volume 12 Number 11 1984
Nucleic Acids Research
Polymer support oligonudeotide synthesis XVffl1-21: use of 0<yanoethyl-N,N-dialkylamino-/Nmorpholino phosphoramidite of deoxynudeosides for the synthesis of DNA fragments simplifying
deprotection and isolation of the final product
N.D.Sinha, J.Biernat, J.McManus and H.KOster
Inslitut fur Organische Chemie und Biochemie, Universitflt 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'-O-N-protected
deoxynucleoside-3'-O-S-cyanoethyl-N,Ndialkylamino-/N-morpholinophosphoramid1tes
were prepared
from
B-cyanoethyl monochiorophosphoramidites o f N,N-dimethylamine,
N , N - d i i s o p r o p y l a m i n e and N-morpholine. These a c t i v e
deoxynucleoside p h o s p h i t e s have s u c c e s s f u l l y been used f o r o l i g o d e o x y n u c l e o t i d e synthessis on c o n t r o l l e d pore glass as polymer
support
and a r e v e r y s u i t a b l e f o r a u t o m a t e d D N A - s y n t h e s i s d u e t o t h e i r
s t a b i l i t y i n s o l u t i o n . The i n t e r m e d i a t e
dichioro-B-cyanoethoxyp h o s p h i n e c a n e a s i l y be p r e p a r e d f r e e f r o m a n y P C I , c o n t a m i n a t i o n . The a c t i v e monomers obtained from B-cyanoethyl
monochloro
N,N-diisopropylaminophosphoramidites
a r e favoured. Cleavage of
t h e o l i g o n u d e o t i d e c h a i n f r o m t h e p o l y m e r s u p p o r t , N - d e a c y l at i on
and d e p r o t e c t i o n o f B - c y a n o e t h y l
group from t h e phosphate t r i e s t e r m o i e t y c a n be p e r f o r m e d i n o n e s t e p w i t h
concentrated
aqueous ammonia. Mixed o l i g o d e o x y n u c l e o t i d e s a r e
characterized
by t h e s e q u e n c i n g m e t h o d o f Maxam a n d G i l b e r t .
INTRODUCTION
Adaptation o f "phosphite triester" approach introduced b y
4 )' t o s o l i d p h a s e s y n t h e s i s o f w e l l - d e f i n e d D N A
Letsinger 3 '
sequences has tremendously reduced the time required for these
syntheses. For the preparation o freactive monomeric intermediates various different types o falkyl/aryl phosphodichi oridites ~ ' ' have been e x p l o r e d . Several d i f f i c u l t i e s g e n e r a l l y
encountered in the preparation and handling o f nucleoside phosp h o r o m o n o c h i or i d i t e s for a u t o m a t e d r o u t i n e s y n t h e s i s o f desired
DNA f r a g m e n t s led t o t h e i n t r o d u c t i o n o f v a r i o u s m o d i f i c a t i o n s
for t h e preparation o f active n u c l e o s i d e p h o s p h i t e interm e d i a t e s " '. T h e c o m m o n r e a c t i v e m o n o m e r i c i n t e r m e d i a t e s
p r e s e n t l y i n u s e f o r s o l i d p h a s e ol 1 g o d e o x y n u c l e o t i d e s y n t h e s i s
f o l l o w i n g p h o s p h i t e t r i e s t e r a p p r o a c h a r e : 5 '-0 , N - p r o t e c t e d d e ox y n u c l e o s i d e - 3 ' - 0 - m e t h y l - N , N - d i m e t h y l a m i n o , -3'-0-methyl-
© IRL Press Limited, Oxford, England.
4539
Nucleic Acids Research
N,N-diisopropylaminophosphoramidites.
Very
amino-/N-morpholino
introduced
These
in
t h epreparation
not
t h esynthesis
of
active
When
protection
tected
In
1 0
e.g.
1 9
"
2 1
) ,
paper,
taken
choice
on solid
phosphite
time
t r i e s t e r
The
phorus
AND
days
approach
f o r d e -
i s i n most
reactions
and
i n solid
pro-
that
these
and r e prior
t o
chromatography
phase
DNA
syn-
approach.
and
o f various
.
amines
blocks
s i g n i f i c a n t l y
simplifies
support
that t h e
protection
a n dw o r k - u p
synthesis
phosphor-
i n polymer
I t i s demonstrated
a s N,N-
a n d their
nucleoside
f o r phosphate
f o rdeprotection
B-cyanosuch
N-morpholine,
reactive
building
group
ammoniurn
o f f u l l y
layer
o f secondary
synthesis
i n
treat-
occasionally
We f e e l
synthesis
g e l thin
a
t r i e t h y l
and
support.
achieved
o f various
protection
includes
and
i n
reduces
o f t h efinal
on polymer
pro-
support.
DISCUSSION
B-cyanoethyl
(V))
reactive
necessary
t h epreparation
o f a n o li g o d e o x y n u c l e o t i d e
RESULTS
with
tl-me
after
t r i e s t e r "
a s nucleotide
necessary
preparation
a t 5 0 °C and
deprotection
b y silica
o f t h eB - c y a n o e t h y l
NH,
several
materials
we report
ol igodeoxynuc 1e o t i d e
work-up
f o r t h esynthesis
N-N-diiso propylamine
serving
present
c h a r a c t e r i -
and
f o r phosphate
Tne
c a nt a k e
monochlorophosphoramidites
amidites
4540
-
t h eimprovements
f o rt h esynthesis
duct
1 1
b y t h e"phosphite
dimethylamine,
the
*
aqueous
o f d i f f e r e n t
p u r i f i c a t i o n
this
ethyl
a t
i s , however,
and
stable
oligonucleotide
t h etime
steps
undermine
thesis
use
linked
o f non-nucleotidic
migth
part
a s t h el a r g e - s c a l e
t h ef i n a l
o li g o d e o x y n u c l e o t i d e s
RP-HPLC
been
t h e e f f i c i e n c y
sequences
p u r i f i c a t i o n
intermediates
approach
i s o l a t i o n
than
improved
time-consuming
synthesis.
