Modulating the Stability of 2-Pyridinyl Thermolabile Hydroxyl Protecting Groups via the "chemical Switch" Approach

Article abstract of DOI:10.1021/acs.joc.5b02033

A novel and effective method is presented for modulating the stability of 2-Pyridinyl Thermolabile Protecting Groups (2-Py TPGs) in the "chemical switch" approach. The main advantage of the discussed approach is the possibility of changing the nucleophili

Full text of DOI:10.1021/acs.joc.5b02033

Article
pubs.acs.org/joc
Modulating the Stability of 2‑Pyridinyl Thermolabile Hydroxyl
Protecting Groups via the “Chemical Switch” Approach
Agnieszka Witkowska, Dominika Krygier, Jolanta Brzezinska, and Marcin K. Chmielewski*
́
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland
S
* Supporting Information
ABSTRACT: A novel and effective method is presented for modulating the stability of 2-Pyridinyl Thermolabile Protecting
Groups (2-Py TPGs) in the “chemical switch” approach. The main advantage of the discussed approach is the possibility of
changing the nucleophilic character of pyridine nitrogen using different switchable factors, which results in an increase or
decrease in the thermal deprotection rate. One of the factors is transformation of a nitro into an amine group via reduction with a
low-valent titanium in mild conditions. The usefulness of our approach is corroborated using 3′-O-acetyl nucleosides as model
compounds. Their stability in various solvents and temperatures before and after reduction is also examined. Pyridine N-oxide
and pH are other factors responsible for the nucleophilicity and stability of 2-Pyridinyl Thermolabile Protecting Groups in
15
thermal deprotection. Protonation of 4-amino 2-Pyridinyl Thermolabile Protecting Groups is demonstrated by 1H N HMBC
and HSQC NMR analysis.
reduces efficiency and results in the formation of byproducts.
However, increasing stability at ambient temperature leads to
an extension of deprotection time and prolonged exposure of
the molecule to heating.
INTRODUCTION
■
Over the past decade, increased interest has been observed in a
novel range of chemical methods which are useful in synthetic
biology (defined as chemistry applied in biology).1 The use of
Thermolabile Protecting Groups (TPGs) in the protection of
functional groups during chemical synthesis of nucleic acids can
serve as a good example of this tendency.2,3 The main
advantage of TPGs is their capability to be removed by thermal
deprotection, which occurs only as a result of increased
temperature in neutral conditions. Such a unique approach
eliminates the need for hazardous reagents used to deprotect
other protecting groups, and complies with the principles of
green chemistry.4 Unlike oligonucleotides carrying the conven-
tional 2-cyanoethyl phosphate/thiophosphate protecting group,
deprotection of TPGs from oligonucleotides does not generate
potent mutagens, such as acrylonitrile, which can alkylate
DNA.5 Thanks to this observation, TPGs have also been
applied in the chemical synthesis of other nucleic acids,6
especially for phosphate,7 hydroxyl,8 and amine groups9 in
nucleotides or DNA microarrays.10 A certain disadvantage of
TPGs lies in their partial instability at ambient temperature.
This poses a serious problem when a TPG is intended to be
used as protection for key functional groups in multistage
chemical transformations. Partial loss of a TPG during synthesis
Useful TPGs are characterized by high stability at ambient
temperature, and fast deprotection after raising the temperature
by approximately 70 °C. This can be attained if TPGs have high
activation energy in the thermal deprotection process. In
contrast, those TPGs that are unstable at room temperature are
marked by lower activation energy. An example of manipulating
the stability of TPGs is the “click-clack” approach.11 This is
based on forming a 3-pyridinyl-[1,3,2]-oxazaphospholidine
ring, which temporarily “freezes” thermolytic properties that
are recovered after opening the oxazaphospholidine under mild
acidic conditions. This method has been used to protect the
phosphate function and for intramolecular oxidation of H-
phosphonate.12
In practice, however, hydroxyl protecting groups are more
significant, and some new protecting groups have been
designed for this function.13 Devising an efficient system of
controlling the stability of TPGs for these functional groups
Received: September 4, 2015
© XXXX American Chemical Society
A
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may significantly increase the popularity of TPGs in modern
chemistry. Protection of the hydroxyl function through a TPG
containing a nucleophilic 2-pyridinyl moiety is very promis-
ing,14 and the TPG is likely to become a comprehensive
protecting group. Application of these groups in the protection
of the phosphate or hydroxyl function has revealed a persistent
problem with attaining high stability at ambient temperature.
The use of a 2-pyridinyl moiety as a nucleophilic agent in TPGs
allows for control of stability by using substituents which
change the electron density of the pyridine ring, and
consequently the control of thermocyclization. This is based
on a nucleophilic attack by the pyridine nitrogen on a carbon
atom directly linked to a carbonate group (Scheme 1). This
Here, we report on an efficient method for increasing or
decreasing the stability of 2-pyridinyl TPGs (2-Py TPGs) at
ambient temperature by switching a nitro into an amine group
by reduction via titanium complexes.
RESULTS AND DISCUSSION
■
The rate of 2-Py TPG deprotection that occurs by
thermocyclization mainly depends on the nucleophilic strength
of pyridine nitrogen. The presence of the nitro group at
position 4 of the pyridine ring results in lowered nucleophilicity
(electron density) of pyridine nitrogen, which stops depro-
tection at ambient temperature and considerably contributes to
their usefulness. However, switching the property may be
possible by modulating the character of TPG using reduction of
a nitro group (electron withdrawing group) into an amine
group (electron donating group) (Scheme 2).
Scheme 1. Mechanism of Thermal Deprotection
Scheme 2. “Chemical switch”general idea
process demonstrates the removal mechanism of 2-Py-TPG
that occurs via intramolecular cyclization. The main advantage
of 2-Py TPGs is that the deprotection restores the free state of
the protected function which can be triggered in a controlled
manner without using external chemical agents.
