i
volume 7 Number 7 1979
».
Nucleic A c i d s Research
The use of barium salts of protected deoxyribonucJeoside-3' p-chlorophenyl phosphates for
construction of oligonucleotides by the phosphotriester method: high-yield synthesis of dinucleotide
blocks
G.R.Gough*, KJ.Colliert, H.L.Weith+ and P.T.Gilham*
Departments of Biological Sciences* and Biochemistry^, Purdue University, West Lafayette, IN
47907, USA
Received 24 September 1979
ABSTRACT
A new experimental approach t o t h e s y n t h e s i s of p o l y d e o x y r i b o n u c l e o t i d e s
v i a the p h o s p h o t r i e s t e r method i n v o l v e s c o n s t r u c t i o n of o l i g o n u c l e o t i d e
blocks by d i r e c t use of t h e e a s i l y prepared barium s a l t s of 0_ 5> ,N-protected
d e o x y r i b o n u c l e o s i d e - 3 ' £ - c h l o r o p h e n y l phosphates aa the
key monomers in
condensation r e a c t i o n s .
The procedure has been demonstrated by t h e
rapid s y n t h e s i s in high y i e l d and p u r i t y of a l l s i x t e e n f u l l y p r o t e c t e d
d i n u c l e o t i d e s [(MeO) 2 Tr]dN'2dN'2(CNEt), (where dN' - dT, dbzC, dbzA, or dibG;
- - d i e s t e r i f l e d £-chlorophenyl p h o s p h a t e ) . This s e t of molecules c o n s t i t u t e s
a " s y l l a b a r y " for t h e p r e p a r a t i o n of defined sequence o l i g o n u c l e o t i d e s .
INTRODUCTION
We r e c e n t l y r e p o r t e d 1 s i m p l i f i e d methods for p r e p a r a t i o n by t h e modified
p h o s p h o t r i e s t e r a p p r o a c h 2 ' 3 of o l i g o n u c l e o t i d e blocks t h a t can be used in t h e
s y n t h e s i s of defined sequence p o l y n u c l e o t i d e s .
new s t r a t e g y a r e b a s e - p r o t e c t e d
The key i n t e r m e d i a t e s in t h e
5'-0-dimethoxytrltyldeoxyribonucleoside-3'
£-chlorophenyl phosphates which a r e i s o l a t e d by a barium s a l t
procedure.
precipitation
These nucleoside p h o s p h o d i e s t e r s , a f t e r conversion to t h e i r
ethylammonium s a l t s , can be employed for t h e phosphorylation of
tetra-
suitable
n u c l e o t i d e d e r i v a t i v e s possessing 5'-hydroxyl groups, under t h e a c t i o n of a
new c l a s s of condensing a g e n t s , t h e a r y l s u l f o n y l n i t r o i m i d a z o l e a .
With t h e s e
techniques we prepared moderately l a r g e q u a n t i t i e s of two d i n u c l e o t i d e blocks
which, In conjunction with a p p r o p r i a t e monomers, provided t h e b a s i c m a t e r i a l s
for s y n t h e s i s of an i c o s a n u c l e o t i d e analogous t o t h e sequence a t t h e ends of
Rous sarcoma v i r u s 35S RNA.
We have now determined t h a t t h e barium s a l t s of
t h e p r o t e c t e d n u c l e o s i d e p h o s p h o d i e s t e r s can be used d i r e c t l y in syntheses of
t h i s t y p e , and have demonstrated t h e e f f i c a c y of t h e new procedure by r a p i d l y
p r e p a r i n g In high y i e l d a l l 16 f u l l y p r o t e c t e d d i n u c l e o t i d e blocks of t h e form
[(MeO) 2 Tr]dN'2dN'-(CKEt).' 1
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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Nucleic Acids Research
SYNTHESIS OF FULLY PROTECTED DINUCLEOTIDES FROM MONONUCLEOTIDE BARIUM SALTS
The pure barium s a l t s of S'-Oj-dlmethoxytrityl protected deoxyribonucleosi d e - 3 1 j)-chlorophenyl phosphates ( I , F i g . l ) were prepared In high y i e l d by our
p r e v i o u s l y reported method,
s l i g h t l y modified t o decrease the time required
for t h e phosphorylatlon r e a c t i o n .