IB)
group
' i s used
a t 4 5 °c
time-consuming
moval
most
as well
concentrated
and
longer
have
DNA
t r i e s t e r "
t-butylamine
phosphoramidites
o f oligodeoxynucleotide
o f t h epolymer
'
phosphites
b u t t h ew o r k - u p ,
t h em e t h y l
1 5
o-chiorophenyl-N,N-dimethyl-
considerably
The
deoxynucleoside
thiophenol ate,
cases
have
methods.
f o rautomated
"phosphite
ment
recently
o f t h esequences
enough
a n d- 3 ' - 0 - m e t h y l - N - m o r p h o l i n o
monomeric
developments
t h esynthetic
'
deoxynuc1eoside
a s reactive
of
zation
1 5 > 1 6
group
protecting
h a sb e e n
group
used
a s a phosphate
i n t h ephosphate
t r i e s t e r
(phosapproach
Nucleic Acids Research
(2 e q u i v .
when Ft = H)
(1 e q u i v .
when R =
NC-CH CH -O-
(CH3)3S1)
NC-CH,C.H,-O-P-<^\
'
(1 equiv. )
^
i
I
Cl
^>R
2
JO-P-N^
a
i X
MTrO-V<y
OH
R1-R2-CH
a)
B =• Thyrai n e , 2 - ( m e t h y l ) b e n z o y l c y t o s i ne ,
benzoyladenine
Scheme
by
RL+R2-
c)
b)
3 8
'
morpholino
1 sobu t y r y l g u a n i ne ,
(I)
Letsinger
' and Cramer
' and was p r e v i o u s l y
introduced
by
24)
f o r t h e phosphodiester method. The B-cynoethyl
group
Tener
has
only
t e c t i n g
pared
p r e l i m i n a r i l y
group
when
as reactive
s t a b i l i t y
o f
phoramidites
reactive
Preparation
Three
compounds
synthesis.
have
very
o f
as a
phosphorous
( I I I )p r o -
monochiorophosphites
.
they
Due t o
have
In c o n t r a s t ,
promising
deoxynucl eoside
deoxynucleotide
used
intermediates
these
oligonuc1eotide
been
r i b o n u c l e o s i d e
n o t found
and low
a p p l i c a t i o n
t h e corresponding
and a t t r a c t i v e
intermediates
a r e p r e -
t h e s e n s i v i t y
f o r
p r o p e r t i e s
polymer
as
support
o l i g o -
synthesis.
monochiorophosphoramidites
d i f f e r e n t
B-cyanoethyl
monochiorophosphoramidites
N,N-dimethylamine,
N-morpholine
and N,N-diiso propylamine
prepared
in
phos-
by t r e a t i n g
B-cyanoethyl
t r i m e t h y l s i l y l - N , N - d i m e t h y l a m i n e ,
N,N-diisopropylamine,
r e s p e c t i v e l y ,
phosphordichi o r i d i t e
o f
were
with
N - t r i m e t h y l s i l y l m o r p h o l i n e
f o l l o w i n g
Scheme
( I )
Nand
accor-
15 2 0 2 1 )
ding t ot h e literature
'
. The B-cyanoethyl monochiorophosphoramidites o f H,N-dimethylamine and N,H-diiso propylamine
were obtained a s clear liquids inhigh yield after distillation
under reduced pressure. The monochloro morpholino derivative o b 4541
Nucleic Acids Research
•Idltti
3b
31
•oiling
90 -92° C/0 (
point
Chealctl shift for
It
II)
J-KMB ! • CHjCH
ChtBlcil
for
l
4 . 0 1 , 4 . 17
thiftt
(t.
I n COClj
2 . 7 (d.
-«(CH,)j
, 6 H)
12 H)
(d.
Ha In p t i k t 1n
182
- ) * .
136
110
'
201
)*. 236 (J)*.
(HIE,
i ) \ 166 (!l£,
136
[ '* )
238
t
145
(t .
3.67
« ( C H ) j . 2H)
3.80 ( • .
-CHjCH,
3.96 , 4 . 1
2H)
4.02 , 4. 2 ( 2 t . f-OCHj
P-OCH?, 2H)
(2t,
168.22
179.82 ppa
p pa
175.97
H-«Nl (pp»)
103-105° C/0.08 • •
u
pp>
(2t.
P-OCHj,
O(CHj)2,
4H)
4H)
3.17 (•
.
IIICHjlj.
2.74 (t
,
C H j - C H . 2H)
2H)
224
. H7
I
CJHJIIO,*
136
-
nitril.11':
Phoiphoraaidtttt of
145.43
144.97
142.57
142.17
145.61
137.51
135.33
147.57
146.01
147.44
142.62
142.44
142.30
142.17
1)
II)
tained
a f t e r
thermal
reaction
t h e t i c
purposes.
cyanoethyl
t i o n
o f B-cyanoethyl
morpholine
They
n o t be d i s t i l l e d
present
phosphorodichloridite
d e r i v a t i v e .
3 1
could
but was s u f f i c i e n t l y
The impurities
l i n e
50
work-up
decomposition,
might
i n t h i s
(<~5I)
be removed
and concentrating
be
^ - N M R ,
stable
under
d e r i v a t i v e
s t r i n g e n t
compared
the
a n d mass
t h e reaction
d r y and inert
was found
anhydrous
with
The
4542
o f
and inert
5'-O-N-protected
r e a c t i v e
c o n d i t i o n s
over
work-up
were
i n
protected
B-
o f t h e morphot h e
propor-
i n vacuo
a t
characterized
I ) . They
The
s y n -
were
N - t r i m e t h y l s i 1 y l -
were
found
by
t o
N,N-dimethylamino
a n d hence
required
f o r i t s handling
two phosphoramidites.
a r e shown
s u i t a b l y
(Table
c o n d i t i o n s .
t o be v e r y
t h e other
m o n o c h l o r i d i t e s
Preparation
spectra
case
a n d traces
phosphorodichloridite
duet o
f o r o u r
by adjusting
C. A l l t h e m o n o c h l o r o p h o s p h o r a m i d i t e s
P-NMR,
pure
P-NMR
when
spectra
o f
Figure 1A.
deoxynucleoside
phosphoramidites
deoxynuc1eoside-3'-O-B-cyanoethyl - N , N -
Nucleic Acids Research
Figure 1A:
P-NMR spectra
phoramid ites
o fmonochloro 3-cyanoethyl
phos-
OMTrdO -OOCH^CH^CM
.b
^
DMTfdQ -O-P
/KIZ(CHICMI>IO
IK
144.000 ppm
WB.4If (Km
Figure
IB:
I4M7I
ppm
ULttt
PP*n
P-NMR s p e c t r a o f d e o x y g u a n o s i n e B - c y a n o e t h y l - N , Ndialkylamino-/N-morpholino
phosphoramidites
dimethyl
amino-/N,N-diisopropylamino-/H-morpholino
tes
prepared
were
dered
form
fication
Thus
TLC
yield
developed
offers
material
phitylated
a simple
and
Scheme
procedure
was
i n d i s t i l l e d
method
and
t o detect
t o follow
quality
using
dried
o f
modi-
these
P-NMR
and
ethylacetate.
t h epresence o f
decomposed
those
i n pow-
( I ) by a slight
. The
controlled
a n dh y d r o l y s e d ,
products
phosphoramidi-
monochiorophosphoramidites
following
phosphoramidites
g e l TLC
starting
high
these
o f t h el i t e r a t u r e
nucleoside
silica
and
from
o r oxidised
reactions.
