Volume 12 Number 11 1984
Nucleic Acids Research
Polymer support oligonucleotide synthesis XVIJI1.2: use of (-cyanoethyl-N,N-dialkylamino-/Nmorpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying
deprotection and isolation of the final product
N.D.Sinha, J.Biernat, J.McManus and H.Koster
Institut fur Organische Chemie und Biochemie, Universitat Hamburg, Martin-Luther-King-Platz 6,
D-2000 Hamburg 13, FRG
Received 9 February 1984; Revised 10 April 1984; Accepted 9 May 1984
ABSTRACT
Various 5'-0-N-protected deoxynucleoside-3'-0-5-cyanoethyl-N,Ndialkylamino-/N-morpholinophosphoramidites were prepared from
3-cyanoethyl monochlorophosphoramidites of N,N-dimethylamine,
N,N-diisopropylamine and N-morpholine. These active deoxynucleoside phosphites have successfully been used for oligodeoxynucleotide synthessis on controlled pore glass as polymer support
and are very sui'table for automated DNA-synthesis due to their
stability in solution. The intermediate dichloro-B-cyanoethoxyphosphine can easily be prepared free from any PCl contamination. The active monomers obtained from B-cyanoeth'l monochloro
N,N-diisopropylaminophosphoramidites are favoured. Cleavage of
the oligonucleotide chain from the polymer support, N-deacylation
and deprotection of 3-cyanoethyl group from the phosphate triester moiety can be performed in one step with concentrated
aqueous ammonia. Mixed oligodeoxynucleotides are characterized
by the sequencing method of Maxam and Gilbert.
INTRODUCTION
Adaptation of "phosphite triester" approach introduced by
Letsinger3,4) to solid phase synthesis of well-defined DNA
sequences has tremendously reduced the time required for these
syntheses. For the preparation of reactive monomeric intermediates various different types of alkyl/aryl phosphodichloridites4 11'17) have been explored. Several difficulties generally
encountered in the preparation and handling of nucleoside phosphoromonochloridites for automated routine synthesis of desired
DNA fragments led to the introduction of various modifications
for the preparation of active nucleoside phosphite intermediates12 17). The common reactive monomeric intermediates
presently in use for solid phase oligodeoxynucleotide synthesis
following phosphite triester approach are: 5'-O,N-protected de-
oxynucleoside-3'-O-methyl-N,N-dimethylamino-14),
© I R L Press Limited, Oxford, England.
-3'-0-methyl4539
Nucleic Acids Research
N,N-diisopropylamino-15 16)
and -3'-0-methyl-N-morphol ino15)
phosphoramidites. Very recently O-chlorophenyl-N,N-dimethylamino-/N-morpholino deoxynucleoside phosphoramidites have been
introduced as reactive monomeric phosphites17).
These developments have considerably improved the efficiency
of the synthetic methods. The most time-consuming part at present
in the preparation of oligodeoxynucleotide sequences is, however,
not the synthesis but the work-up, purification and characterization of the sequences as well as the large-scale preparation
of active deoxynucleoside intermediates stable and reactive
enough for automated DNA synthesis.
When the methyl groupl8) is used for phosphate protection in
"phosphite triester" approach the final work-up includes a treatment of the polymer linked oligonucleotide with triethylammonium
thiophenolate, concentrated aqueous NH3 at 500 C and occasionally
t-butylamine at 450 c'0'1'9 21). The time necessary for deprotection and isolation can take several days and is in most
cases longer than the time taken for the synthesis of fully protected oligodeoxynucleotides on solid support. We feel that these
time-consuming steps of different deprotection reactions and removal of non-nucleotidic materials after synthesis and prior to
RP-HPLC purification e.g. by silica gel thin layer chromatography
migth undermine the improvements achieved in solid phase DNA synthesis by the "phosphite triester" approach.
In this paper, we report the preparation of various 13-cyanoethyl monochlorophosphoramidites of secondary amines such as N,Ndimethylamine, N-N-diisopropylamine and N-morpholine, and their
use for the synthesis of various reactive nucleoside phosphoramidites serving as nucleotide building blocks in polymer support
oligodeoxynucleotide synthesis 1) . It is demonstrated that the
choice of the 13-cyanoethyl group for phosphate protection in
phosphite triester approach significantly simplifies and reduces
the time necessary for deprotection and work-up of the final product of an ol igodeoxynucleotide synthesis on polymer support.
