Novel thermolabile protecting groups with higher stability at ambient temperature

Article abstract of DOI:10.1016/j.tetlet.2011.11.122

Novel thermolabile hydroxyl protecting groups of increased thermostability are proposed. The stability of these groups at different temperatures ranges has been determined. The 2-pyridyl-N-(2,4-difluorobenzyl)aminoethyl unit was selected as stable at ambient temperature and very labile at increased temperature. The half-life times for the best groups were established. Application of this chemistry to the protection of different hydroxyl groups of thymidine was also demonstrated.

Full text of DOI:10.1016/j.tetlet.2011.11.122

Tetrahedron Letters 53 (2012) 666–669
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Novel thermolabile protecting groups with higher stability
at ambient temperature
Marcin K. Chmielewski
´
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel thermolabile hydroxyl protecting groups of increased thermostability are proposed. The stability of
these groups at different temperatures ranges has been determined. The 2-pyridyl-N-(2,4-difluoroben-
zyl)aminoethyl unit was selected as stable at ambient temperature and very labile at increased temper-
ature. The half-life times for the best groups were established. Application of this chemistry to the
protection of different hydroxyl groups of thymidine was also demonstrated.
Received 31 August 2011
Revised 15 November 2011
Accepted 25 November 2011
Available online 3 December 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Thermolabile protecting groups
Thermocyclization
Chemical synthesis of DNA
Thermolytic carbonate
Click–clack
With the development of further applications of synthetic bio-
molecules, for example, nucleic acids, peptides, etc., rapid progress
in devising new systems for protecting reactive sites of molecules
is being made. This trend focuses on the development of groups
whose removal may be carried out in vivo. However, apart from
the protective function most of the popular protecting groups often
find multi-functional uses.1
exhibited near optimal properties for 50-hydroxyl protection by vir-
tue of the mildness of its cleavage conditions.9
Recently, the introduction of the nucleophilic 2-aminopyridyl
system10 has made it possible to develop a new class of thermola-
bile groups which are useful for protection of the hydroxyl func-
tional group.11 Their usefulness as phosphate protecting groups
increased after a ‘click–clack’ concept was devised, a method
which temporarily blocks the thermolabile properties of TPG.12
2-[N-Methyl-N-(2-pyridyl)]amino-1-phenylethanol (1) was se-
lected as a particularly promising precursor of thermolytic carbon-
ate for hydroxyl group protection.13 It was characterized by a total
unblocking time of approximately 15 min at 90 °C. However, par-
tial removal of this group at ambient temperature has been ob-
served (10%, 24 hours in acetonitrile). This limits considerably
the application of protecting groups for multi-stage syntheses
(e.g., during the synthesis of nucleic acids).14 Structural and crys-
tallographic analysis15 showed that its isomer 2 could be a good
candidate for thermolytic carbonates (Scheme 1). These findings
prompted further investigation of N-pyridyl-N-benzylaminoethyl
carbonates for protecting hydroxyl functions. The application of
isomer 2 as a precursor of TPG brings the following advantages:
(i) eliminates the chiral center at the carbon atom; (ii) increases
the nucleophilicity of the pyridine ring, thus affecting the unblock-
ing rate; (iii) reduces the cost of the TPG group synthesis.
Very useful protecting groups have been proposed, which can be
removed only by increasing the temperature. Due to this property,
they have been termed thermolabile protecting groups (TPG). The
first thermal deprotection method was presented for the protection
of a phosphate during the synthesis of nucleic acids.2,3 Nucleophilic
intramolecular thermocyclization was the driving force for removal
of TPGs and the kinetics depend closely on the temperature. This
reaction can be conducted successfully in aqueous environment
at pH 7 as well as in organic/aqueous solvents. 2-(N-Formyl-N-
methyl)aminoethyl,4 3-(N-tert-butylcarboxamido)-1-propyl,5 3-
(2-pyridyl)-1-propyl,6 and 4-methylthio-1-butyl7 were tested as
effective protecting groups for phosphate. Among TPGs, the 4-oxo-
pentyl group deserves special attention as it enables effective and
economical protection of phosphate and thiophosphate centers.
This group can be removed by intramolecular cyclodeesterification
induced by heating or using either pressurized gaseous amines, or
concentrated ammonium hydroxide.8 However, none of these
groups has been used effectively to protect a hydroxyl moiety due
to the high durability of the appropriate carbonate derivatives used.
Also the 2,2,5,5-tetramethylpyrrolidin-3-one-1-sulfinyl group
In an effort to find the optimum medium between shortening
the time of deprotection and increasing the stability of the thermo-
labile protecting group, new TPGs based on N-pyridyl-N-benzyl-
aminoethyl carbonates were investigated (Scheme 1). Their
precursor, N-pyridyl-N-benzylaminoethanol, was prepared from
E-mail address: maro@ibch.poznan.pl
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.11.122