These s t a b l e , e a s i l y handled s o l i d s were
the basis for synthesis of the 16 possible fully protected dinucleotides,
which were prepared in groups of four from a common 3'-terminal nucleotide.
Mononucleotide barium salt (I) was converted in 16 hr to the fully protected
trlester II by reaction with 2-cyanoethanol in the presence of the condensing
agent l-(p_-toluenesulfonyl)-4-nitroimidazole.*
I I , without isolation, was
detritylated using excess benzenesulfonic acid and the resulting 5'-hydroxyl
component III was divided into four equal parts.
These were separately
phosphorylated with different nucleotide barium salts ( I ) , the four reactions
running simultaneously.
The resulting dimere IV were then isolated by s i l i c a
gel column chromatography.
Three more runs, using the other mononucleotide
barium salts as 3' termini, generated the remaining 12 dinucleotides.
Minor
complications arose In syntheses of dinucleotides containing deoxyguanosine
residues.
One problem concerned the deoxyguanosine nucleotide III which
(UlO)ITl
(IM)tTr0
w
HOCHJCHJCM
(IMl.TrO
B\l*
TJX I • M>-TLl
Figure 1.
1956
Hlljl>-4-lllluM*ilu<l
Synthesis of fully protected dinucleotides from protected mononucleotide barium salts.
Nucleic Acids Research
proved to be moderately soluble in water, a property that resulted In some
l o s s of material when the d e t r l t y l a t l o n mixture was subjected to aqueous
e x t r a c t i o n as part of the regular procedure t o remove benzenesulfonic a c i d .
Consequently, for d i n u c l e o t i d e s with deoxyguanosine at the 3 ' terminus, the
general method was modified to include intermediate i s o l a t i o n of the monomer
[(MeO) 2 Tr]dibG i (CNEt).
Less benzenesulfonic acid was then required for d e -
t r i t y l a t i o n , and aqueous extraction was unnecessary s i n c e small q u a n t i t i e s of
the a c i d , as i t s pyridinium s a l t , can be carried through into the subsequent
condensation r e a c t i o n s without apparent adverse e f f e c t s .
A further d i f f i c u l t y was encountered in early t r i a l s of reactions i n volving deoxyguanosine d e r i v a t i v e s , regardless of the method of s y n t h e s i s .
During the second condensation ( I I I + I -*- IV), s i g n i f i c a n t amounts of a s i d e
product were formed.
This material was f i r s t observed in syntheses leading
to the d(NG) s e r i e s of d i n u c l e o t i d e s where i t appeared on thin layer chromatography as a band with a s l i g h t l y higher mobility than the f u l l y protected
dimer.
I t was l a t e r a l s o detected in l e s s e r amounts in the d(GN) s e r i e s .
However, i t proved to be l a b i l e and disappeared when the post-condensation
reaction mixtures were treated for 40 min with aqueous p y r i d i n e .
In order to
determine i t s nature, we i s o l a t e d by s i l i c a g e l column chromatography a small
quantity of the s i d e product generated in a reaction for the s y n t h e s i s of
f u l l y protected d(AG).
On treatment with aqueous pyridine i t broke down into
[(MeO)2Tr]dbzA*dibG-(CNEt) together with a product that contained a dimetho x y t r i t y l group and remained at the o r i g i n on TLC p l a t e s .
Moreover, when the
material was p a r t i a l l y deprotected with ammonia and analyzed as discussed
in the following s e c t i o n , i t was converted to [(MeO)2Tr]dA-dG-(ClPh) and
a substance that had the same s p e c t r a l properties and chromatographic
r e t e n t i o n volume as [(MeO)2Tr]dA-(ClPh).
On the b a s i s of these r e s u l t s we
suggest that the s i d e product a r i s e s from attack of a c t i v a t e d monomer
[(HeO) 2 Tr]dbiA-(ClPh) on the guanine moiety of
[(MeO) 2 Tr]dbzA2dibG i (CNEt),
leading to the formation of a "branched" t r i n u c l e o t i d e . 5 Since related
contaminants can be found in a l l d i n u c l e o t i d e reactions involving deoxyguanosine r e s i d u e s , we have incorporated a post-condensation treatment with
aqueous pyridine i n t o t h e i r work-up as a routine procedure.
Using the above methods the s i x t e e n d i n u c l e o t i d e s were prepared in high
y i e l d as shown in Table 1.
