Improved synthesis of oligodeoxyribonucleotide using 3-methoxy-4-phenoxybenzoyl group for amino protection

Abstract

3-Methoxy-4-phenoxybenzoyl group has been used for the protection of exocyclic amino group of nucleosides. In case of 2′-
deoxycytidine it has been found to be highly selective under controlled conditions. The N-protected derivatives of 2′-deoxyadenosine
and 2′-deoxyguanosine have been found to be sufficiently stable towards acids minimising depurination under conditions of
synthesis of oligodeoxyribonucleotide on solid support via phosphotriester approach. The high lipophilicity of the group and
milder deprotection conditions are additional advantages.

Full text

Improved
synthesis
of
oligodeoxyribonucleotide
using
3-methoxy4-phenoxybenzoyl
group
for
amino
protection
Rakesh
K.Mishra
and
K.Misra
Nucleic
Acids
Research
Laboratory,
Department
of
Chemistry,
University
of
Allahabad,
Allahabad-211
002,
India
Received
18
June
1986;
Accepted
17
July
1986
ABSTRACT
3-Methoxy-4-phenoxybenzoyl
group
has
been
used for
the
pro-
tection
of
exocyclic
amino
group
of
nucleosides.
In
case
of
2'-
deoxycytidine
it
has
been
found
to
be
highly
selective
under
con-
trolled
conditions.
The
N-protected
derivatives
of
2'-deoxyade-
nosine
and
2
'-deoxyguanosine
have
been
found
to
be
suf
ficiently
stable
towards
acids
minimising
depurination
under
conditions
of
synthesis
of
oligodeoxyribonucleotide
on
solid
support
via
phos-
photriester
approach.
The
high
lipophilicity
of
the
group
and
milder
deprotection
conditions
are
additional
advantages.
INTRODUCTION
Although
attempts
have
been
made
from
time
to
time
to
achie-
ve
outright
selective
protection
of
the
amino
function
in
nucleo-
sides,
notable
success
has
only
been
achieved
in
case
of
2'
-deoxy
cytidine
and
cytidine
employing
different
reagents
for
benzoyla-
tion
(1-5).
with
other
groups
success
has
been
achieved
in
vary-
ing degree
(6,7).
Jones,
procedure
is
a
general
one,
whereby
pr-
ior
silylation
of
hydroxy
functions
is
employed
(8).
3-Methoxy-4-phenoxybenzoyl(NPB)
group
has
now
been
used
in
case
of
2'-deoxyadenosine,
2'-deoxycytidine
and
2'-deoxyguanosi-
ne.
In
case
of
2'-deoxycytidine
it
has
been
found
to
be
highly
selective
for
amino
function
under
controlled
conditions,
while
in
case
of
2'-deoxyadenosine
and
2'-deoxyguanosine,
the
N-protec-
ted
derivatives
have
been
prepared
by
full
protection
followed
by
selective
hydrolysis
of
the
o-ester
functions
at
alkaline
pH.
In
the
conventional
routes
of
oligonucleotide
synthesis
di-
methoxytrityl
(DMTr)
or
monomethoxytrityl
(MMrTr)
groups
are
renm-
ved
by
acid
hydrolysis.
This
causes
some
depurination
thereby
lowering
the
yield
of
the
final
product
(9,10).
To
overcome
this
©)
I
RL
Press
Limited,
Oxford,
England.
Nucleic
Acids
Research
Volume
14
Number
15
1986
6197

Nucleic
Acids
Research
problem
efforts
have
been
made
to
facilitate
the
removal
of
DMWr
or
MMTr
groups
by
alternative
reagents
(11,12)
or
new
protecting
groups
for
5-OH
function
have
been
explored
(13,14).
An
alter-
native
solution
lies
in
changing
the
protecting
group
on
amino
function.
Phthaliimide
derivative
of
2'-deoxyadenosine
has
been
found
to
be
useful
in
oligonucleotide
synthesis
via
phosphotries^
ter
approach
(15).
other
bulky
groups
have
been
reported
to
make
better
stable
derivatives.
N-Methyl-2-pyrrolidine
amidine
(16)
and
dialkyl
formamidine
derivatives
of
2'-deoxyadewsine
(17)
have
been
shown
to
be
less
susceptible
to the
protic
acid
depuri-
nation
than
N-benzoyl
derivatives.
N-MPB
derivatives
of
2'-de-
oxyadenosine
and
2'-deoxyguanosine
have
been
found
to
be
highly
stable
and
better
acid
resistant
than
the
comnonly
used
deriva-
tives.
