating hxy7c7Ad residues and dA
(oligomers 31 and 32) are present, stable duplexes with
d(T)12 (31 ´ 26 and 32 ´ 26) are formed. These are stabilized by
a more favorable entropy term (Table 6).
spectra of the alternating oligonucleotide 31 is somewhat
similar to that of d(A)12 (25), which shows the characteristics
of a right-handed B-DNA helix. These spectra are signifi-
cantly different from that of the homooligomer 5'-d(I7c7A11 ±
A) (29). However, the latter is also typical for the CD
spectrum of normal (A/T)-paired duplexes of a homo-
DNA.[35] On the other hand, the oligomer 5'-d[(hxy7c7A)11 ±
A] (30) exhibits a CD spectrum that looks different from the
spectra mentioned above.
As it was of interest whether the temperature-induced
changes of the CD spectra resulted from a cooperative
destacking, the CD values of 5'-d(I7c7A11 ± A) (29) at 283 nm
were plotted against the temperature. The linear dependence
indicates that no cooperative destacking occurred (Figure 8).
It has been reported that base-stacking interactions within
single-stranded oligomers can be quite strong and can lead to
preorganised single-strand helices.[34] This preorganization of
a single strand influences the duplex stability in such a way
that the more stable secondary structure of one of the single
strands determines the secondary structure of the duplex. As
the CD spectra give information on the secondary structure of
such single strands, the spectra of compounds d(A)12 (25), 5'-
d[(I7c7A)11 ± A] (29), 5'-d[(hxy7c7A)11 ± A] (30), and 5'-
d[(hxy7c7A ± A)6] (31) were measured (Figure 7). The CD
Figure 8. Temperature-dependent CD data (ellipticities) of single-strand-
ed 5'-d[(I7c7A)11-A] (29), measured at 283 nm in 1m NaCl, 100mm MgCl2,
and 60mm Na cacodylate (pH ˆ 7.0) with 7.5mm single-strand concentra-
tion.
Non-self-complementary oligonucleotides with mismatches: It
was also of interest to study the base-pair selectivity of the
modified oligonucleotides. For this purpose, a hxy7c7Ad
residue was incorporated into the center of dT12. This
oligomer was hybridized with dA12 homomers containing
the dT, dA, dC, and dG bases opposite to the 7-deazaadenine
moiety (Table 7). The modified duplex (34 ´ 35) showed a
slightly higher stability than the parent duplex (33 ´ 34),
caused by more favorable entropy and a slightly lower
enthalpy change. With dC (35 ´ 36), dG (35 ´ 37), and dA
(25 ´ 35), the duplex stability is significantly decreased. These
results show that only the hxy7c7Ad-dT base pair is stable and
Figure 7. CD spectra of the single-stranded homooligonucleotides d(A)12
(25), 5'-d[(I7c7A)11-A] (29), 5'-d[(hxy7c7A)11-A] (30), and 5'-d[(hxy7c7A-
A)6] (31). Measured at 108C in 1m NaCl, 100mm MgCl2, and 60mm Na
cacodylate (pH ˆ 7.0) with 7.5mm single-strand concentration.
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Duplex Stability
1781±1790
Table 7. Tm values and thermodynamic data of non-self-complementary oligonucleotides containing mismatches
opposite to 7-(hex-1-ynyl)-7-deazaadenine.
standard to study the influence
of modified bases on duplex
structure and stability. The oli-
Tm [8C][a,b]
DH
[kcalmol
DS
[calmol 1 K
DG8
[kcalmol ]
1
1
1
]
]
gonucleotides 39 ± 45 were syn-
thesized. They were then hybri-
dized with the complementary
strand, and the Tm values were
measured. Duplex formation
was observed in all cases (Ta-
ble 8). The overall increase in
stability of the deoxyoligonu-
5'-d[(T)5-A-(T)6] (33)
3'-d[(A)5-T-(A)6] (34)
5'-d[(T)5-hxy7c7A-(T)6] (35)
3'-d[(A)5-T-(A)6] (34)
5'-d[(T)5-hxy7c7A-(T)6] (35)
3'-d[(A)5-C-(A)6] (36)
5'-d[(T)5-hxy7c7A-(T)6] (35)
3'-d[(A)5-G-(A)6] (37)
5'-d[(T)5-hxy7c7A-(T)6] (35)
3'-d(A)12 (25)
39(33)
41(35)
28
97( 110)
81( 90)
44
284( 301)
232( 268)
146
8.4( 6.6)
8.7( 7.1)
6.0
28
46
127
5.7
cleotide duplexes was 1.58C per
I7c7Ad residue, although the du-
27
64
188
5.9
5'-d[(T)5-Br8A-(T)6] (38)
3'-d[(A)5-T-(A)6] (34)
34(28)
77( 81)
226( 245)
7.1( 5.4)
plex stabilization caused by a
hxy7c7Ad residue is somewhat
lower. The highest Tm values
were found for the hybrids 42 ´
43, 44 ´ 45, and 42 ´ 45. When
single-stranded oligomers con-
[a] Measured at 270 nm in 1m NaCl, 100mm MgCl2, and 60mm Na cacodylate (pH ˆ 7.0) with 5mm single-strand
concentration. [b] Values in parentheses measured at 270 nm in 0.1m NaCl, 100mm MgCl2, and 10mm Na
cacodylate (pH ˆ 7.0) with 5mm single-strand concentration.
