Download General scheme of the phosphotriester condensation in the

Volume 12 Number 4 1984
Nucleic Acids Research
Geneml scheme of the phosphotriestef condensation in the oUgodeoxyribonudeotide synthesis with
arylsulfonyl chlorides and arylsulfonyl azoUdes
V.F.Zarytova and D.G.Knorre
Institute of Organic Chemistry, Siberian Division of the Academy of Sciences of the USSR, 630090,
prospekt Akademika Lavrentyeva 9, Novosibirsk, USSR
Received 3 January 1984; Accepted 2 February 1984
ABSTRACT
fHosphotriester condenaation (R0)(R'0)P0p (PDE) + R"0H
(R0)(R'0)(R"0)P0 (PTB) in the presence oJT arylsulfonyl
chloride (ArSOoCl) as well as arylsulfonyl azolides proceeds
in two steps
as revealed by -''P _O_R spectroscopy. Pyrophosphotetraester (PPTE) accumulates in over 80% yield in the
first step and converts to PTE in the second one, Nucleophilic catalysts of pyridine type (Hiv) are necessary in the
first step. The second step is catalyzed by Nu^ aa well as
by catalysts of the tetrazole type (Nu^-H). Base catalysis
operates in the latter case. With Nu"1 catalysts (pyridine,
4-N,N-dimethylaminopyridine, N-methylimidazole) the general
scheme may be presented as follows: ArSOpCl + Hu' -«
—
ArSOpNfi] + Cl~: ArS0-.Uu1 + PDE
(R0)(R'0)P(0)0S0pAr (I);
I
+
Nu1
(RO)(R'O)P(O)N51
(II);
II + PDE
*
-
[(RQ)(R'0)P0]
II + R"0H—•- (R0)(R'0)(R"0)P0.Catalysts of
20;
Hu2H type don't accelerate PPTE formation. In the second2 step
they partacipate2 most probably in the process PPTE + Nu H a=t
(R0KR'0)P(0)Nu (III) + PDE; III + R%_-*-(R0) (R« 0) (R"0)P0 +
+ rP" . The latter step is subjected to strong base catalysis.
INTRODUCTION
A major contribution to the present impressive success
in the chemical synthesis of oligodeoxyribdmucleotides was
made by phosphotriester condensation. The main advantages
of this approach over phosphodiester method elaborated by
Khorana are the possibility to use highly efficient silicagel chromatography for purification of intermediate oligonucleotides as well as the absence of. numerous inherent to phosphodiester condensation side reactions at internucleotide
phosphate groups [j-4]].
Although well known for a lot of years phosphotriester
approach became really efficient after Narang et al, have
proposed arylsulfonyl tetrazolides (ArSOpTet) as condensing
© IRL Press Limited, Oxford, England.
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reagents [5#63 instead of traditionally used arylsulfonyl
chlorides (ArSOpCl). Similar results were further obtained
with arylsulfonyl 3-nitro-1,2,4-triazolides (ArS02lITr) [7,8j.
A bit later mixtures of ArSOgCl with various catalysts tetrazole (TetH) [9-11] , 3-nitro-1,2,4-triazole (NTrH) [10-12],
dimethylaminopyridine (DMAP) [ 1 2 - H ] and N-methylimidazole
(Melm) [i 1,12,15-18J were demonstrated to be powerful condensing reagents in phosphotriester condensations. Some kinetic
measurements of the accumulation of phosphotriesters were
performed and consideration of mechanism of phosphotriester
condensation was presented in these papers P9,11,16].
Pew years ago we have started investigation of the mechanism of phosphotriester condensation by * P M R spectroscopy
previously found to be useful in similar research of phosphodiester synthesis [3,4,14,19]• The main goal of the present
paper is to summarize the most essential results obtained by
this approach and to consider them together with the data and
statements of the other scientific groups.