MATERIAL
AND
METHOD
Nucleosides,
N-protected
deoxyadenosine
and
deoxyguanosine,
5'
-Q-dimethoxytrityl
thymidine-3'
-Q-2
-chlorophenyl
phosphate
(triethylamnoninum
salt),
dimethoxytrityl
chloride,
mesitylene-
sulfonyl-3-nitrotriazole
(MSNT),
1,1,3,
3-tetramethylguanidine
and
4-nitrobenzaldoxirne
were
purchased
from
Cruachan
chemical
Co.
1-Methylimidazole,Dcc,FMoC-glycine
,vanillic
acid
and
ninhydrin
were
purchased
from
Fluka
and
sigma
Chemical
Co.
3-Methoxy-4-
phenoxybenzoic
acid
was
prepared
by
treating
vanillic
acid
with
phenylbromide
in
presence
of
KOH
and
Cu.
It
was
later
converted
to
its
chloride
by
treatment
with
PC13(IS).
Composite
Polydimethylacrylamide/Keiselguhr
support
and
DNA-
bench
synthesiser
(CMNIFIT
Ltd.,Cambridge)
were
used
for
solid
phase
synthesis
(19).
HPLC
was
done
on
LKB
ultrapac
TSK
DEAE-3SW
column
(7.SxlSOmm)
and
W
absorption
was
measured
on
Hitachi
2205
spectrophotometer.
solvents
used
were
duly
purified
prior
to
use.
Pyridine
was
refluxed
with
ninhydrin
and
distilled
over
KOH.
Tlc
was
done
on
silicagel
G
(Merck)
plates
and
sprayed
with
iodine,
H2S04
and
perchloric
acid
for
location
and differentiation
of
spots.
Yield
reported
is
the
percent
of
theoretical.
A
small
portion
of
all
the
new
derivatives
was
hydrolysed
for
confirmation
of
their
structure
(20).
6198

Nucleic
Acids
Research
Preparation
of
N4
-(
3-.methoxy-4.-phenoxybenzoyl
)-2
'-deoxycytidinem
2'-Deoxycytidine
(263.5
mg,;
1
rmnole)
was
dried
by
evapora-
ting
in
vacuo
with
3
x
dry
pyridine
(5
ml).
It
was
then
suspen-
ded
in
dry
pyridine
(10 ml)
and 3-methoxy-4-phenaxybenzoyl
chlo-
ride
(262.5
mg;
1
[mole)
was
added
in
cooled
suspension
(00).
Flask
was
sealed
and
shaken
in
dark
raising
temrperature
to
500
in
4
h.
A
clear
yellowish
solution
was
obtained.
Completion
of
reaction
was
checked
by
tlc
in
benzene/mathanol
(8.5:1.5;
v/v;
new
spot
of
Rf
0.
61).
Reaction
minxture
was
now
evaporated
to
a
gum
in
vacuo.
It
was
taken
in
dry
pyridine,
concentrated
and
ethanol
was
added
gradually
till
slight
turbidity
appeared.
After
few
hours
white
precipitate
settled
dovin.
The
product
thus
obtained
was
filtered,
washed
with
2
x
ether
(
5
ml
)
and
dried.
The
white
powder,
chromatographically
single
(N
max
303
nm
and
250
nm)
was
directly
used
for
further
reactions.
Yield
0.405
g
(90%).
Preparation
of
51-0_
_(4,4
'-dimethoxytrityl
)-N4-(
3-methoxy-4-phe-
nox
ybenzoyl
)-2
'-deoxycytidine
N
-(
3-MKethoxy-4-phenoxybenzoyl
)
-2
'-deoxycytidine
(
360
mg;
0.8
mnole)
was
dried
by
evaporating
with
2
x
dry
pyridine
(3
ml).
It
was
taken
in
dry
pyridine
(
4
ml)
and
4,4'-dimethcocytrityl
chloride
(
298
mg;
0.88
mole)
was
added
in
small
portions.
Flask
was
sealed
and
kept
in
dark
for
4
h.
asaction
was
follow-
ed
by
tlc
in
dichlor6methane/methanol
(9:1;v/v;
new
spot
at
Rf
0.82).
After
completion
of
the
reaction,
mi;xture
was
poured
into
water
(
10
ml)
and
extracted
with
2
x
dichloromethane
(lOmll
It
was
then
washed
with
5%
NaHC03
(
5
ml
)followed
by
water(5ml).
Solution
in
dichloromethane
was
evaporated
in
vacuo
with
6
x
di-
chloromethane
(10
ml).
The
water
free
gum
thus
obtained
was
dissolved
in
mirnimn
amount
of
dichloromethane
and
precipitated
by
gradual
addition
of
petroleum
ether
(40O-60°).
Process
of
precipitation
was
repeated
to
remove
most
of
the
pyridine
and
4,4'
-dimethoxytritylalcohol.