M. K. Chmielewski / Tetrahedron Letters 53 (2012) 666–669
667
hydrogen atom of the alkyl chain (Fig. 1). This kind of interaction
will move the benzyl group toward the alkyl chain, limiting the
thermal deprotection reaction due to the inaccessibility of the elec-
trophilic center. Probably, in the case of compounds 8 and 9 with
shorter half-life times at 20 °C, there is no interaction between
the benzyl ring and the chain.
R'
R
OH
OH
N
N
2 R=H; R'=H
N
N
3 R=H; R'=F
4 R=F; R'=H
5 R=F; R'=F
1
A study of the kinetics of the thermal unblocking reaction was
performed using model 30-O-acetylthymidine where the 50-OH
function was protected by the TPG. The presence of a 30-O-acetyl
group indicates that the deprotection process does not follow the
ester hydrolysis mechanism. The protection of a free 50-hydroxyl
group consists of a two-step reaction of carbonyldiimidazole with
the amino alcohol/TGP precursor and a hydroxyl group, which re-
sults in compounds 7–10. The final products were purified by silica
gel chromatography at 5 °C and characterized by 1H NMR spectros-
copy and high-resolution mass spectrometry. Removal of the pro-
tecting group (Scheme 3) was conducted in acetonitrile/aqueous
phosphate buffer mixture at two different temperatures, 90 and
20 °C. It is proposed that the half-life times can be used for evalu-
ation of the thermolability of TPGs for a given group under defined
conditions.17 The obtained values of half-life times are presented in
Table 1, and the deprotection curve is shown in Figure 2.
From the data presented it can be concluded that fluorine deriv-
atives have higher half-life times at 90 °C. Compounds 7 and 8 are
characterized by shorter half-life times at 90 °C. The situation is
quite different when the stability of these groups at ambient tem-
perature is analyzed. Compounds 8 and 9, which have one fluorine
atom, showed lower stability of the carbonate under these condi-
tions. However, 10 and in particular 15, have increased thermal
stability, allowing their free manipulation. These compounds also
have longer half-life times at 90 °C.
All the analyzed N-benzyl derivatives, compared to previously
published compound 6, are characterized by increased stability
at ambient temperature. It is known from the literature10 that
10% deprotection of compound 6 occurs in anhydrous acetonitrile
at ambient temperature over 24 h. For compounds 7, 8, and 9 the
thermal deprotection level under these conditions did not exceed
7%, and for compound 10 was 4%.
These characteristics may be due to restricted rotation in cer-
tain parts of the molecule. This makes adoption of a convenient
conformation for intramolecular cyclization difficult. It seems that
the presence of the benzyl ring is a significant factor in the thermal
control of the molecule at lower temperatures.
O
NH
O
N
O
O
O
O
N
N
O
O
6
O
R'
NH
O
7 R=H; R'=H
8 R=H; R'=F
9 R=F; R'=H
10 R=F; R'=F
N
O
R
O
O
O
N
N
O
O
Scheme 1. Structures of the precursors of thermolabile protecting groups and their
active carbonates.
commercially available reagents via nucleophilic substitution as
presented in Scheme 2.16 The synthesis of amino alcohols—TPG pre-
cursors—involved reaction of 2-pyridylaminoethanol (13), by
means of microwave irradiation, with fluoro-substituted benzyl
bromides 14. The MW assistance reduced the reaction time and in-
creased the yield of the total process.
Herein is reported the preparation and characterization of novel
precursors 2–5 and their application for the protection of hydroxyl
groups in nucleosides to give thermolabile carbonates 7–10
(Scheme 1). The deprotection kinetics of these groups for the mod-
el 30-O-acetylthymidine and 50-O-DMT thymidine are presented.
A characteristic feature of these groups is the presence of a ben-
zyl group at the exocyclic amine. Preliminary experiments have
shown that fluorine atoms on the aromatic ring stabilize thermal
carbonates.
The usefulness of a protecting group increases when it is possi-
ble to monitor its blocking and unblocking (Fig. 3). The blocked
form of N-benzyl TPG 7–10 has an additional diagnostic absorption
maximum at 308 nm in the UV–vis spectrum. In the UV–vis spec-
trum for bicyclic compound 12, a shift of the absorption band oc-
In particular, the para and ortho positions on the benzyl ring
have a significant impact on the stability of TPGs in acetonitrile.
An attempt to synthesize a TPG containing methyl groups, instead
of fluorine, was unsuccessful.
Additionally, the introduction of two fluorine atoms on the ben-
zyl ring may generate additional types of interactions due to a pos-
sibility of Fꢀ ꢀ ꢀH–C bonds. For compound 5 in which the fluorine
atoms are located at the ortho and para positions the NOESY-type
interactions in the two dimensional 1H–19F NMR HOESY spectrum
at 20 °C were observed between the ortho fluorine atom and
curs to
a new maximum at 337 nm. Measurement of the
absorption intensity at 337 nm can be used as a diagnostic element
to determine the level of TPG unblocking (Fig. 4).
It can be concluded that the TPG based on precursor 5 may have
practical application for the protection of a hydroxyl function. To
demonstrate the usefulness of TPG in protecting a secondary hy-
droxyl, 50-O-DMT thymidine was selected as a model compound.
Protection of this secondary hydroxyl group was performed under
the same conditions as in the previous example. The results of the
kinetic experiments showed that such a protecting group had a
higher stability (higher half-life time—Table 1). In addition, it
was shown that removal of a thermolabile protecting group does
not affect the stability of the acid–labile DMT group. This finding
has extended the scope of applications for TPGs in chemical syn-
thesis of biomolecules where a DMT group is popular.
Br
OH
N
N
R'
MW=350 W
OH
+
N
N
H
R'
R
R
Compounds 12 and 17 are products of intramolecular cycliza-
tion (Scheme 3). Analysis using NMR spectroscopy showed that a
bicyclic thermocyclization product was formed. This proves that
13
14
2-5
Scheme 2. Synthesis of the TPGs precursors.