This “chemical switch” results in an increased nucleophilicity
of the pyridine nitrogen and significantly accelerates
thermocyclization. This transformation requires the application
of a precise agent to reduce the nitro group into the amine
group. Malinowski and co-workers28 showed that low-valent
titanium (LVT) is an efficient reagent to reduce the nitro-
substituted pyridine and benzene derivatives with over 90%
yield. Starting with this observation, we successfully applied a
mixture of TiCl4 with LiAlH4 to selectively reduce the nitro
group at the 2-pyridinyl ring. Moreover, under these conditions,
the carbonate remains unchanged, allowing the TPG to retain
its functionality.
A higher stability of TPGs with a nitro group across a wide
range of temperatures was presented on the model compounds,
3′-O-acetyl nucleosides. Selection of nucleosides was dictated
by two factors: (i) large potential application, (ii) multi-
functionality which gives an opportunity to study the
coexistence of various protecting groups in one molecule.
Therefore, the 3′-O-acetyl thymidine with a free 5′-hydroxyl
group was blocked by different TPGs: one with a nitro
substituent 1, and the second without a nitro substituent group
11, which is known as unstable TPG (Scheme 3).8a The
protected nucleosides were dissolved in anhydrous acetonitrile
and kept at two different temperatures: 25 and 90 °C for 360 h
and 120 min, respectively.29
Currently, developed methods place particular emphasis on
controlling molecular properties via chemical modification.
“Chemical switch” is a term used to denote manipulations and
transformations of molecules between two extreme chemical
properties. One example of reversible off−on switching of the
electron spin has been achieved by nitric oxide (NO)
functioning as an axial ligand of cobalt(II)tetraphenylporphyrin
(CoTPP).15 The reversible off−on switching zinc-dependent
factor in a mutant F1-ATPase motor containing a metal-
binding site has been shown to be a functional and promising
mechanical component in motor proteins.16 Also, a chemical
switch has been used in an optical sensor developed for sensing
nitrite based on a monotonous decrease in absorbance of
Rhodamine 6G at 525 nm (the absorbance maximum of dye)
with increasing concentrations of nitrite.17 This reduction is
one very convenient chemical transformation; in particular, the
reduction of aryl nitro compounds has found numerous
applications in organic chemistry and remains an attractive
method for changing the properties of molecules. Chemical
reduction of anthraquinone has been applied in modulated
switching between two forms of 90 bp DNA in Y-branched
junctions.18
Selecting the proper reductant for nitro groups that would
work efficiently in complex biological systems constitutes a
great challenge. Such reductants as palladium-on-carbon,19 or
Raney nickel,20 sodium hydrosulfite,21 tin(II) chloride,22 iron in
acidic and alkaline media,23 titanium(III) chloride,24 zinc,25 and
samarium26 are used in laboratory organic synthesis to obtain
aromatic amines. However, it appears that the choice of a
proper reductant that might be applied in biological systems
may be based on metal complexes which can act as artificial
enzymes.27
As shown in Chart 1, incorporation of a nitro group into a 2-
Py TPG increases its stability at both analyzed temperatures.
The amount of blocked nucleosides 1 and 11 in the reaction
mixture was calculated based on the RP-HPLC data obtained
over the respective time periods. Nucleosides with a free
hydroxyl group and bicyclic byproduct were observed as
thermocyclization products. This observation suggests that the
nitro group plays a key role as a stabilizing agent in a 2-PyTPG
and almost completely blocks intramolecular cyclization30 at
B
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into the other) enables control of the properties of the
protecting group. The switch from a nitro into an amine group
is possible in mild reduction conditions, such as titanium
complexes. An effective reduction of the nitro group can be
carried out with the use of black slurry produced from
titanium(IV) chloride and LiAlH4 in anhydrous tetrahydrofur-
an, in inert gas atmosphere
Scheme 3. General Method of Synthesis and Structure of 3′-
O-acetyl 5′-TPG-Protected Nucleosides
Under these conditions, due to slower decomposition and
high activity of LiAlH4, reduction is quantitatively completed
within 15 min. Moreover, the reaction analysis has revealed that
pH of the reduction mixture is very low (measured as 2), which
has an impact on higher stability of carbonates (6−10).
Malinowski and co-workers have observed that the LVT
obtained from TiCl4/SnCl2 is a highly selective and mild
reducing agent for nitro compounds, and it does not reduce
carbonyl groups, unlike LVT attained from TiCl4/LiAlH4.
However, our research has proven that the reducing agent
obtained from TiCl4/LiAlH4 (mole ratio: 1/0.8) effectively
reduces a nitro group to a primary amine, even in the presence
of functional groups such as a carbonate or ester (Chart 2).
During the formation of the amine group, charge distribution
in the pyridine ring is changing, which enhances the
nucleophilicity of the pyridine nitrogen. A change in electron
distribution affects the susceptibility of TPG to thermal
deprotection, which is more evident at higher temperatures.
An example of using the switch is depicted in Chart 2. The
effect of reduction is observed as the disappearance of 1
(chromatogram A) and the occurrence of a new mixture of
diastereoisomers 6 (chromatogram B). After heating at 90 °C,
within only 10 min, the TPG is completely removed from 6 and
no traces of the blocked nucleoside are observed (chromato-
gram C). The presented HPLC data from subsequent
transformations indicate that the deprotection mechanism is
based on temperature-induced intramolecular cyclization whose
rate is accelerated by changing the chemical nature of
substituents.
room temperature within a five-day measurement period. This
increased stability is also noticeable at higher temperatures, but
not as distinctly as at room temperature.
Increasing the stability of TPG by introducing a nitro group
allows for easier manipulation at room temperature of the
blocked TPG molecules, but its removal at higher temperatures
requires prolonged heating.
The nitro group increases TPG stability, while the amine
group has the opposite effect. Therefore, connecting these
groups into an interdependent system (a quick switch of one
Moreover, this mechanism is confirmed by the identification
of thermal deprotection products, i.e., cyclic compound 14 and
3′-O-acetyl thymidine with a free hydroxyl group in 5′ position.