In a l l
phosi n -
4543
Nucleic Acids Research
s t a n c e s ,
i t
w a s found
oxynuc 1eosides
side
This
P-N(C H. )side
most
The
were
some
extent
i n less
t h e t w o d i a s t e r e o m e r s
from
were
o f
deoxynuc1eoside
n o n - d i s t i l l e d
group
as i n d i c a t e d
f r e q u e n t
found
d e r i v a t i v e s
exposure
t e m p e r a t u r e
t o
i s
t o
o f
p r e p a r a t i o n
The
3 1
o f
compounds
P-NMR
o f
A f t e r
vacuo,
f o l l o w e d
t h e case
purine
4544
w i t h
t h e a c t i v e
equ.)
t o
o f
a n d 4?
even
argon
these
a t l o w
were
5 ' - 0 - N - p r o -
type
CPG bound
r e a c t o r
be m a i n t a i n e d
washing
n u c l e o s i d e
o r
ZnBr-
p y r i m i d i n e
f i t t e d
a f t e r
be
t h e i r
s a t i s f a c t o r y .
d e r i v a t i v e s .
w i t h
i n
o f
a
( J t o
s i n t e r e d
serum
t o avoid
o f
i n
glass
c a p .
The
i n 1%
u n d e s i r a b l e
syringe
t o
t h e g l a s s
powdered
t e t r a z o l e
w a s 3
a
a c c o r d i n g
I S w a t e r - n i t r o m e t h a n e
o l i g o d e o x y n u c 1 e o t i d e
d e r i v a t i v e
a
3% C C 1 C O O H
i n order
w a s added
m l ) w i t h
w i t h
e i t h e r
a n d d r y i n g
b y a d d i t i o n
( 1 . 5 - 2 . 0
. T h e
was performed
fi ? ft \
n u c l e o s i d e
'
'
a i r t i g h t
using
s o l u t i o n
s a t u r a t e d
o f mixed
s i x months
deoxyguanosine
also
monochloro
w i t h
t o
a f t e r
e o t i d e s
w a s removed
t h o r o u g h
a c e t o n i t r i l e
w a s checked
1 0 0 mg o f
column
could
group
d e p u r i n a t i o n
In
from
o l i g o d e o x y n u c 1 e o t i d e s
met h a n o l / n i t r o m e t h a n e
30
under
and TLC a n d w a s found
P-NMR
( I ) using
i n a
which
5'-0-DMTr
t i o n .
prepared
p r e -
P-NMR.
d e r i v a t i v e s
t o o u r o b s e r v a t i o n
o l iqodeoxynuci
s y n t h e s i s
(imol)
f r i t t
these
b y
Scheme
7.5
when
b y
be s t a b l e
storage
The guanosine
i n c o n t r a s t
I B i n d i c a t e s
Synthesis
to
P-NMR.
g u a n o s i n e - 3 ' - m e t h y l - N - m o r p h o l i n o p h o s p h o r a m i d i t e
s t a b i l i t y
Figure
t o
In
phosphor,
3% 3 ' - 3 ' - d i m e r s
found
a i r . However,
s t a b l e
p h o s p h o r a m i d i t e s
t e c t e d
were
recommended.
be q u i t e
d e r i v a t i v e s
m o n o c h i o r o - N - m o r -
m a t e r i a l s
o x i d i s e d
other
s p e c t r o s c o p y .
B-cyanoethyl
h y d r o l y s e d
these
these
N - m o r p h o l i n o p h o s p h o r a m i d i t e s
(crude)
about
o r
a n d
b y
o n TLC a n d
contained
o f
r e a c t i v e
This
t h e deoxynuc1eoside
separated
p h o l i n o p h o s p h o r a m i d i t e
Most
t h eT L C
i n d i c a t e d
i n
polar
o f
t h e v e r y
5% a s
d e -
d e o x y n u c l e o -
could
l i n e
b y i n f r a r e d
o f
c l e a r l y
o f
g e l p l a t e .
than
d e t e c t a b l e
p r o t e c t e d
t h e case
a t t h e base
o f t h e n i t r i l e
e a s i l y
o f
d e r i v a t i v e s
TLCo n t h e s i l i c a
present
d e r i v a t i v e s
r e a c t i v e
pared
d u r i n g
marker
cases
a m i d i t e
t o
T h e presence
another
i n
m a y be d u e t o t h e h y d r o l y s i s
bond
products
P-NMR.
Only
phosphoramidite
be d e t e c t e d
p l a t e .
t h e p h o s p h o r y l a t i o n
w a s q u a n t i t a t i v e .
N , N - d i m e t h y l a m i n o
m a t e r i a l
is
that
(70-75
under
s o l u -
beads
i n
form ( 2 5 e q u . )
argon
s y n t h e s i s
2 b y w e i g h t .
a n d
atmosphere.
t h e r a t i o
The
o f
c o n -
Nucleic Acids Research
Tabl e II- Suooary
Step
of thi dlfftrtnt
Operation
Sol»tnt/Rtagent
1
Dttr I tyla t Ion
a) 31 C C K C O O H
2
Washing
3
Washing
4
Drying
5
Condensation
perforncd
In o n f a l o n g a t i o n
VoluDe
or
t) 2
cyclf
(al )
Ho. of
or
tlat/Duntion
5 tiats for 1 atnutfl
b) ZnBr ?
b) 3
a) CH-HO^
a) 5
3 tiaes
b) n - B o O H / L u t i d i n t / T H F 1 "
b) 2
2 tiaes
3 tint) for 3 nlnutes
a) CH^Cfl
a) 5
2 tiaes
b) C H ; C 1 ;
b) 10
2
High racoua
tUts
5 ainutts
Actire nucltosidts and
tttrazole
1.5
25-35 ainutes
'
In CH,C H
6
Washing
CH } CH
3
10 tlats
7
Oildation
2
2 tiats for 1 ainute
8
Wishing
0.1 B Ij In T H F / P y r l d 1 m /
HjO (80:40:2. »/•)
a) Hethinol
a) 2
2 tiaes
b) THF
b) 5
2 tiaes
ACjO/ONAP/LutIdine/THF
2.5
2 tiats for 2 ainutts
9
Capping
10
As sttp 1
'' W h e n
Ill)
CCljCOOH
25 alnutes
densation
by
tttpt
Is u s t d .
for pyriaidlnt
yield
"'
and 3$ a i n u t e s
was checked
for purine
after
Whin
ZnBr;
1i u s e d .
nucleotidis.