RESULTS AND DISCUSSION
The f-cyanoethyl group has been used as a phosphate (phosphorus (V)) protecting group in the phosphate triester approach
4540
Nucleic Acids Research
RN<
R1
R2
(2 equiv. when R
(1 equiv. when R
=
H)
=
(CH3)3SO)
Cl
1~ ~ ~ ~ ~
ether
.N- HC NC-CH2CH2--P-N
NC-CH2
2C
.
(1 equiv.)
Cl
1~~~~~~~~~~~~~~~~~~
B
OMl.o0-y
(2qv) Cl 'R2 NTrOY
2
(leqtsv)
a)
R =R2=CH 3
NC-GVCH20-P--N'RI
b)
J
VP-N
R1=R2= 3=CH-
~~~~H3C
c) R1+R2= morpholino
B= Thymine, 2-(methyl)benzoylcytosine, 38) isobutyrylguanine,
benzoyl adenine
Scheme (I)
Letsinger22) and Cramer23) and was previously introduced by
Tener24) for the phosphodiester method. The f-cynoethyl group
by
has only preliminarily been used as a phosphorous (III) protecting group when ribonucleoside monochlorophosphites are prepared as reactive intermediates5)0 Due to the sensivity and low
stability of these compounds they have not found application in
oligonucleotide synthesis. In contrast, the corresponding phosphoramidites have very promising and attractive properties as
reactive deoxynucleoside intermediates for polymer support oligodeoxynucl eotide synthesis.
Preparation of monochlorophosphoramidites
Three different f-cyanoethyl monochlorophosphoramidites of
N,N-dimethylamine, N-morpholine and N,N-diisopropylamine were
prepared by treating 3-cyanoethyl phosphordichloridite with Ntrimethylsilyl-N,N-dimethylamine, N-trimethylsilylmorpholine and
N,N-diisopropylamine, respectively, following Scheme (I) according to the
literature15'20, 21),
The B-cyanoethyl monochloro-
phosphoramidites of N,N-dimethylamine and N,N-diisopropylamine
were obtained as clear liquids in high yield after distillation
under reduced pressure. The monochloro morpholino derivative ob4541
Nucleic Acids Research
Table 1:
a) Physical constants of B-cyanoethyl monochlorophosphoramidites
B-Cyanoethyl monochloro-N,N-dialkyl-/N-morphollno phosphoramidites
90-92° C/0.6
Boiling point
Chemical shift for
in CH3CN ii)
in
CDC13
179.82 ppm
168.22 ppm
4.02, 4.2 (2t, P-OCH2. 2H)
3.80 (n, N(CH)2. 2H)
2.77 (t, -CH2CN, 2H)
1.29 (d. N(C(CH3)2)2. 12 H)
3.96, 4.1 (2t, P-OCH2. 2H)
3.67 (t. 0(CH2)2. 4H)
3.17 (m, N(CH2)2. 4H)
2.74 (t, CH2-CN, 2H)
175.97 pp
31P-NOR
Chemical shifts
for 1H-CHR (ppm)
103-105° C/0.08 mm
mm
4.01, 4.17 (2t, P-OCH2, 2H)
2.81 (t, -CH2CN, 2H)
2.7 (d. -N(CH3)2. 6 H)
(m)4,
Main peaks in
182
(+2).,
mass spectra
145
(lm-C2H6N)+
(-C)+
(.Cl)v
136 (C2H6+
110 (
180
238
2
~~201
136
)
(!2)',
lj-CJ *
236 (!.),
1660
(-C6O1IO)+
224
(!±2),
136
C)+, 1522 (mC
)
(m!S),12(.~40
('mC4H8NO)+
(m C3H4)
222
(ml),
b) 31P-NMR Chemical shift values (ppm) for the various deoxynucleoside phosphoramidites,
measured in
acetonitrile1i):
ibGd
bzAd
tlCd38)
Td
N.N-Dimethylamine
145.43
144.97
145.21
144.49
145.61
137.51
135.33
N,N-Diisopropylamine
147.40
147.44
Phosphoramidites of
N-Mloorpholine
142.57
142.17
i)
i1)
147.53
147.57
147.40
146.01
142.53
141.86
142.62
142.44
142.30
142.17
The crude reaction product after removal of the amine hydrochloride and the volatile material
under reduced pressure at room te.mperature (about 95X pure).