DEPRPTECTION AND ANALYSIS OF THE DINUCLEOTIDES
The purity of the f u l l y blocked d i n u c l e o t i d e s was examined in two ways.
F i r s t , t h i n - l a y e r chromatography on s i l i c a g e l p l a t e s showed each dimer
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Nucleic Acids Research
Table 1.
Yields and Chromatographic Retention Volumes of Dinucleotide Blocks
Nucleotide
Sequence
Fully Protected
Dinucleotide
Yield*
Z
CC
[(MeO)2Tr]dbzC£dbrC-(CNEt)
[(MeO)2Tr]dbzA2dbzC2(CNEt)
93
83
AC
91
80
TC
[(MeO)2Tr]dT*dbzC-(CNEt)
92
82
GC
[(MeO)2Tr]dibG2dbzC£(CNEt)
88
111
CA
[(MeO)2Tr]dbzC*dbzA-(CNEt)
[(MeO)2Tr]dbzA2dbzAS(CNEt)
91
86
92
84
89
86
GA
[(MeO)2Tr]dT*dbzA*(CNEt)
[(MeO)2Tr]dibG2dbzAS(CNEt)
85
109
CT
[(MeO)2Tr]dbzC*dT2(CNEt)
88
88
AT
[(MeO)2Tr]dbzA£dT*(CNEt)
[(MeO)2Tr]dT*dT*(CNEt)
91
87
90
92
[(MeO)2Tr]dibG2dT2(CNEt)
[(MeO)2Tr]dbzC2dibG-(CNEt)
83
111
81
123
83
108
TG
[(MeO)2Tr]dbzA^dibG-(CNEt)
[(MeO)2Tr]dTidibG-(CNEt)
82
116
GG
[(MeO)2Tr]dibG^dlbGi(CNEt)
75
155
AA
TA
TT
GT
CG
AG
a
V (ml) for
[(MeO)2Tr]dN-dN-(ClPh)
Yield of the fully protected dinucleotide is based on the nucleoside
phosphodiester barium s a l t used for the 3 ' end, except in the NG s e r i e s ,
where yield i s based on [(MeO)2Tr]dibG*(CNEt).
Anion-exchange r e t e n t i o n volume of p a r t i a l l y deprotected dinucleotide.
running as a single band or, in some cases, as two or three closely adjacent
bands representing resolution of some of the four possible diastereomers.
A more stringent test of purity was carried out by partially deprotecting
each dinucleotide with ammonia.
This treatment removes the 3'-terminal
cyanoethyl group, the £-chlorophenyl group from the internucleotide phosphotriester linkage, and the blocking groups from the bases.
The resulting
compounds [(MeO)2Tr]dN-dN-(ClPh) were analyzed by high-resolution anionexchange column chromatography.
Each dinucleotide ran as a single peak
accompanied by only trace amounts of material in the mononucleotide elution
range.
This range was established by chromatographing authentic samples of
the various mononucleotides dN-(ClPh) and [(MeO)2Tr]dN-(ClPh), since these
would be the deprotected equivalents of contaminants most likely to be found
in the dinucleotide products. The purities of the dimers, based on optical
density measurements at 254 nm, were estimated to lie between 96 and 99Z.
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Nucleic Acids Research
^,
PREPARATION OF THE BARIUM SALT OF A PROTECTED DINUCLEOTIDE
The f u l l y protected d i n u c l e o t i d e s must be decyanoethylated in every case
before addition to the 5 1 end of a growing o l i g o n u c l e o t i d e .
An obvious e x -
tension of the present work, t h e r e f o r e , r e s i d e s In using the d i n u c l e o t i d e s
as t h e i r preformed 3'-£-chlorophenyl phosphate barium s a l t s for such chain
elongation.
As a preliminary t e s t of the f e a s i b i l i t y of preparing these
compounds, we decyanoethylated [(MeO)2Tr]dT£dT-(CNEt) using triethylamlne In
pyridine and precipitated the r e s u l t i n g [ (MeO) 2Tr]dT2dT-(ClPh) as i t s barium
salt.
The i n t e g r i t y and purity (> 99Z) of t h i s material were demonstrated by
p a r t i a l deprotection followed by ion-exchange chromatographic a n a l y s i s .