We have shown that the “chemical switch” on TPGs provides
efficient hydroxyl protection for four common DNA-series
nucleosides. The 4-nitro 2-Py TPG carbonates (1-4) were
prepared from the new aminoalcohols obtained according to
the literature procedure.8a Table 1 presents the results of
removing the 2-Py TPG to reveal factors influencing thermal
a
Chart 1. Comparison of the Thermal Stability of Two 2-Py TPGs, with (1) and without (11) Nitro Substituent
a
The HPLC data calculated as decreasing the amount of protected nucleosides 1 and 11. A − The analysis was performed at room temperature
(25°C) for 2 weeks. B − The analysis was performed at 90°C for 2 h. All measurements were taken in anhydrous acetonitrile.
C
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a
Chart 2. HPLC Analysis of the “Chemical Switch” Approach in Nucleoside Protection
a
(A) The nucleoside protected by the TPG with a nitro substituent. (B) After reduction with a titanium complex. (C) Thermolytic deprotection at
90 °C (8 min, buffer pH = 7).
a
Table 1. Percentage of TPG-Protected Nucleosides after Deprotection in Differ Solvents
a
b
Analysis was performed both before and after reduction. The deprotection time is showing the state when one of the nucleosides is fully
c
deprotected. In the case of 4-nitro derivatives at 25 °C, we have not observed total deprotection for any of nucleosides.
deprotection other than temperature or deprotection time.
Deprotection times were selected to demonstrate complete
deprotection of at least one of the presented blocked
nucleosides.
Introduction of the 4-nitro 2-Py TPG in compounds 1−4
and 6−9 (after reduction) leads to considerable disadvantages:
the presence of a mixture of diastereoisomers and possible
elimination of the benzyl proton during deprotection which
could reveal a competitive way of removing the TPGs. In order
to prevent this inconvenience, the phenyl group should be
replaced, e.g., by hydrogen. Therefore, we prepared a 2-
pyridinyl-2-(N-benzylamino)ethanol and its corresponding
carbonate 5 following a standard procedure.31 In the case of
5, using a nitro group enhances the stability of TPGs, but not as
significantly as in 1−4.32 This results in increased influence of
the benzyl group on the nucleophilicity of the pyridine nitrogen
and the steric effect that has been described earlier for
substituents of this type.30
We compared (Table 1) stability for 1−4 before reduction
and 6−9 after reduction in various solvents at 90 °C. Our
results indicate that 4-nitro-substituted 2-PyTPG in anhydrous
acetonitrile is marked by high stability in the heating process
independent of the used nucleosides. Moreover, we observed
that stability in aqueous solutions is significantly lowered and
also connected with the type of nucleosides. In the case of Cyt
(2) and Gua (4) derivatives, faster deprotection can be
explained by their higher basicity, which is particularly
significant in aqueous solutions. The same relationship was
also observed where the nitro group is switched into the amine
group (Table 1). Even though using buffer (pH = 7) instead of
water does not considerably affect the deprotection rate (Table
1), it can reduce the probability of unwanted alkaline
hydrolysis. Our experimental data show that the deprotection
rate is influenced not only by the nucleophilicity of the 2-
pyridine nitrogen but also by the composition of the solvents in
which heating occurs, as well as the character of the nucleoside
used.
Reduction with TiCl4/LiAlH4 results in a decrease in the pH
of the reaction mixture to 2. We noticed that nucleosides 6−10
obtained during reduction remain stable in thermal depro-
tection conditions in this pH value. On the basis of these two
observations, we postulate protonation in the pyridine ring
which can be another factor to stop thermal deprotection.
However, changing the pH value to neutral and then basic leads
to deprotonation and consequently initiates TPG thermal
deprotection.33
D
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15
Scheme 5. 2D 1H N HMBC and HSQC NMR Analysis of
These relations have proven that the change of pH is also
important in modulation of the thermal deprotection process
and is presumably related to protonation of a pyridine nitrogen.
In other words, possible protonation of the nucleophilic center
may hamper thermocyclization, which allows one to manipulate
stability and control thermocyclization in certain pH values.
In order to determine how protonation occurs, we used two-
Pyridine Nitrogen before and after Reduction
1
15
dimensional H N NMR analysis of the 2-PyTPG before
and after reduction Scheme 4. Proper assignments of nitrogen
Scheme 4. PKa Values for Precursor of 2PyTPG
(Measurment Was Performed in Acetonitryle/H2O)
a
a
1
15
Number of atoms for two-dimensional H N NMR descripted in
Scheme 5.
resonances before reduction were performed following the
15
analysis of the 1H N HMBC spectrum of 5 which is a model
to present the usefulness of “chemical switch” and protonation
(Scheme 5a). When 5 was completely reduced to 10, aqueous
HCl solution was added and the mixture was evaporated.
15
Comparative analysis of the 1H N HMBC and HSQC NMR
spectra (Scheme 5) for compounds 5 and 10 shows three
observable changes in the chemical shifts of TPG protons: (1)
signal of N1 is shifted downfield, (2) additional resonance of
new hydrogen (δ = 12.5 ppm) and its corresponding cross-peak
to the N1 (pyridine nitrogen) is observed, (3) appearance of
signals attributed to the amino hydrogen correlated to N4. The
correlations between HN1 in HSQC spectra, as well as
chemical shift differences of N1 (about 140 ppm) provided
evidence for protonation.
a
15
1H N HMBC spectra of compound 5, depicts all correlations
b
between nitrogen atoms and protons in molecule. Enlarged region of
1H N HSQC spectra of 5before reduction. This spectrum shows
15
one correlation between proton from thymidine and directly attached
c
nitrogen from nucleobase. Enlarged region of 1H N HSQC
spectra of 10after reduction. This spectrum shows an additional
correlation peaks between H1N1 and H4N4. This observation
confirm a protonation of N1 and present of amine group NH2 (N4) a
result of reduction process.