Finally
the
product
was
purified
on
silica
gel
colum(2.5g
silica
gel;
eluent
petroleum
ether
(40°-60°)
with
increasing
ratio
of
methanol),Nmax
303
nm
and
260
nm;
yield
0.530
g,(88%).
6199

Nucleic
Acids
Research
Preparation
of
56
_O-
4
4'-d.imetho
Ltnity1-N
4...3-methox
--
he-
noxybenzoyl
)-2'
-deoxyctidine-3'
-0-succinate
5
'
Q0-.(4,
48
'_Dimethoxytrityl
)
-N
-
(
3-methoxy-4-phenoxybenzoyl)
-2'-deoxycytidine(378
mg;
0.5
rruKle)
was
dissolved
in
pyridine
(2.5
ml).
Triethylamine(0O.2
ml)
and
succinic
anhydride
(55
mg;
0.55
mmole)
were
added.
Mixture
was
kept
at
roan
temperature
for
12
h
and
then
applied
to
a
Dowex-50
(pyridinium
form)column
(8x3
cm).
Column
was
eluted
slowly
with
pyridine/water(l:4,v/v).
E3lute
was
evaporated
to
dryness,
redissolved
in
dichlororrethane
and
purified
on
silical
gel
colunn(5x2
cm).
Elution
was
first
done
with
dichloromethane
followed
by
ethanol/dichlororrethane
(3:97;
v/v),
Fractions
of
the
latter
eluent
were
pooled
and
trityl
positive,
high
Rf
spot
on
tlc
(low
retention
time)
was
characterised
as
the
desired
product.
Pure
fractions
(as
che-
cked
by
tlc
in
ethanol/dichloromethane;
1:9,v/v;
for
single
trityl
and
sugar
positive
spot)
were
evaporated
to
dryness
in
vacuo
and
precipitated
into
ether/pentane
(3z2,v/v).
Yield
324
ig,
(70/).
Preparation
of
d(TTTC)
5'0o-(4,
4
'-Dinethoxy
trityl)-N4-(3-Methoxy-4-phenoxybenzoyl)
-2'-deoxycytidine-3'-0-succinate
(0.
25
mmrole)
was
linked
to
the
support
(Kieselguhr/polydin'ethylacrylamide,
500
mgr)
with
the
help
of
a
spacer
of
ethylenediamine
and
two
glycine
units(19).
Loading
was
estimated
to
be
68p
nil
g1.
This
derivatised
resin
was
used
for
the
preparation
of
tetranucleotide.
Functionalised
dry
resin
(100
rg)
was
taken
in
the
column
of
the
"DNA-bench
synthesisern
and
allowed
to
stand
in
DMF
for
few
min.
Then
the
cycle
of
solvents
was
followed
in
the
seque-
nce;
pyridine
(5
mmi
)
dichloromethane
(3
main),
10%
TCA
in
di-
chloromethane
(3
irin),
DMF(2
min)
and
finally
pyridine
(5
min).
Flow
rate
was
kept
at
2
ml
min
1.
After
the
conpletion
of
first
cycle
flow
is
stopped
and
a
solution
of
triethylamoniwn
salt
of
56'.Q-DmTr
thymidine-3'-Q-2-chlorophenyl
phosphate
(55
p
mole)
in
anhydrous
pyridine
(0.4
ml)
freshly
activated
with
MST
(82.5
mg;
275
,u
mle)
and
1-methylimidazole
(0.C01
ml)
was
injected
onto
the
column
over
a
three
minute
period
via
a
Hamilton
syringe
(1750
RN-3150801).
Column
is
allowed
to
stand
for
20
min
before
the
second
cycle
of
washing
starts.
After
the
third
coupling
support
was
washed
with
pyridine
and
dichlorome-
6200

Nucleic
Acids
Research
thane
and
then
with
ether.
It
was
then
taken
out
of
the
column
and
dried.
Deprotection
of
the
tetramer;d(TTTC)
support
linked
to
the
tetranucleotide
chain
was
given
following
treatnrnt
to
completely
deprotect
the
tetramer.
(a)
The
support
taken
in
a
3
ml
eppendorf
tube,
was
treated
with
a
solution
of
4-nitrobenzoldoxime
(200
mg)
in
3
ml
of
dioxane/
water
(1:1)
to
which
143
)z
of
1,1,3,3-tetramethylguanidine
had
been
added.
The
mixture
was
left
at
rocm
temperature
for
over-
night
and
filtered
through
a
small
sintered
glass
funnel.
The
support
was
washed
well
with
dioxane/water
(1:1)
and
filterate
collected.
(b)
The
filterate
obtained
as
above
was
dried
in
vacuo
and
taken
in
40%
ammuonia
(6
ml).
