Base-boronated dinucleotides: synthesis and effect of N
7
-cyanoborane substitution on the base protons
Base-boronated dinucleotides: synthesis and effect of N 7 -cyanoborane substitution on the base protons
Ahmad
Hasan
,
Hong
Li
,
Jeno
Tomasz
+
and
Barbara
Ramsay Shaw*
Department of Chemistry, P. M. Gross Chemical Laboratory, Duke University,
Durham
, NC 27708-0346,
USA
Received January 19, 1996;
Revised and Accepted April 16, 1996
ABSTRACT
Boron-modified nucleic acids comprise a new set of DNA mimics that have potential biological and therapeutic applications. A series of nine dinucleotides containing N
7
-cyanoborane-2
'
-deoxyguanosine (
7b
dG) at the 3
'
, 5
'
or both positions of the phosphodiester linkage have been synthesized using solution phase phosphoramidite chemistry.
Fmoc was used as the 5
'
-protecting group because of incompatibility of the cyanoborane moiety with 5
'
-DMT cations generated during the deprotection step. The presence of the cyanoborane group was
confirmed on the basis of Fab-MS and
1
H NMR spectroscopy. The H-8 proton of
7b
dG in the dinucleotides shifted 0.35-0.80 p.p.m. downfield relative to that of unmodified dG. A comparison of the D
2
O exchange kinetics of the H-8 proton at 60
oC showed that H-8 of
7b
dG
is very labile relative to unmodified dG, indicating that the N
7
-cyanoborane modification increases the acidity of the H-8 proton of
7b
dG. These studies illustrate the feasibility of synthesizing boron-containing oligonucleotides which are modified at the N
7
-guanine to block Hoogsteen pairing in the DNA major groove.
INTRODUCTION
Recent advances in the field of nucleic acid chemistry have drawn attention to
the synthesis of oligonucleotides with modified backbones (
1
-
11
). A number of these modified oligonucleotides have resulted in compounds with
potentially useful therapeutic properties, i.e., targeting cellular DNA or RNA
and interfering with gene expression via antisense and antigene therapy (
12
-
15
). Furthermore, modified oligonucleotides have manifold applications for
understanding and controlling biological processes (
16
-
20
). Base-modifications (
21
,
22
) of an oligonucleotide offer the opportunity to enhance the affinity of the
oligonucleotide for a cellular RNA or DNA target. The C-5 propyne pyrimidines, for example, increase the binding affinity with complementary RNA (
23
-
25
), and oligonucleotides containing modified adenine, for example N
7
-deaza-2'-deoxyadenosine, result in DNA duplex stabilization (
26
,
27
).
Our recent research focus has been to develop base-boronated oligonucleotides with unique major groove recognition sites, yet not
affect their base pairing properties and overall stability of duplex formation.
One of the several possible base modifications includes the attachment of a
cyanoborane at the endocyclic nitrogen. Our previous studies (
28
-
31
) have shown that nucleosides containing cyanoborane modification at the
endocyclic nitrogen of the base can exhibit potent anti-tumor activity in mammalian cell lines, and anti-inflammatory and hypolipidemic activities in mice (
30
,
31
). The N
7
-boronated-2'-deoxyguanosine (
7b
dG) is of particular interest because, like the N
7
-deaza analogue, it does not prevent Watson-Crick base pairing (
32
) but rather blocks Hoogsteen base pairing. Furthermore, the 5'-triphosphate-
7b
dG is an excellent substrate for DNA polymerases including the thermostable Vent
®
and Taq
®
polymerases, and it is incorporated within an M13mp2 DNA duplex efficiently (
33
,
34
). The unique properties exhibited by
7b
dG and the ability to enzymatically prepare long DNA polymers of good stability
(
34
) have led us to explore the chemical syntheses of oligonucleotides containing a
cyanoborane moiety on the heterocyclic base.
A series of boron-modified d(
7b
GpX), d(Xp
7b
G) and d(
7b
Gp
7b
G) deoxydinucleotides have been chemically synthesized, wherein
7b
G is N
7
-cyanoborane-dG, and X is either a dA, dC, dG or dT. The structural properties of
all nine dinucleotides containing
7b
dG have been studied using
1
H and
31
P NMR. It was of interest to examine the effect of the cyanoborane moiety on the base stacking interactions
and the sugar-base backbone conformation, as well as the influence of sequence. For
comparison, the corresponding unmodified deoxydinucleotides d(GpX) and d(XpG)
were also included in this study. We report here, for the first time, the
syntheses of all nine possible combinations of dinucleotides containing N
7
-cyanoborane 2'-deoxyguanosine and the effect of cyanoborane substitution on
the base protons of dinucleotides.
MATERIALS AND METHODS
General
All solvents, chemicals and reagents were of analytical grade and used without
further purification unless otherwise indicated. The 3'-
O
-(acetyl)-2'-deoxynucleoside monomers (
11
,
12
and
14
) required for coupling reactions were purchased from ChemGenes Corporation,
Waltham, MA, and monomer
13
was purchased from Sigma Chemical Co. UV spectra were recorded on a Milton Roy
Spectronic 3000 Array spectrometer. Baker analyzed silica gel (60-200 mesh) was used for flash column chromatography. Thin layer
chromatography (TLC) was performed using 250 [mu] layers of silica gel GF precoated glass plates (Analtech, Inc.). Spots on
the TLC plates were detected by visualization under short wave UV light or by
heating the chromatogram at 100oC after spraying with 5% sulfuric acid in methanol.