The
same compound was a l s o conveniently synthesized by phosphorylating the dimer
*•
[(MeO)2Tr]dT2dT and p r e c i p i t a t i n g the product in a manner analogous t o that
used for preparation of the mononucleotide barium s a l t s .
Agarwal and R l f t i n a 6
have recently reported an extremely rapid procedure for synthesizing various
dinucleoside monophosphates of the type [(MeO)2Tr]dN'£dN', which in l i g h t of
the above can be readily adapted to our own s t r a t e g y .
i
CONCLUSION
The r e a d i l y prepared barium s a l t s of 0_ 5 ',N-protected d e o x y r i b o n u c l e o s l d e 3 ' £ - c h l o r o p h e n y l phosphates and t h e i r c a p a c i t y for d i r e c t u s e i n condensation
reactions provide a f a c i l e route to the sixteen fully protected dimer8
[(MeO)2Tr]dN'2dN'-(CNEt) .
.
These dinucleotides, together with the four mono-
nucleotide barium s a l t s and one 3'-terminal blocking group (such as the removable rlbonucleoslde bzC(OBr>21), c o n s t i t u t e a complete and easily acquired set
of synthetic units for rapid construction of oligodeoxyribonucleotidea of any
desired sequence.
EXPERIMENTAL
M a t e r i a l s and Methods
5 ' - 0 - D i m e t h o x y t r i t y l t h y m i d i n e and S ' - O - d i m e t h o x y t r i t y l - N ^ - i s o b u t y r y l deoxyguanosine were s y n t h e s i z e d by p u b l i s h e d p r o c e d u r e s . 7 * 8
The dimethoxy-
t r i t y l d e r i v a t i v e s o f N ^ - b e n z o y l d e o x y c y t i d i n e and N ^ b e n z o y l d e o x y a d e n o s i n e
were purchased from C o l l a b o r a t i v e Research, I n c .
2-Cyanoethanol
(Aldrich
Chemical Co.) was d i s t i l l e d under reduced p r e s s u r e and s t o r e d over molecular
s i e v e s (Type 4A) .
The condensing agent
l-(p_-toluenesulfonyl)-4-nitroimida-
z o l e (TSNI) was prepared a s p r e v i o u s l y d e s c r i b e d . 1
Eastman Chromagram s h e e t s ( s i l i c a g e l w i t h f l u o r e s c e n t I n d i c a t o r ) were
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Nucleic Acids Research
used for thin-layer chromatography. The completeness of condensation reactions was monitored by TLC using 21 or 5Z MeOH In CHC13 (v/v) as developing solvents. Dlmethoxytrityl-contalnlng compounds were detected by exposing
the plates to the vapor from concentrated HC1. S i l i c a gel chromatography of
fully protected dinucleotldes was carried out at 3° on columns (2.4 X 30 cm)
of SillcAR CC-7 Special (Mallinckrodt) packed In chloroform, and partially
deprotected dinucleotldes were chromatographed on the anion-exchange material
AS Pellionex SAX, obtained from Whatman, Inc.
Barium Salts of Protected Deoiyrlbonucleoslde-3' p-Chlorophenyl Phosphates (I)
For 5 rnmol: 1,2,4-Triazole (2.075 g, 30 mmol) was dissolved with
stirring In warm anhydrous dioxane (50 ml). The solution was cooled to 25°
and treated with triethylamine (4.2 ml, 30 mmol) followed by p-chlorophenyl
phosphorodichloridate 9 (2.46 ml, 15 mmol). The thick slurry was stirred for
45 min then filtered to remove Et3NH Cl . The f i l t r a t e was mixed with a
solution of 5 mmol of protected nucleoside (5'-O|-dimethoxytrityl derivative of
dT, dbrC, dbzA, or dibG) In anhydrous 2,6-lutidine (10.5 ml) and the mixture
was allowed to stand at 25° until TLC showed the phosphorylation to be complete (approximately 2 hr). It was then added dropwlse with stirring to
water (250 ml). After standing for 1 hr, the clear solution was poured into
vigorously stirred aqueous barium chloride (10 g of BaCl2.2H20 in 1 l i t e r of
water) at 3°. The resulting suspension was gently warmed to 30° without
allowing the barium s a l t to s e t t l e . Upon formation of a f i l t e r a b l e s o l i d ,
the mixture was again cooled to 3°, stirred for 1 hr, then filtered through
a coarse sinter under gravity flow. The collected precipitate was washed
thoroughly with water and dried in vacuo over P2O5. The barium s a l t s obtained
by this procedure were white, non-hygroscopic powders, chromatographically
homogeneous on TLC in CHCl3-MeOH (80:20), and stable on prolonged storage at
0°. Barium 5'-0-diaethoxytritylthymldine-3' p_-chlorophenyl phosphate: Anal.