15
This experimental proof of pyridine nitrogen protonation
simultaneously decreases its nucleophilicity, which results in
stabilization of the TPG. Since deprotection of the 4-amino 2-
Py TPG does not occur in low pH, it should be triggered in
increased pH. Consistent with this postulate, pH 4 is still too
acidic to run thermal deprotection at higher temperatures,
while increasing the pH to 6.5 is sufficient to totally eliminate
protonation (heated for 7 min) and carry out thermal
deprotection of 2-PyTPG (Schemes 3, 4, and S5 of the SI).34
On the basis of the discussion presented above, thermocyc-
lization of a 2-pyridinyl TPG largely depends on the
nucleophilicity of a nitrogen atom in a pyridine ring. However,
electron distribution in a molecule does not always explain the
rate of intramolecular deprotection.35 Introduction of the 2-
pyridinyl N-oxide (2-PNO) in the structure of a thermolabile
protecting group might be an additional factor influencing
thermocyclization because, according to the dipole-moment
value, the oxygen atom in pyridine N-oxide is more nucleophilic
than the pyridine nitrogen atom.36 Therefore, the presence of
N-oxide in TPGs should significantly accelerate thermocycliza-
tion, but no such relation was observed.37 Impeding the
formation of a 6-membered cyclic product which requires the
high level of activation energy to be exceeded could have an
influence on the high thermal stability of the 2-PNO TPG. This
is noticeable in comparison with 11, where a 5-membered
product is generated more easily (90 °C, 15 min, aqueous
conditions). Analysis of TPG thermolytic properties shows that
nucleophilicity is required but not always decisive during
thermal deprotection. Ring size has a considerable influence on
the temperature necessary for thermocyclization, which has
previously been shown in the case of thermal isomerization of
cyclodienynes leading to corresponding benzocycloalkenes.38
In the case of 2-PNO TPGs, thermocyclization is possible
but needs additional support. Introduction of a nitro group to
11 and its “chemical switch” into an amine group reverses
electron distribution in the molecule, which can result in
thermal deprotection. Consequently, increasing the nucleophil-
icity of the N-oxide by means of an amine group has allowed
the 6-membered ring to be obtained. Thus, it is assumed that
the cyclic product was formed by a nucleophilic attack of an
oxygen atom on carbon located at the carbonate. Such a
reaction sequence has been deduced from the analysis of the
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from 3′-O-Acetyldeoxythymidine (0.1 mmol) with 82% (52 mg) yield
reaction mixture and the postulated structure of the byproduct
(13).
The isolated cyclic byproducts 13 and 14 (Scheme 6) of
thermocyclization were confirmed by UV−Vis and mass
1
following general procedure A. H NMR (400 MHz, DMSO-d6) δ
(ppm) 11.37 (d, J = 6.3, 1H), 8.41−8.37 (m, 1H), 7.42−7.20 (m, 8H),
6.17−6.13 (m, 1H), 5.91−5.85 (m, 1H), 5.11−5.05 (m, 1H), 4.3−3.97
(m, 5H), 3.05 (s, 3H), 2.34−2.21 (m, 2H), 2.04 (s, 3H), 1.72 (s, 3H).
13C NMR (125 MHz, DMSO-d6) δ (ppm) 20.7, 35.3, 35.4, 37.4, 37.5,
54.3, 54.4, 67.2, 77.6, 80.7, 80.8, 83.9, 98.4, 103.7, 109.9, 126.3, 126.4,
128.3, 135.5, 137.4, 150.3, 153.6, 155.2, 159.3, 163.6, 169.9. HRMS
(ESI-q-TOF) m/z: [M + H]+ calcd. for C27H30N5O10 584.1993; found
584.1988.
Scheme 6. Thermocyclization ProductsPossible Structures
N4-Benzoyl-3′-O-acetyl--5′-O-[2-N-methyl-N-(4-nitropyridin-2-
yl)]amino-1-phenylethyloxycarbonyl-2′-deoksycytidine (2). Com-
pound 2 was obtained from 3′-O-Acetyldeoxycytidine (0.1 mmol)
with 79% (57 mg) yield following general procedure A. 1H NMR (400
MHz; DMSO-d6) δ (ppm) 11.28 (s, 1H), 8.38 (t, J = 5.63, 1H), 8.0
(m, 3H), 7.63 (m, 1H), 7.52 (m, 2H), 7.37 (m, 7H), 7.21 (m, 2H),
6.12 (t, J = 6.76, 1H), 5.88 (m, 1H), 5.14 (m, 1H), 4.26 (m, 3H), 4.02
(m, 2H), 3.05 (s, 3H), 2.27 (m, 1H), 2.05 (s, 3H). 13C NMR (100
MHz; DMSO-d6) δ 169.9, 167.3, 163.3, 159.3, 155.2, 154.2, 153.6,
150.3, 144.8, 137.4, 133.1, 132.7, 128.6, 128.4, 128.3, 126.3, 118.1,
103.6, 98.4, 96.3, 86.8, 81.8, 77.6, 73.7, 67.2, 55.9, 54.4, 40.0, 38.9,
37.6, 25.1, 20.7, 18.5. HRMS (ESI-q-TOF) m/z: [M + H]+ calcd. for
C33H33N6O10 673.2258; found 673.2254
analyses for 13 and MS, UV−Vis analysis, and NMR spectra
for 14. The presence of these compounds in the reaction
mixture is a proof of the removal of TPGs by the intramolecular
mechanism.