The
flask
was
sealed
well
and
immersed
in
a
water
bath
at
500
for
4
h.
(c)
The
flask
was
taken
out,
cooled
and
the
liquid
evaporated
in
vacuo
to
dryness.
To
this
was
added
80%
acetic
acid
(
6
ml)
and
left at
room
tempera-
ture for
30
min.
It
was
then
evaporated
in
vacuo,
taken
in
water
(6
ml)
and
washed
with
5
x
ether
(
3
ml).
The
aqueous
solution
was
evaporated
again
and
taken
up
in
water.
Purification
of
the
tetramer,
d(TWTC)
A
gradient
hplc
system
was
set
up
using
a
TSK
DEAE-3SW
colu-
mn
(LKB)
(7.5
x
150
mm)
with
buffers
of
1
nM
and
0.
3
M
KH2PO4,pH
6.3,in
acetonitrile/water
(30:70,v/v)
as
solvents
A
and
B
respectively.
Fractions
were
monitored
at
270
nm
and
pooled.
Pool
containing
tetranucleotide
was
desalted
by
pass-
ing
it
through
Biogel
p2
column,
using
ethanol/water(2s8)
as
eluent.
Yield
of
the
tetramer
was
72.6%.
preratin
of
-(
3-thoxy-4phenoxybenzoyl)
2'
-deoxyguanosine
2'-Deoxyguanosine
(255
mg;
1
rmmole)
was
dried
by
evapora-
ting
with
3
x
dry
pyridine
(
1
ml).
It
was
then
taken
in
dry
pyridine
(2.5
ml)
and
treated
with
3-methoxy-4-phenoxybenzoyl
chloride
(
525
mg;
2
nmole).
After
shaking
for
2
h
in
dark
dichloromethane
(
5
ml)
was
added
to
the
reaction
mixture.After
a
further
shaking
of
2
h
a
clear
solution
was
obtained.
Comple-
tion
of
the
reaction
was
checked
by
tlc
(dichloromethane/ethanol,
9.5:O.5;
v/v).
It
was
then
evaporated
in
vacuo
to
gum.
The
gum
was
dissolved
in
ethanol
(2.5
ml);
2
N
NaOH
was
added
to
bring
the
pH
of
the
solution
to
12,
stirred
in
ice
bath
for
10
6201

Nucleic
Acids
Research
min
and
then
neutralised
by
excess
pyridinum
Dowex-50
resin.
Resin
was
filtered
off
and
washed
with
aqueous
pyridine.
Washin-
gs
and
filterate
were
collected
concentrated
in
vacuo
and
cooled.
white
solid
thus
separated
was
filtered
out,
dissolved
in
dry
benzene(
minimum
volume),
dry
hexane
was
added
dropwise
and
the
product
crystallised
by
leaving
the
solvent
at
room
temperature
after
a
slight
turbidity
appears.
The
crystallisation
was
repe-
ated
till
when
a
chromatographically
single
entity
was
obtained
(Rf
0.5
in
chloroforuVethanol,
9:1,
v/v).
UV
absorption
Amax
270
and
235
nm;
yield
375
rmg,
(70%).
Preparation
of
N6-(
3-methoxy-4-phenoxyenzyl)-2
-deoxydeosine
2'-Deoxyadenosine
(250
mg;
1
rrnole)
was
rendered
dry
by
eva-
poration
with
3
x
dry
pyridine
(
2
ml).
It
was
suspended
in
dry
pyridine
(
3
ml)
and
treated
with
3-methoxy-4-phenoxybenzoyl
chloride
(630
no;
2.4
nmole).
Reaction
was
checked
for
cofple-
tion
by
tlc
(chloroforrv/methanol,
9.5:O.5,
v/v)
after
2
h
shaking
in
dark
at
room
tenperature.
After
corpletion,
the
reaction
mixture
was
poured
into
water
(
4
ml)
and
extracted
with
4
x
di-
chloromethane(
2ml).
organic
solution
was
evaporated
to
gum.
It
was
dissolved
in
2
ml
pyridine/ethanol
(4:l,v/v)
cooled
in
ice
bath
and
2
N
NaOH
was
added
to
bring
the
pH
to
12.
After
10
min,
excess
of
pyridintmDowex-5o
resin
was
introduced
for
neutralisation.
Resin
was
filtered
off
and
washed
with
aqueous
pyridine.
Filtrate
and
washings
were
combined
and
evaporated
in
vacuo
to
half
the
volume.
It
was
washed
with
2
x
ether(l
ml),
further
evaporated
to
reduce
the
volume
and
cooled.
Solid
that
appeared
wvas
filtered
and
recrystallised
from
pyridine/ethanol
to
a
chromatographically
single
(Rf
0.6,
chloroforr/ethanol,9:l,
v/v)
product.
uv
absorption
\
max
282
and
274
nm,
A
min
280
nMn
yield
60%.