The chemical shifts are from down field with respect to 80X H3P04 in acetone d6 as an internal
standard.
tained after reaction work-up could not be distilled due to
thermal decomposition, but was sufficiently pure for our synthetic purposes. The impurities present in thi s case were [3cyanoethyl phosphorodichloridite (r-.--5%) and traces of the morphol ine derivative. They might be removed by adjusting the proportion of 3-cyanoethyl phosphorodichloridite over N-trimethyl silylmorpholine and concentrating the reaction work-up in vacuo at
500 C. All the monochlorophosphoramidites were characterized by
31 P-NMR, 1H-NMR, and mass spectra (Table I). They were found to
be stable under dry and inert conditions. The N,N-dimethylamino
derivative was found to be very reactive and hence required
stringent anhydrous and inert conditions for its handling when
compared with the other two phosphoramidites. 31P-NMR spectra of
the monochloridites are shown in Figure 1A.
Preparation of suitably protected deoxynucleoside phosphoramidites
The 5'-0-N-protected deoxynucl eoside-3 '--13--cyanoethyl -N,N4542
Nucleic Acids Research
- H=(CH12CH42)20
P°
NC (CH2)20
168.12 ppm
Figure 1A:
31P-NMR spectra of monochloro f-cyanoethyl phosphoramidites
Figure 1B:
31P-NMR spectra of deoxyguanosine B-cyanoethyl-N,Ndialkylamino-/N-morpholino phosphoramidites
dimethyl amino-/N,N-diisopropylamino-/N-morpholino phosphoramidites were prepared from these monochlorophosphoramidites in powdered form and high yield following Scheme (I) by a slight modi5
fication of the literature procedure 15.
The quality of these
nucleoside phosphoramidites was controlled using 31P-NMR and
silica gel TLC developed in distilled and dried ethylacetate.
Thus TLC offers a simple method to detect the presence of
starting material and hydrolysed, decomposed or oxidised phosphitylated products and to follow those reactions. In all in4543
Nucleic Acids Research
stances, it was found that the phosphorylation of protected deoxynucleosides was quantitative. Only in the case of deoxynucleoside N,N-dimethylamino phosphoramidite derivatives could polar
material be detected to some extent at the base line of the TLC
plate. This may be due to the hydrolysis of the very reactive
P-N(CH3)2 bond during TLC on the silica gel plate. This and other
side products were present in less than 5% as indicated by
31P-NMR. The presence of the nitrile group in these derivatives
is another marker easily detectable by infrared spectroscopy. In
most cases the two diastereomers of the deoxynucleoside phosphor.
amidite derivatives were clearly separated on TLC and 31P-NMR.
The reactive deoxynucleoside N-morpholinophosphoramidites prepared from non-distilled (crude) 1-cyanoethyl monochloro-N-morpholinophosphoramidite contained about 3% 3'-3'-dimers and 4%
hydrolysed or oxidised materials as indicated by 31P-NMR.
Most of these derivatives were found to be stable even after
frequent exposure to air. However, storage under argon at low
temperature is recommended. The guanosine derivatives were also
found to be quite stable when prepared from these monochloro
phosphoramidites in contrast to our observation with 5'-0-N-protected guanosine-3'-methyl-N-morpholinophosphoramidite2). The
stability of these compounds was checked six months after their
preparation by 31P-NMR and TLC and was found to be satisfactory.
Figure 1B indicates 31P-NMR of deoxyguanosine derivatives.
Synthesis of oligodeoxynucleotides
The synthesis of oligodeoxynucleotides was performed according
to Scheme (I) using 100 mg of CPG bound nucleoside6' 28) (7 to
7.5 pmol) in a column type reactor fitted with a sintered glass
fritt which could be maintained airtight with a serum cap. The
5'-O-DMTr group was removed using either 3% CCl 3COOH in 1%
methanol/nitromethane solution or in order to avoid undesirable
depurination with saturated ZnBr2 in 1% water-nitromethane solution. After thorough washing and drying of the glass beads in
vacuo, the active nucleoside was added in powdered form (2530 equ.) followed by addition of tetrazole (70-75 equ.) and
acetonitrile (1.5-2.0 ml) with a syringe under argon atmosphere.
