Tuning the stability of alkoxyisopropyl protection groups

Article abstract of DOI:10.3762/bjoc.15.70

Five different 2-alkoxypropan-2-yl groups are introduced as acid-labile protecting groups for the 5’- and 3’-hydroxy groups of a 2’-deoxynucleoside. All studied protecting groups were readily introduced with good to excellent yields using the appropriate enol ether as a reagent and 0.5 to 1 mol % p-toluenesulfonic acid as a catalyst. The protected compounds could be purified by silica gel column chromatography without degradation. The compatibility of these protecting groups in parallel use with benzoyl and silyl groups was verified. The stabilities of the different alkoxy acetal protecting groups were compared by following the kinetics of their hydrolysis at 25.0 °C in buffered solutions through an HPLC method. In the pH range 4.94 to 6.82 the hydrolysis reactions are of first order in the hydronium ion. The rate of hydrolysis correlates with the electron-donating or electron-withdrawing ability of the corresponding alkoxy group. The studied 2-alkoxypropan-2-yl groups and the relative rate constants for their cleavage from the 5’-hydroxy group of 2’-deoxythymidine were: cyclohexyloxy (k_rel = 7.7), isopropoxy (7.4), methoxy (1), benzyloxy (0.6) and 2,2,2-trifluoroethyloxy (0.04). The attachment of the same groups to the 3’-hydroxy group are from 1.3 to 1.9-fold more stable. The most reactive of these acetone-based acetal groups are faster removed than a dimethoxytrityl group, and they are easier to cleave completely in solution. The structural variation allows steering of the stability and lipophilicity of the compounds in some range.

Full text of DOI:10.3762/bjoc.15.70

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Tuning the stability of alkoxyisopropyl protection groups
Zehong Liang, Henna Koivikko, Mikko Oivanen and Petri Heinonen*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 746–751.
Department of Chemistry, University of Helsinki, P.O.Box 55
(A.I.Virtasen aukio 1), FIN-00014 Helsinki, Finland
doi:10.3762/bjoc.15.70
Received: 23 January 2019
Accepted: 12 March 2019
Published: 21 March 2019
Email:
Petri Heinonen* - petri.j.heinonen@helsinki.fi
* Corresponding author
Associate Editor: S. Flitsch
Keywords:
© 2019 Liang et al.; licensee Beilstein-Institut.
License and terms: see end of document.
acetal; hydrolysis; protecting groups
Abstract
Five different 2-alkoxypropan-2-yl groups are introduced as acid-labile protecting groups for the 5’- and 3’-hydroxy groups of a
2’-deoxynucleoside. All studied protecting groups were readily introduced with good to excellent yields using the appropriate enol
ether as a reagent and 0.5 to 1 mol % p-toluenesulfonic acid as a catalyst. The protected compounds could be purified by silica gel
column chromatography without degradation. The compatibility of these protecting groups in parallel use with benzoyl and silyl
groups was verified. The stabilities of the different alkoxy acetal protecting groups were compared by following the kinetics of their
hydrolysis at 25.0 °C in buffered solutions through an HPLC method. In the pH range 4.94 to 6.82 the hydrolysis reactions are of
first order in the hydronium ion. The rate of hydrolysis correlates with the electron-donating or electron-withdrawing ability of the
corresponding alkoxy group. The studied 2-alkoxypropan-2-yl groups and the relative rate constants for their cleavage from the
5’-hydroxy group of 2’-deoxythymidine were: cyclohexyloxy (krel = 7.7), isopropoxy (7.4), methoxy (1), benzyloxy (0.6) and
2,2,2-trifluoroethyloxy (0.04). The attachment of the same groups to the 3’-hydroxy group are from 1.3 to 1.9-fold more stable. The
most reactive of these acetone-based acetal groups are faster removed than a dimethoxytrityl group, and they are easier to cleave
completely in solution. The structural variation allows steering of the stability and lipophilicity of the compounds in some range.