Two-step mechanism of phosphotriester condensation
As revealed by ^ P M R spectrum of the reaction mixture
containing equimolar amounts of (Tr)Tp(CgH.Cl) and T(Ac)
and three fold excess of trilsopropylbenzenesulfonyl chloride
(TPS) in pyridine in few minutes new signals appear in the
spectrum at 19.6, 19.9 and 20.2 ppm (Fig.ia). These signals
were assigned to three diastereoisomeric pyrophosphotetraes—
ters (PPTE) in accordance with the structure and position of
the signals. Additional chemical proves of the assignment may
be found in [12,20,21] . Later the signal of PPTE is going to
disappear giving rise to two close signals at 7.3 and 7.6 ppm
(Pig. 1b) in accordance with those expacted for two diastereoisomeric phosphotriesters (PTE) bearing one aromatic substituent [22,23]. The nearly quantitative formation of
(Tr)Tp(CgH.Cl)T(Ac) was demonstrated by TLC and electrophoresis. Thus the kinetics of phosphotriester condensation may
be easily followed by •* P HMR spectroscopy. The same is the
case for another phosphodiester (ClCgH.)pT(Ac).
The kinetic curves of the consumption of starting phos-
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JL
10
15
20
31, HMR spectra of the reaction mixture containing
Pig.1. '"P
0.1 (Tr)Tp(C6H4Cl), 0.1 M T(Ac), 0.3 M TPS in pyridine at 30° a) 6 min of reaction; b) 5 h of reaction.
phodiesters
(Tr)Tp(CgH.Cl) or (ClGgH-)pT(Ac) accumulation
of PPTE and of final PTE are presented in Pig.2 a,b.
It is seen that the overall process is rather sharply divided
into two steps. In the first one rapid accumulation of PPTE
takes place the maximum yield of the latter exceeding 80%.
This step is followed by significantly slower conversion of
PPTE to protected dinucleoside phosphate. Whereas maximum concentration of PPTE is reached within less than one hour its
conversion is still incomplete in 24 hours.
u
\.
to
20
Pig.2. Kinetic curves of the consumption of starting phosphodiester (1), accumulation of pyrophosphotetraester (2)
and phosphotriester (3) in pyridine solution at 30°.
Reaction mixtures contain:
a) 0.1 M (TrjTpCCgH.Cl), 0.1 M T(Ac), 0,3 M TPS
b) 0.1 M (ClC6H4)pT(Ac), 0.1 M Tr(T), 0.3 M TPS
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The stoichiometric equations of these two steps may be
written as follows:
Q
0
0
2 R'O-P-0" + ArS02Cl
•• R'0-P-O-P-OR' + ArS0~ + Cl"
(1)
OR
OR OR
0
0
Q
u
II
n
R'0-P-O-P-OR' + ArS02oCl + 2R"0H—-2 R'O-P-OR" + ArSO, + Cl"
II
I
3
.
OR OR
OR
+ 2H+(2)
R = - p-ClCgH.-
,
R',R" - protected nucleosides
(Here and below ionic species are presented without counterions. Most probably in pyridine and other solvents with low
dielectric constant the ionised particles exist mainly as ion
pairs).
It is obvious that to shorten essentially the time of phosphotriester condensation it is necessary first of all to accelerate the second step of the process.
As it was already mentioned significant hastening of the
process was achieved by change of arylsulfonyl chloride for
several arylsulfonyl azolides \j>,6,10-12], Kinetics of phosphotriester condensation in the presence of triisopropylbenzenesulfonyl 3-nitro-1,2,4-triazolide is presented in Pig.3a.
