The kinetics and mechanism of the acid-catalysed detritylation of nucleotides in non-aqueous solution

Article abstract of DOI:10.1039/b816235b

The kinetics and mechanism of the deprotection (detritylation) of 5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine nucleoside catalysed by dichloroacetic acid to give a 4,4′-dimethoxytrityl carbocation have been studied in toluene, dichloromethane and acetonitrile. There is little or no effect of solvent polarity on the equilibrium and rate constants. Entropies of activation are highly negative ～-105 J K ^-1 mol^-1 and similarly show little variation with solvent. Addition of small amounts of water to the reaction medium reduces the detritylation rate, presumably through its effect on the solution acidity. All observations are compatible with detritylation occurring through a concerted general acid-catalysed mechanism rather than a stepwise A1 process.

Full text of DOI:10.1039/b816235b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
The kinetics and mechanism of the acid-catalysed detritylation of nucleotides
in non-aqueous solution
Mark A. Russell, Andrew P. Laws, John H. Atherton and Michael I. Page*
Received 16th September 2008, Accepted 10th October 2008
First published as an Advance Article on the web 6th November 2008
DOI: 10.1039/b816235b
The kinetics and mechanism of the deprotection (detritylation) of 5¢-O-(4,4¢-dimethoxytrityl)-2¢-
deoxythymidine nucleoside catalysed by dichloroacetic acid to give a 4,4¢-dimethoxytrityl carbocation
have been studied in toluene, dichloromethane and acetonitrile. There is little or no effect of solvent
polarity on the equilibrium and rate constants. Entropies of activation are highly negative ~ -105 J
-
1
-1
K
mol and similarly show little variation with solvent. Addition of small amounts of water to the
reaction medium reduces the detritylation rate, presumably through its effect on the solution acidity. All
observations are compatible with detritylation occurring through a concerted general acid-catalysed
mechanism rather than a stepwise A1 process.
Introduction
The acid-catalysed removal of the 4,4¢-dimethoxytrityl protect-
ing group from the 5¢-oxygen of the solid-support-bound nu-
cleotide during oligonucleotide synthesis is commonly referred
to as ‘detritylation’. The reaction yields a hydroxyl function at
the 5¢-position of the nucleotide and a relatively stable 4,4¢-
dimethoxytrityl carbocation (Scheme 1). The large scale synthesis
Scheme 2
of oligonucleotides requires quantitative deprotection of the nu-
cleotide as incomplete detritylation causes uncoupled nucleosides
in the subsequent coupling steps, reducing the purity and overall
with dichloroacetic acid being amongst the most popular. The
use of dilute solutions of weaker acids minimises the extent of
depurination but often leads to incomplete detritylation.
1,2
yield of the final product.
1–3,6–8
Although there is a large amount of literature describing the
types of acid and conditions required for optimum detritylation
there is little or no mechanistic data available for the deprotection
reaction under the conditions usually adopted for large scale syn-
thesis i.e. in non-aqueous organic solvents. There have been studies
in aqueous and aqueous–acetonitrile solutions by Maskill and co-
9,10
workers on the deamination of 4,4¢-dimethoxytritylamine and
Scheme 1
11,12
on the ionisation of 4,4¢-dimethoxytrityl alcohol
to the trityl
carbocation, but these results are not easily extrapolated to non-
aqueous organic solvents.
Herein we report mechanistic studies of the detritylation reac-
tion in organic solvents and investigate the equilibrium between
various intermediate species.
High concentrations of strong acids are required for rapid and
quantitative removal of the trityl protecting group, but under these
conditions depurination of the guanosine, and to a lesser extent
the adenosine nucleosides, is frequent and highly undesirable
when product purity is extremely important. The acid catalysed
depurination of 2¢-deoxyguanosine cleaves the N-glycosyl bond
between the purine base and the ribose sugar and occurs through
Results and discussion
3–5
the intermediate formation of an oxenium cation (Scheme 2).