Estimation
of
degree
of
depurination
Dilute
solutions
(15
)j
mole
ml1)
of
N2-MPB-dG,N2-ibu-dG,
dG,
N6-BP-dA,
N6-Bz-dA
and
dA
were
prepared
by
dissolving
0.105
nnnole
of
each
in
7
ml
solvent.
solvent
uid
for
N-protected
nucleosides
was
dichloromethane
and
that
for
free
nucleosides
was
water.
All
the
solutions
were
divided
in
seven
portions
1
ml
each.
Each
1
ml
portion
was
treated
with
80%
acetic
acid
(2
ml)
6202

Nucleic
Acids
Research
at
roomn
tenperature.
Reactions
were
quenched
by
triethylamine
methanol
(4sl,v/v)
at
varied
tine
intervals.
In
the
case
of
dG
and
its
derivatives
quenching
tin-e
was
10
min,
20
min,
40
min,lh,
2h,
4h,
and
20h
and
in
the
case
of
dA
and
its
derivatives
it
was
15
min,
30
min,
lh,
4h,
lOh,
24h
and
72h.
After
quenching
each
portion
was
evaporated
to
dryness
and
taken
in
small
amrrnts
of
solvents
(dichloromethane
for
N-protec-
ted
nucleosides
and
water
for
free
nucleosides).
These
solutiors
were
applied
on
preparative
tlc.
solvent
for
tlc
was
chloroformv
methanol
(95:5,v/v)
for
N-protected
nucleosides
and
chloroforv'
methanol
(80:20,
v/v)
for
free
nucleosides.
Two
bands
were
obtained
in
each
plate.
Upper
band
was
the
depurinated
part
(heterocyclic
base
without
sugar)
and
the
lower
was
nondepuri-
nated
one.
These
bands
were
scratched
and
eluted
in
dichloromethane
in
the
case
of
N-protected
nucleosides
and
in
water
in
case
of
free
nucleosides.UV
absorption
was
measured
at
wavelengths
270
nm
for
N2-MPB-dG,
260
nm
for
N2-ibu-dG,
and
dA,
252
run
for
dG,
280
nm
for
N6-_MB-dA
and
N6-Bz-dA.
Total
OD
of
each
band
was
taken
for
the
concentration
and
percentage
of
depurination
was
calculated
by
multiplying
the
ratio
of
the
total
OD
of
upper
and
lower
band
with
100.
Results
are
tabulated
below.
Coprative
stud
Of
de
rtection
of
MPB
and
other
conventional
arouPs
N-MPB
derivatives
of
dC
-,
dG
and
dA
were
taken
(5
x
3
mg
TABLE-1.
Depurination
of
dA,
dG
and
their
derivatives.
Time
D
r
Time
%
Depurination
N2-MPB-dG
N2-ibu-dG
dG
N6-MPB-dA
N
-Bz-dA
dA
10
min
0.8
7.4
0.9
15min
2.2
3.3
0.51
20
min
1.5
18.0
1.6
30min
3.3
4.6
0.82
40
min
5.4
22.0
2.1
1
h
13.2
21.0
1.2
1
h
6.5
34.5
4.0
4
h
34.0
52.5
3.5
2
h
9.6
49.0
7.2
lOh
51.2
66.0
13.0
4
h
33.7
61.6
22.5
24h
81.3
83.0
22.4
20h
88.5
99.0
85.0
72h
100.0
100.0
69.0
6203

Nucleic
Acids
Research
each)
in
5
ml
flasks.
Concentrated
ananonia
(2
ml)
was
added
to
all
the
flasks
and
the
mixture
was
worked
up
after
1
h,
2
h,
3
h,
4
h
and
5
h
at
40°C.
Deprotection
)
was
estimated
by
analysis
on
the
tlc
plats
(dichloromethansethanol,
9sl,
v/v)
.
Same
experiment
was
repeated
at
500C
and
60°C.
Results
obtained
are
tabulated
below
(Table
-
2)
.
Depurinations
studies
under
synthesis
conditions
5'DMTr-N-MPB-nucleoside-3'o-succinates
of
dAK
and
dG
were
prepared
exactly
as
that
of
dc
mentioned
above.
Polyacrylamide/
Kieselguhr
composite
support
was
functionalised
using
these
derivatives
as
with
corresponding
dC
derivative.
Loading
obtained
in
the
case
of
dA
and
dG
was
40
moleg
1
and
35
moleg'1
respectively.
Functionalised
resine
was
loaded
on
to
the
column
of
'DNF
Bench
Synthesiser'
and
synthesis
of
tetra
nucleotides
d(TTM)
and
d(TT2G)
Was
done
as
mentioned
above
(19).