Introduction
Acetals form one of the most common types of protecting is increased markedly by attaching electron-donating alkyl
groups for hydroxy functions. They are easily introduced by a groups on the acetal carbon. The simplest of the formaldehyde-
rapid acid-catalyzed reaction and they are also readily cleaved type acetals, methoxymethyl (MOM) [2], is rather stable to
under mild acidic hydrolytic conditions. These properties make acidic hydrolysis. The acetaldehyde acetals, 1-ethoxyethyl (EE)
the acetal structures usable even in other applications, where [2,3] and tetrahydropyranyl (THP) [2] are widely used as
easily cleavable linkers are needed [1]. The stability and proper- protecting groups, but they have a disadvantage of generating
ties of acetals/ketals widely varies depending on their structure, an additional asymmetric center. Some structural modifications
which widens the scope of their applicability. The acid lability have been introduced to overcome the chirality problem, e.g.,
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Beilstein J. Org. Chem. 2019, 15, 746–751.
the methoxytetrahydropyranyl group suggested in 1970’s for a
substitute of THP in nucleoside chemistry [4,5]. For protection
of highly sensitive compounds acetone-based acetals can be
applied. However, of these only the 2-methoxypropan-2-yl
protecting group (MIP) has been adopted in use [2] and the al-
ternative, 2-benzyloxypropan-2-yl, introduced by Mukaiyama
in the 1980’s [6] has not gained popularity.
The MIP group has been used, e.g., in solution-phase oligo-
nucleotide synthesis for the primary 5’-hydroxy group of a
nucleoside [7]. The acid lability of MIP is comparable to that of
Figure 1: Reagents for acetal protections.
the dimethoxytrityl group (DMTr) that is frequently used to lysis of the protected compounds in buffered solutions between
protect this position. The DMTr group works more or less pH 4 to 6.
perfectly in solid-phase synthesis, however, in solution-phase
processes there are several cases where cleaving of DMTr fails. Protecting reagents and protection
This is due to the equilibrium formed between the protected and Two general routes have been reported for the preparation of
deprotected compounds. During our search for a highly acid- 1b–e [6,8]. The 2-benzyloxypropene (1b) was synthesized
labile protection for the 5’-hydroxy group of a nucleoside we starting from methyl lactate, which was deprotonated and then
noticed that neither the DMTr nor the MIP are fully satisfac- alkylated with benzyl bromide. Hydrolysis, reduction and elimi-
tory. Whereas MIP is easily removable, has good atom nation yielded 1b [6]. A shorter route starts from chlorocro-
economy and has the notable advantage of giving volatile tonic acid, in which the substitution of the chloride with the
hydrolysis products, the small size of this group in some cases appropriate alkoxide is followed by a thermal decarboxylation
leaves the protected compound unnecessarily hydrophilic. This step to afford the desired propene derivative [8]. We followed
causes solubility problems and complicates isolation and purifi- this route to prepare 1c–e and details of the syntheses are given
cation steps in the synthetic procedures [7].
in Supporting Information File 1.
We realized that the lability of the acetal group can be easily The 5’- and 3’-acetal-protected 2’-deoxythymidines were pre-
steered by varying the alkoxy group, while preserving other pared using 7 equiv of the appropriate reagent 1a–e with the
beneficial properties of the protecting groups. On the other suitably protected thymidine 2 or 5 (Scheme 1) in the presence
hand, structural modifications can also affect the lipophilicity of of p-toluenesulfonic acid as the acid catalyst. A suitable amount
the protected compounds. The present study aimed at tuning the of the acid catalyst is 0.5 to 1 mol %, with which the reaction
properties of acetone-based acetal protecting groups at nucleo- takes place practically instantaneous at room temperature with
side hydroxy functions. We have carried out a systematic reagents 1a–d. In order to avoid formation of the vinyl ether
kinetic study on the hydrolytic lability of five different analog as a side product, the reaction time should not exceed
2-alkoxypropan-2-yl groups installed in the 5’- and 3’-hydroxy 5 minutes. The isolated yields of the protected nucleosides are
groups of a 2’-deoxynucleoside. We are well aware of the fact typically between 70–90%. However, reagent 1e forms an
that hydrolysis of acetals has been widely studied over decades exception, as the electron-withdrawing trifluoroethyl group
and the mechanisms are commonly agreed. However, it reduces the reactivity and the reaction time had to be increased
appeared that more comprehensive and systematic data are to several hours. In addition, the yields of the protected prod-
needed to allow the analysis of structural effects in detail.