taining 7-iodo-7-deazaadenine
that mismatches can be observed. Duplexes with 8-bromo-2'-
deoxyadenosine are strongly destabilized (34 ´ 38). In this
case, the 8-substituent forces the nucleoside into the syn
conformation and can therefore interfere with the sugar±
phosphate backbone.[12, 36]
residues are hybridized with those containing a 7-(hex-1-
ynyl) chain, for example in 42 ´ 45, stable duplexes are also
formed. No final decision can be made as to whether duplex
stabilization results from more favorable enthalpic or entropic
terms. The deoxyoligonucleotide single strands 39, 42, and 44
were also hybridized with the ribooligonucleotide 41. In these
cases, a less stable helix was formed than for the oligodeox-
ynucleotide duplexes 39 ´ 40, 40 ´ 42, and 40 ´ 44. This agrees
with observations on regular DNA ± RNA hybrids.[37] The
oligodeoxyheteroduplexes shown in Table 8 form B-DNA
structures, as observed by CD spectroscopy, whereas the
hybrids with the ribooligonucleotide strand 41 form an A-
DNA structure (Figure 9).
Non-self-complementary oligonucleotides with random base
composition: The oligonucleotides discussed above have
special sequences and are not representative of the common
regions of natural DNA. Therefore, two oligonucleotides, 5'-
d(TAGGTCAATACT) (39) and 5'-d(AGTATTGACCTA)
(40), were constructed to form a stable hybrid with a Tm
value of 508C. This duplex is used in our laboratory as a
Table 8. Tm values and thermodynamic data for non-self-complementary oligonucleotides containing 7-iodo- and 7-(hex-1-ynyl)-7-deazaadenine.
1
1
1
Tm [8C][a,b]
DH [kcalmol
]
DS [calmol 1 K
]
DG8 [kcalmol ]
5'-d(TAGGTCAATACT) (39)
3'-d(ATCCAGTTATGA) (40)
50(47)
90( 82)
252( 230)
11.8( 10.4)
5'-d(TAGGTCAATACT) (39)
3'-r(AUCCAGUUAUGA) (41)
5'-d(TI7c7AGGTC(I7c7A)2TI7c7ACT) (42)
48(46)
55
65( 82)
81
176( 230)
221
10.2( 10.1)
12.2
3'-d(ATCCAGTTATGA) (40)
5'-d(TAGGTCAATACT) (39)
3'-d(ATCCI7c7AGTTI7c7ATGA) (43)
54
85
236
12.3
5'-d(TI7c7AGGTC(I7c7A)2TI7c7ACT) (42)
58(54)
48
85( 81)
80
231( 224)
225
13.1( 12.0)
10.1
3'-d(ATCCI7c7AGTTI7c7ATGA) (43)
5'-d(TI7c7AGGTC(I7c7A)2TI7c7ACT) (42)
3'-r(AUCCAGUUAUGA) (41)
5'-d(Thxy7c7AGGTC(hxy7c7A)2Thxy7c7ACT) (44)
54
79
217
11.9
3'-d(ATCCAGTTATGA) (40)
5'-d(TAGGTCAATACT) (39)
52
93
260
12.2
3'-d(ATCChxy7c7AGTThxy7c7ATGA) (45)
5'-d(Thxy7c7AGGTC(hxy7c7A)2Thxy7c7ACT) (44)
58(55)
48(43)
59(54)
103( 91)
96( 74)
82( 81)
285( 253)
273( 209)
223( 222)
14.2( 12.8)
11.5( 9.5)
13.0( 12.0)
3'-d(ATCChxy7c7AGTThxy7c7ATGA) (45)
5'-d(Thxy7c7AGGTC(hxy7c7A)2Thxy7c7ACT) (44)
3'-r(AUCCAGUUAUGA) (41)
5'-d(TI7c7AGGTC(I7c7A)2TI7c7ACT) (42)
3'-d(ATCChxy7c7AGTThxy7c7ATGA) (45)
[a] Measured at 270 nm in 1m NaCl, 100mm MgCl2, and 60mm Na cacodylate (pH ˆ 7.0) with 5mm single-strand concentration. [b] Values in parentheses
measured at 270 nm in 0.1m NaCl, 100mm MgCl2, and 10mm Na cacodylate (pH ˆ 7.0) with 5mm single-strand concentration.
Chem. Eur. J. 1998, 4, No. 9
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F. Seela and M. Zulauf
FULL PAPER
NH2
N
NH2
R
N
N
N
N
Br
N
N
O
P
O
P
O
P
O
P
O
P
O
P
O
O
HO
HO
O
O
O
O
O
O
HO
HO
OH OH
OH OH
HO
HO
46a,b
47
Scheme 4. Triphosphates of 7-deazaadenine and adenine [a: R ˆ I, b: R ˆ
ꢀ
C C(CH2)3CH3].
mation required for hybridization. The bright fluorescence of
7-deaza-7-(hex-1-ynyl)-2'-deoxyadenosine (1) makes this
molecule useful for fluorescence studies on DNA, even on
the single molecule level.