In the case of mixed oligodeoxynucleotide synthesis the ratio of
purine to pyrimidine derivative was 3 to 2 by weight. The con4544
Nucleic Acids Research
Table II: Summary of the different steps performed in one elongation cycle
Step
Operation
Solvent/Reagent
1
Detritylation
a) 3% CC13COOH
b) ZnBr2
2
Washing
a) CH 3 NO 2 i)
ii
b) n-BuOH/Lutidine/THF
3
Washing
or
a) CH3CN
b) CH2C12
4
Drying
High vacuum
5
Condensation
Active nucleosides and
tetrazole in CH3CN
Volume (ml)
No. of time/Duration
a) 2
b) 3
5 times for 1 minute
3 times for 3 minutes
a) 5
b) 2
a) 5
b) 10
or
3 times
2 times
2 times
2 times
5 minutes
1.5
25-35 minutes iii)
6
Washing
CH3CN
3
10 times
7
Oxidation
0.1 M 12 in THF/Pyridine/
H 20 (80:40:2. v/v)
2
2 times for 1 minute
8
Washing
a) Methanol
b) THF
a) 2
b) 5
2 times
2 times
Capping
Ac20/DMAP/Lutidine/THF
2.5
2 times for 2 minutes
9
10
As step 1
i) When CC 3COOH is used.
ii) When ZnBr2 is used.
iii) 25 minutes for pyrimidine and 35 minutes for purine nucleotides.
densation yield was checked after 30 to 40 minutes in each step
by determination of the dimethoxytrityl cation concentration. It
has been possible29) to achieve a shorter condensation time (less
than 5 minutes) by using 5-(4-nitrophenyl)-tetrazole as condensing agent which has very recently been used in condensations
with methyl-5'-0-N-protected deoxynucleoside-3'-0-N-morpholinophosphoramidites25) .
In the case of purine derivatives, coupling was found to require either slightly longer time or higher concentrations than
pyrimidine derivatives in order to afford more than 95% coupling
yield. The steps involved in the synthesis of oligomers of de
sired sequences are summarized in Table (II). The average coupling yield per elongation step was found to be greater than 92%.
The deoxynucleoside N,N-dimethylaminophosphoramidites were
used for the synthesis of d(GGGATCCC) resulting in 63% overall
yield for the synthesized product. N-morpholino derivatives
used for the synthesis of d(GGGATATCCC) gave 55% of the product.
N,N-diisopropylamino derivatives had been used for the synthesis
of d(TCAGTTGCAGTAG) and two mixed oligonucleotides
4545
Nucleic Acids Research
d(GGGTGGATATACAC)
and d(GGATGAATATAAAC) in respective yield of
45%, 40%, 38%.
Although all three types of phosphoramidites have been successfully used for the synthesis of oligodeoxynucleotides, for
routine use, however, we prefer the N,N-diisopropylamino 3-cyanoethyl deoxynucleoside phosphoramidites as active monomers because
of the ease with which they can be obtained in pure and very
stable form. In contrast, the monochloro-r-cyanoethoxy-N-morpholino phosphine could not be obtained in 100% purity and the N,Ndimethylamino 5-cyanoethyl deoxynucleoside phosphoramidites required stringent anhydrous and inert condition to ensure good
condensation and storage.
Deprotection of the synthetic oligodeoxynucleotides
To isolate the desired product after synthesis, various protecting groups must be removed and then cleaved from the polymer
support without causing any side reaction.
The deprotection, in our synthetic strategy with the f-cyanoethyl group as phosphate protection, may be achieved: either by
stepwise deprotections, where t-BuNH2/Pyridine (or Et3N/Pyridine)
mixture is used for the removal of B-cyanoethyl group, followed
by N-deacylation and cleavage from the polymer with conc. aq.
NH3 or one step deprotections by conc. aq. NH3 alone.
To explore the reliability of the deprotections outlined
above, three model experiments were performed using the dimer
DMTr-Ctl T-0-P
tBuNH2/Pyridine27) for 15 minutes followed
by incubation with conc. NH3 for 16 hours at 500 C,
ii) treatment with 20% Et3N/Pyridine26) for 2 hours followed by
conc. aq. NH3 incubation for 16 hours at 500 C, and
iii) incubation with conc. aq. NH3 for 16 hours at 500 C alone.