30 t o 40 minutes
i n each
step
determination
has
been
o f t h ed i m e t h o x y t r i t y l
cation concentration. I t
29)
possible
' t o achieve a shorter condensation time
(less
than
5 minutes)
sing
agent
with
methyl-5'-0-N-protected
which
phosphoramidites
b y using
h a sv e r y
5-(4-nitrophenyl)-tetrazole
recently
been
used
i n
a s conden-
condensations
deoxynucleoside-3'-0-N-morpholino-
25 )
.
In t h e c a s e o f p u r i n e d e r i v a t i v e s , c o u p l i n g w a s f o u n d t o r e quire either slightly longer time o r higher concentrations than
pyrimidine derivatives inorder t oafford more than 95J coupling
yield. T h e steps involved i n t h e synthesis o fo l i g o m e r s o f d e
sired sequences a r e summarized i nTable ( I I ) . T h e average coupling yield per elongation step w a s found t o b egreater than 9 2 % .
The deoxynucleoside N,N-dimethylaminophosphoramidites
were
used for t h e synthesis o fd(GGGATCCC) resulting in 6 3 % overall
yield for the synthesized product. N-morpholino derivatives
used f o r t h e synthesis o fd ( G G G A T A T C C C ) gave 5 5 % o ft h e p r o d u c t .
N,N-diisopropylamino d e r i v a t i v e s had been used f o r t h e synthesis
of d(TCAGTTGCAGTAG) a n d t w o mixed
ol1gonuc1eotides
4545
Nucleic Acids Research
d(GG^TGpAT^TApAC)
b
45*.
u
b
40%,
of
a l l three
used
use,
types
t h e ease
lino
with
form.
we p r e f e r
which
they
could
d i m e t h y l a m i no
n o t be o b t a i n e d
and storage.
t h e desired
groups
support
without
The
ethyl
group
mixture
o r one step
To
explore
above,
three
andcleavage
model
Treatment
incubation
with
cone.
i i )
treatment
with
20%
cone.
i i i )
with
The
each
the
reverse
case
with
phase
suggesting
B-cyanoethyl
thus
causing
to
acertain
of
t h edimer
HPLC
4546
that
were
t l
only
and subjected
'
;
group,
with
by
followed
cone. a q .
outlined
using
a t 50
t h e dimer
when
cone.
venom
followed
C,
f o r 2 hours
f o r 16 hours
profile
only
followed
a t 50
by
C alone.
wasidentical
cone.
removed
was no 3 - 3 ' d i m e r
with
either
Et.N/Pyridine)
f o r 15 minutes
internucleotidie
t o snake
t h e B-cyano-
a t 5 0 ° C ,a n d
is selectively
treated
t h e polymer
a q . NH^ alone.
performed
chromatographic
there
pro-
^T-0-P
a q . NH..
no detectable
( o r
o f t h e deprotections
f o r 16 hours
even
from
with
t h e polymer
Et-N/Pyridine
that
good
various
m a y be a c h i e v e d :
N H ,f o r 1 6 h o u r s
cone.
group
cleaved
strategy
10% t B u N H . / P y r i d i n e
a q .NH-incubation
incubation
synthesis,
by cone.
DHTr-C
i)
t o ensure
o f B-cyanoethyl
from
experiments
by
phosphoramidites r e -
t-BuNH2/Pyridine
deprotections
t h e r e l i a b i l i t y
a n d t h e N,N-
reaction.
protection,
where
f o r t h e removal
N-deacylation
NHj
after
i n our synthetic
as phosphate
i s used
and very
purity
condition
andthen
anyside
deprotections,
because
oligodeoxynuc1eotides
product
be removed
causing
deprotection,
stepwise
by
must
f o r
B-cyano-
monomers
i n pure
i n 100%
and inert
o f t h e synthetic
tecting
a s active
deoxynuc1eoside
anhydrous
Deprotection
isolate
been s u c -
o f oligodeoxynucleotides,
c a n be obtained
condensation
To
o f
t h e monochioro-B-cyanoethoxy-N-morpho-
B-cyanoethyl
stringent
have
t h e N , M - d i i s o p r o p y l a m i no
phosphoramidites
In contrast,
phosphine
quired
o f phosphoramidites
f o r t h e synthesis
however,
deoxynucl eoside
stable
yield
38*.
cessfully
ethyl
i n respective
T
Although
routine
a n d d(GGATG^ATATAAAC)
o
a q . N H ,i s
by
i n
used,
B-elimination,
cleavage.
formation
In
, an
a q . N H ,w a s i s o l a t e d
phosphodiesterase
order
aliquot
from
(SVDP) d i -
Nucleic Acids Research
E
c
1O
IS
2O
29
3O
o
5
1O
t(min)
Figure
19
2O
2S
3O
39
t(min)
a
2 : HPLC chromatograms
b) m i x e d
primer
b
of a ) 13-mer, d(TCAGTTGCAGTAG) , and
G
14., d(GGATG^ATATAAAC).
1
T
T
g e s t i o n a t 37° C for 2 h o u r s , 4 hours and 2 6 h o u r s . The digested
s a m p l e s w e r e c h r o m a t o g r a p h e d u n d e r the same c o n d i t i o n s (RP 1 8 )
as for D M T r - C T . Only two peaks were o b s e r v e d , one having t h e
same r e t e n t i o n time a s d p T , the other o f D M T r - ( d C ) . This d e m o n strates that DMTr-dimer was completely digested b y SVDP. It is
therefore evident, that incubation with cone. aq. NH, alone completely d e p r o t e c t s the o l i g o n u c l e o t i d e under the c o n d i t i o n s used
without any side r e a c t i o n s .