Figure 9. CD spectra of the heteroduplexes (41 ´ 42), (41 ´ 44), (42 ´ 43), and
(44 ´ 45) (for sequences see Table 8),
Conclusion and Outlook
Experimental Section
From these experiments, it can be concluded that bulky iodo
substituents or lipophilic hexynyl residues attached to the 7-
position of a 7-deazaadenine moiety stabilize the oligonu-
cleotide duplex structure. The stabilization occurs both in the
series of self-complementary and non-self-complementary
oligonucleotide duplexes. The increase of Tm is sequence-
dependent and can be due to more favorable enthalpy or
entropy changes.
Monomers: Flash chromatography (FC): at 0.4 bar with silica gel 60
(Merck, Darmstadt, Germany). Solvent systems for FC and TLC: CH2Cl2/
MeOH 9:1 (A), petroleum ether (boiling range 40 ± 608C)/acetone 1:1 (B).
Samples were collected with an UltroRac II fractions collector (LKB
Instruments, Sweden). Melting points: Büchi SMP-20 apparatus (Büchi,
Switzerland). UV spectra: 150-20 spectrometer (Hitachi, Japan). NMR
spectra: AC 250 and AMX-500 spectrometers (Bruker, Germany); d values
are relative to internal Me4Si or external H3PO4. Microanalyses were
performed by Mikroanalytisches Laboratorium Beller (Göttingen, Ger-
many).
In all cases, the bulky 7-substituents have steric freedom
within the grooves of the DNA. This is different from the 8-
substituents of purine residues, which unlike the 7-substitu-
ents interfere with the sugar± phosphate backbone. Further-
more, the halogen and the alkynyl 7-substituents reduce the
basicity of nitrogen 1 of the 7-deazaadenine moiety (pKa
values: c7Ad ˆ 5.3,[38] I7c7Ad ˆ 4.5, hxy7c7Ad ˆ 4.3). At the
same time, the 6-amino group can become a better proton
donor. With regard to these properties, compounds 1 and 2 are
closer analogues of dA (pKa ˆ 3.8[38]) than the nonsubstituted
7-deaza-2'-deoxyadenosine (c7Ad).
The duplex stability of oligonucleotides with 7-substituted
7-deazaadenine residues is significant for hybridization stud-
ies and for the stability of antisense oligonucleotides. It is also
important for the complete replacement of a purine base by
base-modified nucleosides with a large substituent or a
reporter group on the purine base. However, oligonucleotides
with bulky substituents at position 8 of an adenine base cannot
be used for such purposes, as they make the duplex rather
unstable (Table 7). Recently, it was shown that compounds 1
and 2 can be used in the triphosphate form (46a,b, Scheme 4)
as 100% substitutes for dATP during the DNA-polymerase-
catalyzed elongation of a growing oligonucleotide chain on a
single-stranded DNA matrix.[39] The 8-bromo dATP 47 cannot
be incorporated in such a way and will cause chain termi-
nation when used instead of ATP.[39]
Oligonucleotides: Oligonucleotides were synthesized with a 380B DNA
synthesizer (Applied Biosystems, Germany) according to the standard
protocol. The oligonucleotides were purified by oligonucleotide purifica-
tion cartridges.[25] The enzymatic hydrolysis of the oligomers was performed
as described in ref. [40]. Quantification of the constituents was made on the
basis of the peak areas, which were divided by the extinction coefficients of
the nucleoside (e260 values: dA 15400, dC 7300, dG 11400, dT 8800, I7c7Ad
5000, hxy7c7Ad 6600). Snake-venom phosphodiesterase (EC 3.1.15.1,
Crotallus durissus) and alkaline phosphatase (EC 3.1.3.1., E. coli) were
generous gifts of Boehringer Mannheim, Germany). MALDI-TOF spectra
were provided by Dr. S. Hahner and J. Gross (Prof. Hilgenkamp, Institute
of Medicinal Physics and Biophysics, University of Münster, Germany).
RP-18 HPLC: 250 Â 4 mm RP-18 column; Merck-Hitachi HPLC; gradients
of 0.1m (Et3NH)OAc (pH 7.0)/MeCN 95:5 (A) and MeCN (B); gradient I:
50 min 0 ± 50% B in A, flow rate 1 mLmin 1; gradient II: 20 min 0 ± 20% B
1
in A; 40 min 20 ± 40% B in A, flow rate 1 mLmin
.
Determination of melting curves and thermodynamics: Absorbance vs.
temperature profiles were measured on a Cary 1/1E UV/VIS spectropho-
tometer (Varian, Australia) with a Cary thermoelectrical controller. The Tm
values were measured in the reference cell with a Pt-100 resistor, and the
thermodynamic data (DH, DS, DG8) were calculated with the program
MeltWin 3.0.[31] Circular dichroism (CD) spectra were recorded on a Jasco-
600 (Jasco, Japan) spectropolarimeter, a thermostatically controlled bath
(Lauda-RCS-6) and 1 cm cuvettes.
