2-Pyridinyl-N-(2,4-difluorobenzyl)aminoethyl Group As Thermocontrolled Implement for Protection of Carboxylic Acids

Article abstract of DOI:10.1021/acs.orglett.6b01475

A thermolabile protecting group strategy for carboxylic acids is expanded. Thermosensitive esters are readily prepared using a known procedure, and their stability under neutral condition is investigated. Effective thermolytic deprotection initiated only by temperature for different carboxylic acids is demonstrated, and the compatibility of a thermolytic protecting group with acidic and basic protecting groups in an orthogonal protection strategy is also presented. This study showed interesting correlations between the pK_a of acids and the deprotection rate of their well-protected moieties.

Full text of DOI:10.1021/acs.orglett.6b01475

Letter
pubs.acs.org/OrgLett
2‑Pyridinyl‑N‑(2,4-difluorobenzyl)aminoethyl Group As
Thermocontrolled Implement for Protection of Carboxylic Acids
Jolanta Brzezinska, Agnieszka Witkowska, Sandra Bałabanska, and Marcin K. Chmielewski*
́
́
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland
S
* Supporting Information
ABSTRACT: A thermolabile protecting group strategy for
carboxylic acids is expanded. Thermosensitive esters are readily
prepared using a known procedure, and their stability under
neutral condition is investigated. Effective thermolytic depro-
tection initiated only by temperature for different carboxylic acids
is demonstrated, and the compatibility of a thermolytic protecting
group with acidic and basic protecting groups in an orthogonal protection strategy is also presented. This study showed
interesting correlations between the pKa of acids and the deprotection rate of their well-protected moieties.
hemical strategies for synthesizing nature-based biopol-
ymers (peptides,1 nucleic acids,2 or polysaccharides3) still
unstable carbonic ion or phosphate anion (one negative
charge) (Scheme 1). Therefore, we can hypothesize that
C
require a combination of various protecting groups to mask
naturally occurring functions.4 These protecting groups are
stable during biopolymer synthesis or transformation but
should be removable selectively when required. This selectivity
has recently been carried out by applying mild conditions
manifested in enzymatic or thermolabile approaches. Thermo-
labile Protecting Groups (TPGs) are a modern concept where
deprotection takes place in neutral conditions only by
increasing temperature.5 This approach was successfully applied
in phosphate,6 hydroxyl,7 and amine8 protection, which
assumes universality of the thermolytic approach. In order to
demonstrate extension of the mentioned approach, we explored
it for other functional groups. Despite the fact that carboxyl
groups are less present in biopolymers (some peptides or
oligosaccharides, lipids), their dual nature (electrophilicity of a
carbonyl group and nucleophilicity of a hydroxyl group)
requires a specific strategy especially when they must remain
protected until the very last step. In practice, the carboxyl group
is most frequently protected by transforming it into an ester
form.9 For more popular applications, tris(triethylsilyl)silyl,10
tert-butyl,11 benzyl,12 9-fluorenylmethyl,13 1,3-dioxanes,14
ethyl,15 2-trimethylsilylethyl,16 and 2,2,2-trichloroethyl are
applied.17,18 Additionally, selection of the proper strategy for
protecting reactive functions is mainly based on customized sets
of protective groups, when it is necessary to remove one
protecting group without affecting others. Therefore, an
orthogonal protection strategy assumes removal of acid-labile
protecting groups without affecting base- or heat-labile ones
which remain stable under these conditions.19
Scheme 1. General Mechanism for Thermocyclization and
Ion Formation
thermocyclization might occur with a high yield only when
monoanionic acid is formed.23 In the context of carboxylic ions
which can have one negative charge, we can postulate that the
2-pyridinylaminoethyl ester of carboxylic acid will undergo
thermocyclization, thus forming such an ion. In other words,
formation of an ion with one negative charge may be a driving
force of 2-PyTPG thermolytic removal from carbonates,
phosphate triesters, or carboxylic esters.
The present paper is the first report on using a 2-pyridinyl-N-
(2,4-difluorobenzyl)aminoethyl (PydFBz) thermolabile pro-
tecting group for carboxylic acids protection which can be
selectively removed under neutral conditions.