The reaction equilibrium
The rate of detritylation is generally faster than the rate
of depurination and to minimise the amount of depurination,
One obvious potential difference between the reaction in non-
aqueous solvents compared with water is the stability of ions, and
the extent of ion-pair formation. The first step in the investigation
was to explore the equilibrium of the detritylation reaction of
3,6
detritylation is often performed as quickly as possible. To achieve
this objective strong protic acids such as benzenesulfonic acid
and trichloroacetic acid have been used, but due to continuing
complications of depurination and an increasing desire for product
purity over yield, less acidic alternatives have recently been sought
5¢-O-(4,4¢-dimethoxytrityl)-2¢-deoxythymidine (Scheme 3). The
product 4,4¢-dimethoxytrityl carbocation absorbs visible radiation
at a lmax which shows a bathochromic shift on decreasing solvent
polarity if the dielectric constant is taken as a measure of solvent
polarity (Table 1). This implies that the stability of the cation is
Department of Chemical and Biological Sciences, University of Huddersfield,
Queensgate, Huddersfield, UK HD1 3DH
5
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Table 1 Change in lmax with changing solvent polarity for the 4,4¢-
for trityl carbocation formation is dichloromethane and the
most polar solvent, acetonitrile, is intermediate between toluene
and dichloromethane. The concentration of dichloroacetic acid
required to give 50% conversion to the trityl cation is 0.007 M
in dichloromethane, 0.125 M in acetonitrile and 0.230 M in
toluene. As well as the 4,4¢-dimethoxytrityl cation being formed
more readily in dichloromethane with a lower concentration of
dichloroacetic acid required for complete conversion, the molar
extinction coefficient also appears to be slightly greater in this
solvent than in acetonitrile or toluene, in which it is identical. The
dimethoxytrityl carbocation
Solvent
l
max
Dielectric constant
Toluene
Dichloromethane
Acetonitrile
510
505
497
2.4
4.8
37.5
4,4¢-dimethoxytrityl carbocation is a very stable species due to its
highly delocalised charge, and so stabilisation by either solvent or
counter-ion is less important.
Differences also arise because on the varying stabilities of 5¢-O-
4,4¢-dimethoxytrityl)-2¢-deoxythymidine, 2¢-deoxythymidine and
(
Scheme 3
the acid and its conjugate base in different solvents (Scheme 3).
The presence of small amounts of water in the reaction systems
can cause difficulties when comparing the different solvents.
The concentration of water present during detritylation has a
significant effect upon the shape of the absorbance versus acid
concentration profiles (Fig. 2).
related to the solvent polarity as lmax is related to the energy gap
between the HOMO and LUMO, with a lower lmax indicative of
a higher energy gap and therefore a more stable carbocation. The
absorbance is a good measure of concentration of the carbocation
and equilibrium and kinetic studies were undertaken by measuring
changes in the absorbance at the lmax of the carbocation in the
corresponding solvent. As will be described later, the presence
of trace amounts of water in the reaction systems has a profound
effect on the reaction. As a consequence steps were taken to ensure
all solvents were dry and water was excluded. The concentration
of water in the systems was measured by Karl Fischer titration in
each experiment before and after reaction.
The reaction of 5¢-O-(4,4¢-dimethoxytrityl)-2¢-deoxythymidine
togeneratethecarbocation(Scheme3)onlyoccurs innon-aqueous
solvents with sufficiently high concentration of acid.
The extent of reaction is easily measured by titration of the
nucleoside against dichloroacetic acid in toluene, dichloromethane
and acetonitrile, which give sigmoidal absorbance–dichloroacetic
acid profiles (Fig. 1).The relationship between acidity and extent
of reaction was not as may have been expected. Carbocation/ion
formation may be anticipated to be favoured in the more polar
solvent due to charge stabilisation. However, the best solvent
Fig. 2 Plot of absorbance against acid concentration for the detritylation
of 5¢-O-(4,4¢-dimethoxytrityl)-2¢-deoxythymidine (3.6 ¥ 10 M) with
-5
dichloroacetic acid in acetonitrile with increasing concentrations of water
◦
at 30 C. Water concentration increases from left to right (0.011–0.34 M).