Comparison with Pig.2b demonstrates that in spite of lower
temperature (20° instead of 30°) the reaction is nearly accomplished in one hour. This is scarcely due to the presence
of ArS02Tet instead of ArSOgCl in the step (2). As it was
found [9,11,12,20,21] the mixture of ArS02Cl with tetrazole
is at least aa efficient as ArSOpTet. It was shown that no
conversion of ArSOpCl -fcoArSOpTet occurs in the reaction mixture [V]. Therefore, the significant acceleration of the process most likely has to be ascribed to TetH and HTrH liberated in the first rapid step of the process. As is seen in
Pig.3d and Pig.3e phosphotriester condensation proceeds at
high rate when mixture of ArSOgCl with either TetH or NTrH
is used*
Similar hastening of the process was found also when
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5O
to
*{*,,
Fig.3. Kinetic curves of the consumption of starting phosphodiester (1), accumulation of pyrophosphotetraester (2)
and phosphotriester (3) at 20° in pyridine solution
containing 0,1 M (ClCgH,)pT(Lev) and 0,1 II
(HCCgH-^pT in the presense of: a) 0.3 M triisopropylbenzene sulfonyl 3-nitro—1,2,4-triazolide; b) 0,3 M
TPS and 0.3 H N-methylimidazole; c) 0.3 M TPS and
0.3 U dimethylaminopyridine; d) 0.3 M TPS and 0.3 M
3-nitro-1,2,4-triazole; e) 0,3 M TPS and 0.3 M tetrazole.
DMAP or Melm were added (Pig.3b, 3c) . In all cases reaction
proceeds in two steps -the rapid formation of over 80% PPTE
which in conditions of Pig.3 is accomplished in few minutes
and significantly slower conversion of the latter to p-chloro—
phenyl ester of dinucleoside phosphate. Thus irrespective of
condensing reagents and catalysts used PPTE behaves kinetically as intermediate of phosphotriester condensation. To similar
conclusion have come the authors of CioJ«
Mechanism of the pyrophosphotetraeater formation in the
reaction of phosphodiesters with arylsulfonyl chlorides
and tetrazolides
It is commonly accepted that arylsulfonyl chloride reacts
with the esters of phosphoric acid via intermediate formation
of mixed anhydride I [24,25]
0
0
II
II _
R'O-P-OSO2Ar
+ Cl"
(3)
ArS0201 + R'O-P-0
OR
OR
(I)
However no
P HMR signals corresponding to this mixed
anhydride were observed neither for phosphomonoesters nor
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for phosphodiesters. Therefore, one has to conclude that anhydride I reacts immediately with the second phosphate molecule and is present in the reaction mixture in low steady
state concentration. This means that reaction (3) is a rate
limiting step of the overall process of the formation of pyro—
phosphates (1) and should be first order reaction both towards condensing reagent and phosphate. In the alternative
case of concerted transformation of two phosphate molecules
and ArSOgCl to pyrophosphate and arylsulfonic acid the reaction should reveal second order towards phosphate. The kinetic measurements were carried out by the authors using pT(Ac)
and 0[pT(Ac)32 a s phosphate components and the reaction order
towards these compounds was found to be 0,8 and 0.9 respectively [19,263. By analogy we may expect the same to be the
case also for PDEs.
However as one can see in Pig.4 the reaction of
(ClCgH4)pT(Lev) (Lev - levulinyl residue) with TPS proceeds
very slowly in chloroform solution containing 0.4 M triethylamine. Addition of 0.4 M pyridine and especially 0,4 M Melm
to the chloroform solution of the same compounds accelerates
manifold PPTE format ion [17] « As pyridine and Melm are known
to be nucleophilic catalyst (in the contrary to triethylamine)
one may conclude that process (3) is subjected to nucleophilic catalysis and proceeds via two steps
+
1
ArS02Cl + Nu' ~
+
°
ArS02Nu1 + R'O-P-O~
OR
(Nu
1
ArS02Wu'
+
Cl"
(4)
°
— R'O-P-OSOgAr + Nu 1
OR
(1)
- nucleophiles bearing N ^
(5)
atom in heterocycle -
- pyridine, Melm, DMAP). Tetrazole and 3-nitro-1,2,4-triazc—
le (further reffered as Nu &) don't catalyse the process.