We performed the study of their thermal stability in neutral
pH at different temperatures in relation to the acidic character
of examined acids. Additionally, we investigated the orthogon-
ality of PydFBz TPG with other protecting groups: acid-labile
bis(4-methoxyphenyl)phenylmethyl (DMT) and base-labile
2-Pyridinyl Thermolabile Protecting Groups (2-PyTPGs)
have previously been applied successfully as carbonates in
hydroxyl protection, as phosphate esters in phosphate
protection,20 and in polymerization of poly(methacrylic
acid).21 Their deprotection mechanism via specific intra-
molecular cyclization22 is based on nucleophilic pyridine
nitrogen attacks on a carbon atom and formation of an
Received: May 21, 2016
© XXXX American Chemical Society
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Scheme 2. Structure of Investigated Acids and Thermal Deprotection of PydFBzTPG from Their Esters
fluorenylmethyloxycarbonyl (FMOC) as typical representatives
in oligonucleotide24 and peptide synthesis,25 respectively. 2-
Pyridinyl-N-(2,4-difluorobenzyl)aminoethyl esters (2) were
prepared by conventional methods, from the corresponding
carboxylic acid (1) and 2-pyridinyl-N-(2,4-difluorobenzyl)-
aminoethanol (versatile TPG precursor) under mild basic
catalysis (DMAP). Effective transformation into a protected
carboxyl moiety 2 was carried out in the presence of a
condensing agent, i.e. WSC or DIC (Scheme 2).
Because we postulated that the thermal stability of TPG
depends on the ionic strength (i.e., concentration of electrical
charge) of the acid departing anion, we selected carboxylic acids
according to the different pKa values (Table 1). The acids
represent different classes whose properties vary in the
structure (aliphatic 1e or aromatic 1c, 1d), electron distribution
(electron-donating group 1c, electron-withdrawing group 1d),
or their functionality as biologically important compounds, i.e.
nucleoside 1a and amino acid 1b. This juxtaposition allows us
to observe a difference in their thermal deprotection rate and
could be helpful for designing more comprehensive 2PyTPG.
Well-protected carboxylic acids 2a−e were studied to
determine their thermolytic properties in two ways: (i)
determining the stability of 2 under different conditions and
(ii) finding optimal conditions for fast and selective
deprotection.
During the kinetic studies, we observed the formation of only
two main products on HPLC chromatograms which were
identified as recovered carboxylic acids 1, and 1-(2,4-
difluorobenzyl)-2,3-dihydro-1H-imidazo[1,2-a]pyridine-4-ium
3 with a characteristic absorption band at maximum 337 nm.26
The presence of the bicyclic product 3 confirms the mechanism
known from carbonate thermocyclization.27 Table 2 shows the
Table 2. Percentage of Thermodeprotection of 2a−e at
Different Temperatures and Times
2 h, 25 °C
2 h, 60 °C
1 h, 90 °C
2 h, 90 °C
2a
2b
2c
2d
2e
0
8
54
78
3.5
0
29
1.9
31
22
100
14
100
24
Table 1. Carboxylic Acids Used in Thermal Protection
3.2
0
98
100
45
18
data of the deprotection rate for protected carboxyl moieties
2a−e which are presented in time position (1 and 2 h heating)
and include room (25 °C), lower (60 °C), and higher (90 °C)
temperatures.
Under neutral conditions and at room temperature, the 2-
pyridinyl-N-2,4-difluorobenzylaminoethyl group is totally stable
in aqueous solution or organic solvents. We demonstrated that
total deprotection of the 2-pyridinyl-N-2,4-difluorobenzyl-
aminoethyl group is possible and effective in an aqueous
solution of 2 through simply raising the temperature. The
deprotection rate is a function of temperature, but this
dependence is not linear. We noticed that deprotection
significantly accelerates over 60 °C. However, the relation
between thermal deprotection rate and time at 90 °C is
constant (Table 2).
We also found a strong relation between pKa values of
carboxylic acids 1 and the deprotection rate of their protected
moieties 2. For the well-protected acids, whose pKa was below
4, thermal deprotection was quite fast and very effective.
However, for less acidic carboxylic acids total removal of the 2-
pyridinyl-N-(2,4-difluorobenzyl)aminoethyl group from esters
may be difficult, even at high temperatures (Table 2). We
assumed that the deprotection rate is directly proportional to
a
https://www.anaspec.com/html/pK_n_pl_Values_of_AminoAcids.
b
html. Dean, J.A. Handbook of Organic Chemistry; New York, NY:
McGraw-Hill Book Co.: 1987; pp 8−50. Fmoc: fluorenylmethyl-
oxycarbonyl. DMT: bis(4-methoxyphenyl) phenylmethyl. Thy:
thymine.
B
DOI: 10.1021/acs.orglett.6b01475
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Figure 1. HPLC analysis of 2a (left) and 2b (right) shows the compatibility of TPGs with acidic and basic protecting groups.