The oligomer synthesized on the C P Gbeads possessing dimethoxytrityl group a t 5'-end was thus d e p r o t e c t e d and removed from
the polymer b y treatment with cone. aq. ammonia a t room
t e m p e r a t u r e for 3 0 m i n u t e s and f i n a l l y incubated a t 50° C o v e r night under sealed conditions. After cooling to room temperature,
CPG b e a d s w e r e centrifuged and the s u p e r n a t a n t l i q u i d was e v a p o r a ted to d r y n e s s . Removal o f B - c y a n o e t h y l g r o u p s from the intern u c l e o t i d i c p h o s p h a t e t r i e s t e r s , d e - N - a c y l a t i o n and cleavage
from the p o l y m e r i c s u p p o r t were p e r f o r m e d in o n e step taking less
than 1 6 hours.
Purification and analysis o f o l i g o d e o x y n u c l e o t i d e s
Purification
has been
performed
by reversed
phase
(RP 1 8 )
H P L C 30) . T h e c r u d e o l i g o n u c 1 e o t i d e o b t a i n e d a f t e r e v a p o r a t i o n w a s
4547
Nucleic Acids Research
X
L
d(pT).
Figure 3
Figure
F1gure
3:
E l e c t r o p h o r e s i s
o f t h e o l i g o m e r s :
8-mer,
lane 1 ;
10-mer,
lane 3 ;
13-mer,
lane 4 ; 1 4 , - m e r ,
lane 6 ;
14,-mer,
lane 7 a n d 8 i n r e l a t i o n
t o
homo-ol1go-dT
l e n g t h
standard
(lane
2 , 5 a n d 9 r e s p e c t i v e l y ) ,
on 2 0 % p o l y a c r y l a m i d e
g e l a f t e r
HPLC s e p a r a t i o n , d e t r i t y l a t i o n
a n d phos p h o r y 1at i o n w i t h
(.f32_p)ATP a n d
T. p o l y n u c l e o t i d e
k i n a s e .
Lane 7 c o n t a i n s
t h e main
m a t e r i a l
from 1 4 j a n d lane 8 c o n t a i n s
t h e r . !-,. s . o f
the d e s i r e d
peak o n F i g u r e 2 b .
Figure
4 :
Sequence a n a l y s i s
o f 13-mer b y " m o b i l i t y
s h i f t "
method.
+ : x y l e n e cyanol marker.
1 . d i m e n s i o n :
e l e c t r o p h o r e s i s ,
2 . d i m e n s i o n :
homochromatography.
taken
u p i n a p p r o x i m a t e l y
f o l l o w e d
b y f i l t r a t i o n
1 . 0 m l o f b u f f e r
using
The
sample
were
i n j e c t e d
and
e l u t e d
w i t h
a g r a d i e n t
Typical
s e p a r a t i o n s
The
HPLC
p r o f i l e s
the
DMTr
o l i g o m e r i c
ted
n u d e o t i d i c
m i l l i p o r e
onto
shewn
t h e reversed
a c c o r d i n g
a r e shown
well
RP1 8 - H P L C
p u r i f i c a t i o n
A f t e r
c o l l e c t i o n
o f t h e d e s i r e d
pure
4548
sample
phase
a n d t h e sample
s i n g l e
separated
o l i g o m e r ,
from
b i d i s t i l l e d
column
i n Figure
sharp
t h e
n o n t r i t y l a y i e l d s
o f 23%t o 36%
80%a c e t i c
water
2 .
peaks f o r
T h e o v e r a l l
t h e e l u t i o n
w i t h
7 . 0 )
s i z e ) .
a n d Methods.
from
a r e i n t h e range
d e t r i t y l a t e d
w a s l y o p h i l i s e d
(RP 1 8 )
a n d 14-mer
a n d non-nucl eot i di c m a t e r i a l .
a f t e r
e v a p o r a t e d
2 gave
pH
( 1 . 2 u pore
t o M a t e r i a l s
f o r 13-mer
i n Figure
m a t e r i a l s
( 0 . 1 M TEAA,
f i l t e r
4 0
^.
b u f f e r
w a s
a c i d .
T h e
a n d a
small
Nucleic Acids Research
amount
phoretic
phosphoryla ted with ( r - P)ATP. I t s purity a n d electro31 )
mobility
were determined by 2 0 1 polyacrylamide gel
electrophoresis (Figure 3 ) . The presence of trace amounts of
longer chain oligonucleotidic material might be d u e to side r e actions, whereby some DMTr group is removed (acidic effect o f
tetrazole) giving rise to additional condensation reactions
during a single condensation step. This problem might arise
during repetition o r longer condensation time, which we have r e cently overcome with 3 - 4 minutes condensation time using p-nitrophenyltetrazole. The sequence analysis of all t h e oligonucleotides w a s performed using either "mobility shift"
' (Figure 4)
or M a x a m - G i 1 b e r t
' method (Figure 5 ) .
"Haxam-Gi1bert" sequence analysis o f "mixed oligomers"
The sequence analysis o f mixed synthetic
oligodeoxynuc1eotides
is a f o r m i d a b l e t a s k , e s p e c i a l l y i f o n e w a n t s t o r e c e i v e i n f o r m a tion on t h e relative amounts o f t h e different oligomers within a
m i x e d p r o b e . In special c a s e s o n e w a s a b l e t o s e p a r a t e some
oligomers o u t o f t h e mixture by complicated HPLC procedures a n d
to s e q u e n c e t h e m i n d i v i d u a l l y b y t h e " m o b i l i t y s h i f t "
method
* '. W e i n v e s t i g a t e d t h e p o s s i b i l i t y o f u s i n g t h e
"Maxam-Gi1bert" method directly for sequencing t h e oligonucleotide mixture. We were particularly interested in examining whether all required sequences were present in t h e mixture a n d if
the intensity o f t h e bands at t h e mixed positions could be used
to e s t i m a t e t h e r a t i o o f t h e o l i g o n u c l e o t i d e s w i t h i n t h e m i x t u r e .
After HPLC purification o f t h e 5'-dimethoxytrityla ted mixed
oligomers on RP 18 (e.g. Figure 2 b ) , detritylation a n d phosphorylation, t h e mixture w a s isolated from 2 0 %po1yacrylamide gel
electrophoresis (Figure 3) and directly subjected to t h e MaxamGilbert sequencing protocol.