We have succeeded to demonstrate the formation of respec
tive intermediates in the mixture of TPS or TsCl in pyridine
with DHAP by IR spectroacopy [27].
The intense band is observed in IR spectrum of the reaction mixture at 1040 cm" . There is no similar band in IR
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20
10
30
50
70
t(nln)
Pig.4. Kinetic curves of the formation of pyrophosphotetraeater in the reaction of 0.1 11 (ClC/-H.)pT(Lev), triethylammonium salt with 0.2 M TPS in chloroform at
30° in the presence of: 1) 0.4 M triethylamine;
2) 0.4 M pyridine; 3) 0.4 M N-methylimidazole.
spectrum of 4-H,N-dlmethylaminopyridinium salt of arylsulfonic acid. The band disappears at addition of morpholine, diphenylphosphate and 2-cyanoethanol. Intense band at 1632-1645 cm" typical of the quinoide structure is also seen.
We have ascribed these bands to DMAP derivative formed by
reaction
ArS0oCl + N
2
No similar band was found in the case of pyridine thus
indicating that respective equilibrium is strongly displaced
to the left. Most probably the significant amount of the derivative in the case of DMAP is observed due to stabilisation
of the cation by conversion to quinoid structure characteristic for DMAP derivatives.
The significant acceleration of the reaction of ArSOpCl
with phosphate by nucleophilic catalysts bearing aromatic
nitrogen of pyridinium type may be understood as a result
of electrostatic interaction of positively charged arylsulfonyl derivative of the catalyst with negatively charged
phosphate anion. It seems probable that the process proceeds
inside the ion pair
p
• RO-P-OR'
0
•- ArS0o-0-P-0R
OR'
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c.10 2 (M)
10
24
J8
t(min)
Fig* 5"Kinetic curves of the formation of pyrophosphotetraester in the reaction of (ClC 6 H 4 )pT(Ac) with TsTet
in methylene chloride at 30°: 1)without pyridine;
2)in the presence of 0.4 M pyridine.
The presence of pyridine was also found to be necessary
with ArSOgTet as condensing reagent[28]. It is seen in Pig.5
that in the absence of pyridine the initial step of accumulation of PPTB from (ClC^H^)pT(Ac) in the presence of TsTet
in methylene chloride proceeds slowly and is sharply accelerated by addition of pyridine. The S-shaped kinetic curve
without addition of pyridine is due to progressive accumulation of pyridine as pyridinium salt of phosphate was used in
the experiment
0
2 RO-P-OR' • PyH + + ArS0 2 Tet
0"
0
«- RO-P-OR' + Py + ArSO' PyH + +
0
+ TGtH
RO-P-OR.
il
0
The reaction is a bit complicated as compared with ArSOpCl
by involvement of acid catalysis. One may see in Pig.6 that
consumption of PDE is significantly accelerated by toluenesulfonic acid and is completely arrested by triethylamine.
Tnis may be easily understood from the stoichiometry of
the formation of reactive arylsulfonyl derivative
ArS0 2 Tet + PyH + ^=£- ArS0 2 Py + + TetH
The equilibrium may be completely displaced to the left by
addition of tertiary amine.
Thus both with arylaulfonyl chlorides and arylsulfonyl
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-\
10
20
t(mln)
Pig.6. Kinetic curves of the consumption of (ClCgH4)pT(Ac)
(0.06 M) in the presence TsTet (0.06 M)in pyridine
at 2° in the presence of: 1) 0.26 M triethylamine;
2) 0.13 M toluenesulfonic acid; 3) without additions.
azolides nucleophilic catalysts of Nu type are necessary
for efficient conversion of phosphodlesters to respective
symmetric pyrophosphates.
The conversion of mixed anhydride I to PPTE proceeds
outside the rate limiting step and no kinetic evidence can be
presented concerning this step of the overall process (1).