General procedure for the acylation of 4-amino-7-(2-deoxy-b-d-erythro-
pentofuranosyl)-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (2): Me3SiCl (650 mL,
5.1 mmol) was added to a solution of compound 2[13] (188 mg, 0.5 mmol) in
anhydrous pyridine (3 mL), and was stirred at r.t. After 30 min, the acid
chloride (2.5 mmol) was introduced and the solution was kept at r.t. for
another 2 h. The mixture was cooled to 08C, diluted with H2O (2 mL), and
stirred for 10 min. After the addition of 25% aq. NH3 (2 mL), stirring was
continued for 2 h at r.t. The solution was evaporated and the residue was
purified by using flash chromatography (column 10 Â 3 cm, solvent A).
4-(Benzoylamino)-7-(2-deoxy-b-d-erythro-pentofuranosyl)-5-iodo-7H-py-
rrolo[2,3-d]pyrimidine (4a): The reaction was performed according to the
general procedure. When benzoyl chloride (351 mg, 290 mL) was used,
In addition to the observations made for the oligonucleo-
tides, the 7-alkynyl derivatives of 7-deaza-2'-deoxyadenosine
represent a new class of highly fluorescent nucleosides. They
have bulky hydrophobic bases in the favorable anti confor-
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Duplex Stability
1781±1790
colorless needles were obtained, which were crystallized from MeOH
(159 mg, 66%). M.p. 1958C; TLC (A): Rf 0.5; UV (MeOH): lmax (e) ˆ 230
(28400), 273 (6500), 312 nm (6100); 1H NMR (500 MHz, [D6]DMSO, 308C,
TMS): d ˆ 2.24 (m, 1H; Ha-C(2')), 2.47 (m, 1H; Hb-C(2')), 3.55 (m, 2H; 2
H-C(5')), 3.84 (m, 1H; H-C(4')), 4.36 (m, 1H; H-C(3')), 5.03 (t, 3J(H,H) ˆ
for 2 h at 408C. After evaporation, the residue was purified by FC (column
10 Â 5 cm, A), yielding a colorless foam (389 mg, 85%), which was isolated
from the main zone. TLC (A): Rf 0.5; UV (MeOH): lmax (e) ˆ 229 (17400),
277 (10400), 323 nm (19000); 1H NMR (500 MHz, [D6]DMSO, 308C,
TMS): d ˆ 2.18 (m, 1H; Ha-C(2')), 2.47 (m, 1H; Hb-C(2')), 3.18, 3.22 (2s,
6H; Me2N), 3.54 (m, 2H; 2 H-C(5')), 3.81 (m, 1H; H-C(4')), 4.32 (m, 1H;
H-C(3')); 5.00 (t, 3J(H,H) ˆ 5.4 Hz, 1H; OH-C(5')), 5.23 (d, 3J(H,H) ˆ
3.9 Hz, 1H; OH-C(3')), 6.52 (t, 3J(H,H) ˆ 7.0 Hz, 1H; H-C(1')), 7.70 (s,
3
5.3 Hz, 1H; OH-C(5')), 5.33 (d, J(H,H) ˆ 3.9 Hz, 1H; OH-C(3')), 6.66 (t,
3J(H,H) ˆ 6.9 Hz, 1H; H-C(1')), 7.57 (m, 3H;
3 arom. H), 8.05 (d,
3J(H,H) ˆ 8 Hz, 2H; 2 arom. H), 8.05 (s, 1H; H-C(6)), 8.71 (s, 1H; H-
C(2)), 10.90 (s, 1H; NH); C18H17IN4O4 (480.3): calcd. C 45.02, H 3.57, N
11.67; found C 45.10, H 3.62, N 11.85.
ˆ
1H; H-C(6)), 8.30 (s, 1H; H-C(2)), 8.82 (s, 1H; N CH); C14H18IN5O3
(431.2): calcd C 38.99, H 4.21, N 16.24; found C 39.09, H 4.27, N 16.10.
4-(Acetylamino)-7-(2-deoxy-b-d-erythro-pentofuranosyl)-5-iodo-7H-pyr-
rolo[2,3-d]pyrimidine (4b): As described for 4a with acetyl chloride
(196 mg, 178 mL). A 6% aq. NH3 solution (2 mL, 20 min, r.t.) was used.
Crystallization (MeOH/EtOAc, 8:2) produced colorless needles (101 mg,
48%). M.p. 2018C; TLC (A): Rf 0.5; UV (MeOH): lmax (e) ˆ 273 (6500),
7-(2-Deoxy-b-d-erythro-pentofuranosyl)-4-{[(dimethylamino)methylide-
ne]amino}-5-(hex-1-ynyl)-7H-pyrrolo[2,3-d]pyrimidine (5b): Compound
5b was prepared from 7-(2-deoxy-b-d-erythro-pentofuranosyl)-5-(hex-1-
ynyl)-7H-pyrrolo[2,3-d]pyrimidine (1)[13] (400 mg, 1.21 mmol) and N,N-
dimethylformamide dimethylacetal (2.0 g, 16.8 mmol) as described for 5a.
On FC, a colorless foam (373 mg, 80%) was obtained from the main zone.