It c o u l d b e s e e n f r o m r t h e a u t o r a d i o g r a m f o r t h e a n a l y s i s o f
the 14-mer d ( G G A T G T A T A T A A A C ) , that t h e correct sequence had been
established (Figure 5A). T h emixed positions 6 (A,T) a n d 12
(G,A,T) reading from t h e 5'-end a r e also clearly visible a n d it
is e v i d e n t t h a t t h e i n t e n s i t y o f t h e s e b a n d s i s r e l a t i v e l y w e a k e r
than that o f t h e "unmixed" positions d u e to t h e distribution o f
the radioactivity over more than o n e oligonuc1eotide .
In c o m p a r i n g
the intensities
of the bands
for the mixed
posi-
4549
o
01
01
A>G
C
TiC
G
A>G
C
T+C
F i g u r e 5A: S e q u e n c i n g of m i x e d p r o b e
G
A
d ( G G A T G "1 A T A T A A A C ) a c c o r d i n g to M a x a m - G 1 1 b e r t .
T
In t h e l e f t e l e c t r o p h o r e s 1 s b r o m o p h e n o l - b l u e
( B P B ) w a s run f o r 2 0 c m , in t h e r i g h t o n e for
8 cm .
' 'I r
G A A»-
G
B
.G,*
_
A>G
C
"i
T*C
Gm-
G A>G
C
T+C
Gi1bert.
F i g u r e 5A.
S e e a l s o l e g e n d to
Figure 5B: Sequencing of mixed probe
d ( G G r T G r A T r T A p A C ) according to Maxam-
It
G
z
O
3"
V)
•J3
CD
a.
c
Nucleic Acids Research
tion 1 2 , o n e can see that G,A and T n u c l e o t i d e i n t e n s i t i e s a r e
q u i t e s i m i l a r . At p o s i t i o n 6 t h e A n u c l e o t i d e is s l i g h t l y s t r o n g e r t h a n T , b u t in g e n e r a l it m a y be s t a t e d t h a t a l l e x p e c t e d
o l i g o n u c 1 e o t i d e s a c c o r d i n g to the sequence are c l e a r l y p r e s e n t .
In t h e a u t o r a d i o g r a m f o r t h e a n a l y s i s o f t h e 1 4 » - m e r
d ( G G p T G - A T - T A - A C ) , which contains four mixed positions resulting
in a m i x t u r e o f 1 6 o l i g o n u c 1 e o t i d e s , i t m a y b e o b s e r v e d t h a t
bands derived from o l i g o n u c 1 e o t i d e of the same length but whose
base c o m p o s i t i o n is d i f f e r e n t have been s e p a r a t e d ( F i g u r e 5 B ) .
This phenomenon arises from the fact that oligonuc1eotides of
the same c h a i n l e n g t h b u t d i f f e r i n g in b a s e c o m p o s i t i o n s e x h i b i t
d i f f e r e n t m o b i l i t e s . This is d u e to a b a s e - s p e c i f i c m o b i l i t y
u n d e r t h e s e e l e c t r o p h o r e t i c c o n d i t i o n s in t h e o r d e r C > A > T > G
,
t h e e f f e c t b e i n g p a r t i c u l a r l y s t r o n g in s e q u e n c e s w i t h h i g h G
c o n t e n t . From this c o n s i d e r a t i o n one expects separation into two
bands at position 4 , 5 and 6 from 5'-end (mixture of 2 o l i g o n u c i e o t i d e s ) a n d t h r e e b a n d s a t p o s i t i o n 7 , 8 a n d 9. T h e f o u r
hexanucleotides (pGGATGAp, pGGGTGAp, pGGGTGGp) representing
p o s i t i o n 7 d i f f e r in t h e i r G / A c o n t e n t , g i v i n g r i s e t o t h e t h r e e
ratios 3G/2A, 4G/1A, and 5G. As two of the four h e x a n u c 1 e o t i d e s
c o n t a i n t h e s a m e G / A r a t i o o n l y t h r e e b a n d s c a n be e x p e c t e d a s
v i s u a l i s e d . T h e s a m e d i f f e r e n c e in G / A c o n t e n t r e s u l t s 1n t h r e e
b a n d s f o r p o s i t i o n s 8 a n d 9. A p p a r e n t l y t h e b a n d s c o r r e s p o n d i n g
to l o n g e r o l i g o n u c l e o t i d e s a r e n o t so w e l l s e p a r a t e d d u e to t h e
g r e a t e r n u m b e r o f d i f f e r e n t o l i g o n u c 1 e o t i d e s p r e s e n t in t h e
m i x t u r e and the shorter d i s t a n c e which they have travelled on
t h e g e l . S i m i l a r t o 1 4 , - m e r , h o w e v e r , it m a y be p o i n t e d o u t t h a t
again all n u c l e o t i d e s r e p r e s e n t i n g the desired sequences a r e
visible.
CONCLUSIONS
As d e m o n s t r a t e d , the B-cyanoethyl group has many a d v a n t a g e s
w h e n u s e d in t h e p h o s p h i t e t r i e s t e r m e t h o d w i t h C P G a s p o l y m e r
support :
1 ) D i c h l o r o - B - c y a n o e t h o x y p h o s p h i n e ' c a n e a s i l y b e p r e p a r e d in
p u r e f o r m a n d is s t a b l e f o r l o n g e r t i m e s . T h e r e p e a t e d d i s t i l l a t i o n very
often necessary for the complete removal of phosphorus trichloride during preparation of dichloro-methoxyphos-
4551
Nucleic Acids Research
phine is u n n e c e s s a r y in this c a s e .
2)
The B-cyanoethylmonochlorophosphoramidites of N, N-dimethylamine, N,N-diisopropylam1ne and N-morpholine can easily be o b tained.
3)
The 5'-0-,N-protected
deoxynucleoside-3'-O-B-cyanoethyl-N,Ndialkylamino-/N-morpholinophosphoramidites c a n be prepared as
pure white precipitates stable for many months.
Their purity
(and thus their reactivity) c a n be routinely checked simply by
thin layer chromatography.
Especially useful a r e the N,N-diisopropylaminophosphoramidites.
4)
T h e r e a c t i v i t y o f t h e d e o x y n u c l e o s i d e p h o s p h o r a m i d i t e s is
comparable to that of t h e corresponding methoxy derivatives.
With
4 ' -nitrophenyltetrazole
, h o w e v e r , c o u p l i n g times a r e in t h e
range o f 2 to 5 minutes.
5)
The main advantage of these new active deoxynucleoside derivatives is their influence on t h e simplification a n d time-reduction o f t h e final work-up procedure o f synthesized oligodeoxynucleot1des.
can easily be removed
24 2 6 2 7^
by B - e l i m i n a t i o n using mild a l k a l i n e c o n d i t i o n s
'
.