Scheme 3. Compatibility of PydFBz TPG with Acid- and Base-Labile Groups
pKa. This dependence is as follows: the lower the pKa, the more
labile the protecting group is under thermolytic conditions. As
we expected, the formation of a more stable carboxylic anion
during thermocyclization generates a better leaving group
which is the driving force of the deprotection process.
Additionally, we observed that storing esters 2 in anhydrous
organic solvent within 5 days did not result in significant
deprotection, particularly for acids with a pKa over 4. Our
investigations have led to the following conclusions: (i) thermal
stability depends on the pKa of carboxylic acids; (ii) a lower pKa
increases the deprotection rate; (iii) the PydFBz group is not
suitable as a protecting group for acids with a pKa over 4,
because it remains stable for several hours; and (iv) 2-pyridinyl
protecting groups could be suitable for the orthogonal
protection strategy that uses acid and basic labile protecting
groups. Since the deprotection mechanism is based on
intramolecular cyclization, TPGs could be selectively removed
in the presence of other classes of protecting groups.
Therefore, deprotection of thermolabile groups in neutral
conditions is a good reason for testing them in an orthogonal
protection system for nucleic acids or peptide synthesis. We
demonstrated that the acid-labile bis(4-methoxyphenyl)phenyl-
methyl (DMT) and base-labile fluorenylmethyloxycarbonyl
(FMOC) group were successfully removed, while the PydFBz
TPG remained intact (Figure 1). The performed HPLC
analysis revealed that a strong acid (3% TFA/DCM, rt, 5 min)
and basic conditions (morpholine/DMF, rt, 1 h) had no
influence on the stability of TPG protected nucleosides and
amino acid at ambient temperature. Moreover, after removal of
acid- and base-labile groups, full thermodeprotection of 2a−b
with release of the unmasked molecule and bicyclic product is
still possible within 3 h (Scheme 3). These observations show
that the protecting acid-labile (DMT), base-labile (FMOC),
and heat-labile (PydFBzTPG) groups are orthogonal to one
another. The advantage of this strategy is that we can
successfully remove TPG and that PydFBz could be a suitable
participating group for biopolymer synthesis.
We have shown that the 2-pyridinyl-N-(2,4-difluorobenzyl)-
aminoethyl group is a proper, quantitatively removable
protecting group for the acids with a pKa below 5 under
neutral conditions. Selective removal of the PydFBz group can
be also accomplished in the presence of the two most common
biopolymer protecting groups (DMT and Fmoc).
Moreover, we have confirmed that the deprotection
mechanism via intramolecular cyclization of 2-pyridinyl-N-
(2,4-difluorobenzyl)aminoethyl ester is the same as the
mechanism investigated on thermolytic carbonates. Due to its
advantageous properties such as ease of introduction, selective
removal, and orthogonality, the PydFBz is foreseen as a
valuable tool for the protection of carboxyl groups.
Thorough recognition of structural dependencies of
individual TPGs and examination of the deprotection
mechanism will allow us to precisely design a protecting
group for concrete application. We are also planning to study
the stability of thermolitic esters in a wide range of
temperatures and pH’s. In the near future we will be testing
the chemical synthesis of peptides using thermolytically
protected carboxylic acids.
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DOI: 10.1021/acs.orglett.6b01475
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Letter
(20) (a) Ciesl
́
ak, J.; Beaucage, S. L. J. Org. Chem. 2003, 68, 10123−
ASSOCIATED CONTENT
■
10129. (b) Ratajczak, T.; Chmielewski, M. K. J. Org. Chem. 2012, 77,
7866−7872.
S
* Supporting Information
(21) Elladiou, M.; Patrickios, C. S. Polym. Chem. 2012, 3, 3228−
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b01475.
3231.
(22) Chmielewski, M. K.; Tykarska, E.; Markiewicz, W. T.;
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General synthetic procedures, HPLC analysis at different
temperatures and times, and NMR and HRMS data for
all discussed compounds (PDF)
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: maro@ibch.poznan.pl.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Prof. Jacek Stawin
ski (Institute of
Bioorganic Chemistry, PAS, Poland) for his comments and
suggestions, which helped us to considerably improve the
manuscript. This research was supported by the European
Regional Development Fund within the Innovative Economy
Programme [Grant No. POIG.01.03.01-30-045] and was also
supported by the Polish Ministry of Science and Higher
Education, under the KNOW program.
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DOI: 10.1021/acs.orglett.6b01475
Org. Lett. XXXX, XXX, XXX−XXX