N6-Benzoyl-3′-O-acetyl--5′-O-[2-N-methyl-N-(4-nitropyridin-2-
yl)]amino-1-phenylethyloxycarbonyl-2′-deoxyadenosine (3). Com-
pound 3 was obtained from 3′-O-Acetyldeoxyadenosine with 75% (56
mg) yield following general procedure A. 1H NMR (500 MHz,
Cd2Cl2) δ (ppm) 9.03 (s, 1H), 8.74 (s, 1H), 8.37−8.34 (dd, J = 7.9,
5.8 Hz, 1H), 8.01 (d, J = 7.7, 2H), 7.65−7.62 (m, 1H), 7.56 (td, J =
7.6, 4.5, 2H), 7.43−7.31 (m, 5H), 7.20−7.16 (m, 2H), 6.56−6.52 (m,
1H), 5.94−5.91 (m, 1H), 5.41−5.38 (m, 1H), 4.43−4.31 (m, 3H),
4.09−3.92 (m, 2H), 3.06 (d, J = 7.3, 3H), 2.94−2.87 (m, 1H), 2.67−
2.62 (m, 1H), 2.11 (s, 3H). 13C NMR (100 MHz; DMSO-d6) δ
(ppm) 169.9, 169.6, 159.2, 155.2, 153.5, 151.92, 151.67, 150.50,
150.25, 142.9, 137.4, 130.3, 132.4, 128.5, 128.3, 126.3, 125.9, 103.6,
98.4, 83.7, 81.4, 77.5, 74.0, 67.2, 54.5, 37.5, 35.3, 20.7. HRMS (ESI-q-
TOF) m/z: [M + H]+ calcd. for C34H33N8O9 697.2371; found
697.2363.
CONCLUSIONS
■
TPGs in the form of carbonates are easily incorporated into a
molecule and thus protect hydroxyl groups with variable
thermolability. Incorporation of a nitro group stabilizes TPGs
across a wide range of temperatures and opens the way to
modulate TPG properties, which also improves their potential
application in organic chemistry. Thermolability steering is
based on the “chemical switch” approach, i.e., selective
reduction of a nitro group to an amine group by means of
titanium complexes, which results in accelerated thermocycliza-
tion. The LVT was chosen as a reductant for the nitro group to
generate low pH and preserve the carbonyl group in reduction
conditions. Moreover, the highest importance for further
applications is the fact that the pH manipulation provides the
steering for TPG thermolability by protonation of a pyridine
nitrogen.
The strong dependence between thermocyclization and pH
values after reduction may have a very practical biological
application. Since stabilizing 2-PyTPG by protonation is a very
promising method for thermocyclization control, we are
currently searching for cellular and molecular applications for
the discussed approach, also using synthetic nucleic acids. The
results of our future research will be the subject of subsequent
reports.
N2-isobutyryl-3′-O-acetyl-5′-O-[2-N-methyl-N-(4-nitropyridin-2-
yl)]amino-1-phenylethyloxycarbonyl-2′-deoxyguanosine (4). Com-
pound 4 was obtained from 3′-O-Acetyldeoxyguanosine with 86% (56
mg) yield following general procedure A. 1H NMR (400 MHz,
DMSO-d6) δ (ppm) 8.37 (m, 1H), 8.16 (s, 1H), 7.36 (m, 6H), 7.19
(m, 2H), 6.20 (t, J = 6.90, 1H), 5.81 (m, 1H), 5.25 (t, J = 7.64, 1H),
4.21 (m, 3H), 4.03 (m, 2H), 3.03 (d, J = 2.29, 3H), 2.84 (m, 1H), 2.05
(d, J = 3.21, 3H), 1.11 (m, 7H). 13C NMR (100 MHz, DMSO-d6) δ
(ppm) 180.1, 169.8, 159.3, 155.2, 154.8, 153.7, 150.3, 148.55, 148.25,
137.4, 137.2, 128.3,126.8, 126.3, 120.3, 103.6, 98.4, 81.5, 77.6, 74.1,
67.1, 56.0, 54.5, 45.7,, 38.2, 37.5, 35.6, 34.8, 20.7, 18.8, 18.5. HRMS
(ESI-q-TOF) m/z: [M + H]+ calcd. for C31H35N8O10 679.2476; found
679.2474.
3′-O-acetyl-5′-O-[2-N-methyl-N-(pyridin-2-yl)]amino-1-phenyle-
thyloxycarbonyl-thymidine (11). Compound 1139 was obtained from
3′-O-acetylthymidine (0.1 mmol) and ( )-2-[N-Methyl-N-(pyridin-2-
yl)amino]-1-phenylethanol (0.1 mmol, 28.9 mg) with 80% (54 mg)
yield following general procedure A. 1H NMR (400 MHz, DMSO-d6)
δ (ppm) 11.33 (bs, 1H), 8.11−8.08 (m, 1H), 7.53−7.32 (m, 7H),
6.62−6.55 (m, 2H), 6.21−6.13 (m, 2H), 5.88−5.83 (m, 1H), 5.12−
5.08 (m, 1H), 4.35−3.9 (m, 5H), 2.92 (s, 3H), 2.33−2.2 (m, 2H),
1.73−17.2 (dd, J = 5.5, 0.9, 3H). HRMS (ESI-q-TOF) m/z: [M +
H]+calcd. for C27H31N4O8 539.2142; found 539.2136.
3′-O-acetyl-5′-O-[2-N-benzyl-N-(4-nitropyridin-2-yl)]-
aminoethyloxycarbonylthymidine (5). N-benzylethanolamine (81.9
mg, 0.30 mmol) and 1,1′-carbonyldiimidazole (58.3 mg, 0.36 mmol)
were added to a anhydrous acetonitrile (3 mL) in a dried round-
bottom flask. The reaction mixture was stirred at room temperature
(60 min), in inert atmosphere, until starting material was no observed
by thin layer chromatography. Then 3′-O-acetylthymidine (78.9 mg,
0.3 mmol) and 1,1,3,3-tertramethylguanidine (0.0008 mmol, 150 μL)
were added. Reaction was carried out at room temperature for 2 h.
EXPERIMENTAL SECTION
■
General Procedure (A) for Preparation of N-Protected-3′-O-
acetyl-5′-(N-methyl-(4-nitropyridin-2-yl)]amino-1-phenylethy-
loxycarbonyl Nucleosides (1−4). 3′-O-Acetyldeoxynucleosides (0.1
mmol) were dissolved in dry acetonitrile (2 mL) and placed into the
flask with a magnetic stirrer with 1′1′-carbonyldiimidazol (0.36 mM,
58.32 mg). The reaction was carried out for 30 min at room
temperature and analyzed by TLC (ethyl acetate/n-hexane 9/1). Then
N-methyl-(4-nitropyridin-2yl)-1-phenylaminoethyl-1-ol (0.1 mmol,
29.3 mg) and 1,1,3,3 -tetramethylguanidine (0.0008 mmol, 100 μL)
were added. The resulting solution was allowed to stir for 2 h at room
temperature. The reaction was analyzed by TLC analysis (dichloro-
methane/methanol 95:5). The solvent was evaporated under reduced
pressure, and the resulting residue was purified by column
chromatography over silica gel (DCM/MeOH) to obtain pure
product (1-4, 11).