Increasing the concentration of water in the reaction solution
causes the profiles to become more sigmoidal in shape with a
greater initial range of acid concentration where there is apparently
no carbocation formation. As a consequence the concentration of
acid required to cause complete cation formation is increased.
There is also a small decrease in the molar extinction coefficient
at the measured wavelength as the concentration of water is
increased, which is due to a slight shift in the lmax of the 4,4¢-
dimethoxytrityl cation, presumably due to an increase in solvent
polarity on water addition. The effects are the same in all three
solvent systems.
It is apparent that the presence of water in the reaction has a
significant effect on the detritylation equilibrium. One possible
explanation is reaction of water with the 4,4¢-dimethoxytrityl
carbocation to form 4,4¢-dimethoxytrityl alcohol. Evidence for
the presence of this reaction was obtained from treating 4,4¢-
dimethoxytrityl alcohol with dichloroacetic acid in acetonitrile,
which gave absorbance–acid concentration profiles sigmoidal in
Fig. 1 Plot of absorbance against acid concentration for the detritylation
of 5¢-O-(4,4¢-dimethoxytrityl)-2¢-deoxythymidine (3.6 ¥ 10 M) with
-5
dichloroacetic acid in ꢀ toluene (0.035 M water), ꢁ dichloromethane
◦
(
0.034 M water) and ꢀ acetonitrile (0.044 M water) at 30 C.
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shape similar to those observed previously, as expected from
For the dehydration reaction, Kdehyd (Scheme 5) was calculated
the equilibrium formation of the 4,4¢-dimethoxytrityl carbocation
assuming that at high acid concentration complete conversion to
+
(
Scheme 4).
DMTr occurs and its concentration is equal to the concentration
-
5
of the ether DMTrOTh used (3.6 ¥ 10 M). If water is added to
+
reduce the concentration of DMTr by half then there will be equal
+
-5
concentrations of DMTr and alcohol, DMTrOH (1.8 ¥ 10 M).
Eqn (1) is then simplified to eqn (3).
+
ÈDMTr ˘
H O
]50%
2
[
Î
˚
50%
[
HA]50%
=
(
3)
Kdehyd
Using the data in Fig. 2, a plot of [HA]50% as a function
of [H O]50% is linear with a slope of 1.70, which is equal to
2
Scheme 4
+
-5
[
DMTr ]50%/Kdehyd and from which Kdehyd = 1.06 ¥ 10 M.
Reversing the reaction of Scheme 3 by adding thymidine to
Addition of water to this solution reduced the absorbance due to
the carbocation, indicating the reversible formation of the alcohol,
the solution of the 4,4¢-dimethoxytrityl carbocation could not be
achieved due to the poor solubility of thymidine in acetonitrile.
Consequently, an estimate of Kdetrit was determined using the data
obtained from the addition of propan-1-ol to the reaction prior to
acid titration to simulate the reverse reaction of detritylation; the
formation of ether from the cation (Fig. 3).
1
3
which was confirmed by C NMR spectra that show distinct
chemical shifts of the central carbon of the 4,4¢-dimethoxytrityl
group (Table 2).
It appears that the observed ‘lag’ in the absorbance–acid
concentration profiles (Fig. 1 and Fig. 2) at low concentrations
of acid is due to trapping of the cation formed from the detrity-
lation of the trityl ether with any available water to form 4,4¢-
dimethoxytrityl alcohol, thus keeping the measured absorbance
close to zero. This occurs until a sufficient concentration of
acid is added to force the equilibrium towards formation of the
+
carbocation (DMTr ) by removal of both the thymidine group
from the ether (DMTrOTh) and the hydroxyl group from the
alcohol (DMTrOH). If the concentration of water is increased,
a greater concentration of dichloroacetic acid is required to cause
quantitative cation formation (Scheme 5).
Fig. 3 Plot of absorbance against acid concentration for the reaction
-5
of 4,4¢-dimethoxytritylthymidine (3.6 ¥ 10 M) with dichloroacetic acid
in acetonitrile with varying concentrations of propan-1-ol at 30 C.
Scheme 5
◦
Propan-1-ol concentration increases from left to right (0.502–0.903 M).
