Volume 10 Number 221982
Nucleic Acids Research
Silica gel: as unproved support for the solid-phase phosphotriester synthesis of oligonudeotides
V.Kohli*, A.Balland*, M.Wintzerith"1", R.Sauerwald*, A.Staub+ and J.P.Lecocq*
Transgene S.A., and +Laboratoire de Ge'ne'tique Moleculaire des Eucaryotes du CNRS, Unite' 184
de 1'INSERM, Faculty de M6decine de Strasbourg, 11, rue Humann, 67000 Strasbourg, France
Received 24 August 1982; Revised 15 October 1982; Accepted 29 October 1982
ABSTRACT
The phosphotriester method for the stepwise synthesis of deoxyoligonucleotides has been employed using HPLC-grade s i l i c a gel (Porasil B) as
the solid support. The procedure results In a convenient flow-through system for the synthesis of oligomers where a l l the reaction steps Including
the zinc bromide method of d e t r i t y l a t i o n are compatible with the selected
support. Deoxyoligonucleotides of 25-30 nucleotides 1n length can be synthesized In high yields u t i l i s i n g stable phosphotriester Intermediates.
Ease of handling of the solid support allows convenient synthesis of mixed
oligonucleotide sequences.
INTRODUCTION
Recent
advances
In molecular
biology
have exploited
the methods of
recorabinant DNA to obtain peptides and proteins of therapeutic and Industrial
Importance.
chemical
In
many
cases
this
approach
requires
unambiguous
synthesis of DNA fragments with defined sequences of bases. The
phosphotriester
oligonucleotide
method
appears
synthesis
since
to
1t
be
the
method
has resulted
In
of
choice
the synthesis
for
of
various biologically Important sequences. These Include, among others, the
genes coding for somatostatin, Insulin and a-1nterferon as well as several
defined
sequences
useful
for
manipulating
natural
DNA and RNA ( 1 - 4 ) .
Adaptation of nucleotide coupling procedures to sol Id-phase technology has
led
to an enormous reduction
In the time
required
for
oiigonucieotide
synthesis ( 5 - 9 ) .
Numerous sol Id-phase methods have been described for the synthesis of
defined o11gonucleot1des. Classically the major problem with polymer-supported synthesis
strategies
has been Inherent
In nature to the polymer
support. Various polymers used for such synthesis have proved to be Inadequate for reasons such as slow diffusion of activated nucleotides Into the
support, I r r e v e r s i b l e absorption of reagents onto the polymer or excessive
swelling of the polymer. However, a l l these problems can be avoided by the
© IR L Prea Limited, Oxford, England.
0305-1048/82/1022-74398 2JM/0
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Nucleic Acids Research
use of Inorganic carriers as solid supports. The solvents and reactants can
be
easily
flushed
through
Supports based on s i l i c a
the
porous
non-swellable
Inorganic
matrices.
gels have been used for the synthesis of o l i g o -
nucleotides by the phosphodiester and phosphite methods ( 1 0 , 1 1 ) ; a successful attempt to use phosphotriester chemistry with a glass bead support has
been described by Gough et a l . ( 1 2 ) .
We report here that the use of Inorganic s i l i c a gel carriers (HPLC-grade
s i l i c a Porasil B) can be extended to the phosphotriester method of o l i g o nucleotide synthesis. The choice of Porasil B as a solid support was based
on the observation of Majors and Hopper ( 1 3 ) . They showed that with lower
surface area s i l i c a s l i k e Porasil the bonded phase coverage 1s only 1 % or
less when the direct
silylation
reaction 1s performed using a
four-fold
excess of alkoxysilane. Thus problems arising from steric hindrance
lower
reaction
rates,
requirement
for
high reactant
(e.g.
concentrations)
are
unlikely to be encountered during oligonucleotide assembly. The use of low
surface area s i l i c a permits the synthesis of longer oligonucleotides (up to
chain length
30 or
more)
than reported on Inorganic c a r r i e r s .
method u t i l i z e s stable phosphotriester
1n large
quantities
and stored.
The basic
Intermediates which can be prepared
The present
report describes the use of
dinucleotide blocks, but both mononucleotides and trinucleotides have been
used to success.