3′-O-acetyl-5′-O-[2-N-methyl-N-(4-nitropyridin-2-yl)]amino-1-
phenylethyloxycarbonylthymidine (1). Compound 1 was obtained
F
DOI: 10.1021/acs.joc.5b02033
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The Journal of Organic Chemistry
Article
Afterward the solution was concentrated, under reduced pressure, and
applied to the top of a silica gel (15 g) chromatography column,
equilibrated in CH2Cl2. The column was eluted with a linear gradient
of CH2Cl2 (100%) to CH2Cl2: MeOH (95:5) to afford pure 5 (129
[N-methyl-N-(4-nitro-2-pyridyl-N-oxide)amino]-1-phenylethanol (0.3
mmol, 86.73 mg) with 77% (139 mg) yield following general
1
procedure B. H NMR (400 MHz, DMSO-d6) δ (ppm) 8.26 (d, J =
7.2, 1H), 7.64 (m, J = 7.2, 1H), 7.56 (dd, 1H), 7.35 (m, 6H), 6.16 (m,
1H), 5.93 (m, 1H), 5.09 (m, 1H), 4.35 (m, 1H), 4.23 (m, 2H), 4.05
(m, 1H), 3.9 (m, 1H), 3.08 (s, 3H), 2.30 (m, 2H), 2.1 (m, 3H), 1.74
(dd, J = 1.2, 7.0, 3H). 13C NMR (100 MHz, DMSO-d6) δ (ppm)
170.0, 163. 6, 159.3, 155.2, 153.6, 150.4, 142.6, 140.7, 137.3, 135.7,
128.5, 128.3, 126.1, 110.9, 109.9, 108.7, 84.0, 83.6, 80.8, 78.2, 74.7,
73.4, 67.2, 53.9, 35.4, 20.7, 12.0. HRMS (ESI-q-TOF) m/z: [M + H]+
calcd. for C27H30N5O11 600.1942; found 600.1939.
1
mg, 80%). H NMR (500 MHz, DMSO-d6) δ (ppm) 11.36 (s, 1H),
8.37 (d, J = 5.4 Hz, 1H), 7.46 (s, 1H), 7.36 (s, 1H), 7.31 (t, J = 7.4 Hz,
2H), 7.26−7.21 (m, 5H), 6.16 (dd, J = 8.0, 6.5 Hz, 1H), 5.16−5.15
(m, 1H), 4.86 (s, 2H), 4.38−4.30 (m, 2H), 4.13 (dd, J = 7.6, 4.6 Hz,
1H), 3.96 (t, J = 5.3 Hz, 2H), 2.36−2.31 (m, 1H), 2.26 (ddd, J = 14.2,
6.1, 2.4 Hz, 1H), 2.06 (s, 3H), 1.73 (s, 3H). 13C NMR (125 MHz,
DMSO-d6) δ (ppm) 169.9, 163.5, 159.2, 155.3, 154.2, 150.3, 137.6,
135.6, 128.6, 128.3, 127.0, 126.5, 109.9, 104.1, 98.7, 84.0, 80.8, 73.6,
67.2, 65.5, 51.5, 47.0, 35.4, 20.7, 12.0. HRMS (ESI-q-TOF) m/z: [M
+ H]+ calcd. for C27H30N5O10 584.1993; found 584.1984.
General Procedure (B) for Generating of the Reducing
Agent. Anhydrous tetrahydrofuran was placed in a two-neck round-
bottom flask and cooled to 0 °C in water/ice bath. Then the titanium
tetrachloride (0.5 mmol, 55 μL) was added portionwise and kept for
10 min at room temperature. Lithium aluminum hydride (0.35 mmol,
13 mg) was added portionwise, and the resulting black slurry was
stirred in inert atmosphere, at room temperature for 15 min (TiCl4/
LiAlH4 − 1/0.8).
General Procedure for N-Protected-3′-O-acetyl-5′-(N-meth-
yl-(4-aminopyridin-2-yl)]aminoethyloxycarbonyl Nucleosides
(6−10). Compounds (1−4) (0.1 mmol), diluted in a anhydrous
THF, was added portionwise at 0 °C to the stirred black slurry of
titanium reagent. The reaction mixture was stirred at room
temperature for 15−30 min, until the starting material was not
observed by TLC. Traces of LiAlH4 were decomposed with water, the
black slurry was centrifuged, and the solution was flushed through thin
layer of silica gel (DCM/MeOH 95:5).
8-Amino-1-methyl-3-phenyl-2,3-dihydro-1H-pyrido[1,2-b][1,2,4]-
oxadiazin-5-ium Chloride (13). HRMS (ESI-q-TOF) m/z: [M]+
calcd. for C14H16N3O+ 242.1288; found 242.1309.
7-Amino-1-methyl-3-phenyl-2,3-dihydro-1H-imidazo[1,2-a]-
1
pyridin-4-ium Chloride (14). H NMR (400 MHz, DMSO) δ (ppm)
7.71 (s, 2H), 7.47−7.35 (m, 5H), 6.18 (d, J = 7.2 Hz, 1H), 5.91 (s,
1H), 5.72 (dd, J = 9.3, 6.8, 1H), 4.17 (t = , J = 9.7 Hz, 1H), 3.67 (dd, J
= 9.8, 6.7 Hz, 2H), 2.96 (s, 3H). HRMS (ESI-q-TOF) m/z: [M]+
+
calcd. for C14H16N3 226.1339; found 226.1339.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.5b02033.