In [1 ij it is declared that anhydride I reacts preferentially
with nucleophilic catalysts of Nu or NuTJ type rather than
with the second PDE molecule. Thus the mechanism of PPTE
formation in the presence of Nu type catalyst may be written as
0
R'0-P-0S0oAr + Nu1 a1
OR
0 +,
II
0 +
R'0-P-Nu1
^
i
R'0-P-Nu1
OR
+ ArS0~
i
OR
0
+
(R0)(R'0)P02
(6)
3
0
II II
•- R'0-P-O-P-OR'
OR OR
(7)
This statement means that anhydride I reacts more readily
with Nu than with the starting PDE anion. As an argument
in favour of this statement it may be mentioned that reaction (4) proceeds more readily than (3) [17] , i.e. that tetrahedral S atom of ArS0oCl is attacked more efficiently by
Nu than by PDE anion. Quantitatively the same reactivities
ratio may be expected for the attack of tetrahedral P atom
of anhydride I.
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In the same time it was found that TetH does not react
with TP8 in pyridine solution in the absence of strongly
basic amine [3] . By analogyone should expect that TetH does
not attack directly mixed anhydride I as is suggested in the
above mentioned papers [9,1iJ. As the phosphotriester condensations with TetH and NTrH are performed in pyridine solution
it seems reasonable to suggest that pyridine participates in
PPTB formation and the process proceeds mainly via reactions
(6) and (7) irrespective to the presence or absence of Nu a.
type catalysts*
Nucleophllic catalysis of the reaction of pyrophosphotetraesters with alcohols
Comparing the data presented in Fig.2 and Pig*3 one
easily sees that the second step of the overall process namely the conversion of PPTB to PTE is significantly accelerated
by addition of TetH, HTrH, DMAP and Melm. The hastening of
phosphotriester condensation with ArSOgTet and ArSOgNTr as
compared with condensation in the presence of ArSOgCl may be
easily understood as TetH and HTrH are liberated in the first
step. Thus the second step of the process is catalysed by the
above mentioned compounds. In the absence of these components
pyridine may play the role of similar catalyst.
Both nucleophilic and base catalysis may be essential in
this process. Hucleophilic catalyst should convert PPTB to
reactive species whereas base catalyst may help to abstract
protons from OH group of R"OH. Both functions may be inherent
to pyridine, DMAP and Melm. To discriminate these functions
the reaction of pyridinium salt of (ClCgH.)pT(Ac) with (Tr)T
in the presence of TPS was studied in methylene chloride in
the presence of pyridine, ^-picoline and benzo[b]-1,4—diazabicyclo-[2,2,2j-octene. The latter has pK Q 5.6 close to pK &
of pyridine however at aliphatic nitrogens thuB being poor
nucleophile. The data presented in Pig.7 demonstrate that
no reaction with (Tr)T proceeds in the latter case. Therefore
we may conclude that nucleophllic catalysis is necessary to
convert PPTE to PTE. As one can easily see from the stoichiometric equation (2) of the reaction participation of condensing reagent is necessary for complete conversion.
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»
l"ig,7. Kinetic curves of the consumption of (ClCgH.)pT(Ac) (1)
accumulation of pyrophosphotetraester (2) and of phosphotriester (3)in the mixture containing 0.1 M
(ClG6H4)pT(Ac), 0.1 M (Tr)T and 0.3 M TPS in methylene
chloride at 30° in the presence ofJ a) 0.7 M benzo[b]-1,4-diazabicyclo[2,2,2]-octene; b) 0.7 M pyridine;
c) 1.0 M 'f'-Py001!116* Dotted curve of the formation
of phosphotriester in the presence of 1.0 pyridine
instead of 1.0 M
'jf-picoline is shown for comparison.