METHODS AND MATERIALS
Solvents and reagents
All
solvents were dried prior
to use. 5'-d1methoxytr1tyl
N-acyl deoxy-
nucleosides were synthesized by a described procedure (14) and p u r i f i e d by
short
column
chromatography
on
silica
gel.
Mesitylene
sulfonyl
1,2,3-
n i t r o t r i a z o l e (MSNT) and 0-n1trobenzaldox1me were synthesized by the method
of Reese et a l . ( 1 5 , 1 6 ) .
S i l i c a gel support
Waters HPLC-grade s i l i c a
(Porasil B, p a r t i c l e size 37-75
u)
was deriva-
tized to create -NH2 groups as described by Chow et a l . (17) resulting 1n a
functional
support where remaining silanol
by treatment with t r i m e t h y l s i l y l
groups were e f f e c t i v e l y blocked
chloride. Loading of f i r s t nucleoside on
to the NH2-Poras1l was perforned by using pentachlorophenyl esters of 5'-Odiraethoxytrityl N-acyl deoxynucleoside 3'-succ1nates (18) except 1n case of
deoxyguanosine where the NH2-S1l1ca was f i r s t succinylated using sucdnic
anhydride
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In pyridine
In the presence of c a t a l y t i c
amounts of
dimethyl-
aminopyridine
and
deoxyguanosine
then
using
condensed
with
5'-d1methoxytr1tyl
dicyciohexylcarbodiimide
followed
remaining carboxyl groups ( 1 1 ) . Loading of the f i r s t
to
be
In
the
range
dimethoxytrityl
of
190-220
^raoles/g
cation released for a l l
as
by
N-1
blocking''
nucleoside was
determined
by H^gg
nm of
the four nucleosides bound to the
s o l i d support.
Mono- and dinucleoflde blocks
5'-d1methoxytr1tyl
N-acyl deoxynucleoside 3'-0-chlorophenyl
p-cyanoethyl
phosphates were synthesized by direct phosphorylation of
5'-dimethoxytr1tyl
N-acyl deoxynucleosides with O-chiorophenyl p-cyanoethyl
phosphoroonochlori-
date In the presence of N-methyl Iraidazole by modification of the procedure
described by De Bernardini et a l . ( 1 9 ) .
a) Synthesis of a o-chlorophenyl p-cyanoethyl-phosphoraonochloridate
A solution of o-chlorophenyl-phosphod1chlor1date
:
(100 ninol) 1n 20 ml anhy-
drous tetrahydrofuran was cooled to -70*C. To the clear solution a mixture
of cyanoethanol
(110 mnol) and triethylamine
added dropwise over a period of
(110 mnol) 1n 20 ml THF was
1.5 hour under s t i r r i n g . The mixture was
warmed to room temperature during 0.5 hour after
completion of
addition.
Toluene 300 ml was added and solid triethylammonium hydrochloride
filtered
o f f . The f i l t r a t e was evaporated In vacuum to an oil which was used without
any further
purification.
b) General
method for
the synthesis
of
5'-OMTr-N-acyl
deoxynucleoside
3'-phosphotr1esters :
0,5 ml a c e t o n i t r i l e containing 2 mmol o-chlorophenyl
p-cyanoethyl-phospho-
monochi or 1 date was added slowly with s t i r r i n g over 5 min. to 2 ml of I c e cooled
anhydrous
nucleoside
acetonitrile
containing
mmol N-methyl 1m1 dazole.
The reaction
potassium dihydrogen phosphate buffer
porated
water.
and the residue
partitioned
The chloroform layer
phosphate
0,5 ramol 5'-DMTr-N-acyl
(dried by repeated evaporation of anhydrous pyridine)
solution
pH 7.0.
between
The organic
1 molar
phase Is
eva-
10 ml chloroform and 10 ml
was then washed with 0.1 H sodium dihydrogen
thoroughly
dried chloroform layer
was quenched with 2 ml of
deoxyand 2,2
three
tiroes
followed by water
twice.