The reverse phase chromatographic profile was identical in
each case suggesting that even when only conc. aq. NH3 is used,
the R-cyanoethyl group is selectively removed by 3-elimination,
thus causing no detectable internucleotidic cleavage. In order
to acertain that there was no 3-3 dimer formation39), an aliquot
of the dimer only treated with conc. aq. NH3 was isolated from
HPLC and subjected to snake venom phosphodiesterase (SVDP) dii) Treatment with 10%
4546
Nucleic Acids Research
E
LL
kn
0
5
10
15
20
t(min)
Figure 2:
25
30
35
0
5
10
15
20
25
30
35
t(min)
a
b
HPLC chromatograms of a) 13-mer, d(TCAGTTGCAGTAG), and
G
mixd
primr
l4i~
A
mixed
b) b)
primer
141, d(GGATGTATATAAAC).
gestion at 370 C for 2 hours, 4 hours and 26 hours. The digested
samples were chromatographed under the same conditions (RP 18)
as for DMTr-CT. Only two peaks were observed, one having the
same retention time as dpT, the other of DMTr-(dC). This demonstrates that DMTr-dimer was completely digested by SVDP. It is
therefore evident, that incubation with conc. aq. NH3 alone completely deprotects the oligonucleotide under the conditions used
without any side reactions.
The oligomer synthesized on the CPG beads possessing dimethoxytrityl group at 5'-end was thus deprotected and removed from
the polymer by treatment with conc. aq. ammonia at room
temperature for 30 minutes and finally incubated at 50° C overnight under sealed conditions. After cooling to room temperature,
CPG beadswere centrifuged and the supernatant liquid was evaporated to dryness. Removal of 5-cyanoethyl groups from the internucleotidic phosphate triesters, de-N-acylation and cleavage
from the polymeric support were performed in one step taking less
than 16 hours.
Purification and analysis of oligodeoxynucleotides
Purification has been performed by reversed phase (RP 18)
HPLC30). The crude oligonucleotide obtained after evaporation was
4547
Nucleic Acids Research
1
2
3
4
5
6
7 8 9
-~
4-
*F
pdT
~~~~~~~C
F ..
_1
*
d pT)_
0
$T
~~~Al
1
*T
**
2
Figure 3
Figure 4
Figure 3:
Electrophoresis of the oligomers: 8-mer, lane 1;
10-mer, lane 3; 13-mer, lane 4; 142-mer, lane 6;
141-mer, lane 7 and 8 in relation to homo-oligo-dT
length standard (lane 2, 5 and 9 respectively),
on 20% polyacrylamide gel after HPLC separation, detritylation and phosphorylation with ( r32-P)ATP and
T polynucleotide kinase. Lane 7 contains the main
m terial from 141 and lane 8 contains the r.h.s. of
the desired peak on Figure 2b.
Figure 4:
Sequence analysis of 13-mer by "mobility shift" method.
+ : xylene cyanol marker. 1. dimension: electrophoresis, 2. dimension: homochromatography.
taken up in approximately 1.0 ml of buffer (0.1 M TEAA, pH 7.0)
followed by filtration using millipore filter (1.2 u pore size).
The sample were injected onto the reversed phase (RP 18) column
and eluted with a gradient according to Materials and Methods.
Typical separations are shown for 13-mer and 14-mer in Figure 2.
The HPLC profiles shcwn in Figure 2 gave single sharp peaks for
the DMTr oligomeric materials well separated from the nontritylated nucleotidic and non-nucleotidic material. The overall yields
after RP 18-HPLC purification are in the range of 23% to 36%
After collection of the desired oligomer, the elution buffer was
evaporated and the sample detritylated with 80% acetic acid. The
pure sample was lyophilised from bidistilled water and a small
4548
Nucleic Acids Research
amount phosphorylated with (t-32P)ATP. Its purity and electrophoretic mobility31) were determined by 20% polyacrylamide gel
electrophoresis (Figure 3). The presence of trace amounts of
longer chain oligonucleotidic material might be due to side reactions, whereby some DMTr group is removed (acidic effect of
tetrazole) giving rise to additional condensation reactions
during a single condensation step. This problem might arise
during repetition or longer condensation time, which we have recently overcome with 3-4 minutes condensation time using p-nitrophenyltetrazole. The sequence analysis of all the oligonucleotides was performed using either "mobility shift"32) (Figure 4)
or Maxam-Gilbert33) method (Figure 5).
"Maxam-Gilbert" sequence analysis of "mixed oligomers"
The sequence analysis of mixed synthetic oligodeoxynucleotides
is a formidable task, especially if one wants to receive information on the relative amounts of the different oligomers within a
mixed probe. In special cases one was able to separate some
oligomers out of the mixture by complicated HPLC procedures and
to sequence them individually by the "mobility shift"
method1 '34). We investigated the possibility of using the
"Maxam-Gilbert" method directly for sequencing the oligonucleotide mixture. We were particularly interested in examining whether all required sequences were present in the mixture and if
the intensity of the bands at the mixed positions could be used
to estimate the ratio of the oligonucleotides within the mixture.