1
H NMR spectra of protected nucleosides were recorded on a Varian-300 spectrometer and reported in p.p.m. downfield from the internal
tetramethylsilane (TMS=0) standard. The signals are expressed as s (singlet), d
(doublet), t (triplet), m (multiplet) or br (broad). The presence of
exchangeable protons was confirmed by treatment with deuterium oxide followed
by reintegrating the NMR spectrum.
NMR experiments
All
1
H,
31
P and
11
B spectra of dinucleotides were collected using a reverse-detect probe on a Varian Unity-500 MHz NMR spectrometer. All
2
H chemical shifts were measured relative to TSP (3-trimethylsilyl-propionate-2,3,3,3-d
9
sodium salt) as internal reference. The field-frequency lock was provided by deuterium oxide in the solvent. Spectra
were measured at 30 +-
0.2 and 60oC for deuterium exchange experiments. Bulk susceptibility corrections have
not been made for any of the
1
H NMR data.
11
B NMR spectra were acquired at 160 MHz and the
11
B chemical shifts were referenced externally to a solution of
diethylboron trifluoride
Et
2
O
.
BF
3
.
31
P NMR spectra were acquired at 202 MHz and the chemical shifts were referenced
externally to a solution of
85% H
3
PO
4
.
11
B signals of BH
2
CN in the boronated dinucleotides were not observed even in the
1
H-decoupling modes under the conditions used, possibly due to the
11
B broadening effect and asymmetric electronic environments of
11
B in this molecule (
35
). Thus, the
11
B chemical shifts of N
7
-cyanoborane-containing dinucleotides are not reported in this paper.
NMR samples
Purified unmodified dinucleotides were obtained from Sigma Chemical Co. [d(CpG), d(ApG) and d(GpG)], Clontech Laboratories, Inc., Palo Alto, CA [d(TpG)] and ChemGenes Corporation [d(GpG)]. Weighed
dinucleotides for NMR experiments were dissolved in 100 mM NaCl, 0.1 mM EDTA,
10 mM potassium phosphate, pH 7.4. The samples were brought into D
2
O by lyophilizing the solution once or twice with 99.99% D
2
O (Cambridge Isotope Laboratories), and dissolving the mixture in 99.996% D
2
O to a final concentration ~3 mM of dinucleotide. A 5 mm NMR sample tube (Wilmad) was used.
Kinetics of GH-8 proton exchange with D
2
O
In order to examine the ability of GH-8 base protons to exchange with D
2
O, we monitored the GH-8
1
H NMR spectra at different time intervals while incubating an equimolar mixture
of d(
7b
GpG) and d(GpG) in D
2
O at 60oC over 2 h. The deuterium substitution rates were measured by following
changes in intensities of the respective
1
H NMR signals with time and the data was analyzed according to pseudo-first order kinetics.
General procedure for the synthesis of 5
'
-
O
-(Fmoc)-3
'
- phosphoramidite 2
'
-
O
-deoxynucleosides (6-9)
5'-
O
-(Fmoc)-2'-
O
-deoxynucleoside (1 mmol) was dissolved in anhydrous THF (10 ml) under an
argon atmosphere.
N,N
-diisopropylethylamine (4 eqv.) was added in the solution followed by 2-cyanoethyl
N,N
-diisopropylchlorophosphoramidite (2.5 eqv.) under anhydrous conditions.
The mixture was stirred at room temperature under argon atmosphere for 30 min.
The TLC (solvent: TEA:MeOH:CH
2
Cl
2
:1:5:94 v/v/v) showed complete disappearance of the starting material and
formation of a less polar compound. The reaction was quenched by the addition
of methanol (0.1 ml). The solvents and excess of reagents were removed under
reduced pressure. The residue was dissolved in a solution of 2% triethylamine
in ethyl acetate (100 ml) and washed with saturated sodium bicarbonate solution
(2 * 50 ml), saturated sodium chloride solution (2 * 50 ml) followed by water (50 ml). All washings were combined and
re-extracted with 2% triethylamine in ethyl acetate solution (50 ml). The
organic phase was dried (Na
2
SO
4
) and filtered. The solvent was removed under reduced pressure, and the
resulting residue was then taken up in ethyl acetate and precipitated with cold
hexane. The solid was collected by filtration and dried under vacuum to afford
72-90% (depending upon the nucleoside) of the corresponding 5'-Fmoc-deoxynucleoside 3'-phosphoramidite. The purity and
homogeneity of the compound was checked with
31
P and
1
H NMR.
5
'
-
O
-(Fmoc)-thymidine 3
'
-
O
-(
N,N
- diisopropyl- amino)phosphoramidite (6)
Compound
6
was prepared in 74% yield following the general procedure using 5'-
O
-(Fmoc)-thymidine
1
(464 mg, 1 mmol),
N,N
-diisopropylethylamine (0.69 ml, 4 mmol) and 2-cyanoethyl
N,N
-diisopropyl chlorophosphite (0.456 ml, 2 mmol) in anhydrous THF (8.0 ml).