The role of nucleophillc catalyst consists most probably
in the generation of reactive intermediates of the type II
or III
0
0
i
0
RO-P-OR'
n
0
0
II
RO-P-OR'
RO-P-OR
RO-P-OR•
+
i
0
N;
(II)
Wi
0
o
RO-P-OR'
RO-P-OR'
0
RO-P-OR'
n
0
- type Nu
catalyst
RO-P-OR'
(+H + )
(in)
HH:
- type NuTI c a t a l y s t
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l
II + R"OH
•-
RO-P-OR1
+
i
OR"
III + R"OH
— RO-P-OR'
OR"
+
HU'
It is seen that some base is necessary for the reaction
of PPTE with N u ^ .
The PDE formed should rapidly react with the present arylsulfonyl derivative thus regenerating reactive intermediates
II or III
0
RO-P-OR'
1
0"
AXS02H£^
+0
RO-P-OR'
I
OSOgAr
»- II or III
(I)
Thus the following questions arise:
1) whether really the addition of the above mentioned
catalysts to the mixture of PPTE with alcohol catalyses the
PTE formation in the absence of condensing reagent;
2)whether the derivatives of the types II and III are
really highly reactive towards alcohols so that being present
in undetectable amounts atill permit reaction to proceed with
high rate;
3) whether basic catalysis really exist in the formation
of intermediates of type III as well as in the reactions of
intermediates with OH-component.
To answer these questions the model phosphodiester namely
diphenylphosphate was used.
Hucleophllic catalysis of the reaction of tetraphenyl pyrophosphate with alcohols and reactive derivatives of diphenylphoaphoric acid
Theffln-inadvantage of tetraphenylpyrophosphate in the following experiments is that it can be prepared in the absence
of condensing reagent in highly anhydrous state by reaction
of diphenylphosphochloridate and diphenylphosphoric acid. When
(Tr)T is added to the pyridine solution of tetraphenylpyro2102
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0.102(«)
120
240
360
t(mln)
t(mln)
Pig.8. Kinetic curves of the conversion of tetraphenylpyro-
phosphate in pyridine at 30° in the reaction with
(Tr)T
a) 0.05 M tetraphenylpyrophosphate and 0.11M (Tr)T.
In the initial mixture 0,01 M diphenylphosphochloridate is present;
b) 0.04 M tetraphenylpyrophosphate, 0.12 M (Tr)T and
0.25 M tetrazole. 0.01 M diphenylphosphochloridate
is present in the starting reaction mixture.
1) tetraphenylpyrophoephate; 2) (Tr)T0p0(0Ph)2;
3) diphenylphosphorlc acid; 4)P0(0Ph)2Cl.
phosphate containing small amounts of the excess diphenylphosphochloridate the latter disappears in few minutes and subsequent conversion of PPTE to phosphotriester is easily followed. Due to absence of condensing reagent diphenylphosphate
accumulates in parallel with phosphotriester (Fig.8a). When
tetrazole is added simultaneously with (Tr)dT formation of
phoaphotriester proceeds manifold more rapid (Pig.8b) . With
0.25 M tetrazole reaction is accomplished nearly in half an
hour instead of many hours £20], Thus tetrazole really acce lerates reaction of PPTE with OH-component. In the presence of
triethylamine reaction proceeds still more rapid and the time
course of accumulation of phosphotriester can not be followed
by -*1P NMR technique.
To elucidate the existence and reactivity of the intermediates of type II and III diphenylphosphochloridate was treated in methylene chloride respectively with DMAP and TetH.
The ^ P HME signal of diphenyl phosphochloridate disappears
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tm
]?ig.9.
P HMR spectra of the reaction mixture containing
0,2 11 diphenylphosphochloridate and 0.2 DMAP in
methylene chloride in 3 hours after preparation
of the mixture: a) at 20°, b) at -40°. Spectra are
recorded without heteronuclear j Hv- P decoupling.
under addition of DMAP and a new signal appears (Pig.9a) which
is splitted when recorded at -40°C (Pig.9b) to retard the
exchange between the expected DMAP derivative and unreacted
DMAP
f~\
JJH,
° J=\
H
V—N<^
~=~--CgH^O-P-N
V
CH
\=/
3
C6H5-O \ = /
+/CH
N
\
^
In H HMR spectrum H,^ , H« and CH,-group signals are displaced dowtlfield as compared with DMAP (Pig. 10) in accordance
with the expected shift due to positive charge at the nitrogen atom. Some additional details of assignment of the signals are described in Q29l.