■
S
Materials, general methods, characterization of depro-
tection rates and NMR and HRMS data (PDF)
AUTHOR INFORMATION
Corresponding Author
*Phone: +48618528503; e-mail: maro@ibch.poznan.pl
(M.K.C.).
■
3′-O-acetyl-5′-O-[2-N-methyl-N-(4-aminopyridin-2-yl)]amino-1-
phenylethyloxycarbonyl-thymidine (6). Compound 1 was trans-
formed into compound 6 with 68% (38 mg) yield following general
procedure B. 1H NMR (400 MHz, DMSO) δ (ppm) 11.34 (d, J = 6.8,
1H), 11.03 (bs, 2H), 8.92 (d, J = 5.4, 1H), 8.13−8.09 (m, 1H), 7.53
(d, J = 3, 1H), 7.36−7.28 (m, 6H), 6.08 (dd, J = 12.3, 6.1, 1H), 5.84−
5.79 (m, 1H), 5.02 (dd, J = 16.1, 6.5, 1H), 4.27−4.04 (m, 5H), 2.41−
2.29 (m, 1H), 2.16−2.14 (m, 1H), 1.98 (d, J = 1.8, 3H), 1.66 (s, 3H),
1.22 (d, J = 6.4, 3H). 13C NMR (100 MHz, DMSO-d6) δ (ppm) 12.5,
21.2, 35.8, 37.8, 54.7, 67.6, 73.8, 78.1, 81.3, 84.5, 99.0, 104.2, 110.4,
126.9, 18.1, 129.0, 137.8, 136.4, 141.7, 146.3, 150.8, 155.6, 159.6,
164.1, 170.4. HRMS (ESI-q-TOF) m/z: [M + H]+ calcd. for
C27H32N5O8; 554.2251, found 554.2250.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We would like to thank Professor Jacek Stawin
■
ski (IChB PAS)
for reading this manuscript and for his helpful suggestions. This
research was supported by the European Regional Develop-
ment Fund within the Innovative Economy Program (Grant
No. POIG.01.03.01-30-045). This publication was supported
by the Polish Ministry of Science and Higher Education, under
the KNOW program.
N4-Benzoyl-3′-O-acetyl-5′-O-[2-N-methyl-N-(4-aminopyridin-2-
yl)]amino-1-phenylethyloxycarbonyl-2′-deoksycytidine (7). Com-
pound 2 was transformed into compound 7 following general
procedure B. HRMS (ESI-q-TOF) m/z: [M + H]+ calcd. for
C33H35N6O8; 643.2516; found 643.2517.
REFERENCES
■
(1) Keasling, J. D. ACS Chem. Biol. 2008, 3, 64−76.
(2) Grajkowski, A.; Pedras-Vasconcelos, J.; Wang, V. V.; Ausin, C.;
Hess, S.; Verthelyi, D.; Beaucage, S. L. Nucleic Acids Res. 2005, 33,
3550−3560.
(3) Lebedev, A. V.; Paul, N.; Yee, J.; Timoshchuk, V. A.; Shum, J.;
Miyagi, K.; Kellum, J.; Hogrefe, R. I.; Zon, G. Nucleic Acids Res. 2008,
36, 131.
3′-O-acetyl-5′-O-[2-N-benzyl-N-(4-aminopyridin-2-yl)]-
aminoethyloxycarbonyl-thymidine (10). Compound 5 (84 mg, 0.14
mmol) was reduced following the general procedure. To the obtained
precipitate free solution aq 12 M HCl (1 equiv) was added, and the
solvent was evaporated under reduced pressure. Crude product was
diluted in methanol, applied on the PLC and eluted with iPrOH: H2O
(7:3) at 4 °C to afford product 10 (58 mg, 74%). 1H NMR (500 MHz,
MeOd) δ (ppm) 7.45−7.44 (m, 1H), 7.32 (t, J = 7.5 Hz, 2H), 7.24(t, J
= 7.4 Hz, 1H), 7.17 (d, J = 7.2 Hz, 2H), 6.25 (dd, J = 7.2, 2.0 Hz, 1H),
6.18 (dd, J = 8.4, 5.9 Hz, 1H), 5.95 (d, J = 2.0 Hz, 1H), 5.17 (dt, J =
6.5, 2.3 Hz, 1H), 4.72 (s, 2H), 4.42−4.35 (m, 2H), 4.32 (dd, J = 11.8,
3.3 Hz, 2H), 4.20−4.18 (m, 1H), 3.85 (dd, J = 10.7, 5.7 Hz, 2H), 2.37
(ddd, J = 14.3, 5.9, 2.2 Hz, 1H), 2.29−2.26 (m, 1H), 2.06 (s, 3H), 2.00
(s, 3H). 13C NMR (100 MHz, MeOd) δ (ppm) 172.1, 166.2, 161.2,
156.0, 153.7, 152.2, 137.9, 137.3, 136.5, 130.1, 128.9, 127.4, 111.9,
104.3, 89.8, 86.7, 83.5, 75.6, 68.7, 62.9, 56.3, 54.2, 30.7, 20.9, 12.9.
HRMS (ESI-q-TOF) m/z: [M + H]+ calcd. for C27H32N5O8
554.2251, found 554.2245.
(4) Anastas, P. T.; Kirchhoff, M. M. Acc. Chem. Res. 2002, 35, 686−
694.
(5) (a) Wilk, A.; Grajkowski, A.; Philips, L. R.; Beaucage, S. L. J. Org.
Chem. 1999, 64, 7515−7522. (b) Prokopczyk, B.; Bertinato, P.;
Hoffman, D. Carcinogenesis 1988, 9, 2125−2128.
(6) (a) Wilk, A.; Chmielewski, M. K.; Grajkowski, A.; Phillips, L. R.;
Beaucage, S. L. Tetrahedron Lett. 2001, 42, 5635−5639. (b) Grajkow-
ski, A.; Wilk, A.; Chmielewski, M. K.; Phillips, L. R.; Beaucage, S. L.