ucts 3e and 6e also remained rather low, around 35%.
Results and Discussion
If a larger amount than 1 mol % of the acid catalyst is used and
Five acetone-based acetal protecting groups for nucleoside the reaction time extended, an enol ether formation takes place
hydroxy groups were introduced by an acid-catalyzed acetaliza- during the acetalization. We followed the acetal formations by
tion into compounds 1a–e (Figure 1). Three of those (1c–e) 1H NMR spectroscopy, and it was shown that the formed acetal
have not been earlier used for acetal protections, although 1c product starts to degrade in a slower consecutive step, which
and 1d have been synthesized previously [8]. The acetal leads to release of the enol ether derivative, e.g., 8a (Figure 2).
protecting groups were inserted either to the primary Recently, the same kind of enol ether formation was considered
5’-hydroxy or the secondary 3’-hydroxy group in 2’-deoxy- in connection to an optimization of the conditions for MIP
thymidine. The labilities of the protecting groups were com- protection on secondary and benzylic hydroxy groups of
pared by determining the first order rate constants for the hydro- mandelonitrile derivatives in a flow reactor process [9].
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Beilstein J. Org. Chem. 2019, 15, 746–751.
Scheme 1: Synthesis of 2-alkoxyprop-2-yl-protected thymidines. Reagents and conditions (i) 7 equiv 1a–e, 0.5 mol % TsOH in dry THF, 5 min (1a–d),
18 h (1e); (ii) 1:1 conc. aq NH3/MeOH, 18 h; (iii) 2 equiv TBAF in dry THF, 1.5 h. Notation: a: R = Me, b: R = Bn, c: R = cyclohexyl, d: R = isopropyl,
e: R = CH2CF3.
nium ion concentration is observed for each acetal derivative in
this pH region. The methoxy and benzyloxy acetals show rather
similar stabilities, whereas the isopropyloxy and cyclohexyloxy
derivatives are on average 7.5 and 7.7-fold faster cleaved than
the MIP derivative. The more electron-withdrawing trifluo-
roethoxy group strongly stabilizes the acetal, making it 30-fold
more stable compared to MIP protection.
Only minor differences are observed between the stabilities of
acetal protection on the primary 5’- and on the secondary
3’-hydroxy functions. Steric effects on the hydrolysis rate are
supposed not to be important, because the rate-controlling step
Figure 2: Enol ether 8a: R1 = TBS and 8b: R1 = H.
of the hydrolysis is suggested to be the unimolecular degrada-
tion of the protonated substrate. This releases the stabilized
oxocarbenium ion as an intermediate (Scheme 2). Experimental
data on the acidity of the 5’- and 3’-hydroxy groups of thymi-
dine are not available, but computed NBO charges suggest that
the electronic properties of both hydroxy functions are close to
each other [10]. Our DFT level calculations indicated that the
3’-hydroxy function is slightly more acidic than the 5’-hydroxy
group, but absolute values for the acidity constants could not be
reliably determined.
All of the studied acetals are stable toward basic conditions
used to remove benzoyl protecting groups and toward tetra-
butylammonium fluoride used to remove the tert-butyl-
dimethylsilyl (TBS) protection. The products could be purified
by silica gel column chromatography without degradation when
0.5% triethylamine was added to the eluent.