TLC (A): Rf 0.5; UV (MeOH): lmax (e) ˆ 278 (12100), 321 nm (14300); 1H
NMR (500 MHz, [D6]DMSO, 308C, TMS): d ˆ 0.91 (t, 3J(H,H) ˆ 7.3 Hz,
3H; CH3), 1.45 (sextet, 3J(H,H) ˆ 7.2 Hz, 2H; CH2-CH3), 1.53 (quintet,
3J(H,H) ˆ 7.3 Hz, 2H; CH2-CH2-CH3), 2.18 (m, 1H; Ha-C(2')), 2.42 (t,
3J(H,H) ˆ 6.8 Hz, 2H; CH2), 2.47 (m, 1H; Hb-C(2')), 3.16, 3.18 (2s, 6H;
Me2N), 3.56 (m, 2H; 2 H-C(5')), 3.84 (m, 1H; H-C(4')), 4.35 (m, 1H; H-
C(3')), 5.02 (t, 3J(H,H) ˆ 5.3 Hz, 1H; OH-C(5')), 5.24 (d, 3J(H,H) ˆ 3.7 Hz,
1H, OH-C(3')); 6.53 (t, 3J(H,H) ˆ 6.8 Hz, 1H, H-C(1')), 7.71 (s, 1H; H-
1
298 nm (6000); H NMR (500 MHz, [D6]DMSO, 308C, TMS): d ˆ 2.18 (s,
3H; CH3), 2.24 (m, 1H; Ha-C(2')), 2.44 (m, 1H; Hb-C(2')), 3.54 (m, 2H; 2
H-C(5')), 3.83 (m, 1H; H-C(4')), 4.35 (m, 1H; H-C(3')), 4.95 (t, 3J(H,H) ˆ
3
5.3 Hz, 1H; OH-C(5')), 5.29 (d, J(H,H) ˆ 3.9 Hz, 1H; OH-C(3')), 6.62 (t,
3J(H,H) ˆ 6.9 Hz, 1H; H-C(1')), 8.00 (s, 1H; H-C(6)), 8.62 (s, 1H; H-C(2)),
10.21 (s, 1H; NH); C13H15IN4O4 (418.2): calcd C 37.34, H 3.62, N 13.40;
found C 37.37, H 3.74, N 13.48.
7-(2-Deoxy-b-d-erythro-pentofuranosyl)-5-iodo-4-(iso-butyrylamino)-7H-
pyrrolo[2,3-d]pyrimidine (4c): The reaction was performed as described
for 4a but with iso-butyryl chloride (266 mg, 260 mL). In this case, 12% aq.
NH3 (2 mL, r.t., 30 min) was used. Colorless needles from MeOH (114 mg,
51%). M.p. 1928C; TLC (A): Rf 0.5; UV (MeOH): lmax (e) ˆ 272 (6200),
ˆ
C(6)), 8.32 (s, 1H; H-C(2)), 8.76 (s, 1H; N CH); C20H27N5O3 (385.5): calcd
C 62.32, H 7.06, N 18.17; found C 62.48, H 7.03, N 18.53.
7-[2-Deoxy-5-O-(4,4'-dimethoxytriphenylmethyl)-b-d-erythro-pentofura-
nosyl]-4-[{(dimethylamino)methylidene}amino]-5-iodo-7H-pyrrolo-
[2,3-d]pyrimidine (6a): 4,4'-dimethoxytriphenylmethyl chloride (256 mg,
0.76 mmol) was added to a solution of 5a (300 mg, 0.70 mmol) in dry
pyridine (3 mL). After stirring at 508C for 1 h, the mixture was poured into
5% aq. NaHCO3 soln. (10 mL) and extracted with CH2Cl2 (2 Â 50 mL). The
combined organic layers were dried over Na2SO4, filtered, and evaporated.
The residue was analyzed by FC (column 12 Â 4 cm, A), yielding a colorless
foam (360 mg, 70%). TLC (A): Rf 0.6; UV (MeOH): lmax (e) ˆ 236 (29200),
275 (12200), 322 nm (19600); 1H NMR (500 MHz, [D6]DMSO, 308C,
TMS): d ˆ 2.24 (m, 1H; Ha-C(2')), 2.57 (m, 1H; Hb-C(2')), 3.18 (m, 2H; 2
H-C(5')), 3.18, 3.22 (2s, 6H; Me2N), 3.72 (s, 6H; 2 MeO), 3.92 (m, 1H; H-
C(4')), 4.37 (m, 1H; H-C(3')), 5.30 (d, 3J(H,H) ˆ 4.0 Hz, 1H; OH-C(3')),
6.54 (t, 3J(H,H) ˆ 6.6 Hz, 1H; H-C(1')), 6.84 (m, 4H; 4 arom. H), 7.22-7.38
(m, 9H; 9 arom. H), 7.56 (s, 1H; H-C(6)), 8.31 (s, 1H; H-C(2)), 8.82 (s, 1H;
1
299 nm (5700); H NMR (250 MHz, [D6]DMSO, 308C, TMS): d ˆ 1.21 (s,
6H; 2 CH3), 2.24 (m, 1H; Ha-C(2')), 2.44 (m, 1H; Hb-C(2')), 2.80 (m, 1H;
CH), 3.55 (m, 2H; 2 H-C(5')), 3.85 (m, 1H; H-C(4')), 4.37 (m, 1H; H-
C(3')), 4.96 (t, 3J(H,H) ˆ 5.3 Hz, 1H; OH-C(5')), 5.29 (d, 3J(H,H) ˆ 3.9 Hz,
1H; OH-C(3')), 6.64 (t, 3J(H,H) ˆ 6.9 Hz, 1H; H-C(1')), 8.01 (s, 1H; H-
C(6)), 8.63 (s, 1H; H-C(2)), 10.16 (s, 1H; NH); C15H19IN4O4 (446.2): calcd
C 40.37, H 4.29, N 12.56; found C 40.25, H 4.30, N 12.46.