Therefore, t h e deprotection of t h e oligodeoxynucleotide chain can be
performed in o n e step at t h e heterocyclic bases, the phosphotriester moiety and t h e linkage to t h e polymer support by treatment with e.g. concentrated aqueous ammonia.
After evaporation,
the r e s i d u e c a n d i r e c t l y be taken u p in t h e starting buffer f o r
reversed phase (RP 18 ) HPLC, filtrated a n d purified by HPLC.
After detritylat1on with 8 0 S acetic acid and subsequent lyophili z a t i o n t h e o l i g o n u c l e o t i d e is ready f o r p h o s p h o r y l a t 1 o n with T 4
polynucleot1de kinase and ATP.
T h e total time required f o r this
w o r k - u p s e q u e n c e is less t h a n 2 4 h o u r s .
T h e loss o f valuable
oligonucleotidic material is significantly reduced as there a r e
no t r a n s f e r s o f m a t e r i a l a n d n o e x t r a c t i o n o r thin layer o r p a p e r
chromatographic steps necessary prior to HPLC purification.
During t h e whole deprotection a n d purification step only volatile
reagents a r e used.
6)
Mixed sequences c a n also efficiently be prepared by these n e w
reactive
intermediates.
7)
T h e Maxam-G11bert sequencing method c a n successfully be used
4552
The B-cyanoethyl
group
Nucleic Acids Research
to s i m p l y
characterize
synthetic
mixed
ol 1g o d e o x y n u c l e o t i d e
frag-
ments .
We
tide
phite
believe
that
the introduction
phosphoramidites
triester
of B-cyanoethyl
is a s i g n i f i c a n t
improvement
deoxynucleoof the phos-
approach.
MATERIALS AND METHODS
The following chemicals were purchased from commercial sources:
deoxynucleos1des from Pharma Haldhof ( M a n n h e i m ) , CPG from Serva
( H e i d e l b e r g ) , Z n B r 2 from Riedel de Haen ( S e e l z e ) , 4-N, N-dimethylamino pyridine, aminopropyltriethoxysi1 a n e, tetrazole, N,N-diisopropylamine, N , N , N-diisopropylethyl amine from EGA (Steinheim),
N-trimethylsilyl-N,N-dimethylamine from Fluka (Neu-Ulm).
Tetrazole w a s purified by s u b l i m a t i o n , 2 . 6 - l u t i d i n e w a s purified a c cording to the literature
. Ether, THF were distilled from
benzophenone/sodium under nitrogen.
Acetronitri1e was first d i s tilled over P , 0 c and then C a H , under inert atmosphere.
Dimeth3 6 );
o x y t r i t y l c h l o r i d e , N - t r i m e t h y l s i 1 yl m o r p h o l 1 ne
and dimethoxytritylated amino protected deoxynucleosides
were prepared following standard published methods.
The B-cyanoethylphosphord i c h l o r i d i t e w a s p r e p a r e d by m o d i f y i n g t h e p u b l i s h e d m e t h o d
.
Thin l a y e r s i l i c a gel p l a t e s w e r e d e v e l o p e d in e t h y l a c e t a t e .
H-NMR spectra were recorded either with T-60 (Varian) or 270
31
MHz (Bruker).
P-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 U l t r a s p h e r e O D S c o l u m n ( 4 . 6 x 2 5 0 ) o n a B e c k m a n m o d e l 3 4 4 o r
L.D.C. dialog dual pump Instrument.
B-Cyanoethylphosphorodichloridite:
A three-necked flask (1.0 1)
fitted with addition funnel, mechanical stirrer and argon delivery system was charged with phosphorus trichloride (1.0 m o l ) ,
dry e t h e r ( 2 0 0 m l ) a n d d r y p y r i d i n e ( 1 . 0 m o l ) (2 e q u i v a l e n t s o f
p y r i d i n e h a d been u s e d in t h e l i t e r a t u r e ' ) . T h e m i x t u r e w a s
cooled to - 7 8 C with a d r y i c e / a c e t o n e bath under argon a n d
f r e s h l y d i s t i l l e d B - c y a n o e t h a n o l ( 1 . 0 m o l ) d i s s o l v e d in e t h e r
(100 m l ) w a s added d r o p w i s e over 1 to 1 1/2 h o u r s .
After addition
of the alcohol the m i x t u r e w a s stirred at room t e m p e r a t u r e for 3
hours and kept at 5° C overnight.
Pyridinium hydrochi oride was
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removed by filtration, the filtrate w a s concentrated to a small
volume and t h e residue distilled under reduced pressure to give
B - c y a n o e t h y 1 p h o s p h o r o d i c h i o r i d i t e ( 9 5 g ) i n 55% y i e l d .
General method for the preparation of B-cyanoethyl monochlorophosphorami di tes : A two-necked flask fitted with a d d i t i o n funnel,
magnetic stirrer and argon delivery systems w a s charged with Bcyanoethyldichiorophosphite (17.2 g, 100.0 mmol) and d r y ether
(60 m l ) . To this a solution o f N-trimethylsi1yl derivatives of
N , N - d i m e t h y l a m i n e o r N - m o r p h o l i n e (1 e q u . , 1 0 0 m m o l ) o r N - N - d i isopropyl a m i n e (2 equ., 2 0 0 m m o l ) in ether ( 3 0 m l ) w a s a d d e d a t
-20° C over 1.5 hours with constant stirring.
After this a d dition t h e mixture w a s stirred for an additional 20 hours at room
temperature under argon.
In t h e c a s e o f t h e N , N - d i i s o p r o p y l a m i n e
reaction, its hydrochi oride was filtered prior to concentration
of solvent under reduced pressure at room t e m p e r a t u r e .
The concentrates from N,N-dimethylamine and N,N-d1isopropy1 amine reactions were distilled in vacuo to give B-cyanoethyl
monochloro
N,N-dimethylaminophosphoramidite (15.3 g) at 90-92° C/0.6 mm
and B-cyanoethylmonochloro-N.N-diisopropylamino
phosphoramidite
(16.5 g ) a t 103-104° C/0.08 m m , respectively.
N-morpholino derivative w a s used without distillation as attempted distillation
decomposed the product.