The
(with anhydrous sodium sulfate) was concentrated to
a gum and precipitated 1n 100 ml of petroleum ether:ether
( 1 : 1 , v / v ) . The
product was then hydrolysed with 50 % aqueous pyridine for 30 minutes and
the solvents were removed In vacuum. Short column chromatography on s i l i c a
gel gave 60 %-80 % of pure product.
c) General method for the synthesis of dinucleotide blocks :
7441
V-CH,-C«
I
U-o
2
+ it*
—»•
m««<2
°
6—C—
1 - General method for the synthesis of o11gonuc1eot1des on a s i l i c a
support using suitably protected phosphotHester Intermediates.
R : diroethoxytrityl
CE : p-cyanoethanol
R1: O-chiorophenyl
TEA: t r i e t h y l amine
All the 16 dinucleotide blocks could be conveniently synthesized by d e t r i tylation
of one mononucleotide and p-elimination
from the other prior
of the cyanoethyl
group
to condensation of the two components with HSNT (see
F1g. 1 ) .
Oligonucleotide sequence analysis
5'
end l a b e l l i n g ,
chemical cleavage and gel electrophoresis were per-
formed as described by Kaxam and Gilbert
( 2 0 ) . Two-dimensional
"wandering
spot" analysis was carried out according to Tu and Wu ( 2 1 ) .
RESULTS AND DISCUSSION
Porasil B was found to be a suitable solid support for the stepwise syn7442
Nucleic Acids Research
(wih) 1 a1n
DttrityUtion
Pyrtdine (*<ash) 1 sin
5 ain
\
Capping 10 •In
.
y
I—
Pyridiw(wsh) \ ain—1
Coupling 60-90 Bin
/ * ~
^ \
Oetritylition 5 a i n
CHjC^-fPrOH (Mlh) lain
\
<
Pyridine 1(«sh) 1 airi
Quenching 5 «1n
TKF («sh)
1 ain
F1g. 2 - Cycle of reaction steps for the stepwise oligonucleotide synthesis
on Porasil B.
Detr1tylat1on : saturated solution of ZnBr. 1n Isopropanol:d1chlororaethane
2
(3:7, v/v)
Quenching : n-butanol :lut1d1ne:THF (4:1:5, v/v/v)
Coupling : 0.1 M dinucleotide blocks 1n pyridine + 0.5 M MSNT
Capping : acetic anhydride : capping mix (1:4, v/v)
(capping mix » THF:pyr1d1ne:dinethylam1nopyr1d1ne; 9:1:0,6 v/v/W)
thesis of oligonucleotides by the phosphotriester approach. All steps described 1n the cycle (Fig. 2) are compatible with Porasil B. The detritylation reaction with zinc bromide (22) was effective for chain lengths of
up to 17 nucleotides, treating for 5-10 minutes at rooa temperature with a
saturated solution of zinc bromide 1n Isopropanol:d1chloromethane
v/v)
but was retarded with Increasing chain lengths.
(3:7,
OUgonucleotides
longer than 17 units required longer periods of contact with zinc bromide,
usually 20-30 minutes for complete detritylation. Use of zinc bromide as
the detritylating agent allowed successful synthesis of oligonucleotides
ending with N-benzoyl deoxyadenosine at the 3'-end attached via a sucdnate
linkage to the silica
support
(22,23). Each detritylation reaction was
followed by quenching to remove chelated zinc bromide. Each dinucleotide
block was added sequentially to the solid support as a 3'-triethyl ammonium
salt
(F1g. 1) 1n anhydrous pyridine. The reaction was carried out In a
small glass column fitted with a sintered glass f i l t e r and a stop-valve.
After 60-90 nrlnutes of condensation, excess reagents were filtered out by
flushing with dry nitrogen, the solid support washed with pyridine and the
unreacted hydroxyl groups capped before proceeding to the next step of the
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Nucleic Acids Research
cycle (F1g. 2 ) .