Calcd. for C37H35ClN201oPBaa5.H20 : C, 54.14; H, 4.54; N, 3.41; P, 3.77.
Found: C, 53.84; H, 4.67; N, 3.40; P, 3.77. Barium 5'-0-dimethoxytrltyl-N 6 benzoyldeoxyadenoslne-3' p_-chlorophenyl phosphate: Anal. Calcd. for Ci^I^g
ClN5O9PBaa5.4H20: C, 53.49; H, 4.69; N, 7.09; P, 3.14. Found: C, 53.50;
H, 4.63; N, 6.77; P, 2.93. Barium 5'-0-dimethoxytrityl-N lt -benzoyldeoxycytidlne-3' p_-chlorophenyl phosphate: Anal. Calcd. for Ci,3H38ClN30ioPBa(^5.2.5
H20: C, 55.12; H, 4.63; N, 4.49; P, 3.31. Found: C, 55.34; H, 4.92; N,
4.52; P, 3.54. Barium 5'-0-dimethoxytrityl-W 2 -isobutyryldeoxyguanosine-3'
p_-chlorophenyl phosphate: Anal. Calcd. for CitiHitoClN50i0PBa0_5.4H20: C,
50.77; H, 4.98; N, 7.22; P, 3.19. Found: C, 50.54; H, 5.05; N, 7.23; P, 3.55.
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Nucleic Acids Research
General Method for Synthesis of Quartets of Fully Protected Dlnucleotldes (IV)
The barium salt of that protected deoxyrlbonucleoslde-3' £-chlorophenyl
phosphate ( I , 2 mmol) destined to constitute the 3 ' end of a set of four
dlnucleotldes was freed from water by several additions and evaporations of
dry pyrldlne, then dissolved In a total of 5 g of anhydrous pyridine and
treated with 2-cyanoethanol (10 mmol) and TSNI (6 mmol). After 16 hr at 25°,
the mixture, which often set to a g e l , was dissolved In pyridine (25 ml) and
added to ethyl acetate (200 ml). The resulting suspension in ethyl acetate
was extracted with 0.1 N sodium bicarbonate (3 X 120 ml) followed by 101
aqueous NaCl (120 ml). The EtOAc layer was concentrated to an o i l from which
pyrldlne was removed by evaporation with toluene (60 ml) . The residue was
treated at 0° with 60 ml of a 2Z (w/v) solution of benzenesulfonic acid in
chloroform-methanol (7:3, v / v ) . After 25 min, the mixture was neutralized
with 5Z NaHCC>3 an(^ added to ethyl acetate (200 ml) . Following extraction with
51 NaH(X>3 (60 ml) and water (60 ml) , the EtOAc layer was treated with pyridlrie
(30 ml), evaporated to ca 20 ml, and divided into four equal portions, each
representing 0.5 nmol of I I I . To each of the four separate solutions was
added a different protected deoxyribonucleoslde-3' 2 r c h l o r o P b e n y l phosphate
barium s a l t ( I , 0.65 mmol). The solutions were rendered anhydrous by
additions and evaporations of pyridine, then each residue was dissolved In a
total of 2 g of dry pyridine and treated with TSNI (2 mmol). After 16-18 hr
at 25°, the mixtures were diluted with pyridine (6 ml), and, in the case of
dlnucleotides containing deoxyguanosine, the suspensions were further treated
with 3 ml of HjO for 40 min. Each condensation reaction mixture was
separately transferred Into ethyl acetate (75 ml), and washed with 5Z NaHCO3
(30 ml) followed by 10Z NaCl (30 ml). The EtOAc layers were concentrated
to thick o i l s , then dissolved in chloroform (3 ml each) and chromatographed on
s i l i c a gel columns. Elution with 300 ml of CHCI3 followed by 600 ml of CHCI3MeOH (98:2) yielded the fully protected dinucleotidea, which were obtained as
foams after evaporation of appropriate fractions from the columns. Yields
(Table 1) were based on the constant weights of these materials after drying
In vacuo (0.1 mm).