668
M. K. Chmielewski / Tetrahedron Letters 53 (2012) 666–669
Figure 1. Two dimensional 1H–19F HOESY spectra for 5. Interactions between the F and H atoms are observed as strong signals between benzyl and weaker between
methylene hydrogens.
O
O
N
F
NH
O
N
O
N
NH
a
N
O
DMT
O
O
NH
NH
O
DMT
O
F
O
O
+
O
O
a
O
O
HO
N
O
O
N
N
+
O
O
N
N
N
F
OH
N
N
O
O
O
O
O
F
7
11
12
15
16
17
a = MeCN/buffer PBS pH=7.4; 90 °C; 10 min.
Scheme 3. Deprotection of TPG in protected nucleosides, from a primary hydroxyl group in 7 and from a secondary hydroxyl in 15.
Table 1
Half-life times for N-pyridyl-N-benzylaminoethyl carbonatesa
7
8
9
10
15
90 °C (min)
MeCN, 20 °C (h)
MeCN/phosphate buffer, 20 °C (h)
1.8
210
29
1.8
145
26
2.1
170
30
2.7
245
58
2.5
288
57
a
Calculations are based on RP C-18 HPLC analysis.
Figure 3. RP-HPLC profiles for the thermodeprotection of 7. Analyses were
performed using a 3
l
m Luna C(18)2 100 Å column (15 cm ꢁ 4.6 mm) according
to the following conditions: starting from 0.01 M triethylammonium acetate pH 7.0,
a linear gradient of 1% MeCN/min was pumped through at a flow rate of 0.8 mL/min.
intramolecular cyclization occurs according to a mechanism of
nucleophilic attack at the carbon atom adjacent to the carbonyl
group.
Figure 2. Modeled curves for the deprotection of TPGs in buffer pH 7.4/MeCN at
90 °C.

M. K. Chmielewski / Tetrahedron Letters 53 (2012) 666–669
669
mAU
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7
Figure 4. UV–vis profiles of each of the detected peaks. Analyses were performed using a UFLC Shimadzu System equipment with a diode array UV–vis detector.
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with increased stability at ambient temperature have been pre-
sented. The half-life times for these groups do not exceed
2.7 min. The study also shows that these compounds are stable
in anhydrous acetonitrile, but during long term storage in solution
these groups should be kept at 5 °C. It is planned to determine the
factors influencing the higher stability of 2-pyridyl protecting
groups in further research.
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Acknowledgment
This research was supported by a grant from the Polish State
Committee for Scientific Research in the years 2008–2011 (Grant
No. N N302098534).
Supplementary data
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Beaucage, S. L. Ann. N.Y. Acad. Sci. 2003, 1002, 1–11.
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17. Full derivation of the formula and calculation can be found in the
Supplementary data.
Supplementary data (details on the experimental procedures,
NMR and mass spectra as well as calculations of half-life times
and RP-HPLC profiles of the thermodeprotection of 15) associated
with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2011.11.122.
References and notes
1. Wuts, P. G. M.; Greene, T. W.; Haslam, E. Greene’s Protective Groups in Organic
Synthesis; John Wiley & Sons: New York, 2007.