The v a l i d i t y of the cycle and compatibility of each step to Porasil are
demonstrated by the synthesis of following oHgonucleotides:
16mer (A) d-CAGCCATCACGGACCC
16mer (B) d-CAGCCTTCACGGACCC
2liner (C) d-TGATTTCTGCTCTGACAACCT
25mer (D) d-ATGAAGTAAAAGTTCCTTAGGAnT
29mer (E) d-CAAACCCAATGGTCCGAATTCAAACTGCA
31mer (F) d-CTGTTAATGAAGTAAAAGTTCCTTAGGAnT
14mer (G) d-CACCA(A/G)CACTCATA (mix)
The mixture
H0-ABz_ (S\
51 CACCA(A/G)CACTCATA 3' was synthesized by starting with
followed
t
by
condensing
with
suitably
protected
triethyl-
anmonium salts of T, CA, CT and CA. This yielded a support HO-CACTCATA-(V
after d e t r i t y l a t i o n . The total resin was washed, dried and weighed. I t was
then divided Into two equal parts. One part was condensed with the dinucleotide block AA, the other with AG 1n different glass columns and f i n a l l y
d e t r i t y l a t e d . The t r i t a n o l absorbance was measured at 498 run from the total
resin.
Each part
of
the resin now contained sequences,
and HO-AGCACTCATA-(T) 1n the protected triester
HO-AACACTCATA-(V
form, having 0.60 and 0.71
absorbance units at 498 nm respectively of the released dimethoxytritanol
on d e t r i t y l a t i o n .
The two resins were mixed together
with the dinucleotide
and then condensed
blocks CC and CA successively to obtain the mixed
sequence CACCA(A/G)CACTCATA-(T).
Similarly we synthesized several oligonucleotides (A-F) ranging from 1631roer 1n length. The overall
coupling yields were determined by measuring
the t r i t y l cation absorbance spectrophotoraetrically and were found to be 1n
the range of 72 *-96 1, I l l u s t r a t e d by the 29mer synthesis (Table 1 ) . After
completion of synthesis, performed using 100 mg of s i l i c a ,
solid
support
nucleotide
was subjected
o-chlorophenyl
to
oximate-promoted
protecting
groups
(15).
a part of the
deprotection
This
of
1nter-
was followed by
treatment with concentrated anrtonia at 5O*C overnight, f i l t r a t i o n through a
glass f i b r e
filter
(GF/E, Whatman) and concentration
In the presence of
pyridine to avoid Inadvertant cleavage of d1methoxytr1tyl
groups. The pro-
duct was f i r s t p u r i f i e d by preparative thin-layer chroraatography on s i l i c a
gel
In
chlorofonn:raethanol
(9:1, v/v).
The band containing
the
product
(usually at the o r i g i n ) was scratched from the thin layer plate and eluted
with 50 % aqueous ethanoi. The dimethoxytrityl oligonucleotide
(DMTr-DNA)
was then p u r i f i e d over a ^-Bondapak C-18 column (Haters) by high perform-
7444
Nucleic Acids Research
Table 1 - Absorbance units at 498 nm of the released diraethoxytritanol
using ZnBr
after each successive coupling step during the
synthesis or the 29roer.
DHTrOH
Chain length sequence of linked oiigonucleotide
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
DMTr
- A
- GCA
- CTGCA
- AACTGCA
- CAAACTGCA
- TTCAAACTGCA
- AATTCAAACTGCA
- CSAATTCAAACTSCA
- TCCGAATTCAAACTGCA
- GGTCC6AAnCAAACTGCA
- ATG6TCC6AATTCAAACT6CA
- CAATGGTCC6AATTCAAACTSCA
- CCCAATGGTCCSAAnCAAACTSCA
- AACCCAATGGTCC6AATTCAAACTSCA
- CAAACCCAATGGTCC6AATTCAAACT6CA
A
498
3.68
2.35
2.21
1.73
1.67
1.41
1.15
0.83
0.76
0.64
0.52
0.50
0.45
0.40
0.35
Yield per coupling itep
64
94 1
78 1
96 1
84 )
82 1
72 1I
91 X
84 X
81 X
96 X
90 X
89 X
87 I
ance l i q u i d chroroatography using 20 %-3O % a c e t o n i t r i l e gradients 1n 0.1 Kl
t r 1 ethyl ammonium acetate at pH 7.0 ( 2 4 ) . The product peak was usually the
major well defined peak and, being the most retarded, could easily be Iden-
_3_ - HPLC elution p r o f i l e s on ^-Bondapak C,_ column using a 20 %-30 X
a c e t o n i t r i l e gradient 1n O.I M Triethylararaoniura acetate. The peak
marked with an arrow contains the oligonucleotide.