Org. Lett. 2001, 3, 1287−1290. (c) Wilk, A.; Chmielewski, M. K.;
Grajkowski, A.; Phillips, L. R.; Beaucage, S. L. J. Org. Chem. 2002, 67,
6430−6438.
(7) (a) Ciesl
́
ak, J.; Grajkowski, A.; Livengood, V.; Beaucage, S. L. J.
O-5′-(3′-O-acetylthymidinylcarbonyloxy-1-phenylethyl)
(methyl)amino)-4-nitropyridine 1-Oxide (12). Compound 12 was
obtained from 3′-O-acetylthymidine (0.3 mmol, 87.9 mg) and ( )-2-
Org. Chem. 2004, 69, 2509−2515. (b) Kiuru, E.; Ahmed, Z.; Lonnberg,
H.; Beigelman, L.; Ora, M. J. Org. Chem. 2013, 78, 950−959.
̈
G
DOI: 10.1021/acs.joc.5b02033
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
(8) (a) Chmielewski, M. K.; Marchan, V.; Cieslak, J.; Grajkowski, A.;
Livengood, V.; Munch, U.; Wilk, A.; Beaucage, S. L. J. Org. Chem.
2003, 68, 10003−10012. (b) Marchan, V.; Cieslak, J.; Livengood, V.;
́ ́
Beaucage, S. L. J. Am. Chem. Soc. 2004, 126, 9601−9610.
(9) Ohkubo, A.; Kasuya, R.; Miyata, K.; Tsunoda, H.; Seio, K.;
Sekine, M. Org. Biomol. Chem. 2009, 7, 687−694.
(10) Grajkowski, A.; Cieslak, J.; Chmielewski, M. K.; Marchan, V.;
Phillips, L. R.; Wilk, A.; Beaucage, S. L. Ann. N. Y. Acad. Sci. 2003,
1002, 1−22.
(11) Chmielewski, M. K. Org. Lett. 2009, 11, 3742−3745.
(12) Ratajczak, T.; Chmielewski, M. K. J. Org. Chem. 2012, 77,
7866−7872.
(13) Kocienski, P. J. Protecting Groups, 3rd ed.; Georg Thieme Verlag:
New York, 2005.
(14) Beaucage, S. L.; Chmielewski, M. K. Patent No US 7,612,197 B2
Nov 3, 2009.
(15) Wackerlin, C.; Chylarecka, D.; Kleibert, A.; Muller, K.; Nolting,
F.; Jung, T. A.; Ballav, N. Nat. Commun. 2010, 1, 1−7.
(16) Liu, H.; Schmidt, J. J.; Bachand, G. D.; Rizk, S. S.; Looger, L. L.;
Hellinga, H. W.; Montemagno, C. D. Nat. Mater. 2002, 1, 173−177.
(17) Vishnuvardhan, V.; Kala, R.; Rao, T. P. Anal. Chim. Acta 2008,
623, 53−58.
(18) Wettig, S. D.; Bare, G. A.; Skinner, R. J. S.; Lee, J. S. Nano Lett.
2003, 3, 617−622.
(19) Bavin, P. M. G. Org. Synth. 1960, 40, 5.
(20) Allen, C. F. H.; VanAllan, J. Org. Synth. 1942, 22, 9.
(21) Scheuerman, R. A.; Tumelty, D. Tetrahedron Lett. 2000, 41,
6531−6535.
(22) Jiang, Q.; Jiang, D.; Jiang, Y.; Fu, H.; Zhao, Y. Synlett 2007, 12,
1836−1842.
(23) Awad, W. I.; Hassan, S. S. M; Zaki, M. T. M. Anal. Chem. 1972,
44, 911−915.
(24) Ho, T. L.; Wong, C. M. Synthesis 1974, 1, 45−45.
(25) Khan, F. A.; Dash, J.; Sudheer, C.; Gupta, R. K. Tetrahedron Lett.
2003, 44, 7783−7787.
(26) Basu, M. K. Tetrahedron Lett. 2000, 41, 5603−5606.
(27) Breslow, R.; Overman, L. E. J. Am. Chem. Soc. 1970, 92, 1075−
1077.
(28) Malinowski, M.; Kaczmarek, Ł.; Rozpłoch, F. J. Chem. Soc.,
Perkin Trans. 2 1991, 6, 879−883.
(29) In the SI, the full data was presented (Tables S1−S3; page S3)
(30) Preliminary laboratory tests show increased stability in ambient
temperature TPG former from N-oxide derivatives.
(31) Chmielewski, M. K. Tetrahedron Lett. 2012, 53 (6), 666−669.
(32) The charts with kinetic in different temperature and different
solvent mixture was placed in the SI on page S6.
(33) In the SI, pH influence on the stability of TGO is presented
(Scheme S3; page S5).
(34) In the SI, pH influence on stability of TGO is presented
(Scheme S4; page S9).
(35) Chmielewski, M. K.; Tykarska, E.; Markiewicz, W. T.;
Rypniewski, W. R. Engineering N-(2-pyridyl)aminoethyl alcohols as
potential precursor of thermolabile protection groups. New J. Chem.
2012, 36 (3), 603−612.
(36) Higher dipole-moment [4.37 D (PNO) vs2.03 D (pyridine)]-
much weaker base[pKa= 0.79 (PNO) vs pKa= 5.2 (pyridine)].
(37) Initial experiments based mainly on the HPLC analysis. The
obtained results suggested reinforcing nucleophilicity and introducing
the “chemical switch” discussed herein.
(38) Hopf, H.; Kruger, A. Chem. - Eur. J. 2001, 7, 4378−4385.
̈
(39) Chmielewski, M. K.; Marchan, V.; Cieslak, J.; Grajkowski, A.;
Livengood, V.; Munch, U.; Wilk, A.; Beaucage, S. L. J. Org. Chem.
2003, 68, 10003−10012.
H
DOI: 10.1021/acs.joc.5b02033
J. Org. Chem. XXXX, XXX, XXX−XXX