After HPLC purification of the 5'-dimethoxytritylated mixed
oligomers on RP 18 (e.g. Figure 2b), detritylation and phosphorylation, the mixture was isolated from 20% polyacrylamide gel
electrophoresis (Figure 3) and directly subjected to the MaxamGilbert sequencing protocol.
It could be seen from Gthe autoradiogram for the analysis of
the 14-mer d(GGATGAATATAAAC), that the correct sequence had been
established (Figure SA). The mixed positions 6 (A,T) and 12
(G,A,T) reading from the 5'-end are also clearly visible and it
is evident that the intensity of these bands is relatively weaker
than that of the "unmixed" positions due to the distribution of
the radioactivity over more than one oligonucleotide.
In comparing the intensities of the bands for the mixed posi4549
Nucleic Acids Research
WD
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4550
4-
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-
Nucleic Acids Research
tion 12, one can see that G,A and T nucleotide intensities are
quite similar. At position 6 the A nucleotide is slightly stronger than T, but in general it may be stated that all expected
oligonucleotides according to the sequence are clearly present.
In the autoradiogram for the analysis of the 142-mer
d(GGATGAATATAGAC), which contains four mixed positions resulting
in a mixture of 16 oligonucleotides, it may be observed that
bands derived from oligonucleotide of the same length but whose
base composition is different have been separated (Figure 5B).
This phenomenon arises from the fact that oligonucleotides of
the same chain length but differing in base compositions exhibit
different mobilites. This is due to a base-specific mobility
under these electrophoretic conditions in the order
the effect being particularly strong in sequences with high G
content. From this consideration one expects separation into two
bands at position 4, 5 and 6 from 5 '-end (mixture of 2 oligonucl eotides) and three bands at position 7, 8 and 9. The four
hexanucleotides (pGGATGAp, pGGGTGAp, pGGGTGGp) representing
position 7 differ in their G/A content, giving rise to the three
ratios 3G/2A, 4G/1A, and 5G. As two of the four hexanucleotides
contai;n the same G/A ratio only three bands can be expected as
visualised. The same difference in G/A content results in three
bands for positions 8 and 9. Apparently the bands corresponding
to longer oligonucleotides are not so well separated due to the
greater number of different oligonucleotides present in the
mixture and the shorter distance which they have travelled on
the gel. Similar to 141-mer, however, it may be pointed out that
again all nucleotides representing the desired sequences are
C>A>T>G31)
visible.
CONCLUSIONS
As demonstrated, the R-cyanoethyl group has many advantages
when used in the phosphite triester method with CPG as polymer
support:
Dichloro-f-cyanoethoxyphosphine5)
can easily be prepared in
The repeated distiltimes.
for
and
stable
is
longer
pure form
lation very often necessary for the complete removal of phosphorus trichloride during preparation of dichloro-methoxyphos-
1)
4551
Nucleic Acids Research
phine is unnecessary in this case.
2) The B-cyanoethylmonochlorophosphoramidites of N,N-dimethylamine, N,N-diisopropylamine and N-morpholine can easily be obtained.
3) The 5'-0-,N-protected deoxynucleoside-3'-0-1-cyanoethyl-N,Ndialkylamino-/N-morpholinophosphoramidites can be prepared as
pure white precipitates stable for many months. Their purity
(and thus their reactivity) can be routinely checked simply by
thin layer chromatography. Especially useful are the N,N-diisopropylaminophosphoramidites.
4) The reactivity of the deoxynucleoside phosphoramidites is
comparable to that of the corresponding methoxy derivatives. With
4'-nitrophenyltetrazole 25), however, coupling times are in the
range of 2 to 5 minutes.
5) The main advantage of these new active deoxynucleoside derivatives is their influence on the simplification and time-reduction of the final work-up procedure of synthesized oligodeoxynucleotides. The B-cyanoethyl group can easily be removed
by f-elimination using mild alkaline conditions24'26'27). Therefore, the deprotection of the oligodeoxynucleotide chain can be
performed in one step at the heterocyclic bases, the phosphotriester moiety and the linkage to the polymer support by treatment with e.g. concentrated aqueous ammonia. After evaporation,
the residue can directly be taken up in the starting buffer for
reversed phase (RP 18 ) HPLC, filtrated and purified by HPLC.