The two equilibrium constants, Kdetrit and Kdehyd (Scheme 5), are
given by eqn (1) and eqn (2) and were determined for detritylation
in acetonitrile.
Using the same assumptions used for the determination of
K
dehyd, eqn (2) simplifies to eqn (4).
+
-
ÈDMTr ˘ H O ÈA ˘
[
2
]
Î
˚
Î
˚
Kdehyd
=
(1)
(2)
+
[
DMTrOH][HA
]
ÈDMTr ˘
[ROH]50%
Î
˚
50%
(
4)
[
HA]50%
=
Kdetrit
+
-
ÈDMTr ˘ÈA ˘ ThOH
[
]
Î
˚Î
˚
Kdetrit
=
[
DMTrOTh][HA
]
A plot of [HA]50% as a function of [ROH]50% is linear with a
+
slope of 0.65, which is equal to [DMTr ]50%/Kdetrit and from which
1
3
-5
Table 2
C NMR chemical shifts in deuterated acetonitrile for the central
Kdetrit = 2.75 ¥ 10 M.
carbon of the trityl group
The ratio of the two equilibrium constants can then be used
to calculate the equilibrium between the ether and the alcohol,
13
C NMR of central carbon in
deuterated acetonitrile d/ppm
K
trans = 2.59 (Scheme 6). This could indicate that the 4,4¢-
4
,4¢-Dimethoxytrityl species
dimethoxytrityl alcohol is, marginally, thermodynamically more
stable than the ether, but there will also be thermodynamic
contributions from the relative stabilities of the thymidine alcohol
and water in acetonitrile.
Thymidine
Cation
87.3
197.3
81.6
Alcohol
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Table 4 Second-order rate constants for the detritylation of 5¢-O-(4,4¢-
-5
dimethoxytrityl)-2¢-deoxythymidine (3.6 ¥ 10 M) with dichloroacetic acid
◦
in acetonitrile with varying concentrations of water at 30 C
1
-1
Initial water concentration/M
k/M s
0
0
0
.022
.078
.110
4.7
3.7
2.2
1.1
Scheme 6
0.130
The effect of the magnitude of Ktrans is evident by comparing
Fig. 2 and Fig. 3 where, for example, to reduce the amount of
Table 5 Second-order rate constants for reaction of 5¢-O-(4,4¢-
-5
dimethoxytrityl)-2¢-deoxythymidine (3.6 ¥ 10 M) with dichloroacetic acid
4
,4¢-dimethoxytrityl carbocation corresponding to an absorbance
of 0.5 at an acid concentration of 0.2 M, requires approximately
.27 M water or 0.72 M propan-1-ol.
DS /J K mol-1
π
-1
DH /kJ mol
π
-1
Solvent
0
Toluene
Dichloromethane
Acetonitrile
-91
-106
-117
41.3
35.6
38.5
Kinetics
The kinetics of the detritylation of 5¢-O-(4,4¢-dimethoxytrityl)-
of charge on going to the transition state are normally susceptible
to large solvent effects, and entropies of activation normally vary
enormously.
2¢-deoxythymidine (Scheme 3) were studied spectrophotometri-
cally by stopped-flow and conventional techniques in toluene,
dichloromethane and acetonitrile. Pseudo-first-order rate con-
stants kobs were determined from the change in absorbance due to
the 4,4¢-dimethoxytrityl carbocation with time. The concentration
of acid used was greater than that needed to achieve complete
conversion to the carbocation. The dependence of kobs on the
dichloroacetic acid concentration for each solvent gave a good
linear correlation from which the second-order rate constants were
calculated (Table 3).
Mechanism
It is usually assumed that the mechanism for the acid-catalysed
detritylation reaction is A1 in nature with initial protonation of
the 5¢-oxygen followed by unimolecular C–O bond cleavage to give
the carbocation and the displaced alcohol (Scheme 7).
There is a remarkably small dependence of the rate of detrity-
lation on the solvent and, perhaps contrary to expectation, the
second-order rate constant in acetonitrile is the smallest despite the
presumed development of some charge in the transition state and
the better charge stabilisation in the more polar solvent. Similarly
one would expect the acid strength of dichloroacetic acid to vary
with solvent and so the small dependence of rate is indicative of
a small dependence on acidity and possibly an ‘early’ transition
state.