After
NTBLE-2.
Amronolysis
of
N-protected
nucleosides
Temp
Time,_
deprotection
(X)
(0C)
(h)
MPBd
4Bzd
N2MPBdG
NibudG
NrMPBdA
N6BzdA|
1
31
15
28
12
32
16
2
48
34
45
30
49
33
40
3
75
52
70
45
77
51
4
92
70
89
66
93
72
5
100
81
96
75
100
80
1
38
31
37
30
40
31
2
69
54
70
51
73
56
50
3
90
75
88
72
93
76
4
100
92
100
90
100
90
5
-
100
-
100
-
100
1
52
40
52
38
54
41
2
78
64
77
60
79
65
60
3
95
85
93
82
98
86
4
100
100
100
100
100
100
5
_
_
e-
a
m
6,04
5OC)
1.3.
1.6
143
10
1.
1.6
6204

Nucleic
Acids
Research
each
coupling
25
mg
of
resine
was
taken
out
for
the
depurination
studies
and
yield
estimations.
Capping
(21)
was
done
before
the
next
coupling
followed.
Yield
(%)
was
determined,
with
respect
to
starting
monomer,
of
the
di,
tri
and
tetra
nucleotides
direc-
tly
by
trityl
analysis
of
the
support
comparing
with
the
initial
amount
of
trityl
on
the
same
amount
of
resine.
Results
obtained
are
tabulated
below
(Table
-
3)
.
Resine
containing
mono,
di,
tri
and
tetra
nucleotides
were
divided
in
to
four
parts
(5
mg
each)
and
each
part
was
treated
with
10%
trichloro
acetic
acid/dichloromethan
(1
ml)
.
Hydrolysis
was
quenched
with
triethylamin/raethanol
(4sl,
v/v#
2ml)
after
4
min,
10
min,
20
min
and
40
min
in
each
case.
After
the
quench-
ing,
resine
was
thoroughly
washed
with
dichloromethan
followed
by
ether.
It
was
then
dried
in
desiccator
and
treated
with
2ml
of
concentrated
ammonia.
Phials
were
sealed
and
left
at
500C
for
24
h.
After
the
time
specified
resine
was
filtered
and
washed
with
water.
All
the
washings
and
filtrates
were
collect-
ed
and
back
washed
with
ether
(1/4th
the
amount
of
water
soluti-
on)
several
times.
Amount
of
the
non-depurinated
portion
was
estimated
in
CD
units
by
taking
UV
absorption
at
260nm-
It
was
compared
with
the
UV
absorptions
(260
nm)
of
the
aqueous
soluti-
on
obtained
on
delinking
the
resine
first
with
aminonia
then
removing
DMTr
by
80%
acetic
acid
followed
by
ether
washing.
This
represented
the
total
nucleoside
part
(no
depurination
takes
place
as
N-protecting
group
is
removed
before
acid
treatment).
Parallel
studies
were
carried
out
with
N2-ibu-X3
and
N6
-Bz-d
under
identical
conditions,.
Degree
of
depurination
was
calcula-
ted
and
results
obtained
are
given
in
table
-
4.
RESUTLT
AN)
DISCUSSION
3-Methoxy-4-phenoxybenzoyl
(MPB)
has
been
used
as
protecti-
ng
group
for
amino
sites
of
all
the
three
deoxyribonucleosides.
Table
-
3
.
Yield
of
oligodeoxyribonucleotides
6205

Nucleic
Acids
Research
B
-4.
D~
urination
Stid
Under
Sythe
is
Conditiong
Sequence
of
Time
N-f-derivative
N
erivative
nucleotides
(min)
%depurination
t½,
(h)
%depurination
t2
(h)
4
0.41
0.98
d
)
-
P*
10
1.30
8.1
2.45
3.4
20
2.06
4.90
40
4.12
9.80
4
0.34
0.81
d
(TA)
-P*
10
0.85
9.8
2.03
4
1
20
1.70
4.06
40
3.40
8.13
4
0.31
0.74
d
(TIM)
P*
10
0*79
10.6
1.85
4.5
20
1.57
3.70
40
3.14
7.41
4
0.30
0.72
d
(TTTA)-P*
10
o.76
10.9
1.81
4.6
20
1.53
3.62
40
3.06
7.25
4
0.53
1.59
d
(G)-P
10
1.32
6*3
3.98
2.1
20
2.65
7.94
40
5.29
15.87
4
0.44
1.33
d
(TG)-P*
10
1.10
7.S
3*33
2.4
20
2.19
6.67
40
4.39
13.32
4
0.40
1.19
d
(TTG)-P*
10
1.00
8.3
2.98
2.8
20
2.01
5.95
40
4.10
11.91
4
0.38
1.15
d
(T]TTG)-P*
10
0.97
8.6
2.87
2.9
20
0.97
5.75
40
3.88
11.49
tP
represents
the
solid
svwport.)