After detritylation with 80% acetic acid and subsequent lyophilization the oligonucleotide is ready for phosphorylation with T4
polynucleotide kinase and ATP. The total time required for this
work-up sequence is less than 24 hours. The loss of valuable
oligonucleotidic material is significantly reduced as there are
no transfers of material and no extraction or thin layer or paper
chromatographic steps necessary prior to HPLC purification.
During the whole deprotection and purification step only volatile
reagents are used.
6) Mixed sequences can also efficiently be prepared by these new
reactive intermediates.
7) The Maxam-Gilbert sequencing method can successfully be used
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to simply characterize synthetic mixed oligodeoxynucleotide fragments.
We believe that the introduction of B-cyanoethyl deoxynucleotide phosphoramidites is a significant improvement of the phosphite triester approach.
MATERIALS AND METHODS
The following chemicals were purchased from commercial sources:
deoxynucleosides from Pharma Waldhof (Mannheim), CPG from Serva
(Heidelberg), ZnBr2 from Riedel de Haen (Seelze), 4-N,N-dimethylamino pyridine, aminopropyltriethoxysilane, tetrazole, N,N-diisopropylamine, N,N,N-diisopropylethylamine from EGA (Steinheim),
N-trimethylsilyl-N,N-dimethylamine from Fluka (Neu-Ulm). Tetrazole was purified by sublimation, 2.6-lutidine was purified acEther, THF were distilled from
cording to the literature35)
benzophenone/sodium under nitrogen. Acetronitrile was first distilled over P 205 and then CaH2 under inert atmosphere. Dimethoxytritylchloride, N-trimethylsilylmorpholine36) and dimethoxytritylated amino protected deoxynucleosides37) were prepared following standard published methods. The f-cyanoethylphosphordichloridite was prepared by modifying the published method5).
Thin layer silica gel plates were developed in ethylacetate.
1H-NMR spectra were recorded either with T-60 (Varian) or 270
MHz (Bruker). 31P-NMR were measured with 80 mHz (Bruker). Visible and UV-spectroscopic measurements were performed with Beckman model 35. HPLC-purification was carried out with Altex
RP Ultrasphere ODS column (4.6 x 250) on a Beckman model 344 or
L.D.C. dialog dual pump instrument.
f-Cyanoethylphosphorodichloridite: A three-necked flask (1.0 1)
fitted with addition funnel, mechanical stirrer and argon delivery system was charged with phosphorus trichloride (lO mol),
dry ether (200 ml) and dry pyridine (1.0 mol) (2 equivalents of
pyridine had been used in the literature 5)). The mixture was
cooled to -78° C with a dry ice/acetone bath under argon and
freshly distilled B-.cyanoethanol (1.0 mol) dissolved in ether
(100 ml) was added dropwise over 1 to 1 1/2 hours. After addition
of the alcohol the mixture was stirred at room temperature for 3
hours and kept at 50 C overnight. Pyridinium hydrochloride was
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Nucleic Acids Research
removed by filtration, the filtrate was concentrated to a small
volume and the residue distilled under reduced pressure to give
B-cyanoethylphosphorodichloridite (95 g) in 55% yield.
General method for the preparation of 0-cyanoethyl monochlorophosphoramidites: A two-necked flask fitted with addition funnel,
magnetic stirrer and argon delivery systems was charged with Rcyanoethyldichlorophosphite (17.2 g, 100.0 mmol) and dry ether
(60 ml). To this a solution of N-trimethylsilyl derivatives of
N,N-dimethylamine or N-morpholine (1 equ., 100 mmol) or N-N-diisopropyl amine (2 equ., 200 mmol) in ether (30 ml) was added at
-20° C over 1.5 hours with constant stirring. After this addition the mixture was stirred for an additional 20 hours at room
temperature under argon. In the case of the N,N-diisopropylamine
reaction, its hydrochloride was filtered prior to concentration
of solvent under reduced pressure at room temperature. The concentrates from N,N-dimethylamine and N,N-diisopropylamine reactions were distilled in vacuo to give f-cyanoethyl monochloro
N,N-dimethylaminophosphoramidite (15.3 g) at 90-920 C/0.6 mm
and 1-cyanoethylmonochloro-N,N-diisopropylamino phosphoramidite
(16.5 g) at 103-1040 C/0.08 mm, respectively. N-morpholino derivative was used without distillation as attempted distillation
decomposed the product.