7-(2-Deoxy-b-d-erythro-pentofuranosyl)-5-iodo-4-(pivaloylamino)-7H-py-
rrolo[2,3-d]pyrimidine (4d): As described for 4a with 2[13] (94 mg,
0.25 mmol), pivaloyl chloride (150 mg, 150 mL), and treatment with 12%
aq. NH3 (2 mL, r.t. 30 min). An amorphous solid was obtained (58 mg,
51%). TLC (A): Rf 0.5; UV (MeOH): lmax (e) ˆ 274 (5800), 300 nm (5300);
1H NMR (500 MHz, [D6]DMSO, 308C, TMS): d ˆ 1.10 (s, 3H; 3 CH3); 2.23
(m, 1H; Ha-C(2')), 2.47 (m, 1H; Hb-C(2')), 3.56 (m, 2H; 2 H-C(5')), 3.84
(m, 1H; H-C(4')), 4.36 (m, 1H; H-C(3')), 4.98 (br, 1H; OH-C(5')), 5.30 (br,
1H; OH-C(3')), 6.63 (t, 3J(H,H) ˆ 6.9 Hz, 1H; H-C(1')), 8.00 (s, 1H; H-
C(6)), 8.63 (s, 1H; H-C(2)), 9.90 (s, 1H; NH). FAB-MS (NBA): m/z ˆ
ˆ
N CH); C35H36IN5O5 (733.6): calcd C 57.30, H 4.95, N 9.55; found C 57.48,
H 5.12, N 9.44.
7-[2-Deoxy-5-O-(4,4'-dimethoxytriphenylmethyl)-b-d-erythro-pentofura-
nosyl]-4-[{(dimethylamino)methylidene}amino]-5-(hex-1-ynyl)-7H-pyrro-
lo[2,3-d]pyrimidine (6b): Compound 6b was prepared from 5a (300 mg,
0.78 mmol) as described for 6a but with 4,4'-dimethoxytriphenylmethyl
chloride (290 mg, 0.86 mmol). FC (column 12 Â 4 cm, A) produced a
colorless foam (360 mg, 67%). TLC (A): Rf 0.6; UV (MeOH): lmax (e) ˆ
276 (17500), 320 nm (12900); 1H NMR (500 MHz, [D6]DMSO, 308C,
TMS): d ˆ 0.91 (t, 3J(H,H) ˆ 7.3 Hz, 3H; CH3), 1.45 (sextet, 3J(H,H) ˆ
7.2 Hz, 2H; CH2-CH3), 1.53 (quintet, 3J(H,H) ˆ 7.3 Hz, 2H; CH2-CH2-
CH3), 2.18 (m, 1H; Ha-C(2')), 2.41 (t, 3J(H,H) ˆ 6.8 Hz, 2H; CH2), 2.53 (m,
1H; Hb-C(2')), 3.16, 3.18 (2s, 6H; Me2N), 3.18 (m, 2H, H-C(5')), 3.71 (s,
6H; 2 MeO), 3.91 (m, 1H; H-C(4')), 4.34 (m, 1H; H-C(3')), 5.28 (d,
3J(H,H) ˆ 3.9 Hz, 1H; OH-C(3')), 6.53 (t, 3J(H,H) ˆ 7.0 Hz, 1H; H-1'), 6.82
(m, 4H; 4 arom. H), 7.20 ± 7.36 (m, 9H; 9 arom. H), 7.56 (s, 1H; H-C(6)),
‡
461.1 [M‡H] .
7-(2-Deoxy-b-d-erythro-pentofuranosyl)-5-iodo-4-(phenoxyacetylamino)-
7H-pyrrolo[2,3-d]pyrimidine (4e): Phenoxyacetic anhydride (1.7 g,
6.0 mmol) was added to a solution of 2[13] (376 mg, 1.0 mmol) in dry
pyridine (10 mL) under stirring at r.t. After 2 h, H2O (2 mL) was added and
the stirring was continued for another 15 min. The mixture was evaporated
and the residue dissolved in CH2Cl2 (100 mL). The solution was washed
with 5% aq. NaHCO3 (2 Â 25 mL) and H2O (2 Â 25 mL). The organic
layers were combined, dried over Na2SO4, filtered and evaporated to
dryness. The resulting oil was treated with Et3N/pyridine/H2O (1:1:3)
(30 mL) and was stirred (30 min). The solution was evaporated to dryness
and was analyzed using FC (column 10 Â 3 cm, A). Crystallization (MeOH/
EtOAc, 3:1) produced colorless crystals (265 mg, 52%). M.p. 1998C; TLC
(A): Rf 0.5; UV (MeOH): lmax (e) ˆ 236 (21600), 275 (6100), 301 nm (5800);
1H NMR (250 MHz, [D6]DMSO, 308C, TMS): d ˆ 2.23 (m, 1H; Ha-C(2')),
2.47 (m, 1H; Hb-C(2')), 3.53 (m, 2H; 2 H-C(5')), 3.84 (m, 1H; H-C(4')),
4.36 (m, 1H; H-C(3')), 4.88 (s, 2H; CH2OPh), 4.99 (t, 3J(H,H) ˆ 5.3 Hz,
1H; OH-C(5')), 5.32 (d, 3J(H,H) ˆ 3.9 Hz, 1H; OH-C(3')), 6.63 (t,
3J(H,H) ˆ 6.9 Hz, 1H; H-C(1')), 7.01 (m, 3H; 3 arom. H), 7.32 (m, 2H; 2
arom. H), 8.05 (s, 1H; H-C(6)), 8.66 (s, 1H; H-C(2)), 10.37 (s, 1H; NH);
C19H19IN4O5 (510.3): calcd C 44.72, H 3.75, N 10.98; found C 44.75, H 3.61,
N 10.79.