The dinucleotides were stored at <-20°. In the case of the d(GN) s e r i e s ,
slight losses of dimethoxytrityl groups were observed following chromatographic i s o l a t i o n . This breakdown can be prevented by maintaining the
presence of small amounts of pyridine in the samples after their emergence
from the columns and during subsequent storage.
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Modified Procedure for Synthesis of Fully Protected Dlnucleotldea Containing
a 3'-Terr"-I""! dG Residue
5 '-0-Dlmethoiytrltyl-N^ 2 -lsobutyTyldeoxyguano8ine-3' £-chlorophenyl
phosphate barium salt (2 mmol) was converted to I t s fully protected t r i e s t e r
[(MeO)2Tr]dlbG-(CNEt) by treatment with 2-cyanoethanol and TSNI In the usual
manner. Upon completion of the cyanoethylation, the reaction mixture was
dissolved In pyridine (10 ml) and added to ethyl acetate (200 ml) . The
suspension in ethyl acetate was extracted with 0.1 N NaHC03 (3 X 120 ml), then
with 10Z aqueous NaCl (3 X 120 ml). The EtOAc layer was evaporated to a thick
o i l which was dissolved in chloroform (5 ml) and chromatographed on s i l i c a
g e l . Elution of the column with 600 ml of CHC13 followed by 600 ml of CHC13MeOH (98:2) gave pure [(MeO)2Tr]dibG£(CNEt) in 96Z yield.
For conversion to dlnucleotides, this material was detritylated by
treatment with 1.66 equivalents of benrenesulfonic acid, as a 2% solution in
CHCl3-MeOH (7:3), for 25 mln at 0°. The deprotection was terminated by the
addition of pyridine (30 ml). The mixture was then evaporated to ca 20 ml and
divided into four equal portions. These four solutions were each treated with
a different protected nucleoslde phosphodieater barium s a l t (I, 1.3 equiv.)
and, following removal of water by co-evaporation with pyridine, TSNI (2.6
equiv.) was added in the usual way. After 16-18 hr at 25°, the reaction
mixtures were treated with pyrldlne (3 ml) and water (1.5 ml) and allowed to
stand for 40 min. Ethyl acetate extraction and s i l i c a gel chromatography were
then carried out as usual to Isolate the four dinucleotides.
Deprotection and Analysis of the Dinucleotide Blocks
Each fully protected dinucleotide (ca_ 3 mg) was dissolved in pyridine
(2 ml) and treated with 7 M NH,,OH (4 ml). After 3 days at 25°, pyrldlne was
removed by several additions and evaporations of dilute ammonia. Finally, the
deprotected material was stored at -20° In 40Z aqueous ethanol (0.6 ml) containing one drop of concentrated NH,,0H. For analysis, a sample (30-50 yl)
of the solution of dinucleotide was evaporated to dryness in the presence of
triethylamine, redissolved In 40Z ethanol, and chromatographed on a column
(0.4 X 50 cm) of AS Pellionex SAX using a 200 ml gradient of 0-0.2 H NH^Cl
(pH 8) in 40* ethanol at 20 ml/hr. The partially deprotected dinucleotldes
[(MeO)2Tr]dN-dN-(ClPh) appeared as single peaks accompanied by only trace
amounts (< 4Z) of mononucleotlde material, and their retention volumes (V) are
given in Table 1. All possible mononucleotide contaminants, such as dN-(ClPh)
and [(MeO)2Tr]dN-(ClPh), have retention volumes In the range 28 to 58 ml;
representative examples
dC-(ClPh), [(MeO)2Tr]dA-(ClPh), [(MeO)2Tr]dC-(ClPh),
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Nucleic Acids Research
and [(MeO)2Tr]dG-(ClPh) have V values of 28, 42, 44, and 58 ml, respectively.