Increasing the concentration of water in acetonitrile decreases
the rate of formation of the carbocation. As before, good linear
correlations are obtained for plots of kobs against dichloroacetic
acid concentration from which the corresponding second-order
rate constants were obtained (Table 4). As the initial concentration
of water is increased in the reaction medium the rate of reaction
decreases, which is presumably due to a decrease in the acidity of
the solution.
Scheme 7
This assumption is supported by studies on the deamination
9,10
of 4,4¢-dimethoxytritylamine
and on the ionisation of 4,4¢-
11,12
dimethoxytrityl alcohol
to the carbocation in aqueous solu-
The activation energy parameters were determined for detrity-
lation in toluene, dichloromethane and acetonitrile, which all gave
good linear correlations using the Eyring equation. Given the large
differences in polarity of the solvents, the activation parameters are
remarkably similar (Table 5). Reactions that involve the generation
tions. The A1 mechanism is a simple stepwise process and the
second order rate constant kHA is equal to the product of the
equilibrium constant for the first step K
the second step k (Scheme 8).
1
and the rate constant for
2
Table 3 Second-order rate constants for reaction of 5¢-O-(4,4¢-
Scheme 8
-5
dimethoxytrityl)-2¢-deoxythymidine (3.6 ¥ 10 M) with dichloroacetic acid
◦
as a function of solvent at 30 C
In acetonitrile the second-order rate constant is 4.7 M- s .
Estimation of K can be obtained from the ratio of the dissociation
constants of dichloroacetic acid, K
1
-1
k/M-1 s-1
1
Solvent
HA
a
, and that for the protonated
ROR
Toluene
Dichloromethane
Acetonitrile
18.7
18.6
4.7
ether, K
The pK
no pK
a
, as described below.
of dichloroacetic acid in acetonitrile is 15.8. There are
values available for protonated ethers in acetonitrile, but in
13
a
a
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1
4
water they range between -2 and -6. Based on the relationship
between pK values in water and acetonitrile, those for ether
conjugate acids can be estimated to be between 3 and 6, suggesting
proton transfer coupled to C–O bond fission is anticipated for the
formation of the stable 4,4¢-dimethoxytrityl carbocation.
Typically, the formation of relatively unstable carbocations
would be more favoured in polar solvents due to increased
stabilisation of the charged species. The equilibrium and kinetic
studies reported here where solvent polarity is not having these
expected effects on either the rate or equilibrium, are the result of
the 4,4¢-dimethoxytrityl carbocation being a stable species due to
extensive delocalisation.
a
-
10
-13
that K
The value of the unimolecular rate constant for the breakdown
of the protonated trityl ether, k , for the hypothetical A1 mech-
anism (Scheme 8) can be calculated from the ratio of the actual
second-order rate constant for detritylation and K indicating that
= 10 –10 s . This extremely high value indicates
1
= 10 –10
.
2
1
1
1
14 -1
k
2
= kHA/K
1
that the lifetime of the conjugate acid intermediate (Scheme 8) is
15
close to or less than that expected for an intermediate to exist,
Experimental
and therefore the formation of this intermediate and consequently
the A1 mechanism seem unlikely. A more likely mechanism for
detritylation in acetonitrile and in other non-aqueous solvents
is a concerted general acid-catalysed process (Scheme 9). The
microscopic reverse of this reaction is the concerted general
base-catlysed addition of water to the carbocation, which is the
mechanism that has been proposed for triarylmethyl carbocations
Equilibrium studies were performed on a Cary 1E UV-Visible
spectrophotometer with 1 cm quartz cuvettes. All solvents were
˚
dried over 4 A molecular sieves under dry argon for at least
4 hours prior to use. In a typical experiment, solutions of 5¢-
2
-
5
O-(4,4¢-dimethoxytrityl)-2¢-deoxythymidine (3.6 ¥ 10 M, 2 ml)
were pipetted into the cuvette and dichloroacetic acid (12 M,
1–20 ml) was then titrated. After each aliquot of acid was added
the sample was shaken and left to equilibrate for five minutes
prior to recording of the UV spectrum over a wavelength range of
16
in water.