1
H NMR (CDCl
3
): [delta] 8.07 (s, 1H, M
H
), 7.71 (d, J = 7.5 Hz, 2H, Ar-
H
), 7.52 (d, J = 7.5 Hz, 2H, Ar-
H
), 7.36 (d, J = 7.5 Hz, 2H, Ar-
H
), 7.26 (m, 2H, Ar-
H
), 7.19 (s, 1H, H-6), 6.27 (q, J = 6.3 Hz, 1H, H-1'), 4.5-4.32 (m, 5H, H-3', H-4', C
H
CH
2
OCO, OC
H
2
CH
2
CN), 4.20 (m, 2H, H-5'), 3.8-3.51 (3*m, 4H, OC
H
2
CH
2
CN and 2*NC
H
Me
2
), 2.59-2.03 (3*m, 4H, 2*H-2', OCH
2
C
H
2
CN), 1.29-1.17 (m, 12H, 2*NHC
M
e
2
);
31
P NMR (CDCl
3
): [delta] 149.97, 149.82 p.p.m.
N
6
-Benzoyl-5
'
-
O
-(Fmoc)-2
'
-
O
-deoxyadenosine 3
'
-
O
- (
N,N
-diisopropylamino)-phosphoramidite (7)
Compound
7
was prepared in 87.7% yield following the general procedure using compound
2
(575 mg, 1 mmol),
N,N
-diisopropylethylamine (0.69 ml) and 2-cyanoethyl
N,N
-diisopropyl chlorophosphite (0.46 ml, 2 mmol) in anhydrous THF (30 ml).
1
H NMR (CDCl
3
): [delta] 8.94 (s, 1H, N
H
), 8.82 and 8.81 (2*s, 1H, H-8), 8.27 and 8.27 (2*s, 1H, H-2), 7.97 (d, J = 7.2 Hz, 2H, Ar-H), 7.75 (d, J = 7.5 Hz, 2H, Ar-H), 7.56 (m, 3H, Ar-H), 7.50 (t, J = 7.5 Hz, 2H, Ar-H), 7.38 (t, J = 7.2 Hz, 2H,
Ar-H), 7.32 (t, J = 7.2 Hz, 2H, Ar-H), 6.53 (q, J = 6.0 Hz, 1H, H-1'), 4.82 (m, 1H, H-3'), 4.47-4.39 (m, 3H, H-4', H-5'), the
methylene protons were assigned as in compound
6
;
31
P NMR (CDCl
3
): [delta] 149.92, 149.77 p.p.m.
N
4
-Ibu-5
'
-
O
-(Fmoc)-2
'
-
O
-deoxycytidine-3
'
-
O
-(
N,N
-diiso-propylamino)-phosphoramidite (8)
Compound
8
was prepared in 72% yield following the general procedure using compound
3
(519 mg, 1 mmol),
N,N
-diisopropylethylamine (0.69 ml) and 2-cyanoethyl
N,N
-diisopropyl chlorophosphite (0.46 ml, 2 mmol) in anhydrous THF (10 ml).
1
H NMR (CDCl
3
): [delta] 8.09 and 8.03 (2*d, J = 7.5 and 12.6 Hz, 2H, H-5, H-6), 7.77 (d, J = 7.2 Hz, 2H, Ar-H), 7.56 (m, 2H, Ar-H), 7.41 (m, 2H, Ar-H), 7.39 (m, 2H, Ar-H), 6.28 (q, J = 7.5 Hz, 1H, H-1'), the methylene
protons were assigned as in compound
6
;
31
P NMR (CDCl
3
): [delta] 150.13, 149.71 p.p.m.
N
2
-Ibu-5
'
-
O
-(Fmoc)-2
'
-
O
-deoxyguanosine-3
'
-
O
-(
N,N
-diiso- propylamino)-phosphoramidite (9)
Compound
9
was prepared in 82% yield following the general procedure using compound
4
(559 mg, 1 mmol),
N,N
-diisopropyl- ethylamine (0.69 ml) and 2-cyanoethyl
N,N
-diisopropyl chlorophosphite (0.46 ml, 2 mmol) in anhydrous THF.
1
H NMR (CDCl
3
): [delta] 9.02 and 9.01 (2*s, 1H, H-8), 7.77 (t, J = 8.2 Hz, 2H, Ar-H), 7.59 (m, 2H, Ar-H), 7.43-7.38 (m, 2H, Ar-H), 7.37-7.28 (m, 2H, Ar-H), 6.20 (m, 1H, H-1'), 4.83-4.55 (m, 2H, H-3', H-4'), 4.50-4.38 (m, 5H, H-5',OCOC
H
2
C
H
), the methylene protons were assigned as in compound
6
;
31
P NMR (CDCl
3
): [delta] 149.52, 149.27 p.p.m.
N
2
-Ibu-N
7
-cyanoboranyl-5
'
-
O
-(Fmoc) 2
'
-
O
-deoxy- guanosine (5)
5'-
O
-(Fmoc)-N
2
-IbudG (838.5 mg, 1.5 mmol) was dissolved in THF (18 ml) and
triphenylphosphine cyanoborane (1.5 g, 5.0 mmol) was added. The mixture was heated at 85-90oC for 2 h. TLC (solvent MeOH:CH
2
Cl
2
, 5:95 v/v) showed that ~50% of the starting material had been converted to a less polar compound.