Synthesis of B-cyanoethyl-5'-0,N-protected deoxynucleoside-3 ' O-N,N-dialkylamino/-N-morpholinophosphoramidites : T h e 5'-0,Nprotected deoxynuc 1eoside (3.0mmol) was dried by coevapora11 on
with pyridine, toluene and T H F . The dried residue w a s dissolved
in d r y T H F ( 1 5 m l ) i n p r e s e n c e o f N , N , N - d 1 i s o p r o p y l e t h y l a m i n e
(12.0 mmol) a n d e . g . B-cyanoethylmonochioro
N,N-dia1kylaminophosphoramidite ( 6 . 0mmol) w a s added dropwise through a syringe
with constant stirring under argon at room temperature over 2
minutes.
After 35 minutes of stirring, the hydrochi oride, which
precipitated out during reaction was filtered and the filtrate
was concentrated to remove THF and excess amine.
The residue
was d i s s o l v e d 1n a r g o n s a t u r a t e d e t h y l a c e t a t e ( 1 5 0 m l ) , w a s h e d
with ice-cold 1 0 2 N a . C 0 3 solution ( 5 0 ml twice) a n d dried over
Na.SO..
C o n c e n t r a t i o n o f t h e dried o r g a n i c e x t r a c t r e s u l t e d in
a foam, which w a s dissoved in toluene (15-20 m l ) ( f o r p y r i m i d i n e s )
or e t h y l a c e t a t e ( 2 0 m l ) ( f o r p u r i n e s ) , then precipitated into
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Nucleic Acids Research
hexane ( 2 5 0 m l ) at -78 C. T h e precipitates were removed by
f i l t r a t i o n a n d d r i e d i n a d e s i c c a t o r o v e r C a C1 - u n d e r r e d u c e d
pressure overnight.
General coupling procedure on the polymer (CPG):
Usually 100
mg o f t h e controlled pore glass beads ( C P G )containing 5 ' - 0 dimethoxytritylated aminoprotected deoxynucleoside was detrit y l a t e d w i t h e i t h e r 3 % C C 1 - C 0 0 H i n 1% m e t h a n o l - n i t r o m e t h a n e o r
s a t u r a t e d Z n B r - i n 1% w a t e r - n i t r o m e t h a n e a n d a f t e r w a s h i n g a c c o r d i n g to T a b l e ( I I ) w a s dried in v a c u o f o r 5 m i n u t e s . T h e
solid active nucleoside ( 1 5 0 m g , 25 equ.) and tetrazole
(50-60
mg) were placed into t h e column type reactor containing t h e d e tritylated polymer.
T h e reactor w a s capped with a serum c a p ,
flushed with argon and acetonitrile (1.5 m l ) w a s added with a
syringe.
The suspension of CPG beads w a s shaken gently for
25-35 minutes.
The excess of reagents w a s removed by flushing
with argon and washing with suitable solvents (Table II). T h e
condensation yield w a s determined at this stage by taking o u t a
small sample of C P G beads a n d by measuring t h e c o n c e n t r a t i o n of
DMT-cation on the polymer.
When t h e desired high coupling yield
was obtained, oxidation w a s performed with 2 m l , 0.1 M I- s o l u t i o n in a m i x t u r e o f T H F , p y r i d i n e , w a t e r ( 8 0 : 4 0 : 2 , v / v )f o r
2 minutes.
The oxidising reagent was removed with argon pressure
and washing with methanol followed by d i c h i o r o m e t h a n e .
Then t h e
p o l y m e r w a s s u s p e n d e d in a m i x t u r e o f a c e t i c a n h y d r i d e ( 0 . 5 g ) ,
2 . 6 - l u t 1 d i n e ( 0 . 5 5g ) a n d 4 - N , N - d i m e t h y l a m i n o p y r i d i n e ( 0 . 3 g )
in 5 m l T H F f o r 5 m i n u t e s t o b l o c k t h e u n r e a c t e d 5' - O H g r o u p o f
nucleoside or olIgonucleotide linked to CPG. The repetition of
the above cycle with appropriate active nucleotide gave the d e sired sequence of oligonucleotide.
Removal of t h e oligomer from t h e CPG-beads and deprotection of
all p r o t e c t i n g g r o u p s b u t D M T a t 5 ' - e n d :
After the final elongation step of the desired sequence, the CPG beads, which were
thoroughly washed and dried, were transfered to a flask (25 m l )
and treated with c o n c e n t r a t e d aqueous N H - (5 m l ) f o r 3 0 m i n u t e s
at r o o m t e m p e r a t u r e a n d f i n a l l y a t 5 0 C o v e r n i g h t .
After
cooling, the supernatant liquid w a s removed and the polymer was
washed with bidistilled water ( 3 x 1 m l ) . The combined
supernatant a n d washings were c o n c e n t r a t e d to a small volume a n d t h e
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Nucleic Acids Research
insoluble material resulting from t h eB-elimination a n d polymerization of CH?=CH-CN w a s removed by filtering through millipore
filter.
T h e filtrate w a s evaporated to near dryness a n d r e d i s s o l v e d in a small a m o u n t o f 0 . 1 M TEAA b u f f e r , pH 7 . 0 .
Purification of t h e oliqomer by RP-HPLC: T h e chromatographic
p u r i f i c a t i o n o f t h e ol 1 g o n u c l e o t i d e w a s c a r r i e d o u t o n a B e c k man dual pump model 3 4 4 HPLC a p p a r a t u s in c o n j u n c t i o n with an
Altex R P U l t r a s p h e r e O D S c o l u m n a n d a n UV d e t e c t o r (filter
254 n m ) . T h emobile phases used were 0.1 M TEAA buffer pH7.0
in p u m p A a n d C H - C N i n p u m p B . A s t e p l i n e a r g r a d i e n t f r o m
1 0 - 2 5 $ pump B in 5 m i n u t e s f o l l o w e d b y 2 5 - 2 9 1 pump B in 3 0
minutes w a s performed.
I n j e c t i o n v o l u m e s w e r e t y p i c a l l y 2 0 yl
of solution. Polyacrylamide gel electrophoresis a n d sequencing
were performed according to published procedures
"
.
Acknowledgement
This work h a s been financially supported by t h e Deutsche
F o r s c h u n g s g e m e i n s c h a f t a n d t h e B u n d e s m i n i s t e r flir F o r s c h u n g u n d
Technologle.
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Determinations
o f t h e overall
y i e l d s a r e based on t h e
material
isolated
f r o m HPLC a s
5'-DMTr-oligodeoxynucleotides
with respect t o t h e total
material
obtained a f t e r
t h e d e p r o t e c t i o n
from t h e polymer.
4557