6206

Nucleic
Acids
Research
(9
()
~~~Pyridine
(9sa.
Fiqure
1.
Selective
protection
of
amino
function
in
2
'
d
eoxycy
tidi
ne
.
It
has
been
found
to
be
highly
selective
in
the
case
of
2'-deoxy-
cytidine
and
gives
90%
yield
under
controlled
conditions
of
ratio,
time
and
temperature
(figure
-
1).
In
the
case
of
2'-
deoxyadenosine
and
2'
-deoxyguanosine
(figure-2)
yield
is
compar-
able
to
that
with
benzoyl
and
isobuteryl
groups,
respectively,
by
Khoranas
method
(10,20).
MPB
can
work
equally
well
in
ON%K
idA
(60%)
N.
CH3
a2N
Naci
2N
NaCGi
N
/
NH
H~~N~k
(7
C)
Figure
2-.
Protection
of
amino
function
of
2-!deoxyadenosine
ag
2
'
deoxyguanosine.
6207

Nucleic
Acids
Research
lo
2o
30
Time
(ain)
Figure
3.
Ion
exchange
hplc
of
tetrarrer,
d(TTTC),
gradient
-7c%
buffer
B,
45
min,
temperature
200.
Jones
procedure
(8).
Removal
of
MPB
group
from
amino
protected
derivatives
is
comparatively
quick
under
milder
conditions*
It
can
be
removed
by
concentrated
ammonia
(40%)
at
S0°C
in
4
hours,
essentially
quantitatively,
The
half
life
of
the
derivatives
were
found
to
be
N4-MPBdC
1.3
h,
N2-MPBdG
1-3
h
and
N6-tMPBd!,
1.2
h,
whereas
the
half
life
of
N4
-BzdC,
N2-ibd
and
N6-BzcYN
are
1.6
h,
1.7
h
and
1.6
h
respectively.
too]t;
50-
4J~~~~~~~~
50
(
Ai
~(iii)
(Hiii
)
C
=
Li
$4~~~~A
8
18
36
54
72
4
10
15
Tine(h
)
Tine
(h
)
Piure
4.
Depurination
of
dA,
dG
and
their
N-acylated
derivatives
)
BzdA,
tI4h,
(ii)
Ng
MPB-dA,
t
9.5h,
(iii)
dA,t
5o,
t8J
(i)
N-ibu.dG,
t½
2.5
h
(ii)
N2-MPB-dG,
t½7.5
h
(iii)
dG,t
9h.
-15~~
6208

Nucleic
Acids
Research
(B)
Figure
5.
Illustration
of
corf
ormations
around
the
glycosyl
bond,
(A
anti;
B-
syn
).
The
advantages
envisaged
for
this
group
were
its
bulkier
nature
and
more
lipophilicity.
It
is
an
important
consideration
relating
to
N-acyl
protecting
groups.
Phosphotriester
oligomers
have
a
tendency
to
become
less
soluble
in
organic
solvents
with
increasing
chain length
due
to
polar
effects
(22)
.
This
can
make
experimental
procedure
difficult
in
connection
with
solvent
extractions
and
adsorption
chromatographic
methods.
As
evident
from
the
experimental
results
MPB
derivatives
are
obtained
in
better
yields,
procedures
are
simple
and
crystallisation
from
(A)
(
B)
Figure
6.
Stereostructures
of
N-MPB
derivatives
of
(A)
2'deoxy_
adenosine
and
(B)
2
Vdeoxyguanosine
as
in
aqueous
iedium
displaying
predominent
hydrophobic
interactions.
6209

Nucleic
Acids
Research
organic
solvents
is
easy
due
to
lipophilicity
of
the
group
which
is
also
well
suited
for
solvent
extraction
and
reversed
phase
hplc
of
protected
oligonucleotides
(23).
In
order
to
adjudge
the
efficiency
of
the
group
in
oligonu-
cleotide
preparation
by
solid
phase
method,
a
tetranucleotide
d(TT:C)
was
prepared
using
Gait's
procedure
(19).
An
over
all
yield
of
72.6%
was
obtained
which
calculates
to
approximately
90%
yield
per
coupling,
indicating
thereby
the
suitability
of
this
group
in
solid
phase
synthesis
(figure-3).
Good
yields
are
obtained
in
the
case
of
other
oligomers
also
as
shown
in
table-3.
Most
interesting
and
noteworthy
part
of
the
study
was
the
stability
of
the
MPB
derivatives.