Solvent was removed under reduced pressure and the resulting residue was
purified on silica gel column. Elution of the column with 2-3% methanol in methylene chloride gave 550 mg (61.3% yield, 93% based on
recovered starting material) followed by unreacted starting material (228 mg).
FAB-MS [M+H] 599.22 (calculated 599.39).
1
H NMR (DMSO-d
6
): [delta] 12.29 (s, 1H, N
H
), 11.85 (s, 1H, N
H
CO), 8.98 (s, 1H, H-8), 7.87 (d, J = 7.5 Hz, 2H, Ar-
H
), 7.59 (t, J = 6.9 Hz, 2H, Ar-
H
), 7.42 (m, 2H, Ar-
H
), 7.30-7.27 (m, 2H, Ar-
H
), 6.23 (t, J = 6.6 and 6.1 Hz, H-1'), 5.54 (d, J = 4.2 Hz, 1H, 4.51-4.01 (m, 7H, H-5', H-4', H-3', C
H
-C
H
2
OCO), 2.82-2.34 (m, 5H, H-2', B
H
2,
C
H
), 1.12 (d, J = 6.8 Hz, 6H, 2*C
H
3
).
N
2
-Ibu-N
7
-cyanoboranyl-5
'
-
O
-(Fmoc)-2
'
-
O
-deoxyguanosine- 3
'
-
O
-(
N,N
-diisopropylamino)phosphoramidite (10)
Compound
5
(490 mg, 0.82 mmol) was dissolved in anhydrous THF (6 ml, Aldrich Sure/Sealtm) under argon atmosphere. The mixture was stirred at room temperature
until a clear solution was obtained (~15 min).
N,N
-Diisopropylethylamine (0.6 ml, 3.5 mmol) and cyanoethyl
N,N
-diisopropyl chlorophosphite (0.4 ml, 1.75 mmol) were added under argon atmosphere. The TLC (solvent TEA:MeOH:DCM, 1:9:90 v/v/v) of the reaction mixture after 30 min showed that >90% of the starting material was converted to a less polar
compound. Stirring was continued for an additional 30 min and methanol (0.1 ml)
was added. The solvent was removed under reduced pressure and the residue was
dissolved in 2% TEA ethyl acetate solution (50 ml) and washed with saturated
sodium bicarbonate solution (2 * 50 ml), saturated sodium chloride solution (2 * 50 ml) followed by water (50 ml). All the aqueous washings were
combined and re-extracted with 2% TEA ethyl acetate solution (50 ml). The organic phase
was dried (Na
2
SO
4
), filtered, and solvent was removed under reduced pressure. The residue was
dissolved in ethyl acetate and added into cold hexane. The separated solid was
removed via filtration and dried under vacuum to yield compound
10
(534 mg, 81.8%).
1
H NMR (CDCl
3
): [delta] 12.23 (br, s, 1H, NH), 9.59 (bs, s, 1H, NHCO), 8.32, 8.299 (2*s, 1H, H-8). 7.751 (d, J = 7.2 Hz, 2H, Ar-H), 7.59 (d, badly separated, 2H, Ar-H), 7.41-7.38 (m, 2H, Ar-H) 7.33-7.27 (m, 2H, Ar-H), 6.25 (m, 1H, H-1'), 4.84-4.25 (3m, 7H, H-3', H-4', H-5', OCOC
H
2
C
H
), 3.94-3.63 (3m, 4H, 2* N
H
C Me
2,
OC
H
2
CH
2
CN), 2.94-2.55 (2m, 7H, COC
H
Me
2
, H-2', OCH
2
C
H
2
CN, B
H
2
), 1.22-1.19 (m, 18H, COCHMe
2
, 2*NCHMe
2
);
31
P NMR (CDCl
3
): [delta] 150.82, 149.51 p.p.m.
N
2
-Ibu-N
7
-cyanoboranyl 3
'
-
O
-(acetyl)-2
'
-
O
-deoxyguanosine (23)
Compound
11
(700 mg, 1.85 mmol) was dissolved in tetrahydrofuran (40 ml) and
triphenylphosphine cyanoborane (2.2 g, 7.39 mmol) was added in the solution.
The mixture was heated at 85-90oC for 30 min. TLC (solvent: 5% methanol in methylene chloride)
showed ~50% disappearance of the starting material (R
f
= 0.45), and formation of a less polar compound (R
f
= 0.58). The solvent was removed under reduced pressure and the resulting crude
mixture was purified on silica gel column. Elution of the column with 1-1.5% methanol in methylene chloride solution gave compound
23
(348 mg, 45% yield). FAB-MS [M+H] 419.19 (calculated 419.16).
1
H NMR (DMSO-d
6
): [delta] 9.05 (s, 1H, H-8), 6.28 (t, J = 6.34 Hz, 1H, H-1'), 5.33 (d, J = 2.64 Hz, 1H, H-3'), 5.31 (t, J = 1.98, 1H, 5'-OH), 4.14 (d, J = 2.15 Hz,
1H, H-4'), 3.63 (m, 2H, H-5'), 2.88 and 2.78 (2*m, 2H, H-2'), 2.59-249 (m, 3H, Me
2
C
H
, B
H
2
), 1.14 and 1.12 (2*s, 6H,
Me
2
CH). Continued elution of the column gave 320 mg of the unreacted starting
material.