Preparation of the Barium Salt of the Dinucleotide [(MeO)?Tr]dTadT-(ClPh)
(a) Fully protected dinucleotide [(MeO)2Tr]dT2dT*(CNEt) (60 mg, 0.05 mmol)
was dissolved in dry pyridine (2 ml) and treated with anhydrous triethylamine
(1 ml). After 6 hr at 25°, the mixture was evaporated several times with
pyridine to ca^ 1/3 of i t s original volume. The residual solution was added
dropwise to vigorously stirred aqueous barium chloride (200 mg of BaCl2.2H20
in 20 ml water). The suspension of barium s a l t was centrifuged and the precipitate was washed with water (2 X 20 ml). It was dried in vacuo over P2O5
to give an off-white solid (57 mg, > 90Z y i e l d ) . A sample (3 mg) of this
material was deprotected as described above. Ion-exchange chromatography
showed the product to be identical with the dinucleotide generated by direct
ammonia deprotection of [(MeO)2Tr]dT*dT-(CNEt), and indicated a purity of >99X.
(b) 5'-O-Dimethoxytritylthymidine-3' p_-chlorophenyl phosphate barium salt
(1 mmol), rendered anhydrous by several evaporations with pyridine, was
treated in 3-4 g of dry pyridine with thymidine (1.3 mmol) and TSNI (2 mmol).
After 20 hr at 25°, the mixture was transferred into chloroform (100 ml)
which was extracted with 0.1 M triethylammonium bicarbonate (3 X 40 ml).
The organic layer was dried (Na2S0i,) , evaporated, and applied to a 60 ml
sintered glass funnel containing 50 ml of s i l i c a gel. The short column was
washed with CHCI3 (125 ml), then with increasing concentrations of MeOH In
CHCI3; the product (86Z yield) was eluted with CHCI3 containing ca_ 2Z MeOH.
This material was converted to i t s 3'-£-chlorophenyl phosphate barium s a l t by
the same technique used to prepare the mononucleotide barium s a l t s . About
0.7 mmol of [(MeO)2Tr]dT£dT-(ClPh) barium salt was obtained and was shown, by
partial deprotection and ion-exchange chromatography, to be identical with the
material prepared as described in ( a ) , and to have a purity of > 97Z. The
absence of 3 ' - 3 ' phosphodiester linkages in this product was demonstrated by
i t s complete degradation to thymidine upon detritylation followed by treatment
with spleen phosphodiesterase and calf Intestinal phosphatase.
ACKNOWLEDGEMENTS
The work w a s s u p p o r t e d by NIH G r a n t s GM 1 9 3 9 5 , GM 1 1 5 1 8 , and CA 2 3 3 3 2 .
KJC was s u p p o r t e d by a n AES G r a d u a t e R e s e a r c h A s s i s t a n t s h i p , and HLW by NIH
R e s e a r c h C a r e e r D e v e l o p m e n t Award CA 0 0 4 4 7 .
T h i s i s paper No. 7793 from t h e
Purdue U n i v e r s i t y A g r i c u l t u r a l Experiment S t a t i o n .
1963
Nucleic Acids Research
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1964
Gough, G. R. , S i n g l e t o n , C. K., Weith, H. L . , and Gilham, P. T. (1979)
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H . , and Narang, S. A. (1973) Can. J . Chem. 5 1 , 3 6 4 9 - 3 6 5 1 .
dN1 r e p r e s e n t s dT, dbzC, dbzA, or dibG, and £ - c h l o r o p h e n y l p h o s p h o t r i e s t e r
l i n k a g e s a r e denoted by the symbol - ( s e e r e f . 1 ) .
We have a l s o d e l i b e r a t e l y s y n t h e s i z e d a r e p r e s e n t a t i v e example of t h i s
unusual s i d e product by condensing [(MeO) 2 Tr]dT-(ClPh) barium s a l t with
t h e fully
protected
d i n u c l e o t i d e [ (MeO) 2 Tr]dT 2 dibG 2 (CNEt), and are
attempting t o d i s c o v e r the nature and p o s i t i o n of t h e proposed l i n k a g e t o
g u a n i n e . This apparent lack of f u l l p r o t e c t i o n of t h e base i n dibG may
w e l l be a f a c t o r c o n t r i b u t i n g to t h e low y i e l d s commonly encountered i n
chemical s y n t h e s e s of o l i g o n u c l e o t i d e s c o n t a i n i n g guanine r e s i d u e s .
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Chem. Soc. 8 5 , 3821-3827.
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Maguire, M. H. and Shaw, G. (1953) J . Chem. Soc. 1479-1482.