The addition of 2 eqv. of 2-cyanoethanol to diphenylphosphodimethylaminopyridinium (6"= 13.4 ppm) in pyridine solution
results in the immediate disappearance of the J P NMR signal
at 13.4 ppm and new signal appears at 12.5 ppm. This signal
is identical to that of 2-cyanoethyldiphenylphosphate obtained
by reaction of 2-cyanoethanol with diphenylphosphochloridate
in pyridine. Therefore, one may conclude that really derivatives of type II are extremely reactive towards alcohols.
Similar experiments were carried out with tetrazole [20].
1.5 mmole of diphenylphosphochloridate in 5 ml dioxane were
treated with 6 mmole of tetrazole and 1.5 mmole triethylamine.
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Pig.10.
H HMR spectra of the same reaction mixtures as in
Pig.9 at 20°(a) and -40°(b). The spectrum oif DMAP
is recorded for comparison (c).
After 30 min the precipitate of triethylammonium chloride was
filtered off. The signal at 21.5 ppm was present
in ^ 1 P HMR
spectrum of the solution obtained. The position of the signal
differs significantly from that of starting (PhOpPOCl (5.5ppm)
as well as from the position of the signal of tetraphenylpyrophosphate (25.5 ppm). The chemical shift is in agreement with
that expected for tetrazolide of diphenylphosphoric acid (23J.
Thus we could assign the signal to this tetrazolide.
Adding (Tr)T we could follow the disappearance of this
signal and accumulation of the compound with singlet signal
at 13.0 ppm reasonable for (Tr)T0P0(0Ph)2. The kinetic curves
are given in Pig.11. One may see that in the absence of bases
reaction proceeds rather slow. However it is significantly
accelerated by addition as few as 0.5 eqv. of pyridine and
'J'-picoline. The addition of 2 eqv. of triethylamine leads
to complete conversion within 2 m1n. Thus in the presence of
bases phosphodiester tetrazolides efficiently phosphorylate
alcohols.
In the contrary to the statement presented in [1 i] protona2105
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C.1OZ(1)
*
•
'
t(h>
Fig.11. Kinetic curves of accumulation of (Tr)TOPO(OPh)2 in
dioxane at 30° in the reaction mixture containing
0.08 M diphenylphosphotetrazolide and 0.1 II (Tr)T:
1) in the absence of amines; 2) In the presence of
0.04 M pyridlne; 3) in the presence of 0.04 M collidine.
tion of tetrazole moiety is not necessary for reaction of phosphodiester tetrazolides with OH-component and vice versa
this process is subjected to base catalysis.
General scheme of phosphotriester condensation
It is shown that phosphotriester formation in the presence of arylsulfonyl chloride and arylsulfonyl azolides as revealed by ^ P M R spectroscopy proceeds in two steps. In the
first step pyrophosphotetraester accumulates. In the second
PPTE is converted to PTE [12,20,21,28]. Nucleophilic catalysts of the pyridine type (Nu ) are necessary in the first
step. The second step may be catalyzed by Nu catalysts as
well as by catalysts of the tetrazole type (NuT). Base catalysts must be present in the latter case [14,20,28] .