N
7
-Cyanoboranyl-2
'
-deoxyguanylyl (5
' ->
3
'
) 2
'
-deoxy- nucleoside d(
7b
G
p
X): general procedure
N-Protected 3'-
O
-acetyl-2'-deoxynucleoside (
11
-
14
, 1.0 mmol) was dissolved in anhydrous acetonitrile (100 ml) under argon atmosphere and
1H
tetrazole (4.0 eqv.) was added in the reaction mixture. The mixture was stirred
at room temperature until a clear solution was obtained. A solution of
10
(1.2 eqv.) in acetonitrile (2 ml) was added through syringe under argon
atmosphere; Scheme 2 . The TLC (solvent 5% methanol in ethyl acetate) of the
reaction mixture after 30 min showed complete disappearance of the starting
material. A mixture of oxidizing solution (0.1 M iodine in THF: lutidine:water,
7:2:1 v/v/v) was added until iodine color persisted (~3.0 ml). After 1 h stirring at room temperature the TLC (solvent:
TEA:MeOH:DCM:EtOAc, 0.5:6.5:45:48 v/v/v/v) showed complete conversion of the
non-oxidized compound to a more polar product. A saturated solution of sodium
thiosulfate was then added to the reaction mixture to quench the reaction and
the solvent was removed under reduced pressure. The resulting residue was
dissolved in dichloromethane (50 ml) and washed with a saturated solution of
sodium bicarbonate (30 ml), saturated solution of sodium chloride (2 * 30 ml) and water (50 ml). The aqueous washings were combined and re-extracted with dichloromethane (50 ml). The organic phase was dried
over sodium sulfate, filtered, and solvent was removed under reduced pressure
to yield crude product which was purified on silica gel column. Elution of the
column with 2% methanol in ethyl acetate gave pure blocked dinucleotides (
15
-
18
), which were deblocked by treating with concentrated ammonium hydroxide (2 ml)
in a sealed vial at 45-50oC for 4-5 h. The mixture was cooled and the solvent was removed under
reduced pressure. The residue was purified on Sephadex G-10 column (35 * 1 cm), eluted with a linear gradient of water:ammonium bicarbonate (0-0.8 M) using a Tristm pump and Isco fraction collector; 20 ml fractions
were collected. The absorbance at 260 nm of each fraction was recorded,
fractions containing the compound were combined, and solvent was removed under
reduced pressure to yield pure compounds (
19
-
22
). In a similar manner, the compounds containing
7b
dG at either the 3' or both (3' and 5') ends were prepared starting with monomers
23
and
6
-
10
; Scheme 3 . The physical properties and yields of all the nine dinucleotides
containing boronated guanosine are reported in Tables
1
and
1 1
.
All nine possible combinations of dinucleotides containing
7b
dG either at one or both 3'-, 5'- ends of a phosphodiester linkage were synthesized
using solution phase phosphoramidite chemistry (Schemes
1 - 3
). Initially, we
envisioned the use of commercially available 5'-DMT-2'-deoxynucleosides as the viable intermediate and
the N
7
-BH
2
CN precursor was readily prepared by treatment of 5'-DMT-2'-deoxyguanosine with Ph
3
PBH
2
CN (
36
). However, further experiments revealed that standard deprotection conditions
(30 s, 20oC, 2.5 M dichloroacetic acid) for removal of DMT resulted in a complex
mixture, including deboronation. Attempts to remove the 5'-DMT group selectively, without affecting deboronation, using
different acids such as trichloroacetic acid or benzene sulfonic acid in
various solvents (chloroform:methanol, 7:3 v/v, acetonitrile and
tetrahydrofuran) were futile. It should be noted that the cyanoborane guanosine
derivative is quite stable in acidic media. The cyanoborane instability is
caused by the formation of an unstable adduct between the cyanoborane and DMT
cation (
36
-
38
) generated during DMT deprotection. This led us to investigate an alternate
route which would not involve use of the 5'-DMT protecting group.
Monomers required for the coupling reaction were synthesized via the treatment
of commercially available 2'-deoxynucleosides with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) in anhydrous pyridine following the procedure of
Lehmann
et al
. (
39
) to yield the corresponding 5'-
O
-blocked nucleosides (
1
-
4
) in 35-62% yields (Scheme 1 ). The actual yields based on the recovered starting
material were 58-80%. The purity and homogeneity of the compounds were checked by TLC, and
the structures were confirmed on the basis of
1
H NMR spectroscopy and by comparing the melting points with those reported in
the literature. Compounds
1-4
were readily converted to the corresponding phosphoramidite derivatives (
6
-
9
) by treatment with 2-cyanoethyl
N,N
-diisopropylchlorophosphoramidite and diisopropylethylamine in anhydrous
tetrahydrofuran in 72-90% yields. The structures of these compounds were confirmed on the basis
of
1
H and
31
P NMR spectroscopy.