Depurination
in
acid
PH
during
detritylation
(9,10)
is
a
persistent
problem
in
oligonuc-
leotide
synthesis
.
N-MPB
derivatives
of
2
-deoxyguanosine
and
2
-deoxyadenosine
have
been
fournd
to
be
much
more
stable
towards
acid
than
the
commonly
used
N-protected
nucleosides
(figure-4a
and
4b).
Some
groups
have
already
been
tried
for
their
depurin-
ation
resistant
nature
in
the
case
of
2'-deoxyadenosine
(15-17).
We
have
found
MPB
orking
better
for
rendering
acid
resistivity
equally
to
2'-deoxyadenosine
and
2'-deoxyguanosine
derivatives
(half
life;
N6-MPB-dk
9.5
h,
N2-MPB-c;
7.5
h)
.
Depurination
studies
were
also
carried
out
under
synthesis
conditions,
i.e.,
N-protected
nucleoside
on
solid
support
trea-
ted
with
10%
trichloroacetic
acid
in
dichloromethane
as
depuri-
nating
reagent.
Under
these
conditions
monomers,
dimers,
trimers
and
tetramers,
containing
one
adanine
or
guanine
base
each,
sho-
wed
more
emphatic
improvements
as
regards
the
depurination
resistivity
of
their
N-MPB
derivatives.
Half
life
of
the
N-MPB
derivatives
is
considerably
higher
than
corresponding
benzoyl
and
isobuteryl
derivatives
(table-4).
Acylation
of
amino
function
renders
protonation
of
N7
more
facile
(24)
in
the
case
of
purine
nucleosides
which
makes
glyco-
sidic
linkage
more
prone
to
hydrolysis
under
acidic
conditions
(25,26)
.
C.Morin
(27)
has
claimed
that
depurination
is
checked
in
N1-oxidated
(N1-
-
0)
derivative
of
2
'-deoxyadenosine
which
were
reported
earlier
(28,29).
Analogous
procedure
cannot
be
applied
in
the
case
of
2
-deoxyguanosine.
Purine
nucleosides
exist
in
syn
and
anti
forms
(figure-5)
6210

Nucleic
Acids
Research
the
latter
being
more
Orobable
(30)
.
Introduction
of
bulky
grotp
on
amino
functicn
inhibit's
rotation
of
N-glycosidic
linkage
and
il
aces
N
in
the groove
of
5
'-OH
and
heterocyclic
base.
This
is
the
reason
why
bulky
groups
are
sought
for
depurArnation
resistivity
(31).
In
the
case
of
N-MPB
derivatives
of
both
2
'-deoxyadenosine
as
viell
as
2'-deoxyguanosine
(figure-6)
proto-
nation
at
N7
is
inhibited
in
all
probability
due
tQ
two
main
factors
(i)
the
bulky
nature
of
the
group
which
prevents
free
rotation
of
the
glycosyl
bond
and
(ii)
its
hydrophobicity
which
inhibits
approach
of
H
0+
at
N7.
3
N4MPB-derivatives
of
2'-deoxyadenosine
and
2'-deoxyguanos-
ine
are
more
than
twice
and
four
times
stable,
respectively,
than
the
conmionly
used
benzoyl
and
isobuteryl
derivatives
under
the
acidic
pH
(80
acetic
acid)
required
for
detritylation
of
oligonucleotides.
Under
synthesis
condlitions
acid
resitivity
of
N-MPB
derivatives
is
on
an
avrerage
2.4
and
3
times
greater,
in
case
of
2'-deoxyadenosine
and
2'-deoxyguanosine
respectively,
than
the
corresponding
comnon
homologue.
On
considering
average
depurination
per
cycle
in
the
case
of
tetramer
synthesissan
MPM
derivative
gives
around
0.29%
depurination
as
against
O.&8%
when
benzoyl/isobuteryl
is
used
in
place
of
MPB.
Thus,
this
single
factor
increases
the
yield
of
oligonomer
by
0.51%
per
cycle.
In
conclusion
it
can
be
statcd
that
3-methoxy-4-phenoxy-
benzoyl
(MPB)
group
by
virtue
of
its
high
selectivity
in
case
of
21-deoxycytidine
and
comparatively
very
high
depurination
resis-
tivity
in
case
of
2'-deoxyadenosine
and
2'-deoxyguanosine
due
to
its
bulky
nature,
coupled
with
easy
handling
due
to
its
high
lipophilicity
during
crystallisation
of
its
derivatives,
solvent
extraction
and
reverse
phase
hplc
of
the
protected
oligonucleo-
tide
stands
advantageous
application
as
compated
to
otter
groups
so
far
reported.
Ackowleement
The
authors
are
thankful
to
the
department
of
Science
and
techrnlogy
(Government
of
India)
for
financial
assistance
.
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6213