2
50–600 nm.
1
13
H and C NMR spectra were recorded on a Bruker 400 MHz
or Bruker 500 MHz spectrometer with deuterated acetonitrile as
solvent.
Kinetic measurements were performed on an Applied Photo-
physics SX.18 MV-R1 Stop-flow spectrophotometer with a 25 : 1
syringe ratio. In a typical experiment, a solution of 5¢-O-(4,4¢-
-
4
dimethoxytrityl)-2¢-deoxythymidine (9 ¥ 10 M) was loaded into
the smaller of the two syringes and dichloroacetic acid (0.2–3.5 M)
was loaded into the larger syringe. Injection of the contents of the
two syringes into the reaction cell initiated the reaction, which was
followed by measurement of the increase in the absorbance of the
produced carbocation, an average of 20–30 results being taken for
each run.
Scheme 9
17
18,19
Jencks and Fife
have both proposed a number of re-
quirements to determine whether a mechanism will be stepwise
or concerted. Jencks suggests that for a reaction to occur by a
concerted general acid-catalysed mechanism there must be a large
Synthesis of 4,4¢-dimethoxytrityl alcohol
Sodium hydroxide (10 ml, 2.95 mmol) was added to a solution
of 4,4¢-dimethoxytritylchloride (1 g, 2.95 mmol) in acetonitrile
(25 ml). As the reaction proceeded the solution was kept basic with
further additions of sodium hydroxide (approx. 3 ml). On addition
of water (20 ml) to the solution, a yellow–brown oil, which was
found to be the crude form of the 4,4¢-dimethoxytrityl alcohol,
and a white precipitate was formed. The oil was dissolved in ether
(25 ml) and water (25 ml) and the organic layer extracted with
ether (3 ¥ 15 ml). After drying over sodium sulfate and filtration,
the solvent was removed and the resulting very viscous orange
pK
have a pK
the substrate site. During the course of the detritylation reaction
in acetonitrile there is a large change in the pK of the protonated
ether from 3–6 to that of the alcohol liberated (thymidine) of >
2 while the pK of 16 for the proton donor, dichloroacetic acid,
a
change during the course of the reaction and the catalyst must
a
intermediate between the initial and final pK values of
a
a
2
a
is intermediate between those of the ether conjugate acid and the
alcohol. Fife has also proposed a number of requirements for
a concerted general acid-catalysed mechanism based mainly on
the study of the mechanisms of hydrolysis of acetals and ketals.
These state that concerted mechanisms occur when either the
leaving group is very good and/or the product cation is very
stable. A concerted general acid-catalysed mechanism for the
detritylation of 5¢-O-(4,4¢-dimethoxytrityl)-2¢-deoxythymidine is
compatible with all of these criteria.
Experimentally, the small dependence of the rate constants
for the acid-catalysed detritylation of 5¢-O-(4,4¢-dimethoxytrityl)
thymidine on solvent (Table 3), the negative entropies of activation
and their small variation with solvent (Table 4) are all compatible
with a bimolecular process leading to an early transition state with
little charge development (Scheme 9). A concerted mechanism for
1
oil was the 4,4¢-dimethoxytrityl alcohol (0.76 g, 81%). H NMR:
d (CD
H, J = 2.2 Hz), 6.82 (4H, d, aromatic H, J = 2.2 Hz), 4.24
(1H, s, OH) (lost on D O shake), 3.57 (6H, s, OCH ). C NMR: d
(CD CN) 159.9, 149.0, 140.9, 130.0, 128.6, 127.7, 113.8, 81.6, 55.8.
IR: n/cm 3485.0 (OH), 3058.1 (Ph-H), 2835.3 (OCH
3
CN) 7.23 (5H, m, aromatic H), 7.12 (4H, d, aromatic
1
3
2
3
3
-
1
3
), 1607.5,
-
1
1583.1, 1508.7 (Ph) cm .
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