Chemical shifts of
31
P and base protons of d(
7b
GpX), d(Xp
7b
G) and d(
7b
Gp
7b
G) series in 100 mM NaCl, 0.1 mM EDTA, 10 mM phosphate, PH 7.4 at 30oC
No./(compd.)
a
AH-2
AH-8
CH-5
CH-6
GH-8 (5')
GH-8 (3')
TH-6
T-CH
3
31
P
b
19; d(
7b
GpC)
-
-
5.84
7.82
8.55
-
-
-
-2.94
N d(GpC)
-
-
5.94
7.87
7.87
-
-
-
-0.91
20; d(
7b
GpG)
-
-
-
-
8.36
8.00
-
-
-3.65
N d(GpG)
-
-
-
-
8.02
7.78
-
-
-2.97
21; d(
7b
GpA)
8.10
8.38
-
-
8.39
-
-
-
-3.03
N d(GpA)
8.04
8.37
-
-
7.75
-
-
-
-1.10
22; d(
7b
GpT)
-
-
-
-
8.60
-
7.65
1.74
-3.05
N d(GpT)
-
-
-
-
7.94
-
7.56
1.73
-2.79
29; d(Cp
7b
G)
-
-
5.98
7.66
-
8.58
-
-
-2.29
N d(CpG)
-
-
5.97
7.60
-
8.05
-
-
-2.06
30; d(Gp
7b
G)
-
-
-
-
7.81
8.58
-
-
-2.07
N d(GpG)
-
-
-
-
7.78
8.02
-
-
-2.97
31; d(Tp
7b
G)
-
-
-
-
-
8.62
7.50
1.88
-2.46
N d(TpG)
-
-
-
-
-
8.07
7.45
1.86
-1.10
32; d(Ap
7b
G)
8.17
8.15
-
-
-
8.55
-
-
-2.20
N d(ApG)
8.17
8.12
-
-
-
7.99
-
-
-1.96
33; d(
7b
Gp
7b
G)
-
-
-
-
8.46
8.54
-
-
-2.00
N d(GpG)
-
-
-
-
8.02
7.78
-
-
-2.97
a
Compounds in bold letter have at least one N
7
-boronated deoxyguanosine and those numbered N are the corresponding
unmodified oligonucleotides.
b
Referenced internally to the resonance from phosphate buffer.
Physical data of dinucleotides containing N
7
-boronated-deoxyguanosine
Compound
% yield
R
f
a
FAB-MS (m/e)
UV [lambda]
max
(overall)
(M+Na)
+
H
2
O
0.1 N HCl
0.1 N NaOH
19
; d(
7b
GpC)
39
0.61
y; 618.23
262.1
277.0
273.3
z; 618.21
20
; d(
7b
GpG)
31
0.59
y; 658.23
255.0
256.5
268.4
z; 658.21
21
; d(
7b
GpA)
37
0.63
y; 642.23
258.0
257.6
261.7
z; 642.21
22
; d(
7b
GpT)
18
0.62
y; 633.23
260.2
261.0
271.0
z; 633.20
29
; d(Cp
7b
G)
52
0.56
y; 618.23
260.2
277.3
272.2
z; 618.23
30
; d(Gp
7b
G)
39
0.51
y; 658.23
255.0
256.9
268.4
z; 658.00
31
; d(Tp
7b
G)
27
0.60
y; 633.23
259.9
259.9
270.7
z; 633.20
32
; d(Ap
7b
G)
42
0.58
y; 641.23
257.6
257.3
260.2
z; 641.28
33
; d(
7b
Gp
7b
G)
26
0.56
y; 697.07
257.3
257.6
275.5
z; 697.21
a
TLC solvent isopropanol:ammonium hydroxide:water, 7:1:2 v/v/v; y = calculated
values and z = found.
N
7
-Boronated monomers (5
'
- and 3
'
-fragments of dinucleotides)
The boronated monomer, N
2
-Ibu-5'-Fmoc-
7b
dG-3-phosphoramidite (
10
), was prepared starting from compound
4
as outlined in Scheme 1 . Cyanoboronation of N
2
-Ibu-2'-deoxyguanosine is not a suitable alternative since the
loss to some extent of cyanoborane moiety occurs in pyridine. It was important,
therefore, that all reactions involving use of pyridine were carried out prior
to the boronation step. Thus, compound
4
on refluxing with triphenylphosphine cyanoborane (Ph
3
P:BH
2
CN)
in THF for 2 h (heating the reaction mixture for longer time resulted in some
decomposition) gave the N
7
-cyanoborane derivative
5
. In the
1
H NMR spectrum of compound
5
, the H-8 proton shifted downfield to 8.98 p.p.m. compared with unmodified
guanine, and a broad peak for BH
2
at 2.84-2.34 p.p.m. merged with the H-2' proton was observed. Treatment of compound
5
with 2-cyanoethyl
N,N
-diisopropylchlorophosphoramidite and diisopropylethylamine in anhydrous
tetrahydrofuran gave the corresponding phosphoramidite (
10
; Scheme 1 ).
31
P NMR showed phosphorus peaks at 150.82 and 149.51 p.p.m. 3'-
O
-(acetyl)-N
2
-Ibu-
7b
dG (
23
) was prepared in 65% yield starting with the compound
11
as described for
5
, and the structure of compound
23
was confirmed by FAB-MS and NMR spectroscopy analogous to compound
5
.