Hydrolytic stability
The first-order rate constants for the hydrolysis of the acetal
protecting groups in the pH range 4.94 to 6.82 at 25.0 °C were The hydrolysis of the acetals is suggested to follow most often
determined by an HPLC method and the results are collected in the A-1 mechanism via formation of an oxocarbenium ion [11].
Table 1 as half-life data. Details of kinetic analyses are given in Polarity effects and the stability of the formed oxocarbenium
Supporting Information File 1, including a figure of pH–rate ion determine, which one of the alkoxy groups is protonated
profiles. A strictly first-order dependence of the rate on hydro- and released as an alcohol. The two proposed competing path-
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Beilstein J. Org. Chem. 2019, 15, 746–751.
Scheme 2: Proposed acetal hydrolysis pathways.
ways are illustrated in Scheme 2. Salomaa has shown earlier 8b was found to be about 15-fold more stable than the cyclo-
[12] with formaldehyde acetals that the structural effects on the hexyl derivative 7c at pH 4.94, which means that it should have
stability of the oxocarbenium intermediate are directly reflected been accumulating as an intermediate, if formed in a significant
by the observed hydrolysis rate, whereas the changes in polarity amount during hydrolysis of 7c under these conditions. While
of the leaving alcohol do not give marked changes on the rate. going towards neutral conditions, the rate of the vinyl ether
The latter is due to the fact that the kinetic effects on the proton- hydrolysis does not show a linear dependence on the acidity of
ation step and the leaving group ability largely cancel each the solution (see Suppporting Information File 1). Thus, at pH
other.
6.8 the rate of hydrolysis of 8b is close to that of 7c and 7d. The
vinyl ether hydrolysis has earlier shown to be even a subject of
In our study, the observed rate constants are proportional to the general acid catalysis [13,14].
acidity of the alcohol moieties corresponding to the varied
stems of the acetal structures: the hydrolysis rate steadily It is useful to compare the stabilities of the acetal protections to
decreases with increasing electron-withdrawing ability of the those of the 4,4’-dimethoxytrityl protecting group used in oligo-
alkyl group. A comparison of this observation with the results nucleotide synthesis. Directly comparable data are hard to find,
of Salomaa [12] can be taken as support for the upper pathway since hydrolysis in water solution is not often a very practical
outlined in Scheme 2, involving protonation of the nucleoside method for detritylation, due to hydrophobicity of the trityl de-
oxygen and departure of it as alcohol. However, it must be rivative. Nevertheless, half-times for the deprotection of 5’-O-
noted that comparative structural effects were not studied over a dimethoxytrityl oligonucleotides have been determined under
very wide range, and the correlation order may change with several different conditions [15]. The values reported were
using more electron-withdrawing groups than trifluoroethanol. 10 minutes in an acetate buffer at pH 4.18 and approximately
Nevertheless, our interest was in studying the protecting group 4 minutes at pH 3.22. These values are close to an order of
abilities, and the studied compounds fit the most relevant reac- magnitude longer than what we determined for MIP 4a, and two
tivity area in that sense.
orders of magnitude longer than those of the cyclohexyl and
isopropyl analogs 4c, 4d (Table 1). It must be noted, however,
As mentioned above, enol ether derivatives (as 8a) were formed that the removal of the DMTr protection is very dependent on
under more acidic conditions of acetal synthesis. We also pre- the conditions, especially the solvent composition [15]. In solu-
pared the corresponding derivative 8b of thymidine and studied tion chemistry the removal of trityl protection is sometimes
its hydrolytic stability separately. This is of some interest for problematic, since the cleavage reaction via a trityl cation may
discussion of mechanisms of acetal hydrolysis, since the vinyl become reversible, leading to an equilibrium between the pro-
ether intermediate 8b could probably be formed on the pathway tected and deprotected substrates and thus incomplete deprotec-
shown in the lower part of Scheme 2. However, accumulation tion [16]. In those cases an acetal protection may offer a more
of 8b was not observed during our kinetic studies. Compound useful solution.
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