ˆ
8.30 (s, 1H; H-C(2)), 8.73 (s, 1H; N CH); C41H45N5O5 (687.8): calcd C
71.59, H 6.59, N 10.18; found C 71.64, H 6.63, N 10.28.
7-[2-Deoxy-5-O-(4,4'-dimethoxytriphenylmethyl)-b-d-erythro-pentofura-
nosyl]-4-[{(dimethylamino)methylidene}amino]-5-iodo-7H-pyrrolo[2,3-d-
]pyrimidine-3'-[(2-cyanoethyl)-N,N-diisopropyl phosphoramidite] (3a):
Chloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine
(126 mg,
0.53 mmol) was added to a stirred solution of 6a (300 mg, 0.41 mmol)
and anhydrous N,N-diisopropylethylamine (212 mg, 1.64 mmol) in dry
THF (2 mL) under an Ar atmosphere at room temperature. The reaction
mixture was stirred for another 30 min and was filtered. The filtrate was
diluted with ethyl acetate (30 mL) and extracted twice with ice-cold aq.
10% Na2CO3 solution (2 Â 10 mL) and H2O (10 mL). The organic phases
were dried over Na2SO4 and evaporated to dryness. The solid material was
purified by FC (column 8 Â 3 cm, B), yielding a colorless foam (222 mg,
7-(2-Deoxy-b-d-erythro-pentofuranosyl)-4-{[(dimethylamino)methylide-
ne]amino}-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (5a): N,N-dimethylform-
amide dimethylacetal (2.0 g, 16.8 mmol) was added to a solution of 7-(2-
deoxy-b-d-erythro-pentofuranosyl)-5-iodo-7H-pyrrolo[2,3-d]pyrimidine
(2)[13] (400 mg, 1.06 mmol) in methanol (20 mL) and the solution was stirred
Chem. Eur. J. 1998, 4, No. 9
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
0947-6539/98/0409-1789 $ 17.50+.25/0
1789

F. Seela and M. Zulauf
FULL PAPER
61%) from the main zone. TLC (B): Rf 0.4, 0.5; 31P NMR (101 MHz,
CDCl3, 308C, 85% H3PO4): d ˆ 149.0, 149.2.
[14] H. Rosemeyer, M. Zulauf, N. Ramzaeva, G. Becher, E. Feiling, K.
Mühlegger, I. Münster, A. Lohmann, F. Seela, Nucleosides Nucleo-
tides 1997, 16, 821 ± 828.
7-[2-Deoxy-5-O-(4,4'-dimethoxytriphenylmethyl)-b-d-erythro-pentofura-
nosyl]-4-[{(dimethylamino)methylidene}amino]-5-(hex-1-ynyl)-7H-pyrro-
lo[2,3-d]pyrimidine-3'-[(2-cyanoethyl)-N,N-diisopropyl phosphoramidite]
(3b): Compound 6b (300 mg, 0.44 mmol) was treated with anhydrous N,N-
diisopropylethylamine (220 mg, 1.70 mmol) and chloro-(2-cyanoethoxy)-
N,N-diisopropylamino phosphine (135 mg, 0.57 mmol) as described for 3a.
A colorless foam (220 mg, 56%) was obtained. TLC (B): Rf 0.4, 0.5; 31P
NMR (101 MHz, CDCl3, 308C, 85% H3PO4): d ˆ 149.1, 149.3.
[15] M. Sauer, M. Zulauf, F. Seela, unpublished results.
[16] C. Seidel, F. Seela, M. Zulauf, unpublished results.
[17] F. Seela, T. Grein, Nucleic Acids Res. 1992, 20, 2297 ± 2306.
[18] J. Zemlicka, A. Holy, Collect. Czech. Chem. Commun. 1967, 32, 3159 ±
3168.
[19] B. A. Otter, J. J. Fox, in Synthetic Procedures in Nucleic Acid
Chemistry (Eds.: W. W. Zorbach, R. S. Tipson), Wiley, New York,
1968, pp. 285 ± 287.
[20] G. S. Ti, B. L. Gaffney, R. A. Jones, J. Am. Chem. Soc. 1982, 104,
1316 ± 1319.
[21] J. C. Schulhof, D. Molko, R. Teoule, Nucleic Acids Res. 1987, 15, 397 ±
416.
[22] B. C. Froehler, P. G. Ng, M. D. Matteucci, Nucleic Acids Res. 1986, 14,
5399 ± 5407.