5
'
-Boronated dinucleotides d(
7b
dGpX)
Dinucleotides containing a normal dG at the 3'-end and a boronated
7b
dG at the 5'-end were synthesized as shown in Scheme 2 . N-protected-3'-
O
-(acetyl)-2'-deoxynucleoside (1 eqv.) was dissolved in acetonitrile and
1H
-tetrazole (4.0 eqv.) was added to the solution under argon atmosphere. A
solution of compound
10
(1.2 eqv.) in acetonitrile was added and the reaction mixture was stirred at room temperature for 30 min. A mixture of oxidizing
solution (0.1 M iodine in THF:lutidine:water, 7:2:1 v/v/v) was then added until
the iodine color persisted in the solution. The reaction was quenched after 1 h
with saturated sodium thiosulfate solution and solvent was removed. The residue
on extraction with ethyl acetate gave the crude product. The crude mixture on
silica gel column purification gave the corresponding pure compounds (
15
-
18
) which on subsequent treatment with conc. ammonium hydroxide at 50oC for 4-5 h gave dinucleotides (
19
-
22
).
3
'
-Boronated dinucleotides d(Xp
7b
dG)
The synthesis of dinucleotides (
29
-
33
) containing
7b
dG at the 3'-end and either dG or
7b
dG at the 5'-ends were carried out as shown in Scheme 3 . All the reaction
conditions were similar to those described for Scheme 4 except that compound
23
was used as the boron-containing monomer and compounds
6
-
9
were used as the 5'-protected-2'-deoxynucleoside-3'-phosphoramidite. Compounds
23
and
10
were used to synthesize the dinucleotide
33
containing two
7b
dG. The structure of compounds
29
-
33
were confirmed on the basis of NMR (
1
H and
31
P) and FAB-MS (Tables
1
and
2
). The molecular ion peak confirmed the presence of a cyanoborane moiety in the
molecule.
1H NMR spectral assignments
All NMR experiments were done in D
2
O. Protons N2 and N1 of G, N4 of C, N6 of A and N3 of T are exchanged with D
2
O at room temperature (data not included). Assignment of other protons of
dinucleotides is divided into four parts: (i) non-exchangeable endocyclic base protons (H-2 and H-8 of A, H-5 and H-6 of C, H-8 of G and H-6 of T); (ii) cyanoborane protons;
(iii) CH
3
- protons of T; and (iv) sugar ring protons (H-1', H-2', H-3', H-4', H-5'). In
this paper we focus primarily on the
1
H NMR studies of the endocyclic base protons.
The assignment of proton resonances in unmodified dinucleotides, d(GpX) and
d(XpG), is well documented (
40
). Since the
1
H resonance of the modified bases occur in a pattern similar to that of
unmodified dinucleotides, the assignments for
1
H resonances in the boronated-dinucleotides d(
7b
GpX), d(X
7b
G) and d(
7b
Gp
7b
G) were tentatively made analogous to the unmodified dinucleotides, d(GpX),
d(XpG), as shown in Table
1
.
The
1
H resonance of the BH
2
CN moiety can easily be located. When the proton is bonded to a quadrupolar nucleus, like
11
B (I = 3/2) and
10
B (I = 3), a rapid nuclear quadrupolar relaxation of
11
B and
10
B (80.4 and 19.6% natural abundance respectively) will broaden the proton peak (
35
) resulting in a very broad
1
H resonance covering >100 Hz centered at ~2.5-2.6 p.p.m. for all dinucleotides containing N
7
-cyanoborane (data not shown). The assignment was further confirmed by
11
B-decoupling experiments in which the broad peak at this position became sharper relative to the
11
B-decoupling off experiment. The exact chemical shift was not assigned,
however, due to overlap with peaks from the H-2' sugar protons.
31P NMR spectroscopy
31
P chemical shifts of all unmodified and boronated dinucleotides are reported in
Table
1
. The
31
P chemical shifts have very similar values within a range of 2.0 p.p.m.
Exchange of
7b
GH-8 in d(
7b
GpG) with D
2
O
Solvent exchange ability of GH-8 was studied in D
2
O using
1
H NMR; the GH-8 deuteration rate was measured by change in the intensity of the GH-8 proton. Since the dinucleotides were stable to hydrolysis at the
higher temperatures (unpublished data), a higher temperature (60oC) was chosen for convenient NMR studies over a 2 h period. Dinucleotides
d(
7b
GpG) and d(GpG) were selected as being representative for study of the GH-8 exchange rate in the d(
7b
GpX), d(Xp
7b
G) and d(
7b
Gp
7b
G) series. A correlation between time and intensity change of GH-8 resonances of a mixture of d(
7b
GpG) and d(GpG) incubated at 60oC is shown in Figure
3
. We found that the
7b
GH-8 of the 5'-residue
in the d(
7b
GpG) dinucleotide deuterates almost 10-fold faster than the corresponding 5'-residue of unmodified dinucleotide d(GpG), i.e., deuteration
rates of 2.3 * 10
-4
s
-1
versus 2.7 * 10
-5
s
-1
(Fig.
3
) respectively, assuming pseudo-first order reaction kinetics. Unlike the 5'-residue, the GH-8 of 3'-residues in both boronated d(
7b
GpG) and unmodified d(GpG) dinucleotides have comparable deuteration rates (~4.1 * 10
-5
s
-1
).
DISCUSSION
Effect of BH
2
CN on the chemical shifts of non-exchangeable base protons in dinucleotides
BH
2
CN is an electron withdrawing group and is expected to cause a downfield shift
on the neighboring protons. This is reflected (see Table
1
and Fig.