Synthesis of B-cyanoethyl-5'-0,N-protected deoxynucleoside-3'0-N,N-dialkylamino/-N-morpholinophosphoramidites: The 5'-0,Nprotected deoxynucleoside (3.0 mmol) was dried by coevaporation
with pyridine, toluene and THF. The dried residue was dissolved
in dry THF (15 ml) in presence of N,N,N-diisopropylethylamine
(12.0 mmol) and e.g. B-cyanoethylmonochloro N,N-dialkylaminophosphoramidite (6.0 mmol) was added dropwise through a syringe
with constant stirring under argon at room temperature over 2
minutes. After 35 minutes of stirring, the hydrochloride, which
precipitated out during reaction was filtered and the filtrate
was concentrated to remove THF and excess amine. The residue
was dissolved in argon saturated ethylacetate (150 ml), washed
with ice-cold 10% Na2Co3 solution (50 ml twice) and dried over
Na2So4. Concentration of the dried organic extract resulted in
a foam, which was dissoved in toluene (15-20 ml) (for pyrimidines)
or ethylacetate (20 ml) (for purines), then precipitated into
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Nucleic Acids Research
hexane (250 ml) at -78° C. The precipitates were removed by
filtration and dried in a desiccator over CaCl2 under reduced
pressure overnight.
General coupling procedure on the polymer (CPG): Usually 100
mg of the controlled pore glass beads (CPG) containing 5'-0dimethoxytritylated aminoprotected deoxynucleoside was detritylated with either 3% CC13COOH in 1% methanol-nitromethane or
saturated ZnBr2 in 1% water-nitromethane and after washing according to Table (II) was dried in vacuo for 5 minutes. The
solid active nucleoside (150 mg, 25 equ.) and tetrazole (50-60
mg) were placed into the column type reactor containing the detritylated polymer. The reactor was capped with a serum cap,
flushed with argon and acetonitrile (1.5 ml) was added with a
syringe. The suspension of CPG beads was shaken gently for
25-35 minutes. The excess of reagents was removed by flushing
with argon and washing with suitable solvents (Table II). The
condensation yield was determined at this stage by taking out a
small sample of CPG beads and by measuring the concentration of
DMT-cation on the polymer. When the desired high coupling yield
was obtained, oxidation was performed with 2 ml, 0.1 M I2 solution in a mixture of THF, pyridine, water (80:40:2, v/v) for
2 minutes. The oxidising reagent was removed with argon pressur
and washing with methanol followed by dichloromethane. Then the
polymer was suspended in a mixture of acetic anhydride (0.5 g),
2.6-lutidine (0.55 g) and 4-N,N-dimethylamino pyridine (0.3 g)
in 5 ml THF for 5 minutes to block the unreacted 5'-OH group of
nucleoside or oligonucleotide linked to CPG. The repetition of
the above cycle with appropriate active nucleotide gave the desired sequence of oligonucleotide.
Removal of the oligomer from the CPG-beads and deprotection of
all protecting groups but DMT at 5'-end: After the final elongation step of the desired sequence, the CPG beads, which were
thoroughly washed and dried, were transfered to a flask (25 ml)
and treated with concentrated aqueous NH3 (5 ml) for 30 minutes
at room temperature and finally at 500 C overnight. After
cooling, the supernatant liquid was removed and the polymer was
washed with bidistilled water (3 x 1 ml). The combined supernatant and washings were concentrated to a small volume and the
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insoluble material resulting from the B-elimination and polymerization of CH2=CH-CN was removed by filtering through millipore
filter. The filtrate was evaporated to near dryness and redissolved in a small amount of 0.1 M TEAA buffer, pH 7.0.
Purification of the oligomer by RP-HPLC: The chromatographic
purification of the oligonucleotide was carried out on a Beckman dual pump model 344 HPLC apparatus in conjunction with an
Altex RP Ultrasphere ODS column and an UV detector (filter
254 nm). The mobile phases used were 0.1 M TEAA buffer pH 7.0
in pump A and CH3CN in pump B. A step linear gradient from
10-25% pump B in 5 minutes followed by 25-29% pump B in 30
minutes was performed. Injection volumes were typically 20 il
of solution. Polyacrylamide gel electrophoresis and sequencing
were performed according to published procedures
Acknowledgement
This work has been financially supported by the Deutsche
Forschungsgemeinschaft and the Bundesminister fUr Forschung und
Technologie.
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4557