[23] H. Rosemeyer, F. Seela, J. Chem. Soc. Perkin Trans. 2 1997, 2341 ±
2345.
Acknowledgments: We thank Dr. Stephanie Hahner and Julia Gross,
University of Münster, for the MALDI-TOF mass spectra, Drs. Natalya
Ramzaeva and Helmut Rosemeyer for discussion, and Dr. Leonid Beigel-
man, Ribozyme Pharmaceuticals, for the synthesis of the ribooligonucleo-
tide. Financial support by the Bundesministerium für Bildung, Wissen-
schaft, Forschung und Technologie, and the Fonds der Chemischen
Industrie and the Hoechst AG is gratefully acknowledged.
[24] B. C. Froehler in Protocols for Oligonucleotides and Analogs in
Methods in Molecular Biology, Vol. 20 (Ed.: E. S. Agrawal), Humana,
Totowa, New York, 1993, p 63.
Received: December 19, 1997 [F941]
[25] Applied Biosystems, Oligonucleotide Purification Cartridges.
[26] M. A. Viswamitra, Z. Shakked, P. G. Jones, G. M. Sheldrick, S. A.
Salisbury, O. Kennard, Biopolymers 1982, 21, 513 ± 533.
[27] A. Klug, A. Jack, M. A. Viswamitra, O. Kennard, Z. Shakked, T. A.
Steitz, J. Mol. Biol. 1979, 131, 669 ± 680.
[28] F. Seela, A. Kehne, Biochemistry. 1987, 26, 2232 ± 2238.
[29] R. E. Dickerson, H. R. Drew, J. Mol. Biol. 1981, 149, 761 ± 786.
[30] L. A. Marky, K. S. Blumenfeld, S. Kozlowski, K. J. Breslauer, Bio-
polymers 1983, 22, 1247 ± 1257.
[31] J. A. McDowell, D. H. Turner, Biochemistry 1996, 35, 14077 ± 14089.
[32] H. Hashimoto, M. G. Nelson, C. Switzer, J. Am. Chem. Soc. 1993, 115,
7128 ± 7134.
[33] N. Ramzaeva, C. Mittelbach, F. Seela, Helv. Chim. Acta. 1997, 80,
1809 ± 1822.
[34] E. T. Kool, Chem. Rev. 1997, 97, 1473 ± 1487.
[35] J. Hunziker, H.-J. Roth, M. Böhringer, A. Giger, U. Diederichsen, M.
Göbel, R. Krishnan, B. Jaun, C. Leumann, A. Eschenmoser, Helv.
Chim. Acta. 1993, 76, 259 ± 352.
[36] E. Dias, J. L. Battiste, J. R. Williamson, J. Am. Chem. Soc. 1994, 116,
4479 ± 4480.
[37] E. A. Lesnik, S. M. Freier, Biochemistry 1995, 34, 10807 ± 10815.
[38] F. Seela, H. Thomas, Helv. Chim. Acta. 1994, 77, 897 ± 903.
[39] K. Mersmann, F. Seela, unpublished results.
[40] F. Seela, S. Lampe, Helv. Chim. Acta. 1991, 74, 1790 ± 1800.
[1] E. Uhlmann, A. Peyman, Chem. Rev. 1990, 90, 543 ± 584.
[2] A. I. H. Murchie, D. M. J. Lilley, EMBO J. 1994, 13, 993 ± 1001.
[3] F. Seela, K. Mersmann, J. A. Grasby, M. J. Gait, Helv. Chim. Acta
1993, 76, 1809 ± 1820.
[4] S. Mizusawa, S. Nishimura, F. Seela, Nucleic Acids Res. 1986, 14,
1319 ± 1325.
[5] F. Seela, H. Thomas, Helv. Chim. Acta 1995, 78, 94 ± 108.
[6] J. Kypr, J. Sagi, E. Szakonyi, K. Ebinger, H. Penazova, J. Chladkova,
M. Vorlickova, Biochemistry 1994, 33, 3801 ± 3806.
[7] B. C. Froehler, S. Wadwani, T. J. Terhorst, S. R. Gerrard, Tetrahedron
Lett. 1992, 33, 5307 ± 5310.
[8] N. Ramzaeva, F. Seela, Helv. Chim. Acta 1996, 79, 1549 ± 1558.
[9] G. Balow, J. Brugger, E. Lesnik, O. L. Acevedo, Nucleosides Nucleo-
tides 1997, 16, 941 ± 944.
[10] C. A. Buhr, R. W. Wagner, D. Grant, B. C. Froehler, Nucleic Acids
Res. 1996, 24, 2974 ± 2980.
[11] F. Seela, M. Zulauf, H. Rosemeyer, H. Reuter, J. Chem. Soc. Perkin
Trans. 2 1996, 2373 ± 2376.
[12] R. G. Eason, D. M. Burkhardt, S. J. Phillips, D. P. Smith, S. S. David,
Nucleic Acids Res. 1996, 24, 890 ± 897.
[13] F. Seela, M. Zulauf, Synthesis 1996, 726 ± 730.
1790
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
0947-6539/98/0409-1790 $ 17.50+.50/0
Chem. Eur. J. 1998, 4, No. 9