2
) in the GH-8 protons of all the N
7
-boronated guanosines, situated either at the 5'- or 3'-end or at both the ends (3' and 5'), such as d(
7b
GpX), d(Xp
7b
G) and d(
7b
Gp
7b
G) respectively. The
7b
GH-8 proton in these compounds shifted downfield by 0.35-0.8 p.p.m. relative to the corresponding GH-8 proton of unmodified residues; presumably these downfield
shifts are through-bond effects. As expected, the BH
2
CN moiety has a relatively smaller effect (<0.1 p.p.m. in general) on the proton chemical shifts of the neighboring
unmodified base in the boronated-dinucleotide (Table
1
and Fig.
1
). The observed small chemical shift differences relative to unmodified
dinucleotides must be due to base stacking and conformational changes caused by
modification. It is noted that, in d(
7b
GpG), the BH
2
CN had a greater effect on the GH-8 chemical shifts of the neighboring unmodified guanosine residue compared
with d(
7b
GpX) and d(Xp
7b
G), ~0.2 p.p.m. and 0.1 p.p.m. respectively, where X is either a dA, a dC or a
dT. The GH-8 proton shift of the unmodified residue of the d(
7b
GpG) dinucleotide may indicate different base stacking interactions as compared
with the corresponding parent dinucleotide, d(GpG), due to the presence of a BH
2
CN moiety on the adjacent dinucleotide base.
31P chemical shifts provide information about the phosphate backbone
The
31
P chemical shifts and the
1
H,
31
P coupling constants of oligonucleotides provide information about the
phosphodiester backbone (
41
). The internucleotide linkage is defined by six torsion angles from one
phosphate atom to the next along the DNA backbone. Theoretical studies have
shown that the conformation of two of the six torsion angles (O3'-P-O5'-C5' and C3'-O3'-P-O5')
appear to be most important in determining
31
P chemical shifts (
42
,
43
). The small differences of
31
P chemical shifts (<2 p.p.m.) between boron-modified and unmodified dinucleotides (Table
1
) indicate that a cyanoborane moiety at N
7
-guanine in the d(
7b
GpX), d(Xp
7b
G) and d(
7b
Gp
7b
G) series at 30oC did not induce large alterations in the phosphodiester backbone
conformations, which is not surprising because cyanoborane is spatially located
far from the phosphorus atom. However, care should be taken in interpreting the
shifts observed in oligonucleotides, since it is uncertain to what degree the
stacking and base interaction shifts are influenced by the neighboring bases
(sequence effect) in dinucleotides. Detailed conformational studies by
1
H NMR are in progress and will be published elsewhere.
GH-8 proton exchange ability in d(
7b
GpG)
The fact that BH
2
CN, an electron withdrawing group, will generate a partial positive charge on
the neighboring carbon through inductive effects and will make
7b
GH-8 more acidic compared with the unmodified base proton is supported by our
findings. A more acidic GH-8 proton can explain our result of ~10-fold faster deuteration rate of GH-8 (5'-residue,
7b
G) in the dinucleotide, d(
7b
GpG), compared with the unmodified guanosine (5'-residue) of d(GpG) (Fig.
3
).
CONCLUSIONS
The N
7b
-dinucleotides were readily prepared using 5'-Fmoc- 3'-phosphoramidite-2'-deoxynucleosides in
good yields (26-52%, including coupling, oxidation and deblocking steps). The solution
phase synthesis employed for dinucleotides using 5'-Fmoc-3'-phosphoramidite-2'-deoxynucleosides should be
adaptable to the solid phase synthesis for oligonucleotides containing mixed
sequences. The ability to readily prepare dinucleotides modified with
cyanoborane at the N
7
-position of guanosine that are stable to acid and base (
44
) facilitates further studies with these base-boronated compounds.
1
H NMR at 60oC showed that the
7b
GH-8 is more acidic compared with unmodified GH-8 as evidenced by the ~10-fold faster deuterium exchange rate. The increased
acidity of the
7b
GH-8 proton should facilitate alkylation reactions at the C-8 position under mild conditions, and make it possible to generate
new types of site-specific C-8-modified oligonucleotides in DNA.
Until now, the poor hydrolytic stability of N
7
-alkylated nucleosides to depurination has impeded studies of the
interactions of other molecules with N
7
-modified DNA. Interestingly, the N
7
-boronated guanosine is a hydrolytically stable nucleoside (
28
) that facilitates Watson-Crick base pairing (
32
), but blocks Hoogsteen base pairing interactions; it thereby provides a new set
of reagents for probing interactions of nucleic acids with DNA, RNA and
proteins. The
1
H and
31
P NMR studies demonstrate that the N
7
-cyanoborane moiety has minimal influence on the conformation of
dinucleotides, which would agree with physico-chemical studies (
35
) wherein the
T
m
values, melting profiles, and circular dichroism spectra of a N
7b
-modified 14mer oligonucleotide and unmodified oligonucleotide were nearly
identical, showing that a single boronated guanosine perturbs the DNA structure
very slightly.
ACKNOWLEDGEMENTS
This work was supported by grants NP-741 from the American Cancer Society and 5UO1-CA60139 from NIH to B.R.S. NMR spectra were recorded at the Duke
University NMR Center Facility. We thank Drs Edward Budowsky and Prem C. Srivastava for reading the
manuscript and Ms Ying Wang for collecting some of the NMR data.
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3 Cao,X. and Matteuci,M.D. (1994) Tetrahedron Lett.35, 2325-2328.
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