The general scheme of the process in the presence of Nu
with ArSOpCl as condensing reagent may be presented as follows
ArS02Cl +
.1
(4)
0
II
ArSOgNu'
R'0-P-Hu
I
OR
OR
(ID
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1
(5)
(6)
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0+
R'O-P-Nu1
1
OR
0+
R'O-P-Nu1
1
OR
0
+ R'O-P-O" = ^
I
OR
+ R"OH
0 0
R'O-P-O-P-OR'
II
OR OR
0
-R'O-P-OR"
I
OR
+ Nu1
(7)
+
+ HHu1
(8)
The stage (4) was demonstrated by IR-spectroscopy for
Nu1 = DMAP[27]. Stage (5) follows by analogy with phosphodiester condensation from the first order kinetics of pyrophosphodiester formation towards starting phosphomonoester
[19,26J. Stage (6) was postulated in[n] and seems reasonable
by analogywith higher reactivity of Nu1 as compared with PDE
in the reaction with tetrahedral S atom of ArSOpCl. Stage
(7) follows from the direct observation of PPTE accumulation
in the first step of the overall process [12,14,20,21,28],
Stage (8) seems reasonable by analogy with the extreme reactivity of diphenylphosphoryl-4-N3N-dimethylpyridinium towards
alcohols [13,29].
According to presented scheme intermediate II either converts directly to PTE by reaction (8) or reacts with PDE forming PPTE (reaction (7)). In all so far investigated cases
the latter route strongly predominates in the first step of
condensation. This seems quite reasonable as the nucleophilicity of PDE should exceed significantly that of OH group.
Reaction (7) results in the transformation of up to 90% PDE
to PPTE. However one may expect that it is possible to escape
in more or less extent PPTE formation using more reactive
OH-components as well as greater excess of these components.
It seems probable that PPTE formation is partially or nearly
completely omitted in the solid phase synthesis performed
with immobilised P-component and with OH-component in solution. Such scheme of synthesis although less popular than
the opposite one was found to be highly efficient in the
phosphotriester oligonucleotide synthesis [30] .
In the second step the direction of the reversible reaction (7) changes to the opposite one. One PDE moiety of PPTE
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converts directly to intermediate II , the other one namely
PDE liberated converts to the same intermediate via reactions
(5), (6). Due to low steady state concentration of PDE it
seems likely that direct route (5),(6),(8) becomes predominant
in this step.
With the H u H type catalysts the necessity of Hu catalyst
remains and the first step proceeds most probably via the
same stages (4)-(6).
Some participation of NuTI catalyst in the conversion
of (RO)(R'O)P(O)Nu
to PPTE may not be completely excluded.
However there is no data permitting to estimate the contribution of N u H in this step. Quite definite is the participation of N u H catalyst in the subsequent reaction of PPTE
with OH-component most probably described by the scheme
0
0
0
0
I
2
R'0-P-O-P-OR' + N u ^ + B:=^R O-P-Nu + R'O-P-0" + BH+ (9)
ii,,.
i
i
OR OR
(base)
OR
OR
0
B'0-P-Hu2
OR
+ R"0H (+B)
0
-R'0-P-OR" + Hu2!!
OR
(10)
So far tetrazole and nitrotriazole were found to be efficient catalysts only in pyridine solution. Pyridine is
necessary as Nu type catalysts for the conversion of ArSOpCl
to reactive species as well as base catalysts in the stages
(9) and (10). In the same time Hu type catalysts - DMAP and
H-methyl imidazole can operate in all stages requiring nucleo—
philic or base catalysis. Therefore one may perform phosphotriester condensation in the presence of these catalysts in
dioxane Qli], acetonitrile [11], chloroform [16,18,31] and
other halogenated hydrocarbons [323.
Abbreviations used: TetH - tetrazole, HTrH - 3-nitrc—
-1,2,4-triazole, DMAP - N,N -dimethylaminopyridine, Melm - H—methylimidazole, ArSOpTet - arylsulfonyltetrazolide,
TsTet - toluenesulfonyltetrazolide, ArSOpNTr - arylsulfonyl-3-nitro-1,2,4-triazolide, PDE - phosphodiester, PTE - phosphotriester, PPTE - pyrophosphotetraester, TPS - triisopropylbenzenesulfonyl chloride, TsCl - toluenesulfonyl chloride.
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