(a) DMTr-29raer (E)
(b) DMTr-14raer mix (G)
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Nucleic Acids Research
t i f i e d (F1g. 3 ) .
The overall y i e l d of DMTr-29mer was estimated to be 8 I from the total r e covered absorbance
Similarly,
units
of
the
product
based on the
first
nucleoside.
the Isolated yields of DMTr-ol1gonucleot1des A to F were found
to be 32 %, 30 %, 21 %, 16 I , 8 X and 5 % respectively. The product 1n the
peak was concentrated and d e t r i t y l a t e d using 80 % aqueous acetic a d d for
25 minutes at room temperature. Excess acetic acid was removed by d i l u t i n g
the
reaction
evaporation
mixture
and extracting
with
of remaining acid with ethanol
product was labelled at the 5' end with
32
diethylether
followed
by co-
1n vacuum. An aliquot of the
P using polynucleotide kinase and
found to be usually 80 4-90 % pure as estimated by 20 S polyacrylamide gel
electrophoresis under denaturing conditions (F1g. 4 ) .
OUgonucleotides
geneity after
acetonitMle
longer
of
15-16
gradients
In 0.1 ^
oligonucieotides
phoresis.
1n length could be further
purified
to homo-
d e t r i t y l a t i o n by HPLC on (i-Bondapak columns using 5 % -20 %
were
t r i ethyl ammonium acetate buffer,
purified
by
polyacryl amide
However, the products Isolated as DMTr-DNA after
run were found to be of high quality
simply after
gel
whereas
electro-
a single HPLC
detritylation.
OUgo-
nucleotides purified 1n such a way have already been used for further biological
experiments
(25).
In a d d i t i o n ,
the 21mer
has been successfully
used as hybridization probe to I s o l a t e clones containing human a - I n t e r f e r o n
sequences
(C. ttorcamp. Personal
communication).
4
<
7446
Similarly,
the 25mer and
- Sizing of oligonucleotides
by 7 M urea-20 % polyacrylamide gel
electrophoresis
after a single HPLC run.
Nucleic Acids Research
^
B
^
* G
V
G
G
:
•
A
A
*C
T
'
C
^
G
A
^
F1g.
A
5 - Wandering spot analysis of the 16mers (A) and (B) after
labelling.
I . Electrophoresis on cellulose acetate (pH 3 . 5 ) .
II. Homochromatography on DEAE c e l l u l o s e .
5'-end
the 31mer have been used to I s o l a t e the clones coding for p - I n t e r f e r o n (D.
Oupret.
Personal
conmunication).
After
5'-end
labelling
the sequence of
oilgonucieotides up to 16mer was confirmed by wandering spot analysis (21)
(F1g. 5) and the Maxam and G i l b e r t technique was employed for longer o l i g o nucleotides ( 1 9 ) .
CONCLUSION
Our r e s u l t s demonstrate that by judicious choice of s i l i c a the stepwise
chemical synthesis of deoxyoligonucieoti des can be performed u t i l i z i n g the
phosphotriester method of synthesis. Recent r e s u l t s on the synthesis of 12mer oiigonucleotide
published by Ohtsuka et a l .
(26) show that a r e l a t e d
support, Porasil C, can also be used. HPLC-grade s i l i c a I s r e l a t i v e l y I n e x pensive, r e a d i l y accessible and we believe I t 1s an a t t r a c t i v e candidate to
form the basis for an automated system of sol Id-phase oligonucleotides synthesis.
ACKNOWLEDGEMENTS
We wish to thank Or. M.J. Gait for useful discussions, D.
Einhorn,
Y.
7447
Nucleic Acids Research
Cordier
typing
and P. Mensch
the
manuscript.
for
their
technical
We are grateful
assistance
to
Profs.
and I .
P.
Batra for
Chambon
and P.
Kouriisky for their continued Interest 1n this work. We would also l i k e to
thank Dr. R.
Lathe for c r i t i c a l l y reading this paper.
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